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Pharmacogenomics—a New Frontier for Individualized Treatment of Parkinson’s Disease
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a00535ec-c983-4904-93b8-e5533385b63c
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10207905
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Pharmacology[mh]
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INTRODUCTION PD is the second most frequent neurodegenerative disease, affecting approximately 1% of individuals >60 years of age globally . In China, its prevalence rate among people aged over 65 years is about 1.7% . Therefore, as PD is more prevalent in older people, it creates the highest public health burden in the elderly population. It is characterized by a progressive loss of dopamine neurons in the central nervous system. PD manifests clinically as motor symptoms such as bradykinesia, resting tremor, and muscular rigidity, as well as various non-motor symptoms such as autonomic dysfunction, olfactory disturbances, sleep disorders, and cognitive deficits . In recent years, advances in scientific research technologies have provided a new understanding of the occurrence and etiological mechanism of PD. The major pathological manifestations of PD include intracellular aggregation of α-synuclein, which forms Lewy bodies, and loss of dopaminergic neurons, which first occur in the substantia nigra before spreading to other brain parts as the disease progresses . The main mechanism underlying the development of motor symptoms in PD is the loss of dopamine neurons in the substantia nigra. Therefore, exogenous dopamine supplementation is a standard treatment strategy for PD . PD treatment includes both pharmacological therapy and non-pharmacological treatment. Pharmacological therapy remains a crucial treatment method for the current management of PD. Compared with other neurodegenerative diseases, PD symptoms are effectively controlled by drug treatments, thereby improving the quality of life. Based on the mechanism of action, anti-Parkinson's drugs are divided into six categories: dopaminergic drugs, dopamine receptor (DR) agonists, anticholinergic drugs, amantadine, monoamine oxidase (MAO) inhibitors, and catechol-O-methyltransferase (COMT) inhibitors. Unfortunately, these drugs can only alleviate PD symptoms, but cannot delay the progression of the disease, cure it, or reverse its neurodegenerative effects . In clinical settings, patients respond differently to anti-Parkinson's drugs. The following three elements demonstrate the personalized variances in anti-PD medicines . 1) Differences in initial effective dosage: the treatment of anti-PD drugs emphasizes the principle of dose titration. The minimal effective dosage required to induce adequate relief in clinical symptoms varies substantially across people. Some patients achieve considerable relief in symptoms after obtaining a lower dose of medication, whilst others must titrate to a higher dose in order to fulfill their fundamental needs in life. 2) Side effect differences: There is greater individual diversity in the side effects of anti-PD medications. Some people experience no or just slight adverse effects, whilst others experience major side effects such as palpitation, gastrointestinal issues, and impulse control disorder. 3) The disparities in motor problems: most patients who have been on anti-PD medicines for a long time will experience motor complications such as dyskinesia and wearing out, which will have a negative impact on their quality of life. However, the onset of motor problems varies widely between patients, and a small percentage of people who take anti-PD medications for many years still achieve good effectiveness. The mechanism behind this phenomenon remains unclear but many experts suggest a tic basis. In recent decades, mainly due to the development of sequencing technology and increased availability of affordable genetic testing methods, substantial progress has been made in the identification of genetic biomarkers of drug response. Particular gene variants have been associated with drug inefficacy, hypersensitivity, or increased toxicity risk. In addition, there have been efforts to define the role of genetic polymorphisms in optimizing the pharmacotherapy of PD. Studies of the role of genetic variation in human-to-human differences associated with drug responses and adverse reactions may shed light on the mechanisms underlying interindividual variability observed in response to antiparkinsonian agents . Current evidence-based medicine emphasizes drug efficacy in the entire population but ignores individual differences, which makes it less effective in specific subpopulations. Therefore, considering differences in genetic susceptibility, the individualized precision therapy strategy is desirable . The two main research areas in drug response variability are pharmacokinetics and pharmacodynamics. The accumulated to date mainly encompasses three effects of genetic variation on drug response characteristics: affecting serum drug concentration, changing the ability of a drug to cross the blood-brain barrier, and modifying pharmacodynamic characteristics . Pharmacogenomics refers to the study of drug response in various diseases due to genetic variations and the development of new drugs or new drug use methods on this basis. Pharmacogenomic studies have proved that some drugs perform better in certain populations with specific genes, or that the selection of therapeutic drugs based on genes improves the effectiveness of drugs and avoids adverse reactions . Therefore, the ultimate purpose of pharmacogenomics is to identify genetic factors for the different drug responses among individuals, providing a precise personalized medical treatment model. Anti-Parkinson's drugs show 60-90% variability in pharmacokinetics and pharmacodynamics . Transport, metabolic, mechanistic, pathogenic, and polyclonal genes are involved in pharmacogenomics. Recently, increasing evidence suggested that pharmacogenomics affects the efficacy and safety of antiparkinsonian drugs. Therefore, combining personalized treatment of PD with pharmacogenomics could improve the efficacy and safety of drugs. This is a narrative review comprising a comprehensive summary and novel data compiled from all pharmacogenomic studies published in various databases up to May of this year. To get a better understanding of anti-pharmacogenomics Parkinson's research, we analyzed existing pharmacogenomics research based on the categories of anti-drugs Parkinson's and characterized the study hotspot-polymorphisms in levodopa-related genes in terms of medication efficacy and drug adverse effects. This will serve as a significant reference point for future studies and clinical applications of anti-Parkinson pharmacogenomic research. METHODS We collected the studies of PD pharmacogenomics published in multiple databases, critically examined current discoveries, enumerated the recent progress of major genes (Table ), then elaborated on the genetic factors with strong reliability and great clinical value. For a more intuitive understanding of the current research status, we have listed the genetic factors associated with drug response according to the classification of anti-parkinsonian drugs, as shown in Fig. . RESULTS-CURRENT ACHIEVEMENTS IN PD PHARMACOGENOMICS 3.1 Pharmacogenetic Studies of Levodopa Levodopa is one of the most commonly used and effective drugs for PD treatment. Most pharmacogenomic studies have explored polymorphisms associated with its response and adverse effects. The investigated genes were mainly those involved in levodopa metabolism, transport, and excretion, including the catecholamine-o-methyltransferase (COMT) gene, the dopamine receptors (DRD1-5) gene, the dopamine transporter (DAT) gene, the dopa decarboxylase enzyme (DDC) related genes and others . Among them, the COMT gene has been confirmed to play a role in levodopa response. 3.1.1 COMT Genes The COMT gene is localized on chromosome 22q11.1-q11.2. It encodes catecholamine-o-methyltransferase (COMT), which converts levodopa into 3-O-methyldopa in the body . Elevated levels of COMT increase the degradation of L-levodopa, thereby reducing its effectiveness. Conversely, combining levodopa with COMT inhibitors enhances its efficacy. G1947A polymorphism (also called RS4680) in exon 4 of the COMT gene changes valine (Val) to methionine (Met) at amino acid 158 of the COMT enzyme, affecting the thermal instability of the enzyme and decreasing its activity . Studies have found that COMT enzyme activity varies greatly among individuals. Three phenotypes have been characterized: high activity, moderate activity, and low activity in the population, which are determined by co-dominantly inherited COMT-H (high) and COMT-L (low) alleles. Their genotypes include COMT-HH (Val/Val), COMT-HL (Val/Met), and COMT-LL (Met/Met) . Several experiments have confirmed that the COMT genotype affects the efficacy of levodopa. Generally, other conditions held constant, patients with a highly active COMT (COMT-HH) phenotype tend to require a larger dose of levodopa than those with a lowly active COMT (COMT-LL) phenotype. Bialecka and his colleagues divided 95 patients with sporadic PD into two groups based on daily levodopa dose: group 1 received levodopa at dose > 500 mg per day and group 2 received levodopa at dose < 500 mg per day. They found that the COMT-LL genotype was more common in group 2 than in group 1 . A study by Cheshire et al. examined the effect of COMT, MAO-A, and BDNF polymorphisms on levodopa response in 285 patients with PD, establishing that homozygous COMT activity was associated with a higher maximum daily dose of levodopa . These findings were recently corroborated by Sampaio et al. research . Better clinical response to lower L-dopa doses in patients with low COMT activity may be explained by slower catabolism of the drug, more stable serum and CNS (central nervous system) drug concentrations, and lower levels of 3-O-MD. However, some studies showed no significant correlation between COMT gene polymorphism and levodopa use . 3.1.2 DR Genes Dopamine receptor genes DRD1, DRD2, DRD3, DRD4, and DRD5 , encode dopamine receptors D1, D2, D3, D4, and D5, respectively. Current studies about DR gene polymorphism and drug response mainly focus on the effects of DRD2 and DRD3 gene polymorphisms on the severity of the side effects of dopamine preparation. Few studies have found that the DRD2 and DRD3 gene polymorphisms are associated with the maximum daily dose limit of levodopa tolerated by patients. DRD2 is located at 11q22-23, with its main genetic variant, TaqIA (RS1800497) associated with movement effects, including movement fluctuations and disorders induced by L-dopa. TaqIA originally belonged to the DRD2 gene family but was later classified as the ANKK1 gene, which lies downstream of DRD2 and overlaps with DRD2 . Kaiser et al. found no correlation between DRD2/ANKK1 and the clinical characteristics of patients treated with L-dopa. In contrast, Dos Santos et al. found that DRD2/ANKK1 was significantly associated with increased doses of L-dopa in a 2019 study involving 195 patients with sporadic PD in Brazil . 3.1.3 SLC22A1, SLC6A3, and SV2C Genes The solute carrier (SLC) transporter family is the second-largest membrane protein family in human cells, which is distributed on various biofilm structures in cells, including nuclear and cytoplasmic membranes. SLC susceptibility sites are potential therapeutic targets for various diseases, with SLC22A1 and SLC6A3 genes being clinically significant to PD pharmacogenomics. Becker et al. collected levodopa dosage for 7,983 PD patients aged over 55 years and found a higher dosage of levodopa in SLC22A1 gene carriers than that in the control group . The SLC6A3 gene which encodes the dopamine transporter is highly expressed in dopaminergic neurons in the presynaptic midbrain. Previous studies have found that double-allelic mutations in the SLC6A3 gene cause dopamine transporter deficiency syndrome (DTDS), manifested as PD in infants. In a 2018 study, Altmann et al. conducted multiple regression analysis of levodopa dose and SLC6A3 gene in 224 patients demonstrating that SLC6A3 was associated with low doses of levodopa . Similar results were found in the SV2C genome . In contrast, MTHFR TT677 mutants had a lower daily levodopa dose . It is evident that the studies reviewed on the pharmacogenomics of levodopa differ in the ethnicity of patients, choice of study sites, study endpoints, and methods of analysis. In particular, ethnic differences of study participants may explain the contradictory results of different studies. 3.2 Pharmacogenomics of Levodopa Side Effects PD patients taking levodopa for a long period may experience various side effects. The most common side effects are dyskinesia, end-use phenomena, visual hallucinations, daytime lethargy, and impulse control disorders. 3.2.1 COMT Gene Previous studies have found that L-dopa dose is influenced by the COMT genotype. Recent studies have found that COMT polymorphisms are associated with side effects of long-term use of L-dopa. A paper published by Sampaio and his team in 2018 suggested that patients with the COMT-LL phenotype are more prone to levodopa-induced dyskinesia (LID) , which was corroborated by de Lau et al. ’s research . In other words, patients with low COMT enzyme activity are more likely to develop levodopa-induced dyskinesia than those with high COMT enzyme activity. The low-activity COMT enzyme reduces levodopa degradation, increasing dopamine accumulation in the synaptic cleft, which causes dyskinesia. Cheshire et al. and Watanabe et al. also explored this question . However, both studies found no significant correlation between low-activity COMT enzyme and LID, which may be attributed to individual differences between patients (that is, some patients may have multiple genotypes affecting LID at the same time). 3.2.2 DR Gene Currently, the DRD2 gene is the main dopamine receptor gene associated with levodopa side effects. It is highly expressed in the basal ganglia, which is a motor-regulating region of the central nervous system and is crucial to PD. Oliveri is the first researcher to study the role of DRD2 polymorphism in LID . In his study, the frequency of both alleles 13 and 14 of DRD2 gene polymorphism was higher in non-dyskinetic than in dyskinetic PD patients. In addition, carrying at least 1 of the 13 or 14 alleles reduced the risk of developing peak-dose dyskinesias by 72% in PD patients compared to PD patients not carrying these alleles. Further, DRD1 polymorphisms were not associated with the risk of developing PD or peak-dose dyskinesias. Rieck et al. verified this relationship with 199 Brazilian PD patients. Strong and his colleagues found that the DRD2 gene was involved in an early-onset of LID in 92 PD patients and further determined that the early onset PD was due to 14 and 15 DRD2 alleles . Some researchers have also found that DRD3 gene polymorphism is associated with levodopa-induced side effects, but evidence supporting this conclusion is still insufficient. 3.2.3 DAT Genes The dopamine transporter (DAT, SLC6A3) plays an important role in controlling the intensity and duration of dopaminergic neurotransmission by rapid reuptake DA into presynaptic terminals . The single nucleotide polymorphism (RS393795) in the DAT gene is significantly correlated with the onset time of levodopa-induced motor dysfunction, whereas the C allele of DAT is associated with late-onset LID, which may be regulated by a change in dopamine reuptake rate in synaptic cleft . Sossi et al. have shown that greater DAT expression levels are directly associated with lower dopamine turnover and lower changes in synaptic dopamine concentration in PD patients . Troiano et al. subsequently confirmed decreased DAT in the presynaptic membrane using a DAT-PET (Dopamine transporter-Positron Emission Tomography) . In addition to these three genes implicated in dopamine metabolism, the following genes are also potentially involved in LID. LID is a type of motor complication common in patients with PD during chronic levodopa therapy. It is reported that 40% of patients develop LID four years after receiving levodopa therapy. This risk is even higher among younger patients receiving high doses of levodopa therapy. In addition to the early onset age of PD and higher L-dopa total exposure, other studies have found that LID is more common in patients with a longer course of PD, lower body mass index, and female gender . Several researchers have also investigated the potential involvement of genetic factors or individual genetic variations in the development of LID. Analysis of the chi-square correlation between genotype and the presence of multiple levodopa-induced side effects in 205 PD patients by Schumacher-schuh found that rs4704559 G ( HOMER1 ) allele was associated with a lower prevalence of dyskinesia . Contrastingly, a higher prevalence of LID was found in patients with MAO-B (rs1799836, A644G) A allele and AA genotype and in patients with mTOR gene polymorphism after multivariate analysis. A better understanding of the role played by genetic factors in the development of LID may be important to identify patients more likely to develop LID and elucidate the molecular mechanisms underlying this causal link. With the increasing understanding of the adverse side effects of levodopa, more and more studies have focused on the influence of genotypes on side effects other than LID. Some studies have found an association between COMT gene polymorphism and the Epworth Sleepiness Scale (ESS, a scale designed by Johns MW to assess excessive daytime sleepiness state, facilitating quantitative semi-objective evaluation of the drowsiness state). Sleep disturbances are a well-known disabling nonmotor manifestation of PD, affecting almost 80% of patients . Patients with COMT-LH and COMT-LL genotypes had higher ESS scores than patients with COMT-HH, suggesting that these patients were more likely to experience daytime sleepiness . However, Rissling’s study yielded contradictory results . An SNP C667T (rs1801133) in the MTHFR gene was consistently linked to hyperhomocysteinemia (HHcy), which is defined as elevated serum concentrations of homocysteine (Hcy) exceeding 15 µmol/L . Elevated Hcy levels have been associated with increased cardiovascular, cerebrovascular, and thromboembolic diseases due to the L-dopa treatment in several studies. This mutation generates a temperature-labile MTHFR enzyme, which ultimately leads to hyperhomocysteinemia . Regarding visual hallucinations as side effects, current research suggests that the COMT RS165815 C allele is a protective factor against visual hallucinations , as the incidence of visual hallucinations in patients with this allele is relatively low. In contrast, the DRD3 RS6280 C genotype is a risk factor for visual hallucinations , whereas DRD2 TaqIA C has been associated with delayed visual hallucinations . Other studies have confirmed that OPRK1, HTR2a, and DDC genotypes are associated with the incidence of ICD(impulse control disorder) . However, these results have only been confirmed by a few studies focusing on occidental patient samples. It is expected that more studies will be conducted in this area, especially focusing on the Asian population, to support these conclusions. 3.3 Pharmacogenetic Studies on Dopamine Receptor Agonists Dopamine agonists are one of the most commonly used drugs in PD treatment but have lower efficacy than levodopa. Many studies have shown that dopamine agonists could overcome the shortcomings of levodopa, strengthen levodopa’s curative effect and delay complications. The combination of low-dose levodopa and dopamine receptor agonists is as effective as high-dose levodopa alone, but with a significantly lower incidence of side effects. In the later stages of the disease, due to gradual degeneration and loss of dopaminergic neurons in the nigrostriatal system, exogenous levodopa decarboxylation is no longer converted to dopamine. At this point, levodopa becomes ineffective, while dopamine receptor agonists remain effective. Examples of commonly used dopamine receptor agonists include pramipexole, bromocriptine, and rotigotine. Presently, studies examining the relationship between dopamine receptor agonists and genomics mainly focus on the effect of gene polymorphism on the efficacy of dopamine receptor agonists. 3.3.1 DRD2 and DRD3 Gene The DRD2 and DRD3 gene polymorphisms may influence drug efficacy and tolerance. Together with his team, Liu found that DRD3 Ser/Gly polymorphism influenced the efficacy of dopamine receptor agonists in 30 Chinese PD patients , which was corroborated by Xu’s study . To investigate the association between Dopamine receptor D type 2 (DRD2) dinucleotide short tandem repeat (CA(n)-STR) and Dopamine receptor D type 3 (DRD3) Ser9Gly polymorphisms and different doses of Dopamine receptor agonists (DAs) in PD patients, professor Xu recruited 168 idiopathic PD patients and 182 controls. Further exploration showed no association between DRD2 CA(n)-STR polymorphism and DA dosage. Among patients with three different DRD3 Ser9Gly genotypes (Ser/Ser, Ser/Gly, and Gly/Gly), patients carrying Gly/Gly genotype used higher doses of DAs than those with Ser/Gly and Ser/Ser genotypes. DRD3 Ser9Gly Ser/Ser genotype has a higher response rate to dopamine agonists and requires a smaller dose. In a recent study of patients in Brazil, the presence of the TTCTA haplotype, derived from five DRD2 SNPs, was also linked with a high risk of dyskinesia . 3.3.2 DRD4 Gene Sleep attacks in PD were initially reported to occur only with particular dopamine agonists, pramipexole, and ropinirole. The DRD4 48-bp VNTR short/short variant was significantly associated with sleep attacks without warning signs . In general, research in this area is insufficient. Considering the complexity of nervous system diseases, comorbidities in elderly patients, multiple treatment regimens, lack of dose-response relation for many drugs, and other non-genetic factors affecting treatment results, as well as various problems that may be encountered in the design and implementation of PD pharmacogenomics conclusive studies, confirming the clinical utility of pharmacogenomics is challenging. Therefore, the positive results obtained for some candidate genes studied to date indicate that pharmacogenomics of PD warrants more extensive studies involving more uniform, large patient groups to minimize the effect of nongenetic factors. 3.4 Pharmacogenetic Studies on Other Anti-PD Drugs Until now, levodopa has been the most effective treatment for patients with PD. However, with long-term use, the effect of levodopa gradually decreases and contributes to various motor complications. Therefore, for patients with advanced PD, combination therapy including levodopa and other drugs is routinely indicated. Monoamine oxidase (MAO) inhibitor drugs, such as selegiline and rasagiline, could reduce the metabolic degradation of levodopa, thus they are often concurrently administered with it . In later stages of the disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce ‘off’ time and may improve gait and freezing . Monoamine oxidase is an extra-mitochondrial protein found in almost all human tissues . This enzyme regulates neurotransmitter metabolism in the brain and other systems. Therefore, its inhibition can boost neurotransmitter levels in the brain. MAO inhibitors can be divided into two categories according to their pharmacological effects. MAO-A is primarily involved in the oxidative metabolism of tyramine, whereas inhibition of MAO-B primarily reduces the metabolism of dopamine and β-phenylethylamine . Because of their role in levodopa metabolism, selective MAO-B inhibitors are clinically used for PD treatment drugs. Few pharmacogenetic studies have evaluated the effect of DRD2 gene polymorphisms on the clinical response to rasagiline. The results of a large study including 692 PD patients indicated that two SNPs of the DRD2 gene, rs2283265, and rs1076560, were significantly correlated with improved motor function in response to 12-week management with rasagiline after controlling for placebo effects . COMT inhibitors are concomitantly used with levodopa to inhibit levodopa metabolism. PD treatment is based on replacing lost DA with levodopa, which can pass the blood-brain barrier. Administration of COMT inhibitors blocks methylation of levodopa to 3-methyldopa (3-OMD) through the inhibition of the COMT enzyme, thereby preventing levodopa degradation through this major peripheral metabolic pathway and improving its clinical potency efficacy. Presently, COMT inhibitors commonly used in clinical practice include entacapone, tolcapone, and opicapone. Tolcapone and entacapone have mainly been used with patients with more advanced diseases, including chronic motor fluctuations. Whereas tolcapone is associated with hepatotoxicity, entacapone has a short half-life in plasma and requires dosing with every administration of levodopa . However, compared with other COMT inhibitors, opicapone, a third-generation COMT inhibitor, not only reduces the risk of toxicity but also improves COMT inhibitory efficacy and peripheral tissue selectivity . In general, the COMT inhibitors such as entacapone and tolcapone are concomitantly used with levodopa, as they could inhibit the metabolism of levodopa. The effect of genetic variants in the COMT gene on clinical response to entacapone has been evaluated in a clinical trial including 33 PD patients. The results demonstrated that entacapone-treated patients with Val/Val genotype showed a higher levodopa concentration . Besides, the A528G SNP of the UGT1A6 gene is associated with the hepatotoxicity of entacapone and tolcapone in a large European study of 409 PD patients . Pharmacogenetic Studies of Levodopa Levodopa is one of the most commonly used and effective drugs for PD treatment. Most pharmacogenomic studies have explored polymorphisms associated with its response and adverse effects. The investigated genes were mainly those involved in levodopa metabolism, transport, and excretion, including the catecholamine-o-methyltransferase (COMT) gene, the dopamine receptors (DRD1-5) gene, the dopamine transporter (DAT) gene, the dopa decarboxylase enzyme (DDC) related genes and others . Among them, the COMT gene has been confirmed to play a role in levodopa response. 3.1.1 COMT Genes The COMT gene is localized on chromosome 22q11.1-q11.2. It encodes catecholamine-o-methyltransferase (COMT), which converts levodopa into 3-O-methyldopa in the body . Elevated levels of COMT increase the degradation of L-levodopa, thereby reducing its effectiveness. Conversely, combining levodopa with COMT inhibitors enhances its efficacy. G1947A polymorphism (also called RS4680) in exon 4 of the COMT gene changes valine (Val) to methionine (Met) at amino acid 158 of the COMT enzyme, affecting the thermal instability of the enzyme and decreasing its activity . Studies have found that COMT enzyme activity varies greatly among individuals. Three phenotypes have been characterized: high activity, moderate activity, and low activity in the population, which are determined by co-dominantly inherited COMT-H (high) and COMT-L (low) alleles. Their genotypes include COMT-HH (Val/Val), COMT-HL (Val/Met), and COMT-LL (Met/Met) . Several experiments have confirmed that the COMT genotype affects the efficacy of levodopa. Generally, other conditions held constant, patients with a highly active COMT (COMT-HH) phenotype tend to require a larger dose of levodopa than those with a lowly active COMT (COMT-LL) phenotype. Bialecka and his colleagues divided 95 patients with sporadic PD into two groups based on daily levodopa dose: group 1 received levodopa at dose > 500 mg per day and group 2 received levodopa at dose < 500 mg per day. They found that the COMT-LL genotype was more common in group 2 than in group 1 . A study by Cheshire et al. examined the effect of COMT, MAO-A, and BDNF polymorphisms on levodopa response in 285 patients with PD, establishing that homozygous COMT activity was associated with a higher maximum daily dose of levodopa . These findings were recently corroborated by Sampaio et al. research . Better clinical response to lower L-dopa doses in patients with low COMT activity may be explained by slower catabolism of the drug, more stable serum and CNS (central nervous system) drug concentrations, and lower levels of 3-O-MD. However, some studies showed no significant correlation between COMT gene polymorphism and levodopa use . 3.1.2 DR Genes Dopamine receptor genes DRD1, DRD2, DRD3, DRD4, and DRD5 , encode dopamine receptors D1, D2, D3, D4, and D5, respectively. Current studies about DR gene polymorphism and drug response mainly focus on the effects of DRD2 and DRD3 gene polymorphisms on the severity of the side effects of dopamine preparation. Few studies have found that the DRD2 and DRD3 gene polymorphisms are associated with the maximum daily dose limit of levodopa tolerated by patients. DRD2 is located at 11q22-23, with its main genetic variant, TaqIA (RS1800497) associated with movement effects, including movement fluctuations and disorders induced by L-dopa. TaqIA originally belonged to the DRD2 gene family but was later classified as the ANKK1 gene, which lies downstream of DRD2 and overlaps with DRD2 . Kaiser et al. found no correlation between DRD2/ANKK1 and the clinical characteristics of patients treated with L-dopa. In contrast, Dos Santos et al. found that DRD2/ANKK1 was significantly associated with increased doses of L-dopa in a 2019 study involving 195 patients with sporadic PD in Brazil . 3.1.3 SLC22A1, SLC6A3, and SV2C Genes The solute carrier (SLC) transporter family is the second-largest membrane protein family in human cells, which is distributed on various biofilm structures in cells, including nuclear and cytoplasmic membranes. SLC susceptibility sites are potential therapeutic targets for various diseases, with SLC22A1 and SLC6A3 genes being clinically significant to PD pharmacogenomics. Becker et al. collected levodopa dosage for 7,983 PD patients aged over 55 years and found a higher dosage of levodopa in SLC22A1 gene carriers than that in the control group . The SLC6A3 gene which encodes the dopamine transporter is highly expressed in dopaminergic neurons in the presynaptic midbrain. Previous studies have found that double-allelic mutations in the SLC6A3 gene cause dopamine transporter deficiency syndrome (DTDS), manifested as PD in infants. In a 2018 study, Altmann et al. conducted multiple regression analysis of levodopa dose and SLC6A3 gene in 224 patients demonstrating that SLC6A3 was associated with low doses of levodopa . Similar results were found in the SV2C genome . In contrast, MTHFR TT677 mutants had a lower daily levodopa dose . It is evident that the studies reviewed on the pharmacogenomics of levodopa differ in the ethnicity of patients, choice of study sites, study endpoints, and methods of analysis. In particular, ethnic differences of study participants may explain the contradictory results of different studies. COMT Genes The COMT gene is localized on chromosome 22q11.1-q11.2. It encodes catecholamine-o-methyltransferase (COMT), which converts levodopa into 3-O-methyldopa in the body . Elevated levels of COMT increase the degradation of L-levodopa, thereby reducing its effectiveness. Conversely, combining levodopa with COMT inhibitors enhances its efficacy. G1947A polymorphism (also called RS4680) in exon 4 of the COMT gene changes valine (Val) to methionine (Met) at amino acid 158 of the COMT enzyme, affecting the thermal instability of the enzyme and decreasing its activity . Studies have found that COMT enzyme activity varies greatly among individuals. Three phenotypes have been characterized: high activity, moderate activity, and low activity in the population, which are determined by co-dominantly inherited COMT-H (high) and COMT-L (low) alleles. Their genotypes include COMT-HH (Val/Val), COMT-HL (Val/Met), and COMT-LL (Met/Met) . Several experiments have confirmed that the COMT genotype affects the efficacy of levodopa. Generally, other conditions held constant, patients with a highly active COMT (COMT-HH) phenotype tend to require a larger dose of levodopa than those with a lowly active COMT (COMT-LL) phenotype. Bialecka and his colleagues divided 95 patients with sporadic PD into two groups based on daily levodopa dose: group 1 received levodopa at dose > 500 mg per day and group 2 received levodopa at dose < 500 mg per day. They found that the COMT-LL genotype was more common in group 2 than in group 1 . A study by Cheshire et al. examined the effect of COMT, MAO-A, and BDNF polymorphisms on levodopa response in 285 patients with PD, establishing that homozygous COMT activity was associated with a higher maximum daily dose of levodopa . These findings were recently corroborated by Sampaio et al. research . Better clinical response to lower L-dopa doses in patients with low COMT activity may be explained by slower catabolism of the drug, more stable serum and CNS (central nervous system) drug concentrations, and lower levels of 3-O-MD. However, some studies showed no significant correlation between COMT gene polymorphism and levodopa use . DR Genes Dopamine receptor genes DRD1, DRD2, DRD3, DRD4, and DRD5 , encode dopamine receptors D1, D2, D3, D4, and D5, respectively. Current studies about DR gene polymorphism and drug response mainly focus on the effects of DRD2 and DRD3 gene polymorphisms on the severity of the side effects of dopamine preparation. Few studies have found that the DRD2 and DRD3 gene polymorphisms are associated with the maximum daily dose limit of levodopa tolerated by patients. DRD2 is located at 11q22-23, with its main genetic variant, TaqIA (RS1800497) associated with movement effects, including movement fluctuations and disorders induced by L-dopa. TaqIA originally belonged to the DRD2 gene family but was later classified as the ANKK1 gene, which lies downstream of DRD2 and overlaps with DRD2 . Kaiser et al. found no correlation between DRD2/ANKK1 and the clinical characteristics of patients treated with L-dopa. In contrast, Dos Santos et al. found that DRD2/ANKK1 was significantly associated with increased doses of L-dopa in a 2019 study involving 195 patients with sporadic PD in Brazil . SLC22A1, SLC6A3, and SV2C Genes The solute carrier (SLC) transporter family is the second-largest membrane protein family in human cells, which is distributed on various biofilm structures in cells, including nuclear and cytoplasmic membranes. SLC susceptibility sites are potential therapeutic targets for various diseases, with SLC22A1 and SLC6A3 genes being clinically significant to PD pharmacogenomics. Becker et al. collected levodopa dosage for 7,983 PD patients aged over 55 years and found a higher dosage of levodopa in SLC22A1 gene carriers than that in the control group . The SLC6A3 gene which encodes the dopamine transporter is highly expressed in dopaminergic neurons in the presynaptic midbrain. Previous studies have found that double-allelic mutations in the SLC6A3 gene cause dopamine transporter deficiency syndrome (DTDS), manifested as PD in infants. In a 2018 study, Altmann et al. conducted multiple regression analysis of levodopa dose and SLC6A3 gene in 224 patients demonstrating that SLC6A3 was associated with low doses of levodopa . Similar results were found in the SV2C genome . In contrast, MTHFR TT677 mutants had a lower daily levodopa dose . It is evident that the studies reviewed on the pharmacogenomics of levodopa differ in the ethnicity of patients, choice of study sites, study endpoints, and methods of analysis. In particular, ethnic differences of study participants may explain the contradictory results of different studies. Pharmacogenomics of Levodopa Side Effects PD patients taking levodopa for a long period may experience various side effects. The most common side effects are dyskinesia, end-use phenomena, visual hallucinations, daytime lethargy, and impulse control disorders. 3.2.1 COMT Gene Previous studies have found that L-dopa dose is influenced by the COMT genotype. Recent studies have found that COMT polymorphisms are associated with side effects of long-term use of L-dopa. A paper published by Sampaio and his team in 2018 suggested that patients with the COMT-LL phenotype are more prone to levodopa-induced dyskinesia (LID) , which was corroborated by de Lau et al. ’s research . In other words, patients with low COMT enzyme activity are more likely to develop levodopa-induced dyskinesia than those with high COMT enzyme activity. The low-activity COMT enzyme reduces levodopa degradation, increasing dopamine accumulation in the synaptic cleft, which causes dyskinesia. Cheshire et al. and Watanabe et al. also explored this question . However, both studies found no significant correlation between low-activity COMT enzyme and LID, which may be attributed to individual differences between patients (that is, some patients may have multiple genotypes affecting LID at the same time). 3.2.2 DR Gene Currently, the DRD2 gene is the main dopamine receptor gene associated with levodopa side effects. It is highly expressed in the basal ganglia, which is a motor-regulating region of the central nervous system and is crucial to PD. Oliveri is the first researcher to study the role of DRD2 polymorphism in LID . In his study, the frequency of both alleles 13 and 14 of DRD2 gene polymorphism was higher in non-dyskinetic than in dyskinetic PD patients. In addition, carrying at least 1 of the 13 or 14 alleles reduced the risk of developing peak-dose dyskinesias by 72% in PD patients compared to PD patients not carrying these alleles. Further, DRD1 polymorphisms were not associated with the risk of developing PD or peak-dose dyskinesias. Rieck et al. verified this relationship with 199 Brazilian PD patients. Strong and his colleagues found that the DRD2 gene was involved in an early-onset of LID in 92 PD patients and further determined that the early onset PD was due to 14 and 15 DRD2 alleles . Some researchers have also found that DRD3 gene polymorphism is associated with levodopa-induced side effects, but evidence supporting this conclusion is still insufficient. 3.2.3 DAT Genes The dopamine transporter (DAT, SLC6A3) plays an important role in controlling the intensity and duration of dopaminergic neurotransmission by rapid reuptake DA into presynaptic terminals . The single nucleotide polymorphism (RS393795) in the DAT gene is significantly correlated with the onset time of levodopa-induced motor dysfunction, whereas the C allele of DAT is associated with late-onset LID, which may be regulated by a change in dopamine reuptake rate in synaptic cleft . Sossi et al. have shown that greater DAT expression levels are directly associated with lower dopamine turnover and lower changes in synaptic dopamine concentration in PD patients . Troiano et al. subsequently confirmed decreased DAT in the presynaptic membrane using a DAT-PET (Dopamine transporter-Positron Emission Tomography) . In addition to these three genes implicated in dopamine metabolism, the following genes are also potentially involved in LID. LID is a type of motor complication common in patients with PD during chronic levodopa therapy. It is reported that 40% of patients develop LID four years after receiving levodopa therapy. This risk is even higher among younger patients receiving high doses of levodopa therapy. In addition to the early onset age of PD and higher L-dopa total exposure, other studies have found that LID is more common in patients with a longer course of PD, lower body mass index, and female gender . Several researchers have also investigated the potential involvement of genetic factors or individual genetic variations in the development of LID. Analysis of the chi-square correlation between genotype and the presence of multiple levodopa-induced side effects in 205 PD patients by Schumacher-schuh found that rs4704559 G ( HOMER1 ) allele was associated with a lower prevalence of dyskinesia . Contrastingly, a higher prevalence of LID was found in patients with MAO-B (rs1799836, A644G) A allele and AA genotype and in patients with mTOR gene polymorphism after multivariate analysis. A better understanding of the role played by genetic factors in the development of LID may be important to identify patients more likely to develop LID and elucidate the molecular mechanisms underlying this causal link. With the increasing understanding of the adverse side effects of levodopa, more and more studies have focused on the influence of genotypes on side effects other than LID. Some studies have found an association between COMT gene polymorphism and the Epworth Sleepiness Scale (ESS, a scale designed by Johns MW to assess excessive daytime sleepiness state, facilitating quantitative semi-objective evaluation of the drowsiness state). Sleep disturbances are a well-known disabling nonmotor manifestation of PD, affecting almost 80% of patients . Patients with COMT-LH and COMT-LL genotypes had higher ESS scores than patients with COMT-HH, suggesting that these patients were more likely to experience daytime sleepiness . However, Rissling’s study yielded contradictory results . An SNP C667T (rs1801133) in the MTHFR gene was consistently linked to hyperhomocysteinemia (HHcy), which is defined as elevated serum concentrations of homocysteine (Hcy) exceeding 15 µmol/L . Elevated Hcy levels have been associated with increased cardiovascular, cerebrovascular, and thromboembolic diseases due to the L-dopa treatment in several studies. This mutation generates a temperature-labile MTHFR enzyme, which ultimately leads to hyperhomocysteinemia . Regarding visual hallucinations as side effects, current research suggests that the COMT RS165815 C allele is a protective factor against visual hallucinations , as the incidence of visual hallucinations in patients with this allele is relatively low. In contrast, the DRD3 RS6280 C genotype is a risk factor for visual hallucinations , whereas DRD2 TaqIA C has been associated with delayed visual hallucinations . Other studies have confirmed that OPRK1, HTR2a, and DDC genotypes are associated with the incidence of ICD(impulse control disorder) . However, these results have only been confirmed by a few studies focusing on occidental patient samples. It is expected that more studies will be conducted in this area, especially focusing on the Asian population, to support these conclusions. COMT Gene Previous studies have found that L-dopa dose is influenced by the COMT genotype. Recent studies have found that COMT polymorphisms are associated with side effects of long-term use of L-dopa. A paper published by Sampaio and his team in 2018 suggested that patients with the COMT-LL phenotype are more prone to levodopa-induced dyskinesia (LID) , which was corroborated by de Lau et al. ’s research . In other words, patients with low COMT enzyme activity are more likely to develop levodopa-induced dyskinesia than those with high COMT enzyme activity. The low-activity COMT enzyme reduces levodopa degradation, increasing dopamine accumulation in the synaptic cleft, which causes dyskinesia. Cheshire et al. and Watanabe et al. also explored this question . However, both studies found no significant correlation between low-activity COMT enzyme and LID, which may be attributed to individual differences between patients (that is, some patients may have multiple genotypes affecting LID at the same time). DR Gene Currently, the DRD2 gene is the main dopamine receptor gene associated with levodopa side effects. It is highly expressed in the basal ganglia, which is a motor-regulating region of the central nervous system and is crucial to PD. Oliveri is the first researcher to study the role of DRD2 polymorphism in LID . In his study, the frequency of both alleles 13 and 14 of DRD2 gene polymorphism was higher in non-dyskinetic than in dyskinetic PD patients. In addition, carrying at least 1 of the 13 or 14 alleles reduced the risk of developing peak-dose dyskinesias by 72% in PD patients compared to PD patients not carrying these alleles. Further, DRD1 polymorphisms were not associated with the risk of developing PD or peak-dose dyskinesias. Rieck et al. verified this relationship with 199 Brazilian PD patients. Strong and his colleagues found that the DRD2 gene was involved in an early-onset of LID in 92 PD patients and further determined that the early onset PD was due to 14 and 15 DRD2 alleles . Some researchers have also found that DRD3 gene polymorphism is associated with levodopa-induced side effects, but evidence supporting this conclusion is still insufficient. DAT Genes The dopamine transporter (DAT, SLC6A3) plays an important role in controlling the intensity and duration of dopaminergic neurotransmission by rapid reuptake DA into presynaptic terminals . The single nucleotide polymorphism (RS393795) in the DAT gene is significantly correlated with the onset time of levodopa-induced motor dysfunction, whereas the C allele of DAT is associated with late-onset LID, which may be regulated by a change in dopamine reuptake rate in synaptic cleft . Sossi et al. have shown that greater DAT expression levels are directly associated with lower dopamine turnover and lower changes in synaptic dopamine concentration in PD patients . Troiano et al. subsequently confirmed decreased DAT in the presynaptic membrane using a DAT-PET (Dopamine transporter-Positron Emission Tomography) . In addition to these three genes implicated in dopamine metabolism, the following genes are also potentially involved in LID. LID is a type of motor complication common in patients with PD during chronic levodopa therapy. It is reported that 40% of patients develop LID four years after receiving levodopa therapy. This risk is even higher among younger patients receiving high doses of levodopa therapy. In addition to the early onset age of PD and higher L-dopa total exposure, other studies have found that LID is more common in patients with a longer course of PD, lower body mass index, and female gender . Several researchers have also investigated the potential involvement of genetic factors or individual genetic variations in the development of LID. Analysis of the chi-square correlation between genotype and the presence of multiple levodopa-induced side effects in 205 PD patients by Schumacher-schuh found that rs4704559 G ( HOMER1 ) allele was associated with a lower prevalence of dyskinesia . Contrastingly, a higher prevalence of LID was found in patients with MAO-B (rs1799836, A644G) A allele and AA genotype and in patients with mTOR gene polymorphism after multivariate analysis. A better understanding of the role played by genetic factors in the development of LID may be important to identify patients more likely to develop LID and elucidate the molecular mechanisms underlying this causal link. With the increasing understanding of the adverse side effects of levodopa, more and more studies have focused on the influence of genotypes on side effects other than LID. Some studies have found an association between COMT gene polymorphism and the Epworth Sleepiness Scale (ESS, a scale designed by Johns MW to assess excessive daytime sleepiness state, facilitating quantitative semi-objective evaluation of the drowsiness state). Sleep disturbances are a well-known disabling nonmotor manifestation of PD, affecting almost 80% of patients . Patients with COMT-LH and COMT-LL genotypes had higher ESS scores than patients with COMT-HH, suggesting that these patients were more likely to experience daytime sleepiness . However, Rissling’s study yielded contradictory results . An SNP C667T (rs1801133) in the MTHFR gene was consistently linked to hyperhomocysteinemia (HHcy), which is defined as elevated serum concentrations of homocysteine (Hcy) exceeding 15 µmol/L . Elevated Hcy levels have been associated with increased cardiovascular, cerebrovascular, and thromboembolic diseases due to the L-dopa treatment in several studies. This mutation generates a temperature-labile MTHFR enzyme, which ultimately leads to hyperhomocysteinemia . Regarding visual hallucinations as side effects, current research suggests that the COMT RS165815 C allele is a protective factor against visual hallucinations , as the incidence of visual hallucinations in patients with this allele is relatively low. In contrast, the DRD3 RS6280 C genotype is a risk factor for visual hallucinations , whereas DRD2 TaqIA C has been associated with delayed visual hallucinations . Other studies have confirmed that OPRK1, HTR2a, and DDC genotypes are associated with the incidence of ICD(impulse control disorder) . However, these results have only been confirmed by a few studies focusing on occidental patient samples. It is expected that more studies will be conducted in this area, especially focusing on the Asian population, to support these conclusions. Pharmacogenetic Studies on Dopamine Receptor Agonists Dopamine agonists are one of the most commonly used drugs in PD treatment but have lower efficacy than levodopa. Many studies have shown that dopamine agonists could overcome the shortcomings of levodopa, strengthen levodopa’s curative effect and delay complications. The combination of low-dose levodopa and dopamine receptor agonists is as effective as high-dose levodopa alone, but with a significantly lower incidence of side effects. In the later stages of the disease, due to gradual degeneration and loss of dopaminergic neurons in the nigrostriatal system, exogenous levodopa decarboxylation is no longer converted to dopamine. At this point, levodopa becomes ineffective, while dopamine receptor agonists remain effective. Examples of commonly used dopamine receptor agonists include pramipexole, bromocriptine, and rotigotine. Presently, studies examining the relationship between dopamine receptor agonists and genomics mainly focus on the effect of gene polymorphism on the efficacy of dopamine receptor agonists. 3.3.1 DRD2 and DRD3 Gene The DRD2 and DRD3 gene polymorphisms may influence drug efficacy and tolerance. Together with his team, Liu found that DRD3 Ser/Gly polymorphism influenced the efficacy of dopamine receptor agonists in 30 Chinese PD patients , which was corroborated by Xu’s study . To investigate the association between Dopamine receptor D type 2 (DRD2) dinucleotide short tandem repeat (CA(n)-STR) and Dopamine receptor D type 3 (DRD3) Ser9Gly polymorphisms and different doses of Dopamine receptor agonists (DAs) in PD patients, professor Xu recruited 168 idiopathic PD patients and 182 controls. Further exploration showed no association between DRD2 CA(n)-STR polymorphism and DA dosage. Among patients with three different DRD3 Ser9Gly genotypes (Ser/Ser, Ser/Gly, and Gly/Gly), patients carrying Gly/Gly genotype used higher doses of DAs than those with Ser/Gly and Ser/Ser genotypes. DRD3 Ser9Gly Ser/Ser genotype has a higher response rate to dopamine agonists and requires a smaller dose. In a recent study of patients in Brazil, the presence of the TTCTA haplotype, derived from five DRD2 SNPs, was also linked with a high risk of dyskinesia . 3.3.2 DRD4 Gene Sleep attacks in PD were initially reported to occur only with particular dopamine agonists, pramipexole, and ropinirole. The DRD4 48-bp VNTR short/short variant was significantly associated with sleep attacks without warning signs . In general, research in this area is insufficient. Considering the complexity of nervous system diseases, comorbidities in elderly patients, multiple treatment regimens, lack of dose-response relation for many drugs, and other non-genetic factors affecting treatment results, as well as various problems that may be encountered in the design and implementation of PD pharmacogenomics conclusive studies, confirming the clinical utility of pharmacogenomics is challenging. Therefore, the positive results obtained for some candidate genes studied to date indicate that pharmacogenomics of PD warrants more extensive studies involving more uniform, large patient groups to minimize the effect of nongenetic factors. DRD2 and DRD3 Gene The DRD2 and DRD3 gene polymorphisms may influence drug efficacy and tolerance. Together with his team, Liu found that DRD3 Ser/Gly polymorphism influenced the efficacy of dopamine receptor agonists in 30 Chinese PD patients , which was corroborated by Xu’s study . To investigate the association between Dopamine receptor D type 2 (DRD2) dinucleotide short tandem repeat (CA(n)-STR) and Dopamine receptor D type 3 (DRD3) Ser9Gly polymorphisms and different doses of Dopamine receptor agonists (DAs) in PD patients, professor Xu recruited 168 idiopathic PD patients and 182 controls. Further exploration showed no association between DRD2 CA(n)-STR polymorphism and DA dosage. Among patients with three different DRD3 Ser9Gly genotypes (Ser/Ser, Ser/Gly, and Gly/Gly), patients carrying Gly/Gly genotype used higher doses of DAs than those with Ser/Gly and Ser/Ser genotypes. DRD3 Ser9Gly Ser/Ser genotype has a higher response rate to dopamine agonists and requires a smaller dose. In a recent study of patients in Brazil, the presence of the TTCTA haplotype, derived from five DRD2 SNPs, was also linked with a high risk of dyskinesia . DRD4 Gene Sleep attacks in PD were initially reported to occur only with particular dopamine agonists, pramipexole, and ropinirole. The DRD4 48-bp VNTR short/short variant was significantly associated with sleep attacks without warning signs . In general, research in this area is insufficient. Considering the complexity of nervous system diseases, comorbidities in elderly patients, multiple treatment regimens, lack of dose-response relation for many drugs, and other non-genetic factors affecting treatment results, as well as various problems that may be encountered in the design and implementation of PD pharmacogenomics conclusive studies, confirming the clinical utility of pharmacogenomics is challenging. Therefore, the positive results obtained for some candidate genes studied to date indicate that pharmacogenomics of PD warrants more extensive studies involving more uniform, large patient groups to minimize the effect of nongenetic factors. Pharmacogenetic Studies on Other Anti-PD Drugs Until now, levodopa has been the most effective treatment for patients with PD. However, with long-term use, the effect of levodopa gradually decreases and contributes to various motor complications. Therefore, for patients with advanced PD, combination therapy including levodopa and other drugs is routinely indicated. Monoamine oxidase (MAO) inhibitor drugs, such as selegiline and rasagiline, could reduce the metabolic degradation of levodopa, thus they are often concurrently administered with it . In later stages of the disease requiring levodopa, adjunctive monoamine oxidase B inhibitors reduce ‘off’ time and may improve gait and freezing . Monoamine oxidase is an extra-mitochondrial protein found in almost all human tissues . This enzyme regulates neurotransmitter metabolism in the brain and other systems. Therefore, its inhibition can boost neurotransmitter levels in the brain. MAO inhibitors can be divided into two categories according to their pharmacological effects. MAO-A is primarily involved in the oxidative metabolism of tyramine, whereas inhibition of MAO-B primarily reduces the metabolism of dopamine and β-phenylethylamine . Because of their role in levodopa metabolism, selective MAO-B inhibitors are clinically used for PD treatment drugs. Few pharmacogenetic studies have evaluated the effect of DRD2 gene polymorphisms on the clinical response to rasagiline. The results of a large study including 692 PD patients indicated that two SNPs of the DRD2 gene, rs2283265, and rs1076560, were significantly correlated with improved motor function in response to 12-week management with rasagiline after controlling for placebo effects . COMT inhibitors are concomitantly used with levodopa to inhibit levodopa metabolism. PD treatment is based on replacing lost DA with levodopa, which can pass the blood-brain barrier. Administration of COMT inhibitors blocks methylation of levodopa to 3-methyldopa (3-OMD) through the inhibition of the COMT enzyme, thereby preventing levodopa degradation through this major peripheral metabolic pathway and improving its clinical potency efficacy. Presently, COMT inhibitors commonly used in clinical practice include entacapone, tolcapone, and opicapone. Tolcapone and entacapone have mainly been used with patients with more advanced diseases, including chronic motor fluctuations. Whereas tolcapone is associated with hepatotoxicity, entacapone has a short half-life in plasma and requires dosing with every administration of levodopa . However, compared with other COMT inhibitors, opicapone, a third-generation COMT inhibitor, not only reduces the risk of toxicity but also improves COMT inhibitory efficacy and peripheral tissue selectivity . In general, the COMT inhibitors such as entacapone and tolcapone are concomitantly used with levodopa, as they could inhibit the metabolism of levodopa. The effect of genetic variants in the COMT gene on clinical response to entacapone has been evaluated in a clinical trial including 33 PD patients. The results demonstrated that entacapone-treated patients with Val/Val genotype showed a higher levodopa concentration . Besides, the A528G SNP of the UGT1A6 gene is associated with the hepatotoxicity of entacapone and tolcapone in a large European study of 409 PD patients . DISSCUSION The present review gives an overview of the published data on PD pharmacogenetics, shows their limitations and gives insights that may be useful to future studies. Previous studies have explored the effect of gene polymorphism on the efficacy and side effects of levodopa. The efficacy and side effects of levodopa may be connected to the genetic variation of various genes involved in metabolism and transport in vivo , such as the catechol-o-methyltransferase (COMT) gene, the dopamine receptor (DRD 1-5) gene, and the dopamine transporter (SLCA3) gene, among others. Multiple studies have shown that patients with the COMT-LL genotype require less levodopa but are at greater risk for levodopa-induced dyskinesia; Lower frequency of DRD2 13 or 14 alleles in patients with peak-dose dyskinesias; Higher frequency of DAT gene VATR sequence in patients with LID, and so on. The role of many genetic polymorphisms in drug response has been demonstrated by only one or a few studies and is inconclusive, for instance: MAO-A gene, BDNF gene, HOMER1 gene, MTHFR gene, etc . In addition, compared with dopamine agents, pharmacogenomic investigations of other antiparkinson medicines, such as COMT inhibitors, MAO inhibitors, dopamine receptor agonists, anticholinergic pharmaceuticals, and amantadine, have been restricted, and firm findings have not been achieved. In brief, pharmacogenomic investigations have demonstrated that gene polymorphisms are strongly related to disparities in the efficacy and side effects of several anti-PD medications, including levodopa and rasagiline. However, due to the paucity of prospective large-scale clinical studies in China, it cannot be utilized to guide clinical application. As we all know, PD is a neurodegenerative disease manifesting as impaired motor function . Pharmacological treatment of symptoms has shown excellent but highly variable efficacy in PD patients. It can be shown that the efficacy, side effects, and problems of anti-PD medications vary greatly depending on the person, and clinical medication must focus on tailored exact therapy . Unfortunately, appropriate assistance is unavailable due to a lack of professional consensus or recommendations. Thus, studies identifying factors associated with this variability would contribute to more personalized treatment of PD. In the last decade, pharmacogenetic studies in PD have shed light on the role of genetic factors in drug response and the adverse effects of anti-PD drugs . However, results on the link between investigated genes and drug response phenotypes have been inconclusive. The inability to replicate findings among these studies may be due to the small sample size used and the diverse genetic backgrounds of patients. Each genetic variant has a small contribution to the inter-individual variability in drug response . This effect could be missed if a small number of subjects are analyzed. In addition, different ethnicities in the investigated cohort could be another source of variability among different studies . Therefore, genotype groups and genetic backgrounds should be considered when evaluating the effect of genetic factors in pharmacogenetic studies. It is important to note that drug responses or adverse effects are associated with multiple factors, including genetic variants, drug interactions, concomitant therapy, and environmental factors . The effect of a certain genetic variant would be diluted if the other risk factors in the investigated cohort are not adequately considered. Besides, the large heterogeneity of PD in clinical settings is another important issue that should be taken into account. PD manifests as a heterogeneous clinical syndrome, with motor and nonmotor symptoms and variable rates of disease progression . On one hand, although significant strides have been made to improve clinical instruments for accurately assessing the main outcomes of the disease, some characteristics, such as motor fluctuation and some non-motor symptoms, still lack clear definitions and precise scales. On the other hand, identifying PD subtypes may help understand the underlying disease mechanism and design better clinical trials for pharmacogenetic studies. Moreover, most pharmacogenetic studies in PD to date are cross-sectional or retrospective. Given that PD is a progressive disease, longitudinal studies are needed to better identify the effect of genetic factors on drug response and the onset of adverse events . Taken as a whole, PD pharmacogenomics research has become a local and worldwide hotspot, but there are still obstacles in clinical application and directing the customized precision treatment of PD, which may be attributable to the following causes. 1) Most pharmacogenomic studies of Parkinson's disease concentrate on levodopa rather than other regularly prescribed anti-PD medications. 2) The function of the majority of gene polymorphisms is not entirely understood. 3) Existing pharmacogenomics researches on Parkinson's disease concentrate mostly on PD patients in western nations. It must be determined if the research results have reference value for PD patients in China. In order to create personalised precision treatments for PD, it is important to conduct large, homogenous, multi-center, prospective, randomized controlled clinical studies of PD pharmacogenomics encompassing common therapeutic anti-PD medicines. Additionally, to better apply the research results of pharmacogenomics to guide the clinical diagnosis and treatment of PD, we need to incorporate the probable genetic variations of all patients with suspected PD into the diagnostic chain (clinical examination, MRI, LP, DatScan, FDG-PET) for better drug selection. The identification of a genetic variant associated with drug response in PD is a significant step preceding its use in clinical practice. Therefore, the biological functional effect of identified genetic variants and their interactions with other genetic or environmental factors should be explored further. Finally, cost-effectiveness analyses should be conducted to assess the translation of research evidence into clinical practice.
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Feasibility and acceptability of an online intervention to enhance hopefulness among oncology professionals
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a2206bd0-c124-4dd3-b8b3-09ef74e23251
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10208111
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Internal Medicine[mh]
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The present research consisted of a single-arm feasibility and acceptability study. Invitations were emailed to 141 members of the SWOG Cancer Research Network (formerly the Southwestern Oncology Group), which is a member of the National Clinical Trials Network of the National Cancer Institute. Emails were sent to a cross-section of professions within the organization. Enrollment was limited to 1 of 4 available time slots. A total of 29 individuals signed up and attended the intervention, which consisted of a single 2-hour session followed by self-report measures. To optimize fidelity, the same facilitators (B.W.C., D.B.F.) conducted each session and were credentialed with a manualized course. Six to 8 individuals participated in each session, which was delivered live via the Zoom video platform. The workshop was derived from an in-person experience consisting of 3 components. First, a brief didactic presentation on hope was offered. This was oriented around Snyder’s “Hope Theory” , which posits that hope consists of 3 components, known within the workshop as the “three conditions for hope to thrive”: goals (desired ends), pathways (perceived plans or strategies for reaching goals), and agency (motivation or energy to pursue goals). According to 3 decades of research, when these conditions are met, individuals experience a variety of positive psychosocial and physical outcomes . The workshop includes 2 activities to integrate these conditions into participants’ lives: hope mapping and hope-related mental rehearsal. Hope mapping, previously a paper-and-pencil exercise, is now conducted via a smartphone app known as Hopetimize (Life’s Door, Jerusalem, Israel). Users are guided to identify a meaningful goal and a pathway toward that goal (see ). They also identify obstacles that may hamper pursuing that goal along with potential ways to circumvent those obstacles. The mental rehearsal exercise is derived from research showing that such techniques increase performance of skill-related behaviors in various domains, including sports and music . In this 15-minute exercise, participants are guided to close their eyes and imagine executing the pathway from their hope map, encountering each obstacle listed, and motivating themselves to circumnavigate those obstacles. Immediately post workshop, 3 measures were used. First, we administered the Was-It-Worth-It (WIWI) scale , which was developed to assess the degree to which participants in research studies find the experience worthwhile. It consists of 5 questions, 3 answered yes or no and 2 answered on Likert scales. Second, we administered a survey based on the Kirkpatrick Training Evaluation Model (TEM) , one of the widest-used approaches for assessing training experiences like the Hope Workshop. Accordingly, training experiences are assessed on 4 dimensions: general reaction, learning, anticipated behavior change, and outcome. These dimensions are operationalized through individual items rated on Likert-scales. Most items from these measures, along with means and SDs, are found in and . Consistent with past research using these tools, summary scores were not used because items were treated as distinct measures. Finally, a single item prompted participants to rate the degree to which they believed hope concepts from the workshop should be integrated into future SWOG studies involving patients. The study was approved by the institutional review board of Shaare Zedek Medical Center (Jerusalem, Israel).
Twenty-nine SWOG investigators participated in the workshop, and 23 (79%) completed measures. displays sample characteristics. The most common professions were physicians and nursing specialties. Although there was a wide age distribution, the sample was heavily skewed toward female (74%) and White (78%) participants. Because no data were obtained from participants who did not complete measures, it is not known whether they differed on any characteristics. Within the Kirkpatrick TEM instrument, items are rated on 8-point scales, with responses ranging from “definitely false” to “definitely true.” shows that ratings were high, ranging from 6.91 to 7.70. Results on the WIWI were similarly positive (please see ). In particular, nearly all participants answered “yes” to items, including “Was it worthwhile to participate in the Hope Workshop?”, “If you had to do it over, would you participate in the Hope Workshop again?”, and “Would you recommend participating in the Hope Workshop to others?” Two additional WIWI items were reported on 3-point scales, where 1 indicated “it got worse,” 2 indicated “it stayed the same,” and 3 indicated “it improved.” These items were “Overall, how was your experience of participating in the Hope Workshop?” and, “Overall, do you believe your quality of life has increased by participating in the Hope Workshop?”, with means of 2.70 (SD = .64) and 2.52 (SD = .51), respectively. Finally, a single item asked, “To what degree do you think it may be useful to integrate concepts from this workshop into SWOG trials/studies?” On average, this was rated 4.44 (SD = .59) on a scale ranging from 1 (“not at all useful”) to 5 (“extremely useful”).
According to the results of this pilot study, a single-session online hope intervention augmented by a smartphone app appears both feasible and acceptable in a sample of oncology HCPs. Responses to the WIWI and Kirkpatrick TEM items indicate that they not only saw value in this intervention for themselves and fellow HCPs but also potentially for patients in future trials. The SWOG Cancer Research Network has a long-standing commitment to harnessing innovative nonmedical technologies in influencing traditional oncologic endpoints. For instance, Hershman and colleagues rigorously evaluated text messaging to optimize adherence to endocrine therapy among postmenopausal women with breast cancer. In that trial, however, the authors did not find that nonpersonalized text messaging was effective. The hope enhancement intervention described herein is, by definition, a personalized approach and, for this reason, National Clinical Trials Network leadership has expressed interest in studying this approach (Mark O’Rourke, personal communication). Recently, an online questionnaire documented that nearly 25% of Society of Gynecologic Oncology members suffered from burnout . Moreover, those with higher levels of hope were less likely than those with lower hope to meet the criteria for burnout. This relationship is understandable given that burnout can be conceptualized as hopelessness that one’s efforts matter in the work context. This compels us to ask whether professionals can become more hopeful and therefore be less likely to suffer from burnout. Feldman et al. found that hope and social support—both mainstays of the workshop described herein—may mitigate the effects of burnout on general life satisfaction. Moreover, Bareket-Bojmel et al. tested a mediation model among 1200 individuals from 3 countries during the COVID-19 pandemic, concluding that perceived social support facilitates hope during dire times. It is not surprising, therefore, that the professionals in the present sample deemed our brief hope intervention valuable. The COVID-19 pandemic necessitated broad adoption of digital medical options. Virtual platforms allow participation and honing of skills in a context that is not dependent on geographic proximity. Furthermore, the virtual nature of the hope intervention reported here provides for easy scalability, enabling diffusion to a wide audience. Finally, overhead costs are relatively low compared with other interventions, and results appear to be noted relatively quickly. There are limitations of this study. First, the population studied was highly educated. Although we believe the app is intuitive, if it is to be used by patients in addition to HCPs, it is crucial to demonstrate ease of use regardless of level of formal education. Second, there was limited diversity in the present sample, particularly in terms of race and ethnicity. This highlights the necessity of future research using broader samples of individuals who might benefit from hope enhancement interventions . Finally, invitations to participate were accepted at a relatively low rate (20%). Busy clinicians are hard pressed to access training sessions at fixed times. Work is now ongoing to determine if it is feasible to offer a more flexible “self-guided” online training module more adapted to the lives of busy clinicians. The tool described in this report is inexpensive and easily adopted. In an inherently fragile “cancer ecosystem,” which includes patients, caregivers, and HCPs, we dare not forfeit the opportunity enhance hope, reduce burnout, and directly or indirectly optimize oncologic endpoints . We believe that the virtual experience we have developed makes hope—as a pragmatic construct—readily accessible to disparate stakeholders who could benefit from its forward-looking orientation.
pkad030_Supplementary_Data Click here for additional data file.
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The effect of a video-based COVID-19 paediatric patient education on state anxiety in children with suspected COVID-19 admitted to hospital
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03a877b4-15fa-439b-9777-52e50b38eb5a
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10208266
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Patient Education as Topic[mh]
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Introduction The first coronavirus case (2019-nCoV / COVID-19) was reported in Wuhan, the capital of Hubei province in China, in December of 2019 . The World Health Organisation (WHO) declared the coronavirus disease as a pandemic on March 11, 2020. The figures of COVID-19 cases and deaths have been increasing every passing day in the world and in Turkey ( ). COVID-19 is a novel highly contagious virus and so little is known about how to diagnose, treat, or prevent this disease; therefore, families and children are afraid of/worried about it . Moreover, patients suffering from COVID-19 complaints are taken to the paediatric emergency departments of hospitals. In paediatric emergency departments, each nurse provides care to a great number of patients by allocating a very short time for each patient; therefore they have no enough time to prepare the patients. Hence, children and their families cannot be oriented to the hospital setting and the procedures, resulting in an increase in their anxiety , . Nurses need to take an active role in and responsibility for planning, delivering and improving care for children with COVID-19 who are admitted to the hospital. Nurses emphasise the importance of ensuring the patient's compliance with the treatment to enhance the quality and effectiveness of the health service. Hospital settings are the places which are frequently applied for follow-ups of their children as from their birth or when their children get sick. Patients are not only examined but also exposed to painful procedures such as vaccination, venepuncture and cannulation. In addition to procedures at outpatient clinics, paediatric patients may need to stay in the hospitals to undergo some procedures, treatments, and surgeries, which may lead them to feel afraid and anxious. Children’s fears such as hospital, doctor, and treatment prevent them from collaborating with the nurse during the treatment process. This negatively affects the treatment process of the child . To what extent the children are affected by the illness and hospitalisation varies depending on their age, cognitive development level, and previous experiences, duration and type of disease, preparation for hospitalisation, and their parents’ attitudes and cultural characteristics. Children's health-related fears include fear of doctors and nurses, needles, hospitalisation, examination, medication, losing body functions, undergoing surgery, losing control, death and separating from the family . It is important for nurses and families to cope with psychosocial stress and reduce biological stress in children by providing an effective medical treatment in order to prevent or decrease the negative effects of the disease on their development . Methods such as informing the children before going to the hospital, communicating with them and giving rewards to them can be used to reduce their fear of hospital . Preventive health services reduce the death, disability and need for long-term treatment due to diseases. Health education makes it possible to reach large masses to ensure the continuity of health and also raises awareness of people about responsibilities for their own health. If the healthcare professionals communicate with paediatric patients and their families in a reassuring and love-oriented kindness, provide them with health education, and inform them about the examination, procedure or treatment, this plays a major role in easing their fear and anxiety . Thus, considering the long-term psychological effects of the anxiety experienced by children during the COVID-19 pandemic, the aim of this study is to alleviate their anxiety by using the video-based COVID-19 paediatric patient education method and to increase their compliance to treatment.
Methods 2.1 Design and participants This quasi-experimental study with pretest–posttest single-group was carried out in the COVID-19 Paediatric Emergency Department of a training and research hospital in Turkey between May and August 2020. While the population consisted of children admitted to the COVID-19 Paediatric Emergency Department, the sample group consisted of 128 children aged between 4 and 12 years who met the inclusion criteria ( ). Inclusion criteria for the children were determined as follows; being aged between 4 and 12 years, suspected of COVID-19, being able to count to ten, and having the parents who give consent for their participation in the study. One month after the reporting of the first COVID-19 case in Turkey, the study was quickly designed and conducted. Our main aim was to provide benefit to children equally. Therefore, all of the children were trained without being assigned into experimental and control groups. The criteria we paid attention to during the scale selection in the present study: • Being a scale whose validity and reliability has been tested, • Having an age range appropriate for the child age group, • Being capable to assess the state anxiety level. Childhood is a very wide range including 0–18 years. The same method is not used for assessing state anxieties of a four-year-old child and a twelve-year-old child. Therefore, two scales (for 4–10 years and 9–12 years), whose reliability study was conducted, were used in order to make an accurate assessment specific to children's age. 2.2 Instruments The data were collected from the participants through face-to-face interview technique using an introductory information form, the Children’s State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC). Two different scales were used since there is no single scale whose Turkish reliability study was conducted for all age groups. 2.2.1 Introductory information form: This form prepared in accordance with the literature consisted of 22 questions about the patients' socio-demographic characteristics (age, gender, educational background, vaccination status, presence of other diseases, and presence of companion) and disease-related characteristics (COVID symptoms, contact, history of COVID-19 in the family, previous hospitalisation, hospital fear, reasons for the fear, fear of the protective equipment worn by the medical team, fear symptoms). 2.2.2 Children’s State Anxiety (CSA) Scale: Children’s State Anxiety Scale was developed by Ersing et al., in 2013 to assess the stress of children between the ages of 4–10 at the moment when they fill out the scale . The scale was adapted into Turkish by Gerçeker et al., in 2018 , . The content validity index of the scale is 1.00. The scale resembles a thermometer. It has a bulb at the bottom and horizontal lines placed at certain intervals going all the way up. Children are first asked to imagine that all of emotions such as anxiety and anger is at the bottom, and thus to point to them on the scale. They are then instructed to raise their finger upwards if they feel anxious or on edge. If they feel very, very anxious or nervous, the emotions may run high (move their finger to the top). They are asked to put a line showing how anxious or angry they feel “at this moment” to assess state anxiety. Before the children complete the scale, their sequencing ability is monitored. They are asked to count to 10 and respond to the question “Which is bigger, seven? four?” A transparent meter with ½ point increments marked is placed over their rating and then an increment of ½ point is rounded to the nearest number. Total score ranges from 0 to 10 . 2.2.3 State-Trait Anxiety Inventory for children (STAIC) Scale: The State Anxiety Inventory for Children was developed by Spielberg et al., in 1973 to assess how 9–12-year-olds feel at the moment . The State-Trait Anxiety Inventory has 2 sections (“State Anxiety” and “Trait Anxiety”), including 20 items in each. Only the “State Anxiety” section was used in the study. The scale was adapted into Turkish by Özusta et al., in 1995 . In this section, children are asked to assess how they feel at “that moment” and to mark one of the 3 related options. The scale has 20 items that evaluate emotions related to state anxiety such as tension, nervousness, hurry, and uneasiness. If the children mark these emotions as “a lot”, they get 3 points (maximum score). If they mark lack of these emotions, they get 1 point (minimum score).The highest and lowest scores of the State Anxiety Inventory are 60 and 20, respectively. 2.3 Planning of the education For the Video-Based COVID-19 Paediatric Patient Education prepared in line with the literature, a total of ten goals were determined. Before the education video was shot, its scenario was written and revised after taking the opinions of 10 experts. A paediatric nurse and a professional video producer shot the education video in Turkish using a child simulator at the Nursing Faculty Skills Laboratory. The video producer also added special effects, subtitles, and a sign language interpreter (bottom right hand corner). Total duration of the video was determined as 4 min 44 s. Later, the education was uploaded to the tablet in the clinical area. The pilot study of the video education was conducted on 10 children aged 4–12 years who were admitted due to the complaint of COVID-19. These children were not included in the study. The education aimed for the children to comprehend the purpose behind why the paediatric nurse is present there, to identify COVID-19, to describe the reason for using protective equipment, to explain the reason for taking vital signs, to know the purpose of the diagnostic tests, to explain the purpose of the mask put on them and their parents and apply the mask, to know the methods of self-relaxation during hospitalisation, to be aware of the interventions related to respiratory distress, to know the treatment process, and to explain the post-discharge process ( ). 2.4 Data collection 1. The researcher collected the data through face-to-face interviews before and after the application. 2. The pilot study of the education was conducted on 10 children aged 4–12 years who were admitted due to the complaint of COVID-19. These children were not included in the study. 3. The researcher received training on the rules of using protective equipment from the paediatric emergency nurse in charge. 4. After the researcher met the patients, she told to them and their parents about the reason for why he is present there. 5. Consent was obtained from the patients and their parents. 6. The patients’ information was recorded in the introductory information form. 7. The Children's State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC) scale information appropriate for the patient's age were obtained (pre-patient education state stress scores / pretest state stress). 8. The child watched the video-based COVID-19 education. 9. The Children's State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC) scale were completed again after the emergency procedures (fever monitoring, swab sampling, lung tomography, etc.) (post-education state stress scores / posttest state stress). 10. It took a total of 15 min to complete the interview with the patient. 2.5 Ethical consideration In order to conduct the study, written institutional permission was obtained from the hospital's chief physician. Permission was obtained from the Turkey COVID-19 Scientific Research Evaluation Commission. Ethics committee approval was obtained from Istanbul Bakırkoy Dr. Sadi Konuk Training and Research Hospital Ethics Committee. Permission of the scale’s author was obtained via e-mail for the use of the scale in the study. Written consent was obtained from the families of the children participating in the study. In addition, while written consent was obtained from the literate children, verbal consent was obtained from the others. 2.6 Data analysis NCSS (Number Cruncher Statistical System) (Kaysville, Utah, USA) software was used for statistical analysis. Descriptive statistical methods (mean, standard deviation, median, frequency, ratio, minimum, maximum) were used for the data assessment. Kolmogorov-Smirnov and Shapiro-Wilk tests were used to determine whether or not quantitative data were normally distributed. While Student t -Test was used for two-group comparisons of normally distributed quantitative data, Mann-Whitney U test was used for two-group comparisons of non-normally distributed data. While Kruskal Wallis test was used for comparisons of three or more groups that did not show normal distribution, Bonferroni-Dunn test was used for pairwise comparisons. While Paired Sample t -test was used for within-group comparisons of normally distributed parameters, Wilcoxon Signed-Rank test was employed for within-group comparisons of non-normally-distributed parameters. Significance was evaluated at the level of p < 0.05.
Design and participants This quasi-experimental study with pretest–posttest single-group was carried out in the COVID-19 Paediatric Emergency Department of a training and research hospital in Turkey between May and August 2020. While the population consisted of children admitted to the COVID-19 Paediatric Emergency Department, the sample group consisted of 128 children aged between 4 and 12 years who met the inclusion criteria ( ). Inclusion criteria for the children were determined as follows; being aged between 4 and 12 years, suspected of COVID-19, being able to count to ten, and having the parents who give consent for their participation in the study. One month after the reporting of the first COVID-19 case in Turkey, the study was quickly designed and conducted. Our main aim was to provide benefit to children equally. Therefore, all of the children were trained without being assigned into experimental and control groups. The criteria we paid attention to during the scale selection in the present study: • Being a scale whose validity and reliability has been tested, • Having an age range appropriate for the child age group, • Being capable to assess the state anxiety level. Childhood is a very wide range including 0–18 years. The same method is not used for assessing state anxieties of a four-year-old child and a twelve-year-old child. Therefore, two scales (for 4–10 years and 9–12 years), whose reliability study was conducted, were used in order to make an accurate assessment specific to children's age.
Instruments The data were collected from the participants through face-to-face interview technique using an introductory information form, the Children’s State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC). Two different scales were used since there is no single scale whose Turkish reliability study was conducted for all age groups. 2.2.1 Introductory information form: This form prepared in accordance with the literature consisted of 22 questions about the patients' socio-demographic characteristics (age, gender, educational background, vaccination status, presence of other diseases, and presence of companion) and disease-related characteristics (COVID symptoms, contact, history of COVID-19 in the family, previous hospitalisation, hospital fear, reasons for the fear, fear of the protective equipment worn by the medical team, fear symptoms). 2.2.2 Children’s State Anxiety (CSA) Scale: Children’s State Anxiety Scale was developed by Ersing et al., in 2013 to assess the stress of children between the ages of 4–10 at the moment when they fill out the scale . The scale was adapted into Turkish by Gerçeker et al., in 2018 , . The content validity index of the scale is 1.00. The scale resembles a thermometer. It has a bulb at the bottom and horizontal lines placed at certain intervals going all the way up. Children are first asked to imagine that all of emotions such as anxiety and anger is at the bottom, and thus to point to them on the scale. They are then instructed to raise their finger upwards if they feel anxious or on edge. If they feel very, very anxious or nervous, the emotions may run high (move their finger to the top). They are asked to put a line showing how anxious or angry they feel “at this moment” to assess state anxiety. Before the children complete the scale, their sequencing ability is monitored. They are asked to count to 10 and respond to the question “Which is bigger, seven? four?” A transparent meter with ½ point increments marked is placed over their rating and then an increment of ½ point is rounded to the nearest number. Total score ranges from 0 to 10 . 2.2.3 State-Trait Anxiety Inventory for children (STAIC) Scale: The State Anxiety Inventory for Children was developed by Spielberg et al., in 1973 to assess how 9–12-year-olds feel at the moment . The State-Trait Anxiety Inventory has 2 sections (“State Anxiety” and “Trait Anxiety”), including 20 items in each. Only the “State Anxiety” section was used in the study. The scale was adapted into Turkish by Özusta et al., in 1995 . In this section, children are asked to assess how they feel at “that moment” and to mark one of the 3 related options. The scale has 20 items that evaluate emotions related to state anxiety such as tension, nervousness, hurry, and uneasiness. If the children mark these emotions as “a lot”, they get 3 points (maximum score). If they mark lack of these emotions, they get 1 point (minimum score).The highest and lowest scores of the State Anxiety Inventory are 60 and 20, respectively.
Introductory information form: This form prepared in accordance with the literature consisted of 22 questions about the patients' socio-demographic characteristics (age, gender, educational background, vaccination status, presence of other diseases, and presence of companion) and disease-related characteristics (COVID symptoms, contact, history of COVID-19 in the family, previous hospitalisation, hospital fear, reasons for the fear, fear of the protective equipment worn by the medical team, fear symptoms).
Children’s State Anxiety (CSA) Scale: Children’s State Anxiety Scale was developed by Ersing et al., in 2013 to assess the stress of children between the ages of 4–10 at the moment when they fill out the scale . The scale was adapted into Turkish by Gerçeker et al., in 2018 , . The content validity index of the scale is 1.00. The scale resembles a thermometer. It has a bulb at the bottom and horizontal lines placed at certain intervals going all the way up. Children are first asked to imagine that all of emotions such as anxiety and anger is at the bottom, and thus to point to them on the scale. They are then instructed to raise their finger upwards if they feel anxious or on edge. If they feel very, very anxious or nervous, the emotions may run high (move their finger to the top). They are asked to put a line showing how anxious or angry they feel “at this moment” to assess state anxiety. Before the children complete the scale, their sequencing ability is monitored. They are asked to count to 10 and respond to the question “Which is bigger, seven? four?” A transparent meter with ½ point increments marked is placed over their rating and then an increment of ½ point is rounded to the nearest number. Total score ranges from 0 to 10 .
State-Trait Anxiety Inventory for children (STAIC) Scale: The State Anxiety Inventory for Children was developed by Spielberg et al., in 1973 to assess how 9–12-year-olds feel at the moment . The State-Trait Anxiety Inventory has 2 sections (“State Anxiety” and “Trait Anxiety”), including 20 items in each. Only the “State Anxiety” section was used in the study. The scale was adapted into Turkish by Özusta et al., in 1995 . In this section, children are asked to assess how they feel at “that moment” and to mark one of the 3 related options. The scale has 20 items that evaluate emotions related to state anxiety such as tension, nervousness, hurry, and uneasiness. If the children mark these emotions as “a lot”, they get 3 points (maximum score). If they mark lack of these emotions, they get 1 point (minimum score).The highest and lowest scores of the State Anxiety Inventory are 60 and 20, respectively.
Planning of the education For the Video-Based COVID-19 Paediatric Patient Education prepared in line with the literature, a total of ten goals were determined. Before the education video was shot, its scenario was written and revised after taking the opinions of 10 experts. A paediatric nurse and a professional video producer shot the education video in Turkish using a child simulator at the Nursing Faculty Skills Laboratory. The video producer also added special effects, subtitles, and a sign language interpreter (bottom right hand corner). Total duration of the video was determined as 4 min 44 s. Later, the education was uploaded to the tablet in the clinical area. The pilot study of the video education was conducted on 10 children aged 4–12 years who were admitted due to the complaint of COVID-19. These children were not included in the study. The education aimed for the children to comprehend the purpose behind why the paediatric nurse is present there, to identify COVID-19, to describe the reason for using protective equipment, to explain the reason for taking vital signs, to know the purpose of the diagnostic tests, to explain the purpose of the mask put on them and their parents and apply the mask, to know the methods of self-relaxation during hospitalisation, to be aware of the interventions related to respiratory distress, to know the treatment process, and to explain the post-discharge process ( ).
Data collection 1. The researcher collected the data through face-to-face interviews before and after the application. 2. The pilot study of the education was conducted on 10 children aged 4–12 years who were admitted due to the complaint of COVID-19. These children were not included in the study. 3. The researcher received training on the rules of using protective equipment from the paediatric emergency nurse in charge. 4. After the researcher met the patients, she told to them and their parents about the reason for why he is present there. 5. Consent was obtained from the patients and their parents. 6. The patients’ information was recorded in the introductory information form. 7. The Children's State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC) scale information appropriate for the patient's age were obtained (pre-patient education state stress scores / pretest state stress). 8. The child watched the video-based COVID-19 education. 9. The Children's State Anxiety (CSA) and the State-Trait Anxiety Inventory for Children (STAIC) scale were completed again after the emergency procedures (fever monitoring, swab sampling, lung tomography, etc.) (post-education state stress scores / posttest state stress). 10. It took a total of 15 min to complete the interview with the patient.
Ethical consideration In order to conduct the study, written institutional permission was obtained from the hospital's chief physician. Permission was obtained from the Turkey COVID-19 Scientific Research Evaluation Commission. Ethics committee approval was obtained from Istanbul Bakırkoy Dr. Sadi Konuk Training and Research Hospital Ethics Committee. Permission of the scale’s author was obtained via e-mail for the use of the scale in the study. Written consent was obtained from the families of the children participating in the study. In addition, while written consent was obtained from the literate children, verbal consent was obtained from the others.
Data analysis NCSS (Number Cruncher Statistical System) (Kaysville, Utah, USA) software was used for statistical analysis. Descriptive statistical methods (mean, standard deviation, median, frequency, ratio, minimum, maximum) were used for the data assessment. Kolmogorov-Smirnov and Shapiro-Wilk tests were used to determine whether or not quantitative data were normally distributed. While Student t -Test was used for two-group comparisons of normally distributed quantitative data, Mann-Whitney U test was used for two-group comparisons of non-normally distributed data. While Kruskal Wallis test was used for comparisons of three or more groups that did not show normal distribution, Bonferroni-Dunn test was used for pairwise comparisons. While Paired Sample t -test was used for within-group comparisons of normally distributed parameters, Wilcoxon Signed-Rank test was employed for within-group comparisons of non-normally-distributed parameters. Significance was evaluated at the level of p < 0.05.
Results 3.1 Demographic and Disease-Related characteristics The study was conducted with a total of 128 children. While 49.2% (n = 63) of them were girls, 50.8% (n = 65) were boys. The ages of the children ranged from 4 to 12, with a mean age of 8.54 ± 2.62 years. While 74.2% (n = 95) of the children were aged between 4 and 10 years, 25.8% (n = 33) were aged between 11 and 12 years. shows results about demographic characteristics of the participants. When the COVID-19 symptoms observed in children were examined, it was found that 32.0% (n = 41) suffered from coughing, 21.1% (n = 27) had high fever, 18.0% (n = 23) suffered from respiratory distress, 14.1% (n = 18) had headache, and 10.9% (n = 14) had abdominal pain. The total number of symptoms observed in children ranged from 0 to 4; whereas, 46.1% (n = 59) of them had no symptom. shows results related to COVID-19 symptoms, exposures, and fears of the participants. 3.2 Evaluation of state stress scores While the pretest refers to measurement of state stress score of the patient before the patient education, the posttest refers to measurement of state stress score of the patient after the patient education. The decrease in the state stress scores was statistically significant in both genders between the ages of 4–12 in the posttest compared to the pretest (p = 0.001; p < 0.01). The decrease in the state stress scores showed a statistically significant difference between the genders in the age group of 11–12 years in the posttest compared to the pretest (p = 0.044; p < 0.05). The decrease in the state stress scores of girls from age group of 11–12 years was higher when compared to their male counterparts. When the pretest state stress scores of the children in the age group of 4–10 years were examined in terms of their educational level, it was determined that stress scores of those who did not go to school were higher than the scores of those who attended kindergarten, primary school, and secondary school. Regardless of the educational level, the decrease in the posttest state stress scores of all the children from the age group of 4–10 years was statistically significant compared to the pretest (p = 0.001; p < 0.01). The change in the posttest state stress scores of the children in the age group of 11–12 years compared to the pretest showed a statistically significant difference between educational levels (p = 0.003; p < 0.01). The decrease in state stress scores of secondary school students in the age group of 11–12 years was higher than the scores of primary school students. No statistically significant difference was found between the pretest state stress scores of the children in terms of the total number of COVID symptoms (p > 0.05). The decrease in the state stress scores of the children from the age group of 4–10 years in the posttest compared to the pretest showed a statistically significant difference between their fears of hospital (p < 0.01). The decrease in the state stress scores of the 4–10 year-old children who had fear of hospitals was higher. The decrease in the state stress scores of the children in the age group of 11–12 years in the posttest compared to the pretest did not show a statistically significant difference between their fears of hospital (p > 0.05). shows the related results. A statistically significant difference was found between the pretest state stress scores of the children aged 4–10 in terms of the fear of protective equipment used by medical personnel (p = 0.001; p < 0.01). The pretest state stress scores of the children who had fear of protective equipment used by medical personnel were higher. The decrease in the stress score of the 4–10 year-old children, who had fear of protective equipment used by medical personnel, after the education was higher when compared to those who did not have this fear. The decrease in the post-education state stress scores of the children than their pre-education scores was statistically significant (p = 0.001; p < 0.01) Results can be found in .
Demographic and Disease-Related characteristics The study was conducted with a total of 128 children. While 49.2% (n = 63) of them were girls, 50.8% (n = 65) were boys. The ages of the children ranged from 4 to 12, with a mean age of 8.54 ± 2.62 years. While 74.2% (n = 95) of the children were aged between 4 and 10 years, 25.8% (n = 33) were aged between 11 and 12 years. shows results about demographic characteristics of the participants. When the COVID-19 symptoms observed in children were examined, it was found that 32.0% (n = 41) suffered from coughing, 21.1% (n = 27) had high fever, 18.0% (n = 23) suffered from respiratory distress, 14.1% (n = 18) had headache, and 10.9% (n = 14) had abdominal pain. The total number of symptoms observed in children ranged from 0 to 4; whereas, 46.1% (n = 59) of them had no symptom. shows results related to COVID-19 symptoms, exposures, and fears of the participants.
Evaluation of state stress scores While the pretest refers to measurement of state stress score of the patient before the patient education, the posttest refers to measurement of state stress score of the patient after the patient education. The decrease in the state stress scores was statistically significant in both genders between the ages of 4–12 in the posttest compared to the pretest (p = 0.001; p < 0.01). The decrease in the state stress scores showed a statistically significant difference between the genders in the age group of 11–12 years in the posttest compared to the pretest (p = 0.044; p < 0.05). The decrease in the state stress scores of girls from age group of 11–12 years was higher when compared to their male counterparts. When the pretest state stress scores of the children in the age group of 4–10 years were examined in terms of their educational level, it was determined that stress scores of those who did not go to school were higher than the scores of those who attended kindergarten, primary school, and secondary school. Regardless of the educational level, the decrease in the posttest state stress scores of all the children from the age group of 4–10 years was statistically significant compared to the pretest (p = 0.001; p < 0.01). The change in the posttest state stress scores of the children in the age group of 11–12 years compared to the pretest showed a statistically significant difference between educational levels (p = 0.003; p < 0.01). The decrease in state stress scores of secondary school students in the age group of 11–12 years was higher than the scores of primary school students. No statistically significant difference was found between the pretest state stress scores of the children in terms of the total number of COVID symptoms (p > 0.05). The decrease in the state stress scores of the children from the age group of 4–10 years in the posttest compared to the pretest showed a statistically significant difference between their fears of hospital (p < 0.01). The decrease in the state stress scores of the 4–10 year-old children who had fear of hospitals was higher. The decrease in the state stress scores of the children in the age group of 11–12 years in the posttest compared to the pretest did not show a statistically significant difference between their fears of hospital (p > 0.05). shows the related results. A statistically significant difference was found between the pretest state stress scores of the children aged 4–10 in terms of the fear of protective equipment used by medical personnel (p = 0.001; p < 0.01). The pretest state stress scores of the children who had fear of protective equipment used by medical personnel were higher. The decrease in the stress score of the 4–10 year-old children, who had fear of protective equipment used by medical personnel, after the education was higher when compared to those who did not have this fear. The decrease in the post-education state stress scores of the children than their pre-education scores was statistically significant (p = 0.001; p < 0.01) Results can be found in .
Discussions 4.1 Demographic characteristics and Disease-Related characteristics There have been views that children were rarely affected in the early periods of the pandemic. However, later on, it was determined that children were infected as much as adults, although the disease symptoms, severity and mortality rates were less . A systematic review on the clinical findings of COVID-19 in paediatric cases reported that the most common symptoms were fever and cough, followed by nasal discharge, sore throat, headache, fatigue, diarrhoea, and vomiting symptoms . In another study, it was stated that fever (35.7%), coughing (21.4%), and headache (7.1%) were found in children and 57.1% of the children were asymptomatic . In the present study, the complaints of coughing (32.0%), high fever (21.1%), respiratory distress (18.0%), headache (14.1%) and abdominal pain (10.9%) were observed in children; whereas, no symptoms were detected in 46.1% of the children. After the Video-Based COVID-19 Paediatric Patient Education, the decrease in state stress scores of children with two or more symptoms was higher when compared to those without symptoms (p < 0.05). This suggests that children who associated their own symptoms with the video can better manage their state stress control. The extent to which the children are affected by the illness and hospitalisation varies depending on their age, cognitive development and previous experiences, duration and type of the disease, preparation for hospitalisation, and parents’ attitude and cultural characteristics . In the present study, no state stress difference was observed between the children aged 4–10 in terms of genders; whereas, the decrease in the state stress scores of the girls aged 11–12 was higher compared to the boys (p < 0.05). It can be asserted that the video-based patient education was more effective in stress control of girls with increasing age. In their study, Gündüz et al., reported that as the age and educational level of children increased, their fear of hospitals decreased. Likewise, in the present study, the decrease in the post-education state stress scores of 11–12 year-old children, who went to secondary school, was found to be higher when compared to those who went to primary school (p < 0.01). It can be thought that stress control skills increased as the educational level increased. In a study, behavioural changes such as throwing a tantrum when going to bed at night and eating, nocturnal enuresis, fear of new environment/person and objects, fear of doctor/nurse and hospital were observed in 3–6 year-olds after hospitalisation . In the present study, the children admitted to the COVID-19 paediatric emergency department were observed in terms of fear symptoms and it was determined that while crying and parenteral commitment were mostly detected in younger age groups, older age groups became quiet. In the study by Akkavak and Karabudak, children described the negative aspects of the hospital as painful invasive procedures, maltreatment of medical personnel, physical limitation, and separation from loved ones . Przybylo et al., stated that children who wore masks during anaesthesia may tend to fear wearing masks again later on . McLenon et al., stated that children had a great fear of injections, their fear decreased when they got older, and girls felt this fear more than boys . In their study, Gündüz et al., reported that the factors increasing the fear of hospitals included being younger than 36 months, the previous hospitalisation in the emergency department, invasive procedures, the use of tools, the doctor not communicating with the child, not informing the child before the procedure, and forcibly interventions . One study reported that children developed fear against hospitals for several reasons such as separating from their friends, falling behind in their studies, the possibility of dying, and feeling lonely without their family around. They also worried about suffocating whilst wearing an oxygen mask, and being unable to play games . Likewise, the present study revealed that 21.1% of the children were hospitalised before; however, they were afraid of the hospital at a high rate (73.4%). When the reasons behind their fear of hospitals were examined, it was determined that they developed fear due to invasive procedures, tools, separation from family, forcibly intervention and lack of communication ( ). The decrease in the state stress scores of the 4–10 year-old children, who had fear of hospitals, after the Video-Based COVID-19 Paediatric Patient Education was higher than those who did not (p < 0.01). It may be recommended to use Video-Based COVID-19 Patient Education in the stress management of 4–10 year-olds who have fear of hospitals. 4.2 Evaluation of state stress scores Fear of hospital procedures developing in children may be associated with previous negative experiences and lack of communication before the procedure . In the present study, the decrease in state stress scores of children aged 11–12 years, who had previous hospitalisations, after the education was found to be higher than those who did not. This result suggested that the video-based patient education was more effective on children having previous hospital experiences. Although state stress of the children who had no previous hospital experience decreased after the education, this was not statistically significant. The majority of children who had fear of the protective equipment, used by medical personnel during the COVID-19 pandemic were aged between 4 and 10 years. After the Video-Based COVID-19 Paediatric Patient Education, these children's such fear decreased significantly. The video-based education is an effective education method on children because it appeals to the visual and auditory area. In their study, Carpenter et al., aimed to teach the use of inhaler technique to children aged 7–17 using video training and determined that their inhaler usage skill scores increased immediately after the training . Dyson et al., had individuals with type 2 diabetes watched professional training videos (including three parts of 10–15 min), and reported that the knowledge of the participants about diabetes significantly increased . In a video education and consultation-based study conducted by Toledo et al., for diabetic individuals, they stated that hypoglycaemia rates decreased . In the present study, the decrease in the posttest state stress scores of the children compared to the pretest was found to be statistically significant and the video-based COVID-19 education prepared for children was observed to be effective.
Demographic characteristics and Disease-Related characteristics There have been views that children were rarely affected in the early periods of the pandemic. However, later on, it was determined that children were infected as much as adults, although the disease symptoms, severity and mortality rates were less . A systematic review on the clinical findings of COVID-19 in paediatric cases reported that the most common symptoms were fever and cough, followed by nasal discharge, sore throat, headache, fatigue, diarrhoea, and vomiting symptoms . In another study, it was stated that fever (35.7%), coughing (21.4%), and headache (7.1%) were found in children and 57.1% of the children were asymptomatic . In the present study, the complaints of coughing (32.0%), high fever (21.1%), respiratory distress (18.0%), headache (14.1%) and abdominal pain (10.9%) were observed in children; whereas, no symptoms were detected in 46.1% of the children. After the Video-Based COVID-19 Paediatric Patient Education, the decrease in state stress scores of children with two or more symptoms was higher when compared to those without symptoms (p < 0.05). This suggests that children who associated their own symptoms with the video can better manage their state stress control. The extent to which the children are affected by the illness and hospitalisation varies depending on their age, cognitive development and previous experiences, duration and type of the disease, preparation for hospitalisation, and parents’ attitude and cultural characteristics . In the present study, no state stress difference was observed between the children aged 4–10 in terms of genders; whereas, the decrease in the state stress scores of the girls aged 11–12 was higher compared to the boys (p < 0.05). It can be asserted that the video-based patient education was more effective in stress control of girls with increasing age. In their study, Gündüz et al., reported that as the age and educational level of children increased, their fear of hospitals decreased. Likewise, in the present study, the decrease in the post-education state stress scores of 11–12 year-old children, who went to secondary school, was found to be higher when compared to those who went to primary school (p < 0.01). It can be thought that stress control skills increased as the educational level increased. In a study, behavioural changes such as throwing a tantrum when going to bed at night and eating, nocturnal enuresis, fear of new environment/person and objects, fear of doctor/nurse and hospital were observed in 3–6 year-olds after hospitalisation . In the present study, the children admitted to the COVID-19 paediatric emergency department were observed in terms of fear symptoms and it was determined that while crying and parenteral commitment were mostly detected in younger age groups, older age groups became quiet. In the study by Akkavak and Karabudak, children described the negative aspects of the hospital as painful invasive procedures, maltreatment of medical personnel, physical limitation, and separation from loved ones . Przybylo et al., stated that children who wore masks during anaesthesia may tend to fear wearing masks again later on . McLenon et al., stated that children had a great fear of injections, their fear decreased when they got older, and girls felt this fear more than boys . In their study, Gündüz et al., reported that the factors increasing the fear of hospitals included being younger than 36 months, the previous hospitalisation in the emergency department, invasive procedures, the use of tools, the doctor not communicating with the child, not informing the child before the procedure, and forcibly interventions . One study reported that children developed fear against hospitals for several reasons such as separating from their friends, falling behind in their studies, the possibility of dying, and feeling lonely without their family around. They also worried about suffocating whilst wearing an oxygen mask, and being unable to play games . Likewise, the present study revealed that 21.1% of the children were hospitalised before; however, they were afraid of the hospital at a high rate (73.4%). When the reasons behind their fear of hospitals were examined, it was determined that they developed fear due to invasive procedures, tools, separation from family, forcibly intervention and lack of communication ( ). The decrease in the state stress scores of the 4–10 year-old children, who had fear of hospitals, after the Video-Based COVID-19 Paediatric Patient Education was higher than those who did not (p < 0.01). It may be recommended to use Video-Based COVID-19 Patient Education in the stress management of 4–10 year-olds who have fear of hospitals.
Evaluation of state stress scores Fear of hospital procedures developing in children may be associated with previous negative experiences and lack of communication before the procedure . In the present study, the decrease in state stress scores of children aged 11–12 years, who had previous hospitalisations, after the education was found to be higher than those who did not. This result suggested that the video-based patient education was more effective on children having previous hospital experiences. Although state stress of the children who had no previous hospital experience decreased after the education, this was not statistically significant. The majority of children who had fear of the protective equipment, used by medical personnel during the COVID-19 pandemic were aged between 4 and 10 years. After the Video-Based COVID-19 Paediatric Patient Education, these children's such fear decreased significantly. The video-based education is an effective education method on children because it appeals to the visual and auditory area. In their study, Carpenter et al., aimed to teach the use of inhaler technique to children aged 7–17 using video training and determined that their inhaler usage skill scores increased immediately after the training . Dyson et al., had individuals with type 2 diabetes watched professional training videos (including three parts of 10–15 min), and reported that the knowledge of the participants about diabetes significantly increased . In a video education and consultation-based study conducted by Toledo et al., for diabetic individuals, they stated that hypoglycaemia rates decreased . In the present study, the decrease in the posttest state stress scores of the children compared to the pretest was found to be statistically significant and the video-based COVID-19 education prepared for children was observed to be effective.
Limitations The results cannot be generalised because the study was carried out only in one institution. The results reflect self-reports of the children and their parents.
Practice implications The Video-Based COVID-19 Paediatric Patient Education may enable to ease children's fear of hospitals and increase their adherence to care and treatment. The care burden of nurses can also be reduced by increasing the child's adaptation to the COVID-19 pandemic.
Conclusion Illness and hospitalisation pose a significant problem for children since changes in normal health status and environmental routines are stressors for them and they cannot cope with stressful events sufficiently. Hospitalisation is a negative experience that causes anxiety for all children. A conscious preparation for illness and hospitalisation is required in order to make this negative experience the least stressful for the child. When children are hospitalised, the nurse has a great role in reducing the negative effects on them and their families. When the nurse establishes an effective communication with the children, their adaptation to the hospital becomes easier, their parents’ anxiety gets also alleviated and their trust to the nurse increases. A conscious nursing approach helps children and their families feel more positive and comfortable in the hospitals. For this reason, it is recommended for nurses to organise programs that prepare children for hospitals and procedures. One month after the WHO declared COVID-19 as a pandemic, the Video-Based COVID-19 Paediatric Patient Education was used and its effectiveness was proven. This education is a training program that can be used during the COVID-19 pandemic and sets an example for different possible pandemics in the future. It is recommended to manage the pandemic and disease processes by planning similar video-based trainings that increase the children's compliance to the procedure and ease their families.
Yağmur Şancı: Conceptualization, Methodology, Investigation, Writing – original draft, Funding acquisition. Serap Çelik: Investigation, Resources, Funding acquisition. Suzan Yıldız: Methodology, Writing – review & editing, Funding acquisition.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Individual differences in self-reported lie detection abilities
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db9c0710-da80-4ced-9a51-4e845c6fbb70
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10208523
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Forensic Medicine[mh]
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Evolutionary approaches highlight that living in social groups is key to being successful in our environments . Living in groups comes not only with advantages though. Firstly, we must assume that other group members are trustworthy and bare no threat, so that we can engage in effective communication. Without trust, we would constantly experience problematic relationships within and between social groups. Interpersonal trust ensures individuals’ well-being, the achievement of their own aims, and the longevity of social units such as families, communities, and institutions [ – ]. Yet, being excessively trustworthy also bears risks, because others might take advantage for their own benefits . In an ideal world, one would always know who to trust and who not to trust, who and what to believe and not to believe. This ideal world, however, does not exist, because deception occurs daily , on average, at least once or twice a day [ – ]. When people lie, they make completely or partially untrue statements with the intent to deceive, and about 80% of the times, people succeed with lies remaining undetected . Yet, not everybody lies to the same extent with more recent studies showing that a few prolific liars are telling the majority of lies [ , , ]. Then, there is not one type of lies. There are many: lies can be about feelings, opinions, achievements, failures, to name a few examples. We may lie because honesty would hamper social or material goals , for financial or emotional gains, to avoid embarrassment, or to protect the feelings of others [ , – ]. At this point, we wish to highlight a striking paradox between actual (objective) and self-reported (subjective) lie detection abilities. Experimental studies showed that objectively, people perform around chance level when trying to spot somebody else lying . Subjectively, however, people report being able to detect lies successfully [ , , ]. This paradox might originate from the so-called truth-default state, meaning, one assumes that others are honest. Indeed, most everyday situations inspire trust, or at least, do not merit questioning. Such a truth-default state is desirable because trust facilitates cooperation and effective communication . Clare and Levine argued that individuals would only deviate from this truth-default state subsequent to a trigger events, such as suspicious indicators or motives, dishonest behavior, information from third parties, or inconsistencies in statements or known facts. Overall, we can assume that lying is omnipresent, but that most lies remain undetected, because people see few reasons to distrust each other. Consequently, individuals encounter few opportunities to study and learn about others’ successful lies. Thus, we face an imbalance between i) the number of undetected lies by oneself, ii) the number of undetected lies by others, and iii) the number of a few occasions when lies were detected. This imbalance may leave individuals with an inflated estimation of their own lie detection abilities, once a lie had been detected . Moreover, this inflated estimation might be further supported by people’s tendency to think positively about themselves: being easily deceived would not match this positive self-view . When trying to understand this inflated estimation, we noted a surprising void of published studies reporting on reasons or correlates of self-reported lie detection abilities [ – ]. The few available studies focused on the Dark Triad personality traits–Machiavellianism, narcissism, and psychopathy,–because these traits share malevolent behavioral tendencies such as self-promotion, manipulation (including lying), and lack of empathy [ – ]. Wissing and Reinhard showed that higher psychopathy and higher overall scores on Dark Triad personality were associated with higher self-reported confidence in lie detection abilities. Then, Zvi and Elaad showed that higher levels of narcissism were associated with higher production of lies, which in turn was associated with higher self-reported lie detection abilities in comparison to others . The recent study by Elaad supported the association between higher narcissism and higher self-reported lie detection abilities in comparison to others. Interestingly, self-reported confidence in one’s own lie detection abilities was unrelated to individuals’ actual lie detection abilities . Also, variance in Dark Triad traits did not explain variance in actual lie detection abilities either . Results on these few studies were not conclusive. Thus, we designed a new study on individual differences on self-reported lie detection abilities. In addition to the Dark Triad personality traits, we included further measures that had been studied in the context of lie detection abilities and lie production before, though again showing inconclusive results. To this end, we selected a series of different measures, namely gender, social cognition measures, cultural differences, general personality traits, and social desirability. For gender, studies showed no differences between men and women in both lie detection and lie production [ – ]. For social cognition, we considered empathy and emotional intelligence. For instance, Duran et al. found that women with lower as compared to higher levels of empathy performed better at lie detection. Former studies would have predicted the opposite, though . Others argued that higher levels of empathy should link to impaired lie detection performances . In respect to emotional intelligence, higher trait levels might facilitate lie detection . Actual studies, however, showed that higher emotional intelligence, particularly emotionality, yielded impaired lie detection abilities . For cultural differences, the need for lie detection might differ as a function of where you live on the globe, because levels of trust, deception, and corruption vary cross-culturally [ , , ]. For instance, in case of lower national trust levels, people might be more suspicious towards others, having a lower truth-default state . Maybe, they perform above chance in lie detection, or they just think that they do, once the odd lie had been detected. Moreover, individuals from collectivistic societies (prioritizing harmony among group members) seem more inclined to lie for other people’s benefits than those from individualistic societies . To account for individual differences, we performed two consecutive online studies to investigate self-reported lie detection abilities. In Study 1, participants completed self-report measures on the Dark Triad , empathy , cultural values , and national trust levels . Participants also indicated whether they were able to detect lies (yes/no), and at which level of accuracy (0–25%, 25–50%, 50–75%, 75–100%). Around 80% of the sample answered yes , with little variance in the accuracy level ratings. Thus, we made several changes to Study 2. We again asked participants to complete some of the same questionnaires (empathy, cultural values, national trust levels), but exchanged the 12-item Dirty Dozen with the psychometrically superior Dark Triad personality traits questionnaire . Additionally, we assessed participants’ general personality traits such as Agreeableness, Openness to Experience, Extraversion, Conscientiousness, Honesty and Resiliency , due to formerly described relationships with Dark Triad traits . We also assessed emotional intelligence and socially desirable responding . Crucially, participants again completed the yes/no question regarding their self-reported lie detection abilities. This time they rated their estimated accuracy on a scale from 0 ( unable to detect lies ) to 100 ( perfectly able to detect lies ). Moreover, to account for previous studies on the subject (see also [ , – , , ], we also asked participants to judge their lie detection abilities in comparison to others on a scale ranging from 0 ( much worse than others ) to 100 ( much better than others ). Across these two studies, we investigated whether we might observe enhanced self-reported lie detection abilities as a function of i) Dark Triad personality scores (i.e., narcissism, psychopathy, and Machiavellianism), ii) lower empathy scores, iii) lower emotional intelligence scores, and iv) cultural values. Regarding cultural values, we expected to find differences between Hofstede’s cultural values (i.e., collectivism) and self-reported lie detection abilities. Finally, we expected self-reported lie detection abilities to be positively associated with social desirability, in line with people’s tendency to think and show themselves in a positive way .
Method Participants We recruited 764 participants (105 males). After excluding incomplete data and selecting participants between 18 and 31 years old (i.e., the majority), our final sample consisted in 487 participants (99 males) with a mean age of 21.50 years ( SD age = 6.57 years; range = 18–31 years). Of these, 418 (95 males) were undergraduate students at the University of Lausanne, Switzerland, who received course credit for their participation. The remaining 69 participants (4 males) were recruited at the University of Franche-Comté, France. All participants were native French speakers. These studies were conducted in accordance with the principles expressed in the Declaration of Helsinki and received approval from the Research Ethics Commission of the Institute of Psychology, University of Lausanne (C_SSP_052021_00001). We had used the statistical power analysis tool G*Power to estimate the minimum sample size of 473 for a two-tailed binary logistic regression with a small effect size (odds ratio of 1.68) (see), the H 0 probability for Y = 1 of .45, α of 0.05, power (1−β) of 0.80. Material Self-report questionnaires All our questionnaires demonstrated good or acceptable internal consistency (Cronbach’s alpha values between .510 and .817) (see ). Dark Triad Dirty Dozen inventory (French-Canadian version from Savard et al ). This short 12-item scale assesses psychopathy, narcissism and Machiavellianism. Participants indicated their agreement with each statement on a 5-point Likert scale (see ). Narcissism items assess the search for admiration and attention, the importance of status and expectations of special favours from others (e.g., “I tend to want others to admire me ”). Machiavellianism items assess the use of deception, manipulation, flattery, and exploitation of others to achieve own goals (e.g., “ I tend to manipulate others to get my way ”). Psychopathy items assess the lack of remorse, insensitivity, being cynical, and being unconcerned by the morality of own actions (e.g., “ I tend to lack remorse ”). We averaged the scores of the four items of each subscale to obtain the total score on each subscale. Higher scores indicate higher expressions of narcissistic, Machiavellian, or psychopathic traits. Individual Cultural Values Scale (French version from Zheng ) This 26-item scale is based on Hofstede’s cultural dimensions model assessing the five cultural dimensions of i) power distance, ii) uncertainty avoidance, iii) collectivism vs individualism iv) masculinity, and v) long-term vs. short-term orientation (see for details on the scale and sub-scales). Power distance refers to the degree to which less powerful members of institutions and organizations expect and accept that power is unequally distributed. Higher scores indicate a stronger acceptance of this unequal power distribution. Uncertainty avoidance refers to the degree to which societies avoid uncertainty, are anxious, feel threatened by unknown situations, prefer clear procedures, and respect rules. Higher scores indicate a stronger uncertainty avoidance. Collectivism vs. individualism describes the degree to which collectivism (group cohesion and interests) dominates over individualism (one’s own and close kins’ interests). Lower scores indicate a bias towards collectivism, and higher scores–towards individualism. Masculinity describes the degree to which male vs. female role patterns dominate in a society, with higher scores favouring the male pattern. Long-term vs. short term orientation refers to cultures that make long-term or short-term plans, respectively. The scale considers prudence, thrift, persistence, perseverance, and willingness to sacrifice the present to favour future successes. Higher scores indicate a preference for long-term planning. Participants expressed their agreement with each statement on a 7-point Likert scale, and the scores were averaged for each subscale (see ). The World Value Survey 5 This 6-item questionnaire determines in-group and out-group trust levels on a 4-point Likert scale (see ). For the in-group trust level, participants rate their trust of family members, neighbours, and people they know personally. For out-group trust level, participants rate their trust of people they meet for the first time, from another religion, and another nationality. For the two subscales, the Likert scale scores were averaged ( ). Interpersonal Reactivity Index (IRI ; French version from Gilet et al ). The 28-item IRI questionnaire evaluates the following aspects of empathy: i) feelings of compassion and concern for unfortunate others, ii) the ability to consider the perspective of others, iii) participants’ tendencies to transpose themselves imaginatively or to identify with fictional characters, and iv) self-oriented feelings of personal anxiety and distress in difficult interpersonal situations. Participants rated their agreement with items on a 5-point Likert scale. Of the 28 items, eight were reversely coded. We calculated an average empathy score so that higher scores indicated a natural capacity to share and understand the affective state of others (see ). Other questionnaires and measures In Study 1, we also assessed participants’ self-reported trait autism scores and paranormal belief scores . In both studies, we also asked participants to report on cues they rely on to decide that another person is lying (open question added after the questions on self-reported lying detection abilities, ). These data are reported elsewhere. Self-reported lie detection abilities We asked participants to indicate their self-reported lie detection abilities. In other words, we asked whether they were able to recognize somebody lying. Participants answered on a binary yes/no scale. Afterwards, participants indicated their self-reported lie detection abilities on a 4-point Likert-type scale. They had to rate how accurate they think they were at lie detection, choosing from four options: accurate 0–25% of the time (1), 25–50% (2), 50–75% (3), or 75–100% of the time (4). Procedure We used the LimeSurvey platform to prepare and run our online survey. We distributed the online link to potential volunteers. In case of interest, they could complete the survey at their own convenience. On the first two pages, we provided, respectively, written study information and ethical information, such as the right to withdraw from the study at any time, and data confidentiality ( ). We also stated that we treat participants’ continuation as informed consent. Next, participants provided socio-demographic information regarding their age, gender, nationality, and field of studies ( ). Then, they completed the self-reported questionnaires in the following order: The FR-C Dark Triad Dirty Dozen, the Cultural Values Scale, the Interpersonal Reactivity Index, and their level of trust based on the World Value Survey 5 ( -Study 1 and ). Afterwards, they indicated their self-reported lie detection abilities ( -Study 1). Finally, they were fully debriefed and thanked for their participation. The survey took about 20 minutes to complete. Design and statistical analysis To test for the role of individual differences in self-reported lie detection abilities, we considered the self-reported lie detection abilities score as a dependent measure, which was measured on a four-point scale (0–25%; 25–50%; 50–75%; and 75–100%). The histogram showed an uneven distribution. Of the 487 participants, 198 rated their abilities between 25–50% of accuracy and 237 between 50–75%, while only 19 participants self-reported 0–25% of accuracy, and only 33 indicate 75–100% of accuracy. Consequently, we created a dichotomous variable consisting in self-reported lie detection abilities that are below chance level (i.e. scores between 0–25% and 25–50% of accuracy) and above chance level (i.e. scores between 50–75% and 75–100% of accuracy). To test our study question, we conducted a binary logistic regression analysis on this dependent dichotomous variable (below vs. above chance level) using the following 12 continuous predictor variables: psychopathy, narcissism, Machiavellianism, power distance, uncertainty avoidance, collectivism, masculinity, long-term orientation, empathy, ingroup trust level, outgroup trust level, and gender (see also ). We presented the correlations between all the predictor variables as supporting information (see ).
Participants We recruited 764 participants (105 males). After excluding incomplete data and selecting participants between 18 and 31 years old (i.e., the majority), our final sample consisted in 487 participants (99 males) with a mean age of 21.50 years ( SD age = 6.57 years; range = 18–31 years). Of these, 418 (95 males) were undergraduate students at the University of Lausanne, Switzerland, who received course credit for their participation. The remaining 69 participants (4 males) were recruited at the University of Franche-Comté, France. All participants were native French speakers. These studies were conducted in accordance with the principles expressed in the Declaration of Helsinki and received approval from the Research Ethics Commission of the Institute of Psychology, University of Lausanne (C_SSP_052021_00001). We had used the statistical power analysis tool G*Power to estimate the minimum sample size of 473 for a two-tailed binary logistic regression with a small effect size (odds ratio of 1.68) (see), the H 0 probability for Y = 1 of .45, α of 0.05, power (1−β) of 0.80.
We recruited 764 participants (105 males). After excluding incomplete data and selecting participants between 18 and 31 years old (i.e., the majority), our final sample consisted in 487 participants (99 males) with a mean age of 21.50 years ( SD age = 6.57 years; range = 18–31 years). Of these, 418 (95 males) were undergraduate students at the University of Lausanne, Switzerland, who received course credit for their participation. The remaining 69 participants (4 males) were recruited at the University of Franche-Comté, France. All participants were native French speakers. These studies were conducted in accordance with the principles expressed in the Declaration of Helsinki and received approval from the Research Ethics Commission of the Institute of Psychology, University of Lausanne (C_SSP_052021_00001). We had used the statistical power analysis tool G*Power to estimate the minimum sample size of 473 for a two-tailed binary logistic regression with a small effect size (odds ratio of 1.68) (see), the H 0 probability for Y = 1 of .45, α of 0.05, power (1−β) of 0.80.
Self-report questionnaires All our questionnaires demonstrated good or acceptable internal consistency (Cronbach’s alpha values between .510 and .817) (see ). Dark Triad Dirty Dozen inventory (French-Canadian version from Savard et al ). This short 12-item scale assesses psychopathy, narcissism and Machiavellianism. Participants indicated their agreement with each statement on a 5-point Likert scale (see ). Narcissism items assess the search for admiration and attention, the importance of status and expectations of special favours from others (e.g., “I tend to want others to admire me ”). Machiavellianism items assess the use of deception, manipulation, flattery, and exploitation of others to achieve own goals (e.g., “ I tend to manipulate others to get my way ”). Psychopathy items assess the lack of remorse, insensitivity, being cynical, and being unconcerned by the morality of own actions (e.g., “ I tend to lack remorse ”). We averaged the scores of the four items of each subscale to obtain the total score on each subscale. Higher scores indicate higher expressions of narcissistic, Machiavellian, or psychopathic traits. Individual Cultural Values Scale (French version from Zheng ) This 26-item scale is based on Hofstede’s cultural dimensions model assessing the five cultural dimensions of i) power distance, ii) uncertainty avoidance, iii) collectivism vs individualism iv) masculinity, and v) long-term vs. short-term orientation (see for details on the scale and sub-scales). Power distance refers to the degree to which less powerful members of institutions and organizations expect and accept that power is unequally distributed. Higher scores indicate a stronger acceptance of this unequal power distribution. Uncertainty avoidance refers to the degree to which societies avoid uncertainty, are anxious, feel threatened by unknown situations, prefer clear procedures, and respect rules. Higher scores indicate a stronger uncertainty avoidance. Collectivism vs. individualism describes the degree to which collectivism (group cohesion and interests) dominates over individualism (one’s own and close kins’ interests). Lower scores indicate a bias towards collectivism, and higher scores–towards individualism. Masculinity describes the degree to which male vs. female role patterns dominate in a society, with higher scores favouring the male pattern. Long-term vs. short term orientation refers to cultures that make long-term or short-term plans, respectively. The scale considers prudence, thrift, persistence, perseverance, and willingness to sacrifice the present to favour future successes. Higher scores indicate a preference for long-term planning. Participants expressed their agreement with each statement on a 7-point Likert scale, and the scores were averaged for each subscale (see ). The World Value Survey 5 This 6-item questionnaire determines in-group and out-group trust levels on a 4-point Likert scale (see ). For the in-group trust level, participants rate their trust of family members, neighbours, and people they know personally. For out-group trust level, participants rate their trust of people they meet for the first time, from another religion, and another nationality. For the two subscales, the Likert scale scores were averaged ( ). Interpersonal Reactivity Index (IRI ; French version from Gilet et al ). The 28-item IRI questionnaire evaluates the following aspects of empathy: i) feelings of compassion and concern for unfortunate others, ii) the ability to consider the perspective of others, iii) participants’ tendencies to transpose themselves imaginatively or to identify with fictional characters, and iv) self-oriented feelings of personal anxiety and distress in difficult interpersonal situations. Participants rated their agreement with items on a 5-point Likert scale. Of the 28 items, eight were reversely coded. We calculated an average empathy score so that higher scores indicated a natural capacity to share and understand the affective state of others (see ). Other questionnaires and measures In Study 1, we also assessed participants’ self-reported trait autism scores and paranormal belief scores . In both studies, we also asked participants to report on cues they rely on to decide that another person is lying (open question added after the questions on self-reported lying detection abilities, ). These data are reported elsewhere. Self-reported lie detection abilities We asked participants to indicate their self-reported lie detection abilities. In other words, we asked whether they were able to recognize somebody lying. Participants answered on a binary yes/no scale. Afterwards, participants indicated their self-reported lie detection abilities on a 4-point Likert-type scale. They had to rate how accurate they think they were at lie detection, choosing from four options: accurate 0–25% of the time (1), 25–50% (2), 50–75% (3), or 75–100% of the time (4).
All our questionnaires demonstrated good or acceptable internal consistency (Cronbach’s alpha values between .510 and .817) (see ).
] (French-Canadian version from Savard et al ). This short 12-item scale assesses psychopathy, narcissism and Machiavellianism. Participants indicated their agreement with each statement on a 5-point Likert scale (see ). Narcissism items assess the search for admiration and attention, the importance of status and expectations of special favours from others (e.g., “I tend to want others to admire me ”). Machiavellianism items assess the use of deception, manipulation, flattery, and exploitation of others to achieve own goals (e.g., “ I tend to manipulate others to get my way ”). Psychopathy items assess the lack of remorse, insensitivity, being cynical, and being unconcerned by the morality of own actions (e.g., “ I tend to lack remorse ”). We averaged the scores of the four items of each subscale to obtain the total score on each subscale. Higher scores indicate higher expressions of narcissistic, Machiavellian, or psychopathic traits.
] (French version from Zheng ) This 26-item scale is based on Hofstede’s cultural dimensions model assessing the five cultural dimensions of i) power distance, ii) uncertainty avoidance, iii) collectivism vs individualism iv) masculinity, and v) long-term vs. short-term orientation (see for details on the scale and sub-scales). Power distance refers to the degree to which less powerful members of institutions and organizations expect and accept that power is unequally distributed. Higher scores indicate a stronger acceptance of this unequal power distribution. Uncertainty avoidance refers to the degree to which societies avoid uncertainty, are anxious, feel threatened by unknown situations, prefer clear procedures, and respect rules. Higher scores indicate a stronger uncertainty avoidance. Collectivism vs. individualism describes the degree to which collectivism (group cohesion and interests) dominates over individualism (one’s own and close kins’ interests). Lower scores indicate a bias towards collectivism, and higher scores–towards individualism. Masculinity describes the degree to which male vs. female role patterns dominate in a society, with higher scores favouring the male pattern. Long-term vs. short term orientation refers to cultures that make long-term or short-term plans, respectively. The scale considers prudence, thrift, persistence, perseverance, and willingness to sacrifice the present to favour future successes. Higher scores indicate a preference for long-term planning. Participants expressed their agreement with each statement on a 7-point Likert scale, and the scores were averaged for each subscale (see ).
] This 6-item questionnaire determines in-group and out-group trust levels on a 4-point Likert scale (see ). For the in-group trust level, participants rate their trust of family members, neighbours, and people they know personally. For out-group trust level, participants rate their trust of people they meet for the first time, from another religion, and another nationality. For the two subscales, the Likert scale scores were averaged ( ).
]; French version from Gilet et al ). The 28-item IRI questionnaire evaluates the following aspects of empathy: i) feelings of compassion and concern for unfortunate others, ii) the ability to consider the perspective of others, iii) participants’ tendencies to transpose themselves imaginatively or to identify with fictional characters, and iv) self-oriented feelings of personal anxiety and distress in difficult interpersonal situations. Participants rated their agreement with items on a 5-point Likert scale. Of the 28 items, eight were reversely coded. We calculated an average empathy score so that higher scores indicated a natural capacity to share and understand the affective state of others (see ).
In Study 1, we also assessed participants’ self-reported trait autism scores and paranormal belief scores . In both studies, we also asked participants to report on cues they rely on to decide that another person is lying (open question added after the questions on self-reported lying detection abilities, ). These data are reported elsewhere.
We asked participants to indicate their self-reported lie detection abilities. In other words, we asked whether they were able to recognize somebody lying. Participants answered on a binary yes/no scale. Afterwards, participants indicated their self-reported lie detection abilities on a 4-point Likert-type scale. They had to rate how accurate they think they were at lie detection, choosing from four options: accurate 0–25% of the time (1), 25–50% (2), 50–75% (3), or 75–100% of the time (4).
We used the LimeSurvey platform to prepare and run our online survey. We distributed the online link to potential volunteers. In case of interest, they could complete the survey at their own convenience. On the first two pages, we provided, respectively, written study information and ethical information, such as the right to withdraw from the study at any time, and data confidentiality ( ). We also stated that we treat participants’ continuation as informed consent. Next, participants provided socio-demographic information regarding their age, gender, nationality, and field of studies ( ). Then, they completed the self-reported questionnaires in the following order: The FR-C Dark Triad Dirty Dozen, the Cultural Values Scale, the Interpersonal Reactivity Index, and their level of trust based on the World Value Survey 5 ( -Study 1 and ). Afterwards, they indicated their self-reported lie detection abilities ( -Study 1). Finally, they were fully debriefed and thanked for their participation. The survey took about 20 minutes to complete. Design and statistical analysis To test for the role of individual differences in self-reported lie detection abilities, we considered the self-reported lie detection abilities score as a dependent measure, which was measured on a four-point scale (0–25%; 25–50%; 50–75%; and 75–100%). The histogram showed an uneven distribution. Of the 487 participants, 198 rated their abilities between 25–50% of accuracy and 237 between 50–75%, while only 19 participants self-reported 0–25% of accuracy, and only 33 indicate 75–100% of accuracy. Consequently, we created a dichotomous variable consisting in self-reported lie detection abilities that are below chance level (i.e. scores between 0–25% and 25–50% of accuracy) and above chance level (i.e. scores between 50–75% and 75–100% of accuracy). To test our study question, we conducted a binary logistic regression analysis on this dependent dichotomous variable (below vs. above chance level) using the following 12 continuous predictor variables: psychopathy, narcissism, Machiavellianism, power distance, uncertainty avoidance, collectivism, masculinity, long-term orientation, empathy, ingroup trust level, outgroup trust level, and gender (see also ). We presented the correlations between all the predictor variables as supporting information (see ).
To test for the role of individual differences in self-reported lie detection abilities, we considered the self-reported lie detection abilities score as a dependent measure, which was measured on a four-point scale (0–25%; 25–50%; 50–75%; and 75–100%). The histogram showed an uneven distribution. Of the 487 participants, 198 rated their abilities between 25–50% of accuracy and 237 between 50–75%, while only 19 participants self-reported 0–25% of accuracy, and only 33 indicate 75–100% of accuracy. Consequently, we created a dichotomous variable consisting in self-reported lie detection abilities that are below chance level (i.e. scores between 0–25% and 25–50% of accuracy) and above chance level (i.e. scores between 50–75% and 75–100% of accuracy). To test our study question, we conducted a binary logistic regression analysis on this dependent dichotomous variable (below vs. above chance level) using the following 12 continuous predictor variables: psychopathy, narcissism, Machiavellianism, power distance, uncertainty avoidance, collectivism, masculinity, long-term orientation, empathy, ingroup trust level, outgroup trust level, and gender (see also ). We presented the correlations between all the predictor variables as supporting information (see ).
Overall, most participants (81.52%) reported being able to detect lies (yes/no answer) and many participants (55.44%) self-reported that their lie detection abilities ranged between 50% and 100% (i.e. above chance level self-reported lie detection abilities). The likelihood ratio test showed that the overall binary logistic regression model on self-reported lie detection abilities was not significant; LR (12) = 656.23, p =. 360, AIC = 682.23, pseudo R 2 = .025 (Cox & Snell), .034 (Nagelkerke) (see ).
Method Participants We used the statistical power analysis tool G*Power to estimate our sample size. We determined a minimum sample size of 231 for a linear multiple regression test with a small effect size of 0.1, 21 predictors, α of 0.05, and power (1−β) of 0.80. We recruited a new sample of French speaking undergraduate psychology students ( N = 700, 90 males) at the University of Lausanne. After excluding incomplete data and matching participants’ age to those of Study 1, we were left with 386 participants (72 males; M age = 20.21, SD age = 2.22, range = 18 to 31 years). One academic year separated the data collection for Study 1 and Study 2. All participants received course credit for their participation. Material Self-report questionnaires. Short Dark Triad (SD3) (French version from Gamache et al. ) This 27-item version measures psychopathy , narcissism and Machiavellianism using a 5-point Likert scale. We used this questionnaire, because the one we had used in Study 1 showed weaker psychometric properties . Big-Six questionnaire (29QB6) (The French translation was done through translation and back translation, the report on the formal validation is currently prepared for publication. In the meantime, the translation can be retrieved here . The 29QB6 assesses personality along six dimensions: i) Extraversion, ii) Conscientiousness, iii) Honesty/Propriety, iv) Resiliency vs. Internalizing Negative Emotionality, v) Agreeableness, and vi) Originality/Talent. Higher Extraversion scores indicate that participants score higher on traits like talkativeness, sociability, assertiveness., gregariousness, and positive emotionality measures. Conscientiousness items refer to being organized, purposeful, and self-controlled. Honesty/Propriety items measure ethical behavior, integrity and deceit, instrumental use of others as well as aspects related to negative valence. Resiliency items capture the ability to internalize negative emotionality as a reverse of Neuroticism. Lower scores on resiliency resemble higher scores on neuroticism Agreeableness items measure kindness and even temper. Originality/Talent items measure perceived talents and abilities, intellectual and aesthetic interest. They also include positive valence content. The 29 items are rated on a 6-point Likert scale, with 14 being reversely coded (see ). Higher scores indicate higher level of expression of each personality trait (see ). Trait Meta Mood Scale (French version from Maria et al ). This 30-item questionnaire measures emotional intelligence by assessing the ability to monitor and manage one’s own emotions. This scale consists in three subscales: i) emotional attention –the extent to which individuals attend to and value their feelings; ii) emotional clarity –the extent to which they feel clear about their feelings; and iii) emotional repair –the extent to which they use positive thinking to repair their negative mood. Participants rated each item on a 5-point Likert scale. Higher scores indicated higher levels of emotional attention, emotional clarity, and emotional repair (see ). Balanced Inventory of Desirable Responding (French version from D’Amours-Raymond ) This 21-item questionnaire measures socially desirable responding along two dimensions: i) self-deceptive positivity– the tendency to give self-reports believed to have a positivity bias, and ii) impression management –deliberate effort at deception. We calculated the total score by summing all the responses on the 7-point Likert scale. We followed authors’ recommendations and coded presence of social desirability (yes) when participants scored between 6 and 7, and absence of social desirability (no) for values below 5 (see ). Other questionnaires The remaining self-reported questionnaires were the same as in Study 1: i) General empathy score using the 28-item Interpersonal Reactivity Index, ii) Cultural dimensions using the 26-item Individual Cultural Values Scale, and iii) trust levels using the World Value Survey 5. All our questionnaires demonstrated good or acceptable internal consistency (see ). Self-reported lie detection abilities We asked participants to indicate if they were able to recognize somebody lying (yes/no answer) and rate their self-reported lie detection abilities on a continuous scale ranging from 0 ( unable to detect lies ) to 100 ( perfectly able to detect lies ). Afterwards, we asked participants to report their lie detection abilities in comparison to others from 0 ( much worse than others ) to 100 ( much better than others) (see also [ – , ]). Procedure The procedure was comparable to Study 1 in recruitment and programming, as well as in the sequence of events (see ). Participants completed the Cultural Values Scale, the Interpersonal Reactivity Index, their level of trust, the Dark Triad, the Balanced Inventory of Desirable Responding, the Trait and Meta Mood Scale, and the Big-Six questionnaires ( -Study 2). Afterwards, participants self-reported their lie detection abilities ( -Study 2). The entire study took about 20 minutes to complete. Statistical analysis We performed two multiple regression analyses to test whether our individual difference measures predicted i) participants’ self-reported lie detection abilities score (0–100), and ii) participants’ self-reported lie detection abilities in comparison to others (see ). We present results of the correlations between all predictor variables in supporting information (see ).
Participants We used the statistical power analysis tool G*Power to estimate our sample size. We determined a minimum sample size of 231 for a linear multiple regression test with a small effect size of 0.1, 21 predictors, α of 0.05, and power (1−β) of 0.80. We recruited a new sample of French speaking undergraduate psychology students ( N = 700, 90 males) at the University of Lausanne. After excluding incomplete data and matching participants’ age to those of Study 1, we were left with 386 participants (72 males; M age = 20.21, SD age = 2.22, range = 18 to 31 years). One academic year separated the data collection for Study 1 and Study 2. All participants received course credit for their participation.
We used the statistical power analysis tool G*Power to estimate our sample size. We determined a minimum sample size of 231 for a linear multiple regression test with a small effect size of 0.1, 21 predictors, α of 0.05, and power (1−β) of 0.80. We recruited a new sample of French speaking undergraduate psychology students ( N = 700, 90 males) at the University of Lausanne. After excluding incomplete data and matching participants’ age to those of Study 1, we were left with 386 participants (72 males; M age = 20.21, SD age = 2.22, range = 18 to 31 years). One academic year separated the data collection for Study 1 and Study 2. All participants received course credit for their participation.
Self-report questionnaires. Short Dark Triad (SD3) (French version from Gamache et al. ) This 27-item version measures psychopathy , narcissism and Machiavellianism using a 5-point Likert scale. We used this questionnaire, because the one we had used in Study 1 showed weaker psychometric properties . Big-Six questionnaire (29QB6) (The French translation was done through translation and back translation, the report on the formal validation is currently prepared for publication. In the meantime, the translation can be retrieved here . The 29QB6 assesses personality along six dimensions: i) Extraversion, ii) Conscientiousness, iii) Honesty/Propriety, iv) Resiliency vs. Internalizing Negative Emotionality, v) Agreeableness, and vi) Originality/Talent. Higher Extraversion scores indicate that participants score higher on traits like talkativeness, sociability, assertiveness., gregariousness, and positive emotionality measures. Conscientiousness items refer to being organized, purposeful, and self-controlled. Honesty/Propriety items measure ethical behavior, integrity and deceit, instrumental use of others as well as aspects related to negative valence. Resiliency items capture the ability to internalize negative emotionality as a reverse of Neuroticism. Lower scores on resiliency resemble higher scores on neuroticism Agreeableness items measure kindness and even temper. Originality/Talent items measure perceived talents and abilities, intellectual and aesthetic interest. They also include positive valence content. The 29 items are rated on a 6-point Likert scale, with 14 being reversely coded (see ). Higher scores indicate higher level of expression of each personality trait (see ). Trait Meta Mood Scale (French version from Maria et al ). This 30-item questionnaire measures emotional intelligence by assessing the ability to monitor and manage one’s own emotions. This scale consists in three subscales: i) emotional attention –the extent to which individuals attend to and value their feelings; ii) emotional clarity –the extent to which they feel clear about their feelings; and iii) emotional repair –the extent to which they use positive thinking to repair their negative mood. Participants rated each item on a 5-point Likert scale. Higher scores indicated higher levels of emotional attention, emotional clarity, and emotional repair (see ). Balanced Inventory of Desirable Responding (French version from D’Amours-Raymond ) This 21-item questionnaire measures socially desirable responding along two dimensions: i) self-deceptive positivity– the tendency to give self-reports believed to have a positivity bias, and ii) impression management –deliberate effort at deception. We calculated the total score by summing all the responses on the 7-point Likert scale. We followed authors’ recommendations and coded presence of social desirability (yes) when participants scored between 6 and 7, and absence of social desirability (no) for values below 5 (see ). Other questionnaires The remaining self-reported questionnaires were the same as in Study 1: i) General empathy score using the 28-item Interpersonal Reactivity Index, ii) Cultural dimensions using the 26-item Individual Cultural Values Scale, and iii) trust levels using the World Value Survey 5. All our questionnaires demonstrated good or acceptable internal consistency (see ). Self-reported lie detection abilities We asked participants to indicate if they were able to recognize somebody lying (yes/no answer) and rate their self-reported lie detection abilities on a continuous scale ranging from 0 ( unable to detect lies ) to 100 ( perfectly able to detect lies ). Afterwards, we asked participants to report their lie detection abilities in comparison to others from 0 ( much worse than others ) to 100 ( much better than others) (see also [ – , ]).
] (French version from Gamache et al. ) This 27-item version measures psychopathy , narcissism and Machiavellianism using a 5-point Likert scale. We used this questionnaire, because the one we had used in Study 1 showed weaker psychometric properties . Big-Six questionnaire (29QB6) (The French translation was done through translation and back translation, the report on the formal validation is currently prepared for publication. In the meantime, the translation can be retrieved here . The 29QB6 assesses personality along six dimensions: i) Extraversion, ii) Conscientiousness, iii) Honesty/Propriety, iv) Resiliency vs. Internalizing Negative Emotionality, v) Agreeableness, and vi) Originality/Talent. Higher Extraversion scores indicate that participants score higher on traits like talkativeness, sociability, assertiveness., gregariousness, and positive emotionality measures. Conscientiousness items refer to being organized, purposeful, and self-controlled. Honesty/Propriety items measure ethical behavior, integrity and deceit, instrumental use of others as well as aspects related to negative valence. Resiliency items capture the ability to internalize negative emotionality as a reverse of Neuroticism. Lower scores on resiliency resemble higher scores on neuroticism Agreeableness items measure kindness and even temper. Originality/Talent items measure perceived talents and abilities, intellectual and aesthetic interest. They also include positive valence content. The 29 items are rated on a 6-point Likert scale, with 14 being reversely coded (see ). Higher scores indicate higher level of expression of each personality trait (see ).
] (French version from Maria et al ). This 30-item questionnaire measures emotional intelligence by assessing the ability to monitor and manage one’s own emotions. This scale consists in three subscales: i) emotional attention –the extent to which individuals attend to and value their feelings; ii) emotional clarity –the extent to which they feel clear about their feelings; and iii) emotional repair –the extent to which they use positive thinking to repair their negative mood. Participants rated each item on a 5-point Likert scale. Higher scores indicated higher levels of emotional attention, emotional clarity, and emotional repair (see ).
] (French version from D’Amours-Raymond ) This 21-item questionnaire measures socially desirable responding along two dimensions: i) self-deceptive positivity– the tendency to give self-reports believed to have a positivity bias, and ii) impression management –deliberate effort at deception. We calculated the total score by summing all the responses on the 7-point Likert scale. We followed authors’ recommendations and coded presence of social desirability (yes) when participants scored between 6 and 7, and absence of social desirability (no) for values below 5 (see ).
The remaining self-reported questionnaires were the same as in Study 1: i) General empathy score using the 28-item Interpersonal Reactivity Index, ii) Cultural dimensions using the 26-item Individual Cultural Values Scale, and iii) trust levels using the World Value Survey 5. All our questionnaires demonstrated good or acceptable internal consistency (see ).
We asked participants to indicate if they were able to recognize somebody lying (yes/no answer) and rate their self-reported lie detection abilities on a continuous scale ranging from 0 ( unable to detect lies ) to 100 ( perfectly able to detect lies ). Afterwards, we asked participants to report their lie detection abilities in comparison to others from 0 ( much worse than others ) to 100 ( much better than others) (see also [ – , ]).
The procedure was comparable to Study 1 in recruitment and programming, as well as in the sequence of events (see ). Participants completed the Cultural Values Scale, the Interpersonal Reactivity Index, their level of trust, the Dark Triad, the Balanced Inventory of Desirable Responding, the Trait and Meta Mood Scale, and the Big-Six questionnaires ( -Study 2). Afterwards, participants self-reported their lie detection abilities ( -Study 2). The entire study took about 20 minutes to complete.
We performed two multiple regression analyses to test whether our individual difference measures predicted i) participants’ self-reported lie detection abilities score (0–100), and ii) participants’ self-reported lie detection abilities in comparison to others (see ). We present results of the correlations between all predictor variables in supporting information (see ).
Most participants (76.94%) reported being able to detect lies (yes/no answer). Many participants (62.69%) reported that their abilities to detect lies was above chance level (i.e., higher than 50% of accuracy). However, when asked to rate their abilities in comparison to others, only 20.46% of the participants reported being better than others at detecting lies. Most participants (59.59%) estimated their abilities to be below those of others. However, a Pearson correlation on these two self-report measures was significant, r (386) = .683, p < .001 (see ), higher self-reported lie detection abilities correlated with higher self-reported lie detection abilities in comparison to others. The overall multiple regression model on self-reported lie detection abilities was significant, F (363, 22) = 2.49, p < .001, R adj 2 = .078. Results indicated that the model explained 7.8% of the variance (see ). As shown in , lower out-group trust levels and higher social desirability predicted higher self-reported lie detection abilities. The remaining predictor variables were not significant (see ). The multiple linear regression analysis on self-reported lie detection abilities in comparison to others was significant, F (363, 22) = 3.57, p < .001, R adj 2 = .128. The model explained 12.8% of the variance (see ). Again, enhanced social desirability significantly predicted enhanced self-reported lie detection abilities in comparison to others (see ). We also found that higher originality and psychopathy predicted higher self-reported lie detection abilities in comparison to others (see ). The remaining predictor variables were not significant (see ).
The literature on lie detection demonstrates a little understood paradox. Subjectively, many people self-report being above average in lie detection abilities [ , – , ], while objectively, at least on a group level, people perform around chance level . In two successive online studies, we investigated if and how individual differences can predict self-reported lie detection abilities. In Study 1, we measured Dark Triad personality traits, empathy, cultural values, and trust levels. In Study 2, we additionally accounted for conventional personality traits, emotional intelligence, and social desirability. As outcome measures, participants indicated whether they could detect lies (dichotomous yes/no answer), and to what extent. In Study 1, we coded extent as above versus below chance level, and in Study 2 as scores on a 100-point rating scale. In Study 2, we also asked participants to rate their self-reported lie detection abilities in comparison to others (see also [ – , ]). In both studies, over 75% of our participants indicated being able to detect lies. When asked about extent, most participants rated their accuracy above chance level (i.e., being 50% of the time correct). Thus, we replicated that individuals on the group level consider themselves able to detect lies (e.g., [ , – , ]), which is well above the performance one could expect from actual lie detection tasks . When asked to rate their abilities in comparison to others, we found, however, that only about 20% of our participants indicated yes , they judged themselves to be superior to others at detecting lies. For extent, the numbers were comparatively low too; participants indicated being only 40% of the time better than others at detecting lies. Our primary study goal was to investigate individual differences in self-reported lie detection abilities. Contrary to our expectations, we found few systematic relationships. In Study 1, the overall model included 12 predictor variables and was not significant. In Study 2, the overall model included 22 predictor variables and the model was significant. We observed enhanced self-reported lie detection abilities with i) decreasing out-group trust level scores, and ii) increasing social desirability scores. Neither gender, personality traits (i.e., Dark Triad, Big-Six personality traits), social cognition measures (i.e., empathy and emotional intelligence), nor cultural values predicted self-reported lie detection abilities. Interestingly, our individual difference measures were better predictors of self-reported lie detection abilities in comparison to others. Namely, enhanced psychopathy, enhanced Originality/Talent, and enhanced social desirability predicted enhanced self-reported lie detection abilities in comparison to others. The findings on psychopathy and Originality/Talent (i.e., Openness) have been reported previously . Given our unexpected results, we decided to focus on three major observations. First, very few of our individual difference measures predicted self-reported lie detection abilities. We discuss in detail results on the Dark triad personality traits, because we had the strongest predictions for these traits [ – ]. Second, we replicated two previous results, the one on psychopathy and the one Originality/Talent . Yet, these replications were observed when looking at self-reported lie detection abilities in comparison to others. Third, we observed that increased social desirability scores linked to enhanced self-reported lie detection abilities in general as well as in comparison to others. When designing the studies, we selected individual difference measures that have been mentioned in the lying literature before [ , , , , , , , ]. Most obvious was the Dark Triad, because of what it stands for, malevolent behavioral tendencies such as self-promotion, manipulation (including lying), and lack of empathy [ – ]. Moreover, the Dark Triad had been previously linked to self-reported lie detection abilities [ – ]. We found that higher psychopathy scores were associated with higher self-reported lie detection abilities, but only when participants were asked to indicate their abilities in comparison to others. Wissing and Reinhard found a similar relationship, but for higher confidence in self-reported lie detection performances. Thus, we found similar results despite studies using different response modes, suggesting that response mode does not matter. Against this suggestion, we did not replicate that higher levels of narcissism were associated with a higher self-reported lie detection abilities in comparison to others [ – ]. Likewise, we did not find that higher levels of narcissism were associated with higher self-reported lie detection abilities. It is possible that differences between Dark Triad studies are due to differences in populations and measurements. For populations, this possibility is unlikely, because all relevant Dark Triad studies tested undergraduate university students from Western countries. In our case, we tested French speakers, largely in Switzerland, once 487 (99 males) participants and once 386 participants (72 males). Another study tested 207 participants (83 males) from Germany , and still other studies tested, respectively, 125 males , and 100 undergraduate students (15 males) from Israel. These populations were comparable in gender composition and age, several being biased towards females and others towards males, with the mean population age not exceeding 30 years old. For measurements, studies differed. We used different Dark Triad questionnaires in Study 1 and Study 2, both being shortened versions [ , , , ]. In Study 1, we had used the 12-items Dirty Dozen questionnaire , and did not replicate previous findings [ – ]. As questionnaire brevity comes with costs, such as reduced construct validity , we used the longer and psychometrically superior 27-items Short Dark Triad questionnaire in Study 2 . However, results were comparable for Study 1 and Study 2. One possible explanation could be questionnaire length, because relationships with narcissism scores were found for the 40-item Narcissistic Personality Inventory [ – , , ]. Yet, length cannot explain it all, because relationships between elevated psychopathy scores, overall Dark Triad scores, and confidence in lie detection abilities had been found using an even shorter 9-item scale . As of now, we have insufficient evidence to argue that divergent findings were due to populations or measurements. Overall, we found hardly any individual difference measures, significantly predicting self-reported lie detection abilities. The situation was not really different when participants rated their lie detection abilities in comparison to others. We found that social desirability scores predicted enhanced ratings of both self-reported lie detection measures. Thus, self-reported lie detection abilities, whether in general or in comparison to others, tap into something that is akin to an attitude or a social value. Maybe, this latter proposition might also explain why lower levels of out-group trust predicted higher self-reported lie detection abilities. In this regard, self-reported lie detection abilities might reflect people’s truth-default state which implies that we assume that others are trustworthy. In addition, indicating that one is able to detect lies reflects on people’s favorable self-view [ , , ]. In other words, it is better for one’s self-esteem to think that one is not easily deceived. Study limitations, challenges and future directions We worked on self-reported lie detection abilities without assessing people’s actual lie detection performance. Notwithstanding, on a group level, we assume that our populations’ actual lie detection performances would mirror results of previous studies showing that lie detection performance is around chance level . Future studies should assess both actual and self-reported lie detection abilities in a within-subjects design. As with most studies in psychology, we tested undergraduate psychology students. They are not representative of the general population, whether within or between cultures (see e.g., [ , , ]. Therefore, the generalizability of our results remains questionable. To encompass this limitation, we are currently running further studies testing alternative populations such as particular groups of professionals who have a relatively enhanced probability encountering deception (i.e., police officers, teacher, and insurers). We will report these results in the future. Moreover, further studies could consider age, because studies showed that lie detection abilities decreased with age [ – ] and lies were spotted more easily in older than younger people . Worth noting, we did not replicate that our participants reported being above average in self-reported lie detection abilities, i.e., being in average better when comparing oneself to others (e.g., [ , – , ]). This observation reflects the better-than-average effect . On the contrary, only 20% of our participants thought they were better than others. When asked about extent, only 40% of them indicated being better than others at detecting lies. We could have expected this better-than-average effect , because it seems more pronounced for young people, and for dimensions that lack external verification . These conditions both apply to our study. Another observation, we might have found comparable results with previous studies using self-reported lie detection abilities in comparison to others, if we would have highlighted the middle point of the rating scale, just like the other studied did. Previous studies indicated 50 as the point at which participants rated themselves as good as others [ – ]. In contrast, we asked participants to use the scale from 0 ( much worse than others ) to 100 ( much better than others ). Therefore, the average is not explicitly presented to the participants. As a final critical point, some of the self-report questionnaires lacked validity [ , , ]. Returning to the Dark Triad, these self-report questionnaires seemed to extract the uniqueness between narcissism, psychopathy, and Machiavellianism . Several studies, however, reported an overlap between the three constructs and claimed they were not independent . Kajonius et al. even argued that the Dirty Dozen questionnaire assesses only two constructs, namely, narcissism and an anti-social trait (i.e., Machiavellianism and psychopathy combined). Consistent with this, Persson et al. showed that Machiavellianism and psychopathy, measured by the Short Dark Triad, were similar concepts. Therefore, future studies investigating similar questions to ours should include different measures, such as the Narcissistic Personality Inventory , because the latter resulted in comparable findings between studies [ – ]. Regarding social desirability, several studies emphasized the lack of validity of the BIDR (Balanced Inventory of Desirable Responding) . To address this limitation, further studies are needed to measure social desirability as a trait, using validated measures of related dimensions such as self-control (e.g. ).
We worked on self-reported lie detection abilities without assessing people’s actual lie detection performance. Notwithstanding, on a group level, we assume that our populations’ actual lie detection performances would mirror results of previous studies showing that lie detection performance is around chance level . Future studies should assess both actual and self-reported lie detection abilities in a within-subjects design. As with most studies in psychology, we tested undergraduate psychology students. They are not representative of the general population, whether within or between cultures (see e.g., [ , , ]. Therefore, the generalizability of our results remains questionable. To encompass this limitation, we are currently running further studies testing alternative populations such as particular groups of professionals who have a relatively enhanced probability encountering deception (i.e., police officers, teacher, and insurers). We will report these results in the future. Moreover, further studies could consider age, because studies showed that lie detection abilities decreased with age [ – ] and lies were spotted more easily in older than younger people . Worth noting, we did not replicate that our participants reported being above average in self-reported lie detection abilities, i.e., being in average better when comparing oneself to others (e.g., [ , – , ]). This observation reflects the better-than-average effect . On the contrary, only 20% of our participants thought they were better than others. When asked about extent, only 40% of them indicated being better than others at detecting lies. We could have expected this better-than-average effect , because it seems more pronounced for young people, and for dimensions that lack external verification . These conditions both apply to our study. Another observation, we might have found comparable results with previous studies using self-reported lie detection abilities in comparison to others, if we would have highlighted the middle point of the rating scale, just like the other studied did. Previous studies indicated 50 as the point at which participants rated themselves as good as others [ – ]. In contrast, we asked participants to use the scale from 0 ( much worse than others ) to 100 ( much better than others ). Therefore, the average is not explicitly presented to the participants. As a final critical point, some of the self-report questionnaires lacked validity [ , , ]. Returning to the Dark Triad, these self-report questionnaires seemed to extract the uniqueness between narcissism, psychopathy, and Machiavellianism . Several studies, however, reported an overlap between the three constructs and claimed they were not independent . Kajonius et al. even argued that the Dirty Dozen questionnaire assesses only two constructs, namely, narcissism and an anti-social trait (i.e., Machiavellianism and psychopathy combined). Consistent with this, Persson et al. showed that Machiavellianism and psychopathy, measured by the Short Dark Triad, were similar concepts. Therefore, future studies investigating similar questions to ours should include different measures, such as the Narcissistic Personality Inventory , because the latter resulted in comparable findings between studies [ – ]. Regarding social desirability, several studies emphasized the lack of validity of the BIDR (Balanced Inventory of Desirable Responding) . To address this limitation, further studies are needed to measure social desirability as a trait, using validated measures of related dimensions such as self-control (e.g. ).
In two online studies, we tested self-reported lie detection abilities. On the group level, we replicated that people reported being above chance in lie detection. For individual difference measures, we observed that participants had higher lie detection abilities with increasing social desirability and decreasing out-group trust levels, respectively. Unlike our predictions, all other predictor variables were not significant, including the Dark triad and conventional personality traits, empathy, emotional intelligence, and cultural values. When participants rated their lie detection abilities in comparison to others, we did not find the better-than-average effect . Yet, we replicated that enhanced psychopathy and Originality/Talent predicted the latter measure. Our results indicated that it was socially desirable to trust others and to believe being able to detect lies. Future studies should test whether self-reported and actual lie detection abilities are associated and in which way, as well as assess these relationships in different professional groups, different age groups, and in populations beyond the frequently researched Western societies.
S1 Table Results of the binary logistic regression analysis on the continuous 12 predictor variables for self-reported lie detection abilities (below vs above chance level). (TIF) Click here for additional data file. S2 Table Pearson’s correlations between individual characteristics and self-reported lie detection abilities for Study 1. (TIF) Click here for additional data file. S3 Table Pearson’s correlations between individual characteristics and self-reported lie detection abilities for Study 2. (TIF) Click here for additional data file.
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COVID-19 Cluster in the Hematology/Respirology Ward of a University Hospital during the Seventh Wave of the SARS-CoV-2 Pandemic in Japan: A Descriptive Study
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6d7d9017-7871-46da-80b7-ee127c17dbd0
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10208769
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Internal Medicine[mh]
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Japan faced a sharp increase in the coronavirus disease 2019 (COVID-19) incidence during the summer of 2022, which peaked on August 19, 2022, with 260,923 newly diagnosed cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection . At that time, the BA.5 variant was the dominant strain of COVID-19 in Japan. The increase in the number of patients with SARS-CoV-2 infection appeared to be accelerated by the Bon festival, a major holiday accompanied by intensive domestic travel and increased accommodation rates that occurred from August 13 to 16, 2022. In addition, for the first time in three years, Kochi Prefecture organized the annual Yosakoi dance festival on August 10 and 11, which is characterized by a carnival-like energetic style of group dancing. Furthermore, the occurrence of nosocomial COVID-19 clusters is reportedly closely associated with the increased trend in COVID-19 community incidence . The present study therefore retrospectively described the characteristics of COVID-19 clusters involving patients in the hematology/respirology ward of Kochi Medical School Hospital during the seventh wave of the pandemic and discuss the findings.
Kochi Medical School Hospital is a 613-bed university-affiliated hospital in Japan, and its hematology/respirology ward is a 46-bed facility comprising 8 single rooms, 5 two-bed rooms, 7 four-bed rooms, common restrooms (men's and women's), and a washroom. Each room is separated by a floor-to-ceiling wall and connected to a shared indoor hallway through a sliding door. To reduce the risk of nosocomial SARS-CoV-2 infection in the ward, universal reverse transcription-polymerase chain reaction (RT-PCR) screening was performed for all pre-admission patients. Inpatients were required to wear a face mask, and no family members or other visitors were allowed to visit patients in the ward. In addition, universal masking (surgical masks; N95 masks were used in cases where SARS-CoV-2 infection was observed in inpatients) and eye protection were implemented for healthcare workers in the hospital. Close contacts, risk factors for severe COVID-19, and COVID-19 severity were defined according to the Ministry of Health, Labour and Welfare (MHLW) guidelines (version 8.0) that are widely used in Japan . Patients sharing a room with those with confirmed to have SARS-CoV-2 infection were considered to be close contacts. Risk factors for severe COVID-19 include older age (≥65 years old) and comorbidities such as malignancies, chronic lung disease, chronic kidney disease, diabetes, hypertension, hyperlipidemia, cardiovascular disease, cerebrovascular disease, obesity (body mass index ≥30 kg/m 2 ), smoking, solid organ transplantation, pregnancy, and human immunodeficiency virus infection, as well as the use of corticosteroids or other immunosuppressive medications. In the present study, individuals with SARS-CoV-2 infection were divided into symptomatic and asymptomatic groups. COVID-19 symptoms were recorded at the time of the diagnosis in symptomatic individuals with SARS-CoV-2 infection or at the time of the clinical manifestation of COVID-19 in asymptomatic individuals at the time of SARS-CoV-2 detection. Upper respiratory tract symptoms were defined as sore throat, anosmia, loss of taste, or cough. Lower respiratory tract symptoms were defined as dyspnea or symptoms associated with bronchitis or pneumonia. A COVID-19 cluster was defined as five or more individuals with an epidemiological link in the same ward. The duration of the cluster was defined as the time between the first and last nosocomial cases, which was consequently 11 days in late August. The COVID-19 diagnosis was based on RT-PCR performed on nasopharyngeal samples. Nasopharyngeal specimens were collected using the swab technique, and ribonucleic acid (RNA) was extracted and subjected to RT-PCR targeting the SARS-CoV-2 viral gene using the Cobas Ⓡ Liat SARS-CoV-2 test or the Cobas Ⓡ 6800 SARS-CoV-2 test (Roche Molecular Systems, Indianapolis, USA). The cycle threshold (Ct) values were used as indicators of the viral load of SARS-CoV-2 RNA, with lower Ct values corresponding to higher viral copy numbers. A Ct value <40 was considered positive for SARS-CoV-2 RNA. For each patient with SARS-CoV-2 infection, RT-PCR was performed once at the time of the COVID-19 diagnosis in symptomatic individuals or during periodic screening in asymptomatic individuals. Data analyses were performed using the GraphPad Prism 7 software program (GraphPad Software, La Jolla, USA). Average values are expressed as the mean±standard error of the mean. Statistical significance was set at p<0.05. The institutional review board approved this study on October 18, 2022, and waived the need for informed consent, as tests were performed on samples required for routine clinical care (No. 2022-73).
Time-course of the ward cluster A total of 40 individuals, including 25 patients and 15 healthcare workers (3 physicians, 11 nurses, and 1 hospital housekeeping staff member), were found to be infected by SARS-CoV-2 during the cluster . One healthcare worker who had not had close contact in the ward developed COVID-19 (day 0; the day of initial identification of SARS-CoV-2 infection in the ward cluster was considered day 0), presumably because of household transmission. On day 1, a patient who presented with a sore throat immediately underwent a nasopharyngeal swab test for SARS-CoV-2 RT-PCR, and the result was positive. Three other patients who had shared the same four-bed room were negative for SARS-CoV-2. On day 3, three patients in two separate four-bedrooms developed a fever associated with COVID-19. One close contact tested positive for SARS-CoV-2. On day 4, all patients (n=30) and healthcare workers (n=54) in the ward underwent PCR screening (the first active screening), and two asymptomatic physicians tested positive. Both physicians later developed COVID-19 symptoms and presented with a fever. The patients were negative for SARS-CoV-2 on screening. However, on day 5, three patients in separate four-bed rooms developed a fever and tested positive. Two asymptomatic close contacts were also positive for SARS-CoV-2. In addition, five former close contacts and four healthcare workers developed COVID-19. On day 6, two patients (one in the residual four-bed room and one in a two-bed room) developed a fever and were positive for SARS-CoV-2. Two asymptomatic close contacts were also positive. Two healthcare workers developed COVID-19. By day 6, all seven four-bed rooms and one two-bed room were closed due to the spread of COVID-19. On day 7, one patient and two healthcare workers developed COVID-19. The second active screening was performed in residual patients (n=17) and healthcare workers (n=57) in the ward, and 2 (one close contact and one healthcare worker) were found to be positive for SARS-CoV-2. On day 9, one patient and one healthcare worker developed COVID-19. On day 10, two patients (one in a two-bed room and another in a single room) developed COVID-19. The third active screening was performed in residual patients (n=13) and healthcare workers (n=55) in the ward, and 1 close contact and 2 healthcare workers were positive. No individuals with SARS-CoV-2 infection were recorded thereafter. On day 14, the fourth active screening was performed in 9 patients and 45 healthcare workers in the ward, with negative results. As a result, the Department of Infection Control and Prevention declared an end to the ward cluster. Patient characteristics and clinical courses The basic characteristics of inpatients with SARS-CoV-2 infection are summarized in and 2. Among the patients with SARS-CoV-2 infection, 11 had hematological diseases (9 developed COVID-19, and 2 remained asymptomatic), and 14 had respiratory diseases (all developed COVID-19) . Most infected patients (88%, 22/25) had hematological or solid cancers. Except for 1 patient with renal cell carcinoma, the remaining 21 patients with hematological or solid cancers had received anti-cancer agents. The median age was 72 (range, 28-86) years old in patients with hematological diseases and 71.5 (range, 45-82) years old in those with respiratory diseases . Almost all patients (96%, 24/25) had risk factors for severe COVID-19, and the majority (80%, 20/25) had ≥3 risk factors. A fever was the most common initial symptom of COVID-19, presenting in 19 patients, followed by upper respiratory tract symptoms, including sore throat in 10 patients and cough in 2. One patient presented with a headache. Two patients developed COVID-19 pneumonia. All patients with SARS-CoV-2 infection received virus-specific treatment, as they were at a high risk of severe COVID-19. All infected patients received the antivirals remdesivir or nirmatrelvir/ritonavir at a physician's discretion. In general, asymptomatic or mild cases received a three-day course of remdesivir or nirmatrelvir/ritonavir, whereas moderate or severe cases or patients at high risk for severe illness received a five-day course of remdesivir. Among the 25 infected patients, 23 (92%) eventually developed COVID-19. The clinical severity of COVID-19 was mild in 20 patients, moderate in 3, and severe in 0. At a median follow-up of 70 days, the overall mortality was 0%, and none of the patients required invasive mechanical ventilation. All infected patients recovered. Eighteen patients had received three or more doses of a COVID-19 mRNA vaccine [BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna)], and two had received two doses. Five patients had not received the COVID-19 vaccine before the infection. As all patients eventually recovered from SARS-CoV-2 infection, and their disease severities were mostly mild, the effectiveness of the vaccines was not evaluated. RT-PCR for SARS-CoV-2 The Ct values in nasopharyngeal samples from symptomatic and asymptomatic patients with SARS-CoV-2 infection are shown in . Since the Cobas Ⓡ Liat SARS-CoV-2 test was used with most patient samples (n=20), these data were used for the statistical analysis. The Ct values were 18.8±1.8 in patients with COVID-19 (n=14) and 28.0±3.0 in those with SARS-CoV-2 infection (n=6), showing a significant difference (p=0.015). The Ct values appear to be lower in symptomatic patients than in asymptomatic patients in each disease severity subgroup, although the sample sizes were too small for a statistical analysis. Healthcare workers infected with SARS-CoV-2 A total of 15 ward healthcare workers, including 3 physicians, 11 nurses, and 1 housekeeper, were infected with SARS-CoV-2. SARS-CoV-2 infection was confirmed in 8 symptomatic individuals presenting with a fever (n=8), sore throat (n=4), anosmia (n=1), and/or cough (n=1). All seven initially asymptomatic individuals, in whom active PCR screening determined SARS-CoV-2 infection, eventually developed symptoms of COVID-19, presenting with a fever (n=4) and/or sore throat (n=6). COVID-19 in all healthcare workers remained mildly severe, and the individuals recovered without any antivirals.
A total of 40 individuals, including 25 patients and 15 healthcare workers (3 physicians, 11 nurses, and 1 hospital housekeeping staff member), were found to be infected by SARS-CoV-2 during the cluster . One healthcare worker who had not had close contact in the ward developed COVID-19 (day 0; the day of initial identification of SARS-CoV-2 infection in the ward cluster was considered day 0), presumably because of household transmission. On day 1, a patient who presented with a sore throat immediately underwent a nasopharyngeal swab test for SARS-CoV-2 RT-PCR, and the result was positive. Three other patients who had shared the same four-bed room were negative for SARS-CoV-2. On day 3, three patients in two separate four-bedrooms developed a fever associated with COVID-19. One close contact tested positive for SARS-CoV-2. On day 4, all patients (n=30) and healthcare workers (n=54) in the ward underwent PCR screening (the first active screening), and two asymptomatic physicians tested positive. Both physicians later developed COVID-19 symptoms and presented with a fever. The patients were negative for SARS-CoV-2 on screening. However, on day 5, three patients in separate four-bed rooms developed a fever and tested positive. Two asymptomatic close contacts were also positive for SARS-CoV-2. In addition, five former close contacts and four healthcare workers developed COVID-19. On day 6, two patients (one in the residual four-bed room and one in a two-bed room) developed a fever and were positive for SARS-CoV-2. Two asymptomatic close contacts were also positive. Two healthcare workers developed COVID-19. By day 6, all seven four-bed rooms and one two-bed room were closed due to the spread of COVID-19. On day 7, one patient and two healthcare workers developed COVID-19. The second active screening was performed in residual patients (n=17) and healthcare workers (n=57) in the ward, and 2 (one close contact and one healthcare worker) were found to be positive for SARS-CoV-2. On day 9, one patient and one healthcare worker developed COVID-19. On day 10, two patients (one in a two-bed room and another in a single room) developed COVID-19. The third active screening was performed in residual patients (n=13) and healthcare workers (n=55) in the ward, and 1 close contact and 2 healthcare workers were positive. No individuals with SARS-CoV-2 infection were recorded thereafter. On day 14, the fourth active screening was performed in 9 patients and 45 healthcare workers in the ward, with negative results. As a result, the Department of Infection Control and Prevention declared an end to the ward cluster.
The basic characteristics of inpatients with SARS-CoV-2 infection are summarized in and 2. Among the patients with SARS-CoV-2 infection, 11 had hematological diseases (9 developed COVID-19, and 2 remained asymptomatic), and 14 had respiratory diseases (all developed COVID-19) . Most infected patients (88%, 22/25) had hematological or solid cancers. Except for 1 patient with renal cell carcinoma, the remaining 21 patients with hematological or solid cancers had received anti-cancer agents. The median age was 72 (range, 28-86) years old in patients with hematological diseases and 71.5 (range, 45-82) years old in those with respiratory diseases . Almost all patients (96%, 24/25) had risk factors for severe COVID-19, and the majority (80%, 20/25) had ≥3 risk factors. A fever was the most common initial symptom of COVID-19, presenting in 19 patients, followed by upper respiratory tract symptoms, including sore throat in 10 patients and cough in 2. One patient presented with a headache. Two patients developed COVID-19 pneumonia. All patients with SARS-CoV-2 infection received virus-specific treatment, as they were at a high risk of severe COVID-19. All infected patients received the antivirals remdesivir or nirmatrelvir/ritonavir at a physician's discretion. In general, asymptomatic or mild cases received a three-day course of remdesivir or nirmatrelvir/ritonavir, whereas moderate or severe cases or patients at high risk for severe illness received a five-day course of remdesivir. Among the 25 infected patients, 23 (92%) eventually developed COVID-19. The clinical severity of COVID-19 was mild in 20 patients, moderate in 3, and severe in 0. At a median follow-up of 70 days, the overall mortality was 0%, and none of the patients required invasive mechanical ventilation. All infected patients recovered. Eighteen patients had received three or more doses of a COVID-19 mRNA vaccine [BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna)], and two had received two doses. Five patients had not received the COVID-19 vaccine before the infection. As all patients eventually recovered from SARS-CoV-2 infection, and their disease severities were mostly mild, the effectiveness of the vaccines was not evaluated.
The Ct values in nasopharyngeal samples from symptomatic and asymptomatic patients with SARS-CoV-2 infection are shown in . Since the Cobas Ⓡ Liat SARS-CoV-2 test was used with most patient samples (n=20), these data were used for the statistical analysis. The Ct values were 18.8±1.8 in patients with COVID-19 (n=14) and 28.0±3.0 in those with SARS-CoV-2 infection (n=6), showing a significant difference (p=0.015). The Ct values appear to be lower in symptomatic patients than in asymptomatic patients in each disease severity subgroup, although the sample sizes were too small for a statistical analysis.
A total of 15 ward healthcare workers, including 3 physicians, 11 nurses, and 1 housekeeper, were infected with SARS-CoV-2. SARS-CoV-2 infection was confirmed in 8 symptomatic individuals presenting with a fever (n=8), sore throat (n=4), anosmia (n=1), and/or cough (n=1). All seven initially asymptomatic individuals, in whom active PCR screening determined SARS-CoV-2 infection, eventually developed symptoms of COVID-19, presenting with a fever (n=4) and/or sore throat (n=6). COVID-19 in all healthcare workers remained mildly severe, and the individuals recovered without any antivirals.
In our cluster, the spread of SARS-CoV-2 mostly involved shared rooms. Among the 25 patients with SARS-CoV-2 infection, 21 were from four-bed rooms, 2 were from two-bed rooms, and 1 was from a single room. The spread initially occurred in a non-contiguous fashion, i.e. infected inpatients were simultaneously identified in separate rooms rather than in shared rooms. SARS-CoV-2 transmission usually occurs through close contact with respiratory droplets. The possibility of healthcare-associated SARS-CoV-2 transmission was unlikely, since most infected patients identified by day 5 did not share the same physicians, nurses, or other healthcare workers. One possibility is that the washroom played a role in SARS-CoV-2 transmission. While almost all infected patients used a common washroom, patients using personal equipment inside single rooms were generally not infected. Since three inpatients simultaneously brushed their teeth and gargled in the washroom without masks, our updated rules after the cluster required inpatients to use the washroom in turns. Shared rooms require patients to eat and drink in the same room under limited ventilation or aeration, so such hospital environmental factors that are difficult to prevent may have contributed to the spread. Some studies have shown that airborne transmission can occur in specific circumstances . Before and during the cluster, no patients received aerosol-generating procedures, including endotracheal intubation or high-flow nasal cannula oxygen or standard oxygen therapy . Considering the non-contiguous occurrence of newly diagnosed patients with SARS-CoV-2 infection in our cluster, airborne transmission might not have been the major route of spread. Nevertheless, the World Health Organization recommends droplet and contact precautions be practiced by healthcare workers caring for patients with COVID-19. SARS-CoV-2 was first recognized in an outbreak of pneumonia in Wuhan, Hubei Province, China, in December 2019 . In March 2020, 4 months after the initial outbreak in Wuhan, a large cluster of nosocomial SARS-CoV-2 infections occurred at the Eiju General Hospital in Tokyo , involving 109 inpatients and 83 healthcare workers (according to the final report from the Eiju General Hospital issued on May 16, 2020). In a detailed study , the mortality rate was strikingly high in patients with hematological diseases (19/31, 61%). Patients with hematological malignancies reportedly tend to have a more severe COVID-19 trajectory than do those with solid organ tumors or other diseases . A retrospective study using nationwide data from the COVID-19 Registry Japan identified 10 risk factors for severe illness in older patients hospitalized during the early (first, second, and third) waves, among which the presence of hematological malignancies (leukemia and lymphoma) was the most significant . In our series with 11 cases of hematological malignancies, no patients died of COVID-19, although 2 patients had moderate II COVID-19, requiring oxygen. The severity of COVID-19 was generally mild, irrespective of patient characteristics, sharply contrasting with previously reported nosocomial clusters occurring during the earlier waves of SARS-CoV-2. The precise reasons are unknown, but COVID-19 vaccination, early or preemptive treatment with antivirals, and intrinsic changes in SARS-CoV-2 might be associated with better outcomes in our series. The SARS-CoV-2 Omicron variant reportedly shows slow replication and low pathogenicity in human lungs, which may contribute to a lower prevalence of pneumonia and a lower risk of hospitalization than with other variants . Further studies are needed to determine whether or not the prevailing SARS-CoV-2 variants have a lower pathogenicity of COVID-19 than do their ancestors. This practice-based retrospective study had a few limitations. First, viral sequencing was not performed in samples from patients or healthcare workers involved in this outbreak, limiting the establishment of phylogenetic relationships. Second, we were unable to precisely track the modes of transmission from one individual to another. Daily monitoring of SARS-CoV-2 PCR results and clinical symptoms in all admitted patients and healthcare workers, if available, may help clarify the transmission modes in the ward. However, despite these potential limitations, we believe that our study provides important insight into the clinical features of nosocomial SARS-CoV-2 infection during the seventh wave of SARS-CoV-2. In conclusion, the SARS-CoV-2 cluster occurred in our hospital during the seventh wave of the SARS-CoV-2 pandemic in Japan, overcoming the enhanced infection control measures that were implemented including case isolation, extensive contact tracing, quarantine measures, and active screening by PCR. Fortunately, all SARS-CoV-2-infected inpatients with hematological or respiratory diseases recovered or did not develop symptoms of COVID-19 at all. COVID-19 vaccination, early or preemptive treatment with antivirals, and intrinsic changes in SARS-CoV-2 might have contributed to the better outcomes in our series than in previously reported nosocomial clusters.
This study was supported in part by a grant from the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (JSPS KAKENHI, 21K083960A; to Kensuke Kojima).
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Changes in the epidemiology of invasive fungal disease in a Pediatric Hematology and Oncology Unit: the relevance of breakthrough infections
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1c8d9991-b8e9-467c-9c71-1d938cb1381e
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10210274
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Internal Medicine[mh]
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Invasive fungal disease (IFD) is one of the leading causes of morbidity and mortality among immunocompromised children. In recent years, there has been a significant increase in pediatric patients at risk, due to the extended use of immunosuppressive medications and a rising complexity of the baseline pathologies . Children who receive chemotherapy for malignancy or undergo hematopoietic stem cell transplant (HSCT) are at the highest risk for an IFD, particularly invasive candidiasis and aspergillosis . In December 2019, the European Organization for Research and Treatment of Cancer and the Mycoses Study Group Education and Research Consortium (EORTC/MSGERC) published an update on the definitions of IFD, including specific considerations for pediatric patients . Monitoring the local epidemiology is essential to understand the pediatric IFD burden to set up preventive measures, rationalize resources and implement institution-based infection control strategies . The management of IFD, especially mold infections, in immunocompromised children is a challenge. Signs and symptoms may be non-specific and often develop late during the disease progression . Data on imaging tools for the diagnosis of IFD are scarce and the role of fungal biomarkers may differ from those used in adults . Randomized clinical trials evaluating antifungal drugs rarely involve children; therefore, these drugs are often used "off-label" for compassionate use. Antifungal prophylaxis (AFP) has shown to improve outcomes of these patients, but, as well as for adults, breakthrough invasive fungal infections have emerged as a significant problem . The Pediatric Hematology-Oncology Unit (PHOU) of Hospital General Universitario Gregorio Marañón (HGUGM) in Madrid (Spain) has experienced a rising activity and an increase in the complexity of patients’ pathologies in recent years. This study aims to describe IFD in children admitted to the PHOU, according to the latest EORTC criteria , analysing the breakthrough IFD rate and evaluating the changes over the study period. Secondary objectives were to compare yeast and mold infections and the characteristics of surviving and non-surviving patients.
Study design and population Clinical data of all pediatric patients (from 6 months to 18 years old) diagnosed with IFD at the PHOU of the HGUGM from January 2006 to December 2019 were retrospectively reviewed. HGUGM is a tertiary hospital located in Madrid (Spain) with 120 pediatric beds and around 7000 pediatric inpatient admissions per year. The PHOU of HGUGM counts with 18 hospitalizations beds and has experienced a rising activity in recent years, due, partially, to the creation of an adolescent unit and the accreditation as a National Reference Center for hereditary erythropathologies. The number of admissions per year in the PHOU has progressively increased from 343 in 2006 to 853 in 2019. The information collected from the patients included: demographics, underlying condition, IFD host factors, AFP, laboratory, microbiological and radiological findings, antifungal therapy and clinical outcome. Deaths observed during the IFD treatment period were analyzed. Definitions Cases were defined as proven, probable and possible IFD based on the last update in 2019 of the consensus definitions of IFD from the EORTC/MSGERC. Proven disease required histopathologic or microbiologic documentation of infection from tissue obtained by biopsy or autopsy, or an isolation from a culture sample obtained from a normally sterile site. Probable disease was defined as the presence of host factors, clinical features and mycological evidence of an IFD. Possible disease required proper host factors and sufficient clinical evidence compatible with IFD . Host factors for IFD were those defined by the EORTC/MSGERC consensus: a recent history of prolonged neutropenia (<500 neutrophils/µL for >10 days), hematologic malignancy, allogeneic HSCT or solid organ transplant, prolonged use of corticosteroids (≥0.3 mg/kg for ≥ three weeks in the past 60 days), treatment with B-cell o T-cell immunosuppressants, severe hereditary immunodeficiency and graft-versus-host disease (GVHD) . The risk for IFD represented the probability of IFD for each underlying condition. Patients considered at high-risk (≥10%) for IFD were those with acute myeloid leukemia (AML), high-risk or relapsed or acute lymphoblastic leukemia (ALL), severe aplastic anemia, myelodysplastic syndrome or those who underwent an allogeneic HSCT or developed a GVHD. Patients with non-Hodgkin lymphoma, standard-risk ALL or autologous HSCT were considered at low-risk (≤5%) for IFD. In opposition, patients with solid organ tumors (SOT), like brain tumors or Hodgkin lymphoma, were classified as sporadic risk . The global prevalence of IFD was calculated dividing the number of IFD cases by the total number of patients at risk. Moreover, the ratio of IFD cases per 1000 admissions in the PHOU was calculated globally and by time periods. The activity in the PHOU was measured using the number of admissions and HSCT carried out among periods. Breakthrough IFD was defined as any IFD occurring during exposure to an antifungal drug, according to the recent definitions by the MSGERC . In our institution protocol, used along the study period, oral or intravenous posaconazole, with therapeutic drug monitoring, and intravenous micafungin are considered the first choices for AFP in patients at high-risk for IFD. Liposomal amphotericin B is usually used for empirical or pre-emptive treatment in the setting of persistent neutropenic fever over 96 hours of significant respiratory symptoms, and voriconazole is the treatment of choice if an invasive aspergillosis is proven or probable. Ethics This study was conducted in accordance with the 1964 Declaration of Helsinki and its subsequent amendments. Ethics approval was obtained from the Clinical Research Ethics Committee at HGUGM (date 5 th October 2020/ Number IFI-HOI-2020). Statistical analyses Descriptive analyses were performed using frequencies and proportions for categorical variables and medians and interquartile ranges (IQR) for continuous variables. Comparative analyses were conducted according to time periods (three 56-months periods: January 2006 to August 2010 vs September 2010 to April 2015 vs May 2015 to December 2019), the type of infection (yeast vs mold infections), and outcome (survivors vs non-survivors). Categorical variables were compared using the chi-square or the Fisher exact test, as appropriate. Continuous dicotomic variables were evaluated with the Mann-Whitney U test. Kruskal-Wallis test, followed by posthoc analyses, was used when comparing variables among the three time periods. Statistical analyses were performed using IBM SPSS Statistics software (Statistical Package for the Social Sciences) version 23.0. The statistical significance level was defined as a two-tailed p- value <0.05.
Clinical data of all pediatric patients (from 6 months to 18 years old) diagnosed with IFD at the PHOU of the HGUGM from January 2006 to December 2019 were retrospectively reviewed. HGUGM is a tertiary hospital located in Madrid (Spain) with 120 pediatric beds and around 7000 pediatric inpatient admissions per year. The PHOU of HGUGM counts with 18 hospitalizations beds and has experienced a rising activity in recent years, due, partially, to the creation of an adolescent unit and the accreditation as a National Reference Center for hereditary erythropathologies. The number of admissions per year in the PHOU has progressively increased from 343 in 2006 to 853 in 2019. The information collected from the patients included: demographics, underlying condition, IFD host factors, AFP, laboratory, microbiological and radiological findings, antifungal therapy and clinical outcome. Deaths observed during the IFD treatment period were analyzed.
Cases were defined as proven, probable and possible IFD based on the last update in 2019 of the consensus definitions of IFD from the EORTC/MSGERC. Proven disease required histopathologic or microbiologic documentation of infection from tissue obtained by biopsy or autopsy, or an isolation from a culture sample obtained from a normally sterile site. Probable disease was defined as the presence of host factors, clinical features and mycological evidence of an IFD. Possible disease required proper host factors and sufficient clinical evidence compatible with IFD . Host factors for IFD were those defined by the EORTC/MSGERC consensus: a recent history of prolonged neutropenia (<500 neutrophils/µL for >10 days), hematologic malignancy, allogeneic HSCT or solid organ transplant, prolonged use of corticosteroids (≥0.3 mg/kg for ≥ three weeks in the past 60 days), treatment with B-cell o T-cell immunosuppressants, severe hereditary immunodeficiency and graft-versus-host disease (GVHD) . The risk for IFD represented the probability of IFD for each underlying condition. Patients considered at high-risk (≥10%) for IFD were those with acute myeloid leukemia (AML), high-risk or relapsed or acute lymphoblastic leukemia (ALL), severe aplastic anemia, myelodysplastic syndrome or those who underwent an allogeneic HSCT or developed a GVHD. Patients with non-Hodgkin lymphoma, standard-risk ALL or autologous HSCT were considered at low-risk (≤5%) for IFD. In opposition, patients with solid organ tumors (SOT), like brain tumors or Hodgkin lymphoma, were classified as sporadic risk . The global prevalence of IFD was calculated dividing the number of IFD cases by the total number of patients at risk. Moreover, the ratio of IFD cases per 1000 admissions in the PHOU was calculated globally and by time periods. The activity in the PHOU was measured using the number of admissions and HSCT carried out among periods. Breakthrough IFD was defined as any IFD occurring during exposure to an antifungal drug, according to the recent definitions by the MSGERC . In our institution protocol, used along the study period, oral or intravenous posaconazole, with therapeutic drug monitoring, and intravenous micafungin are considered the first choices for AFP in patients at high-risk for IFD. Liposomal amphotericin B is usually used for empirical or pre-emptive treatment in the setting of persistent neutropenic fever over 96 hours of significant respiratory symptoms, and voriconazole is the treatment of choice if an invasive aspergillosis is proven or probable.
This study was conducted in accordance with the 1964 Declaration of Helsinki and its subsequent amendments. Ethics approval was obtained from the Clinical Research Ethics Committee at HGUGM (date 5 th October 2020/ Number IFI-HOI-2020).
Descriptive analyses were performed using frequencies and proportions for categorical variables and medians and interquartile ranges (IQR) for continuous variables. Comparative analyses were conducted according to time periods (three 56-months periods: January 2006 to August 2010 vs September 2010 to April 2015 vs May 2015 to December 2019), the type of infection (yeast vs mold infections), and outcome (survivors vs non-survivors). Categorical variables were compared using the chi-square or the Fisher exact test, as appropriate. Continuous dicotomic variables were evaluated with the Mann-Whitney U test. Kruskal-Wallis test, followed by posthoc analyses, was used when comparing variables among the three time periods. Statistical analyses were performed using IBM SPSS Statistics software (Statistical Package for the Social Sciences) version 23.0. The statistical significance level was defined as a two-tailed p- value <0.05.
From January 2006 to December 2019, a total of 471 children at risk for an IFD were followed and 90 HSCT (65 allogeneic, 25 autologous) were carried out at the PHOU. The underlying condition of these children at risk were: 26 AML, 66 ALL, 333 SOT and 46 HSCT performed in non-malignant diseases. Twenty-six children had one episode of IFD and one child had two episodes separated by four years, corresponding to 28 episodes overall. The median age at diagnosis was 9.8 years (IQR 4.9-15.1 years) and 50% were males. The global prevalence of IFD was 5.9%, and differed according to the underlying condition: 23.1% (6/26) for AML, 12.1% (8/66) for ALL and 1.8% (6/333) for SOT. The prevalence in those who required a HSCT was 13.3% (12/90), being 16.9% (11/65) for allogeneic and 4% (1/25) for autologous HSCT. Twenty-three were mold and five were yeast infections, with a prevalence of 4.9% and 1.1%, respectively. There was a global ratio of 4.1 cases of IFD per 1000 admissions in the PHOU and decreased by 25% ( p =0.674) between the first and the last study periods (Table ). In contrast, there was a significant rising activity in the PHOU, with a 64% increase in the number of admissions ( p <0.001) and 277% in the number of HSCT carried out ( p =0.008). In the last period, children had more IFD host factors ( p =0.028) and high-risk underlying disorders ( p =0.012), being the cases of breakthrough IFD more frequent ( p =0.012). All yeast infections took place during the first period (55.6% of cases in period 1), whereas in the second and third periods all IFD episodes were mold infections ( p =0.002). Most episodes (71.4%) occurred in children with underlying conditions considered at high-risk for IFD. Fourteen (50%) corresponded to children with blood malignancies: ALL n= 8 (standard-risk ALL n= 2; relapsed ALL n= 3; high-risk ALL n= 3) and AML n= 6. Eight (28.6%) occurred in children with non-malignant blood disorders, all of them after allogeneic HSCT (sickle cell disease n= 4, aplastic anemia n= 2, Fanconi anemia n= 1, β-thalassemia major n= 1), and six (21.4%) were children with SOT (neuroblastoma n= 3, osteosarcoma n= 2, sarcoma n= 1). One of the IFD episodes in patients diagnosed with ALL occurred during the induction treatment (standard-risk ALL), and the 3 cases in high-risk ALL occurred after a HSCT. The most common host factors for IFD were blood malignancies (50%), prolonged use of corticosteroids (50%), allogeneic HSCT (39.3%; median time between HSCT and IFD 43 days [IQR 14 - 253 days]) and prolonged neutropenia (39.1%); median time of neutropenia 16 days [IQR 12.5 - 23 day]). Eight patients (28.6%) were not neutropenic at the time they developed the IFD. Other host factors were: treatment with T-cell immunosuppressants (28.6%), acute GVHD (17.9%) and chronic GVHD (14.3%). More than a third of the patients (39.3%) had three or four IFD host factors and 7.1% had five or more. Twenty children (71.4%) were receiving AFP, including 19/20 high-risk children and 1/3 low-risk children. The characteristics of these 20 breakthrough IFD episodes are described in Table . Levels of posaconazole were subtherapeutic in both patients at the time they developed the IFD. The global prevalence of breakthrough IFD was 4.2%, and according to the underlying condition, it was 23.1%, 9.1%, 0.3% and 12.2% for AML, ALL, SOT and HSCT groups, respectively. All the breakthrough episodes were mold infections. Forty percent of these episodes were defined as probable and 60% as possible disease. In 11 episodes (39.3%), a clinically relevant pathogen was identified by culture or polymerase chain reaction (PCR): five culture-positive yeast infections ( Candida albicans n= 2; C. parapsilosis n= 2; C. kefyr n= 1), one culture-positive mold infection ( Aspergillus ustus ) and five PCR-positive mold infections ( Aspergillus spp n= 3; Cunninghamella spp n= 1; Myriangiales spp n= 1). According to EORTC criteria, the final diagnosis was proven IFD in six episodes (21.4%; prevalence 0.2%), probable in eight (28.6%; prevalence 1.7%), and possible in 14 (50%; prevalence 3%). Table shows the differences between yeast and mold infections. All yeast infections were candidemia ( n= 5; 17.9%), whereas all mold infections caused bronchopulmonary disease ( n= 23; 82.1%), being associated with rhino-sinusitis in two cases. Children with mold infections had a higher number of IFD host factors (median 3 vs 1; p =0.001) and had more frequently underlying high-risk disorders (87% vs 0%; p =0.001). All yeast and only one mold infection met the criteria for proven IFD (100% vs 4.3%, p =0.001), the last one due to the findings in a necropsy compatible with an invasive aspergillosis. There was a non-significant trend towards higher mortality in mold than yeast infections (26.1% vs 0%, p= 0.553). Table shows the radiological and microbiological findings of mold IFD. Liposomal amphotericin B and voriconazole were the first options for IFD treatment in 85.7% ( n= 24) and 25% ( n= 7) of cases, respectively, being used in combination in 14.2% of cases. Two-thirds of the children experienced a change of treatment (67.9%), mostly based on voriconazole (68.4%), and 10.7% needed a third-line option. The median duration of treatment was 43 days (IQR 19.5-69 days). Regarding outcomes, 8 patients (28.6%) required admission to the Pediatric Intensive Care Unit (PICU), 4 (14.3%) needed mechanical ventilation and 3 (10.7%), inotropic support. One patient required pleural effusion drainage but no cases required surgery. Six patients (21.4%) died during the IFD episode, all of which had mold infections, although death was directly attributed to the IFD in only one of them. Other causes of mortality were: disease progression ( n= 2), refractory GVHD ( n= 2) and a bacterial sepsis with multi-organic failure ( n= 1). Table compares the characteristics between surviving and non-surviving children.
The present study describes 28 episodes of IFD in 27 children out of 471 patients at risk in a PHOU during 14 years. Data about global prevalence, the breakthrough IFD rate, clinical characteristics, diagnosis and treatment of IFD in these children receiving chemotherapy or undergoing HSCT were described. Yeast infections decreased over time, whereas mold infections increased, being breakthrough IFD the majority of them, occurring in children with high-risk pathologies and numerous host factors. A rising activity in the PHOU and an increasing complexity of patients’ pathologies over the study period were not followed by an increase in the rate of IFD cases per 1000 admissions in the PHOU or in the mortality rates. The global prevalence of IFD was 5.9%, being higher in children with AML (23.1%) and in those who underwent allogeneic HSCT (16.9%). The global prevalence of IFD in children with cancer and HSCT recipients ranges from 3.4-7.2% . The wide variety of immune dysfunctions related to underlying conditions, institutional variations in diagnostic and supportive care practices and the inconsistencies in diagnostic criteria make the estimation of this prevalence very challenging . Our study´s prevalence in different groups of patients was similar to that reported recently by Bartlett et al . Other studies have also shown that AML, allogeneic HSCT and high-risk ALL have an exceptionally high risk for IFD . Several patients in our study, especially those with mold infections, had numerous host factors for developing an IFD. It is known that IFD in children rarely occurs in the presence of an isolated host factor . Notably, we detected 20 episodes of breakthrough IFD, with an overall rate of 4.2%. All of them were bronchopulmonary mold infections and 80% of these patients were receiving a mold-active agent. Breakthrough IFD is an emerging significant problem in patients who receive systemic antifungals, ranging from 1.6 to 7.7% for proven and possible breakthrough infections . Nevertheless, the validity of fungal biomarkers may differ in such cases and rates can increase up to 13% when including possible infections . Invasive mold infection are the most common breakthrough IFD . Primary AFP is mainly based on posaconazole, but the concern about pharmacological interactions and toxicity of triazoles has led to a search for alternatives . Liposomal amphotericin and micafungin have been shown to be safe and effective options . Prevalence of yeast and mold infections was 1.1% and 4.9%, respectively. We identified five episodes of proven bloodstream candidemia; all of them took place during the first study period in children with a central venous catheter, and 60% were non- C. albicans species. These findings are consistent with recent reports, which describe a decrease in yeast infections in the past decade, attributed to the extended use of AFP, improved environmental strategies and infection control measures during line emplacement. This fact, together with the increasing number of children under immunosuppressive medications or HSCT, have led mold infections to replace invasive candidiasis as the most frequent IFD . The predominance of non- C. albicans in children has been attributed to the affinity of C. parapsilosis for central venous catheter . Twenty-three episodes were bronchopulmonary mold infections and two were associated with rhino-sinusitis. Most of them were possible or probable infections. The diagnosis of mold IFD in children is a challenge, as only 30-50% of cases meet the criteria for a proven or probable disease . In our study, pulmonary infiltrates and consolidations were the most frequent radiological signs whereas the halo sign and cavities appeared in 17.4% and 8.7% of cases. Other studies have shown that radiological findings in pediatric IFD are unspecific and adult hallmarks are less common, with the halo sign appearing in less than 15% of images and the air crescent sign being very rarely observed . In our study, only one child met the criteria for proven mold disease (based on necropsy findings), 8 were classified as probable disease (based on seric galactomannan [GM; 5/8 cases] or GM in bronchoalveolar lavage [BAL; 3/8]) and 14 as possible cases (including four children with a single positive PCR in BAL and two with positive serum (1-3)-β-D-glucan [BDG]). Reaching a proven mold disease diagnosis is generally unfeasible to pediatric patients . BAL has positioned itself as a safe technique, useful for culture, GM and PCR testing , but the detection of Aspergillus by PCR requires several positive tests to support the diagnosis of probable IFD . The threshold of the promising BDG may vary according to the age, etiology, specimens and manufacturers, and it is not currently recommended to provide evidence of an invasive mold disease in pediatric patients . The exposure to mold-active AFP reduces the sensitivity of GM assay and it may be a reason for the high proportion of possible IFD . Antifungal agents and treatment duration in our study followed pediatric guidelines . The high variability in days of therapy may be explained because the length of treatment is not well defined in mold infections . The prompt initiation of empiric antifungal therapy is critical in a suspected IFD to reduce mortality, being a pre-emptive treatment is a safe strategy to avoid overuse of antifungals . In our study, six patients (21.4%) died during antifungal treatment, all diagnosed with mold infections, one of these cases was directly related to the IFD. Despite the rising activity in the PHOU at HGUGM and an increase in the complexity of pathologies and in the breakthrough IFD rates, mortality rates did not increased. Mortality in the second period was higher but no differences in the comparison between groups were found. The overall case-fatality rate attributable to IFD is very variable, reaching 10-25% in yeast infections and 20-50% in mold infections . The patients who died without completing treatment can explain the shorter duration of treatment in period 2. This study has several limitations. First of all, it is a retrospective study and data are limited to the information available in medical records. Secondly, it is a single-center study with a relatively small sample size; however, these data are probably representative of the characteristics of IFD in pediatric oncohematologic patients, considering that it was done in a tertiary hospital during a long study period. The activity of the PHOU was analysed by non-specific measurements, as the total number of admissions and the HSCT performed. In addition, the incidence of IFD in each risk category was not calculated considering that the risk of each patient can change during the treatment, but prevalence was estimated according to the underlying condition. Finally, a multidisciplinary management between experts in pediatric oncologic and infectious diseases, that maintain a high level of suspicion of IFD in children with numerous host factors, could have led to an over-diagnosis of the cases of possible IFD, taking into account the low specificity of radiological findings in children. However, a prompt treatment of IFD suspected cases is justified given the severity and the mortality rates of this entity and can explain the stable mortality rates within the study period. In conclusion, this study offers a good picture of IFD in children receiving chemotherapy or undergoing HSCT. We observed a decrease in yeast infections during the study time with an increase in the proportion of mold infections. The rising activity and complexity in our PHOU led to an increase in the breakthrough IFD rates, but not in the number of IFD/1000 admissions in the PHOU or in the mortality rates. Local epidemiology knowledge of IFD is essential to implement appropriate therapeutic interventions early and improve survival in these children.
Additional file 1.
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Comparing oral case presentation formats on internal medicine inpatient rounds: a survey study
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246311b9-1f8e-477a-828d-3aa8780a3337
|
10210329
|
Internal Medicine[mh]
|
Excellent inter-physician communication is fundamental to both providing high-quality patient care and promoting learner education , and has been recognized as an important educational goal by the Clerkship Directors in Internal Medicine, the Association of American Medical Colleges, and the Accreditation Council for Graduate Medical Education . Oral case presentations, structured verbal reports of clinical cases , have been referred to as the “currency with which clinicians communicate” . Oral case presentations are a key element of experiential learning in clinical medicine, requiring learners to synthesize, assess, and convey pertinent patient information and to formulate care plans. Furthermore, oral case presentations allow supervising clinicians to identify gaps in knowledge or clinical reasoning and enable team members to learn from one another. Despite modernization in much of medicine, oral case presentation formats have remained largely unchanged, based on the traditional Subjective, Objective, Assessment, Plan (SOAP) format developed by Dr. Lawrence Weed in his Problem Oriented Medical Record in 1968 . Given that the goals of a medical record are different than those of oral case presentations, it should not be assumed that they should share the same format. While Dr. Weed sought to make the medical record as “complete as possible,” internal medicine education leaders have expressed desire for oral case presentations that are succinct, with an emphasis on select relevant details . Using a common SOAP format between the medical record and oral case presentations risks conflating the distinct goals for each of these communication methods. Indeed, in studying how learners gain oral case presentation skills, Haber and Lingard found differences in understanding of the fundamental purpose of oral case presentations between medical students and experienced physicians. While students believed the purpose of oral case presentations was to organize the large amount of data they collected about their patients, experienced physicians saw oral case presentations as a method of telling a story to make an argument for a particular conclusion . In accordance with Dr. Weed’s “problem-oriented approach to data organization,” but with an eye toward optimizing for oral case presentations, we developed an alternative to SOAP known as the Events, Assessment, Plan (EAP) format. The EAP format is used for patients who are already known to the inpatient team, and may also be utilized for newly admitted patients for whom the attending physician already has context (e.g., via handoff or review of an admission note). As the EAP approach is utilized by a subset of attending physicians at our academic hospital, we sought to understand the perceived effectiveness of the EAP format in comparison to the traditional SOAP format among learners (i.e., medical students and resident physicians).
EAP format EAP is a problem-based format used at the discretion of the attending physician. In line with suggested best practices , the EAP structure aims to facilitate transmission of data integrated within the context of clinical problem solving. In this format, significant interval events are discussed first (e.g., a fall, new-onset abdominal pain), followed by a prioritized assessment and plan for each relevant active problem. Subjective and objective findings are integrated into the assessment and plan as relevant to a particular problem. This integration of subjective and objective findings by problem is distinct from SOAP, where subjective and objective findings are presented separately as their own sections, with each section often containing information that is relevant to several problems (Fig. , Additional file : Appendix A). Settings and participants We surveyed third- and fourth-year medical students, and first- through fourth-year internal medicine and internal medicine-pediatrics residents, caring for patients at a large, academic, tertiary care hospital and an affiliated Veterans Affairs medical center. Internal medicine is a 12-week core clerkship for all medical students in their second year, with 8 weeks spent on the inpatient wards. All student participants had completed their internal medicine clerkship rotation at the time of the survey. We did not conduct a sample size calculation at the outset of this study. Data collection methods and processes An anonymous, electronic survey (Qualtrics, Provo, UT) was created to assess student and resident experience with and preference between EAP and SOAP oral case presentation formats during inpatient internal medicine rounds (Additional file : Appendix B). Ten domains were assessed via 5-point Likert scale (1 [strongly disagree] to 5 [strongly agree]), including the ability of the format to incorporate the patient’s subjective experience, the extent to which the format encouraged distillation and integration of information, the extent to which the format focused on the assessment and plan, the format’s ability to help trainees learn from their own patients and those of their peers, time efficiency, and ease of use. Duration of exposure to each format was also assessed, as were basic demographic data for the purposes of understanding outcome differences among respondents (e.g., students versus residents). For those who had experienced both formats, preference between formats was recorded as a binary choice. Participants additionally had the opportunity to provide explanation via free text. For participants with experience in both formats, the order of evaluation of EAP and SOAP formats were randomized by participant. For questions comparing EAP and SOAP formats directly, choice order was randomized. The survey was distributed via official medical school email in October 2021 and was available to be completed for 20 days. Email reminders were distributed approximately one week after distribution and again 48 h prior to survey conclusion. Outcomes The primary outcome was trainee preference in oral case presentation format. Secondary outcomes included comparison between EAP and SOAP on content inclusion/focus, data integration, learning, time efficiency, and ease of use. Statistical analyses Descriptive statistics were used to describe the results (proportion and mean). For comparative analysis between EAP and SOAP, responses from respondents who had experience with both formats were compared using the Wilcoxon Signed Rank Test to evaluate differences. All statistical analyses were done using SAS V9.4 (SAS Institute, Cary, NC). We considered p < 0.05 to be statistically significant.
EAP is a problem-based format used at the discretion of the attending physician. In line with suggested best practices , the EAP structure aims to facilitate transmission of data integrated within the context of clinical problem solving. In this format, significant interval events are discussed first (e.g., a fall, new-onset abdominal pain), followed by a prioritized assessment and plan for each relevant active problem. Subjective and objective findings are integrated into the assessment and plan as relevant to a particular problem. This integration of subjective and objective findings by problem is distinct from SOAP, where subjective and objective findings are presented separately as their own sections, with each section often containing information that is relevant to several problems (Fig. , Additional file : Appendix A).
We surveyed third- and fourth-year medical students, and first- through fourth-year internal medicine and internal medicine-pediatrics residents, caring for patients at a large, academic, tertiary care hospital and an affiliated Veterans Affairs medical center. Internal medicine is a 12-week core clerkship for all medical students in their second year, with 8 weeks spent on the inpatient wards. All student participants had completed their internal medicine clerkship rotation at the time of the survey. We did not conduct a sample size calculation at the outset of this study.
An anonymous, electronic survey (Qualtrics, Provo, UT) was created to assess student and resident experience with and preference between EAP and SOAP oral case presentation formats during inpatient internal medicine rounds (Additional file : Appendix B). Ten domains were assessed via 5-point Likert scale (1 [strongly disagree] to 5 [strongly agree]), including the ability of the format to incorporate the patient’s subjective experience, the extent to which the format encouraged distillation and integration of information, the extent to which the format focused on the assessment and plan, the format’s ability to help trainees learn from their own patients and those of their peers, time efficiency, and ease of use. Duration of exposure to each format was also assessed, as were basic demographic data for the purposes of understanding outcome differences among respondents (e.g., students versus residents). For those who had experienced both formats, preference between formats was recorded as a binary choice. Participants additionally had the opportunity to provide explanation via free text. For participants with experience in both formats, the order of evaluation of EAP and SOAP formats were randomized by participant. For questions comparing EAP and SOAP formats directly, choice order was randomized. The survey was distributed via official medical school email in October 2021 and was available to be completed for 20 days. Email reminders were distributed approximately one week after distribution and again 48 h prior to survey conclusion.
The primary outcome was trainee preference in oral case presentation format. Secondary outcomes included comparison between EAP and SOAP on content inclusion/focus, data integration, learning, time efficiency, and ease of use.
Descriptive statistics were used to describe the results (proportion and mean). For comparative analysis between EAP and SOAP, responses from respondents who had experience with both formats were compared using the Wilcoxon Signed Rank Test to evaluate differences. All statistical analyses were done using SAS V9.4 (SAS Institute, Cary, NC). We considered p < 0.05 to be statistically significant.
The overall response rate was 21% (118/563). The response rate was 14% ( n = 62/441) among medical students and 46% ( n = 56/122) among residents. Respondents were 61% ( n = 72) female. A total of 98% ( n = 116) and 52% ( n = 61) of respondents reported experience with SOAP and EAP formats, respectively. Among medical students, 60% ( n = 37) reported experience with SOAP only while 39% ( n = 24) had experience with both formats. Among residents, 36% ( n = 20) and 63% ( n = 35) had experience with SOAP only and both formats, respectively (Table ). Most students (93%) and residents (96%) reported > 8 weeks of exposure to the SOAP format. Duration of exposure to the EAP format varied (0 to 2 weeks [32% of students, 17% of residents], 2 to 4 weeks [36% of students, 47% of residents], 4 to 8 weeks [16% of students, 25% of residents], and > 8 weeks [16% of students, 11% of residents]). Of the 59 respondents with exposure to both the SOAP and EAP formats, 69% ( n = 41) preferred the EAP format as compared to 19% ( n = 11) preferring SOAP ( p < 0.001). The remainder ( n = 7, 12%) indicated either no preference between formats or indicated another preference. Among residents, 66% ( n = 23) favored EAP, whereas 20% ( n = 7) and 14% ( n = 5) preferred SOAP or had no preference, respectively ( p < 0.001). Among students, 75% ( n = 18) favored EAP, whereas 17% ( n = 4) and 8% ( n = 2) favored SOAP or had no preference, respectively ( p < 0.001). Likert scale ratings for domains assessed by trainees who had experience in either format are shown in Table . In general, scores for each domain were higher for EAP than SOAP, with the exception of perceived ease of use among students. Among those with experience using both formats, EAP outperformed SOAP most prominently in time efficiency (mean 4.39 vs 2.59, p < 0.001) and encouragement to: focus on assessment and plan (4.64 vs 3.05, p < 0.001), distill pertinent information (4.63 vs 3.17, p < 0.001), and integrate data (4.58 vs 3.31, p < 0.001) (Table ). Respondents also ranked EAP higher in its effectiveness at advancing patient care (4.31 vs 3.71, p < 0.001), its capacity to convey one’s thinking (4.53 vs 3.95, p < 0.001), and its ability to facilitate learning from peers (4.10 vs 3.58, p < 0.001) and one’s own patients (4.24 vs 3.78, p = 0.003). There were no significant differences in the amount of time allotted for discussing the patient’s subjective experience or in ease of use. Evaluation of trainee free text responses regarding oral case presentation preference revealed several general themes (Table ). First, respondents generally felt that EAP was more time efficient and less repetitive, allowing for additional time to be spent discussing pertinent patient care decisions. Second, several respondents indicated that EAP aligns well with how trainees consider problems naturally (as a single problem in completion). Finally, respondents generally believed that EAP allowed learners to effectively communicate their thinking and demonstrate their knowledge. Those preferring SOAP most often cited format familiarity and the difficulty in switching between formats in describing their preference, though some also believed SOAP was more effective in describing a patient’s current status.
Our single site survey comparing 2 oral case presentation formats revealed a preference among respondents for EAP over SOAP for those medical students and internal medicine residents who had experience with both formats. Furthermore, EAP outperformed SOAP in 8 out of 10 of the functionality domains assessed, including areas such as advancing patient care, learning from patients, and, particularly, time efficiency. Such a constellation of findings implies that EAP may not only be a more effective means to accomplish the key goals of oral case presentations, but it may also provide an opportunity to save time in the process. In line with SOAP’s current de facto status as an oral case presentation format, almost all respondents reported exposure to the SOAP format. Still, indicative of EAP’s growing presence at our academic system, more than one third of medical students and more than one half of residents also reported having experience with the EAP format. While limited data exist that compare alternative oral case presentations to SOAP on inpatient medicine rounds, such alternatives have been previously trialed in other clinical venues. One such format, the multiple mini-SOAP, developed for complex outpatient visits, encourages each problem to be addressed “in its entirety” before presenting subsequent problems, and emphasizes prioritization by problem pertinency . The creators suggest that this approach encourages more active trainee participation in formulating the assessment and plan for each problem, by helping the trainee to avoid getting lost in an “undifferentiated jumble of problems and possibilities” that accumulate when multiple problems are presented all at once. On the receiving end, the multiple mini-SOAP enables faculty to assess student understanding of specific clinical problems one at a time and facilitates focused teaching accordingly. Another approach has been assessed in the emergency department. Specifically, Maddow and colleagues explored assessment-oriented oral case presentations to increase efficiency in communication between residents and faculty at the University of Chicago . In the assessment-oriented format, instead of being presented in a stylized order, pertinent information was integrated into the analysis. The authors found that assessment-oriented oral case presentations were about 40% faster than traditional presentations without significant differences in case presentation effectiveness. Prior to our study, the nature of the format for inpatient medicine oral case presentations had thus far escaped scrutiny. This is despite the fact that oral case presentations are time (and therefore resource) intensive, and that they play an integral role in patient care and learner education. Our study demonstrates that learners favor the EAP format, which has the potential to increase both the effectiveness and efficiency of rounding. Still, it should be noted that a transition to EAP does present challenges. Implementing this problem-based presentation format requires a conscious effort to ensure a continued holistic approach to patient care: active problems should be defined and addressed in accordance with patient preferences, and the patient’s subjective experience should be meaningfully incorporated into the assessment and plan for each problem. During initial implementation, attending physicians and learners must internalize this new format, often through trial and error. From there, on an ongoing basis, EAP may require more upfront preparation by attending physicians as compared to SOAP. While chart review by attendings in advance of rounding is useful regardless of the format utilized, this practice is especially important for the EAP format, where trainees are empowered to interpret and distill – rather than simply report a complete set of – information. Therefore, the attending physician must be aware of pertinent data prior to rounds to ensure that key information is not neglected. Specifically, attendings should pre-orient themselves with laboratory values, imaging, and other studies completed, and new suggestions from consultants. More extensive pre-work may be required if teams wish to employ the EAP format for newly admitted patients, as attending physicians must also familiarize themselves with a patient’s medical history and their current presentation prior to initial team rounds. Our findings should be interpreted within the context of specific limitations. First, low response rates may have led to selection bias within our surveyed population. For instance, learners who desired change in the oral case presentation format may have been more motivated to engage with our survey. Second, there could be unmeasured confounding variables that could have skewed our results in favor of the EAP format. For example, attendings who utilized the EAP format may have been more likely to innovate in other ways to create a more positive experience for learners, which may have influenced the scoring of the oral case presentation format. Third, our findings were largely based on subjective experience. Objective measurement (e.g., duration of rounds, patient care outcomes) may lend additional credibility to our findings. Lastly, our study included only a single site, limiting our ability to generalize our findings. Our study also had several strengths. Our learner participant pool was broad and included all third- and fourth-year medical students and all internal medicine residents at a major academic hospital. Participation was encouraged regardless of the nature of a participant’s prior exposure to different oral case presentation formats. Our survey was anonymous with randomization to mitigate order bias, and we focused our comparison analysis on those who had exposure to both the EAP and SOAP formats. We collected data to compare EAP with SOAP in 2 distinct ways: head-to-head preference and numeric ratings amongst key domains. Both of these methods demonstrated a significant preference for EAP among learners in aggregate, as well as for students and residents analyzed independently. Our findings suggest a preference for the EAP format over SOAP, and that EAP may facilitate clearer and more efficient communication on rounds. These improvements may in turn enhance patient care and learner education. While our preliminary data are compelling, a broader, multi-center study of the EAP oral case presentation is necessary to better understand preferences, outcomes, and barriers to implementation. Further studies should seek to improve response rates, for the data to represent a larger proportion of trainees. One potential strategy to improve response rates among medical students and residents is to survey them directly at the end of each internal medicine clerkship period or rotation, respectively. Ultimately, EAP may prove to be a much-needed update to the “currency with which clinicians communicate.”
Additional file 1: Appendix A. Exemplar Transcripts (EAP, SOAP). Additional file 2: Appendix B. Survey Instrument.
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The relationship between personality traits and individual factors with perinatal depressive symptoms: a cross-sectional study
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31a8cd9b-5693-4bcb-bbd4-16883338133a
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10210385
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Gynaecology[mh]
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Pregnancy is a major event involving neuroendocrine, anatomical, psychological and relational changes in the life of women. This complex transition can be characterized by vulnerability for mental disorders. Many clinical conditions such as anxiety, mood and psychotic disorders can affect women in the perinatal period, and perinatal depression (PND) shows a high prevalence : across nations and cultures PND has raised significant concerns due to prevalence rates ranging from 5 to 25% of women in the perinatal period . Differently from the common, mild, and self-limiting “baby/maternity blues”, PND is an impairing depressive episode occurring during pregnancy or up to 12 months after childbirth . PND is especially alarming due to its multiple consequences affecting the mother as well as the couple and the mother-child relationship, with potentially severe consequences for the child’s development. Extensive studies have shown a causal relationship of symptoms of PND with child’s emotional development disorders and lower emotion recognition , neuropsychological and cognitive deficits , internalizing and externalizing disorders , sleep disturbances , persistent lower growth and worse long-term mental health outcomes . Ultimately, severe untreated PND can lead to self-harm, harming of the infant and, in tragic cases, to suicide and infanticide . Existing evidence highlighted several factors which are related to PND. As well known by researchers and clinicians, the major neuroendocrine modifications happening during and after pregnancy play a role in the onset of PND . In addition to such biological factors, a growing body of evidence highlights the role of psychological aspects in PND. Patients’ remote and recent anamnesis have been related to PND in well-designed multivariate models. Specifically, a history of childhood trauma, poor marital quality, singlehood, domestic abuse, younger age, poor social support, and low socioeconomic status have been observed to be linked to PND symptoms . Such evidence is consistent with epidemiological data on adverse childhood experiences, showing that certain early experiences (including exposure to psychological, physical, and sexual abuse as well as household dysfunction such as domestic violence, substance use, mental diseases, incarceration) are cumulatively related to greater degrees of adult illness burden . Interestingly, patients’ personality has also been related to the onset of PND . As frequently observed by specialists in the clinical practice, certain personality traits such as dependence, obsession, neuroticism, and severe self-criticism, are related to PND symptomatology . Emerging evidence also focused on the partner’s role and on how several aspects of the future father are closely intertwined to maternal mental health. The evidence shows a meaningful impact of fathers’ anxiety and depression, romantic and father-infant attachment style, material support, and co-parenting skills on the risk and evolution of maternal PND . Only little research tested mediating and moderating etiopathogenetic models leading to PND, and no studies up to date (to the best of our knowledge) examined the possible mediating role of personality traits in the relation between patients’ family of origin history and PND symptoms. More specifically, early experiences and personality features are thought to be interrelated , and it is possible that the experience of motherhood may interact with the women’s own experience of being a child taken care of . Existing research accounting for the role of the family of origin in the prediction of PND suggests that having poor family relationships during childhood, as well as in their married life, and insufficient support from their families during pregnancy, increases odds of PND in multivariate models . Considering the above, it is possible that certain characteristics of the family of origin can influence women’s personality, and that the interaction between such early experience and the occurrence of maladaptive forms of personality can ultimately lead to more severe depressive symptoms in the perinatal period. In order to elucidate the relationship between personality and depression in the perinatal period and in order to test the mentioned hypotheses, the first aim of the present study on women in their perinatal period was to explore personality traits and individual factors independently associated with depressive symptoms. The second aim of the present study was to test the mediating role of dysfunctional personality features in the relation between dysfunctional characteristics of the family of origin and severity of depressive symptoms; the findings of the first objective led us to consider the constructs of neuroticism and of having a parent affected by a psychiatric disorder within the analyses related to the second objective. Subjects The current study represents part of a larger clinical collaboration between the Perinatal Psychiatry out-patients Service and the Child-Neuropsychiatry Unit of “Umberto I” hospital of Rome. The project focuses on prevention and treatment of maternal mental disorders . Recruitment started in February 2019 and ended in February 2020. It took place in the gynecology department of Policlinico Umberto I University Hospital, which is the largest public hospital of the metropolitan city of Rome. Recruitment was mainly performed by two psychiatrists from the Perinatal Psychiatry Service of the same Hospital: once a week, every woman consecutively admitted to the gynecology department for routine visits in the perinatal period were presented the project and proposed to take part in the study. Also, a minority of cases were proposed to participate in the study if the personnel of the gynecology unit asked for a psychiatric evaluation of specific cases. Inclusion criteria were: (1) being pregnant or within six months of giving birth; (2) accepting to take part in the research and give informed consent. Exclusion criteria were: (1) Diagnosis of moderate/severe cognitive impairment (2) insufficient Italian language skills (3) presence of severe acute psychiatric conditions requiring hospitalization (including suicidality). More in detail, eligible women were indicated by the gynaecologist and approached by the psychiatrist, following the gynaecological assessment, who explained the study aims and procedures and gathered the informed consent in case of acceptance of participation. Participants were administered the assessment tools as one bundle, to fill in privately as part of the gynaecological visit in one of the ward’s rooms. In case of doubts on specific items or other questions, participants could refer to the psychiatrist. Sociodemographic variables and pregnancy related variables were gathered from the gynaecological clinical chart as well as from a specifically designed sociodemographic data collection sheet. A statistical power analysis was performed to determine the sample size using G*Power 3.1 software based on the study objectives. The first objective of the study was to explore personality traits and individual factors independently associated with depressive symptoms; considering a potential amount of 24 predictors (as described below), the calculation indicated that, given a probability level of 0.05, a sample size of 169 was needed to provide a satisfactory statistical power (1–β = 0.80) to identify a medium effect size (f²=0.15) in a linear multiple regression model. The second objective of the study was to test the mediating role of neuroticism personality features in the relation between characteristics of the woman’s family of origin and severity of depressive symptoms; according to the sample size guidelines of Fritz and MacKinnon , in the mediation model, assuming medium effect sizes for two distinct pathways of the model (i.e., the effect of the independent variable on the mediator, and the effect of the mediator on the dependent variable), the analyses required a minimum sample size of 78 with the bootstrapping procedure to provide a statistical power of 0.80. Therefore, the current sample size (n = 241) has been chosen as it was considered to provide satisfactory statistical power in relation to the study objectives. All participants gave their written informed consent to participate. The study received the approval of the Local Ethical Committee. At the end of the assessment, patients showing clinical signs of significant mental distress/mental disorders were proposed further clinical assessment in the out-patients service for perinatal psychiatry. Measures The following information were collected: - Sociodemographic variables: age , education (elementary-, middle-, high-school, and higher education), relational status (together, separated/alone), professional status (employed/unemployed); - clinical variables (yes/no): anamnesis of chronic medical illnesses , anamnesis of mental disorders , ongoing psychotherapy , use of psychiatric pharmacological therapy , recent significant griefs , psychiatric disorders in the family of origin (parents only), history of family conflict , partner’s mental disorders , and couple conflict ; - pregnancy related variables: number previous pregnancies , previous voluntary interruption of pregnancy (VIP) (yes/no) or miscarriages (yes/no), use of alcohol (yes/no) or tobacco (yes/no) during pregnancy, pregnancy complications (yes/no). The Edinburgh Postnatal Depression Scale (EPDS) was used to assess the presence and severity of PND symptoms. The EPDS is a cross-culturally validated 10-question form that a woman can complete in 2 to 3 min . Total score can vary between 0 and 30. A score above 10 indicates “possible depression”, while patients scoring 12 or more are considered as suffering from a depressive illness of varying severity. The last item of the test enquires about suicidality and is used to identify patients with any degree of this symptom (i.e., scoring > 0). Cronbach’s α of EPDS for this sample was 0.835, indicating a good reliability . The Big Five Inventory (BFI) was used to assess main personality traits. It is a self-administered questionnaire, consisting of 44 items measuring the main characterizing traits according to the “Five Factor Model of Personality” . They include: extraversion (8 items, which allow to identify traits such as sociability, assertiveness and positive emotionality) (Cronbach’s α = 0.756), agreeableness (9 items, referring to characteristics such as altruism, trust and tendency to provide support) (Cronbach’s α = 0.655), conscientiousness (9 items, referring to the ability to control impulses and self-discipline) (Cronbach’s α = 0.771), neuroticism (8 items, referring to traits of anxiety and negative emotionality) (Cronbach’s α = 0.755), and openness (10 items, identifying characteristics such as openness to experience, intellectual curiosity, aesthetic sense) (Cronbach’s α = 0.780). Each item consists of short sentences, patients were asked to assign a score on a scale from 1 = not at all agree to 5 = completely agree. Also, due to the different number of questions for each trait, each one has a different maximum score . Statistical analysis Descriptive statistics are reported as numbers and proportions for categorical variables, and as means and standard deviations for continuous variables. Prior to analysis, data were screened for missingness. For the 39 cases showing some missing values (0.263% of the dataset, with big-5 and EPDS being 100% complete), automatic multiple imputation analysis was used to impute missing data. An exploratory bivariate Spearman correlation analysis was performed to select, among the 24 individual and clinical collected variables, those variables significantly ( p < .05) related to EPDS. Such variables were then used to construct a multivariate linear regression model to investigate factors independently associated with EPDS total score (dependent variable, objective 1). Data used in the regression respected the basic assumptions associated with a linear regression model that were tested (i.e., homoscedasticity, normal distribution of errors, multicollinearity). Finally, based on the above-mentioned hypothesis and on the results of the correlation and regression analyses, we evaluated (using the original, non-imputed dataset) whether neuroticism (which is a dysfunctional personality feature found to be independently associated with EPDS) can operate as mediator in the relationship between history of a parent psychiatric disorder in the family of origin (which is a dysfunctional characteristic of the family of origin found to be bivariately, but not independently, associated with EPDS) and depressive symptoms (objective 2). Such analysis was performed using the extension “process” (model number four) of the Statistical Package for Social Science, with 10’000 bias-corrected bootstrapping procedure . In the model, EPDS total score was the dependent variable, neuroticism was the mediator, and parent’s psychiatric disorder in the family of origin was the independent variable. Those variables which were independently associated with EPDS total score in the regression model (or which approached significance, p < .06) were included as covariates in the model. The current study represents part of a larger clinical collaboration between the Perinatal Psychiatry out-patients Service and the Child-Neuropsychiatry Unit of “Umberto I” hospital of Rome. The project focuses on prevention and treatment of maternal mental disorders . Recruitment started in February 2019 and ended in February 2020. It took place in the gynecology department of Policlinico Umberto I University Hospital, which is the largest public hospital of the metropolitan city of Rome. Recruitment was mainly performed by two psychiatrists from the Perinatal Psychiatry Service of the same Hospital: once a week, every woman consecutively admitted to the gynecology department for routine visits in the perinatal period were presented the project and proposed to take part in the study. Also, a minority of cases were proposed to participate in the study if the personnel of the gynecology unit asked for a psychiatric evaluation of specific cases. Inclusion criteria were: (1) being pregnant or within six months of giving birth; (2) accepting to take part in the research and give informed consent. Exclusion criteria were: (1) Diagnosis of moderate/severe cognitive impairment (2) insufficient Italian language skills (3) presence of severe acute psychiatric conditions requiring hospitalization (including suicidality). More in detail, eligible women were indicated by the gynaecologist and approached by the psychiatrist, following the gynaecological assessment, who explained the study aims and procedures and gathered the informed consent in case of acceptance of participation. Participants were administered the assessment tools as one bundle, to fill in privately as part of the gynaecological visit in one of the ward’s rooms. In case of doubts on specific items or other questions, participants could refer to the psychiatrist. Sociodemographic variables and pregnancy related variables were gathered from the gynaecological clinical chart as well as from a specifically designed sociodemographic data collection sheet. A statistical power analysis was performed to determine the sample size using G*Power 3.1 software based on the study objectives. The first objective of the study was to explore personality traits and individual factors independently associated with depressive symptoms; considering a potential amount of 24 predictors (as described below), the calculation indicated that, given a probability level of 0.05, a sample size of 169 was needed to provide a satisfactory statistical power (1–β = 0.80) to identify a medium effect size (f²=0.15) in a linear multiple regression model. The second objective of the study was to test the mediating role of neuroticism personality features in the relation between characteristics of the woman’s family of origin and severity of depressive symptoms; according to the sample size guidelines of Fritz and MacKinnon , in the mediation model, assuming medium effect sizes for two distinct pathways of the model (i.e., the effect of the independent variable on the mediator, and the effect of the mediator on the dependent variable), the analyses required a minimum sample size of 78 with the bootstrapping procedure to provide a statistical power of 0.80. Therefore, the current sample size (n = 241) has been chosen as it was considered to provide satisfactory statistical power in relation to the study objectives. All participants gave their written informed consent to participate. The study received the approval of the Local Ethical Committee. At the end of the assessment, patients showing clinical signs of significant mental distress/mental disorders were proposed further clinical assessment in the out-patients service for perinatal psychiatry. The following information were collected: - Sociodemographic variables: age , education (elementary-, middle-, high-school, and higher education), relational status (together, separated/alone), professional status (employed/unemployed); - clinical variables (yes/no): anamnesis of chronic medical illnesses , anamnesis of mental disorders , ongoing psychotherapy , use of psychiatric pharmacological therapy , recent significant griefs , psychiatric disorders in the family of origin (parents only), history of family conflict , partner’s mental disorders , and couple conflict ; - pregnancy related variables: number previous pregnancies , previous voluntary interruption of pregnancy (VIP) (yes/no) or miscarriages (yes/no), use of alcohol (yes/no) or tobacco (yes/no) during pregnancy, pregnancy complications (yes/no). The Edinburgh Postnatal Depression Scale (EPDS) was used to assess the presence and severity of PND symptoms. The EPDS is a cross-culturally validated 10-question form that a woman can complete in 2 to 3 min . Total score can vary between 0 and 30. A score above 10 indicates “possible depression”, while patients scoring 12 or more are considered as suffering from a depressive illness of varying severity. The last item of the test enquires about suicidality and is used to identify patients with any degree of this symptom (i.e., scoring > 0). Cronbach’s α of EPDS for this sample was 0.835, indicating a good reliability . The Big Five Inventory (BFI) was used to assess main personality traits. It is a self-administered questionnaire, consisting of 44 items measuring the main characterizing traits according to the “Five Factor Model of Personality” . They include: extraversion (8 items, which allow to identify traits such as sociability, assertiveness and positive emotionality) (Cronbach’s α = 0.756), agreeableness (9 items, referring to characteristics such as altruism, trust and tendency to provide support) (Cronbach’s α = 0.655), conscientiousness (9 items, referring to the ability to control impulses and self-discipline) (Cronbach’s α = 0.771), neuroticism (8 items, referring to traits of anxiety and negative emotionality) (Cronbach’s α = 0.755), and openness (10 items, identifying characteristics such as openness to experience, intellectual curiosity, aesthetic sense) (Cronbach’s α = 0.780). Each item consists of short sentences, patients were asked to assign a score on a scale from 1 = not at all agree to 5 = completely agree. Also, due to the different number of questions for each trait, each one has a different maximum score . Descriptive statistics are reported as numbers and proportions for categorical variables, and as means and standard deviations for continuous variables. Prior to analysis, data were screened for missingness. For the 39 cases showing some missing values (0.263% of the dataset, with big-5 and EPDS being 100% complete), automatic multiple imputation analysis was used to impute missing data. An exploratory bivariate Spearman correlation analysis was performed to select, among the 24 individual and clinical collected variables, those variables significantly ( p < .05) related to EPDS. Such variables were then used to construct a multivariate linear regression model to investigate factors independently associated with EPDS total score (dependent variable, objective 1). Data used in the regression respected the basic assumptions associated with a linear regression model that were tested (i.e., homoscedasticity, normal distribution of errors, multicollinearity). Finally, based on the above-mentioned hypothesis and on the results of the correlation and regression analyses, we evaluated (using the original, non-imputed dataset) whether neuroticism (which is a dysfunctional personality feature found to be independently associated with EPDS) can operate as mediator in the relationship between history of a parent psychiatric disorder in the family of origin (which is a dysfunctional characteristic of the family of origin found to be bivariately, but not independently, associated with EPDS) and depressive symptoms (objective 2). Such analysis was performed using the extension “process” (model number four) of the Statistical Package for Social Science, with 10’000 bias-corrected bootstrapping procedure . In the model, EPDS total score was the dependent variable, neuroticism was the mediator, and parent’s psychiatric disorder in the family of origin was the independent variable. Those variables which were independently associated with EPDS total score in the regression model (or which approached significance, p < .06) were included as covariates in the model. The total sample consisted of n = 241 women in their perinatal period. Table shows descriptive statistics of the sample. Mean age was 33.88 years (SD = 5.41; min = 16; max = 54). Only four (1.7%) patients were fully single mothers. Roughly half of the sample had a higher education, and the vast majority (82.2%) was employed. Although almost one out of five had a history of mental disorders, only few were under treatment with either psychotherapy or pharmacological therapy (7.9% and 6.2%, respectively). One in four patients reported a history of parent’s psychiatric disorder in the family of origin with one patient out of ten describing conflictual relations in the original family. Differently, 21 (8.7%) patients reported a partner’s psychiatric disorder, and the same percentage reported a conflictual relation with their partner. Table shows descriptive results related to the outcomes of interest. Overall, EPDS total score had a mean of 5.85 (SD = 4.63; min = 0; max = 23). A total of 31 patients (12.9) had an EPDS total score of 12 or more, suggestive of PND, and 12 (5%) reported suicidality as part of their condition. Personality traits are also presented in Table . As already mentioned, bivariate associations were tested with Spearman correlation test. The following variables were significantly correlated to EPDS total score: previous psychiatric disorders ( ρ = 0.165; p = .013); parent’s psychiatric disorder in the family of origin ( ρ = 0.223; p = .001); conflict in the family of origin ( ρ = 0.151; p = .020); conflictuality with the partner ( ρ = 0.232; p < .001); partner’s psychiatric disorder ( ρ = 0.137; p = .039); extraversion ( ρ=- 0.172; p = .007); agreeableness ( ρ=- 0.252; p < .001); conscientiousness ( ρ=- 0.243; p < .001); neuroticism ( ρ = 0.463; p < .001). A table presenting all bivariate correlations is available upon request. Such variables significantly associated with EPDS total score were then included in the multivariate regression model showed in Table . In the regression model with EPDS total score as dependent variable (Table ), having couple conflict, and neurotic personality traits were directly, independently correlated with EPDS total score (respectively: B = 2.337; p = .017; B = 0.303; p < .001), i.e., they were associated with EPDS also when controlling for the other variables (previous psychiatric disorder, a history of parent’s psychiatric disorder in the family of origin, conflict in the family of origin, partner psychiatric disorder, other analyzed personality traits). Having a partner with a psychiatric disorder approached significance (B = 0.82; p = .05). In order to test our hypothesis that personality features mediate the relation between characteristics of the family of origin and depressive symptoms, we built a mediation model using EPDS total score as a dependent variable, neuroticism (i.e. a dysfunctional form of personality which was independently associated with EPDS in the regression model) as mediator, and parent’s psychiatric disorder in the family of origin (i.e. a dysfunctional characteristic of the family of origin which was significantly associated with EPDS by Spearman correlation but was not independently associated with EPDS in the regression model) as predictor. The model was controlled for the other variables independently significantly associated with EPDS total score (or which approached significance) in the regression model (i.e., conflictuality with the partner, partner’s psychiatric disorder). Figure graphically shows the results of the mediation model. The total effect of parent’s psychiatric disorder in the family of origin on EPDS total score was significant (b = 2.3332; S.E.=0.6487; p = .0004; BCCI95%=1.0545—3.6119), and neuroticism partially mediated the relation between parent’s psychiatric disorder in the family of origin and EPDS total score with an indirect effect of b = 0.9688 (S.E.= 0.3154; BCCI95%=0.3655—1.6074). In this research we studied individual features, personality traits and depressive symptoms in a group of women in their perinatal period. Results showed that the personality trait neuroticism (measured by means of the BIG5) and having a conflictual relation with the partner were directly and independently associated with PND symptoms (measured through the EPDS). Further, our hypothesis that personality (i.e., neuroticism) plays a mediating role in the relation between issues related to the family of origin and PND symptoms was supported; indeed, in the tested mediation model the effect of the presence of a parent’s psychiatric disorder in the family of origin on EPDS total score was significantly mediated by the personality trait neuroticism, even when the effect of partner-related factors were controlled for. In relation to the first objective of the study, the results of the regression model suggested that neuroticism is strongly related to PND symptoms. Consistently, in our pool that there was no case of a participant scoring above the EPDS cut-off score for depression showing relatively low score in BIG-5 neuroticism scale. Building on this, couple conflict was also significantly related to EPDS, over and above confounding factors including a partner’s psychiatric disorder, possibly suggesting that conflictuality rather than a partner’s psychiatric disorder per se can play a role in perinatal depression. These findings raise the hypothesis that perinatal depression is a sui generis form of depression in which the symptomatology is closely related to personality and couple functioning. It is possible that, since pregnancy implies a profound redefinition of identity, dysfunctional aspects of personality in this period can facilitate the emergence of emotional distress. This is especially true when the partner is unable to function as containment and support within the couple. When these dysfunctional aspects are elicitated, depressive symptoms can worsen as maternal anxieties do not find adequate support neither at intrapsychic level (due to maladaptive personality traits such as neuroticism), nor at relational level (due to a problematic relation with the partner). Confirming the relevance of the family of origin for the structuring of personality and relational style and for PND-related symptoms, the mediation model which was implemented to address the second objective of the study suggested that having a parent affected by a psychiatric disorder can have a role in explaining higher degree of neuroticism and, indirectly, increased depressive symptoms. As already mentioned in the Introduction, early experiences and personality features are interrelated, and it is possible that the psychological personality features related to the experience of pregnancy/motherhood may interact with the women’s own experience of being a child taken care of in the potential development of depressive symptoms. Of relevance, other factors involved in the complex picture of peri-natal psychopathology, such as neuroendocrinological modifications happening during each phase of the pregnancy and in the pos-natal period , have not been investigated in the present study, and future research is needed to link available knowledge on psychological and biological factors to understand the etiopathogenesis of PND and help clinicians in its prevention and treatment. The present results need to be interpreted in the light of some limitations. First, the lack of longitudinal data does not allow to draw conclusions on risk factors leading to depressive symptoms, but it only allows to elucidate correlates of this condition; it would be of value to conduct long-term research on motherhood starting from the formation of the couple onward. Second, all anamnestic information provided by participants (e.g. presence or not of mental illnesses in the family of origin, conflicts with the partner) have not been verified and have been assessed without specific quantitative tools; also, EPDS and BIG5 scales are self-report measures, and this can be a potential source of bias, such as social desirability and inaccuracy biases; subsequently, the data included in the current study more closely show subjective rather than objective evaluations of participants on their own individual and psychological features, and should thus be interpreted with caution. Third, although the sample size is sufficient for the aims of our study, as suggested by a priory analyses, a multi-center study on a larger group could offer better representativity of the general population. Fourth, the present study is on depressive symptoms among women in the perinatal period, and it is thus not specifically focused on PND (i.e., the sample also included subjects without PND). Lastly, no information was available on the patients who refused to participate in the study, therefore no analyses of possible group differences between participants and non-participants could be performed. The present study also has some strengths: there was not a priori selection of patients, bringing to a more representative pool of women turning to a gynecology clinic; the assessment tools (EPDS and BIG5 scales) are widely validated and showed a good Cronbach’s α in the current sample; the analyses were performed statistically controlling for certain confounding factors. In conclusions, this study contributes to elucidate the complex relationships linking personal risk factors, personality, and depressive symptoms in women in the perinatal period. The study highlights the relevance of the couple relation and of neuroticism traits as factors related to depressive symptoms in women in the perinatal period, as well as the role of parent’s psychiatric disorders in the family of origin on neuroticism and indirectly on PND symptoms. The present evidence highlights some relational, psychological, and familiar features which could be object of early screening and recognition of cases at greater risk of PND . It is important to recognize such cases and to offer effective treatments, possibly involving the partner (such as couple therapy), in order to avoid negative outcomes on the entire family system with potential short- and long-term effects also on the development of the newborn.
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Study protocol for a randomised controlled trial to determine the effectiveness of a mHealth application as a family supportive tool in paediatric otolaryngology perioperative process (TONAPP)
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bf29438a-ecab-49a0-8b3a-b94ee1ef6b76
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10210442
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Otolaryngology[mh]
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Note: the numbers in curly brackets in this protocol refer to SPIRIT checklist item numbers. The order of the items has been modified to group similar items (see http://www.equator-network.org/reporting-guidelines/spirit-2013-statement-defining-standard-protocol-items-for-clinical-trials/ ).
Background and rationale {6a} Tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube are common surgeries in children. However, authors report children and their family are distressed by these perioperative processes. Fear of the unknown may challenge the preoperative period, while pain and other possible complications such as fever, vomiting, limited oral intake, or bleeding may complicate the postoperative management at home . Moreover, parental anxiety has been found to worsen children’s perceptions of pain, perioperative distress, and recovery . Preparing children and families for hospitalisation, surgery, and postoperative management at home has been shown to improve perioperative outcomes . However, not all individuals understand and can benefit from the information provided by healthcarers. In fact, higher levels of anxiety in the perioperative process have been associated with individuals with low health literacy . According to Sørensen and colleagues , “Health literacy is linked to literacy and entails people’s knowledge, motivation and competences to access, understand, appraise, and apply health information in order to make judgements and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life during the life course”. In paediatric surgery, this specifically includes the ability of parents or other caregivers to understand the rationale for surgery to treat their child’s condition, consent forms, preoperative and postoperative instructions, and/or drugs prescriptions. Low health literacy in paediatrics has also been found to affect health-related outcomes and child safety, as well as with preventable hospitalisations . Therefore, authors advocate the health literacy benefits of a family-centred perioperative care and education. The information needs of children and their caregivers, their health literacy, learning and coping styles, and the family’s experiences in the hospital, such as with invasive or painful procedures, are all factors to consider in this process for each family unit . However, patient- and family-centred education and support is a complex and time-consuming care interventions, whereas some surgical procedures, such as tonsillectomy, are characterised by a short hospital stay, thus limiting the time that healthcare providers can devote to this program . Moreover, unmet information needs may lead parents or other caregivers to expose themselves to health-related misinformation through autonomous web and common social media resources investigation . Therefore, health systems have tested different types of formats, content, and delivery of education to meet client needs, time availability, and effectiveness. A systematic review that aimed at exploring the types and benefits of patient/parent education programs prior to tonsillectomy identified several educational methods for tonsillectomy management, such as verbal information for caregivers, informational brochures, videos, Internet resources, mobile health applications (mHealth apps), and mobile reminder text messages. In particular, mHealth apps and text messaging were consistently shown to be beneficial to patients in terms of pain, stress and compliance, and the authors recommend their use in clinical settings where other types of perioperative education are lacking or do not lead to positive outcomes . In particular, mHealth apps are an essential element of electronic health and consist of medical information available through mobile phones or other wireless devices that can be used by patients or healthcarers. Their use is increasing and evolving in a variety of functions and positive outcomes related to the improving the well-being of individuals, including diagnostics and clinical decision making, healthy lifestyles interventions, disease management, and self-care . However, findings from literature reviews addressing current research on the use of clinical apps indicate that further randomised controlled trials with larger sample size (> 60) and improved rigour in study design and methods are needed to confirm positive outcomes . Moreover, in a literature review of qualitative studies exploring patients’ perceptions of mHealth apps, results reported customer satisfaction and engagement in their own care through the use of apps. However, weaknesses of mHealth apps have also been reported, including lack of disease-specific, cultural, and health literacy level personalisation, accessibility, and evidence-based information . Furthermore, to our knowledge, there are few studies testing mHealth apps for family-centred perioperative tonsillectomy and/or adenoidectomy with or without tympanostomy tube insertion. Therefore, this study aims to investigate the effectiveness of an mHealth app in supporting caregivers of children undergoing tonsillectomy and/or adenoidectomy, with or without insertion of a tympanostomy tube, in a family-centred and health literacy-focused manner in an Italian maternal and child health hospital compared to standard care. Moreover, objective of this study is also to evaluate the impact of the app on the surgical ward and other organisational aspects of the hospital compared to standard processes. Objectives {7} Based on these premises, the following research questions will be addressed: Does an mHealth app designed to support caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without tympanostomy tube insertion in the perioperative process improve family-health related outcomes compared to standard care? Does an mHealth app designed to support caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube improve organisational issues of the surgical ward in the perioperative process compared to standard care? Therefore, the primary objective of the study is to determine the effectiveness of a plain-language mHealth app compared with other standard supportive and educational methods on anxiety of caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube. Secondary objectives are to determine the effectiveness of a plain language mHealth app compared with other standard supportive and educational methods in terms of family preparation for hospitalisation and surgery, child distress, family satisfaction with care, and post-discharge management at home. Trial design {8} A two-parallel arm, open randomised controlled trial is being conducted to evaluate the superiority of an mHealth app in reducing anxiety in caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube in the perioperative period compared to standard care. Standard care in the control group consists of information and education provided by nurses and physicians during preoperative visits and hospitalisation about the ORL perioperative process. The information and education are provided orally or through printed brochures. Eligible participants are randomly allocated in a 1:1 ratio to the intervention group (use of mHealth app) or the control group (standard care). Our study protocol followed the SPIRIT guidelines .
Tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube are common surgeries in children. However, authors report children and their family are distressed by these perioperative processes. Fear of the unknown may challenge the preoperative period, while pain and other possible complications such as fever, vomiting, limited oral intake, or bleeding may complicate the postoperative management at home . Moreover, parental anxiety has been found to worsen children’s perceptions of pain, perioperative distress, and recovery . Preparing children and families for hospitalisation, surgery, and postoperative management at home has been shown to improve perioperative outcomes . However, not all individuals understand and can benefit from the information provided by healthcarers. In fact, higher levels of anxiety in the perioperative process have been associated with individuals with low health literacy . According to Sørensen and colleagues , “Health literacy is linked to literacy and entails people’s knowledge, motivation and competences to access, understand, appraise, and apply health information in order to make judgements and take decisions in everyday life concerning healthcare, disease prevention and health promotion to maintain or improve quality of life during the life course”. In paediatric surgery, this specifically includes the ability of parents or other caregivers to understand the rationale for surgery to treat their child’s condition, consent forms, preoperative and postoperative instructions, and/or drugs prescriptions. Low health literacy in paediatrics has also been found to affect health-related outcomes and child safety, as well as with preventable hospitalisations . Therefore, authors advocate the health literacy benefits of a family-centred perioperative care and education. The information needs of children and their caregivers, their health literacy, learning and coping styles, and the family’s experiences in the hospital, such as with invasive or painful procedures, are all factors to consider in this process for each family unit . However, patient- and family-centred education and support is a complex and time-consuming care interventions, whereas some surgical procedures, such as tonsillectomy, are characterised by a short hospital stay, thus limiting the time that healthcare providers can devote to this program . Moreover, unmet information needs may lead parents or other caregivers to expose themselves to health-related misinformation through autonomous web and common social media resources investigation . Therefore, health systems have tested different types of formats, content, and delivery of education to meet client needs, time availability, and effectiveness. A systematic review that aimed at exploring the types and benefits of patient/parent education programs prior to tonsillectomy identified several educational methods for tonsillectomy management, such as verbal information for caregivers, informational brochures, videos, Internet resources, mobile health applications (mHealth apps), and mobile reminder text messages. In particular, mHealth apps and text messaging were consistently shown to be beneficial to patients in terms of pain, stress and compliance, and the authors recommend their use in clinical settings where other types of perioperative education are lacking or do not lead to positive outcomes . In particular, mHealth apps are an essential element of electronic health and consist of medical information available through mobile phones or other wireless devices that can be used by patients or healthcarers. Their use is increasing and evolving in a variety of functions and positive outcomes related to the improving the well-being of individuals, including diagnostics and clinical decision making, healthy lifestyles interventions, disease management, and self-care . However, findings from literature reviews addressing current research on the use of clinical apps indicate that further randomised controlled trials with larger sample size (> 60) and improved rigour in study design and methods are needed to confirm positive outcomes . Moreover, in a literature review of qualitative studies exploring patients’ perceptions of mHealth apps, results reported customer satisfaction and engagement in their own care through the use of apps. However, weaknesses of mHealth apps have also been reported, including lack of disease-specific, cultural, and health literacy level personalisation, accessibility, and evidence-based information . Furthermore, to our knowledge, there are few studies testing mHealth apps for family-centred perioperative tonsillectomy and/or adenoidectomy with or without tympanostomy tube insertion. Therefore, this study aims to investigate the effectiveness of an mHealth app in supporting caregivers of children undergoing tonsillectomy and/or adenoidectomy, with or without insertion of a tympanostomy tube, in a family-centred and health literacy-focused manner in an Italian maternal and child health hospital compared to standard care. Moreover, objective of this study is also to evaluate the impact of the app on the surgical ward and other organisational aspects of the hospital compared to standard processes.
Based on these premises, the following research questions will be addressed: Does an mHealth app designed to support caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without tympanostomy tube insertion in the perioperative process improve family-health related outcomes compared to standard care? Does an mHealth app designed to support caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube improve organisational issues of the surgical ward in the perioperative process compared to standard care? Therefore, the primary objective of the study is to determine the effectiveness of a plain-language mHealth app compared with other standard supportive and educational methods on anxiety of caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube. Secondary objectives are to determine the effectiveness of a plain language mHealth app compared with other standard supportive and educational methods in terms of family preparation for hospitalisation and surgery, child distress, family satisfaction with care, and post-discharge management at home.
A two-parallel arm, open randomised controlled trial is being conducted to evaluate the superiority of an mHealth app in reducing anxiety in caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube in the perioperative period compared to standard care. Standard care in the control group consists of information and education provided by nurses and physicians during preoperative visits and hospitalisation about the ORL perioperative process. The information and education are provided orally or through printed brochures. Eligible participants are randomly allocated in a 1:1 ratio to the intervention group (use of mHealth app) or the control group (standard care). Our study protocol followed the SPIRIT guidelines .
Study setting {9} The study is monocentric and is being conducted in a 136-bed maternal and child health hospital in Northern Italy. In this hospital, a number of approximately 450 tonsillectomies or adeno-tonsillectomies are performed each year. Specifically, enrolment started December 16, 2022, and is taking place in the consulting rooms of the paediatric surgery ORL department. Data collection is being performed in the paediatric surgery ward and in the ORL department. Statistical data analysis will be held at the clinical epidemiology and public health research unit of the same hospital. Eligibility criteria {10} The inclusion criteria are as follows: (1) caregivers of male and female children aged at least 2 years and no more than 10 years who are scheduled for tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube, (2) caregivers who are capable of oral and written Italian communication without impairment, and (3) caregivers who guarantee access to a smartphone and Internet connection. The exclusion criteria will be as follows: (1) caregivers with cognitive impairment,,(2) caregivers of children with cognitive impairment, (3) caregivers with visual impairment, (4) caregivers of children affected by chronic pain, (5) caregivers of children who had another surgical procedure in the previous month, and (6) caregivers who have never used at least one smartphone app. Who will take informed consent? {26a} Nursing staff in the consultation rooms of the paediatric surgery ORL department are in charge of informing eligible caregivers about the study and obtaining signed informed consent from caregivers to voluntarily participate in the study. Additional consent provisions for collection and use of participant data and biological specimens {26b} The present study does not involve the collection or derivation of data and biological specimen for purposes that are separate from the main trial.
The study is monocentric and is being conducted in a 136-bed maternal and child health hospital in Northern Italy. In this hospital, a number of approximately 450 tonsillectomies or adeno-tonsillectomies are performed each year. Specifically, enrolment started December 16, 2022, and is taking place in the consulting rooms of the paediatric surgery ORL department. Data collection is being performed in the paediatric surgery ward and in the ORL department. Statistical data analysis will be held at the clinical epidemiology and public health research unit of the same hospital.
The inclusion criteria are as follows: (1) caregivers of male and female children aged at least 2 years and no more than 10 years who are scheduled for tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube, (2) caregivers who are capable of oral and written Italian communication without impairment, and (3) caregivers who guarantee access to a smartphone and Internet connection. The exclusion criteria will be as follows: (1) caregivers with cognitive impairment,,(2) caregivers of children with cognitive impairment, (3) caregivers with visual impairment, (4) caregivers of children affected by chronic pain, (5) caregivers of children who had another surgical procedure in the previous month, and (6) caregivers who have never used at least one smartphone app.
Nursing staff in the consultation rooms of the paediatric surgery ORL department are in charge of informing eligible caregivers about the study and obtaining signed informed consent from caregivers to voluntarily participate in the study.
The present study does not involve the collection or derivation of data and biological specimen for purposes that are separate from the main trial.
Explanation for the choice of comparators {6b} Standard care was chosen as comparator. Standard care given to caregivers in the ORL perioperative process consists of information and education provided by nurses and physicians during preoperative visits and hospitalisation about the ORL perioperative process. Standard information and education include topics such as ORL surgical and anaesthesiologic techniques that will be performed or strategies for pain control at home after surgery. Information and education is usually provided orally and through printed brochures. Intervention description {11a} The intervention group (group A) uses an a mHealth app for information and education to caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube in the perioperative process. Group A has the app in use from the day of enrolment to the day of follow-up visit. Group B receives standard care. The content and functions of the mHealth app were developed in collaboration with an institute of maternal and child health and Area Science Park, both in Northeastern Italy. The methodological steps followed to develop the mHealth app were guided by the three cycles of the Information System Research Framework . The mHealth app was developed using a user-centred participatory design approach that involved both caregivers of ORL surgical children (primary users) and hospital staff (secondary users) to understand their needs and expectations related to content topics and functionalities . To promote an equally accessible understanding of the mHealth app content, a team of external communication experts was also consulted, and content, such as terminology, was simplified where necessary. The information and education content provided in standard care to the control group by healthcare providers during hospital visits, either orally or in the form of brochures are similar to that provided to the intervention group through the medium of the mHealth app. Information topics included, for example, explaining of the surgical procedure chosen, the type of anaesthesia used, strategies for preparing children for hospitalisation and surgery, or how to manage pain at home after discharge. Because healthcare providers have little time available to inform the family about safe surgery and other topics due to the short hospital stay that characterises this type of surgery , the content in the app is expanded and caregivers have the opportunity to read it at the time that best suits them and with the time they need. In fact, all content is available to users at any time in a library section of the app. Moreover, in accordance with the principles of adult learning and its application in telemedicine , the content of the application is adapted to the needs of the primary end-users and delivered “just in time” . In particular, content is suggested to participants in the form of new content notifications that are available based on the specific perioperative period the child is in, such as the day of prehospital visits, the prehospital period, the day of hospitalisation and surgery, and the post-discharge period at home. Moreover, pop-up messages are displayed to the user as reminders of visits or documents to bring to the hospital. All the content is provided in text format and in Italian. This mHealth app has the potential to enable communication between primary and secondary end-users, but these features are not used by study participants and are not the subject of this study experiment. The nurses from the ORL paediatric surgical department are in charge of caregivers of ORL surgical children enrolment. These nurses, who are familiar with all the content, functions and features of the mHealth app, are also responsible for instructing group A participants on (1) how to download the app to their smartphone or tablet and (2) how to use the mHealth app and what functions and content it offers. The app developers also created a three-minute presentation of the mHealth app, which is shown by the nurses on a tablet to the group A participants after enrolment. The mHealth app is available for group A from the day of the pre-surgery visit until the seventh day after surgery or follow-up, which usually totals 14 days. The iOS and Android app provided to group A complies with technical and sectoral regulations such as the GDPR and the NIS directive, meets standards such as ISO/IEC 27,001 and the CE label, and consists of the following: a web-based platform with a modular architecture for information support and dialogue with users, certified according to DM 93/42/CE in the class II A and meets the main sectoral requirements for functional and reliability. Moreover, the app platform and all related services are developed in accordance with the rules of the Three-Year Plan for Information Technology for Public Administration (2019/2021), as far as they are compatible, and in accordance to the “Cloud First” principle. The app is available free of charge and is available to the hospital’s end-users. However, until the end of the trial, the app will only be available to group A study participants. Criteria for discontinuing or modifying allocated interventions {11b} In the event of a child’s fever or other issues causing a delay in surgery, the possibility to use the app and its content in the correct time frames will be adjusted to reflect the new surgery date selected. Strategies to improve adherence to interventions {11c} Considering the functionality of the app, for example, allowing content to be delivered “just in time” and depending on the specific perioperative period and potential needs of the user, not only is caregiver education made more effective but also their interest in continuing to use the app is stimulated. In addition, pop-up notifications and reminders from the app assist group A participants in using the app and reading its content. Moreover, groups A and B participants are encouraged to attend scheduled visits where healthcare providers give ORL perioperative information and education. Relevant concomitant care permitted or prohibited during the trial {11d} We consider and permit that participants of group A and B may be exposed during the study to information derived from consultation of other health care experts, books, or web and social media. Provisions for post-trial care {30} If this study demonstrates evidence of the effectiveness of the mHealth app, the app will be available for free download and use by caregivers of children undergoing tonsillectomy and/or adenoidectomy at this hospital, with or without the insertion of a tympanostomy tube. Outcomes {12} The primary outcome of the study is the difference between the intervention and control groups in terms of caregivers’ self-reported state anxiety. It is be measured using the State-Trait Anxiety Inventory form Y questionnaire in the surgical department, before the caregiver and child go into the operating theatre on the day of surgery, approximately seven days after enrolment. Secondary outcomes are: The difference between the intervention and control groups in caregiver preparation for hospitalisation and surgery, evaluated at arrival at the hospital using a checklist completed by the administrative nurse (e.g. number of documents missing on arrival) approximately 5 days after enrolment; The difference between the intervention and control groups in preparing children for surgery, evaluated by ORL surgical nurses on arrival at the surgical department, approximately 5 days after enrolment; The difference between the intervention and control groups in terms of children’s distress. It is assessed by ORL surgical nurses in the surgical department using the modified Yale Preoperative Anxiety Scale ; The difference between the intervention and control groups in self-reported primary caregiver anxiety as measured by the State-Trait Anxiety Inventory form Y questionnaire on the day of follow-up, around the seventh day after surgery and 13 days after enrolment. The difference between the intervention and control groups in terms of the social and health effects of the introduction of an mHealth app in a maternal and child hospital. It is evaluated on the day of follow-up, approximately 14 days after enrolment. Participant timeline {13} Caregivers participate in the study from the day of enrolment to day 7 after surgery or follow-up. The assessment time points (T) for caregivers and children after enrolment and signing of the informed consent form are as follows: (T0) after enrolment and allocation, data collection at time zero—(a) demographic data and (b) caregiver anxiety trait and state; (c) caregiver health literacy. At T0, participants in group A receive the app (intervention), while group B continue to receive standard care; (T1) at hospital admission on the day of surgery—IRCCS administrative office (“Punto benvenuto”)—documents required for hospital admission and surgery; (T2) upon admission to the surgical department ORL—(a) caregiver’s state anxiety level; (b) child’s preparation for surgery; (c) child distress; (T3) at follow-up—caregiver’s state anxiety; social impact. Participant timeline is shown schematically in Fig. . Sample size {14} Assuming a mean score of preoperative anxiety (main outcome of the study) of 50 for the control group, with a standard deviation (SD) of 13 , and a mean of 45 for the intervention group, with SD of 10 (effect size = 0.43), an alpha value of 0.05 and a beta value of 0.20, 180 subjects (90 per group) are needed to conduct the study (G*power, Wilcoxon-Mann–Whitney test, two groups). Recruitment {15} Caregivers of children selected for ORL surgery and accessing the hospital are screened according to inclusion and exclusion criteria. The caregivers are offered to participate in this study. Recruitment is expected to last 8 months and will continue until the estimated sample size is reached. Assignment of interventions: allocation Sequence generation {16a} Enrolled caregivers are randomly assigned into two groups (experimental group A and control group B) using a computerised number generation with simple randomisation and block randomisation with a block size of four to ensure a good balance of participants between the two groups. Concealment mechanism {16b} The allocation process is undertaken by the hospital’s Clinical Epidemiology and Public Health Research Unit to ensure random allocation and concealment. The allocation sequence is hidden from the researchers and other staff involved in the study in sealed, opaque, sequentially numbered envelopes. Implementation {16c} The nurse in the consultation rooms of the ORL paediatric surgery department is in charge to open the envelopes and give access to the app to group A, according to the number extracted and corresponding to the study arm. Assignment of interventions: blinding Who will be blinded {17a} Healthcare providers and participants could not be blinded in this study because the app in use is visible to caregivers and staff. Outcome assessors could not be blinded in this study. Procedure for unblinding if needed {17b} Because this study is an open-label study, no unblinding procedures are used.
Standard care was chosen as comparator. Standard care given to caregivers in the ORL perioperative process consists of information and education provided by nurses and physicians during preoperative visits and hospitalisation about the ORL perioperative process. Standard information and education include topics such as ORL surgical and anaesthesiologic techniques that will be performed or strategies for pain control at home after surgery. Information and education is usually provided orally and through printed brochures.
The intervention group (group A) uses an a mHealth app for information and education to caregivers of children undergoing tonsillectomy and/or adenoidectomy with or without insertion of a tympanostomy tube in the perioperative process. Group A has the app in use from the day of enrolment to the day of follow-up visit. Group B receives standard care. The content and functions of the mHealth app were developed in collaboration with an institute of maternal and child health and Area Science Park, both in Northeastern Italy. The methodological steps followed to develop the mHealth app were guided by the three cycles of the Information System Research Framework . The mHealth app was developed using a user-centred participatory design approach that involved both caregivers of ORL surgical children (primary users) and hospital staff (secondary users) to understand their needs and expectations related to content topics and functionalities . To promote an equally accessible understanding of the mHealth app content, a team of external communication experts was also consulted, and content, such as terminology, was simplified where necessary. The information and education content provided in standard care to the control group by healthcare providers during hospital visits, either orally or in the form of brochures are similar to that provided to the intervention group through the medium of the mHealth app. Information topics included, for example, explaining of the surgical procedure chosen, the type of anaesthesia used, strategies for preparing children for hospitalisation and surgery, or how to manage pain at home after discharge. Because healthcare providers have little time available to inform the family about safe surgery and other topics due to the short hospital stay that characterises this type of surgery , the content in the app is expanded and caregivers have the opportunity to read it at the time that best suits them and with the time they need. In fact, all content is available to users at any time in a library section of the app. Moreover, in accordance with the principles of adult learning and its application in telemedicine , the content of the application is adapted to the needs of the primary end-users and delivered “just in time” . In particular, content is suggested to participants in the form of new content notifications that are available based on the specific perioperative period the child is in, such as the day of prehospital visits, the prehospital period, the day of hospitalisation and surgery, and the post-discharge period at home. Moreover, pop-up messages are displayed to the user as reminders of visits or documents to bring to the hospital. All the content is provided in text format and in Italian. This mHealth app has the potential to enable communication between primary and secondary end-users, but these features are not used by study participants and are not the subject of this study experiment. The nurses from the ORL paediatric surgical department are in charge of caregivers of ORL surgical children enrolment. These nurses, who are familiar with all the content, functions and features of the mHealth app, are also responsible for instructing group A participants on (1) how to download the app to their smartphone or tablet and (2) how to use the mHealth app and what functions and content it offers. The app developers also created a three-minute presentation of the mHealth app, which is shown by the nurses on a tablet to the group A participants after enrolment. The mHealth app is available for group A from the day of the pre-surgery visit until the seventh day after surgery or follow-up, which usually totals 14 days. The iOS and Android app provided to group A complies with technical and sectoral regulations such as the GDPR and the NIS directive, meets standards such as ISO/IEC 27,001 and the CE label, and consists of the following: a web-based platform with a modular architecture for information support and dialogue with users, certified according to DM 93/42/CE in the class II A and meets the main sectoral requirements for functional and reliability. Moreover, the app platform and all related services are developed in accordance with the rules of the Three-Year Plan for Information Technology for Public Administration (2019/2021), as far as they are compatible, and in accordance to the “Cloud First” principle. The app is available free of charge and is available to the hospital’s end-users. However, until the end of the trial, the app will only be available to group A study participants.
In the event of a child’s fever or other issues causing a delay in surgery, the possibility to use the app and its content in the correct time frames will be adjusted to reflect the new surgery date selected.
Considering the functionality of the app, for example, allowing content to be delivered “just in time” and depending on the specific perioperative period and potential needs of the user, not only is caregiver education made more effective but also their interest in continuing to use the app is stimulated. In addition, pop-up notifications and reminders from the app assist group A participants in using the app and reading its content. Moreover, groups A and B participants are encouraged to attend scheduled visits where healthcare providers give ORL perioperative information and education.
We consider and permit that participants of group A and B may be exposed during the study to information derived from consultation of other health care experts, books, or web and social media.
If this study demonstrates evidence of the effectiveness of the mHealth app, the app will be available for free download and use by caregivers of children undergoing tonsillectomy and/or adenoidectomy at this hospital, with or without the insertion of a tympanostomy tube.
The primary outcome of the study is the difference between the intervention and control groups in terms of caregivers’ self-reported state anxiety. It is be measured using the State-Trait Anxiety Inventory form Y questionnaire in the surgical department, before the caregiver and child go into the operating theatre on the day of surgery, approximately seven days after enrolment. Secondary outcomes are: The difference between the intervention and control groups in caregiver preparation for hospitalisation and surgery, evaluated at arrival at the hospital using a checklist completed by the administrative nurse (e.g. number of documents missing on arrival) approximately 5 days after enrolment; The difference between the intervention and control groups in preparing children for surgery, evaluated by ORL surgical nurses on arrival at the surgical department, approximately 5 days after enrolment; The difference between the intervention and control groups in terms of children’s distress. It is assessed by ORL surgical nurses in the surgical department using the modified Yale Preoperative Anxiety Scale ; The difference between the intervention and control groups in self-reported primary caregiver anxiety as measured by the State-Trait Anxiety Inventory form Y questionnaire on the day of follow-up, around the seventh day after surgery and 13 days after enrolment. The difference between the intervention and control groups in terms of the social and health effects of the introduction of an mHealth app in a maternal and child hospital. It is evaluated on the day of follow-up, approximately 14 days after enrolment.
Caregivers participate in the study from the day of enrolment to day 7 after surgery or follow-up. The assessment time points (T) for caregivers and children after enrolment and signing of the informed consent form are as follows: (T0) after enrolment and allocation, data collection at time zero—(a) demographic data and (b) caregiver anxiety trait and state; (c) caregiver health literacy. At T0, participants in group A receive the app (intervention), while group B continue to receive standard care; (T1) at hospital admission on the day of surgery—IRCCS administrative office (“Punto benvenuto”)—documents required for hospital admission and surgery; (T2) upon admission to the surgical department ORL—(a) caregiver’s state anxiety level; (b) child’s preparation for surgery; (c) child distress; (T3) at follow-up—caregiver’s state anxiety; social impact. Participant timeline is shown schematically in Fig. .
Assuming a mean score of preoperative anxiety (main outcome of the study) of 50 for the control group, with a standard deviation (SD) of 13 , and a mean of 45 for the intervention group, with SD of 10 (effect size = 0.43), an alpha value of 0.05 and a beta value of 0.20, 180 subjects (90 per group) are needed to conduct the study (G*power, Wilcoxon-Mann–Whitney test, two groups).
Caregivers of children selected for ORL surgery and accessing the hospital are screened according to inclusion and exclusion criteria. The caregivers are offered to participate in this study. Recruitment is expected to last 8 months and will continue until the estimated sample size is reached.
Sequence generation {16a} Enrolled caregivers are randomly assigned into two groups (experimental group A and control group B) using a computerised number generation with simple randomisation and block randomisation with a block size of four to ensure a good balance of participants between the two groups. Concealment mechanism {16b} The allocation process is undertaken by the hospital’s Clinical Epidemiology and Public Health Research Unit to ensure random allocation and concealment. The allocation sequence is hidden from the researchers and other staff involved in the study in sealed, opaque, sequentially numbered envelopes. Implementation {16c} The nurse in the consultation rooms of the ORL paediatric surgery department is in charge to open the envelopes and give access to the app to group A, according to the number extracted and corresponding to the study arm.
Enrolled caregivers are randomly assigned into two groups (experimental group A and control group B) using a computerised number generation with simple randomisation and block randomisation with a block size of four to ensure a good balance of participants between the two groups.
The allocation process is undertaken by the hospital’s Clinical Epidemiology and Public Health Research Unit to ensure random allocation and concealment. The allocation sequence is hidden from the researchers and other staff involved in the study in sealed, opaque, sequentially numbered envelopes.
The nurse in the consultation rooms of the ORL paediatric surgery department is in charge to open the envelopes and give access to the app to group A, according to the number extracted and corresponding to the study arm.
Who will be blinded {17a} Healthcare providers and participants could not be blinded in this study because the app in use is visible to caregivers and staff. Outcome assessors could not be blinded in this study. Procedure for unblinding if needed {17b} Because this study is an open-label study, no unblinding procedures are used.
Healthcare providers and participants could not be blinded in this study because the app in use is visible to caregivers and staff. Outcome assessors could not be blinded in this study.
Because this study is an open-label study, no unblinding procedures are used.
Plans for assessment and collection of outcomes {18a} Assessment tools (groups A and B) A sociodemographic questionnaire is used to collect the following data at T0: age, sex, educational level of caregiver, current occupational status (employed, unemployed, retired, homemaker, student), relationship to child (mother, father, other guardian); being a healthcare professional; previous experience caring for others with health problems; age and sex of child; child’s planned surgery at this hospitalisation; child’s previous surgical experience. The level of health literacy is evaluated at T0 using the European Health Literacy Survey Questionnaire . The HLS-EU-Q16 is a 16-item self-assessment tool with five possible answers on a Likert scale ranging from 1 (very difficult to) 4 (very easy), with the fifth possible answer being “I don’t know.” To determine the score, the possible answers HLS-EU-Q16 are dichotomised (‘don’t know’ answers are coded as missing values). ‘Fairly difficult’ and ‘very difficult’ are both coded 0 (zero), while ‘fairly easy’ and ‘very easy’ are both coded 1. HLS-EU-Q16 is a summated score with a range of 0–16. Based on the final score, three levels of health literacy (HL) can be defined: insufficient HL (0–8), problematic HL (9–12), sufficient HL (13–16) . The documents required for hospital admission and surgery is assessed at T1 using a checklist completed by administrative nurses (number of documents missing when the family arrives at the hospital, e.g. identity card, healthcare card). The child’s preparation for surgery is assessed at T2 by the nurse in the surgical department using a checklist that evaluates whether the child has adequate hygiene, is not wearing nail polish, is fasting, and is not wearing jewellery as prescribed by staff for standard surgical preparation. Caregivers’ anxiety is measured using the Italian version of the State-Trait Anxiety Inventory questionnaire . The questionnaire consists of two self-report scales for measuring state anxiety and trait anxiety. The S-Anxiety scale (STAI Form Y-1) consists of twenty statements that evaluate how the respondent feels “now, at this moment,” on a 4 items Likert scale (from “not at all” to “very much”, attached). The T-Anxiety scale (STAI Form Y-2) consists of twenty statements that evaluate how the respondent feels “generally” on a four-point Likert scale, ranging from “almost never” to “almost always.” The total score ranges from 20 to 80 points, with lower scores indicating higher trait and state anxiety. Caregivers are asked to complete both forms at T0 to determine anxiety trait and baseline levels. The state form of the STAI Form Y-1 is completed in the hospital surgical department prior to surgery at T2 and again on the seventh day after surgery (follow-up) at T3 to measure study outcomes . The modified Yale Preoperative Anxiety Scale (mYPAS) is used as an observational tool to measure anxiety in children in the preoperative period at T2. The mYPAS consists of 27 items that examine five domains, such as child activity, emotional expressiveness, arousal state, vocalisation, and caregiver engagement. Scores range from 23.33 to 100, with higher scores indicating higher levels of anxiety. The tool is completed by nurses. Since there is no validated Italian version of the questionnaire, only English-speaking nurses are responsible for completing the instrument, according to Liguori and colleagues . The social and health impact of the study is self-assessed by caregivers at follow-up (T3) through outcome measures, such as how often caregivers had to call the hospital for additional information, had to visit the paediatric Emergency department, or had other complications at home after surgery (e.g. bleeding, uncontrolled pain). The questionnaires at T0 and T2 are completed online via a tablet available to participants in groups A and B and to nurses in the surgical department. The assessment at T1 is completed in paper form. Moreover, considering that in this hospital not all caregivers bring their child for follow-up, sometimes preferring to see their general paediatrician, data collection at T3 is performed online after sending caregivers an email with a link to fill out the questionnaires. Prior to the start of the study, nurses in the surgical department participating in the study were trained in the use of the assessment tools (e.g. mYPAS) with standardised instructions. The training was conducted by a nurse from the research ream in a meeting room of the department. Other assessments (only group A) Data on app usage (number of logins; quality of content consulted, time spent on app) will be collected during experimentation of the app. At the end of the trial, participants are asked to provide an evaluation over the app’s content and features in digital form through the app itself. Plans to promote participant retention and complete follow-up {18b} Data collection is conducted during the perioperative period when caregivers are in the hospital for visits and surgery, so participant presence in the hospital of participants eases data collection. As with any ORL surgical process at this hospital, participants in groups A and B are encouraged to attend the scheduled follow-up visit and are reminded by email to complete the questionnaires for the final assessments on T3, the day of follow-up. Data management {19} Most data are collected electronically through an interface that complies with European (GDPR No. 679/2016) and Italian (D.L. 101/2018) data protection guidelines. Participants are pseudo-anonymised and given a ID number. Data collected at T1 on a paper form will be entered into an electronic record protected by a password. Two nurses will enter the data collected at T1 to verify correct entry. The data will be processed by the Clinical Epidemiology and Public Health Research Unit of the hospital using the SAS software, Version 9.4 (SAS Institute Inc., Cary, NC, USA) for data analysis. Access to the data will be restricted, and researchers and statisticians will be assigned a user ID and password. Confidentiality {27} Confidentiality is ensured by assigning a unique participant code number to each participant to ensure pseudo-anonymisation. Confidentiality and anonymity of study participants are ensured according to European (GDPR No. 679/2016) and Italian (D.L. 101/2018) regulations. Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} No biological specimens will be collected.
Assessment tools (groups A and B) A sociodemographic questionnaire is used to collect the following data at T0: age, sex, educational level of caregiver, current occupational status (employed, unemployed, retired, homemaker, student), relationship to child (mother, father, other guardian); being a healthcare professional; previous experience caring for others with health problems; age and sex of child; child’s planned surgery at this hospitalisation; child’s previous surgical experience. The level of health literacy is evaluated at T0 using the European Health Literacy Survey Questionnaire . The HLS-EU-Q16 is a 16-item self-assessment tool with five possible answers on a Likert scale ranging from 1 (very difficult to) 4 (very easy), with the fifth possible answer being “I don’t know.” To determine the score, the possible answers HLS-EU-Q16 are dichotomised (‘don’t know’ answers are coded as missing values). ‘Fairly difficult’ and ‘very difficult’ are both coded 0 (zero), while ‘fairly easy’ and ‘very easy’ are both coded 1. HLS-EU-Q16 is a summated score with a range of 0–16. Based on the final score, three levels of health literacy (HL) can be defined: insufficient HL (0–8), problematic HL (9–12), sufficient HL (13–16) . The documents required for hospital admission and surgery is assessed at T1 using a checklist completed by administrative nurses (number of documents missing when the family arrives at the hospital, e.g. identity card, healthcare card). The child’s preparation for surgery is assessed at T2 by the nurse in the surgical department using a checklist that evaluates whether the child has adequate hygiene, is not wearing nail polish, is fasting, and is not wearing jewellery as prescribed by staff for standard surgical preparation. Caregivers’ anxiety is measured using the Italian version of the State-Trait Anxiety Inventory questionnaire . The questionnaire consists of two self-report scales for measuring state anxiety and trait anxiety. The S-Anxiety scale (STAI Form Y-1) consists of twenty statements that evaluate how the respondent feels “now, at this moment,” on a 4 items Likert scale (from “not at all” to “very much”, attached). The T-Anxiety scale (STAI Form Y-2) consists of twenty statements that evaluate how the respondent feels “generally” on a four-point Likert scale, ranging from “almost never” to “almost always.” The total score ranges from 20 to 80 points, with lower scores indicating higher trait and state anxiety. Caregivers are asked to complete both forms at T0 to determine anxiety trait and baseline levels. The state form of the STAI Form Y-1 is completed in the hospital surgical department prior to surgery at T2 and again on the seventh day after surgery (follow-up) at T3 to measure study outcomes . The modified Yale Preoperative Anxiety Scale (mYPAS) is used as an observational tool to measure anxiety in children in the preoperative period at T2. The mYPAS consists of 27 items that examine five domains, such as child activity, emotional expressiveness, arousal state, vocalisation, and caregiver engagement. Scores range from 23.33 to 100, with higher scores indicating higher levels of anxiety. The tool is completed by nurses. Since there is no validated Italian version of the questionnaire, only English-speaking nurses are responsible for completing the instrument, according to Liguori and colleagues . The social and health impact of the study is self-assessed by caregivers at follow-up (T3) through outcome measures, such as how often caregivers had to call the hospital for additional information, had to visit the paediatric Emergency department, or had other complications at home after surgery (e.g. bleeding, uncontrolled pain). The questionnaires at T0 and T2 are completed online via a tablet available to participants in groups A and B and to nurses in the surgical department. The assessment at T1 is completed in paper form. Moreover, considering that in this hospital not all caregivers bring their child for follow-up, sometimes preferring to see their general paediatrician, data collection at T3 is performed online after sending caregivers an email with a link to fill out the questionnaires. Prior to the start of the study, nurses in the surgical department participating in the study were trained in the use of the assessment tools (e.g. mYPAS) with standardised instructions. The training was conducted by a nurse from the research ream in a meeting room of the department. Other assessments (only group A) Data on app usage (number of logins; quality of content consulted, time spent on app) will be collected during experimentation of the app. At the end of the trial, participants are asked to provide an evaluation over the app’s content and features in digital form through the app itself.
A sociodemographic questionnaire is used to collect the following data at T0: age, sex, educational level of caregiver, current occupational status (employed, unemployed, retired, homemaker, student), relationship to child (mother, father, other guardian); being a healthcare professional; previous experience caring for others with health problems; age and sex of child; child’s planned surgery at this hospitalisation; child’s previous surgical experience. The level of health literacy is evaluated at T0 using the European Health Literacy Survey Questionnaire . The HLS-EU-Q16 is a 16-item self-assessment tool with five possible answers on a Likert scale ranging from 1 (very difficult to) 4 (very easy), with the fifth possible answer being “I don’t know.” To determine the score, the possible answers HLS-EU-Q16 are dichotomised (‘don’t know’ answers are coded as missing values). ‘Fairly difficult’ and ‘very difficult’ are both coded 0 (zero), while ‘fairly easy’ and ‘very easy’ are both coded 1. HLS-EU-Q16 is a summated score with a range of 0–16. Based on the final score, three levels of health literacy (HL) can be defined: insufficient HL (0–8), problematic HL (9–12), sufficient HL (13–16) . The documents required for hospital admission and surgery is assessed at T1 using a checklist completed by administrative nurses (number of documents missing when the family arrives at the hospital, e.g. identity card, healthcare card). The child’s preparation for surgery is assessed at T2 by the nurse in the surgical department using a checklist that evaluates whether the child has adequate hygiene, is not wearing nail polish, is fasting, and is not wearing jewellery as prescribed by staff for standard surgical preparation. Caregivers’ anxiety is measured using the Italian version of the State-Trait Anxiety Inventory questionnaire . The questionnaire consists of two self-report scales for measuring state anxiety and trait anxiety. The S-Anxiety scale (STAI Form Y-1) consists of twenty statements that evaluate how the respondent feels “now, at this moment,” on a 4 items Likert scale (from “not at all” to “very much”, attached). The T-Anxiety scale (STAI Form Y-2) consists of twenty statements that evaluate how the respondent feels “generally” on a four-point Likert scale, ranging from “almost never” to “almost always.” The total score ranges from 20 to 80 points, with lower scores indicating higher trait and state anxiety. Caregivers are asked to complete both forms at T0 to determine anxiety trait and baseline levels. The state form of the STAI Form Y-1 is completed in the hospital surgical department prior to surgery at T2 and again on the seventh day after surgery (follow-up) at T3 to measure study outcomes . The modified Yale Preoperative Anxiety Scale (mYPAS) is used as an observational tool to measure anxiety in children in the preoperative period at T2. The mYPAS consists of 27 items that examine five domains, such as child activity, emotional expressiveness, arousal state, vocalisation, and caregiver engagement. Scores range from 23.33 to 100, with higher scores indicating higher levels of anxiety. The tool is completed by nurses. Since there is no validated Italian version of the questionnaire, only English-speaking nurses are responsible for completing the instrument, according to Liguori and colleagues . The social and health impact of the study is self-assessed by caregivers at follow-up (T3) through outcome measures, such as how often caregivers had to call the hospital for additional information, had to visit the paediatric Emergency department, or had other complications at home after surgery (e.g. bleeding, uncontrolled pain). The questionnaires at T0 and T2 are completed online via a tablet available to participants in groups A and B and to nurses in the surgical department. The assessment at T1 is completed in paper form. Moreover, considering that in this hospital not all caregivers bring their child for follow-up, sometimes preferring to see their general paediatrician, data collection at T3 is performed online after sending caregivers an email with a link to fill out the questionnaires. Prior to the start of the study, nurses in the surgical department participating in the study were trained in the use of the assessment tools (e.g. mYPAS) with standardised instructions. The training was conducted by a nurse from the research ream in a meeting room of the department.
Data on app usage (number of logins; quality of content consulted, time spent on app) will be collected during experimentation of the app. At the end of the trial, participants are asked to provide an evaluation over the app’s content and features in digital form through the app itself.
Data collection is conducted during the perioperative period when caregivers are in the hospital for visits and surgery, so participant presence in the hospital of participants eases data collection. As with any ORL surgical process at this hospital, participants in groups A and B are encouraged to attend the scheduled follow-up visit and are reminded by email to complete the questionnaires for the final assessments on T3, the day of follow-up.
Most data are collected electronically through an interface that complies with European (GDPR No. 679/2016) and Italian (D.L. 101/2018) data protection guidelines. Participants are pseudo-anonymised and given a ID number. Data collected at T1 on a paper form will be entered into an electronic record protected by a password. Two nurses will enter the data collected at T1 to verify correct entry. The data will be processed by the Clinical Epidemiology and Public Health Research Unit of the hospital using the SAS software, Version 9.4 (SAS Institute Inc., Cary, NC, USA) for data analysis. Access to the data will be restricted, and researchers and statisticians will be assigned a user ID and password.
Confidentiality is ensured by assigning a unique participant code number to each participant to ensure pseudo-anonymisation. Confidentiality and anonymity of study participants are ensured according to European (GDPR No. 679/2016) and Italian (D.L. 101/2018) regulations.
No biological specimens will be collected.
Statistical methods for primary and secondary outcomes {20a} Description of the categorical variables will be made using frequency and percentage while mean and standard deviation (or median and interquartile range if variables are not normal) will be reported for continuous variables. Data will be analysed by intention-to-treat. A per-protocol analysis will be also performed. To evaluate the difference in the score of preoperative anxiety between intervention and control group, Wilcoxon-Mann–Whitney non-parametric test (or t -test, as necessary) will be applied. The same test will be calculated to evaluate the difference in the anxiety score between intervention and control group at T0 and T3. Wilcoxon-Mann–Whitney will be also used to establish if a difference in the children distress will be between intervention and control group. Chi-square test or exact Fisher test will be applied to evaluate difference in the family preparation for hospitalisation and surgery at T1 between group A and B. Interim analyses {21b} Interim analyses are not planned in this low-risk intervention consisting of providing information through a device. Methods for additional analyses (e.g. subgroup analyses) {20b} No other additional or subgroup analyses are planned in this study. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} The main analysis is performed as “intention to treat.” All randomised subjects with an available main outcome will be analysed in the group to which they were randomised, regardless of whether they received the assigned intervention. Reasons for dropping out of the study are recorded in a data file and described in detail. A multiple imputation technique will be used for missing data. Plans to give access to the full protocol, participant-level data, and statistical code {31c} The study protocol and data analysis will be available from the corresponding author on request.
Description of the categorical variables will be made using frequency and percentage while mean and standard deviation (or median and interquartile range if variables are not normal) will be reported for continuous variables. Data will be analysed by intention-to-treat. A per-protocol analysis will be also performed. To evaluate the difference in the score of preoperative anxiety between intervention and control group, Wilcoxon-Mann–Whitney non-parametric test (or t -test, as necessary) will be applied. The same test will be calculated to evaluate the difference in the anxiety score between intervention and control group at T0 and T3. Wilcoxon-Mann–Whitney will be also used to establish if a difference in the children distress will be between intervention and control group. Chi-square test or exact Fisher test will be applied to evaluate difference in the family preparation for hospitalisation and surgery at T1 between group A and B.
Interim analyses are not planned in this low-risk intervention consisting of providing information through a device.
No other additional or subgroup analyses are planned in this study.
The main analysis is performed as “intention to treat.” All randomised subjects with an available main outcome will be analysed in the group to which they were randomised, regardless of whether they received the assigned intervention. Reasons for dropping out of the study are recorded in a data file and described in detail. A multiple imputation technique will be used for missing data.
The study protocol and data analysis will be available from the corresponding author on request.
Composition of the coordinating centre and trial steering committee {5d} The IRCCS Burlo Garofolo is the coordinating centre and is responsible for providing day-to-day support to the surgical department staff and administrative nurses in organising the data collection and management of the study. The trial steering committee is composed of the study investigators and an epidemiologist from IRCCS Burlo Garofolo and a researcher from the Area Science Park. The members of the committee agreed on the final protocol and ensure that the study is conducted rigorously. They are responsible for all aspects of study management, from supervising participant enrolment to reviewing data quality. They have also trained the paediatric surgery nurses and administrative nurses in data collection. They monitor the overall conduct of the clinical trial and meet once a month to oversee the conduct and progress of the study. Composition of the data monitoring committee, its role and reporting structure {21a} Considering the low risks of the study, no external data monitoring committee was appointed. The trial steering committee monitor the conduct of the study and ensures that all phases of the trial are conducted with rigour. Adverse event reporting and harms {22} The study aims to evaluate a low-risk intervention, and no serious adverse events are expected because the intervention consists of providing selected information through a device. Potential risks due incorrect use of the app were not considered relevant to mention. Frequency and plans for auditing trial conduct {23} Considering the low-risk and brief intervention, no audits are planned for this study. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} If important protocol amendments are required, a request for approval of the changes will be forwarded to the regional ethics committee for approval. After approval, the new version of the protocol will be sent to ClinicalTrials.org. Finally, the entire research team will be informed of the changes at a meeting in a surgical department meeting room and through written information sent via institutional e-mail. Trial participants will be informed from the surgical ward nurses and study informed consent forms. Dissemination plans {31a} The results of the study will be disseminated to the scientific community through scientific publications and presentations at conferences. In addition, the results of the study will be shared with citizens through the hospital’s website and through the media and social media.
The IRCCS Burlo Garofolo is the coordinating centre and is responsible for providing day-to-day support to the surgical department staff and administrative nurses in organising the data collection and management of the study. The trial steering committee is composed of the study investigators and an epidemiologist from IRCCS Burlo Garofolo and a researcher from the Area Science Park. The members of the committee agreed on the final protocol and ensure that the study is conducted rigorously. They are responsible for all aspects of study management, from supervising participant enrolment to reviewing data quality. They have also trained the paediatric surgery nurses and administrative nurses in data collection. They monitor the overall conduct of the clinical trial and meet once a month to oversee the conduct and progress of the study.
Considering the low risks of the study, no external data monitoring committee was appointed. The trial steering committee monitor the conduct of the study and ensures that all phases of the trial are conducted with rigour.
The study aims to evaluate a low-risk intervention, and no serious adverse events are expected because the intervention consists of providing selected information through a device. Potential risks due incorrect use of the app were not considered relevant to mention.
Considering the low-risk and brief intervention, no audits are planned for this study.
If important protocol amendments are required, a request for approval of the changes will be forwarded to the regional ethics committee for approval. After approval, the new version of the protocol will be sent to ClinicalTrials.org. Finally, the entire research team will be informed of the changes at a meeting in a surgical department meeting room and through written information sent via institutional e-mail. Trial participants will be informed from the surgical ward nurses and study informed consent forms.
The results of the study will be disseminated to the scientific community through scientific publications and presentations at conferences. In addition, the results of the study will be shared with citizens through the hospital’s website and through the media and social media.
Tonsillectomies and adenotonsillectomies are characterised by a short hospital stay and little time for healthcare providers to inform and educate families in a family-centred manner. To our knowledge, no mobile health application has been tested in Italy to support children, their caregivers, and, consequently, their healthcare professionals in the ORL perioperative period. Therefore, if the developed app proves successful, it could be useful for other Italian children undergoing these procedures and their families. Furthermore, it can be later translated into other languages to be available and helpful for other countries or foreigners in Italy. Such a solution can introduce a new and safe management model in paediatric healthcare. It provides families with access to an easily consumable format with evidence-based health content as part of the ORL operation process, leading to continuity of care and empowerment of citizens in terms of positive health management and access to health services. In general, the app to be implemented aims to strengthen the offer of services to support collaboration between professionals and the interaction between professionals and citizens; to promote the empowerment of citizens in relation to health services; to propose and validate a new management model the health and social care. The presented solution can enable a family-centred and easily consumable format of the presented evidence-based content and introduce a new management model in paediatric healthcare and education.
Study protocol RC 03/2022. Recruitment started on 16 December 2022. Time for enrolment since starting: 1 year . The estimated end date of the study is December 31, 2023.
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International practice patterns of dyslipidemia management in patients with chronic kidney disease under nephrology care: is it time to review guideline recommendations?
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fceea79d-3f0d-45f6-bcd8-393dfa4aca76
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10210460
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Internal Medicine[mh]
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Patients with chronic kidney disease (CKD) have an extremely high cardiovascular disease (CVD) burden, which increases as CKD progresses. CKD may represent the kidney manifestation of the systemic impact of vascular disease under the influence of exposure to risk factors such as dyslipidemia. Despite the lack of evidence of benefit from lipid-lowering therapies (LLT) to reduce the progression of CKD, dyslipidemia is considered a modifiable CVD risk factor in this high-risk population , and LLT has been shown to reduce the risk of atherosclerotic cardiovascular events . Based on the analysis of the evidence specific to CKD patients , the 2012 Kidney Disease: Improving Global Outcomes (KDIGO) Clinical Practice Guideline for Lipid Management in CKD recommended conducting a lipid profile upon diagnosis for CKD to establish the diagnosis of severe hypercholesterolemia and/or hypertriglyceridemia and potentially rule out a remediable (secondary) cause if present. These global nephrology guidelines also recommend that all CKD patients ≥ 50 years, and high-risk younger adult patients, should be treated with a statin with or without ezetimibe regardless of lipid levels. In addition, the guidelines did not recommend a follow-up measurement of lipid levels for most patients. These recommendations were based on the results of several clinical studies but principally the SHARP trial results and two meta-analyses . By contrast, different cardiology society guidelines worldwide have provided LDL-cholesterol (LDL-C) targets for CKD patients according to CKD stages and cardiovascular (CV) patients’ risk, which range from < 55 mg/dL for those considered very high risk (CKD stage 4–5) moving to < 70 mg/dL for those at high risk (CKD 3a-3b) and rising to a maximum of 189 mg/dL for those CKD patients between 40–75 years old and with a 10-year atherosclerotic CVD risk of ≥ 7.5% . However, international variations in practice patterns and adherence to these guidelines have not been described until the present. To evaluate the implementation of these recommendations in real-world clinical practice, we aimed to assess current practice patterns for lipid management in an international cohort of non-dialysis CKD patients under nephrology care. Objectives include describing the prevalence and intensity of statin/ezetimibe prescription, achieved levels of LDL-C, and clinicians’ perceptions of LDL-C goals.
Aim, design, and study setting With this multinational cross-sectional analysis of baseline data from the Chronic Kidney Disease Outcomes and Practice Patterns Study (CKDopps), we aimed to demonstrate the variation between clinical practices regarding lipid management in CKD-non-dialysis patients, including prescription patterns, achieved LDL-C levels, and nephrologists’ targets for LDL-C. Data source The CKDopps is an ongoing prospective cohort study of Stage 3–5 CKD (eGFR < 60 ml/min) patients treated in nephrologist-led CKD clinics in Brazil, France, Germany, and the United States (US) (2013–2019). Unfortunately, data from Japan were unavailable at the time of this analysis. CKDopps sites were randomly selected from CKD clinics after stratification by region and clinic profile (academic vs. private). The criteria used for clinic selection regarding the geographic region, key clinic characteristics, inclusion and exclusion criteria, and study design, details, and objectives have previously been published . No clinical data were collected beyond those performed as part of usual care, as the aim is to evaluate standard nephrology clinic practices. One exception was laboratory measurements in France, where a standard set of urine and blood tests was requested at baseline, including lipids. CKDopps was approved by national and/or local ethics committees, and patient consent was obtained as required by local ethics regulations. Cardiovascular disease and lipids therapy stratification We categorized all cardiovascular diseases as either atherosclerotic CVD (ASCVD) or non-atherosclerotic CVD (referred to as “other CVD”, meaning CVD that is not atherosclerotic). ASCVD was defined by the following diagnoses/events and procedures: angina (stable or unstable), acute myocardial infarction, transient ischemic attack, claudication/rest pain, aortic aneurysm, stroke (ischemic), renal artery stenting and/or angioplasty, cardiac catheterization, coronary angioplasty, coronary bypass graft, carotid endarterectomy, angiogram, arterial bypass surgery, coronary angiogram, percutaneous transluminal angioplasty, and renal angioplasty and/or stenting. Other CVD was defined by the following diagnoses/events and procedures: cardiac arrest/sudden death, congestive heart failure, cardiomyopathy, valvular heart disease, atrial fibrillation, other arrhythmia, pericarditis and/or tamponade, deep vein thrombosis, tachycardia, pulmonary edema due to exogenous fluid, cerebral hemorrhage, ischemic brain damage/anoxic encephalopathy, hemorrhage from a ruptured vascular aneurysm, valve repair or replacement, aortic aneurysm repair, cardioversion, defibrillator placement, pacemaker placement, and pericardial procedure . The composite CV risk is based on comorbidity burden (any history of coronary disease, diabetes, or ischemic stroke) and age. In addition, LLT intensity was categorized into two categories: atorvastatin and rosuvastatin were classified as high intensity, and all other statins were categorized as low intensity: simvastatin, lovastatin, pravastatin, fluvastatin, cerivastatin, and pitavastatin. This classification was chosen due to the lack of statin doses in the CKDopps database. Thus, the definition suggested by most guidelines of considering statin doses to classify them as low vs. moderate vs. high intensity could not be applied here. Statistical analysis We reported the mean or percentages of patient characteristics at enrollment into CKDopps. These are presented for socio-demographics, laboratory values, dyslipidemia prescriptions, and comorbidities, all presented by CKD stage and country. For LDL-C levels, we also presented the results stratified based on a composite measure of CV risk: diabetes, any history of coronary disease, and ischemic stroke and further stratified by age < 50 versus age ≥ 50. The CV risk factors were based on some of the factors listed in the KDIGO recommendations regarding statin use among patients aged 18–49, such as a) known coronary disease (myocardial infarction or coronary revascularization), b) diabetes mellitus, c) prior ischemic stroke and, d) estimated 10-year incidence of coronary death or non-fatal myocardial infarction > 10% . We assessed country-level patterns of care for lipid management, including (a) prevalence and intensity of statin use (high intensity: atorvastatin and rosuvastatin; low intensity: all other statins), (b) frequency of lipid testing, (c) mean LDL-C levels during CKD progression, (d) the distribution of LDL-C by statin use, and (e) nephrologist-reported LDL-C goal upper limits. Models were adjusted for CV risk factor, CKD stage, country, sex, and age. Statins were classified as high intensity (atorvastatin or rosuvastatin) and low intensity (all other types). Linear and logistic regression models were used to obtain p values for comparisons of LDL-C levels and the prevalence of statin and/or ezetimibe treatments. In addition, comparisons were made between age groups (< or ≥ 50), countries, and CKD stages. Linear regression models were used on the mean LDL-C by treatment (including statin intensity), country, and CKD stage. Models used generalized estimating equations with an exchangeable working correlation structure to account for patient clustering by the clinic.
With this multinational cross-sectional analysis of baseline data from the Chronic Kidney Disease Outcomes and Practice Patterns Study (CKDopps), we aimed to demonstrate the variation between clinical practices regarding lipid management in CKD-non-dialysis patients, including prescription patterns, achieved LDL-C levels, and nephrologists’ targets for LDL-C.
The CKDopps is an ongoing prospective cohort study of Stage 3–5 CKD (eGFR < 60 ml/min) patients treated in nephrologist-led CKD clinics in Brazil, France, Germany, and the United States (US) (2013–2019). Unfortunately, data from Japan were unavailable at the time of this analysis. CKDopps sites were randomly selected from CKD clinics after stratification by region and clinic profile (academic vs. private). The criteria used for clinic selection regarding the geographic region, key clinic characteristics, inclusion and exclusion criteria, and study design, details, and objectives have previously been published . No clinical data were collected beyond those performed as part of usual care, as the aim is to evaluate standard nephrology clinic practices. One exception was laboratory measurements in France, where a standard set of urine and blood tests was requested at baseline, including lipids. CKDopps was approved by national and/or local ethics committees, and patient consent was obtained as required by local ethics regulations.
We categorized all cardiovascular diseases as either atherosclerotic CVD (ASCVD) or non-atherosclerotic CVD (referred to as “other CVD”, meaning CVD that is not atherosclerotic). ASCVD was defined by the following diagnoses/events and procedures: angina (stable or unstable), acute myocardial infarction, transient ischemic attack, claudication/rest pain, aortic aneurysm, stroke (ischemic), renal artery stenting and/or angioplasty, cardiac catheterization, coronary angioplasty, coronary bypass graft, carotid endarterectomy, angiogram, arterial bypass surgery, coronary angiogram, percutaneous transluminal angioplasty, and renal angioplasty and/or stenting. Other CVD was defined by the following diagnoses/events and procedures: cardiac arrest/sudden death, congestive heart failure, cardiomyopathy, valvular heart disease, atrial fibrillation, other arrhythmia, pericarditis and/or tamponade, deep vein thrombosis, tachycardia, pulmonary edema due to exogenous fluid, cerebral hemorrhage, ischemic brain damage/anoxic encephalopathy, hemorrhage from a ruptured vascular aneurysm, valve repair or replacement, aortic aneurysm repair, cardioversion, defibrillator placement, pacemaker placement, and pericardial procedure . The composite CV risk is based on comorbidity burden (any history of coronary disease, diabetes, or ischemic stroke) and age. In addition, LLT intensity was categorized into two categories: atorvastatin and rosuvastatin were classified as high intensity, and all other statins were categorized as low intensity: simvastatin, lovastatin, pravastatin, fluvastatin, cerivastatin, and pitavastatin. This classification was chosen due to the lack of statin doses in the CKDopps database. Thus, the definition suggested by most guidelines of considering statin doses to classify them as low vs. moderate vs. high intensity could not be applied here.
We reported the mean or percentages of patient characteristics at enrollment into CKDopps. These are presented for socio-demographics, laboratory values, dyslipidemia prescriptions, and comorbidities, all presented by CKD stage and country. For LDL-C levels, we also presented the results stratified based on a composite measure of CV risk: diabetes, any history of coronary disease, and ischemic stroke and further stratified by age < 50 versus age ≥ 50. The CV risk factors were based on some of the factors listed in the KDIGO recommendations regarding statin use among patients aged 18–49, such as a) known coronary disease (myocardial infarction or coronary revascularization), b) diabetes mellitus, c) prior ischemic stroke and, d) estimated 10-year incidence of coronary death or non-fatal myocardial infarction > 10% . We assessed country-level patterns of care for lipid management, including (a) prevalence and intensity of statin use (high intensity: atorvastatin and rosuvastatin; low intensity: all other statins), (b) frequency of lipid testing, (c) mean LDL-C levels during CKD progression, (d) the distribution of LDL-C by statin use, and (e) nephrologist-reported LDL-C goal upper limits. Models were adjusted for CV risk factor, CKD stage, country, sex, and age. Statins were classified as high intensity (atorvastatin or rosuvastatin) and low intensity (all other types). Linear and logistic regression models were used to obtain p values for comparisons of LDL-C levels and the prevalence of statin and/or ezetimibe treatments. In addition, comparisons were made between age groups (< or ≥ 50), countries, and CKD stages. Linear regression models were used on the mean LDL-C by treatment (including statin intensity), country, and CKD stage. Models used generalized estimating equations with an exchangeable working correlation structure to account for patient clustering by the clinic.
We analyzed 8,194 CKD patients in Brazil (912), France (2,969), Germany (2,761), and the US (1,552) (Table ). The patients were generally similar, but there was some variation by country. Patients from Brazil were usually younger, more often Black, and had more peripheral artery disease. Patients from France had less severe disease (more CKD stage 3 than stage 4), a slightly higher smoking prevalence, and were prescribed ezetimibe more often. Patients from Germany were generally older and had higher HDL-C levels, LDL-C levels, and prevalence of cardiovascular diseases. US patients had lower HDL-C, LDL-C, and hemoglobin levels and a higher prevalence of diabetes. Within each country, the percentage of CKD patients who were female, diabetic, or had high triglyceride levels was higher in stages 4/5 than in stage 3. In contrast, hemoglobin and LDL-C levels tended to be lower in stages 4/5. Overall statin use was similar by country and CKD stage, but the types of statins differed between countries (Fig. A). Among statin users, patients in France and the US used more high-intensity statins (39% and 30% overall) than patients in Brazil (9%) and Germany (4%). Patients with ASCVD, diabetes, or peripheral artery disease had slightly higher statin use within each country, but there was no consistent pattern in the use of high- versus low-intensity statins between patients with versus without these comorbidities (Supplemental Fig. ; see file Supplementary Fig. ). Statin use was higher among patients over the age of 50 compared to patients under the age of 50. Within these age groups, statin use was higher among patients with CV comorbidities or diabetes (Fig. B). Outside Germany, where high-intensity statin use was rare, high-intensity statin use was also generally higher among patients aged 50 + and with CV comorbidities. The upper limit of fasting LDL-C goals by nephrologists varied from country to country (Fig. ). Consistent with the higher LDL-C levels found among their patients, 50–53% (depending on CKD stage) of the German nephrologists surveyed specified an upper LDL-C level of 130 or 160 mg/dL, compared to 13–18% of US nephrologists, 27–33% of French nephrologists, and 41–47% of Brazilian nephrologists. No US nephrologists specified an upper LDL-C limit of 160; only 3–6% of Brazilian and French nephrologists did, while 15–21% of German nephrologists selected this high level. Only 7–17% of nephrologists believed that LDL-C should be < 70 mg/dL, and 38- 68% would choose LDL < 100 mg/dl as a threshold. Within each country, the adjusted difference in mean LDL-C between high- and low-intensity statin categories never exceeded 5.5 mg/dL and was statistically significant only in France. In contrast, the combined high and low categories had an adjusted mean LDL-C that was always significantly lower than the “no statin use” category: -7.6 mg/dL for Brazil ( p -value = 0.019), -25.8 for France ( p -value < 0.001), -19.9 for Germany ( p -value < 0.001), and -21.2 for the US ( p -value < 0.001) (Supplemental Fig. ; see file Supplementary Fig. ). The statin intensity-by-country interaction p-value (6 degrees of freedom) was 0.015, implying that mean LDL-C may vary by country and statin intensity and that the effect of statin intensity may differ across the countries. In each country, LDL-C levels were higher among patients not treated with statins (Fig. ). There was no consistent trend in LDL-C across patients with different eGFR levels (Fig. and Supplemental Fig. ; see file Supplementary Fig. ). German patients had higher LDL-C levels, and US patients had lower LDL-C levels across all eGFR levels independent of serum albumin levels. These observations did not vary by the status of comorbidities such as ASCVD, diabetes, or peripheral artery disease (Supplemental Fig. ; see file Supplementary Fig. ).
In the present study, although there were no major differences in demographic and clinic characteristics between countries, there was a substantial variation in dyslipidemia management across geographies. A similar pattern of LLT underutilization across countries and CKD stages was observed, and different statins (high vs. low intensity) were prescribed in each country. In this group of CKD patients under nephrology care, LDL-C was lower among treated patients and differed significantly by country, with the highest LDL-C levels detected in Germany and the lowest in the US. Also, statin use was higher among patients over 50 and in patients with CV comorbidities or diabetes. These findings reflect a low adherence to evidence-based recommendations in real-world nephrological practice. It is important to mention that, in part, this low-adherence may be a result of the differences in access to treatment due to insurance coverage of medications, particularly in the US. At the patient level, LDL-C did not vary significantly by CKD stage, regardless of statin use. LDL-C levels were also consistently higher for non-statin users in each country (+ 22 mg/dL overall in adjusted models), while low-intensity statin users had higher LDL-C levels than high-intensity statin users (+ 4 mg/dL overall in adjusted models). Different results were also demonstrated in the literature. The Pravastatin Pooling Project demonstrated pravastatin, in our study considered as a low-intensity statin, reduced LDL-C levels by 47.9 ± 24.1 mg/dL and triglyceride levels by 17.3 ± 56.3 mg/dL and raised HDL cholesterol by 2.3 ± 6.0 mg/dL at 12 months . On the other hand, in the Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin (JUPITER), rosuvastatin (high-intensity statin) reduced LDL-C by 52% . It is important to remember that patients with moderate to advanced CKD present a mixed dyslipidemia pattern, with a combination of hypertriglyceridemia, low levels of HDL cholesterol, and variable levels of LDL cholesterol and total cholesterol . In general, the progression of CKD to later stages impacts the lipid profile’s composition, resulting in a more atherogenic profile . Because of the combination of this atherogenic dyslipidemia profile with multiple comorbidities and extremely high CV risk, cardiology guidelines tend to recommend an aggressive and inclusive treatment regimen with statins in patients with CKD not on dialysis. However, all dialysis studies failed to demonstrate a significant reduction in cardiovascular events or mortality with LLT despite significant LDL cholesterol lowering, regardless of different treatment strategies applied . In contrast, the overall and non-dialysis subgroup analysis of the SHARP trial supports the aggressive treatment for CKD non-dialysis patients to reduce CV events. Recently, the International Study of Comparative Health Effectiveness with Medical and Invasive Approaches (ISCHEMIA) and International Study of Comparative Health Effectiveness with Medical and Invasive Approaches—Chronic Kidney Disease (ISCHEMIA-CKD) trials found similar effects in reducing all-cause death or myocardial infarction between initial invasive management compared to initial conservative management, which includes aggressive LDL-C therapies as a standard of care, of patients with chronic coronary disease and moderate to severe ischemia on stress testing without or with advanced CKD . Also, a post-hoc analysis of the Aggressive Lipid-Lowering Initiation Abates New Cardiac Events (ALLIANCE) Study demonstrated that in patients with coronary heart disease and CKD, intensive LLT with higher doses of atorvastatin to achieve a target < 80 mg/dL reduced the relative risk of time to the first cardiovascular event by 28% in patients with CKD (HR, 0.72; 95% CI, 0.54 to 0.97; P = 0.02) and 11% in patients without CKD (HR, 0.89; 95% CI, 0.74 to 1.07; P = 0.3) . In combination, these studies provide additional evidence to the potential benefits of LLT in CKD patients and raises the question: should nephrologists adopt the guidelines from other areas and redefine triggers and targets for LLT? On the other hand, the CKD-REIN study found that lipid goal achievement was not associated with risk of fatal/non-fatal atheromatous CVD or non-atheromatous CVD in patients with non-dialysis CKD . Safety considerations may also temper the use of intensive/high-dose LLT in patients with CKD, as statin-related toxicity is dose-related. An observational study in the general population demonstrated that in comparison with atorvastatin, rosuvastatin was associated with an increased risk of hematuria (HR, 1.08; 95% confidence interval [95% CI], 1.04 to 1.11), proteinuria (HR, 1.17; 95% CI, 1.10 to 1.25), and kidney failure with replacement therapy (HR, 1.15; 95% CI, 1.02 to 1.30) . The risk was higher with a higher rosuvastatin dose . Among patients with eGFR < 30 ml/min per 1.73 m 2 , 44% were prescribed daily high dose rosuvastatin (20 or 40 mg daily), which exceeds the FDA’s recommended 10 mg daily dose. These findings suggest the need for caution in prescribing and monitoring rosuvastatin, especially in patients receiving high doses or who have severe CKD. In this case, the difference in doses of statin LLT seems to influence patient outcomes . Another important point to be considered regarding safety, is the potential statins have of inducing rhabdomyolysis , because in patients with CKD it is important to avoid measures that could exacerbate kidney disfunction. Individualized treatment is a keystone to minimize those safety issues and improve outcomes in our patients. Our study demonstrated that a substantial proportion of hyperlipidemic CKD patients are not receiving LLT, and 7–23% of untreated patients in each country had LDL-C ≥ 160 mg/dL. The kidney-focused KDIGO guidelines recommend no lipid goals or follow-up lipid testing, instead espousing a ‘fire-and-forget’ strategy of fixed-dose, moderate-intensity statin, or statin-ezetimibe therapy . In contrast, the American Heart Association/American College of Cardiology (AHA/ACC) guidelines utilize LDL-based and ASCVD-risk-based thresholds for statin therapy among CKD patients but also do not stipulate treatment goals; they recommend a moderate-intensity statin alone or combined with ezetimibe for adults 40–75 years of age with LDL-C 70–189 mg/dL (1.7–4.8 mmol/L) who are at 10-year atherosclerotic CVD risk of ≥ 7.5% . Conversely, European and UK guidelines recommends treat-to-goals strategies. For example, the European Society of Cardiology/European Atherosclerosis Society (ESC/EAS) guidelines distinguish patients with G3a-3b as high risk, and eGFR G4-5ND as very high risk, with a treatment goal of ≥ 50% LDL-C reduction from baseline and an LDL-C goal of < 1.8 mmol/L (< 70 mg/dL) for high-risk patients, and a goal of < 1.4 mmol/L (< 55 mg/dL) for very high-risk patients . The UK Renal Association guidelines recommend goals for total cholesterol (≤ 4 mmol/L), LDL (≤ 2 mmol/L), and non-HDL (≤ 2.5 mmol/L) . Our CKDopps nephrologist survey demonstrated the most common LDL-C goal was < 100 mg/dL across regions, regardless of CKD stage, but only 8–19% of nephrologists would aim for < 70 mg/dL for G4-5 patients, as per the current ESC guidelines for ‘very high risk’ patients. LDL-C levels did not appear to vary with eGFR in our analysis. The CKD-REIN study previously demonstrated that only 45% of ‘high-risk’ patients and 29–38% of patients at ‘very high risk’ achieved the LDL-C goal, based on the 2016 ESC/EAS guidelines, which recommended less aggressive targets than the current guidelines . In the current study, overall statin use ranged from 51–61% of patients in countries included in this analysis. These findings are similar to a recent Canadian cross-sectional study of CKD patients, in which approximately 63% of statin-eligible patients were taking a statin . This study also demonstrated CKD patients had about five times the odds of receiving statin therapy for secondary vs. primary prevention. Patients planning for conservative care had lower odds of being prescribed a statin than patients planning for dialysis . There are several possible reasons why nephrologists may not prescribe statins for CKD patients, particularly for primary prevention. Respondents to a Canadian nephrologist survey cited some reasons, including disagreement with KDIGO guidelines in favor of a patient-individualized approach that considers life expectancy and the cause of CKD . For example, for older patients with limited life expectancy, some nephrologists were concerned with the lack of evidence of benefit with statins, higher risk of adverse effects, and increased pill burden. The AHA/ACC does indicate it may be reasonable to stop statin therapy among patients > 75 years of age who have functional decline, multimorbidity, frailty, or reduced life expectancy . While the SHARP trial did show evidence of a benefit for statin plus ezetimibe among the subgroup of CKD patients > 70 years of age (risk ratio 0.78, 95% CI 0.65–0.89) , there remains uncertainty about the benefit-risk ratio of statin use for primary prevention in people > 70 years of age in the general population. Our study has some limitations. First, there is some heterogeneity in study protocols. For example, in France, lab data were drawn according to a country-specific protocol and, therefore, not included in the analysis of lipid monitoring which may compromise the results’ generalizability. Another limitation of this study is the absence of statin dose data, which made necessary an adjustment on the statin intensity classification compared to what is suggested by the guidelines. On the other hand, this study’s strengths are that this is a large, international cohort in academic and community settings and, therefore, may reflect the real-world dyslipidemia management in CKD patients under nephrology care. In conclusion, there is substantial variation in practice patterns regarding lipid-lowering therapies across countries but not across CKD stages. Patients on LLT have lower LDL-C, yet in contrast to the evidence-based guideline recommendations, a significant proportion of CKD patients with dyslipidemia managed by nephrologists are not receiving LLT. Despite the literature supporting LLT in the CKD population with care in dose prescription, our study suggests that the nephrology community needs to review the recommendations and improve the implementation of these guidelines in clinical practice, which may lead to a reduction in the burden of CVD in the CKD population. Further comparative effectiveness studies assessing the effects of a ‘fire-and-forget’ strategy vs. a treat-to-target strategy on patient outcomes are needed to inform the optimal approach to lipid management in patients with non-dialysis CKD.
Additional file 1: Supplementary Figure 1. Prevalence and intensity of statin use by country and other patient strata. Atorvastatin and rosuvastatin are categorized as high intensity; all other statins are categorized as low intensity: simvastatin, lovastatin, pravastatin, fluvastatin, cerivastatin, and pitavastatin. Additional file 2: Supplementary Figure 2. Mean LDL-C during CKD progression, by country and other patient strata. Additional file 3: Supplementary Figure 3. Distribution of LDL-C (mg/dL) by statin use and by other patient strata. Additional file 4: Supplementary Figure 4. Adjusted mean LDL-C (mg/dL) by country and statin use. Legend: LDL-C levels adjusted to average age, sex, CKD stage, and comorbid risk status through a linear regression model.
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Insights into artificial intelligence in myopia management: from a data perspective
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a5fd25c1-7af6-47ad-8076-25fc471c0a3d
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10212230
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Ophthalmology[mh]
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Myopia, which is defined as a spherical equivalent (SE) ≤ − 0.5 diopters (D), is a substantial global health issue. The global prevalence of myopia is estimated to be 49.8% (4.758 billion) of the world’s population, of which 9.8% (938 million) will suffer from high myopia by 2050 . In some Asian countries, more than 80% of high school students are myopic , and a greater proportion of young individuals develop high myopia (spherical equivalent (SE) ≤ − 6.0 diopters) , which further results in a higher risk of developing visually impairing and blinding complications . Although the causes of this pandemic remain unknown, strategies for coping with the myopia pandemic such as early identification, regular follow-up, and timely intervention of high-risk groups of myopia, are essential and are gaining more social attention . In recent years, artificial intelligence has been advancing at an unprecedented rate, showing great potential for the automated analysis of medical information and images. In the field of ophthalmology, due to the wide application of various imaging technologies in eye diseases, many studies have applied AI methods to different ophthalmology diseases, such as diabetic retinopathy (DR) , age-related macular degeneration (AMD) , cataract , dry eye syndrome (DES) , and glaucoma . For myopia, research efforts are still relatively insufficient compared to other subspecialties, even though AI has shown the potential to address urgent needs in the field of myopia. The clinical tasks of managing myopia include early screening, risk stratification, progression prediction, timely and individualized intervention, and ongoing management . Relevant data modalities produced during the process can be classified into two categories: clinical data and imaging data. As a concept in AI, machine learning (ML) is deeply entwined with statistics and is powerful for working with numerical or categorical data . Commonly used ML techniques in myopia include support vector machine (SVM), linear regression, random forest (RF), naive Bayes, k-nearest neighbor (KNN), and extreme gradient boosting (XGBoost) . As a subset of ML, deep learning (DL) has performed well in many image-based applications, such as object recognition and semantic segmentation . Convolutional neural networks (CNNs) are the foundation of image-driven applications in myopia and the use of recurrent neural networks (RNNs) is still at an early stage. The abundant datasets with adjunctive AI analysis have led to improvements in myopia management. As an emerging research field, there are currently only few reviews summarizing the application scenarios of AI in myopia , and none has focused on the data modalities available and the AI methods appropriate for each type of data, which is insufficient as data continue to grow in variety and quantity. Therefore, in this review, we examine how AI methods have been applied in analyzing different data modalities generated from clinical practice in myopia. We conducted a comprehensive literature review using two databases (i.e., PubMed and IEEE Xplore) in August 2022 and March 2023. Our search terms included a combination of relevant keywords, such as “myopia” and “artificial intelligence” and Boolean operators to ensure a comprehensive search. We also reviewed the reference lists of relevant articles to identify additional studies that may have been missed in our initial search. Our review is focused on the use of AI in the risk identification, screening, detection, classification, and treatment of myopia. Accordingly, we considered research articles that utilized AI for these purposes to be appropriate for inclusion in our review and have incorporated relevant studies in this article. When facing a patient with myopia in the clinic, clinician considerations usually follow the sequence of risk factor identification, the examination process, selection of treatment strategies, and ongoing management , as shown in Fig. . Myopia has been traditionally viewed as a consequence of the sophisticated interaction of lifestyle, genetics, and environmental factors. Therefore, detailed history taking is routinely conducted at the very beginning, and risk factors for a given individual are identified. Then, simple clinical tests such as cycloplegic and/or noncycloplegic refraction, best-corrected visual acuity, binocular vision and accommodative tests, anterior eye health evaluation, and corneal topography are taken for all visits. Measurement of axial length is optional, and currently, there is no established standard for normal or accelerated axial elongation. For patients who need further examination, especially those with a high degree of refraction, fundus imaging and examination are performed if indicated. After all the examinations, for patients who may result in low vision and blindness, selection of treatment strategies should be considered and in an individual way. For those possessing multiple risk factors, it will be helpful to predict the prognosis and carry out regular follow-up. During the whole process, a large amount of meaningful data is generated. Since the key components of developing an AI application can be concluded as “MDT,” that is “Model, Data and Target”, the abundant datasets make it possible for AI to assist in many tasks of myopia management based on each type of data modality. At nearly all steps of the clinical practice mentioned above, a considerable amount of clinical data is generated. These data can include basic ophthalmologic information, such as cycloplegic and noncycloplegic refraction, axial length (AL), corneal curvature radius (CR), best-corrected visual acuity (BCVA), and intraocular pressure (IOP); behavioral and environmental data, such as eye habits, reading distance, illumination conditions, and outdoor activity; and personal information related to diseases, such as demographics, heredity, and psychological state. All of the basic ophthalmologic information is numerical data, which is a data type expressed in quantitative numbers. For others, most of them are categorical data, a collection of information that is divided into groups and can take on numerical values, although meaningless. In brief, all of these clinical data can be expressed in a numeric form, which is different from imaging data generated from fundus examination. Considering the size, especially for a complex disease such as myopia where numerous codependent factors are involved in the causes, epidemiology, diagnostics, and progression, it is almost impossible to manually analyze the clinical data. Therefore, ML methods, with the capability of handling large amounts of data in a nonlinear way and extracting large numbers of potential predictive parameters, even when it outnumbers observations , are suitable for applications in myopia (Table ). Applications based on this type of data mainly include prognosis prediction, refractive surgery assistance, and remote monitoring. Prognosis prediction By constructing risk models with various variables in this data modality, many studies have determined the capability of ML methods for prognosis prediction. The random forest model is shown to predict the onset of high myopia at 18 years of age as early as 8 years in advance at a clinically acceptable accuracy by using long-term refraction data . Comprehensively assessing the physiological elongation of axial length, a key indicator for high myopia, by SVM and GBRT, instead of mydriatic optometry can be used to predict myopia progression . Additionally, the probability produced by these models can help persuade patients for further referral . Refractive surgery ML methods have been applied to eye parameters obtained from advanced instruments to screen candidates for refractive surgery , detect corneal ectasia susceptibility , and distinguish healthy corneas from diseased one . One of the earliest studies on AI applications in myopia focused on using ML methods and data collected by the GALILEI Dual Scheimpflug Analyzer to automate the detection of subclinical keratoconus, which is a contraindication for refractive surgery . Additionally, different ML models trained on medical records of myopia patients have been reported to improve the accuracy of intraocular lens (IOLs) power selection, which is crucial for reducing postoperative refraction errors, especially in highly myopic eyes that have undergone cataract surgery. The integration of AI into IOL power calculation formulas, such as Hill-RBF3.0 and Kane, has shown to produce more accurate prediction results compared to traditional formulas, including Barrett Universal II, Haigis, and SRK/T . Monitoring Environmental risk factors, such as working at a close range (< 20 cm) and excessive continuous close working time (> 30 min), are considered to be factors relevant to the development of myopia, and increasing effective outdoor exposure time is an independent protective factor against myopia. However, it is difficult to monitor them widely in the public. Various smart wearable devices have been developed for monitoring working distance or outdoor exposure time, such as RangeLife , FitSight , and Cloud clips . Through the data collected by wearable devices, an SVM model has been trained for distinguishing indoor and outdoor locations . It may further combine with Internet apps, encouraging children to spend more time outdoors . By constructing risk models with various variables in this data modality, many studies have determined the capability of ML methods for prognosis prediction. The random forest model is shown to predict the onset of high myopia at 18 years of age as early as 8 years in advance at a clinically acceptable accuracy by using long-term refraction data . Comprehensively assessing the physiological elongation of axial length, a key indicator for high myopia, by SVM and GBRT, instead of mydriatic optometry can be used to predict myopia progression . Additionally, the probability produced by these models can help persuade patients for further referral . ML methods have been applied to eye parameters obtained from advanced instruments to screen candidates for refractive surgery , detect corneal ectasia susceptibility , and distinguish healthy corneas from diseased one . One of the earliest studies on AI applications in myopia focused on using ML methods and data collected by the GALILEI Dual Scheimpflug Analyzer to automate the detection of subclinical keratoconus, which is a contraindication for refractive surgery . Additionally, different ML models trained on medical records of myopia patients have been reported to improve the accuracy of intraocular lens (IOLs) power selection, which is crucial for reducing postoperative refraction errors, especially in highly myopic eyes that have undergone cataract surgery. The integration of AI into IOL power calculation formulas, such as Hill-RBF3.0 and Kane, has shown to produce more accurate prediction results compared to traditional formulas, including Barrett Universal II, Haigis, and SRK/T . Environmental risk factors, such as working at a close range (< 20 cm) and excessive continuous close working time (> 30 min), are considered to be factors relevant to the development of myopia, and increasing effective outdoor exposure time is an independent protective factor against myopia. However, it is difficult to monitor them widely in the public. Various smart wearable devices have been developed for monitoring working distance or outdoor exposure time, such as RangeLife , FitSight , and Cloud clips . Through the data collected by wearable devices, an SVM model has been trained for distinguishing indoor and outdoor locations . It may further combine with Internet apps, encouraging children to spend more time outdoors . Fundus examination, which is recommended annually in high myopes, provides a visualization of both the central and peripheral retina under dilation and generates a considerable amount of imaging data. Among the different imaging methods, fundus photography (FP) and optical coherence tomography (OCT) are most commonly used for the assessment of myopia-related fundus changes. By fundus examination, optic disc tilt and arc-shaped spots can be found in simple high myopia. For the fundus of pathologic myopia, a severe form of high myopia, posterior staphyloma, myopic traction maculopathy (MTM), myopic choroidal neovascularization (mCNV), dome-shape macula (DSM), and high myopia-related optic neuropathy can be seen, with a high specificity. These pathologic changes usually lead to irreversible damage to the retina, choroid, and other tissues, which will seriously affect the visual function of patients, but may present insidiously. Thus, timely imaging as well as accurate interpretation with the help of AI is important in detecting early complications and monitoring progression . Fundus photography-based applications FP is routinely ordered in a wide variety of ophthalmic conditions . It documents the retina, macula, optic nerve, and main retinal blood vessels in our eyes by using a highly specialized camera with high-powered lenses designed to visualize the pattern of the back of the eye . It is often referred to as retinal fundus photography (RFP), highlighting the fact that an ophthalmologist’s primary goal is typically to identify the appearance of the retina. Based on this data modality, several studies have reported the use of machine learning methods for myopia and associated complications, as shown in Table . Unlike prognosis prediction, which is based on clinical data, the prediction task based on FP images mainly aims at predicting refractive error with ResNet , a famous CNN model used for feature extraction, which is surprising given that this was not a task thought to be possible manually. This might be useful for studying possible morphological changes in myopic eyes and can also help in epidemiologic research of myopia from large fundus image datasets where refraction labels are unavailable. In addition, fundus images can be investigated by fully convolutional networks (FCNs), a model modified from CNNs, and the semantic segmentation, or pixelwise classification, of these myopia-related fundus changes is possible . For different types of myopic maculopathy, CNN-based models have been exploited to perform the classification task according to the META-PM classification system . In addition to private datasets, the utilization of a public dataset for training UNet + + to detect pathologic myopia and highlight the areas of lesions is also possible . Traditional fundus cameras only capture images at an angle of 30 to 60° . Therefore, combined with UWF imaging techniques, a novel form of FP, namely UWF fundus images (UWF-FP), enables ophthalmologists to observe the peripheral retina without pupillary dilation , with up to a 200° view of the ocular fundus in a single exposure. The employment of artificial intelligence in this data modality has achieved promising results, such as detecting glaucomatous optic neuropathy , identifying lattice degeneration , and even screening anemia . For myopia, UWF-FP enables ophthalmologists to screen notable peripheral retinal lesions (NPRLs), the clinically significant peripheral retinal lesions that are more frequently seen in myopic eyes than normal eyes . If kept untreated, patients with NPRLs will likely result in rhegmatogenous retinal detachment (RRD), an important cause of visual loss . Based on the peripheral retinal information provided by UWF-FP, a customized CNN network has achieved satisfying accuracy in automatically identifying the NPRLs. Similar to FP, UWF-FP can also be used by CNN-based models to predict refractive error, with an MAE of predicted spherical equivalent (SE) equal to 1.1150D . Although surprisingly, this accuracy is inferior to the result of Varadarajan et al. , which is based on FP. This might result from the difference in the quality and quantity of the training and validation datasets. Further comparison of the performance of AI applications between FP and UWF-FP is needed. Optical coherence tomography-based applications To analyze the fundus changes associated with myopia, OCT is another widely used method. It is carried out for detecting myopia-related vision-threatening conditions, such as retinal detachment, pathological mCNV, macular hole, and retinoschisis . The characteristics of OCT enable ophthalmologists to see a myriad of pathologies in the anterior and posterior segments of myopic eyes, including the cornea, sclera, anterior chamber, vitreous, choroid, retina, and optic nerve, which can only be seen in enucleated eyes before . Based on OCT, deep learning has been extensively studied for the detection of AMD and glaucoma . Regarding myopia and associated complications, 12 studies reported the use of CNN-based models (Table ). It can help ophthalmologists identify myopic maculopathy in patients with high myopia and the presence of pathologic myopia . Four vision-threatening conditions associated with myopia can also be automatically detected with InceptionResNetV2 . Since OCT images contain layer information, which is its unique characteristic, studies have demonstrated the potential for segmenting and analyzing the choroidal sublayers by using U-Net and mask R-CNN , and further utilization of this in myopia is expected. It can also be helpful in automatic screening for high myopia and estimating uncorrected refractive error . Apart from applications that produce actual outputs, there are other ways to use AI methods in myopia. The ATN classification and grading system is a widely applicable clinical diagnostic criterion for myopic maculopathy . While atrophy (A) can be judged based on FP only, determining the categories of traction (T) and neovascularization (N) requires FP together with OCT images. Apparently, OCT examination is much more difficult to adopt than FP. Therefore, a study built a multibranch ResNet with FP and OCT images to achieve ATN grades based on FP only, and the performance was superior to that of ophthalmologists who are not retinal specialists . FP is routinely ordered in a wide variety of ophthalmic conditions . It documents the retina, macula, optic nerve, and main retinal blood vessels in our eyes by using a highly specialized camera with high-powered lenses designed to visualize the pattern of the back of the eye . It is often referred to as retinal fundus photography (RFP), highlighting the fact that an ophthalmologist’s primary goal is typically to identify the appearance of the retina. Based on this data modality, several studies have reported the use of machine learning methods for myopia and associated complications, as shown in Table . Unlike prognosis prediction, which is based on clinical data, the prediction task based on FP images mainly aims at predicting refractive error with ResNet , a famous CNN model used for feature extraction, which is surprising given that this was not a task thought to be possible manually. This might be useful for studying possible morphological changes in myopic eyes and can also help in epidemiologic research of myopia from large fundus image datasets where refraction labels are unavailable. In addition, fundus images can be investigated by fully convolutional networks (FCNs), a model modified from CNNs, and the semantic segmentation, or pixelwise classification, of these myopia-related fundus changes is possible . For different types of myopic maculopathy, CNN-based models have been exploited to perform the classification task according to the META-PM classification system . In addition to private datasets, the utilization of a public dataset for training UNet + + to detect pathologic myopia and highlight the areas of lesions is also possible . Traditional fundus cameras only capture images at an angle of 30 to 60° . Therefore, combined with UWF imaging techniques, a novel form of FP, namely UWF fundus images (UWF-FP), enables ophthalmologists to observe the peripheral retina without pupillary dilation , with up to a 200° view of the ocular fundus in a single exposure. The employment of artificial intelligence in this data modality has achieved promising results, such as detecting glaucomatous optic neuropathy , identifying lattice degeneration , and even screening anemia . For myopia, UWF-FP enables ophthalmologists to screen notable peripheral retinal lesions (NPRLs), the clinically significant peripheral retinal lesions that are more frequently seen in myopic eyes than normal eyes . If kept untreated, patients with NPRLs will likely result in rhegmatogenous retinal detachment (RRD), an important cause of visual loss . Based on the peripheral retinal information provided by UWF-FP, a customized CNN network has achieved satisfying accuracy in automatically identifying the NPRLs. Similar to FP, UWF-FP can also be used by CNN-based models to predict refractive error, with an MAE of predicted spherical equivalent (SE) equal to 1.1150D . Although surprisingly, this accuracy is inferior to the result of Varadarajan et al. , which is based on FP. This might result from the difference in the quality and quantity of the training and validation datasets. Further comparison of the performance of AI applications between FP and UWF-FP is needed. To analyze the fundus changes associated with myopia, OCT is another widely used method. It is carried out for detecting myopia-related vision-threatening conditions, such as retinal detachment, pathological mCNV, macular hole, and retinoschisis . The characteristics of OCT enable ophthalmologists to see a myriad of pathologies in the anterior and posterior segments of myopic eyes, including the cornea, sclera, anterior chamber, vitreous, choroid, retina, and optic nerve, which can only be seen in enucleated eyes before . Based on OCT, deep learning has been extensively studied for the detection of AMD and glaucoma . Regarding myopia and associated complications, 12 studies reported the use of CNN-based models (Table ). It can help ophthalmologists identify myopic maculopathy in patients with high myopia and the presence of pathologic myopia . Four vision-threatening conditions associated with myopia can also be automatically detected with InceptionResNetV2 . Since OCT images contain layer information, which is its unique characteristic, studies have demonstrated the potential for segmenting and analyzing the choroidal sublayers by using U-Net and mask R-CNN , and further utilization of this in myopia is expected. It can also be helpful in automatic screening for high myopia and estimating uncorrected refractive error . Apart from applications that produce actual outputs, there are other ways to use AI methods in myopia. The ATN classification and grading system is a widely applicable clinical diagnostic criterion for myopic maculopathy . While atrophy (A) can be judged based on FP only, determining the categories of traction (T) and neovascularization (N) requires FP together with OCT images. Apparently, OCT examination is much more difficult to adopt than FP. Therefore, a study built a multibranch ResNet with FP and OCT images to achieve ATN grades based on FP only, and the performance was superior to that of ophthalmologists who are not retinal specialists . Despite the reported effective implementation of AI in the clinical practice of myopia, problems and roadblocks remain. Prior to the general adoption of AI, critical technical and clinical restrictions must be overcome. Establishment of a solid data foundation The quality and quantity of data are extremely important to the applications of AI. The majority of the aforementioned AI applications in myopia use datasets collected by ophthalmologists during their clinical practice, which are usually on one or a few population groups exclusively. This might result in poor generalizability and makes it difficult to determine whether the poor performance is attributable to spectrum bias . The disparity of imaging systems, discrepancy in imaging and postprocessing protocols, and lack of computing power also hinder the implementation of these algorithms into clinical practice. People who are in low-resource environments are frequently undercounted because it can be challenging to obtain medical attention and thus capture their data . In this sense, public ophthalmological datasets are essential and provide an equal platform for comparing the outcomes of AI models in ophthalmology. There are some popular public datasets established by ophthalmologists from multiple centers for other ophthalmopathies, but none of them focus on myopia alone . AI research in myopia might consider the feasibility of utilizing these public datasets in the future. The establishment of a large-scale public myopia dataset is also possible with novel AI technologies. A generative adversarial network (GAN) can be used in the generation of a large number of random and diverse images, and You et al. determined its application on FP and OCT images in ophthalmology, offering a new way to enlarge datasets. Federated learning and swarm learning have emerged as potential methods to cope with privacy problems, providing a decentralized and secure method of data management. Handle multitasks with multimodal data The complexity of clinical manifestations in diseases and the diversity of data obtained through different examination modalities present a challenge in AI applications. In myopia, current AI models are typically designed for specific data modalities and purposes, resulting in high accuracy in distinguishing between “disease-free” and “diseased” cases, but poor performance in more complex tasks such as distinguishing between multiple diseases . This challenge arises from the fact that some pathological changes in myopia can also occur in other ophthalmic conditions. One approach to address this is to exclude patients with comorbidities or group together a range of diseases. Alternatively, multimodal medical data fusion techniques can be employed by extracting relevant features from images and processing them with AI algorithms . These features are not limited to geometric measurements but may also include characteristic lesion regions . Additionally, image data can be processed to obtain a virtual score, which can be predicted together with clinical data by AI algorithms . While new developments in this area continue to emerge, it is important to note that multimodal data do not consistently outperform unimodal data, as demonstrated in a study on diabetic retinopathy staging . Explore the potential of novel data modalities Novel forms of OCT images, such as UWF optical coherence tomography (UWF OCT) and OCT angiography (OCTA), can be seen in the clinical practice of myopia. UWF OCT, instead of the traditionally used 3D-MRI, can be helpful for better visualization of the posterior staphyloma in myopic eyes , which is “an outpouching of the wall of the eye with a radius of curvature less than the radius of curvature of the surrounding eye wall” and usually results in poorer vision and more anatomical anomalies . OCTA is very helpful for detecting retinal microvasculature in a noninvasive and depth-resolved way , thus providing a way of detecting mCNV with high sensitivity and specificity. Based on UWF OCT and OCTA, AI research is still restricted to improving the quality of images, such as image reconstruction and denoising , and there is currently a dearth of research exploiting the possibility of developing DL models to detect posterior staphyloma or mCNV. The quality and quantity of data are extremely important to the applications of AI. The majority of the aforementioned AI applications in myopia use datasets collected by ophthalmologists during their clinical practice, which are usually on one or a few population groups exclusively. This might result in poor generalizability and makes it difficult to determine whether the poor performance is attributable to spectrum bias . The disparity of imaging systems, discrepancy in imaging and postprocessing protocols, and lack of computing power also hinder the implementation of these algorithms into clinical practice. People who are in low-resource environments are frequently undercounted because it can be challenging to obtain medical attention and thus capture their data . In this sense, public ophthalmological datasets are essential and provide an equal platform for comparing the outcomes of AI models in ophthalmology. There are some popular public datasets established by ophthalmologists from multiple centers for other ophthalmopathies, but none of them focus on myopia alone . AI research in myopia might consider the feasibility of utilizing these public datasets in the future. The establishment of a large-scale public myopia dataset is also possible with novel AI technologies. A generative adversarial network (GAN) can be used in the generation of a large number of random and diverse images, and You et al. determined its application on FP and OCT images in ophthalmology, offering a new way to enlarge datasets. Federated learning and swarm learning have emerged as potential methods to cope with privacy problems, providing a decentralized and secure method of data management. The complexity of clinical manifestations in diseases and the diversity of data obtained through different examination modalities present a challenge in AI applications. In myopia, current AI models are typically designed for specific data modalities and purposes, resulting in high accuracy in distinguishing between “disease-free” and “diseased” cases, but poor performance in more complex tasks such as distinguishing between multiple diseases . This challenge arises from the fact that some pathological changes in myopia can also occur in other ophthalmic conditions. One approach to address this is to exclude patients with comorbidities or group together a range of diseases. Alternatively, multimodal medical data fusion techniques can be employed by extracting relevant features from images and processing them with AI algorithms . These features are not limited to geometric measurements but may also include characteristic lesion regions . Additionally, image data can be processed to obtain a virtual score, which can be predicted together with clinical data by AI algorithms . While new developments in this area continue to emerge, it is important to note that multimodal data do not consistently outperform unimodal data, as demonstrated in a study on diabetic retinopathy staging . Novel forms of OCT images, such as UWF optical coherence tomography (UWF OCT) and OCT angiography (OCTA), can be seen in the clinical practice of myopia. UWF OCT, instead of the traditionally used 3D-MRI, can be helpful for better visualization of the posterior staphyloma in myopic eyes , which is “an outpouching of the wall of the eye with a radius of curvature less than the radius of curvature of the surrounding eye wall” and usually results in poorer vision and more anatomical anomalies . OCTA is very helpful for detecting retinal microvasculature in a noninvasive and depth-resolved way , thus providing a way of detecting mCNV with high sensitivity and specificity. Based on UWF OCT and OCTA, AI research is still restricted to improving the quality of images, such as image reconstruction and denoising , and there is currently a dearth of research exploiting the possibility of developing DL models to detect posterior staphyloma or mCNV. The advent of AI is expected to transform the management of myopia. The findings of this review suggest that AI has been applied to most parts of the clinical practice of myopia and is built mainly on three types of data: clinical data, FP, and UWF FP, OCT. Image-driven AI applications account for the majority. However, compared with other ophthalmic diseases, AI research in myopia is still in its early stages, and these results are far from clinically viable. It is necessary to establish large public datasets with high quality and improve the capability of handling multimodal input. Exploring novel data modalities, designing advanced algorithms, and finding additional application scenarios could also be of great significance.
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Sex, strain, and lateral differences in brain cytoarchitecture across a large mouse population
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6e674aa8-63f9-410c-aae8-f75313008080
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10212558
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Anatomy[mh]
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The mammalian brain can be divided into neuroanatomical units (i.e., brain regions) characterized by a shared function, connectivity, developmental origin, and/or cytoarchitecture (i.e., number and density of cells it contains). The mouse brain is the most extensively studied in mammals and its regions are well characterized. Although cytoarchitecture is one of the most prominent features of a brain region, few studies have systematically mapped cell bodies or quantified cell densities in mouse brains, compared to the early, detailed cell mapping of the nematode Caenorhabditis elegans . On the other hand, extensive literature explored scaling of cell numbers and densities among mammalian brains . This however was performed by counting dissociated nuclei at the loss of deeper region-specific resolution. Obtaining an accurate cell count for a brain region is technically challenging. Previous estimates relied heavily on extrapolation from manual counting of 2D sections (stereology), making cell-resolved data for subcortical regions sparse . Analyzing complete brains using 2D histological sections remains labor-intensive because it requires sectioning, mounting, and accurate alignment with a reference atlas. Furthermore, automated cell counting proved particularly difficult in dense regions, such as the hippocampal formation (HPF) and the cerebellum . Automated block-face imaging methods solved several of these issues and drastically improved throughput , For instance, serial two-photon tomography (STPT) was a technological breakthrough that integrated tissue sectioning with top-view light microscopy. STPT provided high-quality imaging in an optical plane below the sectioning surface and solved many problems of section distortion and atlas alignment, further easing downstream analysis. Yet, STPT typically represents a subsample of the complete volume, and some interpolation is needed. Because of their limited throughput, histological studies cannot supply the number of analyzed brains needed to uncover potential variability between individuals, experimental conditions, and populations. Complementary approaches aimed at evaluating variability, for example, magnetic resonance imaging (MRI), can measure some features, such as the volume of given brain regions, and can even track individuals over time in a noninvasive manner. Yet, MRI lacks the accuracy needed for counting cells or assessing cell densities, and it remains difficult to simultaneously analyze regional volume and cell density with high accuracy brain-wide, especially in the large throughput required for comparing two experimental populations (such as two strains, or males vs. females). Therefore, there is a need for systematic measurement of all cells over hundreds of brains from multiple experimental groups. To address this knowledge gap, we harnessed the Allen Mouse Brain Connectivity Project (AMBCA) dataset, which is largest existing cohort of whole-brain STPT images, produced by the Allen Institute for the different purpose of mapping mouse regional connectivity ( ; http://help.brain-map.org/display/api/Allen%2BBrain%2BAtlas%2BAPI ). We applied a deep neural network (DNN) to discern cell nuclei using the AMBCA background autofluorescence channel. This enabled us to perform a systematic brain-wide cell density estimation across hundreds of mouse brains. Based on the alignment with the Allen Mouse Brain Atlas (AMBA), we were able to simultaneously measure volume and density for each brain, for each region, over a large population. We constructed a comprehensive database that aggregates these results and provides them as an accessible resource to the community. We also discovered nontrivial relationships between densities and volumes, and gained insights into strain- and sex-dependent characteristics across various brain regions.
Autofluorescence of STPT images displays cell nuclei The AMBCA project was first published in 2014 . The project has systematically imaged 2992 full brains, using serial two-photon tomography, for the purpose of tracing neuronal projections and mapping regional (mesoscale) connectivity with GFP-labeled viral tracers. Each brain in the dataset is covered by 130–140 (median 137) serial coronal sections, with a gap of 100 μm, as reported in the AMBCA study . We noticed that the red (background) channel of STPT images, taken for the purpose of atlas alignment, typically features dark, round-like objects resembling cell nuclei. This phenomenon was described in previous literature . In particular, characterized the use of multiphoton-excited native florescence and second harmonic generation for the purpose of staining-free tissue imaging. To confirm that these dark objects indeed represent cell nuclei with lower autofluorescence intensity than the surrounding lipid-rich brain tissue, we performed a standard 4% paraformaldehyde (PFA) perfusion-fixation followed by cryosectioning and nucleus (DAPI) counterstaining. In these sections, we observed the same low-autofluorescent objects using epifluorescence microscopy. 91–98% of detected objects were also marked by DAPI, confirming that dark objects in STPT indeed represent cell nuclei . About 2–9% of detected low-autofluorescent objects were additional ‘false positive’ detections that were not obvious in the DAPI image. On the other hand, 26–45% of DAPI-detected nuclei were not observed in the autofluorescent images (false negatives), pointing to an underestimate of cell counts based on low-autofluorescence objects, compared to nuclear staining. A more systematic comparison between autofluorescence images and nuclear staining appears in the next section. Population-wide, regionally resolved exploration of neuroanatomical features To automatically collect cytoarchitecture data for each brain, we trained a DNN model to detect and segment the nuclei (low-autofluorescent objects) in all brain regions, including those of the highest density, such as the dentate gyrus (DG). Because of computing constraints, we applied the model systematically to segment a subset of the AMBCA dataset comprising 507 brains ( and ‘Methods’). The model performed with an estimated 97% cell detection accuracy on a test set, with a false positive rate of <0.01 (see ‘Methods’) whenever image quality was sufficient (for exclusion criteria of whole brains or certain regions within sections, see ‘Methods’). Using detected cells in each section, we obtained a local estimate of the volumetric cell density (see ‘Methods’), which, combined with the pixel-wise registration of brain regions provided by the AMBA, allowed us to estimate the average cell density per region for each brain. Similarly, we evaluated the per-region volume of each brain by linear interpolation across all sections (see ‘Methods’). The anterior-most olfactory bulb (MOB) and posterior-most cerebellum (CB) were truncated in imaging, which likely led to an underestimate in their quantifications, and slightly higher variance compared to other regions of similar volume . We further corrected a batch effect in the AMBCA dataset showing a small difference in overall brain volume across experimental batches (see ‘Methods,’ ). In sum, we simultaneously estimated the 3D cell density ( D ) and volume ( V ) of each region for each brain (see ‘Methods’). In total, we estimated per-region D and V for 532 basic regions annotated in the AMBA, which corresponds to levels 6–8 of the AMBA region hierarchy. Cell count ( N ) is the product V × D ; therefore, it was not considered an independent variable. The median male C57BL/6J mouse brain contained a total of 76 ± 11 × 10 6 cells, in 380 mm 3 of gray matter, at a density of 2.05 × 10 5 cells/mm 3 . A pie chart of the volume and cell count of the main regions (level 4 of region hierarchy) calculated across 507 brains appear in , and absolute cell counts for C57BL/6J male mouse representative regions are shown in . We quantified each level of the hierarchical tree structure of the AMBA and found good correlation (ρ = 0.86 and ρ = 0.98 for log and linear scale, respectively; interclass correlation coefficient 0.98–0.99) with a recent 3D whole-brain single-cell resolved light-sheet microscopy study . The diameter of detected objects (nuclei) varied between 7 and 9.5 μm ( , left), which at a nucleus/soma volumetric ratio of 0.08 corresponds to median cell body diameters from 16.25 μm in the RSPv6a, to 22 μm in the ENTl3. The regional variability of cell densities was high, ranging from 1 × 10 5 mm –3 in layer 1 isocortex (e.g., Mos1) to 6 × 10 5 mm –3 in the dentate gyrus granule layer (DG-sg). We show examples of regional distributions across the full cohort of 507 brains in the inset of , right. The large number of AMBCA brains in our analysis enabled us to compare variabilities of macroscopic properties between subsets of the cohort, for example, to compare strains. We compared distributions of volume, cell density, and cell count at the coarsest hierarchical atlas level, that is, across gray matter cell groups in the brains of male C57BL/6J vs. male FVB.CD1 mice . Median cell density was similar for the two strains, with considerably larger variance in FVB.CD1 males. FVB.CD1, however, had a 7.6% larger gray matter volume (GMV) than C57BL/6J. Combining these two features revealed an ~10% increase in the median cell count in FVB.CD1 vs. C57BL/6J ( , right panel). These results suggest that (1) there is no simple relationship between volume and density, therefore, both properties should be measured simultaneously; and (2) a large cohort enables detection of relatively small differences. To expand on our technical comparison between quantification based on autofluorescence vs. staining , we performed nuclear staining (Hoechst 33342) and whole-brain STPT imaging on nine brains of female C57BL/6J mice. We trained a DNN of the same topology using tiles from the resulting Hoechst images, registered with the AMBA coordinates, and repeated our per-region estimates of volume, density, and cell count (see ‘Methods’). As expected, correlation between this in-house dataset and the AMBCA analysis was very high when comparing the volume across regions (ρ = 0.99, ). In addition, cell count correlations were also high (ρ = 0.99, ), but median cell count in Hoechst was 65% higher; recapitulating the trend toward false negatives observed in (epifluorescence microscopy). However, agreement in cell density varied by region. The correlations in density were fair across cortical regions of layers 2/3, 4, 5, and 6a (ρ = 0.78, ): they displayed comparable densities between the methods . The correlation value across cortical regions 1 and 6b was similar (ρ = 0.79), yet the error in absolute values was significantly higher , at about twofold higher density using Hoechst . The correlation across brain stem regions was also lower (ρ = 0.56, ), yet rank order across regions was similar to cortical regions 1 and 6b. Cerebellar regions, which are significantly denser, displayed a twofold density difference (ρ = 0.65, ). This detection bias may therefore stem from several underlying causes; (1) inaccurate registration of border regions (CTX L1 and 6b); (2) physical detection limits of the low-autofluorescent objects compared to stained objects; and (3) region or cell type-inherent differences in autofluorescence, resulting in lower detection of cells in glial-rich (e.g., CTX L1, brainstem), compared to neuron-rich (e.g., other cortex layers) regions. To test the power of our model, we explored the densities and nucleus diameter of cortical regions . First, we considered the HPF because imaging-based quantification of its denser regions (pyramidal layers of Ammon’s horn and the granule layer of the dentate gyrus) has been difficult and was achieved only recently . Analyzing 195 C57BL/6J male brains, we found that the pyramidal layer of CA1 was denser than that of CA3 and CA2, whereas nucleus size was larger in CA3. In the dentate gyrus, the granule layer had the highest density of all regions, with >6.5 × 10 5 cells/mm 3 , and nuclei were largest in the polymorph layer ( , upper panels). In the isocortex, we examined the extent to which the cortical layers across cortical divisions differed in density and size ( , lower panels). Layer 1 was consistently underpopulated, having a density of about 10 5 cells/mm 3 . The overall rank order from densest to sparsest was maintained, with layer 4 > layer 6a > layer 5 > layer 2/3 > layer 1, suggesting a similarity in cytoarchitecture between cortical regions. Layer 4 of the primary visual and somatosensory cortices had higher density than did the auditory and visceral cortices. Nucleus diameters showed less distinct distributions between layers, although layer 2/3 and layer 5 tended to have larger nuclei than did layers 4 and 6a. Density differences between left and right hemispheres Brain laterality has been discussed in the literature since Broca and Wernike found language dominance on the left side of the brain. Although evidence for brain laterality in mice is more scarce, have suggested functional and circuit differences in the auditory cortex. We performed a systematic comparison of the left and right hemispheres seeking differences in region-wise volume and/or cell density. In C57BL/6J (n = 369), we found 229 regions with lateral cell density bias of over 5%, and up to 30%. Both sides showed similar numbers of biased regions in density (left, 100; right, 129, ). Neighboring regions in prefrontal cortex ACAv2/3, ACAd2/3, ORBl2/3, and PL/23 were up to 30% denser in the right hemisphere. In addition, a more posterior part of the HPF, the parasubiculum (PAR) also showed a consistent per-brain higher cell density in the right hemisphere . In contrast, cortical areas GU2/3, RSPv6a, VISC2/3, and AUDv2/3 showed more than 20% higher density in the left hemisphere. Strikingly, we found consistent density bias in left hemisphere cortical regions, specifically in layers 2/3 ( , upper inset). This bias was both most consistent and pronounced in a group of neighboring ventrolateral areas: visceral, gustatory, temporal association, and auditory , where the latter may be consistent with the Levy et al. findings. For example, in we show higher density in the right hemisphere visceral cortex layer 2/3, while corresponding layers 4, 5, and 6a were not biased. Laterally biased regions were consistent across strains (e.g., C57BL/6J vs. FVB.CD1, ). We note that volume laterality was less pronounced with some regions slightly larger in the right hemisphere . Most regions did not show laterality in both density and volume (e.g., PAR and VISC2/3 in display density laterality, while no bias in volume is observed). Regions with volume/density sexual dimorphism in C57BL/6J mice To examine whether differences in overall brain volume or density are isotropic, we conducted a region-specific analysis of volume, density, and cell count. Differences between males and females in regional neuroanatomy have been extensively described, including dimorphic volume and cell count in the medial amygdala (MEA) and in the bed nuclei of the stria terminalis (BST) . We first compared C57BL/6J males (n = 152) with females (n = 140). Although at the global level (whole-brain gray matter), males and females had similar volume (380 ± 17 mm 3 and 380 ± 14 mm 3 , for females and males, respectively), in density (1.97 ± 0.28 × 10 5 cells/mm 3 and 1.97 ± 0.28 × 10 5 cells/mm 3 for females and males, respectively) and total numbers of cells (75 ± 10 × 10 6 and 77 ± 9 × 10 6 for females and males, respectively) , certain regions displayed sexual dimorphism in one or more property. We conducted rank-sum testing on each region that passed QC (see ‘Methods’) for sex differences, in volume, density, or count . Most regions were consistent with the overall trend of equal volume, yet MEA and BST, but also the ventral cochlear nucleus (VCO), taenia tecta ventral (TTv), and the medial preoptic nucleus (MPN) were >5% larger in males. A handful of regions, for example, ventrolateral orbital area layer 2/3 (ORBvl2/3), displayed the opposite effect and had larger volume in females. ORBvl2/3 was the only region to also display significantly higher density and cell count in females. In contrast, many regions were 5–20% denser in males, which also resulted in higher cell counts. These regions include other parts of the amygdala (BMA, BLA, and the CEA), hypothalamic regions LPO and AHN, and cortical regions such as the primary auditory layer 2/3 (AUDv2/3). specifies whether each region is significantly dimorphic in volume, density, or count. Next, we looked beyond the rank-sum statistical test, governed by the median of the distribution, at examples of how distributions differ in volume and/or density. For example, ORBvl2/3 showed both larger volumes and slightly higher density in females ( , left), resulting in significantly more cells in females . The opposite was the case for BST, where males had larger volume yet similar density ( , middle). As a third example, we showed the case of AUDv2/3, which displayed no difference in region volume, yet density in the male brains was higher ( , right). In an independent AMBCA cohort of additional 663 males and 166 females, we could robustly replicate our findings regarding region-specific sexual dimorphism: volumes of MEA, BST, and ORBvl2/3 displayed sex differences of consistent effect size and variance . In sum, the population-wide survey revealed a number of sexually dimorphic areas; Yet, to what extent were region volumes or densities predictors of the individual’s sex? To answer this question, we trained linear support vector machine (SVM) classifiers. In brief, we randomly selected 2/3 of the C57BL/6J brains (n = 184), where each brain was represented by a 532-dimensional vector of either volume or density across regions, and trained the SVM. We then tested its performance on the remaining 1/3 of brains (n = 97). This process was repeated 100 times and resulted in an average accuracy of 78 and 90% for density and volume, respectively. From this, we identified the regions that had the highest contribution to the separating hyperplane and trained new classifiers based on these top regions, adding one region at a time. Using volume data, MEA and BST alone performed classification to >78% accuracy. Adding the postpiriform transitional area (TR) and the rostral lateral septal nucleus (LSr), the classifier’s performance saturated to about 90% . shows the SVM separating line based on four pairs of regions (e.g., BST and LSr) yielding an accuracy of 79–86%. In contrast, the density-based classifier showed only incremental improvements when adding almost any of the top 10 regions . Together, these results suggest that although many brain regions show significant differences between males and females both in volume and density , the considerable overlap between the male and female population distributions in each of these regions hampers classification. Apart from MEA and BST, the classifier revealed TR, LSr, MPN, and ORBl2/3 as predictors for sex based on volume, while density-based classification was based on amygdala-related regions (COApl, BMAp) and frontal cortical regions (ILA and ORB). Strain differences in volume and density We next investigated the relation between recorded body weight and GMV. To this end, we added the cohort of outbred FVB.CD1 mice, a strain with 40–50% higher body weight than C57BL/6J. As expected, in both strains, males and females showed distinct distributions for body weight, and males were larger than females . Within each strain, body weight did not correlate with gray volume. We next quantified sex and strain differences in brain volume and density, resolved to neuroanatomical regions. First, we compared strain differences in females with those in males, showing concordance/discordance patterns between males and females (sex) ( and schematic to the right). Second, we compared sex differences in FVB.CD1 with those in C57BL/6J, showing concordance/discordance patterns between strains ( and schematic to the right). Strain-wise analysis FVB.CD1 brains were overall larger, but the volume expansion with respect to C57BL/6J was not uniform across regions. Region volumes ranged up to 30% differences, with the extreme example of the cerebellum (CENT2), whose size increased by 45% in both FVB.CD1 males and females . Moreover, per-region volume differences between strains were, in general, larger in males (i.e., most data points in quadrant III are above the diagonal). Only two regions showed larger volumes in C57BL/6J: the main olfactory bulb (MOB) and the lateral amygdalar nucleus (LA) ( , quadrant I). A similar comparison for cell density per region suggests nonuniform density differences, with most regions being denser in FVB.CD1 ( , quadrant III). Notably, olfaction-related regions (AOB and MOB) showed higher density in C57BL/6J. Sex-wise analysis Differences in volume confirmed sexual dimorphism in MEA and BST, which were larger in males for both strains. These differences were more pronounced in FVB.CD1 than in C57BL/6J ( , quadrant I). Many brain regions showed ‘strain-discordant’ dimorphism, with females having a larger volume in C57BL/6J and males in FVB.CD1 ( , quadrant II). Although total brain volume in FVB.CD1 males was larger, some regions showed larger volume in females (e.g., the previously mentioned orbital cortex ORB, , quadrant III). Comparing sexual dimorphism in density , we found a simpler and more consistent picture: in both strains, males had higher density in most regions (except for, e.g., ORBvl2/3). Note that in density as well, sex differences were found to be larger in FVB.CD1 (most data points in , quadrant I, are above the diagonal). Region-wise correlations between volume and density across brains To the best of our knowledge, no previous study simultaneously quantified cell density ( D ) and brain region volume ( V ). We therefore sought to investigate whether constraints exist between D and V . For example, if the number of cells in a region is constant across brains, D and V must be negatively correlated. If, by contrast, the number of cells in a region, N , scales with the volume while D remains constant, D and V display zero correlation. If a positive correlation exists between D and V , the number of cells N grows faster than linear with respect to either D or V . Based on per-region measurements of both V and D , we calculated regionally resolved Pearson correlations between volume and density in C57BL/6J . In 79% of regions (356/451), cell density was negatively correlated with volume , with a median correlation of –0.14. For example, we showed two regions where N was positively correlated with both D and V , yet the correlation between D and V was either positive (AAA, ) or negative (SSs2/3, ). This suggests that across regions cell count does not scale uniformly with volume. Inter-brain similarity between regions based on volume and density Finally, we assessed similarity between regions, based on volume or density. We used tSNE as a 2D embedding method over the density data ( ; the similarity matrix that served as input to tSNE appears in ). Briefly, each region is characterized by a vector of 204 components, each representing its density across one brain. 2D embedding aims to preserve the local similarity between regions. The density-based tSNE embedding map in reveals clear 2D ‘clusters,’ largely consistent with neuroanatomical classification. Cortical regions appear in the upper part of the map (colored green), and cerebellum (yellow), midbrain, and hindbrain in the lower part. We further explored whether the order within the cortical part may be explained by layer structure or by cortical division, but found no clear structure . Compared to , tSNE embedding based on volume was more ‘dispersed’ and displayed disorder with respect to neuroanatomical classification . To demonstrate that the tSNE map is indeed based on true variations in region-to-region correlations, we compared density-based and volume-based correlations. First, for each region we identified its 10 most correlated regions based on either density or volume. These correlation values were higher for density-based correlations across almost all regions , supporting the observed density-based ‘order.’ Second, we selected 10 representative regions across the brain and calculated all their pairwise correlations , showing that even for distant regions, density-based correlations remain much higher than volume-based correlations. Thus, similarity in volume across regions is less ‘preserved’ brain-wide than similarity in cell density.
The AMBCA project was first published in 2014 . The project has systematically imaged 2992 full brains, using serial two-photon tomography, for the purpose of tracing neuronal projections and mapping regional (mesoscale) connectivity with GFP-labeled viral tracers. Each brain in the dataset is covered by 130–140 (median 137) serial coronal sections, with a gap of 100 μm, as reported in the AMBCA study . We noticed that the red (background) channel of STPT images, taken for the purpose of atlas alignment, typically features dark, round-like objects resembling cell nuclei. This phenomenon was described in previous literature . In particular, characterized the use of multiphoton-excited native florescence and second harmonic generation for the purpose of staining-free tissue imaging. To confirm that these dark objects indeed represent cell nuclei with lower autofluorescence intensity than the surrounding lipid-rich brain tissue, we performed a standard 4% paraformaldehyde (PFA) perfusion-fixation followed by cryosectioning and nucleus (DAPI) counterstaining. In these sections, we observed the same low-autofluorescent objects using epifluorescence microscopy. 91–98% of detected objects were also marked by DAPI, confirming that dark objects in STPT indeed represent cell nuclei . About 2–9% of detected low-autofluorescent objects were additional ‘false positive’ detections that were not obvious in the DAPI image. On the other hand, 26–45% of DAPI-detected nuclei were not observed in the autofluorescent images (false negatives), pointing to an underestimate of cell counts based on low-autofluorescence objects, compared to nuclear staining. A more systematic comparison between autofluorescence images and nuclear staining appears in the next section.
To automatically collect cytoarchitecture data for each brain, we trained a DNN model to detect and segment the nuclei (low-autofluorescent objects) in all brain regions, including those of the highest density, such as the dentate gyrus (DG). Because of computing constraints, we applied the model systematically to segment a subset of the AMBCA dataset comprising 507 brains ( and ‘Methods’). The model performed with an estimated 97% cell detection accuracy on a test set, with a false positive rate of <0.01 (see ‘Methods’) whenever image quality was sufficient (for exclusion criteria of whole brains or certain regions within sections, see ‘Methods’). Using detected cells in each section, we obtained a local estimate of the volumetric cell density (see ‘Methods’), which, combined with the pixel-wise registration of brain regions provided by the AMBA, allowed us to estimate the average cell density per region for each brain. Similarly, we evaluated the per-region volume of each brain by linear interpolation across all sections (see ‘Methods’). The anterior-most olfactory bulb (MOB) and posterior-most cerebellum (CB) were truncated in imaging, which likely led to an underestimate in their quantifications, and slightly higher variance compared to other regions of similar volume . We further corrected a batch effect in the AMBCA dataset showing a small difference in overall brain volume across experimental batches (see ‘Methods,’ ). In sum, we simultaneously estimated the 3D cell density ( D ) and volume ( V ) of each region for each brain (see ‘Methods’). In total, we estimated per-region D and V for 532 basic regions annotated in the AMBA, which corresponds to levels 6–8 of the AMBA region hierarchy. Cell count ( N ) is the product V × D ; therefore, it was not considered an independent variable. The median male C57BL/6J mouse brain contained a total of 76 ± 11 × 10 6 cells, in 380 mm 3 of gray matter, at a density of 2.05 × 10 5 cells/mm 3 . A pie chart of the volume and cell count of the main regions (level 4 of region hierarchy) calculated across 507 brains appear in , and absolute cell counts for C57BL/6J male mouse representative regions are shown in . We quantified each level of the hierarchical tree structure of the AMBA and found good correlation (ρ = 0.86 and ρ = 0.98 for log and linear scale, respectively; interclass correlation coefficient 0.98–0.99) with a recent 3D whole-brain single-cell resolved light-sheet microscopy study . The diameter of detected objects (nuclei) varied between 7 and 9.5 μm ( , left), which at a nucleus/soma volumetric ratio of 0.08 corresponds to median cell body diameters from 16.25 μm in the RSPv6a, to 22 μm in the ENTl3. The regional variability of cell densities was high, ranging from 1 × 10 5 mm –3 in layer 1 isocortex (e.g., Mos1) to 6 × 10 5 mm –3 in the dentate gyrus granule layer (DG-sg). We show examples of regional distributions across the full cohort of 507 brains in the inset of , right. The large number of AMBCA brains in our analysis enabled us to compare variabilities of macroscopic properties between subsets of the cohort, for example, to compare strains. We compared distributions of volume, cell density, and cell count at the coarsest hierarchical atlas level, that is, across gray matter cell groups in the brains of male C57BL/6J vs. male FVB.CD1 mice . Median cell density was similar for the two strains, with considerably larger variance in FVB.CD1 males. FVB.CD1, however, had a 7.6% larger gray matter volume (GMV) than C57BL/6J. Combining these two features revealed an ~10% increase in the median cell count in FVB.CD1 vs. C57BL/6J ( , right panel). These results suggest that (1) there is no simple relationship between volume and density, therefore, both properties should be measured simultaneously; and (2) a large cohort enables detection of relatively small differences. To expand on our technical comparison between quantification based on autofluorescence vs. staining , we performed nuclear staining (Hoechst 33342) and whole-brain STPT imaging on nine brains of female C57BL/6J mice. We trained a DNN of the same topology using tiles from the resulting Hoechst images, registered with the AMBA coordinates, and repeated our per-region estimates of volume, density, and cell count (see ‘Methods’). As expected, correlation between this in-house dataset and the AMBCA analysis was very high when comparing the volume across regions (ρ = 0.99, ). In addition, cell count correlations were also high (ρ = 0.99, ), but median cell count in Hoechst was 65% higher; recapitulating the trend toward false negatives observed in (epifluorescence microscopy). However, agreement in cell density varied by region. The correlations in density were fair across cortical regions of layers 2/3, 4, 5, and 6a (ρ = 0.78, ): they displayed comparable densities between the methods . The correlation value across cortical regions 1 and 6b was similar (ρ = 0.79), yet the error in absolute values was significantly higher , at about twofold higher density using Hoechst . The correlation across brain stem regions was also lower (ρ = 0.56, ), yet rank order across regions was similar to cortical regions 1 and 6b. Cerebellar regions, which are significantly denser, displayed a twofold density difference (ρ = 0.65, ). This detection bias may therefore stem from several underlying causes; (1) inaccurate registration of border regions (CTX L1 and 6b); (2) physical detection limits of the low-autofluorescent objects compared to stained objects; and (3) region or cell type-inherent differences in autofluorescence, resulting in lower detection of cells in glial-rich (e.g., CTX L1, brainstem), compared to neuron-rich (e.g., other cortex layers) regions. To test the power of our model, we explored the densities and nucleus diameter of cortical regions . First, we considered the HPF because imaging-based quantification of its denser regions (pyramidal layers of Ammon’s horn and the granule layer of the dentate gyrus) has been difficult and was achieved only recently . Analyzing 195 C57BL/6J male brains, we found that the pyramidal layer of CA1 was denser than that of CA3 and CA2, whereas nucleus size was larger in CA3. In the dentate gyrus, the granule layer had the highest density of all regions, with >6.5 × 10 5 cells/mm 3 , and nuclei were largest in the polymorph layer ( , upper panels). In the isocortex, we examined the extent to which the cortical layers across cortical divisions differed in density and size ( , lower panels). Layer 1 was consistently underpopulated, having a density of about 10 5 cells/mm 3 . The overall rank order from densest to sparsest was maintained, with layer 4 > layer 6a > layer 5 > layer 2/3 > layer 1, suggesting a similarity in cytoarchitecture between cortical regions. Layer 4 of the primary visual and somatosensory cortices had higher density than did the auditory and visceral cortices. Nucleus diameters showed less distinct distributions between layers, although layer 2/3 and layer 5 tended to have larger nuclei than did layers 4 and 6a.
Brain laterality has been discussed in the literature since Broca and Wernike found language dominance on the left side of the brain. Although evidence for brain laterality in mice is more scarce, have suggested functional and circuit differences in the auditory cortex. We performed a systematic comparison of the left and right hemispheres seeking differences in region-wise volume and/or cell density. In C57BL/6J (n = 369), we found 229 regions with lateral cell density bias of over 5%, and up to 30%. Both sides showed similar numbers of biased regions in density (left, 100; right, 129, ). Neighboring regions in prefrontal cortex ACAv2/3, ACAd2/3, ORBl2/3, and PL/23 were up to 30% denser in the right hemisphere. In addition, a more posterior part of the HPF, the parasubiculum (PAR) also showed a consistent per-brain higher cell density in the right hemisphere . In contrast, cortical areas GU2/3, RSPv6a, VISC2/3, and AUDv2/3 showed more than 20% higher density in the left hemisphere. Strikingly, we found consistent density bias in left hemisphere cortical regions, specifically in layers 2/3 ( , upper inset). This bias was both most consistent and pronounced in a group of neighboring ventrolateral areas: visceral, gustatory, temporal association, and auditory , where the latter may be consistent with the Levy et al. findings. For example, in we show higher density in the right hemisphere visceral cortex layer 2/3, while corresponding layers 4, 5, and 6a were not biased. Laterally biased regions were consistent across strains (e.g., C57BL/6J vs. FVB.CD1, ). We note that volume laterality was less pronounced with some regions slightly larger in the right hemisphere . Most regions did not show laterality in both density and volume (e.g., PAR and VISC2/3 in display density laterality, while no bias in volume is observed).
To examine whether differences in overall brain volume or density are isotropic, we conducted a region-specific analysis of volume, density, and cell count. Differences between males and females in regional neuroanatomy have been extensively described, including dimorphic volume and cell count in the medial amygdala (MEA) and in the bed nuclei of the stria terminalis (BST) . We first compared C57BL/6J males (n = 152) with females (n = 140). Although at the global level (whole-brain gray matter), males and females had similar volume (380 ± 17 mm 3 and 380 ± 14 mm 3 , for females and males, respectively), in density (1.97 ± 0.28 × 10 5 cells/mm 3 and 1.97 ± 0.28 × 10 5 cells/mm 3 for females and males, respectively) and total numbers of cells (75 ± 10 × 10 6 and 77 ± 9 × 10 6 for females and males, respectively) , certain regions displayed sexual dimorphism in one or more property. We conducted rank-sum testing on each region that passed QC (see ‘Methods’) for sex differences, in volume, density, or count . Most regions were consistent with the overall trend of equal volume, yet MEA and BST, but also the ventral cochlear nucleus (VCO), taenia tecta ventral (TTv), and the medial preoptic nucleus (MPN) were >5% larger in males. A handful of regions, for example, ventrolateral orbital area layer 2/3 (ORBvl2/3), displayed the opposite effect and had larger volume in females. ORBvl2/3 was the only region to also display significantly higher density and cell count in females. In contrast, many regions were 5–20% denser in males, which also resulted in higher cell counts. These regions include other parts of the amygdala (BMA, BLA, and the CEA), hypothalamic regions LPO and AHN, and cortical regions such as the primary auditory layer 2/3 (AUDv2/3). specifies whether each region is significantly dimorphic in volume, density, or count. Next, we looked beyond the rank-sum statistical test, governed by the median of the distribution, at examples of how distributions differ in volume and/or density. For example, ORBvl2/3 showed both larger volumes and slightly higher density in females ( , left), resulting in significantly more cells in females . The opposite was the case for BST, where males had larger volume yet similar density ( , middle). As a third example, we showed the case of AUDv2/3, which displayed no difference in region volume, yet density in the male brains was higher ( , right). In an independent AMBCA cohort of additional 663 males and 166 females, we could robustly replicate our findings regarding region-specific sexual dimorphism: volumes of MEA, BST, and ORBvl2/3 displayed sex differences of consistent effect size and variance . In sum, the population-wide survey revealed a number of sexually dimorphic areas; Yet, to what extent were region volumes or densities predictors of the individual’s sex? To answer this question, we trained linear support vector machine (SVM) classifiers. In brief, we randomly selected 2/3 of the C57BL/6J brains (n = 184), where each brain was represented by a 532-dimensional vector of either volume or density across regions, and trained the SVM. We then tested its performance on the remaining 1/3 of brains (n = 97). This process was repeated 100 times and resulted in an average accuracy of 78 and 90% for density and volume, respectively. From this, we identified the regions that had the highest contribution to the separating hyperplane and trained new classifiers based on these top regions, adding one region at a time. Using volume data, MEA and BST alone performed classification to >78% accuracy. Adding the postpiriform transitional area (TR) and the rostral lateral septal nucleus (LSr), the classifier’s performance saturated to about 90% . shows the SVM separating line based on four pairs of regions (e.g., BST and LSr) yielding an accuracy of 79–86%. In contrast, the density-based classifier showed only incremental improvements when adding almost any of the top 10 regions . Together, these results suggest that although many brain regions show significant differences between males and females both in volume and density , the considerable overlap between the male and female population distributions in each of these regions hampers classification. Apart from MEA and BST, the classifier revealed TR, LSr, MPN, and ORBl2/3 as predictors for sex based on volume, while density-based classification was based on amygdala-related regions (COApl, BMAp) and frontal cortical regions (ILA and ORB).
We next investigated the relation between recorded body weight and GMV. To this end, we added the cohort of outbred FVB.CD1 mice, a strain with 40–50% higher body weight than C57BL/6J. As expected, in both strains, males and females showed distinct distributions for body weight, and males were larger than females . Within each strain, body weight did not correlate with gray volume. We next quantified sex and strain differences in brain volume and density, resolved to neuroanatomical regions. First, we compared strain differences in females with those in males, showing concordance/discordance patterns between males and females (sex) ( and schematic to the right). Second, we compared sex differences in FVB.CD1 with those in C57BL/6J, showing concordance/discordance patterns between strains ( and schematic to the right). Strain-wise analysis FVB.CD1 brains were overall larger, but the volume expansion with respect to C57BL/6J was not uniform across regions. Region volumes ranged up to 30% differences, with the extreme example of the cerebellum (CENT2), whose size increased by 45% in both FVB.CD1 males and females . Moreover, per-region volume differences between strains were, in general, larger in males (i.e., most data points in quadrant III are above the diagonal). Only two regions showed larger volumes in C57BL/6J: the main olfactory bulb (MOB) and the lateral amygdalar nucleus (LA) ( , quadrant I). A similar comparison for cell density per region suggests nonuniform density differences, with most regions being denser in FVB.CD1 ( , quadrant III). Notably, olfaction-related regions (AOB and MOB) showed higher density in C57BL/6J. Sex-wise analysis Differences in volume confirmed sexual dimorphism in MEA and BST, which were larger in males for both strains. These differences were more pronounced in FVB.CD1 than in C57BL/6J ( , quadrant I). Many brain regions showed ‘strain-discordant’ dimorphism, with females having a larger volume in C57BL/6J and males in FVB.CD1 ( , quadrant II). Although total brain volume in FVB.CD1 males was larger, some regions showed larger volume in females (e.g., the previously mentioned orbital cortex ORB, , quadrant III). Comparing sexual dimorphism in density , we found a simpler and more consistent picture: in both strains, males had higher density in most regions (except for, e.g., ORBvl2/3). Note that in density as well, sex differences were found to be larger in FVB.CD1 (most data points in , quadrant I, are above the diagonal).
FVB.CD1 brains were overall larger, but the volume expansion with respect to C57BL/6J was not uniform across regions. Region volumes ranged up to 30% differences, with the extreme example of the cerebellum (CENT2), whose size increased by 45% in both FVB.CD1 males and females . Moreover, per-region volume differences between strains were, in general, larger in males (i.e., most data points in quadrant III are above the diagonal). Only two regions showed larger volumes in C57BL/6J: the main olfactory bulb (MOB) and the lateral amygdalar nucleus (LA) ( , quadrant I). A similar comparison for cell density per region suggests nonuniform density differences, with most regions being denser in FVB.CD1 ( , quadrant III). Notably, olfaction-related regions (AOB and MOB) showed higher density in C57BL/6J.
Differences in volume confirmed sexual dimorphism in MEA and BST, which were larger in males for both strains. These differences were more pronounced in FVB.CD1 than in C57BL/6J ( , quadrant I). Many brain regions showed ‘strain-discordant’ dimorphism, with females having a larger volume in C57BL/6J and males in FVB.CD1 ( , quadrant II). Although total brain volume in FVB.CD1 males was larger, some regions showed larger volume in females (e.g., the previously mentioned orbital cortex ORB, , quadrant III). Comparing sexual dimorphism in density , we found a simpler and more consistent picture: in both strains, males had higher density in most regions (except for, e.g., ORBvl2/3). Note that in density as well, sex differences were found to be larger in FVB.CD1 (most data points in , quadrant I, are above the diagonal).
To the best of our knowledge, no previous study simultaneously quantified cell density ( D ) and brain region volume ( V ). We therefore sought to investigate whether constraints exist between D and V . For example, if the number of cells in a region is constant across brains, D and V must be negatively correlated. If, by contrast, the number of cells in a region, N , scales with the volume while D remains constant, D and V display zero correlation. If a positive correlation exists between D and V , the number of cells N grows faster than linear with respect to either D or V . Based on per-region measurements of both V and D , we calculated regionally resolved Pearson correlations between volume and density in C57BL/6J . In 79% of regions (356/451), cell density was negatively correlated with volume , with a median correlation of –0.14. For example, we showed two regions where N was positively correlated with both D and V , yet the correlation between D and V was either positive (AAA, ) or negative (SSs2/3, ). This suggests that across regions cell count does not scale uniformly with volume.
Finally, we assessed similarity between regions, based on volume or density. We used tSNE as a 2D embedding method over the density data ( ; the similarity matrix that served as input to tSNE appears in ). Briefly, each region is characterized by a vector of 204 components, each representing its density across one brain. 2D embedding aims to preserve the local similarity between regions. The density-based tSNE embedding map in reveals clear 2D ‘clusters,’ largely consistent with neuroanatomical classification. Cortical regions appear in the upper part of the map (colored green), and cerebellum (yellow), midbrain, and hindbrain in the lower part. We further explored whether the order within the cortical part may be explained by layer structure or by cortical division, but found no clear structure . Compared to , tSNE embedding based on volume was more ‘dispersed’ and displayed disorder with respect to neuroanatomical classification . To demonstrate that the tSNE map is indeed based on true variations in region-to-region correlations, we compared density-based and volume-based correlations. First, for each region we identified its 10 most correlated regions based on either density or volume. These correlation values were higher for density-based correlations across almost all regions , supporting the observed density-based ‘order.’ Second, we selected 10 representative regions across the brain and calculated all their pairwise correlations , showing that even for distant regions, density-based correlations remain much higher than volume-based correlations. Thus, similarity in volume across regions is less ‘preserved’ brain-wide than similarity in cell density.
We presented an automated, imaging-based, staining-free study of neuroanatomy and cytoarchitecture in the mouse brain. We conducted our measurements on a massive, high-quality dataset of serial two-photon tomography , aligned with a well-annotated reference atlas . This made possible, for the first time, a detailed population-wide analysis of two important neuroanatomical variables simultaneously: cell density and volume, resolved for 532 regions. The data spans an unprecedented cohort of 507 mice of two strains, the inbred C57BL/6J, and the hybrid FVB1.CD-1, each represented by both females and males. Our high-throughput measurements of cell densities were achieved by using a DNN trained to detect low-autofluorescent cell nuclei with high accuracy, even in the most cell-dense regions of the brain. The study has several limitations. First, the model is sensitive to the contrast between dark nuclei and autofluorescent surroundings, which can be limited by image quality and tissue composition. In particular, the staining-free approach depends on the presence of intrinsic molecular indicators such as NADH, retinol, or collagen , which may vary between cell or tissue components, even within the brain. In the hindbrain (pons, medulla), contrast was exceedingly weak, and we expect our quantifications in this region to be 66% of the value estimated by nuclear staining . Second, AMBA annotations were not always resolved to the most refined level of the atlas hierarchy. For example, density values for the cerebellum appear to be uncharacteristic because the cell-dense granule layer and sparse molecular layer were not distinguished at the deepest level of annotation (e.g., CENT3 included the granular and molecular layers). The same is true for the hippocampus CA1-2-3, where we used cell density-based clustering to distinguish the pyramidal layer (sp) from its surrounding sparse layers (slm, so, sr, see ‘Methods’). Therefore, although the model performed exceedingly well even in these cell-dense regions, the absence of annotations stood occasionally in the way of making biologically meaningful distinctions. Third, regions at the anterior–posterior edges, that is, olfactory bulb (MOB) and cerebellum (CB), appear partially truncated, and we likely report an underestimate of their parameters. MOB and CB volumes also displayed slightly higher variance than more interior regions with similar volume. Therefore, their population comparisons would appear noisier (although likely not biased), compared to other regions. We further attempted to estimate the region-specific accuracy of our cell counting by comparing autofluorescence STPT with brain-wide imaging of nuclear-stained STPT. However, this comparison is technically nontrivial because of the native physical properties of direct staining vs. autofluorescence. For example, stained nuclei located off the focal plane may appear in the image, yet remain undetected by autofluorescence. In addition, tissue composition (e.g., cell types, extracellular matrix) may affect the imaged region. Indeed, in regions rich with non-neuronal cells the error of autofluorescent-based counting was larger compared to nuclear staining. Hence, one may speculate that autofluorescent-based detection is biased for neurons. Nevertheless, we provided key statistics that help answer fundamental, recurring questions in neuroanatomy. Although no other study presented simultaneous measurements of volume and cell density, our data correlate well with a wealth of literature in the field. We achieved good region-wise correlation with full 3D volumetric cell counts by expansion microscopy . Our derived cell count of mouse brain gray matter ( 76 ± 11 × 10 6 for male C57BL/6J) is well within the range of existing cell count estimates for adult males ( 67 - 150 × 10 6 cells) . And although the current mouse brain dataset is unique in its scale, we foresee the application of full-brain, staining-free STPT and DNN-led neuroanatomical characterization could benefit the study of disease models, or be applied to other species for comparative neurobiology, as has been done in dissociation-based studies that revealed mammalian brain scaling rules . By measuring the largest cohort to date, we provided partial support for the notion that this extreme range in the literature may not stem from variation in strain or sex, but rather from individual differences . The median cell counts between sexes and strains differed no more than 13% overall, or ~40% for the most deviant individual structures (MOB and CENT2), while the standard deviation across individuals was ~10 million cells (for C57BL/6J male alone). Hence, values between 55 × 10 6 - 95 × 10 6 are within ±2 SDs of the distribution of total grey matter cell counts. We claim that the notion of ‘ground truth’ values of brain cell number can be reached, yet are best reflected by a population distribution. Our dataset provides a large, important corpus toward this ‘ground truth,’ and similar studies can further help distinguish technical biases from biological variation. For example, our cohort provides the first brain-wide survey of cell architecture laterality, with numerous regions showing density bias to the right or left hemisphere; and some volume bias in favor of the right hemisphere. We describe a remarkable phenomenon of ventrolateral cortical areas increasing cell density in left hemisphere layer 2/3, but no other layers, that may be interesting to follow up functionally. We further validated and expanded on the existing literature describing examples of regions that show sex- or strain-based differences. Sexual dimorphism of brain regions was significant, as exemplified by the success of our SVM-classifier, yet the ability to separate males from females was limited due to population’s interindividual differences. For instance, medial amygdala and bed nuclei of the stria terminalis were both larger and denser in males, but to a lesser extent than reported in smaller studies and to a similar extent to what was reported in MRI-based studies . By contrast, in females, several prefrontal cortex structures were larger (e.g., ORBvl2/3), which resulted in higher cell counts. We found no evidence of this phenomenon in the literature on mice, but an MRI population study of 2838 human individuals found higher GMV in prefrontal areas in women . Between the strains, we found considerable differences in the olfactory system, which was larger and denser in C57BL/6J, and in the cerebellum, which was larger in FVB.CD-1. Finally, we provide an accessible, web-based platform for open exploration of the data. The web application allows researchers to freely compute distributions of any measured neuroanatomical features, for any brain region, and across the entire population or specific subsets. This exploratory resource can be of great use for experimental design and lead to more accurate brain modeling.
Data The AMBCA dataset consists of 2992 brains, of which we processed 507 and eventually used 378 in our analysis (the strain and sex breakdown of the brains are shown in ). Each brain consisted of ~140 section images captured every 100 μ m along the anterior–posterior axis using two-photon tomography . Image resolution was 0.35 μ m per pixel. AMBCA post-processed section images for noise removal. Rather than using the red, green, and blue channels occupied by the fluorescent reporter for anterograde connectivity tracing, we used the background channel of the images, as provided by AMBCA, without additional processing, except for converting the RGB image to grayscale. Training a deep neural network for cell segmentation To detect cells in an image and mark their contour, we used the Detectron2 deep neural network library , which relies on a Mask R-CNN image segmentation model with the ResNet-101 as its backbone. Model training and validation Training the model required three rounds of manual annotation and training. Initial manual annotation of the dataset and model training We annotated cell contours manually using the VGG Image Annotator software . Initially, we annotated only the hippocampus, which is relatively large and easily discernible. The hippocampus contains subregions of different densities, which we believed would adequately represent the variety of cell densities across the mouse brain. We manually annotated tiles of 312 × 312 pixels ( 109 × 109 μ m ), randomly selected from the hippocampus in five sections of three brains (55 tiles in total). We provided these tiles to the network as training data, together with basic data augmentation (e.g., rotation and brightness changes) . Retraining on hippocampal sections We then applied the trained model to detect cells on a new set of 55 randomly selected hippocampus tiles. We manually corrected the results produced by the network to create a new set of ground truth annotations. Next, we retrained the model from scratch over a combined training set of 110 tiles. Retraining on other regions We subsequently used the trained model to detect cells on random sections of three selected brains. Visual inspection enabled us to select a set of 64 tiles that displayed the least accurate results and annotate them manually. Final training We retrained the model from scratch on the resulting training set of 174 tiles (selected from ~15 sections of ~10 brains). The total number of cells across the training set tiles was 6247, corresponding to 0.008% of the estimated 77 million cells in the whole brain. Technical details We conducted the training with a batch of size 2, a learning rate of 0.00025, with decay, using the Adam optimizer . Training over 174 tiles required ~395,000 iterations and took ~36 hr using a Linux server with 160 Intel Xeon Gold 6248 2.5 GHz CPUs and a Tesla V100S-PCIE-32GB GPU. Evaluating model performance The training process completed when the model converged. The accuracy of the model on the training data was 99.8%, with a false negative rate of 0.4%. To evaluate model performance, we manually annotated 30 additional tiles from the isocortex, medial amygdala (MEA), hypothalamus (HY), and hippocampus (HIP) of 27 brains and compared them with model prediction . We obtained highly accurate results, comparable to the performance over the training data, for segmentation scores such as Jaccard measure , F1 score (harmonic mean of precision and recall), and total errors (i.e., percentage of mislabeled pixels), as well as for detection scores such as accuracy (detected cells divided by total cells) and false positive rate (false positives divided by total cells). Brain-wide automatic segmentation The trained DNN was applied to 507 brains, as described in detail below. Extracting cell information per section We divided each section into overlapping tiles sized 312 × 312 pixels, with an overlap of 20 pixels on each side (thus mitigating potential artifacts close to the borders of the tiles). We then applied the trained DNN to detect cells in each tile, resulting in a cell mask (i.e., a Boolean 312 × 312 matrix whose entries are true if the corresponding pixel is part of a detected cell and false otherwise). Next, we stitched the tiles together using a logical OR over overlapping areas, resulting in a single cell mask per section. Subsequently, we performed contour detection to obtain the coordinates of each cell in a section and computed the morphological properties of each cell (i.e., circumference, diameter, and area). Following this analysis step, each section image was represented by a table containing the coordinates and morphological properties of its cells. Assigning cells to regions We used the AMBA to assign the coordinates of detected cells in each section to their corresponding brain region. But the atlas annotation was too coarse for several regions of interest, that is, CA1, CA2, and CA3 of the hippocampus. The common denominator of these regions was the presence of a dense and a sparse region that were not separated by the atlas (e.g., the pyramidal and stratum regions of CA1, CA2, and CA3). To provide the coordinates of these subregions, we defined a local measure of density referred to as cell ‘coverage’ and used it to cluster the relevant cells into a dense and a sparse region. Briefly, in a window of 64 ×64 pixels centered around each cell we counted the number of pixels that belong to cells, thus assigning a local ‘coverage’ measure (the median cell area was 80 pixels, much smaller than the window around it). We then detected the subregions by clustering the cells according to their ‘coverage’ values. For example, we took the ‘coverage’ values of all CA1 cells and used K-means clustering to split them into two clusters of high and low ‘coverage’ values. In this way, the coordinate of each cell center was assigned to either cluster. We then drew the circumference of the subregions by applying a standard morphological closing operation and discarded spurious small regions. Estimating volumes, 3D densities, and cell counts Until this stage, the analysis provided local, that is, microscopic properties for each detected cell, and assigned cells to a brain region. The next step was to collect cells that belong to each region and estimate their density, the volume of the region, and the total cell count. This required calculating 3D estimates based on the relevant 2D data using the following steps: Estimating cell density per section We used AMBA to label the area of a given region in a section. We assumed that cells belonging to a region are equi-radius spheres whose projection on the 2D section depends on the distance between their centers and the optical plane, and on the optical depth of field . Hence, detected cells on a 2D section s originate from a slab whose volume is v s = a s ∙ 2 R + d , where a s is the area of a region, R is the radius of the cells in the region, and d is the optical depth of field. Cell density per section, ρ s , is given by dividing the number of detected cells by v s . The value of a s is measured by pixels whose size is 0.35 μ m , and d = 1.5 μ m . The value of R was taken as the 90th percentile of measured cell radii in a s . The distribution of cell radii corresponds to the ‘projection’ of the cells on the measured section, together with the optical depth of field. Downstream results of cell count and density significantly depend of the value of R , for example, using the 50th percentile would provide larger estimated cell counts. Yet, rank order of cell counts and densities across regions is independent of the selected value of R . Calculating region volume AMBA provides pixel-wise region annotation for each section, making it possible to calculate the area of a region per section (which is independent of cell segmentation). The 3D volume of a region is given by the sum of region volumes between adjacent sections, estimated by the average of its areas over each section. For example, if a region appears in sections 1, 2, 3, and 4, its volume is the sum of average volumes between sections 1 and 2, 2 and 3, and 3 and 4. Calculating cell counts across adjacent sections and in total Cell counts between the adjacent sections of each region are given by the average densities in those slides multiplied by the volume of the region between these sections. The total cell count of a region is provided by a sum across all relevant sections. Calculating cell densities per region The overall density of each region is given by the total cell count divided by the volume of the region. Discarding whole brains or particular regions of lower technical quality After calculating the 3D counts and densities across all regions in all brains, we excluded from subsequent analysis regions and whole brains that displayed potentially flawed estimates. We applied the following criteria: We discarded brains displaying dark images We filtered out brains whose median brightness across the whole brain gray matter (‘gray’ region) was lower than 25 (on a scale between 0 and 255). In such cases, all ~140 sections of the brain were excluded from downstream analysis because DNN cell detection was either impossible or provided significantly lower estimates . In total, 51 brains were discarded due to low brightness. We discarded brains displaying an optical or physical artifact resulting in outliers in cell count We noticed that a common optical or physical artifact of resolution degradation caused the DNN to falsely detect large amounts of excess cells (see distinct ‘columns’ in ). We marked cases in which cell count in a region was 3 SDs larger than the median for the region across brains (calculated as M A D ∙ 1.4826 , assuming normal distribution). We discarded brains that included more than three such outlier regions. 81 brains were discarded due to such artifacts. We discarded regions of small volume We filtered out regions whose median volume across brains was smaller than 0.3 m m 3 , or whose median cell count across sections was smaller than 500 . In total, 283 (out of 690 regions in AMBA) were discarded and excluded from all brains (data of these regions do appear in our online tool described below). We discarded regions displaying different estimates in right vs. left hemispheres Cell count estimates in the right and left hemispheres served as a proxy for technical noise. We calculated cell counts per region using each hemisphere independently. If the average difference in cell count between hemispheres was higher than 15.5% of the total cell count for that region, we excluded the case from downstream analysis . 33 regions were omitted due to right–left differences. We discarded regions displaying a correlation between cell count and image brightness We excluded regions exhibiting strong correlation (>0.3) between brightness and cell count because we assumed that in this case cell count was affected by the inability of the model to discern the cells when the brightness was too low. 30 regions were discarded due to such correlations. In sum, we processed 507 brains, of which 132 were fully discarded. Of 690 regions in AMBA, 346 were discarded completely and another 107 were partially discarded. Correcting a batch effect in volume While analyzing the Allen images, we noticed that the experiments were performed in several batches (based on experiment ID), and that there was a small difference in the total brain volume among batches, yet the total cell did not display a batch effect . In order to correct this effect, we multiplied each brain volume by a batch-specific factor such that the batch median volume 8 will be 380 mm 3 . These factors were 1.064, 1.032, and 1.008 for batches 1, 2, and 3, respectively. This correction subsequently effected the density and cell area estimates but did change cell counts. Predicting sex based on brain region volume or density Performance estimates based on a linear SVM We randomly selected 2/3 of the C57BL/6J brains (n = 184), where each brain is represented by a 532-dimensional vector of either volume or density across regions, and trained the SVM (C = 100). We then tested its performance on the left out 1/3 of brains (n = 97). These random splitting into a training and test sets was repeated 100 times. The performance was 0.910 ± 0.022 and 0.780 ± 0.039 for volume and density, respectively. SVM based on specific regions Each of the regions was ranked according to the average absolute value of its weight in the separating hyperplane across the 100 SVMs for either volume or density. The top 10 regions (i.e., SVM features) were selected and an SVM was trained on the whole dataset (i.e., without a train-test split) while adding one region at a time. Mice Adult, C57BL/6JOlaHsd wildtype mice (Envigo) were housed under standard conditions and provided chow and water ad libitum. Experimental procedures followed the legislation under the Israel Ministry of Health – Animal Experiments Council and were approved by the institutional Animal Experiments Ethics Committees at Technion Israel Institute of Technology (Permit #IL1190819). Histology One male mouse was sacrificed by an overdose of ketamine/xylazine, followed by transcardial perfusion with phosphate-buffered saline (PBS), then 4% PFA in 1× PBS. The brain was extracted, post-fixed 24 hr in 4% PFA at 4°C, cryoprotected in 15%, then 30% sucrose, and finally OCT-embedded, frozen and cryosectioned (20 μm). Sections were collected free-floating to PBS. For staining, we first incubated sections 1 hr in blocking buffer (5% normal goat serum, 3% BSA, 0.3% Triton X-100, in PBS with 0.05% sodium azide), followed by overnight incubation in 1:500 anti-NeuN-PE (Milli-Mark A60, #FCMAB317PE), in the same blocking buffer. After three 10 min washes in PBS, we counterstained 3 min with DAPI (NucBlue, Thermo #R37606), mounted, and coverslipped sections (Fluoromount-G, Invitrogen #00-4958-02), and imaged on a Nikon Ti2 Eclipse inverted fluorescence microscope (×20 objective). STPT on nuclei-stained brains Perfusion Nine adult female C57BL/6JOlaHsd mice were euthanized via intraperitoneal injection with an overdose of ketamine/xylazine mixture (100 and 10 mg/kg, respectively). The mice were transcardially perfused with PBS followed by freshly prepared PFA 4%. Subsequently, brains were removed and postfixed in PFA 4% solution at 4°C for 24 hr and then transferred to PBS containing 0.05% sodium azide at 4°C until used. Nuclear staining The brains were Hoechst stained according to . Briefly, the brains were immersed in staining solution (PBS with 0.05% sodium azide containing 10 ug/ml Hoechst 33342 Trihydrochloride, Trihydrate [Invitrogen #H3570], with 0.1% Triton X-100, and 0–30% w/v sucrose) for 8 d (sucrose concentrations: day 1–10%, day 2–20%, days 3–7–30%, and day 8–0%) at 55°C with gentle shaking and then returned to 4% PFA in PBS for 24 hr, then kept in PBS with 0.05% sodium azide until used. Brains were sent to TissueVision Inc (Boston) for imaging on a TissueCyte 1000 system with ×16 objective (estimated resolution of 0.7 μm per pixel), with a gap of 100 μm between coronal sections. Statistical tests All statistical tests were based on the nonparametric Wilcoxon rank-sum test, thus not assuming a certain underlying distribution. Resulting p-values were then corrected for multiple testing by the Benjamini–Hochberg procedure. Materials availability statement The images and the corresponding atlas registration data were retrieved from the Allen Connectivity Project portal using Allen SDK for Python at https://allensdk.readthedocs.io/en/latest/ . Segmentation-related code is available at https://github.com/delkind/detectron_segmentation (copy archived at ). Brain Explorer: Data viewer for mouse brain cell data We provide a tool for examining analyzed AMBCA brain images. The tool allows visualizing distributions of macroscopic parameters (i.e., density, volume, cell count, etc.) for any region across any subset of the brains, as well as performing statistical tests. Subsets of brains can be based on any combination of strain and sex. Resulting charts can be exported as CSV or PDF files. The viewer is available in https://github.com/delkind/mouse-brain-cell-counting (copy archived at ).
The AMBCA dataset consists of 2992 brains, of which we processed 507 and eventually used 378 in our analysis (the strain and sex breakdown of the brains are shown in ). Each brain consisted of ~140 section images captured every 100 μ m along the anterior–posterior axis using two-photon tomography . Image resolution was 0.35 μ m per pixel. AMBCA post-processed section images for noise removal. Rather than using the red, green, and blue channels occupied by the fluorescent reporter for anterograde connectivity tracing, we used the background channel of the images, as provided by AMBCA, without additional processing, except for converting the RGB image to grayscale.
To detect cells in an image and mark their contour, we used the Detectron2 deep neural network library , which relies on a Mask R-CNN image segmentation model with the ResNet-101 as its backbone.
Training the model required three rounds of manual annotation and training. Initial manual annotation of the dataset and model training We annotated cell contours manually using the VGG Image Annotator software . Initially, we annotated only the hippocampus, which is relatively large and easily discernible. The hippocampus contains subregions of different densities, which we believed would adequately represent the variety of cell densities across the mouse brain. We manually annotated tiles of 312 × 312 pixels ( 109 × 109 μ m ), randomly selected from the hippocampus in five sections of three brains (55 tiles in total). We provided these tiles to the network as training data, together with basic data augmentation (e.g., rotation and brightness changes) . Retraining on hippocampal sections We then applied the trained model to detect cells on a new set of 55 randomly selected hippocampus tiles. We manually corrected the results produced by the network to create a new set of ground truth annotations. Next, we retrained the model from scratch over a combined training set of 110 tiles. Retraining on other regions We subsequently used the trained model to detect cells on random sections of three selected brains. Visual inspection enabled us to select a set of 64 tiles that displayed the least accurate results and annotate them manually. Final training We retrained the model from scratch on the resulting training set of 174 tiles (selected from ~15 sections of ~10 brains). The total number of cells across the training set tiles was 6247, corresponding to 0.008% of the estimated 77 million cells in the whole brain. Technical details We conducted the training with a batch of size 2, a learning rate of 0.00025, with decay, using the Adam optimizer . Training over 174 tiles required ~395,000 iterations and took ~36 hr using a Linux server with 160 Intel Xeon Gold 6248 2.5 GHz CPUs and a Tesla V100S-PCIE-32GB GPU. Evaluating model performance The training process completed when the model converged. The accuracy of the model on the training data was 99.8%, with a false negative rate of 0.4%. To evaluate model performance, we manually annotated 30 additional tiles from the isocortex, medial amygdala (MEA), hypothalamus (HY), and hippocampus (HIP) of 27 brains and compared them with model prediction . We obtained highly accurate results, comparable to the performance over the training data, for segmentation scores such as Jaccard measure , F1 score (harmonic mean of precision and recall), and total errors (i.e., percentage of mislabeled pixels), as well as for detection scores such as accuracy (detected cells divided by total cells) and false positive rate (false positives divided by total cells).
We annotated cell contours manually using the VGG Image Annotator software . Initially, we annotated only the hippocampus, which is relatively large and easily discernible. The hippocampus contains subregions of different densities, which we believed would adequately represent the variety of cell densities across the mouse brain. We manually annotated tiles of 312 × 312 pixels ( 109 × 109 μ m ), randomly selected from the hippocampus in five sections of three brains (55 tiles in total). We provided these tiles to the network as training data, together with basic data augmentation (e.g., rotation and brightness changes) .
We then applied the trained model to detect cells on a new set of 55 randomly selected hippocampus tiles. We manually corrected the results produced by the network to create a new set of ground truth annotations. Next, we retrained the model from scratch over a combined training set of 110 tiles.
We subsequently used the trained model to detect cells on random sections of three selected brains. Visual inspection enabled us to select a set of 64 tiles that displayed the least accurate results and annotate them manually.
We retrained the model from scratch on the resulting training set of 174 tiles (selected from ~15 sections of ~10 brains). The total number of cells across the training set tiles was 6247, corresponding to 0.008% of the estimated 77 million cells in the whole brain.
We conducted the training with a batch of size 2, a learning rate of 0.00025, with decay, using the Adam optimizer . Training over 174 tiles required ~395,000 iterations and took ~36 hr using a Linux server with 160 Intel Xeon Gold 6248 2.5 GHz CPUs and a Tesla V100S-PCIE-32GB GPU.
The training process completed when the model converged. The accuracy of the model on the training data was 99.8%, with a false negative rate of 0.4%. To evaluate model performance, we manually annotated 30 additional tiles from the isocortex, medial amygdala (MEA), hypothalamus (HY), and hippocampus (HIP) of 27 brains and compared them with model prediction . We obtained highly accurate results, comparable to the performance over the training data, for segmentation scores such as Jaccard measure , F1 score (harmonic mean of precision and recall), and total errors (i.e., percentage of mislabeled pixels), as well as for detection scores such as accuracy (detected cells divided by total cells) and false positive rate (false positives divided by total cells).
The trained DNN was applied to 507 brains, as described in detail below.
We divided each section into overlapping tiles sized 312 × 312 pixels, with an overlap of 20 pixels on each side (thus mitigating potential artifacts close to the borders of the tiles). We then applied the trained DNN to detect cells in each tile, resulting in a cell mask (i.e., a Boolean 312 × 312 matrix whose entries are true if the corresponding pixel is part of a detected cell and false otherwise). Next, we stitched the tiles together using a logical OR over overlapping areas, resulting in a single cell mask per section. Subsequently, we performed contour detection to obtain the coordinates of each cell in a section and computed the morphological properties of each cell (i.e., circumference, diameter, and area). Following this analysis step, each section image was represented by a table containing the coordinates and morphological properties of its cells.
We used the AMBA to assign the coordinates of detected cells in each section to their corresponding brain region. But the atlas annotation was too coarse for several regions of interest, that is, CA1, CA2, and CA3 of the hippocampus. The common denominator of these regions was the presence of a dense and a sparse region that were not separated by the atlas (e.g., the pyramidal and stratum regions of CA1, CA2, and CA3). To provide the coordinates of these subregions, we defined a local measure of density referred to as cell ‘coverage’ and used it to cluster the relevant cells into a dense and a sparse region. Briefly, in a window of 64 ×64 pixels centered around each cell we counted the number of pixels that belong to cells, thus assigning a local ‘coverage’ measure (the median cell area was 80 pixels, much smaller than the window around it). We then detected the subregions by clustering the cells according to their ‘coverage’ values. For example, we took the ‘coverage’ values of all CA1 cells and used K-means clustering to split them into two clusters of high and low ‘coverage’ values. In this way, the coordinate of each cell center was assigned to either cluster. We then drew the circumference of the subregions by applying a standard morphological closing operation and discarded spurious small regions.
Until this stage, the analysis provided local, that is, microscopic properties for each detected cell, and assigned cells to a brain region. The next step was to collect cells that belong to each region and estimate their density, the volume of the region, and the total cell count. This required calculating 3D estimates based on the relevant 2D data using the following steps: Estimating cell density per section We used AMBA to label the area of a given region in a section. We assumed that cells belonging to a region are equi-radius spheres whose projection on the 2D section depends on the distance between their centers and the optical plane, and on the optical depth of field . Hence, detected cells on a 2D section s originate from a slab whose volume is v s = a s ∙ 2 R + d , where a s is the area of a region, R is the radius of the cells in the region, and d is the optical depth of field. Cell density per section, ρ s , is given by dividing the number of detected cells by v s . The value of a s is measured by pixels whose size is 0.35 μ m , and d = 1.5 μ m . The value of R was taken as the 90th percentile of measured cell radii in a s . The distribution of cell radii corresponds to the ‘projection’ of the cells on the measured section, together with the optical depth of field. Downstream results of cell count and density significantly depend of the value of R , for example, using the 50th percentile would provide larger estimated cell counts. Yet, rank order of cell counts and densities across regions is independent of the selected value of R . Calculating region volume AMBA provides pixel-wise region annotation for each section, making it possible to calculate the area of a region per section (which is independent of cell segmentation). The 3D volume of a region is given by the sum of region volumes between adjacent sections, estimated by the average of its areas over each section. For example, if a region appears in sections 1, 2, 3, and 4, its volume is the sum of average volumes between sections 1 and 2, 2 and 3, and 3 and 4. Calculating cell counts across adjacent sections and in total Cell counts between the adjacent sections of each region are given by the average densities in those slides multiplied by the volume of the region between these sections. The total cell count of a region is provided by a sum across all relevant sections. Calculating cell densities per region The overall density of each region is given by the total cell count divided by the volume of the region.
We used AMBA to label the area of a given region in a section. We assumed that cells belonging to a region are equi-radius spheres whose projection on the 2D section depends on the distance between their centers and the optical plane, and on the optical depth of field . Hence, detected cells on a 2D section s originate from a slab whose volume is v s = a s ∙ 2 R + d , where a s is the area of a region, R is the radius of the cells in the region, and d is the optical depth of field. Cell density per section, ρ s , is given by dividing the number of detected cells by v s . The value of a s is measured by pixels whose size is 0.35 μ m , and d = 1.5 μ m . The value of R was taken as the 90th percentile of measured cell radii in a s . The distribution of cell radii corresponds to the ‘projection’ of the cells on the measured section, together with the optical depth of field. Downstream results of cell count and density significantly depend of the value of R , for example, using the 50th percentile would provide larger estimated cell counts. Yet, rank order of cell counts and densities across regions is independent of the selected value of R .
AMBA provides pixel-wise region annotation for each section, making it possible to calculate the area of a region per section (which is independent of cell segmentation). The 3D volume of a region is given by the sum of region volumes between adjacent sections, estimated by the average of its areas over each section. For example, if a region appears in sections 1, 2, 3, and 4, its volume is the sum of average volumes between sections 1 and 2, 2 and 3, and 3 and 4.
Cell counts between the adjacent sections of each region are given by the average densities in those slides multiplied by the volume of the region between these sections. The total cell count of a region is provided by a sum across all relevant sections.
The overall density of each region is given by the total cell count divided by the volume of the region.
After calculating the 3D counts and densities across all regions in all brains, we excluded from subsequent analysis regions and whole brains that displayed potentially flawed estimates. We applied the following criteria: We discarded brains displaying dark images We filtered out brains whose median brightness across the whole brain gray matter (‘gray’ region) was lower than 25 (on a scale between 0 and 255). In such cases, all ~140 sections of the brain were excluded from downstream analysis because DNN cell detection was either impossible or provided significantly lower estimates . In total, 51 brains were discarded due to low brightness. We discarded brains displaying an optical or physical artifact resulting in outliers in cell count We noticed that a common optical or physical artifact of resolution degradation caused the DNN to falsely detect large amounts of excess cells (see distinct ‘columns’ in ). We marked cases in which cell count in a region was 3 SDs larger than the median for the region across brains (calculated as M A D ∙ 1.4826 , assuming normal distribution). We discarded brains that included more than three such outlier regions. 81 brains were discarded due to such artifacts. We discarded regions of small volume We filtered out regions whose median volume across brains was smaller than 0.3 m m 3 , or whose median cell count across sections was smaller than 500 . In total, 283 (out of 690 regions in AMBA) were discarded and excluded from all brains (data of these regions do appear in our online tool described below). We discarded regions displaying different estimates in right vs. left hemispheres Cell count estimates in the right and left hemispheres served as a proxy for technical noise. We calculated cell counts per region using each hemisphere independently. If the average difference in cell count between hemispheres was higher than 15.5% of the total cell count for that region, we excluded the case from downstream analysis . 33 regions were omitted due to right–left differences. We discarded regions displaying a correlation between cell count and image brightness We excluded regions exhibiting strong correlation (>0.3) between brightness and cell count because we assumed that in this case cell count was affected by the inability of the model to discern the cells when the brightness was too low. 30 regions were discarded due to such correlations. In sum, we processed 507 brains, of which 132 were fully discarded. Of 690 regions in AMBA, 346 were discarded completely and another 107 were partially discarded.
We filtered out brains whose median brightness across the whole brain gray matter (‘gray’ region) was lower than 25 (on a scale between 0 and 255). In such cases, all ~140 sections of the brain were excluded from downstream analysis because DNN cell detection was either impossible or provided significantly lower estimates . In total, 51 brains were discarded due to low brightness.
We noticed that a common optical or physical artifact of resolution degradation caused the DNN to falsely detect large amounts of excess cells (see distinct ‘columns’ in ). We marked cases in which cell count in a region was 3 SDs larger than the median for the region across brains (calculated as M A D ∙ 1.4826 , assuming normal distribution). We discarded brains that included more than three such outlier regions. 81 brains were discarded due to such artifacts.
We filtered out regions whose median volume across brains was smaller than 0.3 m m 3 , or whose median cell count across sections was smaller than 500 . In total, 283 (out of 690 regions in AMBA) were discarded and excluded from all brains (data of these regions do appear in our online tool described below).
Cell count estimates in the right and left hemispheres served as a proxy for technical noise. We calculated cell counts per region using each hemisphere independently. If the average difference in cell count between hemispheres was higher than 15.5% of the total cell count for that region, we excluded the case from downstream analysis . 33 regions were omitted due to right–left differences.
We excluded regions exhibiting strong correlation (>0.3) between brightness and cell count because we assumed that in this case cell count was affected by the inability of the model to discern the cells when the brightness was too low. 30 regions were discarded due to such correlations. In sum, we processed 507 brains, of which 132 were fully discarded. Of 690 regions in AMBA, 346 were discarded completely and another 107 were partially discarded.
While analyzing the Allen images, we noticed that the experiments were performed in several batches (based on experiment ID), and that there was a small difference in the total brain volume among batches, yet the total cell did not display a batch effect . In order to correct this effect, we multiplied each brain volume by a batch-specific factor such that the batch median volume 8 will be 380 mm 3 . These factors were 1.064, 1.032, and 1.008 for batches 1, 2, and 3, respectively. This correction subsequently effected the density and cell area estimates but did change cell counts.
Performance estimates based on a linear SVM We randomly selected 2/3 of the C57BL/6J brains (n = 184), where each brain is represented by a 532-dimensional vector of either volume or density across regions, and trained the SVM (C = 100). We then tested its performance on the left out 1/3 of brains (n = 97). These random splitting into a training and test sets was repeated 100 times. The performance was 0.910 ± 0.022 and 0.780 ± 0.039 for volume and density, respectively. SVM based on specific regions Each of the regions was ranked according to the average absolute value of its weight in the separating hyperplane across the 100 SVMs for either volume or density. The top 10 regions (i.e., SVM features) were selected and an SVM was trained on the whole dataset (i.e., without a train-test split) while adding one region at a time.
We randomly selected 2/3 of the C57BL/6J brains (n = 184), where each brain is represented by a 532-dimensional vector of either volume or density across regions, and trained the SVM (C = 100). We then tested its performance on the left out 1/3 of brains (n = 97). These random splitting into a training and test sets was repeated 100 times. The performance was 0.910 ± 0.022 and 0.780 ± 0.039 for volume and density, respectively.
Each of the regions was ranked according to the average absolute value of its weight in the separating hyperplane across the 100 SVMs for either volume or density. The top 10 regions (i.e., SVM features) were selected and an SVM was trained on the whole dataset (i.e., without a train-test split) while adding one region at a time.
Adult, C57BL/6JOlaHsd wildtype mice (Envigo) were housed under standard conditions and provided chow and water ad libitum. Experimental procedures followed the legislation under the Israel Ministry of Health – Animal Experiments Council and were approved by the institutional Animal Experiments Ethics Committees at Technion Israel Institute of Technology (Permit #IL1190819).
One male mouse was sacrificed by an overdose of ketamine/xylazine, followed by transcardial perfusion with phosphate-buffered saline (PBS), then 4% PFA in 1× PBS. The brain was extracted, post-fixed 24 hr in 4% PFA at 4°C, cryoprotected in 15%, then 30% sucrose, and finally OCT-embedded, frozen and cryosectioned (20 μm). Sections were collected free-floating to PBS. For staining, we first incubated sections 1 hr in blocking buffer (5% normal goat serum, 3% BSA, 0.3% Triton X-100, in PBS with 0.05% sodium azide), followed by overnight incubation in 1:500 anti-NeuN-PE (Milli-Mark A60, #FCMAB317PE), in the same blocking buffer. After three 10 min washes in PBS, we counterstained 3 min with DAPI (NucBlue, Thermo #R37606), mounted, and coverslipped sections (Fluoromount-G, Invitrogen #00-4958-02), and imaged on a Nikon Ti2 Eclipse inverted fluorescence microscope (×20 objective).
Perfusion Nine adult female C57BL/6JOlaHsd mice were euthanized via intraperitoneal injection with an overdose of ketamine/xylazine mixture (100 and 10 mg/kg, respectively). The mice were transcardially perfused with PBS followed by freshly prepared PFA 4%. Subsequently, brains were removed and postfixed in PFA 4% solution at 4°C for 24 hr and then transferred to PBS containing 0.05% sodium azide at 4°C until used. Nuclear staining The brains were Hoechst stained according to . Briefly, the brains were immersed in staining solution (PBS with 0.05% sodium azide containing 10 ug/ml Hoechst 33342 Trihydrochloride, Trihydrate [Invitrogen #H3570], with 0.1% Triton X-100, and 0–30% w/v sucrose) for 8 d (sucrose concentrations: day 1–10%, day 2–20%, days 3–7–30%, and day 8–0%) at 55°C with gentle shaking and then returned to 4% PFA in PBS for 24 hr, then kept in PBS with 0.05% sodium azide until used. Brains were sent to TissueVision Inc (Boston) for imaging on a TissueCyte 1000 system with ×16 objective (estimated resolution of 0.7 μm per pixel), with a gap of 100 μm between coronal sections.
Nine adult female C57BL/6JOlaHsd mice were euthanized via intraperitoneal injection with an overdose of ketamine/xylazine mixture (100 and 10 mg/kg, respectively). The mice were transcardially perfused with PBS followed by freshly prepared PFA 4%. Subsequently, brains were removed and postfixed in PFA 4% solution at 4°C for 24 hr and then transferred to PBS containing 0.05% sodium azide at 4°C until used.
The brains were Hoechst stained according to . Briefly, the brains were immersed in staining solution (PBS with 0.05% sodium azide containing 10 ug/ml Hoechst 33342 Trihydrochloride, Trihydrate [Invitrogen #H3570], with 0.1% Triton X-100, and 0–30% w/v sucrose) for 8 d (sucrose concentrations: day 1–10%, day 2–20%, days 3–7–30%, and day 8–0%) at 55°C with gentle shaking and then returned to 4% PFA in PBS for 24 hr, then kept in PBS with 0.05% sodium azide until used. Brains were sent to TissueVision Inc (Boston) for imaging on a TissueCyte 1000 system with ×16 objective (estimated resolution of 0.7 μm per pixel), with a gap of 100 μm between coronal sections.
All statistical tests were based on the nonparametric Wilcoxon rank-sum test, thus not assuming a certain underlying distribution. Resulting p-values were then corrected for multiple testing by the Benjamini–Hochberg procedure.
The images and the corresponding atlas registration data were retrieved from the Allen Connectivity Project portal using Allen SDK for Python at https://allensdk.readthedocs.io/en/latest/ . Segmentation-related code is available at https://github.com/delkind/detectron_segmentation (copy archived at ).
We provide a tool for examining analyzed AMBCA brain images. The tool allows visualizing distributions of macroscopic parameters (i.e., density, volume, cell count, etc.) for any region across any subset of the brains, as well as performing statistical tests. Subsets of brains can be based on any combination of strain and sex. Resulting charts can be exported as CSV or PDF files. The viewer is available in https://github.com/delkind/mouse-brain-cell-counting (copy archived at ).
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Tridimensional Analysis of Incisive Canal and Upper Central Incisor Approximation
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28cdc630-cdda-4533-b024-849f297e0d09
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10213738
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Dental[mh]
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The localisation of the upper incisors might be considered an important point of interest in orthodontic diagnosis and treatment planning. The posterior anatomical limit for retracting upper incisors has generally been viewed as the palatal cortical plate. , Nevertheless, recent craniofacial anatomical investigations have demonstrated that the cortical bone surrounding the incisive canal (IC) is dense and has a closer relationship with roots of upper central incisors (U1) than does the palatal cortical plate. Due to IC closeness to the U1, the potential of IC surgical invasion during dental procedures has been recorded in previous studies. , The idea of the “envelope of discrepancy” presented by Proffit and Ackerman clarifies the sequences conceivable with various sorts of orthodontic treatments, including camouflage orthodontics, growth modification, and surgical orthodontics. , It has been imagined that the measure of changes workable for the upper incisors with orthodontic treatment alone are more in retraction and extrusion than protraction and intrusion (7, 4, 2, and 2 mm, respectively). Despite that the morphologic parameters of IC are common, its exact position corresponding to the U1 is not well reported in the 3-dimensional (3D) era. Nakada et al found that posterior movement of the root peak during orthodontic retraction may prompt its contact with the IC cortical plate and cause apical root resorption. Chung et al found the root of one of the U1 was in contact with the cortical plate of the IC, and broad root resorption was noticed after en-masse retraction. Cho et al found that more than 60% of studied cases have recorded greater linear width of IC as compared to the inter-root distance of the U1. A recent study found 53% of studied clinical cases that required U1 retraction greater than 4 mm have shown invasion of IC by the U1 roots following the retraction. Moreover, this process resulted in about a 2.39-mm root resorption, and the severe invasion revealed a 6.2-mm root resorption. Vertical facial morphology is significant for orthodontists because it influences the objectives and the manner of orthodontic treatment by affecting the growth forecast, the anchorage framework, bite power, and other functions. The aforementioned findings considering the U1 and IC relationship raised the importance of reevaluating the broadened dynamics of the envelope of discrepancy using modern 3-D technologies. Further, reevaluation is improved by including segregation of this envelope according to facial type, as it is one of the determinants of orthodontic diagnosis and treatment planning as well as orthodontic biomechanics. Therefore, the present study sought to estimate the morphologic parameters and the approximate location of the incisive canal regarding the upper central incisors and, hence, evaluate the safety zone of orthodontic retraction mentioned in the envelope of discrepancy as per the different vertical facial growth patterns of Chinese people.
Study design and samples Cone-beam computed tomography (CBCT) scans analysed in this cross-sectional study were derived consecutively from the archive of the Hospital of Stomatology, Lanzhou University, between 2014 and 2020, after receiving ethical committee approval from the clinical scientific research of the School of Stomatology, Lanzhou University (No. LZUKQ-2020-20). The data that support the findings of this study are available from the corresponding author upon reasonable request. The sample size was determined at alpha 5% and power of 90% utilising G power software (University of Dusseldorf) and one previous study. As a result, 207 cases were required to be included in the present study. Patient selection was established according to the following inclusion criteria: (1) 18–30 years old, (2) availability of high-quality lateral cephalograms and CBCT images, and (3) skeletal class I relationship (Point A to Nasion to Point B angle from 0° to 4°). Patients who had any of the following criteria were excluded: (1) large maxillary midline diastema, (2) maxillary midline shift ≥2 mm, (3) facial asymmetries (deviation of the mandible), (4) respiratory problems, (5) previous orthodontic/prosthetic treatment, (6) evidence of root resorption, (7) missing teeth (except third molars), and (8) tooth/bone anomalies in the upper jaw midline area. All patients signed consent forms after being given written information. The facial groups were identified according to the parameters of Fields et al. Patients were included in the normodivergent group when the mandibular plane to SN plane angle (GoMe-SN) is 27° to 37°, palatal plane to mandibular plane angle (PP-MP) is 19° to 31°, and lower facial height (ANS-Me) is between 64 and 72 mm. For patients to be included in the hypodivergent group, GoMe-SN is ˂27°, PP-MP is ˂19°, and ANS-Me is ˂64 mm. The hyperdivergent group was identified based on the following: GoMe-SN >37°, PP-MP >31°, and ANS-Me >72 mm. CBCT analysis The CBCT images were obtained using I-CAT Imaging System (Imaging Sciences International Inc.) under standard protocols by a specialist radiographer; field of view was 17 cm; exposure parameter setting was 120 kVp, 20.27 MAs, and 14.9 s; and voxel size was 0.4 mm. All the images were realigned corresponding to Frankfort-horizontal (FH) plane in the sagittal plane. The internal volume of the IC was segmented using 3D segmentation and image analysis software (Mimics 21, Materialise) as described elsewhere, and 3D volumetric models of the IC were generated from these segmentations ( , part A). The other 3D parameters and linear measurements were measured utilising Invivo Dental imaging software (version 6, Anatomage) as described by Al-Rokhami et al. The linear measurements were taken at 3 levels situated on the sagittal plane: (1) the lowest point of the incisive canal buccal wall (opening level, H1), (2) midlevel between the opening level and the root apex of the upper central incisors (midlevel, H2), and (3) the root apex of the upper central incisors (root apex level, H3) ( , part B). These landmarks and measurements are described in the (partsC and D). Statistical analysis The statistical analysis was calculated utilising IBM SPSS Statistics V. 25.0 (IBM Corp.). Descriptive data were presented as mean and standard deviation (SD) or as frequency and percentage where appropriate. Since the data were normally distributed, one-way analysis of variance followed by Tukey test were applied to estimate the differences amongst the facial groups. Independent t test was used to compare variances between sexes. A P value <.05 was considered to represent statistical significance. All measures were conducted by 2 trained investigators. The intra-class correlation coefficient by Cronbach's alpha test has ensured interobserver reliability, which ranged from 0.980 to 0.999.
Cone-beam computed tomography (CBCT) scans analysed in this cross-sectional study were derived consecutively from the archive of the Hospital of Stomatology, Lanzhou University, between 2014 and 2020, after receiving ethical committee approval from the clinical scientific research of the School of Stomatology, Lanzhou University (No. LZUKQ-2020-20). The data that support the findings of this study are available from the corresponding author upon reasonable request. The sample size was determined at alpha 5% and power of 90% utilising G power software (University of Dusseldorf) and one previous study. As a result, 207 cases were required to be included in the present study. Patient selection was established according to the following inclusion criteria: (1) 18–30 years old, (2) availability of high-quality lateral cephalograms and CBCT images, and (3) skeletal class I relationship (Point A to Nasion to Point B angle from 0° to 4°). Patients who had any of the following criteria were excluded: (1) large maxillary midline diastema, (2) maxillary midline shift ≥2 mm, (3) facial asymmetries (deviation of the mandible), (4) respiratory problems, (5) previous orthodontic/prosthetic treatment, (6) evidence of root resorption, (7) missing teeth (except third molars), and (8) tooth/bone anomalies in the upper jaw midline area. All patients signed consent forms after being given written information. The facial groups were identified according to the parameters of Fields et al. Patients were included in the normodivergent group when the mandibular plane to SN plane angle (GoMe-SN) is 27° to 37°, palatal plane to mandibular plane angle (PP-MP) is 19° to 31°, and lower facial height (ANS-Me) is between 64 and 72 mm. For patients to be included in the hypodivergent group, GoMe-SN is ˂27°, PP-MP is ˂19°, and ANS-Me is ˂64 mm. The hyperdivergent group was identified based on the following: GoMe-SN >37°, PP-MP >31°, and ANS-Me >72 mm.
The CBCT images were obtained using I-CAT Imaging System (Imaging Sciences International Inc.) under standard protocols by a specialist radiographer; field of view was 17 cm; exposure parameter setting was 120 kVp, 20.27 MAs, and 14.9 s; and voxel size was 0.4 mm. All the images were realigned corresponding to Frankfort-horizontal (FH) plane in the sagittal plane. The internal volume of the IC was segmented using 3D segmentation and image analysis software (Mimics 21, Materialise) as described elsewhere, and 3D volumetric models of the IC were generated from these segmentations ( , part A). The other 3D parameters and linear measurements were measured utilising Invivo Dental imaging software (version 6, Anatomage) as described by Al-Rokhami et al. The linear measurements were taken at 3 levels situated on the sagittal plane: (1) the lowest point of the incisive canal buccal wall (opening level, H1), (2) midlevel between the opening level and the root apex of the upper central incisors (midlevel, H2), and (3) the root apex of the upper central incisors (root apex level, H3) ( , part B). These landmarks and measurements are described in the (partsC and D).
The statistical analysis was calculated utilising IBM SPSS Statistics V. 25.0 (IBM Corp.). Descriptive data were presented as mean and standard deviation (SD) or as frequency and percentage where appropriate. Since the data were normally distributed, one-way analysis of variance followed by Tukey test were applied to estimate the differences amongst the facial groups. Independent t test was used to compare variances between sexes. A P value <.05 was considered to represent statistical significance. All measures were conducted by 2 trained investigators. The intra-class correlation coefficient by Cronbach's alpha test has ensured interobserver reliability, which ranged from 0.980 to 0.999.
Descriptive statistics A total of 207 adults were enrolled in the present study, with a mean age of 23.41 ± 3.51 years, comprising 104 (50.2%) male and 103 (49.8%) female patients. An equal number of cases were included in each facial group (n = 69, 33.3%). The distribution of male and female patients amongst the normodivergent, hypodivergent, and hyperdivergent groups were 36 (52.5%) and 33 (47.8%), 37 (53.6%) and 32 (46.4%), and 31 (44.9%) and 38 (55.1), respectively. The sagittal skeletal relationship, U1-SN angle, and vertical facial measures per each facial group are reported in . Measures of IC and U1 inter-root distances The IC has recorded volumetric values around 117.9 ± 25.97, 117.7 ± 29.68, and 130.6 ± 50.74 mm 3 amongst the normodivergent, hypodivergent, and hyperdivergent groups, respectively. The IC width (Cl-Cl) showed nonsignificant differences amongst the normodivergent, hypodivergent, and hyperdivergent groups ( ). The sexes also showed nonsignificant differences in terms of IC volume and width ( ). The IC width was decreased from H1 to H3 ( P ˂ .001) similarly in the facial groups and sexes ( ). No significant differences were found in either the inter-root distance (Rm-Rm) or posterior inter-root distance (Rp-Rp) amongst the facial groups, except at Rp-Rp-H1 ( ). However, the male patients in the hypodivergent group showed significantly higher Rm-Rm and Rp-Rp ( P ˂ .05) than female patients at both the H1 and H2 levels ( ). On the other hand, the Rm-Rm was increased from H1 to H3 ( P ˂ .001), whereas the Rp-Rp was decreased ( P ˂ .001) ( ). At H1, the rate of cases with IC width greater than the inter-root distance (Rm-Rm) was comparable in the hyperdivergent and hypodivergent groups (94.4%), whilst the normodivergent group showed a slightly lower rate (86.1%). On the other hand, at H2, the hyperdivergent group recorded a higher rate of IC width greater than Rm-Rm (43.1%), whilst the normodivergent and hypodivergent groups have recorded rates around 32% and 37.5%, respectively. At both H1 and H2 levels, male patients showed almost lower rate of IC width greater than Rm-Rm in all the facial groups ( ). U1 and IC sagittal distances The Cl-Root, Rm-Canal, and Rm-Cat measures were considerably higher in the hypodivergent group (4.60 ± 0.83 mm) than in the normodivergent and hyperdivergent groups (4.00 ± 0.82 and 3.60 ± 0.80 mm, respectively) at all the vertical levels ( P ˂ .001) ( ). Besides, the male patients showed significantly higher Cl-Root, Rm-Canal, and Rm-Cat distances compared with female patients only within the hypodivergent group ( ). Amongst all the facial groups and both sexes, the Cl-Root was significantly higher at H2 than at H1, whilst the Rm-Cat was significantly higher at H1 and H2 than at H3 ( ).
A total of 207 adults were enrolled in the present study, with a mean age of 23.41 ± 3.51 years, comprising 104 (50.2%) male and 103 (49.8%) female patients. An equal number of cases were included in each facial group (n = 69, 33.3%). The distribution of male and female patients amongst the normodivergent, hypodivergent, and hyperdivergent groups were 36 (52.5%) and 33 (47.8%), 37 (53.6%) and 32 (46.4%), and 31 (44.9%) and 38 (55.1), respectively. The sagittal skeletal relationship, U1-SN angle, and vertical facial measures per each facial group are reported in .
The IC has recorded volumetric values around 117.9 ± 25.97, 117.7 ± 29.68, and 130.6 ± 50.74 mm 3 amongst the normodivergent, hypodivergent, and hyperdivergent groups, respectively. The IC width (Cl-Cl) showed nonsignificant differences amongst the normodivergent, hypodivergent, and hyperdivergent groups ( ). The sexes also showed nonsignificant differences in terms of IC volume and width ( ). The IC width was decreased from H1 to H3 ( P ˂ .001) similarly in the facial groups and sexes ( ). No significant differences were found in either the inter-root distance (Rm-Rm) or posterior inter-root distance (Rp-Rp) amongst the facial groups, except at Rp-Rp-H1 ( ). However, the male patients in the hypodivergent group showed significantly higher Rm-Rm and Rp-Rp ( P ˂ .05) than female patients at both the H1 and H2 levels ( ). On the other hand, the Rm-Rm was increased from H1 to H3 ( P ˂ .001), whereas the Rp-Rp was decreased ( P ˂ .001) ( ). At H1, the rate of cases with IC width greater than the inter-root distance (Rm-Rm) was comparable in the hyperdivergent and hypodivergent groups (94.4%), whilst the normodivergent group showed a slightly lower rate (86.1%). On the other hand, at H2, the hyperdivergent group recorded a higher rate of IC width greater than Rm-Rm (43.1%), whilst the normodivergent and hypodivergent groups have recorded rates around 32% and 37.5%, respectively. At both H1 and H2 levels, male patients showed almost lower rate of IC width greater than Rm-Rm in all the facial groups ( ).
The Cl-Root, Rm-Canal, and Rm-Cat measures were considerably higher in the hypodivergent group (4.60 ± 0.83 mm) than in the normodivergent and hyperdivergent groups (4.00 ± 0.82 and 3.60 ± 0.80 mm, respectively) at all the vertical levels ( P ˂ .001) ( ). Besides, the male patients showed significantly higher Cl-Root, Rm-Canal, and Rm-Cat distances compared with female patients only within the hypodivergent group ( ). Amongst all the facial groups and both sexes, the Cl-Root was significantly higher at H2 than at H1, whilst the Rm-Cat was significantly higher at H1 and H2 than at H3 ( ).
There is always a need for continuous updating of the main parameters that limit orthodontic tooth movement, especially with the vast development of 3D imaging. One of these anatomical parameters, which was ignored during the 2D era, is the IC proximity to the upper incisors. The possible effect of the IC and U1 approximation was at the assumption level a few years ago. However, with emergence of 3D analysis and the surprising results found in the literature concerning the U1-IC relationship, it is time to step back and reevaluate the limits of maxillary dentoalveolar retraction mentioned in the envelope of discrepancy. Overall, from H1 to H3, the IC width was considerably decreased, whereas the inter-root distance was significantly increased, similarly amongst the 3 facial groups and sexes. Similar findings were reported in other studies. , The IC 3D volume and linear width showed nonsignificant differences between the facial groups and sexes. However, the rates of cases with IC width larger than U1 inter-root distance were slightly higher in the hyperdivergent group and female patients than in the other facial groups and male patients, which increases the risk of U1 and IC approximation or invasion. A previous study reported that in cases with larger pretreatment IC volume, there has been demonstrated IC invasion after retraction of upper incisors. Additionally, during treatment, changes in root parallelism due to poor bracket positioning or addition of root torque during retraction may likewise lead to root convergence of the U1 and then reducing the inter-root distances. The maximum amount of upper central incisors retraction is 7 mm, as reported in the “envelope of discrepancy,” and the modern improvement of skeletal anchorage has also extended the limits of orthodontic tooth movement. However, a recent study had contrary and shocking findings: 53% of cases of maximum retraction (>4 mm) showed IC invasion by the incisor roots. As a result, a different degree of root resorption was observed with an average of 2.39 mm, and the largest degree of invasion demonstrated 6.2 mm of root resorption. Further, the amount of root resorption was significantly higher in the IC invasion group than non-invasion group (2.39 mm vs 0.82 mm, P < .0001). Nonetheless, the facial type of the included patients was not reported in their study. The present findings revealed that the maximum biological (safe) sagittal distances between the U1 and IC were 4.8, 5.4, and 4.4 mm amongst the normodivergent, hypodivergent, and hyperdivergent groups, respectively: somewhat less than the traditional guideline of envelope of discrepancy (7 mm). , Besides, the normodivergent, hypodivergent, and hyperdivergent groups have recorded various overall mean sagittal distances between the U1 and IC: 4.00 ± 0.82, 4.60 ± 0.83, and 3.60 ± 0.80 mm, respectively. Hence, the hypodivergent group reported greater sagittal distance between the IC and upper centrals. This finding could be illustrated as the hypodivergent facial growth pattern having thicker alveolar bone than the other facial patterns, as reported in previous studies. , , Although the sagittal distance between the U1 roots and the IC has yet to be identified, 3D assessments during orthodontic diagnosis and close observation of the incisor roots throughout treatment would be beneficial in preventing possible complications, particularly in patients requiring maximum retraction. Among all the facial groups, the male patients have shown almost greater sagittal distances between the ICs and upper centrals as compared to females, which appeared signifcant only in the hypodivergent facial group. Hence, the female patients, even those with a hypodivergent facial pattern, were more likely to have UI and IC approximation with subsequent sequelae than were male patients. Overall, the present results were consistent with the findings of Klinge et al, who reported that the male patients had wider alveolar bone cross-sections than female patients. External apical root resorption is one of the most common potential issues provoked during orthodontic therapy, and it has challenged orthodontists for a long time. The radiographic estimation has revealed the incidence of root resorption in a range from 48% to 66%. , Interestingly, the incidence of orthodontically induced root resorption is far greater in the upper centrals, even with their larger tooth dimensions. Multiple factors have been implicated in the incidence of orthodontically induced root resorption, including hereditary impact, age, sex, root morphology, root vicinity to the cortical bone, alveolar bone density, type of malocclusion, treatment length, level of applied force, and extent of apical root movement. However, no clear reasons for resorption were truly identified. Even though remodelling of IC was reported in some previous studies, rates of contact or invasion of the U1 roots to IC were fairly high after maximum anterior retraction. Further, contact of upper central roots and IC was also found after minor incisor movements. Chung et al reported that 24% of participants showed IC remodelling after maximum retraction, and a more recent study by Yu et al reported that 11.4% of participants showed some signs of IC remodelling. Moreover, the remodelling group still demonstrated apical root resorption, even if it was less than in the nonremodelling group. One of the limitations of this study is its cross-sectional nature and the associated limitations. Thereby, further interventional prospective clinical studies are recommended to 3-dimensionally evaluate the possible side effects of IC invasion and maximum incisor retraction to comprehensively determine the limits in the envelope of discrepancy in the 3D era. Another limitation was the small range of malocclusion types evaluated. Hence further studies that encompass different variants of malocclusion, particularly those needing camouflaged treatment, is needed to shed further light on the remdelling capacity of the IC wall after orthodontic tooth movement.
The maximum sagittal distances between the U1 and IC were 4.8, 5.4, and 4.4 mm amongst the normodivergent, hypodivergent, and hyperdivergent groups, respectively. The sagittal distances between U1 and IC were associated with the facial growth pattern, which was significantly higher in the hypodivergent group than in the normodivergent and hyperdivergent groups. Besides, there were relative differences in the sagittal distances between sexes, particularly in the hypodivergent group. The present CBCT-based findings have revised the traditional envelope of discrepancy and provided some insights for clinical practice, particularly amongst Chinese populations.
This research was supported by The Natural Foundation (1208RJZA236), The Key Technology Plan Program (20YF8FA071), and The Key Technology Support Program (1604FKCA089), of Gansu Province, China. The aforementioned funds have no any role in any aspect of the present study. All authors declare that they have no conflict of interest or any financial relationship related to this study.
RKA and KAS contributed equally to this work.
None disclosed.
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National Oral Health Policy and Financing and Dental Health Status in 19 Countries
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dc16a644-a8de-4a13-8f36-098ad16ebe67
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10213794
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Dental[mh]
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Dental caries in permanent teeth is one of the most common global health issues despite being preventable and treatable in early stages. , The high prevalence of caries is partly because many dental services have often been dismissed as a nonessential component of health systems. Consequently, these services are excluded from the global movement towards universal health coverage in most countries. , Ample evidence supports the positive effect of preventive measures on improving oral health outcomes. Preventive oral health services—such as dental screenings, counseling, and application of topical fluoride—reduce kindergarteners’ decayed, missing, and filled teeth (DMFT) indexes in the United States. Policy reform to increase the coverage for dental sealants in children resulted in a reduction in DMFT indexes in children in South Korea. Preventive dental measures reduce the progress of caries development and invasive treatments needed for more extensive decay and may be cost-effective and cost-saving. For example, Medicare beneficiaries who used preventive dental care had more dental visits but lower dental expenses than beneficiaries who sought treatment solely for problems. A cost-effectiveness analysis evaluated sealing all permanent molars (SA), sealing based on levels of risk (RBS), and sealing none (SN) and found that the RBS strategy improved clinical outcomes—in terms of cavity-free months—and saved money, when compared to the SN strategy. The SA strategy improved outcomes further but with additional costs. Given the evidence on the benefits of preventive dental care, health systems should promote disease prevention, allow for early detection, and provide suitable intervention. However, the absence of preventive dental care regulation combined with inadequate insurance coverage contributes to a low utilisation rate of preventive dental services in many countries. National and subnational regulation of the frequency and extent of preventive dental care should be associated with an improvement in dental outcomes. Some studies have examined this relationship between regulation and dental outcomes in the adult population. , , However, no quantitative dental policy analysis of paediatric oral health care has been conducted. This mixed-methods study has 2 objectives: (1) to evaluate the roles of legal policy, access, and regulation in improving countries’ oral health systems and outcomes and (2) to assess the association between national oral health policies and guidelines—specifically for preventive care—and oral health expenditure and outcomes in selected member countries of the Organisation for Economic Co-operation and Development (OECD). This group of high-income and mostly democratic countries is equipped with the financial resources to provide well-rounded preventive oral health care. We hypothesize that countries with more regulations requiring preventive care are more likely to be associated with better oral health outcomes and lower oral health expenditures. Findings on the association between national oral health policies and outcomes will enable governments and policymakers to make evidence-based decisions when implementing health system reforms.
Variables and data sources DMFT indexes We utilised countries’ DMFT indexes as proxy variables for oral health outcomes. DMFT is the sum of a person's decayed, missing, and filled permanent teeth. The DMFT index is a well-established measure of caries burden in oral epidemiology. A higher DMFT index indicates worse caries burden and further deterioration or oral health. , We extracted DMFT data for children aged 12 to 18 years, allowing assessment of national regulations and guidelines which target preventive care for children up to the age of 18, from the WHO Oral Health Country/Area Profile Project (CAPP). See for more details. Oral health expenditure We extracted oral health expenditure data from the OECD Health Expenditure and Financing database. The oral health expenditure considers all dental-sector curative care services provided in an outpatient setting, measured as a percentage of each country's gross domestic product (GDP). The OECD database characterises dental-sector curative care services as all services related to oral health, gums, and teeth and other related disorders; this group of services includes most dental services provided in an outpatient setting. Details of the extraction and missing observations can be found in . Oral Health Policy and Guidelines Country-specific research was conducted for each of the OECD member countries, and countries were assessed based on their level of oral health prevention policy for children. All searches were first conducted in English and then in each country's native language to ensure that all the available data were being accessed; translations for search terms, websites, and documents were conducted using Google Translate. See for search terms. Evaluation of all the relevant policy and guideline documents indicated that some countries have legal policies mandating dental care for children, some have insurance schemes ensuring free services for children, and some have published clinical guidelines on providing preventive care. Therefore, we generated binary variables to indicate the availability of the following types of policies: mandatory dental services for children; availability of free dental services for children; and available guidelines for children's preventive services . The category mandatory dental services for children signifies whether a particular country has an existing law or act that specifically mandates children receive dental care including preventive services like oral health screenings, instruction, and fluoridation. This category indicates that a specific law indicating that the entire population should have access these services but does not measure the actual frequency of utilisation amongst the population. The category of availability of free dental service for children signifies whether a particular country's mandatory national insurance scheme or national public service includes preventive services for children in its basic dental coverage. The category of available guidelines for children's preventive services signifies whether a particular country's government or health ministry has published clinical practice guidelines for dentists on providing preventive care to children. This categorisation system allowed us to assess each country's level of regulation of children's dental services, predominantly preventive. Examples of these services are application of sealants, oral health screenings, fluoride varnish, and oral hygiene instruction. See for additional details. Countries We focused on OECD member countries as these are high-income countries are more likley to have the resources to provide comprehensive dental care. The OECD works with governments and policymakers to establish evidence-based international standards ; we expected these countries to have standardised and accessible policy documents. A list of OECD countries excluded from the study due to insufficient public data can be found in and . After accounting for these limitations, we included 19 of the 38 OECD member countries. Limitations When analysing oral health expenditure data, we focused on dental outpatient care and excluded dental inpatient procedures; the OECD database does not distinguish between dental inpatient services and nondental inpatient services. Whilst some costs may not be captured, most dental care—especially preventive care and caries treatment—are outpatient procedures. Additionally, health ministry websites often listed existing laws and regulations without indicating how these specific policies were enforced. This study therefore does not account for the frequency of utilisation of the services and only captures the fact that a country addresses the need for the services in its legislation. The policy data collection component of this study was conducted utilising Google Translate, and there is the possibility that certain text was not translated accurately. A more comprehensive database that monitors international oral health policy updates across languages is needed to allow for more detailed analysis. This study averaged the DMFT indexes of children from the age of 12 to 18 years and assumed that caries experience does not change that significantly over this time period. , We reasoned that this age range is sufficiently narrow, encompassing only children within similar life stages and allowing for inclusion of additional data points for analysis. As caries experience does not develop steadily over one's lifetime, many argue that DMFT cannot be averaged for the entire population of a country. This argument stems from the significant differences in the caries experience of children and of an older population, especially because DMFT is an irreversible index. Nevertheless, despite its limitations, DMFT has been regarded as one of the most prominent indicators of caries experience. , , Both the OECD Health Expenditure and Financing and the WHO CAPP databases had missing values for certain years (detailed in ). Dental public health research particularly suffers from incomplete datasets, and more standardised data collection methods for oral health measures are needed globally. We recognise that our results may be biased towards countries that devoted more resources to dental care and country-level data collection. It is critical to underline that the trends may be different depending on a country's income level and regime type. We recognize that in most developed countries, dental caries has a skewed distribution, affecting vulnerable groups more severely. The within-country variation of DMFT is an important topic that is beyond scope of this study and should be further examined in the future; in this study, we focused our efforts on investigating country-level policy and DMFT variations. There are many confounding variables that affect the population-level DMFT index, such as socioeconomic status, race, and education level; these variables and their relationship with DMFT should be systematically examined when data become available. Statistical methods The statistical software STATA version 17.0 was used to code, clean, and analyse the datasets. Descriptive statistics were generated for all indicator variables. We calculated bivariate regression analysis between each variable to examine the strength of associations between all indicator variables. Scatterplots were used to evaluate the trends amongst DMFT, oral health expenditure, and level of oral policy.
DMFT indexes We utilised countries’ DMFT indexes as proxy variables for oral health outcomes. DMFT is the sum of a person's decayed, missing, and filled permanent teeth. The DMFT index is a well-established measure of caries burden in oral epidemiology. A higher DMFT index indicates worse caries burden and further deterioration or oral health. , We extracted DMFT data for children aged 12 to 18 years, allowing assessment of national regulations and guidelines which target preventive care for children up to the age of 18, from the WHO Oral Health Country/Area Profile Project (CAPP). See for more details. Oral health expenditure We extracted oral health expenditure data from the OECD Health Expenditure and Financing database. The oral health expenditure considers all dental-sector curative care services provided in an outpatient setting, measured as a percentage of each country's gross domestic product (GDP). The OECD database characterises dental-sector curative care services as all services related to oral health, gums, and teeth and other related disorders; this group of services includes most dental services provided in an outpatient setting. Details of the extraction and missing observations can be found in . Oral Health Policy and Guidelines Country-specific research was conducted for each of the OECD member countries, and countries were assessed based on their level of oral health prevention policy for children. All searches were first conducted in English and then in each country's native language to ensure that all the available data were being accessed; translations for search terms, websites, and documents were conducted using Google Translate. See for search terms. Evaluation of all the relevant policy and guideline documents indicated that some countries have legal policies mandating dental care for children, some have insurance schemes ensuring free services for children, and some have published clinical guidelines on providing preventive care. Therefore, we generated binary variables to indicate the availability of the following types of policies: mandatory dental services for children; availability of free dental services for children; and available guidelines for children's preventive services . The category mandatory dental services for children signifies whether a particular country has an existing law or act that specifically mandates children receive dental care including preventive services like oral health screenings, instruction, and fluoridation. This category indicates that a specific law indicating that the entire population should have access these services but does not measure the actual frequency of utilisation amongst the population. The category of availability of free dental service for children signifies whether a particular country's mandatory national insurance scheme or national public service includes preventive services for children in its basic dental coverage. The category of available guidelines for children's preventive services signifies whether a particular country's government or health ministry has published clinical practice guidelines for dentists on providing preventive care to children. This categorisation system allowed us to assess each country's level of regulation of children's dental services, predominantly preventive. Examples of these services are application of sealants, oral health screenings, fluoride varnish, and oral hygiene instruction. See for additional details. Countries We focused on OECD member countries as these are high-income countries are more likley to have the resources to provide comprehensive dental care. The OECD works with governments and policymakers to establish evidence-based international standards ; we expected these countries to have standardised and accessible policy documents. A list of OECD countries excluded from the study due to insufficient public data can be found in and . After accounting for these limitations, we included 19 of the 38 OECD member countries. Limitations When analysing oral health expenditure data, we focused on dental outpatient care and excluded dental inpatient procedures; the OECD database does not distinguish between dental inpatient services and nondental inpatient services. Whilst some costs may not be captured, most dental care—especially preventive care and caries treatment—are outpatient procedures. Additionally, health ministry websites often listed existing laws and regulations without indicating how these specific policies were enforced. This study therefore does not account for the frequency of utilisation of the services and only captures the fact that a country addresses the need for the services in its legislation. The policy data collection component of this study was conducted utilising Google Translate, and there is the possibility that certain text was not translated accurately. A more comprehensive database that monitors international oral health policy updates across languages is needed to allow for more detailed analysis. This study averaged the DMFT indexes of children from the age of 12 to 18 years and assumed that caries experience does not change that significantly over this time period. , We reasoned that this age range is sufficiently narrow, encompassing only children within similar life stages and allowing for inclusion of additional data points for analysis. As caries experience does not develop steadily over one's lifetime, many argue that DMFT cannot be averaged for the entire population of a country. This argument stems from the significant differences in the caries experience of children and of an older population, especially because DMFT is an irreversible index. Nevertheless, despite its limitations, DMFT has been regarded as one of the most prominent indicators of caries experience. , , Both the OECD Health Expenditure and Financing and the WHO CAPP databases had missing values for certain years (detailed in ). Dental public health research particularly suffers from incomplete datasets, and more standardised data collection methods for oral health measures are needed globally. We recognise that our results may be biased towards countries that devoted more resources to dental care and country-level data collection. It is critical to underline that the trends may be different depending on a country's income level and regime type. We recognize that in most developed countries, dental caries has a skewed distribution, affecting vulnerable groups more severely. The within-country variation of DMFT is an important topic that is beyond scope of this study and should be further examined in the future; in this study, we focused our efforts on investigating country-level policy and DMFT variations. There are many confounding variables that affect the population-level DMFT index, such as socioeconomic status, race, and education level; these variables and their relationship with DMFT should be systematically examined when data become available. Statistical methods The statistical software STATA version 17.0 was used to code, clean, and analyse the datasets. Descriptive statistics were generated for all indicator variables. We calculated bivariate regression analysis between each variable to examine the strength of associations between all indicator variables. Scatterplots were used to evaluate the trends amongst DMFT, oral health expenditure, and level of oral policy.
We utilised countries’ DMFT indexes as proxy variables for oral health outcomes. DMFT is the sum of a person's decayed, missing, and filled permanent teeth. The DMFT index is a well-established measure of caries burden in oral epidemiology. A higher DMFT index indicates worse caries burden and further deterioration or oral health. , We extracted DMFT data for children aged 12 to 18 years, allowing assessment of national regulations and guidelines which target preventive care for children up to the age of 18, from the WHO Oral Health Country/Area Profile Project (CAPP). See for more details.
We extracted oral health expenditure data from the OECD Health Expenditure and Financing database. The oral health expenditure considers all dental-sector curative care services provided in an outpatient setting, measured as a percentage of each country's gross domestic product (GDP). The OECD database characterises dental-sector curative care services as all services related to oral health, gums, and teeth and other related disorders; this group of services includes most dental services provided in an outpatient setting. Details of the extraction and missing observations can be found in .
Country-specific research was conducted for each of the OECD member countries, and countries were assessed based on their level of oral health prevention policy for children. All searches were first conducted in English and then in each country's native language to ensure that all the available data were being accessed; translations for search terms, websites, and documents were conducted using Google Translate. See for search terms. Evaluation of all the relevant policy and guideline documents indicated that some countries have legal policies mandating dental care for children, some have insurance schemes ensuring free services for children, and some have published clinical guidelines on providing preventive care. Therefore, we generated binary variables to indicate the availability of the following types of policies: mandatory dental services for children; availability of free dental services for children; and available guidelines for children's preventive services . The category mandatory dental services for children signifies whether a particular country has an existing law or act that specifically mandates children receive dental care including preventive services like oral health screenings, instruction, and fluoridation. This category indicates that a specific law indicating that the entire population should have access these services but does not measure the actual frequency of utilisation amongst the population. The category of availability of free dental service for children signifies whether a particular country's mandatory national insurance scheme or national public service includes preventive services for children in its basic dental coverage. The category of available guidelines for children's preventive services signifies whether a particular country's government or health ministry has published clinical practice guidelines for dentists on providing preventive care to children. This categorisation system allowed us to assess each country's level of regulation of children's dental services, predominantly preventive. Examples of these services are application of sealants, oral health screenings, fluoride varnish, and oral hygiene instruction. See for additional details.
We focused on OECD member countries as these are high-income countries are more likley to have the resources to provide comprehensive dental care. The OECD works with governments and policymakers to establish evidence-based international standards ; we expected these countries to have standardised and accessible policy documents. A list of OECD countries excluded from the study due to insufficient public data can be found in and . After accounting for these limitations, we included 19 of the 38 OECD member countries.
When analysing oral health expenditure data, we focused on dental outpatient care and excluded dental inpatient procedures; the OECD database does not distinguish between dental inpatient services and nondental inpatient services. Whilst some costs may not be captured, most dental care—especially preventive care and caries treatment—are outpatient procedures. Additionally, health ministry websites often listed existing laws and regulations without indicating how these specific policies were enforced. This study therefore does not account for the frequency of utilisation of the services and only captures the fact that a country addresses the need for the services in its legislation. The policy data collection component of this study was conducted utilising Google Translate, and there is the possibility that certain text was not translated accurately. A more comprehensive database that monitors international oral health policy updates across languages is needed to allow for more detailed analysis. This study averaged the DMFT indexes of children from the age of 12 to 18 years and assumed that caries experience does not change that significantly over this time period. , We reasoned that this age range is sufficiently narrow, encompassing only children within similar life stages and allowing for inclusion of additional data points for analysis. As caries experience does not develop steadily over one's lifetime, many argue that DMFT cannot be averaged for the entire population of a country. This argument stems from the significant differences in the caries experience of children and of an older population, especially because DMFT is an irreversible index. Nevertheless, despite its limitations, DMFT has been regarded as one of the most prominent indicators of caries experience. , , Both the OECD Health Expenditure and Financing and the WHO CAPP databases had missing values for certain years (detailed in ). Dental public health research particularly suffers from incomplete datasets, and more standardised data collection methods for oral health measures are needed globally. We recognise that our results may be biased towards countries that devoted more resources to dental care and country-level data collection. It is critical to underline that the trends may be different depending on a country's income level and regime type. We recognize that in most developed countries, dental caries has a skewed distribution, affecting vulnerable groups more severely. The within-country variation of DMFT is an important topic that is beyond scope of this study and should be further examined in the future; in this study, we focused our efforts on investigating country-level policy and DMFT variations. There are many confounding variables that affect the population-level DMFT index, such as socioeconomic status, race, and education level; these variables and their relationship with DMFT should be systematically examined when data become available.
The statistical software STATA version 17.0 was used to code, clean, and analyse the datasets. Descriptive statistics were generated for all indicator variables. We calculated bivariate regression analysis between each variable to examine the strength of associations between all indicator variables. Scatterplots were used to evaluate the trends amongst DMFT, oral health expenditure, and level of oral policy.
summarises country characteristics with the mean (SD), minimum, maximum, and number of observations of average oral health expenditure and average DMFT as well as the data for each policy category, presenting the percentages of countries that implemented each policy. The least common policy implemented is mandatory dental services for children (26.32%), and the most common policy is available dental services for children (78.95%). is a scatterplot illustrating the relationship amongst average DMFT index, average oral health expenditure, and presence of a legal policy that mandates dental care for children. Countries with legal policies mandating dental care for children have lower DMFT indexes but slightly higher oral health expenditures. A sensitivity analysis was conducted; further information can be found in . presents information on policies implemented in each of the 19 countries included in this study. Denmark and Japan are the only 2 countries that not only mandate dental services for children but also have available dental services for children and guidelines for preventive dental care. Spain and Switzerland were the only 2 countries with no policies in any category. Mexico and Colombia have technical standards and guidelines, which were not commonly found in other countries. Most countries included in this study have available dental services for children, except Spain, Switzerland, Mexico, and Colombia. presents findings on bivariate regression analysis. Oral health expenditure was negatively correlated with DMFT index (−4.42, P < 0.05), suggesting that a 1% increase in oral health expenditure is correlated with a 4.42 reduction in DMFT. This finding signifies a correlation of a child having approximately 4 fewer decayed teeth for each percentage point increase in spending. The average DMFT indexes of countries and regions vary significantly due to resource allocation and socioeconomic factors, but a decrease of 4 units in DMFT score would move most countries from a moderate to low DMFT. The legal policy for mandatory dental services for children was negatively correlated with DMFT index (−1.32, P < 0.05), suggesting that the existence of such a legal policy is correlated with a 1.32 reduction in mean DMFT score. The legal policy for mandatory dental services for children was also positively correlated with average oral health expenditure (0.16, P < 0.05), suggesting that the existence of such a legal policy is correlated with a 0.16% increase in oral health expenditure. illustrates the relationship between the presence of legal policy for mandatory dental services for children and country-level DMFT index and average oral health expenditure as a percentage of GDP. Countries with legal policy for mandatory dental services for children all have a significantly lower average DMFT index than countries without. Countries with a legal policy to mandate oral health care for children tend to have higher oral health expenditure (as a percentage of GDP).
Countries’ policies on child dental care and preventive measures vary. This study found statistically significant bivariate correlations amongst legal policy that mandates dental care for children, the respective country's oral health expenditure, and DMFT index of children aged 12 to 18 years. The presence of legal policy mandating dental care for children is associated with a reduction in the respective country's DMFT index, but a slight increase in oral health expenditure. Although the presence of legal policy mandating dental care for children is not associated with lower oral health expenditures, the benefit of the significant reduction in DMFT indexes may be worth the slightly higher spending. Most countries with legal policy mandating dental care for children are countries that were ranked with the highest efficiency indexes in a study evaluating the relationship between oral health expenditure and oral health rankings of European countries. This pattern adds to the evidence that mandating dental care through legislation increases the efficiency of the services. Each one of the countries that has legal policies mandating dental care for children and free municipal basic and preventive services for children to prevent dental diseases also has dental services tied very closely to their schooling systems. For instance, since 1972, Denmark has implemented the Child Dental Health Care Act mandating that municipalities build dental clinics and provide basic and preventive services to children free of charge. , Because Danish children receive compulsory education, free dental services tied to the school system are efficient. Spain and Switzerland were the only 2 countries with no policies for any category included in our study. These countries both operate under privatised oral health care models; dental care is largely excluded from mandatory health insurance and provided by private practitioners, paid directly by patients. , In Spain, primary health care providers offer some preventive care for children, and the government provides some basic coverage to children aged 6 to 15 years. The services provided vary drastically by region and there is no national programme/oral health coverage for children. Whilst both Spain and Switzerland share relatively low DMFT index averages compared to other countries, Spain has moderate oral health expenditures, whilst Switzerland has the second highest oral health expenditure of the countries studied. It is possible that Switzerland's high oral health expenditure is due to the lack of focus on prevention and dental treatment aimed towards children. Studies show that low-income countries are more heavily affected by dental diseases and suffer from underutilisation of available resources, likely and partially attributed to knowledge gaps. Whilst low-income countries were not included in our analysis, we reason that these countries will likely benefit from an increase in regulation of oral health policy, provided that resources are available to implement and enforce these policies. Our study only investigated the existence of policies and not the actual scope of coverage. Future studies should further examine the laws mandating dental care, the scope of services available to children, and the details of technical guidelines/standards, allowing for examination of the effects of preventive measures on oral health outcomes and cost of care. A database that contains systematic coding of country-level oral health policies and legislations would contribute to more extensive and standardised research and our understanding on the impact of specific dental policies in different contexts as well.
None disclosed.
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Use of a multi-gene pharmacogenetic panel reduces adverse drug effects
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a601fd35-40f9-46ba-ad9e-2b28dd7d8fb0
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10213803
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Pharmacology[mh]
|
Variation in genes encoding for drug-metabolizing enzymes, drug transporters, and drug targets is well recognized to contribute to inter-patient differences in drug response. A number of medical centers have implemented pharmacogenetic testing into practice to help guide drug therapy. To facilitate implementation, the Clinical Pharmacogenetics Implementation Consortium (CPIC) and Dutch Pharmacogenomics Working Group (DPWG) provide genotype-based prescribing recommendations, which address approximately 20 genes and over 100 drugs to date, including very commonly used drugs, e.g., antidepressants, opioids, antiplatelets/anticoagulants, proton pump inhibitors, and statins. , , Data suggest over 90% of individuals have at least onegenetic variant likely to influence drug response (i.e., actionable pharmacogenetic variant), and over 60% of patients in primary care clinics are prescribed at least one drug with pharmacogenetic guidance. These data suggest the potential population impact of pharmacogenetic testing is high, and it would follow that genotyping patients using a multi-gene panel that captures variants with implications for responses to multiple medications across the patient's lifetime could potentially improve drug-related outcomes. While data support the feasibility of pharmacogenetic testing of a single drug-gene combination for improved clinical outcomes, less is known about the feasibility and clinical utility of multi-gene testing to guide prescribing of multiple medications. Swen et al. describe the effect of pharmacogenetic testing, using a 12-gene panel, on preventing adverse drug reactions in patients enrolled across seven countries and a wide range of settings. The Preemptive Pharmacogenomic Testing for Preventing Adverse Drug Reactions (PREPARE) study, published in The Lancet , was a cluster-design crossover study in which participating countries were randomized to genotype-guided prescribing or standard clinical care with crossover after 19 months. The study enrolled adult patients newly starting a drug addressed in DPWG guidelines, referred to as the index drug. Patients enrolled from countries randomized to genotype-guided prescribing had their pharmacogenetic results relevant to the index drug returned within seven days. Once available, remaining results were delivered to the provider through a decision support solution and to the patient via a card with results embedded in a QR code. While DPWG recommendations were provided, the ultimate prescribing decision was left to the provider. Patients in the control arm underwent pharmacogenetic testing at the end of their study participation. The study’s primary endpoint was the occurrence of an adverse reaction to the index drug, assessed via questionnaire 12 weeks after drug initiation. A total of 6,944 patients (97.7% of European, Mediterranean, or Middle Eastern ancestry) were enrolled from 55 hospitals, clinics, community health centers, or pharmacies; 93.5% had at least one actionable variant, 75.4% had >1 actionable variant, and 25.2% had an actionable variant relevant to their index drug. Among patients with an actionable variant, 21% in the genotype-guided group and 28% in the control group had an adverse reaction considered drug-related and clinically relevant over the 12-week follow-up period, equating to 30% lower risk (odds ratio [OR] 0.70, 95% confidence interval 0.54–0.91, p = 0.0075) in the genotype-guided group. In the population overall, 21% in the genotype group and 29% in the control group had an adverse drug reaction (p < 0.0001). During the up-to-18-month follow-up period, 14% of patients were prescribed a second drug with DPWG recommendations. The investigators should be congratulated for completing this large, complex, multi-national trial. Likely owing to their effective educational strategy and mechanism for returning pharmacogenetic results, there was a high recommendation acceptance rate by providers, demonstrating the feasibility of the investigator's approach to pharmacogenetic implementation. The study also demonstrated a significant reduction in adverse drug reactions in the genotype group, which was observed in both the subset of participants with an actionable genotype and the study population overall. Given nearly identical adverse event reductions in the genotype-guided arm in those with an actionable variant and the overall population, one must assume there was a significant reduction in adverse events in those lacking an actionable genotype. Such a finding is unexpected and raises the question about whether the pharmacogenetic testing directly led to reduction of adverse drug events or rather to a more careful approach to drug therapy overall, thus reducing adverse drug events. Future studies are needed to evaluate whether pharmacogenetic testing is associated with the equivalent of a placebo effect or the findings of Swen and colleagues in their non-actionable group have a different explanation, which they propose in their paper. Also unknown is whether study results extend to individuals of non-European ancestry, though data are clear that the associations between pharmacogenes and drug responses are common across populations. Nonetheless, this is the first large multi-national pharmacogenetic implementation study to show clinical benefit with broad implementation of panel-based pharmacogenetic testing. It provides a framework and argument for the feasibility and clinical utility of widespread clinical implementation of pharmacogenetics. The investigators referred to their testing strategy as preemptive. However, the major eligibility criterion was that patients were starting a drug addressed in DPWG guidelines, and testing in this regard is considered reactive versus preemptive. Preemptive testing (i.e., testing prior to medication decisions) offers the advantage of having results available at the point of prescribing. Because rapid return of results is not needed, samples can be batched for genotyping, reducing technical time and cost. However, it may be months or years before results are used for prescribing decisions. Indeed, only 14% of PREPARE study patients were prescribed a second drug addressed by pharmacogenetic guidelines over the up-to-18-month follow-up. The approach taken by the PREPARE investigators, whereby testing is ordered to guide prescribing of a specific medication but includes genotypes useful for guiding future prescribing of multiple medications, may be the most clinically feasible and defensible approach to advancing the clinical utility of pharmacogenetic testing at present, particularly in the absence of clear data on outcomes and questionable reimbursement with preemptive testing. Findings from the PREPARE study provide some confidence that this approach will improve drug-related outcomes, and the authors should be congratulated for this important study that significantly advances the field.
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Nitrate-dependent anaerobic methane oxidation (N-DAMO) as a bioremediation strategy for waters affected by agricultural runoff
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7817f901-da44-4253-a9a0-59d6c5d1533f
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10214460
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Microbiology[mh]
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Lakes, rivers, wetlands, ditches, and ponds are strongly affected by agricultural practices, becoming more eutrophic and anoxic, consequently releasing a substantial amount of nitrous oxide (N 2 O) and methane (CH 4 ) into the atmosphere (Walter et al. , O’Connor et al. , Peacock et al. ). Both N 2 O and CH 4 are potent greenhouse gases, significantly contributing to the global temperature increase (Jackson et al. , Rosentreter et al. ). It has been estimated that in The Netherlands, 330 000 km of ditches may be responsible for 16% of national CH 4 emissions (Koschorreck et al. ). Methane produced in anoxic environments via methanogenesis can be consumed via either aerobic or anaerobic methanotrophs, which are thus important players to lower emissions. Especially, nitrate-dependent anaerobic methane oxidation (N-DAMO) is relevant in this respect, as this process removes both CH 4 and nitrogen compounds at the same time. N-DAMO was first discovered in 2006 in the sediment of a Dutch freshwater canal (Raghoebarsing et al. ). Research on enrichment cultures showed that Candidatus Methanoperedens nitroreducens was coupling anaerobic CH 4 oxidation to the reduction of NO 3 − (Equation ; Haroon et al. ). In this process, produced nitrite (NO 2 − ) can be further reduced to dinitrogen gas (N 2 ) by Candidatus Methylomirabilis oxyfera while oxidizing CH 4 through an intra-aerobic methane oxidation pathway (Ettwig et al. ; Equation ). (1) [12pt]{minimal}
}{} {}{{}}_4 + 4{}{{{}}_3}^- {}{{}}_2 + 4{}{{{}}_2}^ - + 2{{}}_2{} (2) [12pt]{minimal}
}{} 3{}{{}}_4 + 8{}{{{}}_2}^ - + 8{{}}^ + 3{}{{}}_2 + 4{{}}_2 + 10{{}}_2{} Several freshwater eutrophic ecosystems and peatlands have been shown to harbor N-DAMO communities (Liu et al. , Shen et al. , Welte et al. , Yang et al. , Shi et al. ), indicating that fertilization may stimulate the abundance of Ca . Methylomirabilis sp. in rice paddy fields, potentially decreasing CH 4 emissions from these methanogenic environments (Shen et al. , Wang et al. , Yang et al. ). Consequently, the N-DAMO process gained a lot of attention in recent years as a potential mitigation strategy to decrease both greenhouse gas emissions and the accumulation of nitrogen originating from agricultural activities (Contreras et al. , Gómez-Gallego ). Until today, however, no laboratory studies had been conducted to evaluate the potential of the N-DAMO process in CH 4 and NO 3 − removal as a bioremediation strategy. Therefore, this work aimed to evaluate whether bioaugmentation with an N-DAMO enrichment culture consisting of Ca . Methanoperedens and Ca . Methylomirabilis could decrease CH 4 emissions and NO 3 − concentrations. For that purpose, microcosm experiments supplemented with 13 CH 4 and 15 NO 3 were conducted using sediments and surface water collected from three different drainage ditches influenced by agricultural runoff. The nitrogen species evolution, CH 4 concentration, and 13 CO 2 formation were followed over time. Additionally, the genomic potential of the inoculum and microbial community composition in the original sediment and the microcosm sediment was analyzed using metagenomics and 16S rRNA amplicon sequencing, respectively.
Study sites, sampling, and field data collection Sediment and surface water samples were collected from three agriculture-impacted areas in The Netherlands. The selected sites consisted of a sandy-clay ditch (SCD) located in the Ooijpolder, a former floodplain of the River Rhine, a freshwater-fed peatland ditch (FPD) surrounded by peat meadows in agricultural use in Aldeboarn, and a brackish peatland ditch (BPD) located in Polder Westzaan that is within a “Natura 2000” protected area under agricultural influence and subjected to re-salinization to restore brackish ecological values (Fig. ). The three selected sites differ substantially due to their location, history, sediment type and composition, land use, and yearly N regime (Table ); nevertheless, all three sites are under strong anthropogenic pressure mainly due to intensive agriculture activities. In each of the sites, we collected sediment samples ( n = 9) from the upper part of the sediment profile targeting organic layers in 1.8-mm-thick transparent PVC tubes (diameter 6 cm and height 60 cm), using a UWITEC sediment corer (UWITEC, Mondsee, Austria), while the surface water samples were collected from the water column above the sediment. In each of the sites, we measured CH 4 and CO 2 fluxes using a transparent closed chamber (height 30 cm and diameter 28.8 cm) floating on top of the water column connected to a Los Gatos (LGR ® ) Ultra-Portable Greenhouse Gas Analyzer. A fraction of the surface water (40 mL) and well-mixed sediment samples (15 mL) of each site were frozen (−20°C), to measure the nutrient concentration in the water column and to extract DNA, respectively. The water was filtered through 0.45 μm pore size filters before chemical analysis. Dissolved N species (NO 3 − , NO 2 − , NH 4 + ) were measured colorimetrically using a Bran + Luebbe system and Seal Model III system continuous auto-analyzer. Inoculum source and characteristic The N-DAMO enrichment culture was propagated from the original enrichment using sediment from Twentekanaal canal (The Netherlands; Raghoebarsing et al. ) fed with extra NO 3 − in bioreactor systems (Arshad et al. ). In April 2021, the culture mainly consisted of Ca . Methanoperedens nitroreducens BLZ2 (∼33%) and Ca . Methylomirabilis oxyfera (∼27%; ). To determine the CH 4 oxidation and NO 3 − reduction potential of the culture, we screened for the functional genes involved in CH 4 and N cycling of the top (>1% mapped reads) most abundant metagenome-assembled genomes ( ). The analyzed metagenomic dataset was first reported in Schoemerich et al. ( ; “Bioreactor 1”), available under NCBI Bioproject ID PRJNA850006. See for more details on metagenomic analysis. For the microcosm’s inoculation, 30 mL of culture was anoxically withdrawn from the bioreactor and washed three times with sterile medium (without NO 3 − ) in an anaerobic glovebox. Washed biomass was then used to inoculate the microcosm. Microcosm setup and sampling procedure To measure microbial activity and assess the effect of N-DAMO addition on the N-concentration and CH 4 emissions, we set up microcosms experiment with three different treatments: (1) Control—sediment without any additions; (2) NO 3 − —sediments supplemented with 3 mM Na 15 NO 3 − and 10 mL 13 CH 4 ; and (3) NO 3 − + N-DAMO—sediment supplemented with 3 mM Na 15 NO 3 − , 10 mL 13 CH 4 , and 0.40 ± 0.0081 g (dry weight) of N-DAMO enrichment culture. All the treatments were prepared in quadruplicates using 30 g of homogenized slurry sediment and 40 mL of surface water mixed in 120 mL sterile serum bottles. The microcosms were set up in the anoxic glovebox (<10 ppm O 2 ), where anoxic Na 15 NO 3 − and inoculum were added. The bottles were closed and crimped to ensure anoxic conditions and prevent oxygen intrusion. Afterward, the headspace was exchanged with N 2 /CO 2 and 13 CH 4 was injected into all treatments except the control. All the microcosms were kept in the dark at 17°C. Sampling was performed using a sterile syringe and needle. The first time point was measured immediately after inoculation and supplementation of 13 CH 4 and 15 NO 3 − . Measurements continued two to three times a day while NO 3 − was being actively reduced. Reduction of NO 3 − was followed using NO 3 − strips (MQuant, STEP Systems) and later measured spectrophotometrically. Depending on the NO 3 − reduction rate, the incubation time varied between sediment types from lasting several days (FPD and SCD) to several weeks (BPD). To capture the microbial community actively involved in NO 3 − reduction and CH 4 oxidation, halfway through the experiment, one bottle of each treatment was sacrificed for DNA extraction. Consequently, the final number of replicates per treatment was three at the end of the incubations. Gas measurements The 13 CO 2 / 12 CO 2 , 46 N 2 O/ 44 N 2 O, and 30 N 2 / 28 N 2 ratios were determined by gas chromatography coupled to mass spectrometry (Trace DSQ II, Thermo Finnigan, Austin, TX, USA), and the concentration of CH 4 was quantified by gas chromatography with flame ionization detection (Hewlett Packard HP 5890 Series II Gas Chromatograph, Agilent Technologies, CA, USA). The pressure was measured at each time point using a portable pressure meter (GMH 3100, GHM Messtechnik, Regenstauf, Germany) to account for the compressed gas in the headspace. Water chemistry One milliliter of liquid sample was withdrawn anoxically at each time point for the NO 3 − , NO 2 − , and NH 4 + quantification using colorimetric methods. The Griess assay was used to quantify NO 3 − and NO 2 − , while the o -phthalaldehyde assay was used to determine the concentration of NH 4 + (Meseguer-Lloret et al. , Sun et al. ). Both assays were performed using 96-well plates and absorbance was measured on the spectrophotometer (Molecular Devices, Spectramax 190 Microplate Reader). DNA extraction and amplicon sequencing The sediment samples for the DNA extraction were collected in the field ( in-situ sediment) and from all microcosms in the middle of incubation. DNA was extracted from one bottle of each treatment across all sediment types. In total, 15 DNA samples were obtained. The DNA extraction was performed using the PowerSoil DNA Extraction Kit (DNeasy PowerSoil Pro Kit, QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. The concentration of the DNA was quantified using the Qubit® 2.0 Fluorometer with DNA HS kits (Life Technologies, Carlsbad, CA, USA). 16S rRNA gene amplicon sequencing was done by Macrogen (Macrogen, Amsterdam, The Netherlands) using the Illumina MiSeq Next Generation Sequencing platform. Paired-end libraries were constructed using the Illumina Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Illumina, Eindhoven, The Netherlands). Detailed information on primers and data analysis is provided in the Supplementary Materials. The raw sequence data and metadata of the microcosms experiment have been deposited in the sequence read archive (SRA) database of the NCBI under the BioProject ID PRJNA918998.
Sediment and surface water samples were collected from three agriculture-impacted areas in The Netherlands. The selected sites consisted of a sandy-clay ditch (SCD) located in the Ooijpolder, a former floodplain of the River Rhine, a freshwater-fed peatland ditch (FPD) surrounded by peat meadows in agricultural use in Aldeboarn, and a brackish peatland ditch (BPD) located in Polder Westzaan that is within a “Natura 2000” protected area under agricultural influence and subjected to re-salinization to restore brackish ecological values (Fig. ). The three selected sites differ substantially due to their location, history, sediment type and composition, land use, and yearly N regime (Table ); nevertheless, all three sites are under strong anthropogenic pressure mainly due to intensive agriculture activities. In each of the sites, we collected sediment samples ( n = 9) from the upper part of the sediment profile targeting organic layers in 1.8-mm-thick transparent PVC tubes (diameter 6 cm and height 60 cm), using a UWITEC sediment corer (UWITEC, Mondsee, Austria), while the surface water samples were collected from the water column above the sediment. In each of the sites, we measured CH 4 and CO 2 fluxes using a transparent closed chamber (height 30 cm and diameter 28.8 cm) floating on top of the water column connected to a Los Gatos (LGR ® ) Ultra-Portable Greenhouse Gas Analyzer. A fraction of the surface water (40 mL) and well-mixed sediment samples (15 mL) of each site were frozen (−20°C), to measure the nutrient concentration in the water column and to extract DNA, respectively. The water was filtered through 0.45 μm pore size filters before chemical analysis. Dissolved N species (NO 3 − , NO 2 − , NH 4 + ) were measured colorimetrically using a Bran + Luebbe system and Seal Model III system continuous auto-analyzer.
The N-DAMO enrichment culture was propagated from the original enrichment using sediment from Twentekanaal canal (The Netherlands; Raghoebarsing et al. ) fed with extra NO 3 − in bioreactor systems (Arshad et al. ). In April 2021, the culture mainly consisted of Ca . Methanoperedens nitroreducens BLZ2 (∼33%) and Ca . Methylomirabilis oxyfera (∼27%; ). To determine the CH 4 oxidation and NO 3 − reduction potential of the culture, we screened for the functional genes involved in CH 4 and N cycling of the top (>1% mapped reads) most abundant metagenome-assembled genomes ( ). The analyzed metagenomic dataset was first reported in Schoemerich et al. ( ; “Bioreactor 1”), available under NCBI Bioproject ID PRJNA850006. See for more details on metagenomic analysis. For the microcosm’s inoculation, 30 mL of culture was anoxically withdrawn from the bioreactor and washed three times with sterile medium (without NO 3 − ) in an anaerobic glovebox. Washed biomass was then used to inoculate the microcosm.
To measure microbial activity and assess the effect of N-DAMO addition on the N-concentration and CH 4 emissions, we set up microcosms experiment with three different treatments: (1) Control—sediment without any additions; (2) NO 3 − —sediments supplemented with 3 mM Na 15 NO 3 − and 10 mL 13 CH 4 ; and (3) NO 3 − + N-DAMO—sediment supplemented with 3 mM Na 15 NO 3 − , 10 mL 13 CH 4 , and 0.40 ± 0.0081 g (dry weight) of N-DAMO enrichment culture. All the treatments were prepared in quadruplicates using 30 g of homogenized slurry sediment and 40 mL of surface water mixed in 120 mL sterile serum bottles. The microcosms were set up in the anoxic glovebox (<10 ppm O 2 ), where anoxic Na 15 NO 3 − and inoculum were added. The bottles were closed and crimped to ensure anoxic conditions and prevent oxygen intrusion. Afterward, the headspace was exchanged with N 2 /CO 2 and 13 CH 4 was injected into all treatments except the control. All the microcosms were kept in the dark at 17°C. Sampling was performed using a sterile syringe and needle. The first time point was measured immediately after inoculation and supplementation of 13 CH 4 and 15 NO 3 − . Measurements continued two to three times a day while NO 3 − was being actively reduced. Reduction of NO 3 − was followed using NO 3 − strips (MQuant, STEP Systems) and later measured spectrophotometrically. Depending on the NO 3 − reduction rate, the incubation time varied between sediment types from lasting several days (FPD and SCD) to several weeks (BPD). To capture the microbial community actively involved in NO 3 − reduction and CH 4 oxidation, halfway through the experiment, one bottle of each treatment was sacrificed for DNA extraction. Consequently, the final number of replicates per treatment was three at the end of the incubations. Gas measurements The 13 CO 2 / 12 CO 2 , 46 N 2 O/ 44 N 2 O, and 30 N 2 / 28 N 2 ratios were determined by gas chromatography coupled to mass spectrometry (Trace DSQ II, Thermo Finnigan, Austin, TX, USA), and the concentration of CH 4 was quantified by gas chromatography with flame ionization detection (Hewlett Packard HP 5890 Series II Gas Chromatograph, Agilent Technologies, CA, USA). The pressure was measured at each time point using a portable pressure meter (GMH 3100, GHM Messtechnik, Regenstauf, Germany) to account for the compressed gas in the headspace. Water chemistry One milliliter of liquid sample was withdrawn anoxically at each time point for the NO 3 − , NO 2 − , and NH 4 + quantification using colorimetric methods. The Griess assay was used to quantify NO 3 − and NO 2 − , while the o -phthalaldehyde assay was used to determine the concentration of NH 4 + (Meseguer-Lloret et al. , Sun et al. ). Both assays were performed using 96-well plates and absorbance was measured on the spectrophotometer (Molecular Devices, Spectramax 190 Microplate Reader). DNA extraction and amplicon sequencing The sediment samples for the DNA extraction were collected in the field ( in-situ sediment) and from all microcosms in the middle of incubation. DNA was extracted from one bottle of each treatment across all sediment types. In total, 15 DNA samples were obtained. The DNA extraction was performed using the PowerSoil DNA Extraction Kit (DNeasy PowerSoil Pro Kit, QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. The concentration of the DNA was quantified using the Qubit® 2.0 Fluorometer with DNA HS kits (Life Technologies, Carlsbad, CA, USA). 16S rRNA gene amplicon sequencing was done by Macrogen (Macrogen, Amsterdam, The Netherlands) using the Illumina MiSeq Next Generation Sequencing platform. Paired-end libraries were constructed using the Illumina Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Illumina, Eindhoven, The Netherlands). Detailed information on primers and data analysis is provided in the Supplementary Materials. The raw sequence data and metadata of the microcosms experiment have been deposited in the sequence read archive (SRA) database of the NCBI under the BioProject ID PRJNA918998.
The 13 CO 2 / 12 CO 2 , 46 N 2 O/ 44 N 2 O, and 30 N 2 / 28 N 2 ratios were determined by gas chromatography coupled to mass spectrometry (Trace DSQ II, Thermo Finnigan, Austin, TX, USA), and the concentration of CH 4 was quantified by gas chromatography with flame ionization detection (Hewlett Packard HP 5890 Series II Gas Chromatograph, Agilent Technologies, CA, USA). The pressure was measured at each time point using a portable pressure meter (GMH 3100, GHM Messtechnik, Regenstauf, Germany) to account for the compressed gas in the headspace.
One milliliter of liquid sample was withdrawn anoxically at each time point for the NO 3 − , NO 2 − , and NH 4 + quantification using colorimetric methods. The Griess assay was used to quantify NO 3 − and NO 2 − , while the o -phthalaldehyde assay was used to determine the concentration of NH 4 + (Meseguer-Lloret et al. , Sun et al. ). Both assays were performed using 96-well plates and absorbance was measured on the spectrophotometer (Molecular Devices, Spectramax 190 Microplate Reader).
The sediment samples for the DNA extraction were collected in the field ( in-situ sediment) and from all microcosms in the middle of incubation. DNA was extracted from one bottle of each treatment across all sediment types. In total, 15 DNA samples were obtained. The DNA extraction was performed using the PowerSoil DNA Extraction Kit (DNeasy PowerSoil Pro Kit, QIAGEN, Hilden, Germany), according to the manufacturer’s protocol. The concentration of the DNA was quantified using the Qubit® 2.0 Fluorometer with DNA HS kits (Life Technologies, Carlsbad, CA, USA). 16S rRNA gene amplicon sequencing was done by Macrogen (Macrogen, Amsterdam, The Netherlands) using the Illumina MiSeq Next Generation Sequencing platform. Paired-end libraries were constructed using the Illumina Herculase II Fusion DNA Polymerase Nextera XT Index Kit V2 (Illumina, Eindhoven, The Netherlands). Detailed information on primers and data analysis is provided in the Supplementary Materials. The raw sequence data and metadata of the microcosms experiment have been deposited in the sequence read archive (SRA) database of the NCBI under the BioProject ID PRJNA918998.
In-situ nitrogen measurements At the time of measurement (May 2022), NO 3 − was only detected in water columns of BPD and FPD (3.0 and 2.5 µM, respectively), while no detectable NO 3 − was reported in SCD. Moreover, BPD had the highest NH 4 + concentration (12 µM) compared to FPD (4.7 µM) and SCD (5.7 µM). As the samples were collected at the beginning of the growing season, the application of N-fertilizers was still rather low. Nevertheless, it is assumed that the majority of NO 3 − and NH 4 + originated from agricultural runoff. Besides fertilizer application, organic matter degradation and mineralization in the sediment likely also contributed to high NH 4 + concentrations in all ditches. In-situ methane emission Late spring CH 4 emissions varied greatly across the three different ditches (Fig. ). The highest measured CH 4 fluxes were observed in FPD (36.5 ± 6.4 mg m 2 d −1 ), followed by the ditch BPD in Westzaan (14.5 ± 0.60 mg m 2 d −1 ) and SCD in the Ooijpolder (7.7 ± 1.6 mg m 2 d −1 ). The Ooijpolder SCD is characterized by a mineral sandy-clay layer with a relatively thin (15–30 cm) organic-rich top layer. The lower emission of CH 4 in this ditch was likely due to lower carbon (C) availability compared to FPD and BPD. Also, the SCD and BPD were probably subjected to an aerobic breakdown of organic matter as a large amount of CO 2 was released from the water column. The FPD was a sink of CO 2 suggesting that in this ditch, a photosynthetic carbon fixation might have been greater than the decomposition of organic matter (Fig. ). The FPD sediment released most CH 4 . This is not surprising as freshwater peatlands are known to be the largest natural source of atmospheric CH 4 worldwide and store disproportionate amounts of global soil carbon (Feng et al. , Peacock et al. ). The high abundance of sulfate (SO 4 2− ) and increased salinity was likely a reason why the CH 4 release was much lower in the BPD compared to the FPD. A previous study conducted in the same region with a similar sediment type demonstrated that with increasing sea salt concentration (combination of salt and SO 4 2− ), the porewater CH 4 concentrations significantly decrease (van Dijk et al. ). It was hypothesized that increased surface water salinity changed the conditions from methanogenic to SO 4 2− reducing as thermodynamically more favorable, decreasing the availability of fermentation products to fuel CH 4 production. Overall, the three ditches selected for this study represent distinct conditions suitable to study how N-DAMO activity may affect CH 4 emission and NO 3 − removal under various environmental conditions and whether inoculation with N-DAMO enrichment cultures has potential as a bioremediation strategy. N-DAMO bioaugmentation promotes N removal in brackish sediment The effect of N-DAMO inoculation on NO 3 − concentration was the most pronounced in the BPD microcosms. Nitrate reduction in microcosms supplemented with the N-DAMO enrichment culture proceeded much faster compared to uninoculated microcosms, completely removing added NO 3 − (3 mM) within 19 days of incubation (Fig. ). On the other hand, only one-third of the added NO 3 − (1 mM) was consumed in uninoculated microcosms after nearly 20 days of incubation. Therefore, it is evident that N-DAMO was the main process responsible for the complete removal of NO 3 − . The NO 3 − reduction driven by N-DAMO was, however, much slower in BPD compared to FPD and SCD where NO 3 − was removed within 80 and 60 hours after starting the incubation, respectively, and was much less dependent on the N-DAMO process as only a small fraction of CH 4 was oxidized (Figs. and ). The slow NO 3 − reduction rate in BPD is likely because of the high salinity, which was previously shown to negatively affect N-DAMO activity, substantially decreasing denitrification potential (Chen et al. , ). It was also shown that the intrusion of seawater negatively influences the denitrifying microbial community, decreasing the denitrification rate in freshwater ecosystems (Neubauer et al. ). It was hypothesized that the negative correlation between salinity and denitrification might be caused by higher sulfide and/or chloride levels (Rysgaard et al. , Craft et al. ). The concentration of NO 2 − in uninoculated BPD microcosms increased at the beginning of the incubation reaching its maximum at day 8 (98 ± 20 µM), however by the end of the experiment, it was completely gone. The N-DAMO inoculation clearly prevented the formation and accumulation of NO 2 − in water as it was quickly consumed at the beginning of the experiment (Fig. ). The NH 4 + concentration was highest in N-DAMO inoculated microcosms compared to uninoculated microcosms, reaching nearly 790 ± 70 µM at the end of the experiment compared to uninoculated microcosms where it was 145 ± 45 µM (Fig. ). It must be noted, however, that the N-DAMO inoculated microcosms had a higher concentration of NH 4 + from the beginning of the experiment, although it is not clear why. It might be related to decaying organic matter and biomass added with the N-DAMO inoculum and subsequent mineralization processes. Moreover, it has been previously shown that N-DAMO communities dominated by ANME archaea are also capable of dissimilatory nitrate reduction to ammonium (DNRA; Arshad et al. , Nie et al. ). Also, the N-DAMO enrichment culture used here appeared to be capable of DNRA as confirmed by the metagenomic analysis ( ). Therefore, it is possible that some of this NH 4 + originated from NO 3 − reduction. A substantial fraction of added NO 3 − in inoculated BPD microcosms was converted into N 2 gas as the ratio of 30 N 2 / 28 N 2 increased from 1% to 6%, while in uninoculated microcosms, this ratio increased only to 1.3% ( ). No NO 3 − was converted into N 2 O, which was deduced from the ratio of 46 N 2 O/ 44 N 2 O ( ). No increase of 46 N 2 O/ 44 N 2 O ratio suggests steady NO–N 2 O–N 2 reduction mediated by Ca . Methanoperedens and side N 2 O reducing community, or by Ca . Methylomirabilis oxyfera, via the conversion of two nitric oxide (NO) molecules to N 2 and O 2 bypassing N 2 O formation ( ). Freshwater ecosystems are known to have higher denitrification rates compared to brackish ones (Wang et al. ) and this was also found in our experiment. The freshwater SCD from Ooijpolder showed a very high denitrification potential as all NO 3 − was nearly completely depleted within 70 hours of incubation. The addition of the N-DAMO culture did not affect the denitrification rate as both inoculated and uninoculated microcosms performed similarly (Fig. ). In both cases, NO 2 − initially increased in concentration to 35 µM and afterward continuously decreased until it was nearly completely depleted after 53 hours (Fig. ). This is not surprising as the Ooijpolder from where the SCD sediment was collected is an agricultural area where denitrifying communities most likely are already well established. The initial NH 4 + concentration in SCD microcosms inoculated with the N-DAMO enrichment cultures was higher (128 µM) compared to values in uninoculated microcosms (81 µM). By the end of the incubation, the NH 4 + in bioaugmented microcosms was however very low (9.3 µM), while in control and NO 3 − supplemented microcosms it was still above 40 µM (Fig. ). The ratio of 30 N 2 / 28 N 2 increased from 0% to 3%, in both inoculated and uninoculated microcosms ( ) implying that similar denitrification rates occurred in both treatments. Inoculation of FPD sediment with the N-DAMO enrichment culture slightly accelerated the removal of NO 3 − (Fig. ) but did not affect the final concentration of NO 2 − (Fig. ) and NH 4 + (Fig. ). The ratio of 30 N 2 / 28 N 2 in FPD increased from 0% to 3%, in both inoculated and uninoculated, microcosms indicating conversion of 15 NO 3 − into 15 N 2 ( ). The ratio of 46 N 2 O/ 44 N 2 O increased, however, only in uninoculated microcosms supplemented with NO 3 − , reaching its maximum of 2.3% after 71 hours ( ). The production of N 2 O implies that incomplete denitrification or NH 4 + oxidation took place when NO 3 − was supplied to FPD sediment (Hallin et al. ). This is concerning as N 2 O is a potent greenhouse gas, third in the rank after CO 2 and CH 4 . The N-DAMO bioaugmentation prevented the formation of N 2 O possibly because Ca . Methylomirabilis can bypass N 2 O production by reducing NO intra-aerobically (Ettwig et al. ; ). N-DAMO bioaugmentation increases CH 4 oxidation in brackish sediment It has been suggested that increasing N input to various ecosystems may stimulate N-DAMO activity and reduce CH 4 emission to the atmosphere (Shi et al. , Wang et al. ). In our experiment, 13 CO 2 was produced only in the presence of the N-DAMO enrichment culture (Fig. ), implying that the native microbial community was not actively oxidizing CH 4 . Most CH 4 was oxidized in the BPD microcosms, which was reflected in the highest increase of 13 CO 2 / 12 CO 2 ratio from 1% to >10% (Fig. ). This was further confirmed by the decrease in the total amount of CH 4 , which dropped from 0.42 to 0.38 mmol (Fig. ). Overall, 10% of the added CH 4 was consumed after 20 days of incubation. The concentration of CH 4 in uninoculated microcosms however slightly increased over time, although in the control setup, we did not observe methanogenic activity and build-up of CH 4 . Overall, it can be concluded that bioaugmentation with the N-DAMO enrichment culture indeed promoted CH 4 oxidation and resulted in lower emissions of this greenhouse gas in BPD. In the incubation with SCD and FPD sediment inoculated with N-DAMO, only a small amount of 13 CO 2 was produced as the ratio of 13 CO 2 / 12 CO 2 increased from 1.2% to 1.8% and to 3.2%, respectively (Fig. and ). Similarly, there was no difference in CH 4 concentration between inoculated and uninoculated microcosms (Fig. and ). This implies that heterotrophic NO 3 − reduction by the native microbial community, in both FPD and SCD sediments microcosm incubations, outcompeted N-DAMO microorganisms. The rapid depletion of NO 3 − and lack of alternatives led to a shortage of electron acceptors and consequently, CH 4 could not be oxidized. Previous studies showed that Ca . Methanoperedens and Ca . Methylomirabilis mediating N-DAMO have a high doubling time in the order of several weeks (Raghoebarsing et al. , Vaksmaa et al. ), resulting in lower competitiveness compared to fast-growing denitrifiers. N-DAMO archaea and bacteria persisted in the microbial community The bioaugmentation with N-DAMO appeared to be persistent as introduced species were still present in the microbial community and survived over the incubation time, in all microcosms (Fig. ). This is important as many bioinoculants used in agriculture have been shown to have low efficiency and survival rate over time (O’Callaghan ). Generally, the core microbial taxa were similar between all sediment types (Fig. ). All three sediments showed the presence of similar methanogenic archaea such as Methanosaeta, Methanoregula , and Methanobacterium . However, methanogenic archaea affiliating with Methanosarcina were only abundant in Ooijpolder SCD sediment where this taxon represented >9% of microbial community, while in peatland sediments of BPD and FPD its relative abundance was only 0.8% and 1.3%, respectively. Furthermore, the microbial community in BPD was characterized by a much higher relative abundance of Bathyarchaeia (∼50%) compared to SCD (∼20%) and FPD (∼20%). The SCD was also the only sediment type that had a high relative abundance of Nitrososphaera , among which most genera are known ammonia oxidizers (Cao et al. ). It can also be noticed that for each treatment, the original sediment collected in the field, control sediment (unamended), and NO 3 − amended sediment had nearly identical composition, meaning that the microbial community did not change much during the incubation and adding NO 3 − did not trigger changes in the microbial community. However, the inoculation with N-DAMO substantially changed the composition of the archaeal community. Specifically, in all sediments, Ca . Methanoperedens was by far the most dominating taxon (Fig. ). In the BPD microcosms, in the middle of the experiment, it represented >90% of the archaeal community. This suggests that (1) the abundance of archaea was generally low in BPD and (2) Ca . Methanoperedens adapted to the elevated salinity, which was previously suggested by Li et al. ( ). The relative abundance of Ca . Methanoperedens in the archaeal communities of SCD and FPD was also high, 73% and 47%, respectively, implying that these archaea were responsible for the CH 4 oxidation and 13 CO 2 formation in all inoculated sediments. Similarly to the archaeal microbial community, the bacterial community was also very similar across all sediment types but also between the original sediment collected in the field, control sediment (unamended), and NO 3 − amended sediment, halfway through the incubation (Fig. ). Although it was expected that denitrifying microorganisms would increase in relative abundance, it appeared that adding NO 3 − did not trigger changes in the microbial community. This may imply that the denitrifying community was already well established. Indeed, many microorganisms known to be involved in denitrification such as Dechloromonas, Thiobacillus, Bacillus , and Denitratisoma were detected in the microbial community in all sediment types. The inoculation with the N-DAMO enrichment culture led to the introduction of Ca . Methylomirabilis, which represented 10%, 3%, and 3% of the bacterial community in BPD, SCD, and FPD, respectively. Therefore, it can be assumed that the conditions for Ca . Methylomirabilis were the most suitable in BPD, where the denitrification rate was the lowest. The concentration of NO 2 − , a necessary electron acceptor for Ca . Methylomirabilis, was for the most time nearly undetectable in BPD microcosms. This is most likely because NO 2 − was continuously consumed by Ca . Methylomirabilis as soon as NO 3 − was reduced, consequently keeping its concentration at a very low level. Candidatus Methylomirabilis probably also contributed to CH 4 oxidation and 13 CO 2 formation in all inoculated microcosms. It appeared that Methylocystis was introduced together with the N-DAMO enrichment culture as its abundance increased in all inoculated microcosms. This type II methanotroph was particularly abundant in the BPD setup where it represented 6.5% of the bacterial community in inoculated microcosms, while it was nearly undetectable in control and NO 3 − supplemented setups. Also, Rhizobiales and Denitratisoma were found to be particularly enriched in N-DAMO inoculated BPD sediment, possibly contributing to N cycling, as many Denitratisoma species are known to be involved in NO 3 − reduction.
nitrogen measurements At the time of measurement (May 2022), NO 3 − was only detected in water columns of BPD and FPD (3.0 and 2.5 µM, respectively), while no detectable NO 3 − was reported in SCD. Moreover, BPD had the highest NH 4 + concentration (12 µM) compared to FPD (4.7 µM) and SCD (5.7 µM). As the samples were collected at the beginning of the growing season, the application of N-fertilizers was still rather low. Nevertheless, it is assumed that the majority of NO 3 − and NH 4 + originated from agricultural runoff. Besides fertilizer application, organic matter degradation and mineralization in the sediment likely also contributed to high NH 4 + concentrations in all ditches.
methane emission Late spring CH 4 emissions varied greatly across the three different ditches (Fig. ). The highest measured CH 4 fluxes were observed in FPD (36.5 ± 6.4 mg m 2 d −1 ), followed by the ditch BPD in Westzaan (14.5 ± 0.60 mg m 2 d −1 ) and SCD in the Ooijpolder (7.7 ± 1.6 mg m 2 d −1 ). The Ooijpolder SCD is characterized by a mineral sandy-clay layer with a relatively thin (15–30 cm) organic-rich top layer. The lower emission of CH 4 in this ditch was likely due to lower carbon (C) availability compared to FPD and BPD. Also, the SCD and BPD were probably subjected to an aerobic breakdown of organic matter as a large amount of CO 2 was released from the water column. The FPD was a sink of CO 2 suggesting that in this ditch, a photosynthetic carbon fixation might have been greater than the decomposition of organic matter (Fig. ). The FPD sediment released most CH 4 . This is not surprising as freshwater peatlands are known to be the largest natural source of atmospheric CH 4 worldwide and store disproportionate amounts of global soil carbon (Feng et al. , Peacock et al. ). The high abundance of sulfate (SO 4 2− ) and increased salinity was likely a reason why the CH 4 release was much lower in the BPD compared to the FPD. A previous study conducted in the same region with a similar sediment type demonstrated that with increasing sea salt concentration (combination of salt and SO 4 2− ), the porewater CH 4 concentrations significantly decrease (van Dijk et al. ). It was hypothesized that increased surface water salinity changed the conditions from methanogenic to SO 4 2− reducing as thermodynamically more favorable, decreasing the availability of fermentation products to fuel CH 4 production. Overall, the three ditches selected for this study represent distinct conditions suitable to study how N-DAMO activity may affect CH 4 emission and NO 3 − removal under various environmental conditions and whether inoculation with N-DAMO enrichment cultures has potential as a bioremediation strategy.
The effect of N-DAMO inoculation on NO 3 − concentration was the most pronounced in the BPD microcosms. Nitrate reduction in microcosms supplemented with the N-DAMO enrichment culture proceeded much faster compared to uninoculated microcosms, completely removing added NO 3 − (3 mM) within 19 days of incubation (Fig. ). On the other hand, only one-third of the added NO 3 − (1 mM) was consumed in uninoculated microcosms after nearly 20 days of incubation. Therefore, it is evident that N-DAMO was the main process responsible for the complete removal of NO 3 − . The NO 3 − reduction driven by N-DAMO was, however, much slower in BPD compared to FPD and SCD where NO 3 − was removed within 80 and 60 hours after starting the incubation, respectively, and was much less dependent on the N-DAMO process as only a small fraction of CH 4 was oxidized (Figs. and ). The slow NO 3 − reduction rate in BPD is likely because of the high salinity, which was previously shown to negatively affect N-DAMO activity, substantially decreasing denitrification potential (Chen et al. , ). It was also shown that the intrusion of seawater negatively influences the denitrifying microbial community, decreasing the denitrification rate in freshwater ecosystems (Neubauer et al. ). It was hypothesized that the negative correlation between salinity and denitrification might be caused by higher sulfide and/or chloride levels (Rysgaard et al. , Craft et al. ). The concentration of NO 2 − in uninoculated BPD microcosms increased at the beginning of the incubation reaching its maximum at day 8 (98 ± 20 µM), however by the end of the experiment, it was completely gone. The N-DAMO inoculation clearly prevented the formation and accumulation of NO 2 − in water as it was quickly consumed at the beginning of the experiment (Fig. ). The NH 4 + concentration was highest in N-DAMO inoculated microcosms compared to uninoculated microcosms, reaching nearly 790 ± 70 µM at the end of the experiment compared to uninoculated microcosms where it was 145 ± 45 µM (Fig. ). It must be noted, however, that the N-DAMO inoculated microcosms had a higher concentration of NH 4 + from the beginning of the experiment, although it is not clear why. It might be related to decaying organic matter and biomass added with the N-DAMO inoculum and subsequent mineralization processes. Moreover, it has been previously shown that N-DAMO communities dominated by ANME archaea are also capable of dissimilatory nitrate reduction to ammonium (DNRA; Arshad et al. , Nie et al. ). Also, the N-DAMO enrichment culture used here appeared to be capable of DNRA as confirmed by the metagenomic analysis ( ). Therefore, it is possible that some of this NH 4 + originated from NO 3 − reduction. A substantial fraction of added NO 3 − in inoculated BPD microcosms was converted into N 2 gas as the ratio of 30 N 2 / 28 N 2 increased from 1% to 6%, while in uninoculated microcosms, this ratio increased only to 1.3% ( ). No NO 3 − was converted into N 2 O, which was deduced from the ratio of 46 N 2 O/ 44 N 2 O ( ). No increase of 46 N 2 O/ 44 N 2 O ratio suggests steady NO–N 2 O–N 2 reduction mediated by Ca . Methanoperedens and side N 2 O reducing community, or by Ca . Methylomirabilis oxyfera, via the conversion of two nitric oxide (NO) molecules to N 2 and O 2 bypassing N 2 O formation ( ). Freshwater ecosystems are known to have higher denitrification rates compared to brackish ones (Wang et al. ) and this was also found in our experiment. The freshwater SCD from Ooijpolder showed a very high denitrification potential as all NO 3 − was nearly completely depleted within 70 hours of incubation. The addition of the N-DAMO culture did not affect the denitrification rate as both inoculated and uninoculated microcosms performed similarly (Fig. ). In both cases, NO 2 − initially increased in concentration to 35 µM and afterward continuously decreased until it was nearly completely depleted after 53 hours (Fig. ). This is not surprising as the Ooijpolder from where the SCD sediment was collected is an agricultural area where denitrifying communities most likely are already well established. The initial NH 4 + concentration in SCD microcosms inoculated with the N-DAMO enrichment cultures was higher (128 µM) compared to values in uninoculated microcosms (81 µM). By the end of the incubation, the NH 4 + in bioaugmented microcosms was however very low (9.3 µM), while in control and NO 3 − supplemented microcosms it was still above 40 µM (Fig. ). The ratio of 30 N 2 / 28 N 2 increased from 0% to 3%, in both inoculated and uninoculated microcosms ( ) implying that similar denitrification rates occurred in both treatments. Inoculation of FPD sediment with the N-DAMO enrichment culture slightly accelerated the removal of NO 3 − (Fig. ) but did not affect the final concentration of NO 2 − (Fig. ) and NH 4 + (Fig. ). The ratio of 30 N 2 / 28 N 2 in FPD increased from 0% to 3%, in both inoculated and uninoculated, microcosms indicating conversion of 15 NO 3 − into 15 N 2 ( ). The ratio of 46 N 2 O/ 44 N 2 O increased, however, only in uninoculated microcosms supplemented with NO 3 − , reaching its maximum of 2.3% after 71 hours ( ). The production of N 2 O implies that incomplete denitrification or NH 4 + oxidation took place when NO 3 − was supplied to FPD sediment (Hallin et al. ). This is concerning as N 2 O is a potent greenhouse gas, third in the rank after CO 2 and CH 4 . The N-DAMO bioaugmentation prevented the formation of N 2 O possibly because Ca . Methylomirabilis can bypass N 2 O production by reducing NO intra-aerobically (Ettwig et al. ; ).
4 oxidation in brackish sediment It has been suggested that increasing N input to various ecosystems may stimulate N-DAMO activity and reduce CH 4 emission to the atmosphere (Shi et al. , Wang et al. ). In our experiment, 13 CO 2 was produced only in the presence of the N-DAMO enrichment culture (Fig. ), implying that the native microbial community was not actively oxidizing CH 4 . Most CH 4 was oxidized in the BPD microcosms, which was reflected in the highest increase of 13 CO 2 / 12 CO 2 ratio from 1% to >10% (Fig. ). This was further confirmed by the decrease in the total amount of CH 4 , which dropped from 0.42 to 0.38 mmol (Fig. ). Overall, 10% of the added CH 4 was consumed after 20 days of incubation. The concentration of CH 4 in uninoculated microcosms however slightly increased over time, although in the control setup, we did not observe methanogenic activity and build-up of CH 4 . Overall, it can be concluded that bioaugmentation with the N-DAMO enrichment culture indeed promoted CH 4 oxidation and resulted in lower emissions of this greenhouse gas in BPD. In the incubation with SCD and FPD sediment inoculated with N-DAMO, only a small amount of 13 CO 2 was produced as the ratio of 13 CO 2 / 12 CO 2 increased from 1.2% to 1.8% and to 3.2%, respectively (Fig. and ). Similarly, there was no difference in CH 4 concentration between inoculated and uninoculated microcosms (Fig. and ). This implies that heterotrophic NO 3 − reduction by the native microbial community, in both FPD and SCD sediments microcosm incubations, outcompeted N-DAMO microorganisms. The rapid depletion of NO 3 − and lack of alternatives led to a shortage of electron acceptors and consequently, CH 4 could not be oxidized. Previous studies showed that Ca . Methanoperedens and Ca . Methylomirabilis mediating N-DAMO have a high doubling time in the order of several weeks (Raghoebarsing et al. , Vaksmaa et al. ), resulting in lower competitiveness compared to fast-growing denitrifiers.
The bioaugmentation with N-DAMO appeared to be persistent as introduced species were still present in the microbial community and survived over the incubation time, in all microcosms (Fig. ). This is important as many bioinoculants used in agriculture have been shown to have low efficiency and survival rate over time (O’Callaghan ). Generally, the core microbial taxa were similar between all sediment types (Fig. ). All three sediments showed the presence of similar methanogenic archaea such as Methanosaeta, Methanoregula , and Methanobacterium . However, methanogenic archaea affiliating with Methanosarcina were only abundant in Ooijpolder SCD sediment where this taxon represented >9% of microbial community, while in peatland sediments of BPD and FPD its relative abundance was only 0.8% and 1.3%, respectively. Furthermore, the microbial community in BPD was characterized by a much higher relative abundance of Bathyarchaeia (∼50%) compared to SCD (∼20%) and FPD (∼20%). The SCD was also the only sediment type that had a high relative abundance of Nitrososphaera , among which most genera are known ammonia oxidizers (Cao et al. ). It can also be noticed that for each treatment, the original sediment collected in the field, control sediment (unamended), and NO 3 − amended sediment had nearly identical composition, meaning that the microbial community did not change much during the incubation and adding NO 3 − did not trigger changes in the microbial community. However, the inoculation with N-DAMO substantially changed the composition of the archaeal community. Specifically, in all sediments, Ca . Methanoperedens was by far the most dominating taxon (Fig. ). In the BPD microcosms, in the middle of the experiment, it represented >90% of the archaeal community. This suggests that (1) the abundance of archaea was generally low in BPD and (2) Ca . Methanoperedens adapted to the elevated salinity, which was previously suggested by Li et al. ( ). The relative abundance of Ca . Methanoperedens in the archaeal communities of SCD and FPD was also high, 73% and 47%, respectively, implying that these archaea were responsible for the CH 4 oxidation and 13 CO 2 formation in all inoculated sediments. Similarly to the archaeal microbial community, the bacterial community was also very similar across all sediment types but also between the original sediment collected in the field, control sediment (unamended), and NO 3 − amended sediment, halfway through the incubation (Fig. ). Although it was expected that denitrifying microorganisms would increase in relative abundance, it appeared that adding NO 3 − did not trigger changes in the microbial community. This may imply that the denitrifying community was already well established. Indeed, many microorganisms known to be involved in denitrification such as Dechloromonas, Thiobacillus, Bacillus , and Denitratisoma were detected in the microbial community in all sediment types. The inoculation with the N-DAMO enrichment culture led to the introduction of Ca . Methylomirabilis, which represented 10%, 3%, and 3% of the bacterial community in BPD, SCD, and FPD, respectively. Therefore, it can be assumed that the conditions for Ca . Methylomirabilis were the most suitable in BPD, where the denitrification rate was the lowest. The concentration of NO 2 − , a necessary electron acceptor for Ca . Methylomirabilis, was for the most time nearly undetectable in BPD microcosms. This is most likely because NO 2 − was continuously consumed by Ca . Methylomirabilis as soon as NO 3 − was reduced, consequently keeping its concentration at a very low level. Candidatus Methylomirabilis probably also contributed to CH 4 oxidation and 13 CO 2 formation in all inoculated microcosms. It appeared that Methylocystis was introduced together with the N-DAMO enrichment culture as its abundance increased in all inoculated microcosms. This type II methanotroph was particularly abundant in the BPD setup where it represented 6.5% of the bacterial community in inoculated microcosms, while it was nearly undetectable in control and NO 3 − supplemented setups. Also, Rhizobiales and Denitratisoma were found to be particularly enriched in N-DAMO inoculated BPD sediment, possibly contributing to N cycling, as many Denitratisoma species are known to be involved in NO 3 − reduction.
Agricultural runoff leads to increasing N content in surface waters. The input of nutrients further stimulates biomass production, eutrophication, fermentation, and, consequently, methanogenesis. As a result, agriculture-affected waters are strongly methanogenic and loaded with N compounds. There is an urgent need to restore the quality of these surface waters and decrease CH 4 emissions. Although many studies suggested that N-DAMO is an important sink of CH 4 , and might be a good bioremediation strategy, our study demonstrates that the effect of N-DAMO bioaugmentation is system specific. Inoculation with N-DAMO substantially contributed to the removal of NO 3 − and the decrease of CH 4 emission in brackish microcosms. It appeared that Ca . Methanoperedens’ and Ca . Methylomirabilis’ relative abundances were higher in the inoculated brackish sediment than in freshwater ones, implying their adaptability to saline conditions and competitiveness with the native microbial community. By contrast, in freshwater ecosystems, it appears that N-DAMO microorganisms are outcompeted by heterotrophic denitrifiers. Here, inoculation with N-DAMO microorganisms did not improve NO 3 − and CH 4 removal. This is most likely because in these freshwater ditches the denitrifying community is already well developed and outcompetes N-DAMO bacteria and archaea for the N substrates, thereby preventing the mitigation of CH 4 emissions. However, under more realistic, environmental conditions where continuous but rather low NO 3 − supplies from agricultural runoff will take place, the N-DAMO community might be able to access some of the leached NO 3 − , possibly decreasing CH 4 emission. To test this hypothesis, a mesocosm or field experiment should be conducted over a prolonged period. Moreover, the introduction of anammox bacteria together with N-DAMO could further contribute to the removal of NH 4 + and NO 2 − via conversion to N 2 . Overall, our results suggest that N-DAMO inoculation might be a potential strategy for climate-smart water quality protection and restoration in areas with increasing agricultural activities where the native microbial community is incapable of denitrification.
fnad041_Supplemental_File Click here for additional data file.
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Understanding the association between unmet dental care needs and household food security status among older people in Ghana
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af731be2-c125-4fe0-8f7f-0ca18d330ac0
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10214539
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Dental[mh]
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Defined as the “limitation or uncertain availability of nutritionally adequate and safe foods or limited or uncertain ability to acquire food in socially acceptable ways” (p.2330), food insecurity is a persistent public health issue facing older adults in sub-Saharan Africa (SSA) including Ghana . About 66% of people in SSA experienced food insecurity in 2020, which was considerably higher than estimates in other world regions such as Central and Southern Asia (43%) as well as Latin America and Caribbean (41%) . Among people within SSA, research suggests that older people are more likely to experience food insecurity in comparison to their younger counterparts . This trend seems to be extended to older people in Ghana. For example, Gyasi et al. find that 44% and 9% of people aged 50 or older experienced moderate and severe food insecurity, respectively. Blankson and Hall also find among women aged 60 and older that 42% and 20% missed a meal the day before and went to bed hungry, respectively. Research identifies food insecurity as a critical barrier to health care utilization. For instance, it has been found in the United States that people who experience food insecurity, compared to their food-secure counterparts, were less likely to have a usual source of care and more likely to postpone needed medical care and medication . Bhargava and Lee also observe in the United States that food-insecure older adults are less likely to have a family physician or receive home health visits compared to those who do not experience food insecurity. Similar findings have been shown in low- and middle-income countries. In Indonesia, for example, food insecurity was also observed as a risk of restricted access to modern health care for outpatient care . Research also shows in the Democratic Republic of Congo that the odds of adhering to antiretroviral therapy were lower among patients with food insecurity in comparison to those without any food insecurity . Although these studies are useful, we know very little about the relationship between food insecurity and dental care utilization among older people in SSA including Ghana. This void in the literature may be problematic, considering that evidence shows food insecurity as a risk factor of poor oral health. For example, it has been shown that adults aged 60 or older with food insecurity are more likely to have untreated tooth decay, compared to their counterparts with food security . Similarly, poor self-reported oral health is more prevalent among older adults with food insecurity . In Ghana, Amoak et al. find that older adults who report experiencing mild, moderate, and severe food insecurity are more likely to report poor oral health compared to those who were food secure. As food insecurity has been identified as a barrier to the utilization of health care services, it is concerning that older adults who experience food insecurity may be facing a unique barrier to dental care utilization in Ghana, despite their possible exposure to heightened oral health risks. The oral health sector in Ghana is experiencing growth, but there is little investment in both private and public practice [ – ]. The sector’s underdevelopment is evident in the low ratio of dental practitioners to patients, estimated at 1 in 104, 000 . The dental sector is further characterized by spatial inequality whereby 75% of dentists operate in urban areas, with an estimated 70% situated in the two largest cities in the country, that is Accra and Kumasi . Reports further indicate that less than 30% of government hospitals and clinics have dental facilities, of which a third are ill-equipped or dilapidated . While the National Health Insurance Scheme (NHIS) covers various oral health services such as pain relief, tooth extraction, dental restoration, and fillings , these services are also unevenly accessible . Specifically, inequities in healthcare access in Ghana, including low enrollment of the NHIS, shortage of personnel, unavailability and ill-equipped health facilities and an inability to pay for services, may compel vulnerable groups such as older adults to often delay visiting a dentist to address oral health problems, or leave them untreated . This problem of unmet oral care is compounded by the unavailability of procedures for detecting, managing, and treating oral diseases in government-run primary healthcare facilities, such as the community health planning and services (CHPS) compounds that are prominent in rural areas of Ghana. These include inadequate screening for early detection of oral diseases and the lack of basic restorative dental procedures to treat existing dental decay . While oral health is linked to social, psychological, and economic well-being, research also shows that oral health is systemically linked to chronic diseases such as diabetes and heart disease . In this context, it is important to highlight that about 400 million people in SSA have been affected by oral health issues, thereby receiving a growing attention as a public health concern. This situation may be particularly relevant among older people in Ghana where the prevalence of caries and periodontitis is higher among those aged 55 years or older . To this end, the current study uses a representative survey of adults aged 60 or older from three regions of Ghana to examine the association between unmet dental care needs and household food security status.
This study used data from a cross-sectional survey of older adults (people aged 60 years and over) in Ghana. Data were collected from June to August 2019 with the assistance of experienced research assistants from the University of Ghana, the University for Development Studies, and the Catholic University College, Ghana. Research assistants were trained on the survey and various ethical issues and were each made to sign a confidentiality agreement prior to the data collection. Ethical approval for the study was obtained from the Queen’s University General Research Ethics Board (GGEOPL-277-19). In addition, verbal and written informed consent was sought from the participants. Legally Authorized Representatives of illiterate participants (e.g., family members) also provided informed consent for the study. The data were collected using a multi-stage sampling framework. We randomly sampled one region each from three major agroecological zones in Ghana, enabling us to select three regions including Greater Accra Region from the Coastal Zone, Bono East Region from the Forest Zone, and Upper West Region from the Savannah Zone. Furthermore, we randomly drew two districts from each region making 6 districts in all. Ten enumeration areas were then randomly sampled from each district. Finally, we identified and interviewed one respondent each from randomly chosen households within these enumeration areas. Standardized data collection instruments on food security, health, health behaviours, and access to care were adapted from the Ghana Living Standards Survey and the WHO Study on Global Ageing and Adult Health . The total sample is 1,073. However, for the purpose of this study, we restricted the sample to 352 older adults who had any problems with mouth and teeth in the last 12 months. Dependent variable Among respondents who had any oral and dental problem in the last 12 months, the survey asked whether they received any treatment from a dentist or other oral health specialist, with yes, no or can’t remember as possible responses. Based on this question, we created the binary dependent variable called ‘unmet dental care needs’ where they were coded as ‘unmet’ when they did not receive any treatment (0 = met; 1 = unmet). Independent variable Food security status is our focal explanatory variable, which is constructed using the nine-item Household Food Insecurity Access Scale (HFIAS) developed by Coates et al. . Respondents were asked to answer questions about the frequency of occurrence of each food insecurity situation on a Likert scale (0 = rarely; 1 = sometimes; 2 = often) when they experienced each situation Based on the scores from the HFIAS questionnaire, households were divided into four categories of food insecurity: food secure (i.e., scores 0–1), mildly food insecure (i.e., scores 2–7), moderately food insecure (i.e., scores 8–14), and severely food insecure (i.e., scores 15–27). Considering that the number of respondents who experienced mild insecurity was very small, we combined this category with moderate insecurity, resulting in that we have three categories for the dependent variable (0 = food security; 1 = mild/moderate insecurity; 2 = severe insecurity). Control variables The relationship between household food security status and unmet dental care needs may be confounded by other factors. To account for potential founding factors, we introduced predisposing and enabling factors as control variables informed by the Andersen healthcare utilization model . We added predisposing factors such as place of residence (0 = urban; 1 = rural), gender (0 = male; 1 = female), education (0 = secondary/higher education; 1 = primary education; 2 = no education), marital status (0 = currently married; 1 = formerly married; 2 = never married), age (0 = 80+; 1 = 70–79; 2 = 60–69), and religion (0 = Muslim; 1 = Christian; 2 = traditionalist; 4 = no religion). We included two enabling factors, namely health insurance enrolment (0 = yes; 1 = no) and household wealth (0 = richest; 1 = richer; 2 = poorer; 3 = poorest). Household wealth quintiles are measured by utilizing a composite index based on the presence of various household assets such as a vehicle, television, tractor, refrigerator, mobile phone, hoe, and radio. Statistical analysis There were two separate analyses for this study. First, we employed univariate analysis to understand the characteristics of analytical sample. Second, due to the binary nature of the dependent variable, logistic regression analysis was applied to understand the relationship between household food security status and unmet dental care needs. Given the multi-stage sampling strategy, our analyses were weighted with sampling weights constructed for each stage of sampling based on the probabilities of each sampling unit being selected as part of the sample. Models were built sequentially. We explored the bivariate association between food security status and unmet dental care needs in Model 1 while Models 2 and 3 further accounted for predisposing and enabling factors, respectively. Results were shown with odds ratios (ORs). ORs larger than 1 indicate that people were more likely to experience unmet dental care needs while those lower than 1 have lower odds of doing so.
Among respondents who had any oral and dental problem in the last 12 months, the survey asked whether they received any treatment from a dentist or other oral health specialist, with yes, no or can’t remember as possible responses. Based on this question, we created the binary dependent variable called ‘unmet dental care needs’ where they were coded as ‘unmet’ when they did not receive any treatment (0 = met; 1 = unmet).
Food security status is our focal explanatory variable, which is constructed using the nine-item Household Food Insecurity Access Scale (HFIAS) developed by Coates et al. . Respondents were asked to answer questions about the frequency of occurrence of each food insecurity situation on a Likert scale (0 = rarely; 1 = sometimes; 2 = often) when they experienced each situation Based on the scores from the HFIAS questionnaire, households were divided into four categories of food insecurity: food secure (i.e., scores 0–1), mildly food insecure (i.e., scores 2–7), moderately food insecure (i.e., scores 8–14), and severely food insecure (i.e., scores 15–27). Considering that the number of respondents who experienced mild insecurity was very small, we combined this category with moderate insecurity, resulting in that we have three categories for the dependent variable (0 = food security; 1 = mild/moderate insecurity; 2 = severe insecurity).
The relationship between household food security status and unmet dental care needs may be confounded by other factors. To account for potential founding factors, we introduced predisposing and enabling factors as control variables informed by the Andersen healthcare utilization model . We added predisposing factors such as place of residence (0 = urban; 1 = rural), gender (0 = male; 1 = female), education (0 = secondary/higher education; 1 = primary education; 2 = no education), marital status (0 = currently married; 1 = formerly married; 2 = never married), age (0 = 80+; 1 = 70–79; 2 = 60–69), and religion (0 = Muslim; 1 = Christian; 2 = traditionalist; 4 = no religion). We included two enabling factors, namely health insurance enrolment (0 = yes; 1 = no) and household wealth (0 = richest; 1 = richer; 2 = poorer; 3 = poorest). Household wealth quintiles are measured by utilizing a composite index based on the presence of various household assets such as a vehicle, television, tractor, refrigerator, mobile phone, hoe, and radio.
There were two separate analyses for this study. First, we employed univariate analysis to understand the characteristics of analytical sample. Second, due to the binary nature of the dependent variable, logistic regression analysis was applied to understand the relationship between household food security status and unmet dental care needs. Given the multi-stage sampling strategy, our analyses were weighted with sampling weights constructed for each stage of sampling based on the probabilities of each sampling unit being selected as part of the sample. Models were built sequentially. We explored the bivariate association between food security status and unmet dental care needs in Model 1 while Models 2 and 3 further accounted for predisposing and enabling factors, respectively. Results were shown with odds ratios (ORs). ORs larger than 1 indicate that people were more likely to experience unmet dental care needs while those lower than 1 have lower odds of doing so.
Table shows sample characteristics. We find that 40% of older people with dental problems had unmet dental care needs. In addition, about half of the sample experienced severe food insecurity (49%) while slightly more than one-quarter did not experience food insecurity (28%). We also find that about half of the sample lived in rural areas (55%) and were female (48%), currently married (48%), and Christian (56%). About one in three older people had secondary or higher education (32%) although 44% did not have any formal education. It is also noteworthy that 76% of the sample did not have health insurance. Table shows findings from logistic regression analysis. In Model 1, we find at the bivariate level that older people who experienced severe food insecurity had higher odds of reporting unmet dental care need, compared to their counterparts who did not experience any food insecurity (OR = 2.08, p < 0.01). This significant relationship remained largely robust, even after accounting for predisposing factors in Model 2 (OR = 2.07, p < 0.05) and enabling factors in Model 3 (OR = 1.94, p < 0.05). In addition to household food security status, a range of predisposing and enabling factors were significantly associated with unmet dental care needs (see Model 3 of Table ). For example, female older people were more likely to report unmet dental care needs in comparison to their male counterparts (OR = 4.13, p < 0.01). Similarly, older people who have never been married were more likely to report unmet dental care needs in comparison to those who were married at the time of the survey (OR = 2.57, p < 0.05). In addition, older people who were affiliated with traditional religions were more likely to report unmet dental care needs, compared to their Muslim counterparts (OR = 4.42, p < 0.01). Compared to their richest counterparts, older people whose household wealth belonged to the ‘poorest’ category were also more likely to report unmet dental care needs (OR = 2.49, p < 0.05). Finally, older people without any health insurance had higher odds of reporting unmet dental care need in comparison to those who did (OR = 2.60, p < 0.05).
Older adults from food-insecure households are often exposed to heightened risks of oral health issues, which are systemically linked to chronic diseases such as diabetes and heart diseases. Access to dental care services for timely diagnosis and treatment is particularly important among this population. As a barrier to health care utilization in general, however, dental care utilization may be restricted due to food insecurity. Using a representative survey of people aged 60 or older from three regions of Ghana, the current study aims to advance the literature by examining whether food security status is associated with unmet dental care needs among older adults. We find that household food insecurity is a significant predictor of unmet dental care needs. Specifically, having an “unmet dental need” in the context of our study refers to respondents who self-reported a dental health problem but did not seek treatment for it. Thus, older people who experienced severe food insecurity were more likely to report unmet dental care needs compared to their counterparts who did not experience any food insecurity, even after accounting for a range of predisposing and enabling factors. This finding is largely consistent with Giannoni and Grignon , observing that food insecurity is associated with higher odds of reporting lack of access to dental care in Canada. Similarly, it is found in the United States that adults with low food security were more likely to have unmet dental care needs as compared to their counterparts with full food security . In the Ghanaian context, Amoak et al. highlighted that older people who experienced severe food insecurity were more likely to rate their oral-health as poor, pointing to possible unmet dental needs. Although the association between dental care utilization and food security status is largely explored in high-income countries such as the United States and Canada, it is imperative that food insecurity as a potential barrier to meeting dental care needs may be extended to older people in the LMICs including Ghana. There are potential reasons to explain higher odds of reporting unmet dental care needs among older people with severe household food insecurity in Ghana. As explained by Weiner et al. , food insecurity is situated within the wider social context of the food system, which encompasses factors such as the availability of fresh and nutritious food. Older individuals, for example, may encounter difficulties in accessing such foods, which can compromise their dental health and diminish the benefits that are typically associated with a healthy diet. Other studies have also highlighted that food insecure adults are more likely to consume poor quality diets and employ coping strategies that may include lower intake of vegetables, fruits, dairy products, zinc, calcium and magnesium [ – ]. While this association is established, earlier studies such as Barret posits that the concept of food security is complex and multi-dimensional as it goes beyond measures of financial poverty to capture other household shocks including periodic unemployment and loss of productive assets that reflect the social and structural vulnerabilities of individuals and households. Indeed, food security is not only argued to represent the social vulnerability of households but may unveil their heightened exposure to economic and political disenfranchisement [ – ]. Furthermore, it has been argued that households may not prioritize health care utilization including dental care utilization when basic resources required for survival such as food and shelter are not accessible . In addition to treatment cost, the cost of travelling to dental care facilities may be perceived as an extra burden on meeting household basic needs, considering that dental care resources are largely scarce in many SSA countries, often requiring people to travel far to have access to treatment from dental care facilities . Previous research also points to the possibility that food-insecure households actively seek for cheaper treatment options . In other words, older people with dental problems may rely on other alternative, cost-effective, and home-based remedies, instead of receiving formal treatment from dental care facilities. These arguments largely underline possible pathways where older people with food insecurity may redistribute limited household resources through avoiding dental care utilization, which may be perceived as less essential for survival. Although not the focus of this study, we also find that some predisposing and enabling factors were significantly associated with unmet dental care needs. For example, we find that marital status was associated with unmet dental care need, showing that never married people were more likely to report unmet dental care needs, compared to their currently married counterparts. This finding is consistent with previous research, which argues that marriage can serve as social capital that promotes desirable health-related behaviours including access to dental care services . We also find that traditionalists were more likely to report unmet dental care needs in comparison to Muslims. This finding may be explained by Peprah et al. who reveal that some traditionalists can use self-treatment prescribed by faith healers. Finally, we find that low household wealth and lack of insurance enrolment were positively associated with unmet dental care needs. These findings are consistent with previous studies, suggesting that there are unequal financial barriers to health care utilization including dental care utilization in Ghana . Based on these findings, there are several policy implications. The literature highlights that food insecurity possibly makes it difficult for older people to spend scarce household resources on items that are often perceived to be less essential for survival including access to dental care services [ , , ]. This possible mechanism may be useful for informing food-related policies to pay attention beyond nutritional values of food and introduce policy strategies that enable food-insecure households to flexibly allocate material resources, which may increase their chance of receiving required health care attention including dental care attention. From the perspective of health-related policies, however, it is equally critical to review and potentially revise the NHIS in Ghana. Specifically, although the NHIS’s focus is to protect socially and financially vulnerable groups from being excluded from access to health care including dental care, some important measures of vulnerability such as food insecurity are not integrated as part of the policy . These policy suggestions may be helpful for reducing the burden of unmet dental care needs among older people. There are some limitations to this study. For example, we use a cross-sectional survey. Therefore, our results are limited to statistical association and cannot claim any causal relationship. In addition, we rely on the self-reported indicator of unmet dental care needs. Considering that we do not have any clinical and biomedical indicators of oral health, the accuracy of dental care needs is unknown and unmeasurable. For this reason, unmet dental care needs may be underreported or overreported. Thirdly, limiting our sample to individuals who self-reported oral health issues may have underestimated the actual magnitude of oral health problems in Ghana, as some people may not be aware of their oral health issues. Prospective studies should move beyond self-reporting and incorporate clinical measures to obtain a more accurate assessment of oral health issues among older adults in Ghana. Furthermore, in community-level studies in Ghana where access to regular dental check-ups remain low, we recommend future studies to include dental examinations as part of community-level health surveys. Additionally, the data also do not include other oral health-related behavioural factors such as tooth brushing, which may have been a predictor of unmet dental care needs. To overcome these limitations, incorporating clinical measures of oral health, such as the index of decayed, missing, and filled teeth, untreated cavities, and salivary flow, should be considered in future research . We also recommend future research to employ longitudinal analysis as well as in-depth qualitative analysis, which may be useful for deconstructing the underlying processes that inform unmet dental care needs among older people in Ghana.
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Relationship between dental anxiety levels and oral health among dental patients in Turkey: a cross-sectional study
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923bb2b5-35cc-4aa2-af0e-764bba9a5dbe
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10214591
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Dental[mh]
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Dental anxiety is widespread and poses an important problem during the management of patients in different age groups . In developed countries, patients are affected by dental anxiety . Dental hygiene habits are affected by dental fear . Studies have shown that patients with dental fear commonly ignore dental care, and dental anxiety can be an important cause of missed or cancelled dental appointments . Dental anxiety is a multifactorial condition . Previous dental experiences play a major role in developing dental anxiety, which is exhibited by painful and traumatic experiences regarding dental treatment, unpleasant dentist contact and general fearfulness . Enhancing dental anxiety includes fear of pain, introversion, weakness, loss of control, fear of unexpected events and shame . Children with parents with dental anxiety can be affected and are highly prone to dental fear . Studies have shown that the prevalence of dental anxiety ranges from 11% to 91.2%. These differences can be attributed to the subjectivity of the diagnostic criteria, assessment methods and sampling techniques of the studies. A study has reported a high prevalence of dental anxiety in a group of adult patients in India at 46% . Other studies have reported that the prevalence rates of dental anxiety are at 61.3%, 72% and 91.2% [ – ]. In Turkey, the reported prevalence rates of dental anxiety are at 5.1%, 21.3-23.5%, and 42.1% [ – ]. A vicious cycle exists between dental anxiety, which leads to delayed dental care, and oral health, which causes dental deterioration. Individuals entering this vicious circle may have increased dental anxiety and may require more dental treatment for an existing oral problem than persons who routinely visit the dentist for regular control . In addition, a great functional disturbance may occur in patients with dental anxiety, and their quality of life can decrease . This condition can damage their psychological affluence, social function and vitality. Several studies have reported that dental anxiety causes poor oral health, with more decayed, missing and fewer filled teeth and worse periodontal health [ , , ]. The oral health of patients with dental anxiety still has inconclusive findings . At present, the public’s dental anxiety should be evaluated, as this causes management problems during dental treatment and to determine its relationship with oral health status to improve the condition . This study aimed to determine the relationship between dental anxiety and sociodemographic factors and oral health involving dental and periodontal status in patients attending the Suleyman Demirel University in Isparta, Turkey.
Ethics The present cross-sectional study was performed in the clinics of the Restorative Dentistry Department at the Faculty of Dentistry at Suleyman Demirel University. The ethics committee of Suleyman Demirel University, Faculty of Medicine, verified the study protocol (SDU.05.05.2021-11/193) and written informed consent was acquired from each patient before the study. The present study was conducted from May 2021 to August 2021. Sample G-Power software package programme (G*Power Ver. 3.0.10, Germany) was used for the power analysis. At least 459 patients were essential for the study, with 90% power in the d = 0.15 impact width. To avoid possible data loss, we added 41 patients and included a total of 500 patients in the study. Adult patients aged between 18 and 84 years and without any physical or mental illness were included in the study. All patients were reported with a standardised structured questionnaire. The questionnaire provided information regarding sociodemographic characteristics, oral care, dental visits, parafunctions, smoking habits, diet, postponing dental appointment habits, dental fillings and painful experiences. The questionnaires were immediately completed under supervision. Intraoral examination The intraoral dental examination of all patients was performed by the same dentist using a conventional dental probe, mirror, and overhead dental lamp . Digital panoramic radiographs (Planmeca, Helsinki, Finland) were used to detect proximal caries lesions. Bite-wing radiographs were taken in addition to panoramic radiographs in cases where interproximal contact areas could not be recognized alone due to superpositions. The total number of teeth, decayed, missing, or filled teeth (DMFT), decayed, missing, or filled surfaces (DMFS) and gingival index (GI) scores were determined. After air drying the teeth, the DMFT and DMFS scores were calculated. Caries were identified according to the criteria of the World Health Organization (WHO 1997) . Teeth with a carious lesion in a pit or fissure, or on the tooth surface, and teeth with a softened floor, softened wall, or undermined enamel are recorded as carious. A filled tooth containing decayed areas was also recorded as carious. The restored teeth due to caries were recorded as filling. The extracted teeth due to caries were recorded as missed. Teeth restored for causes other than caries were not counted as filling. Third molars were not included in the current study. Periodontal status was identified according to GI. The six index teeth (i.e. teeth 44, 32, 36, 24, 12 and 16) were assessed with GI of Silness and Löe . Dental anxiety score A modified dental anxiety scale (MDAS) was used to evaluate dental anxiety in the current study . The scale is comprised of five questions to measure the anxiety level in a certain dental condition before the clinical examination. The scale consists of the following questions: (Q1) If you had to visit the dentist tomorrow, how would you feel about it? (Q2) While waiting for your turn in the dental office, how would you feel? (Q3) How would you feel if you knew that a tooth drill would be used? (Q4) How would you feel if you knew that your teeth would be scaled and polished? (Q5) How would you feel if you knew that a local anaesthetic injection would be administered in your gingiva or over an upper back tooth? The answers were calculated based on the MDAS, and ratings of 1–5 demonstrated not anxious , slightly anxious , fairly anxious , very anxious, and extremely anxious , correspondingly. Therefore, the maximum score that can be calculated from each question was 5. In addition, the minimum and maximum scores of the entire scale were rated 5 and 25, respectively. MDAS scores of 5–9, 10–18, and ≥19 were defined as less anxiety, moderate anxiety, and high dental anxiety, respectively . Statistical analysis The statistical program SPSS (version 25.0) was used to evaluate the data. The normality distribution of the data was checked using the Shapiro–Wilk and Kolmogorov–Smirnov tests. The relationships between the MDAS and categorical variables were determined using the Chi-square test. The relationships between the MDAS and GI scores were evaluated using the Chi-square test. Group differences were analysed using the Mann–Whitney U or Kruskal–Wallis tests and Bonferroni post hoc correction, as necessary. The correlation between the scores of the MDAS and DMFT or DMFS indexes were assessed with Spearman’s correlation analysis. The level of significance was 0.05 for each statistical analysis.
The present cross-sectional study was performed in the clinics of the Restorative Dentistry Department at the Faculty of Dentistry at Suleyman Demirel University. The ethics committee of Suleyman Demirel University, Faculty of Medicine, verified the study protocol (SDU.05.05.2021-11/193) and written informed consent was acquired from each patient before the study. The present study was conducted from May 2021 to August 2021.
G-Power software package programme (G*Power Ver. 3.0.10, Germany) was used for the power analysis. At least 459 patients were essential for the study, with 90% power in the d = 0.15 impact width. To avoid possible data loss, we added 41 patients and included a total of 500 patients in the study. Adult patients aged between 18 and 84 years and without any physical or mental illness were included in the study. All patients were reported with a standardised structured questionnaire. The questionnaire provided information regarding sociodemographic characteristics, oral care, dental visits, parafunctions, smoking habits, diet, postponing dental appointment habits, dental fillings and painful experiences. The questionnaires were immediately completed under supervision.
The intraoral dental examination of all patients was performed by the same dentist using a conventional dental probe, mirror, and overhead dental lamp . Digital panoramic radiographs (Planmeca, Helsinki, Finland) were used to detect proximal caries lesions. Bite-wing radiographs were taken in addition to panoramic radiographs in cases where interproximal contact areas could not be recognized alone due to superpositions. The total number of teeth, decayed, missing, or filled teeth (DMFT), decayed, missing, or filled surfaces (DMFS) and gingival index (GI) scores were determined. After air drying the teeth, the DMFT and DMFS scores were calculated. Caries were identified according to the criteria of the World Health Organization (WHO 1997) . Teeth with a carious lesion in a pit or fissure, or on the tooth surface, and teeth with a softened floor, softened wall, or undermined enamel are recorded as carious. A filled tooth containing decayed areas was also recorded as carious. The restored teeth due to caries were recorded as filling. The extracted teeth due to caries were recorded as missed. Teeth restored for causes other than caries were not counted as filling. Third molars were not included in the current study. Periodontal status was identified according to GI. The six index teeth (i.e. teeth 44, 32, 36, 24, 12 and 16) were assessed with GI of Silness and Löe .
A modified dental anxiety scale (MDAS) was used to evaluate dental anxiety in the current study . The scale is comprised of five questions to measure the anxiety level in a certain dental condition before the clinical examination. The scale consists of the following questions: (Q1) If you had to visit the dentist tomorrow, how would you feel about it? (Q2) While waiting for your turn in the dental office, how would you feel? (Q3) How would you feel if you knew that a tooth drill would be used? (Q4) How would you feel if you knew that your teeth would be scaled and polished? (Q5) How would you feel if you knew that a local anaesthetic injection would be administered in your gingiva or over an upper back tooth? The answers were calculated based on the MDAS, and ratings of 1–5 demonstrated not anxious , slightly anxious , fairly anxious , very anxious, and extremely anxious , correspondingly. Therefore, the maximum score that can be calculated from each question was 5. In addition, the minimum and maximum scores of the entire scale were rated 5 and 25, respectively. MDAS scores of 5–9, 10–18, and ≥19 were defined as less anxiety, moderate anxiety, and high dental anxiety, respectively .
The statistical program SPSS (version 25.0) was used to evaluate the data. The normality distribution of the data was checked using the Shapiro–Wilk and Kolmogorov–Smirnov tests. The relationships between the MDAS and categorical variables were determined using the Chi-square test. The relationships between the MDAS and GI scores were evaluated using the Chi-square test. Group differences were analysed using the Mann–Whitney U or Kruskal–Wallis tests and Bonferroni post hoc correction, as necessary. The correlation between the scores of the MDAS and DMFT or DMFS indexes were assessed with Spearman’s correlation analysis. The level of significance was 0.05 for each statistical analysis.
The present study included 500 participants, with a median MDAS value of 9.00 (Table ). The Cronbach’s alpha of the MDAS was 0.898 for the current study, which indicated good internal consistency and reliability ( N = 500). Furthermore, 51 (10.2%) of the 500 patients may be categorized with a high MDAS score of 19 or greater (Table ). The median MDAS value of patients in the age group of 20–34 years was higher than that of the individuals in the age group of 50–64 years (Kruskal–Wallis test, p < 0.05). A significant relationship was found between dental anxiety (MDAS) and gender (Chi-square test, p < 0.05). The median MDAS value in women was higher than that in men in the paired comparisons (Mann–Whitney U test, p < 0.05; Table ). On the one hand, patients with no regular income had higher MDAS median values than those with regular income. On the other hand, the median MDAS value of persons with health insurance was higher than that of those without health insurance. In addition, the median value of the dental anxiety score (MDAS) of individuals who visited the dentist with a companion was higher than that of those who visited the dentist without a companion. A statistical difference was found among dental anxiety levels according to the reason for dental visits (Mann–Whitney U, p < 0.05). The median MDAS values of individuals who did not remember the reason for the dental visit were higher than the median value of those who visited the dentist for routine control (Kruskal–Wallis test, p < 0.05; Table ). No statistically significant relationship was found between the dental anxiety score (MDAS) of individuals and the educational status, marital status, smoking habit, and frequency of dental visits (Chi-square test, p > 0.05; Table ). A statistically significant relationship was found between dental anxiety (MDAS) and the total number of teeth in individuals. The dental anxiety rate was higher in participants with 1–9 teeth than in participants with more than 20 teeth (Chi-square test, p = 0.05). The MDAS values of individuals who experienced pain at the first and current filling treatments were higher than those who did not experience any pain (Mann–Whitney U test, p < 0.05). Individuals who postponed current appointments had higher MDAS values than those who did not. In addition, the median MDAS value of individuals who received local anaesthesia during treatment was higher than those who were not applied (Mann–Whitney U test, p < 0.05). Patients with self-reported bad sensation at the current filling treatment had the highest median MDAS value in the present study compared with the other self-reported sensation groups (Kruskal–Wallis test, p < 0.05; Table ).
No statistically significant relationship was found between the MDAS and DMFT and DMFS indexes in the individuals (Spearman correlation analysis, p > 0.05; Table ). Furthermore, no statistically significant relationship was observed between the dental anxiety levels (MDAS) of patients and GI scores (Chi-square test, p > 0.05; Table ).
In the present study, MDAS was used to assess anxiety levels because it is a concise, easy-to-use, and accepted scale . Dou et al. found that the mean for the MDAS score is 14.17 higher than our scores . A study in South India that included 1,148 adults has reported that the mean dental anxiety level is 10.4, which was consistent with our findings . The median value of MDAS is 9.00, indicating that most participants in the current study exhibited less anxiety . In the literature, traumatic dental experiences, personal characteristics, gender, age and education levels may affect the dental anxiety level of patients [ – ]. Compared with the current study, a few studies in the literature have shown an opposite association between age and dental anxiety levels [ , , ]. A study has shown that the anxiety level of patients in the age group of 20–39 years was higher than in younger and older groups, which was in accordance with our findings . In the current study, although no statistical difference was found between all age groups in terms of dental anxiety level, the median value of MDAS of patients in the age group of 20–34 years was higher than the median value of MDAS in the age group of 50–64 years. This result may be because over time and with age, enhanced exposures have allowed the patients to indulge in the treatment and have less anxiety. Gender differences can be a determining factor in dental anxiety levels [ – ]. Previous studies have reported that during dental treatment, women experience more anxiety than men, which was in accordance with the current study [ , – ]. Although no statistical difference according to gender was found, the level of anxiety in women is higher than in men in one study . Notably, the difference in dental anxiety between the sexes may be due to structural and functional alterations in the brain and hormonal changes. Furthermore, this difference may depend on the higher stress levels and phobias of women compared with men in the present study . A few studies have examined the relationship between income levels and anxiety levels . Tunç et al. found no significant relationship between MDAS scores and income levels, which was in contrast to our findings . Melchior et al. found that individuals in low-income families are highly prone to anxiety and depression, which was in accordance with our findings . This study found a statistically significant relationship between anxiety levels and monthly income. Furthermore, in this study, those with a regular monthly income had lower anxiety scores than those without a regular monthly income. High levels of dental anxiety can be considered the primary reason for tooth loss, treatment cancellation or missed dental appointments . In support of our findings, previous studies have reported that patients with anxiety about visiting the dentist tend to postpone their treatment unless during an emergency [ – ]. Furthermore, people with dental anxiety have irregular visits to dentists [ , , ]. Restorative treatments or tooth loss management may be considered with regular dental attendees; however, patients with dental anxiety may perceive that dentists only merely offer symptomatic care for their declining oral health rather than a comprehensive treatment . Haggling et al. reported that individuals with high anxiety have fewer teeth than those with lower anxiety, which supported our results . The anxiety rate of patients with 1–9 teeth was higher than that of the patients with more than 20 teeth in the current study. Unfortunately, this may be due to delayed dental visits, and the existing teeth often become incurable or untreatable anymore . Previous studies, as well as the current study, have reported a significant relationship between dental anxiety and pain . Dou et al. reported that during the most recent dental examination, patients who felt a high rate of pain tended to have high anxiety . Patients who desire the administration of anaesthesia during treatment have signs of dental fear or anxiety . In our study, similar to the findings of the previous study, patients who experienced pain during filling had higher anxiety rates than patients without pain. Furthermore, the dental anxiety rate of individuals who received local anaesthesia during treatment was higher than that of those without any anaesthesia. This was considered to provide effective treatment in dentally anxious patients; particularly in dentally phobic individuals, more effective local anaesthesia and anti-anxiety strategies should be developed in the future for dental treatment. In the literature, several studies have compared dental anxiety levels with the DMFT index . A study has found a significant and inversely proportional relationship between the number of filled teeth and anxiety, which was in contrast to our findings. Zinke et al. found a significant relationship between dental anxiety levels and the DMFS index. In addition, the rate of hard tissue destruction due to caries is higher in individuals with high dental anxiety than in individuals with low dental anxiety levels . Folayan et al. also showed that the number of decayed teeth in patients with high anxiety levels is higher than in those with low anxiety levels . Some studies have reported no significant relationship between dental anxiety and the DMFT index, which was consistent with our results [ , , , ]. A previous study has reported no statistically significant relationship between the number of teeth extracted (MT) and the number of filled teeth (FT) and dental anxiety, which was in accordance with our results . The present study found no significant relationship between MDAS and DMFT and DMFS scores, which are the indices associated with dental caries prevalence employed in previous studies. No correlation exists between the anxiety level of patients and periodontal parameters in several studies [ , , ]. Similarly, no relationship was found between MDAS and GI, which is a periodontal parameter in the present study. The fact that the research group consisted of individuals who applied to the Faculty of Dentistry for treatment can be considered a limitation of the present study. A small sample group in the study cannot be entirely representative of the community regarding dental anxiety levels. In addition, periodontal evaluation performed with only the gingival index and not using the plaque index can be considered another limitation of the study. The gingival health level of individuals could not be completely revealed with a single periodontal parameter in the study.
The findings of the study indicated that dental anxiety negatively affected the dental visiting habits of patients. In this cross-sectional study, patients with dental anxiety had more missing teeth and more frequently postponed their dental appointments. Further studies on the relationship between dental anxiety and oral health are necessary to ensure the benefit of dental services and to create an effective treatment for persons with dental anxiety.
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Influence of design implant and apical depth in post-extraction sockets: an in vitro simulated study
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ccc8ae1f-2f16-437e-a9a8-44ea872b35ea
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10214678
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Dental[mh]
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The introduction of osseointegrated implants has marked a turning point in dental practice. Immediate loading of dental implants has become more popular, which is due to several factors that include reduced treatment time, and aesthetic and psychological benefits for the patient. A fundamental prerequisite for implant success is primary stability at insertion . This can be indirectly measured by recording insertion torque data or by measuring the implant stability quotient (ISQ) using resonance frequency analysis (RFA) . Studies have shown that primary stability is influenced by factors such as implant length and diameter, design, insertion technique, and compatibility between the implant and the surrounding bone [ – ]. Procedures for placement of implants in post-extraction sockets require excellent apical stabilization , while both bone quality and quantity remain key stabilization factors. Low bone density and/or reduced supporting bone tissue remain the greatest risk factors for implant loss . With regard to design, tapered implants are reported to be more stable than cylindrical implants [ – ]. Implant stability is also associated with thread design, and thus thread depth, thickness, angle, pitch, and helix angle all affect the biomechanical loading distribution of the implant . The success of immediate loading protocols depends strictly on the clinician’s ability to ensure the stability of the primary implant and then to monitor changes in stability and healing time. Our study, based on the hypothesis that implants of different shapes and placed at greater depths may provide greater primary stability, (allowing implants to receive an occlusal load earlier), evaluated the mechanical stability of tapered implants, when placed in simulated anterior maxilla region post-extraction bone cavities.
A controlled in vitro laboratory mechanical test regime involving 72 titanium tapered implants (dimension: 3.5 × 13 mm) was performed (Neodent, Brazil). The implants were not experimental; i.e., they were available on the market at the time: “Drive” and “Helix”. The implants presented two macrogeometries with different thread designs (Groups A and B). Group A implants presented tapered bodies, with cylindrical coronal portions, and tapered apical portions. The apices were active and included smooth, rounded tips, and helical chambers. The threads were compressive in the coronal portions, and triangular and self-cutting in the apical regions. Group B implants presented double square main threads, with counterclockwise cutting chambers distributed along the implant bodies, and rounded apices with cutting edges. Six polyurethane blocks (Nacional Ossos, Jaú, São Paulo, Brazil) were used, presenting the following specifications: 60 mm wide × 140 mm long × 33 mm thick. The block density was PCF 30, corresponding to 0.48 g/cm 3 , and simulated bone conditions in the anterior maxillary region. Twelve pyramidal simulations of the upper central incisor sockets (Fig. ) were performed with the insertion of 12 implants (1 in each simulation). The simulations are demonstrated in Fig. . To simulate post-extraction sockets, twelve defects were created in the 6 polyurethane blocks produced during the manufacture of the material. The defects in these sockets are presented in Fig. . All simulations were performed by the same surgeon, with extensive experience in implant dentistry. Two types of implants were evaluated: 36 Helix Gran Morse implants (Group A), and 36 Drive Gran Morse implants (Group B); both produced by Neodent, Curitiba, Brazil. The implants presented similar patterns, 3.5-mm diameters, and 13-mm heights. Drive implants are most commonly used for type III and IV bones and allow for a greater bone expansion. Helix implants are generally used for denser bones (type I and II), and present greater drilling capacity. The milling sequence used for the implants was: drill bit, 2-mm helical drill, pilot drill for cervical enlargement, and a 3.3-mm tapered drill at 5-mm, 7-mm, 9-mm depths (Fig. ), observing the depths of 3 mm, 5 mm, 7 mm. The bits were marked to define drilling depth. Implant insertion torque measurement A TQ 680 digital torque wrench, manufactured by Instrutherm, Instrumentos de Medição Ltda, (São Paulo, SP, Brazil), was used for measuring the insertion torque. The torque wrench was attached to an adjustable guiding base using a spindle and a hand wrench, which allowed it to be stably positioned at 90º angle. After placing the implant at a predetermined height, a hand wrench (Neodent Grand Morse Implant Kit), was placed in the torque wrench spindle, and was then adjusted for rigid attachment. The implant head was then connected to the insertion wrench, and the procedure for measuring the implant torque began with the torque wrench being turned clockwise, with same surgeon noting the values indicated on the equipment. The electronic equipment, consisting of a digital torque wrench (Tohnichi Series QSP Model QSP25N3) also connected to a computer for reading the peak value of insertion torque every millisecond, was customized for this study. The resulting values were then compared (Fig. ). The dental implants used as Group A (Helix) and Group B (Drive) (Fig. a and b). Statistical analysis The data, expressed as mean and standard error, were subjected to the Kolmogorov–Smirnov normality test, and compared using the Mann–Whitney and Kruskal–Wallis/Dunn tests (Non-parametric data) ( p < 0.05), GraphpAdPrism5.0).
A TQ 680 digital torque wrench, manufactured by Instrutherm, Instrumentos de Medição Ltda, (São Paulo, SP, Brazil), was used for measuring the insertion torque. The torque wrench was attached to an adjustable guiding base using a spindle and a hand wrench, which allowed it to be stably positioned at 90º angle. After placing the implant at a predetermined height, a hand wrench (Neodent Grand Morse Implant Kit), was placed in the torque wrench spindle, and was then adjusted for rigid attachment. The implant head was then connected to the insertion wrench, and the procedure for measuring the implant torque began with the torque wrench being turned clockwise, with same surgeon noting the values indicated on the equipment. The electronic equipment, consisting of a digital torque wrench (Tohnichi Series QSP Model QSP25N3) also connected to a computer for reading the peak value of insertion torque every millisecond, was customized for this study. The resulting values were then compared (Fig. ). The dental implants used as Group A (Helix) and Group B (Drive) (Fig. a and b).
The data, expressed as mean and standard error, were subjected to the Kolmogorov–Smirnov normality test, and compared using the Mann–Whitney and Kruskal–Wallis/Dunn tests (Non-parametric data) ( p < 0.05), GraphpAdPrism5.0).
As shown in Table , the insertion torque of the implants was first obtained at different depths, followed by the calculation of the mean standard deviations for the Group (A and B) data at 5-mm, 7-mm, and 9-mm depths. It can be observed in the column for implants placed at 5 mm apical to the socket that the torque of Group B implants was significantly higher than that of Group A implants ( P < 0.01). The same was observed in the column of the implants placed 7 mm apical to the socket, since the internal value torque of Group B implants (32.67 Ncm) was significantly higher than that of Group A (22.83 Ncm). When we compared the groups at the 9 mm depth, we observed no significant difference between them (Drive GM 34.92 Ncm and Helix GM 32.33 Ncm) ( P > 0.001). When analyzing Group A implants at 5-mm, 7-mm, 9-mm depths, the results reveal that the insertion torque was significantly lower at the 5-mm (10.42 Ncm) depth than at the 7-mm (22.83 Ncm) and 9-mm (32.33 Ncm) depths. Although in the Group B implants, the insertion torque at the 5-mm depth was lower than at the 7-mm depth (22.08 Ncm vs 32.67 Ncm) ( P < 0.01). No statistically significant differences were observed between the 7-mm and 9-mm depths (32.67 Ncm vs 34.92 Ncm) ( P > 0.01).
The mechanical stability of the implant at the time of insertion, defined as primary stability, is one of the most important factors for implant success. Micro-movement at the bone-implant interface that exceeds a threshold of 50 to 150 µm can lead to the formation of fibrous instead of bone tissue. This impairs osseointegration. It was found that with immediate loading of the implants, insertion torque scores below 20 Ncm were indicative of higher failure rates . According to Romanos , the apical portion may play an important role in implant stability, and based on current data, the apical third of the implant length contributes to between 30 and 43% of the stability of the entire implant . This was also observed in the present study, which showed that insertion torque is greater with greater apical depth, and implants positioned at more than 7-mm deep may permit clinically safe immediate loading. Chang et al. investigated the effect of self-tapping blades on the initial stability of tapered implants in polyurethane bone blocks. Their findings indicated that tapered implants without self-tapping blades have the same primary stability as implants with self-tapping blades when 1/3 to 2/3 covered by bone, which differs from the results of our study. In our study, 30 to 50% of the implant was submerged, and the Drive GM Implant (Group B), with square and double primary threads, presented a higher insertion torque for all measurements at the same depths, than the Helix GM implant (Group A), which possessed compressive trapezoidal threads in the coronal portion, and self-drilling-self-tapping triangular threads in the apical region. Threads maximize initial contact, improve initial stability, increase implant surface area, and favor stress dissipation throughout the implant. The standard V-shaped thread promotes 10 times more shear loading on the bone than a square thread with similar diameters . Hybrid implant designs present both conical and cylindrical segments, and at 5 mm, the possibility of locking (in both portions) favors the stability of the implant. Falco et al. observed that implants with large and self-cutting blades display greater primary stability than implants with small blades, especially in cases of low quantity or poor quality bone. If only a few millimeters of apical bone are available to stabilize the implant (as in post-extraction implants), the implant macrogeometry becomes critical for achieving sufficient primary stability. Three-dimensional optical tests using different implant thread geometries has revealed increased apical direction stress in implants with lower coronal region stress. Elastic studies reveal that the geometry of the apical thread is a key factor in bone remodeling, and support the hypothesis that new bone formation results from loading forces acting in this region of the implant . In cases of low bone density, incorrect implant geometry may result in insufficient stability for immediate loading . This was observed in this study; implants with thinner blades (Group A) presented poorer results than Group B in all measures evaluated. Tapered implants were initially designed for immediate loading after dental extraction. Tapered implants provide cortical bone compression in regions with poor quality bone tissue, or in post-extraction sockets . Full-body cylindrical implants increase the risk of lip perforation because of buccal concavities; the decrease in the diameter of tapered implants towards the apical region takes lip concavity into consideration . Some studies have shown higher insertion torque values in tapered implants than in cylindrical implants, suggesting greater stability . To reduce the shear force component, by moving axial loading from the prosthesis to the apex of the implant; a square-thread tapered implant is suggested. This would transfer more of the axial load throughout the implant body, and compress the bone . The implants with square threads , such as Drive GM, presented higher success rates in simulations with post-extraction sockets. Obtaining primary stability during implant insertion is essential for achieving osteointegration throughout the entire healing phase. However, the performance of immediate loading procedures in post-extraction sockets depends on variables that are often difficult to assess, such as the general state of health of the patient, bone quality, the implant edge and material used, and the surgical ability of the surgeon [ , , ]. There are various tests which evaluate implant stability, including percussion, digital pressure, radiographs, RF analysis (RFA) using the Periotest (Medizintechnik Gulden, Modautal, Germany), and insertion torque. According to Andreaza da Cunha et al. , RFA and insertion torque methods are more efficient and present fewer contraindications . Radio Frequency Analysis (RFA), which is measured using the Osstell device and insertion torque are the most often used test procedures [ , , ]. An aggressive implant insertion with greater thread depth, which provides close contact between the implant surface and the bone, also generates insertion torque. If excessive force is used during implant insertion, the compression may exceed physiological limits and trigger bone resorption, leading to necrosis and implant failure . The disadvantage of using insertion torque is that it is a single parameter which can only be measured once at the time of implant placement, while RFA can be performed during all phases of implant treatment . The Osstell device can be used at the time of implant placement, during the healing period, and when the dental prosthesis is in use . However, it has the disadvantages of not providing an absolute value, and it does not allow comparison between the stabilities of different implants. Insertion torque was used in this study because it is easy to manage, and it allows predicting results and comparing values at the time of implant placement, as well as comparing implants in the same bone conditions. The bone cavity model developed in this study demonstrated clinically realistic levels of insertion torque and implant stability, while simulating the low bone quality typically found at an extraction site. Moreover, this model enabled simulating the presence of cortical bone on the inner side of the socket, which is an important clinical challenge faced when studying bone defects after extraction. As a limitation, this study did not address the relationship between density and depth of the placed implant, but rather the variation of implant depth when placed in post-extraction socket areas, (which is a very common condition in patients who present the need for both extraction and immediate placement of implants) and did not evaluate: marginal bone loss around neck implants, dental higyene procedures or microleakage and connection with dental prosthetics [ – ]. In this study, digital workflow can benefit the process of rehabilitation . This is common in the anterior region of the maxilla, which has a density similar to the polyurethane block used in this study. When assessing bone depth, especially in post-extraction sockets, the apical portion of the implant contributes to implant stability. We conclude that for immediate loading, a minimum of 7 mm of apical anchorage is required, and that implants with square threads provide additional stability when placing immediate loading implants.
Additional file 1.
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Awareness and perceptions of Filipino obstetrician-gynecologists on fertility preservation: a cross-sectional survey
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d5f20862-8e55-45a2-b882-9969634fd4b5
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10214684
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Gynaecology[mh]
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One of the most critical milestones in reproductive medicine is the advent of fertility preservation. Various fertility preservation techniques allow men and women with compromised fertility a chance to achieve reproductive capacity at a later time. While advances in cancer therapy have led to an increasing number of young patients who survive, a crucial sequela is loss of fertility due to the gonadotoxic profile of current regimens . The field of Oncofertility is a network of different subspecialties focused on techniques to restore reproductive function in patients with malignancies . Aside from cancer patients, fertility preservation has been widely applied to patients with benign conditions such as genetic disorders, autoimmune disorders, and other diseases predisposing to premature gonadal failure. Women who wish to postpone childbearing for social and professional reasons likewise benefit from fertility preservation . Age is a critical factor in the Patient-Oriented Strategies Encompassing Individualized Oocyte Number (POSEIDON) for women with poor ovarian response to stimulation . Age directly affects oocyte quality and embryo ploidy. Studies have shown that the number of euploid blastocysts decline after 34 years . Advanced female age and decreased ovarian reserve were shown to be prevalent in POSEIDON patients. This emphasizes the need for counseling on the importance of age and ovarian reserve on the prospects of future fertility . Women at risk of infertility should be identified and provided information specific to their needs. Information regarding the impact of malignancy and other diseases on reproductive function, the effect of treatment on fertility, fertility preservation options, issues relating to cryopreservation storage, infertility and fertility treatments, pregnancy after gonadotoxic treatment, and other childbearing and parenting options should be presented to patients . The Philippine Society for Fertility Preservation was established in 2019, reflecting the growing need and interest to improve and promote its practice. Awareness of established fertility preservation techniques is essential to ensure appropriate counseling of patients and referral to a specialist. Currently available fertility preservation strategies in females include embryo cryopreservation, mature oocyte cryopreservation, ovarian tissue cryopreservation, ovarian suppression with GnRH analogs, ovarian transposition, and fertility-sparing surgeries . Meanwhile, sperm cryopreservation is the only established fertility preservation method in adolescent and adult males . In the Philippines, fertility preservation techniques are only offered in private centers and paid through out-of-pocket expenses. At the time of writing, no government-funded facility offer these procedures. Expenses are not covered by the Philippine Health Insurance Corporation nor by health maintenance organizations. Despite the rapid progress of fertility preservation in clinical practice, knowledge of its availability is lacking among clinicians . This paucity of knowledge from healthcare providers on the protection of reproductive function certainly affects the patient’s knowledge, attitude, behavior, and perspective. The current study aimed to assess obstetrician-gynecologists awareness and perception of fertility preservation. It was timely and relevant to conduct this study to determine the current status and barriers to improving the practice of fertility preservation in the country. This was the first study among post-residency obstetrician-gynecologists in the Philippines. A cross-sectional survey was conducted among diplomates and fellows of the Philippine Obstetrical and Gynecological Society (POGS) from September to December 2021. A hyperlink to the online survey was sent by electronic mail to the target participants with society’s support and distributed over social media. The minimum required sample was 209 of the 4500 accredited obstetrician-gynecologists in the country. This was computed using the Cochran formula and based on the study by Fritz et al. , which reported that 82.8% of obstetricians believe that fertility discussions should be routinely part of the examinations. The sample size was computed with a 5% margin of error and a design effect of 1.0. Non-probability sampling and consecutive enrollment of participants were done until the sample size was achieved. A self-administered survey patterned from the study of Chung et al. . was utilized. The questionnaire was composed of 24 items divided into two sections. The first section included questions on the demographic profile of the participants. The second section assessed the awareness and perception of fertility preservation. Pilot testing of the questionnaire to 15 subjects was performed before the survey proper. Univariate descriptive statistics were reported as mean for continuous variables and frequency with percentage for categorical variables. Differences in responses were tested using the chi-square test. A P value of < 0.05 was considered statistically significant. All analyses used STATA 14 (Stata Corp Inc). A total of 215 participants accomplished the online questionnaire. The mean age of the respondents was 42.98 ± 10.59 years. The majority were female (94.88%), Catholic (80.94%), married (68.37%), and had children (65.12%). The geographical regions were represented, with the National Capital Region (NCR) being the most represented. The sociodemographic data of the respondents are summarized in Table . Most of the participants belonged to private non-university affiliated hospitals (27.91%) and had practiced for one to five years (44.65%). General obstetrician-gynecologists constituted 57.21% of the study population. Of the 42.79% specialists, the most frequently identified specialties were Ultrasound (12.56%) and Reproductive Endocrinology (10.70%). Table provides an occupational summary of the population. Majority of the respondents agreed that obstetrician-gynecologists should initiate discussions with patients about their childbearing intentions (98.60%) and age-related fertility decline (97.67%). Obstetrician-gynecologists largely believed that discussion of natural fertility decline should be part of a well-woman annual examination, with agreement by 96.28%. Almost all participants (98.60%) were aware of fertility preservation and were familiar with at least one method or procedure. Only 32.56% were familiar with all techniques, including fertility-sparing surgeries, the use of GnRH agonists, sperm freezing, oocyte freezing, embryo freezing, and ovarian or testicular tissue freezing. Most respondents (81.40%) were aware of fertility-sparing surgeries. Approximately half (45.12%) of the participants have not referred patients for fertility preservation in the twelve months before the study proper. Only seven respondents were able to refer patients for all the mentioned procedures (Table ). Respondents were largely aware (86.98%) of a particular clinic or specialist who can accept referrals for fertility preservation. Of note, 28 respondents (13.02%) were unaware of any facility or specialist. In 43.72%, the patient’s desire to have children was identified as the most critical factor when deciding on fertility preservation in medical indications, followed by age (35.81%) and prognosis (10.23%). Majority of the participants (93.49%) deemed it necessary to set up dedicated centers for fertility preservation. About 91.63% think it should be offered as a public health service. Standard educational materials were deemed essential in enhancing patient understanding of fertility preservation. More than half (59.07%) are unaware of regulations relating to fertility preservation, but 98.14% support establishing guidelines. Three respondents did not wish to know more about fertility preservation (Table ). The likelihood of discussing fertility-related practices was not different across characteristics of fellows. However, the analysis is limited by the inadequate number of participants per characteristic category. Cells with a frequency of less than five were merged with other cells to ensure adequacy for analysis. The likelihood of having an awareness of fertility-related practices was not different across characteristics of fellows except for a few geographic locations and subspecialties. Those in Luzon are 2.26 times more likely to be aware of regulations on fertility preservation than those in the NCR. The Philippines is composed of three major islands known as Luzon, Visayas, and Mindanao. For the analysis, the National Capital Region was separated from Luzon because it houses most of the centers able to provide fertility preservation techniques and has the most number of specialists in the country. Provinces included in Luzon were Regions I, II, III, IV-A, MIMAROPA, V, and CAR. Luzon is generally considered to be more urbanized than provinces in Visayas and Mindanao. Distribution of health infrastructures and human resources is skewed toward Luzon and the National Capital Region. Respondents with subspecialties other than Reproductive Endocrinology have a 51% reduced odds of having an awareness of these regulations than general obstetrician-gynecologists. Reproductive endocrinologists have 80% reduced odds of agreeing on setting up fertility preservation counseling compared to general obstetricians. On the other hand, Christians have 20% reduced odds of agreeing on the need for practice guidelines than Roman Catholics. Fertility preservation has continued to gain worldwide attention over the years. A local study conducted by Factor and Novero was the first attempt to examine Filipino practitioners’ knowledge, attitudes, and practices on fertility preservation . The study included 213 surgical oncologists, medical oncologists, and radiation oncologists. Majority of their study participants acknowledged knowing only minimal information. Only 38% have referred patients to fertility specialists, citing lack of knowledge, poor success rates of fertility preservation, poor patient prognosis, and high costs . The current study is the first to describe the awareness and perceptions of Filipino obstetrician-gynecologists about reproductive aging and fertility preservation. The majority of the study respondents were female because they comprise 95% of the diplomates and fellows of the Philippine Obstetrical and Gynecological Society. Being the primary provider of reproductive healthcare, it is reassuring that majority of the respondents agreed that discussions about potential childbearing intentions and age-related fertility decline should be initiated during an annual examination. The International Fertility Decision-Making Study highlighted the lack of knowledge about fertility in 10,045 reproductive-aged men and women in over 79 countries . Counseling increases patient understanding, allows informed decisions about her future reproductive plans, and encourages better patient participation. There is a high awareness of fertility preservation among the respondents. One of the main objectives of the Philippine Society for Fertility Preservation (PSFP) is to promote the science and practice of fertility preservation. The society conducts regular conferences, meetings, and discussions on scientific information and treatment advances. There were varying levels of awareness of the different techniques. Most were familiar with at least one fertility preservation technique. Meanwhile, only a third of the study population knew all methods. Unawareness may lead to the underutilization of available methods of fertility preservation. This emphasizes the need to educate more obstetrician-gynecologists through fertility preservation awareness campaigns and continuing medical education activities, including seminars and workshops. Not surprisingly, the highest level of awareness was associated with fertility-sparing surgeries. Fertility-sparing surgery entails preserving at least a portion of an ovary and the uterus. These are limited to early-stage malignancies and include conization or trachelectomy for cervical cancer and unilateral salpingo-oophorectomy for ovarian cancer. Clinicians should provide appropriate information about oncologic and pregnancy outcomes through an individualized patient approach . Obstetrician-gynecologists were likely to be most aware of fertility-sparing surgeries as they perform the surgeries themselves, and specialists provide further treatment. Despite the high level of awareness, half of the respondents had not referred patients for fertility preservation, and majority desired to know more information. The study’s findings were similar to the reports of Harzif et al. among obstetrician-gynecologists in Indonesia . Identified hindrances were financial constraints, poor success rates of fertility preservation techniques, poor prognosis of patients, and lack of physician knowledge. These underscore that information among obstetrician-gynecologists is lacking. Aside from these, the European Society of Human Reproduction and Embryology (ESHRE) listed limited public awareness of fertility and fertility preservation, limited awareness of oncologists on fertility preservation options, lack of referral pathways, and unavailability of every technique as barriers to access to fertility preservation . Further local studies on the knowledge, attitudes, and practices of Filipino obstetrician-gynecologist may be undertaken to examine the perceived barriers to the provision of much-needed fertility preservation techniques. Most respondents saw setting up dedicated centers for fertility preservation as necessary. The study shows that reproductive endocrinologists have 80% lower odds of agreeing on this than general obstetrician-gynecologists. A small proportion of the study population was unaware of any facility or specialist. In the Philippines, fertility preservation techniques are mainly performed in reproductive centers offering in vitro fertilization. There are only eight centers and 147 infertility specialists able to provide these services in the country. Access to these centers is available to reproductive endocrinologists, which may explain the decreased support for establishing dedicated facilities. Encompassing help from all specialists should be elicited to promote fertility preservation. Early referral of women with malignancy at the time of diagnosis and before treatment commencement is the key to maximizing the success of fertility preservation and allows a greater window of opportunity for preserving fertility . As primary doctors of women with gynecologic malignancies, gynecologic oncologists should refer them for reproductive counseling as soon as the diagnosis is made. The ESHRE advocates a model of care for patients eligible for fertility preservation. Central to this model is the awareness of fertility preservation options and the training of healthcare providers. The clinical care team should provide essential information and referrals for fertility preservation consultation. Fertility preservation counseling is provided by specialists after a thorough patient assessment . There is a need for quick and efficient referral systems. The high cost of most fertility preservation techniques and patient financial constraints have impeded widespread local use. Most respondents agreed that these techniques should be offered as a public health service to mitigate access issues. A multilevel approach is essential to address issues specific to patients and their families, clinicians, organizations, policymakers, and the general population . Fertility preservation is a significant issue in women diagnosed with malignancy. A survey of young women undergoing therapy showed that childbearing remains a priority . Diminished reproductive capacity and fertility loss are leading causes of anxiety and depression among this population. Studies suggest that the risk of infertility has a significant impact on the decision-making process of young cancer patients . In a prospective cohort study among 425 women with newly diagnosed breast cancer, 1% decided not to receive chemotherapy, 2% chose one chemotherapy regimen over another, 1% considered not receiving endocrine therapy, 3% chose not to receive endocrine therapy, and 11% considered receiving endocrine therapy for five years due to concerns in fertility . Similarly, the study respondents deemed a patient’s desire to have children the most important factor when deciding on fertility preservation in medical conditions. It is, therefore, worthwhile to investigate patient perceptions and access to the different techniques in the local setting. Patient age and prognosis were among the top considerations. These again stress the need for timely counseling. Comprehensive recommendations and clinical guidelines on fertility preservation should be established and communicated. ESHRE published its first evidence-based guideline on female fertility preservation for healthcare professionals in 2020 . Socio-economic factors relating to the respondents’ place of practice and affiliation could influence their knowledge, attitudes, and practices on fertility preservation. Private practitioners manage a different subset of patients compared to those in public facilities. Their patients are better able to afford fertility preservation techniques. As such, they are more exposed and knowledgeable on fertility preservation. Considering the current laws, patient population, and socioeconomic factors, these guidelines need to be optimized in the local setting. Interestingly, practitioners in Luzon were 2.26 times more likely to be aware of regulations on fertility preservation than those in the NCR. Subgroup analysis of participants in Luzon showed that the majority have been practicing for one to five years, while most of those in NCR has been practicing for more than 16 years. This may be due to more active personal inquiry by younger clinicians or better participation in regional campaigns. The availability of fertility preservation techniques in the NCR should be an impetus for practitioners in this area to improve awareness. Subspecialties other than reproductive endocrinology had 51% reduced odds of awareness of existing guidelines compared to general obstetrician-gynecologists. Their specialized practices may deter them from acquiring further information in this growing field. As primary reproductive healthcare providers, all obstetrician-gynecologists should be knowledgeable about recommendations and guidelines. Overall, Filipino obstetrician-gynecologists have an encouraging positive perception of fertility preservation. There is a need for further education on the locally available techniques. The information presented by this study can be applied in the framework of establishing local guidelines and designing a curriculum for training. A multidisciplinary team with reproductive specialists, an insurance coverage system, comprehensive laws, and practice guidelines should be prioritized. Reproductive aging and fertility preservation are emerging fields in managing reproductive-aged women. This is the first local study that evaluated the awareness and perceptions of post-residency obstetrician-gynecologists on fertility preservation. The study showed a reassuring positive perception of fertility preservation but a gap in the awareness of different approved methods. A multidisciplinary approach and dedicated facilities should be established for fertility consultation, risk assessment, and counseling. Healthcare delivery should be organized to meet the increasing need for fertility preservation. The study employed non-probability sampling and consecutive enrolment of participants until the sample size was met. Selection and response bias may have influenced the results of the study. Participation in a self-directed online questionnaire entails the awareness of the sample to the existence of the survey. The number of physicians who actually received the survey is uncertain. This was minimized by distribution of the questionnaire by the Philippine Obstetrical and Gynecological Society to its registered members. Regular posting of the survey to various social media platforms was also conducted to improve visibility and response. To minimize response bias, the period of data collection was extended after the minimum sample size was met. The number of Gynecologic Oncology specialists who completed the survey was only eight due to the sampling method employed. The study did not assess the specific reasons for non-referral for fertility preservation techniques. Further studies with the recruitment of gynecologic oncologists may be undertaken. Another vital area of research is the investigation of perceived barriers to the provision of timely and appropriate fertility preservation techniques. The knowledge and perceptions of patients on fertility preservation should also be investigated.
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Safe, fast, and minimally-assisted microsurgical anastomosis with combined open-loop suturing and airborne tying: a clinical and experimental study
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565d6f0c-bf3d-4cb6-8e81-ba66baa2e001
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10214895
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Suturing[mh]
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Flawless microvascular anastomosis is one of the most important steps, if not the most important, for tissue survival in free flap transfers and composite tissue allotransplantation, as well as for limb survival in replantation and revascularization cases. An ideal microvascular anastomosis technique, in terms of both suturing and knot tying, should be easy to perform and teach, fast, easy to control for technical failures, and should reduce operative time and costs while providing stable patency rates. Various techniques have been described in the literature to come up with an ideal solution to address these features simultaneously.[ - ] The simple interrupted suture with conventional knot tying is considered by many microsurgeons as the gold standard technique for performing microsurgical anastomosis. However, proper application of traction and intermittent irrigation of the lumen with the help of an assistant is usually needed to perform a safe anastomosis. If this is not the case, inadvertent catching of the back wall or inaccurate placement of sutures will not be uncommon when the simple interrupted technique is used, especially for inexperienced surgeons and residents continuing their microsurgical training. Moreover, each suture is placed and tied in order in this technique. Therefore, this could potentially increase the ischemia time and compromise outcomes when performing multiple flap transfers, multi-digit replantation, or microsurgical revisions. Similarly, a transfer of a bowel flap, such as jejunum or colon, should be completed in a shorter time due to reduced tolerance to ischemia. In order to reduce the total microsurgical anastomosis time, several alternative suturing and knot tying techniques have been proposed in the literature. The continuous open-loop technique was first described by Lee et al. in 1984, primarily to eliminate the risk of inadvertently catching the back wall, which is the major cause of technical anastomotic failure when using the interrupted suture technique. Correct application of the loose suture loops allows the surgeon to easily manipulate the vessel edges and creates the greatest advantage of the open-loop suture technique, which is the continuous visualization of the lumen during microsurgical anastomosis. Therefore, lower technical anastomotic failure rates can be expected with the open-loop technique when compared to the simple interrupted suture technique. Moreover, continuous placement of the sutures shares the time advantage of the standard continuous suture technique without the concerns of stricture formation or reduced blood flow rate with the latter. Airborne suture tying was first described by Chen et al. in 2003 to allow quicker completion of a microvascular anastomosis. Although this technique does not contribute to improved patency rates of the anastomosis, as it is not related to suture placement, it does significantly speed up the anastomosis by preventing the suture ends from sticking to the tissue and alleviating the cumbersome process of picking the suture end off the surrounding structures. With the utilization of the airborne knot tying technique, the total anastomotic time would be much less with the open-loop technique, which is a modification of continuous suturing. The open-loop suture and airborne tying techniques are not innovations, and either technique or a combination is commonly preferred in some areas, such as Taiwan, where both authors completed their fellowships. However, these techniques have not been widely adopted around the world, and evidence of the benefits of this combination is scarce in the literature. Our aim was to demonstrate the advantages of this combination through an experimental and clinical study and to increase awareness of these useful, fast, and secure techniques.
An experimental study was conducted to compare the combined open-loop suture and airborne knot tying technique with the simple interrupted suture and conventional knot tying technique in terms of total anastomosis time and patency rates. To demonstrate the clinical benefits and feasibility of the open-loop suture with the airborne technique, the outcomes of patients who underwent free flap surgery utilizing the proposed combination in microvascular anastomoses were analyzed and discussed. The experiments were conducted in the Ankara Training and Research Hospital Animal Laboratory, and ethical permission was obtained from the Ethics Board of Animal Experiments (approval no: 70/690-2022). The experimental study and postoperative animal care were conducted in compliance with the institution’s ethical guidelines. The clinical study underwent ethical review and approval by the Ethical Board of the Provincial Health Department (approval no: 2020/1), and written consent was obtained from all the patients involved for their participation in the study. Experimental Study Twenty adult male Wistar-Albino rats, weighing 300-350g, were randomized and divided into two groups according to the chosen anastomosis technique. Microsurgical anastomoses were performed on the bilateral femoral arteries of each rat, with a total of twenty anastomoses performed in each group (n=20). In the control group, the simple interrupted suture with conventional knot tying technique was used. In the experimental group, the open-loop suture with airborne knot tying technique was employed for microsurgical anastomosis. Anastomoses were performed at a level where the femoral artery’s diameter was 0.60 mm ( ). Therefore, the total number of sutures was even for each anastomosis, with a total of eight sutures. The total time for completion of the anastomosis was recorded individually using a digital timer, starting with the passage of the first suture and ending with the tying of the last knot. All of the animals were kept on a twelve-hour day and night cycle, in plastic cages with sawdust bedding in a standardized temperature (24°C) and humidity-controlled environment, and fed ad libitum. All the experimental cases were performed by the same author (G.S). Surgical Technique The rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). Bilateral inguinal regions were shaved and cleaned with an antiseptic solution. A 2.5 cm skin incision was used to expose the femoral artery and vein. The superficial epigastric artery and vein were ligated and cut after complete mobilization of the fat pad. The artery was dissected off the vein, and limited adventitiectomy was performed with sharp micro-scissors. The artery was sharply cut after defining the level with a microscale ruler and placing double approximator Acland clamps. Intraluminal irrigation was performed using a diluted heparinized solution to wash out the remaining blood. Microsurgical anastomosis was completed with the specific technique using a 10-0 nylon micro suture on a 3/8 of a circle needle (Ethilon, Ethicon, United States) under a surgical microscope (16x magnification). Early patency was checked with a milking test, and the procedure was repeated on the contralateral side. Finally, the incisions were closed primarily with a running non-absorbable suture. On postoperative day twenty-eight, bilateral inguinal scars were re-incised, and the femoral arteries were exposed. Following this, long-term patency was checked using the milking test. Simple interrupted suture and conventional knot tying technique (Control Group) Two stay sutures were placed at 0 and 180 degrees to ensure even spacing between the additional sutures on either wall. Simple interrupted sutures were placed on the opposing vessel ends, and knots were tied conventionally with an initial double throw, followed by an additional two single throws. Once suturing of the anterior wall was completed with three additional sutures, the clamp was rotated 180 degrees to suture the back wall in the same manner. Combined open-loop suture and airborne knot tying technique (Experimental Group) Two stay sutures were placed at 0 and 180 degrees. After this, the suture was passed continuously along the anterior wall to create two loops, starting from the far end and moving towards the surgeon ( and ). All the sutures, including the stay sutures, were tied with the airborne technique in order, by cutting the loops sequentially. In the airborne knot tying technique, the suture is pulled through the vessel end so that the leading suture end is about three times longer than the free end of the thread. The free end is passed behind the opposite end while the right forceps forms the knot and the free end leans on the adjacent loop. The free end is then picked up at its tip and pulled through the loop, completing the knot. Technical Notes and Tips: We would like to emphasize several technical tips to facilitate the use of the open-loop suture technique and ease microvascular anastomosis. The authors prefer to create small loops and leave the long thread in the first loop if the vessel size is relatively small, magnification is high, and the space is not wide enough to accommodate the redundant suture that tends to get stuck. However, in free flap surgeries with larger vessels and spaces, larger loops and leaving a short thread end in the first loop can be preferred because this would accelerate the anastomosis without having to pull the suture each time for each loop. Loops should be created from further to closer to the surgeon, or from left to right in vertical anastomosis if the surgeon prefers left micro forceps to tie the suture, and preferably each loop being larger or smaller to avoid suture entanglement. The clockwise rotation of the needle holder while pulling out the needle from the vessel edge is followed by a counterclockwise rotation to hold and reposition the needle back to continue for the next loop. The environment should be moist but not too wet. If it is too wet, loops can stick back to the pooled area. Irrigation after completing the loops helps the loops fix closer to the surgeon and ease the suture tying. Moist gauze around the surgical area helps to avoid the suture sticking or being trapped in unintended areas. If the loops are tangled and stuck, be gentle and patient when untangling. The airborne technique is much easier to apply under less magnification with larger sutures. The authors suggest practicing in these conditions first. The long thread is generally preferred to be at least twice as long as the short thread. If the length of both threads is similar, more vertical use of the left forceps (for a right-handed surgeon) with more rotation would be needed. Lastly, microsurgical instruments should be in good condition and clean for a higher level of precision. Statistical Analysis Statistical analysis was performed using Windows SPSS 23.0 (IBM Corporation, Armonk, New York, United States). Both groups were analyzed for normal distribution using the Shapiro-Wilk test, and the homogeneity of the variances was analyzed with the Levene’s test. The student t-test was used for the comparison of the quantitative data (total anastomosis time) of two independent groups. The chi-square test was used to compare the categorical data (patency of the anastomoses). Descriptive statistics of the quantitative variables were presented as mean ± standard deviation and distribution range (minimum-maximum). Categorical variables are reported as frequency (percentage) in the tables. All of the variables were examined at a 99% confidence level, and p<0.01 were considered statistically significant.
Twenty adult male Wistar-Albino rats, weighing 300-350g, were randomized and divided into two groups according to the chosen anastomosis technique. Microsurgical anastomoses were performed on the bilateral femoral arteries of each rat, with a total of twenty anastomoses performed in each group (n=20). In the control group, the simple interrupted suture with conventional knot tying technique was used. In the experimental group, the open-loop suture with airborne knot tying technique was employed for microsurgical anastomosis. Anastomoses were performed at a level where the femoral artery’s diameter was 0.60 mm ( ). Therefore, the total number of sutures was even for each anastomosis, with a total of eight sutures. The total time for completion of the anastomosis was recorded individually using a digital timer, starting with the passage of the first suture and ending with the tying of the last knot. All of the animals were kept on a twelve-hour day and night cycle, in plastic cages with sawdust bedding in a standardized temperature (24°C) and humidity-controlled environment, and fed ad libitum. All the experimental cases were performed by the same author (G.S).
The rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (80 mg/kg) and xylazine (10 mg/kg). Bilateral inguinal regions were shaved and cleaned with an antiseptic solution. A 2.5 cm skin incision was used to expose the femoral artery and vein. The superficial epigastric artery and vein were ligated and cut after complete mobilization of the fat pad. The artery was dissected off the vein, and limited adventitiectomy was performed with sharp micro-scissors. The artery was sharply cut after defining the level with a microscale ruler and placing double approximator Acland clamps. Intraluminal irrigation was performed using a diluted heparinized solution to wash out the remaining blood. Microsurgical anastomosis was completed with the specific technique using a 10-0 nylon micro suture on a 3/8 of a circle needle (Ethilon, Ethicon, United States) under a surgical microscope (16x magnification). Early patency was checked with a milking test, and the procedure was repeated on the contralateral side. Finally, the incisions were closed primarily with a running non-absorbable suture. On postoperative day twenty-eight, bilateral inguinal scars were re-incised, and the femoral arteries were exposed. Following this, long-term patency was checked using the milking test.
Two stay sutures were placed at 0 and 180 degrees to ensure even spacing between the additional sutures on either wall. Simple interrupted sutures were placed on the opposing vessel ends, and knots were tied conventionally with an initial double throw, followed by an additional two single throws. Once suturing of the anterior wall was completed with three additional sutures, the clamp was rotated 180 degrees to suture the back wall in the same manner.
Two stay sutures were placed at 0 and 180 degrees. After this, the suture was passed continuously along the anterior wall to create two loops, starting from the far end and moving towards the surgeon ( and ). All the sutures, including the stay sutures, were tied with the airborne technique in order, by cutting the loops sequentially. In the airborne knot tying technique, the suture is pulled through the vessel end so that the leading suture end is about three times longer than the free end of the thread. The free end is passed behind the opposite end while the right forceps forms the knot and the free end leans on the adjacent loop. The free end is then picked up at its tip and pulled through the loop, completing the knot.
We would like to emphasize several technical tips to facilitate the use of the open-loop suture technique and ease microvascular anastomosis. The authors prefer to create small loops and leave the long thread in the first loop if the vessel size is relatively small, magnification is high, and the space is not wide enough to accommodate the redundant suture that tends to get stuck. However, in free flap surgeries with larger vessels and spaces, larger loops and leaving a short thread end in the first loop can be preferred because this would accelerate the anastomosis without having to pull the suture each time for each loop. Loops should be created from further to closer to the surgeon, or from left to right in vertical anastomosis if the surgeon prefers left micro forceps to tie the suture, and preferably each loop being larger or smaller to avoid suture entanglement. The clockwise rotation of the needle holder while pulling out the needle from the vessel edge is followed by a counterclockwise rotation to hold and reposition the needle back to continue for the next loop. The environment should be moist but not too wet. If it is too wet, loops can stick back to the pooled area. Irrigation after completing the loops helps the loops fix closer to the surgeon and ease the suture tying. Moist gauze around the surgical area helps to avoid the suture sticking or being trapped in unintended areas. If the loops are tangled and stuck, be gentle and patient when untangling. The airborne technique is much easier to apply under less magnification with larger sutures. The authors suggest practicing in these conditions first. The long thread is generally preferred to be at least twice as long as the short thread. If the length of both threads is similar, more vertical use of the left forceps (for a right-handed surgeon) with more rotation would be needed. Lastly, microsurgical instruments should be in good condition and clean for a higher level of precision.
Statistical analysis was performed using Windows SPSS 23.0 (IBM Corporation, Armonk, New York, United States). Both groups were analyzed for normal distribution using the Shapiro-Wilk test, and the homogeneity of the variances was analyzed with the Levene’s test. The student t-test was used for the comparison of the quantitative data (total anastomosis time) of two independent groups. The chi-square test was used to compare the categorical data (patency of the anastomoses). Descriptive statistics of the quantitative variables were presented as mean ± standard deviation and distribution range (minimum-maximum). Categorical variables are reported as frequency (percentage) in the tables. All of the variables were examined at a 99% confidence level, and p<0.01 were considered statistically significant.
Anastomosis Time The mean time required for completion of an anastomosis was 779.65 seconds (13 mins, range: 706-848 s) in the control group and 527.4 seconds (8.7 mins, range: 462-592 s) in the experimental group ( ). The difference in total anastomosis time between the two groups was statistically significant (p<0.001). Thrombosis Rates A milking test conducted immediately after completion of the anastomosis revealed 100% patency rates in both groups ( ). Two anastomoses in group I and one anastomosis in group II were recorded as occluded when the milking test was repeated on postoperative day 28. The comparison of long-term patency rates of the two groups revealed no statistically significant difference (p=0.5483). Clinical Outcomes The proposed technique was used in 35 microsurgical procedures, in a total of 31 patients. Eighteen replantations were performed in 16 patients, and 17 free flap transfers were performed in 15 patients. A total of 104 anastomoses were performed. The cases and outcomes are presented in detail in . Free Flap Transfers (n=17) Three of the 15 patients were female and 12 were male. The patients’ ages ranged between 4-47 years (mean 26.4, median 24). Five patients were in the pediatric group (4-17 years, mean 11, median 10). Only one patient was operated on for a congenital defect, while the remaining 16 free flaps were used for treatment of traumatic defects. Thirteen of the 16 free flaps were performed for acute traumatic defects, such as gunshot injuries, mine explosions, traffic accidents, and landslide injuries. The remaining three flaps were used for treatment of chronic sequelae after an initial trauma: burn contracture, scaphoid nonunion, and Volkmann ischemic contracture. Six superficial circumflex iliac artery perforator flaps (SCIP), five anterolateral thigh flaps (ALT), one gracilis muscle flap, one osteocutaneous fibula flap, one peroneal artery perforator flap, one deep circumflex iliac artery perforator flap (DCIAP), one iliac crest bone flap, and one femoral condyle chimeric cutaneous-osteoperichondrial flap were used in 15 patients. Eight flaps (one ALT, six SCIP, and one DCIAP) were harvested in a thin fashion; four of these eight flaps (three SCIP and one DCIAP) were elevated in a super-thin fashion. One patient was operated on with two free flaps due to a wide defect, and another patient was operated on with a secondary free flap to salvage the total loss of the first flap. All venous anastomoses and 11 arterial anastomoses were performed in an end-to-end configuration, whereas 5 arterial anastomoses were performed in an end-to-side fashion. One arterial anastomosis was performed in a ‘T’ shape in order to restore distal vascularization to the affected limb. One vein graft was used for a vein defect. Sixteen of the 17 flaps (94.1%) fully survived; however, one thin SCIP flap failed due to congestion. One end-to-side arterial anastomosis was revised and converted to an end-to-end fashion because of kinking and subsequent thrombosis. Another revision was conducted to salvage venous compression due to a hematoma without revising the anastomosis. In total, 37 anastomoses (19 veins, 18 arteries) were performed in 17 free flap transfers. Anastomoses were technically successful in 33 of 35 cases, representing an anastomosis success rate of 94.2%; double artery repair for flow-through reconstruction and the use of a vein graft were counted as double anastomoses, yet double vein repair in one case was excluded. The mean diameter of the anastomotic site was 1.8 mm (1.2-2.4 mm) for arterial anastomoses and 1.7 mm (1.1-2.6 mm) for venous anastomoses in free flap transfers. The mean time needed to complete a single arterial anastomosis was 551.9 seconds (9.1 minutes, range: 385-730 s), whereas 594.2 seconds (9.9 minutes, range: 435-745 s) were needed to complete a single venous anastomosis. Replantations (n=18) A total of 18 replantations were performed on 16 patients ( ). The patients’ ages ranged from 4 to 69 years (mean 35, median 33). Four patients were in the pediatric group (4-17 years, mean 10.75, median 11.5). All of the patients were male, and the etiology was trauma in all cases. Four patients had clean-cut injuries, while the remaining patients had different types of crush-avulsion injuries. Fourteen patients underwent surgery for digital amputation, one patient for near-total ear amputation, and one patient for toe amputation. A total of 5 vein grafts were used, two for vein defects and three for artery defects. Two of the 18 replantations failed, while the remaining 16 cases resulted in success without any anastomosis revision, representing an 88.8% success rate. The mean diameter of the vessels was 0.75 mm (0.60-1.2 mm) for arterial anastomoses and 0.70 mm (0.5-1.1 mm) for venous anastomoses in replantation cases. The mean time needed to complete a single arterial anastomosis was 426.4 seconds (7.1 minutes, range: 385-540 seconds), whereas 465.6 seconds (7.7 minutes, range: 420-615 seconds) were required to complete a single venous anastomosis. In total, 67 anastomoses (39 veins, 28 arteries) were performed in 18 replantations, including vein grafts, multi-vein repairs, and double artery repairs. Excluding double vein and artery repairs but counting the vital vein grafts as two anastomoses, the known successful patency rate was 39/41 (95.1%) ( ).
The mean time required for completion of an anastomosis was 779.65 seconds (13 mins, range: 706-848 s) in the control group and 527.4 seconds (8.7 mins, range: 462-592 s) in the experimental group ( ). The difference in total anastomosis time between the two groups was statistically significant (p<0.001).
A milking test conducted immediately after completion of the anastomosis revealed 100% patency rates in both groups ( ). Two anastomoses in group I and one anastomosis in group II were recorded as occluded when the milking test was repeated on postoperative day 28. The comparison of long-term patency rates of the two groups revealed no statistically significant difference (p=0.5483).
The proposed technique was used in 35 microsurgical procedures, in a total of 31 patients. Eighteen replantations were performed in 16 patients, and 17 free flap transfers were performed in 15 patients. A total of 104 anastomoses were performed. The cases and outcomes are presented in detail in .
Three of the 15 patients were female and 12 were male. The patients’ ages ranged between 4-47 years (mean 26.4, median 24). Five patients were in the pediatric group (4-17 years, mean 11, median 10). Only one patient was operated on for a congenital defect, while the remaining 16 free flaps were used for treatment of traumatic defects. Thirteen of the 16 free flaps were performed for acute traumatic defects, such as gunshot injuries, mine explosions, traffic accidents, and landslide injuries. The remaining three flaps were used for treatment of chronic sequelae after an initial trauma: burn contracture, scaphoid nonunion, and Volkmann ischemic contracture. Six superficial circumflex iliac artery perforator flaps (SCIP), five anterolateral thigh flaps (ALT), one gracilis muscle flap, one osteocutaneous fibula flap, one peroneal artery perforator flap, one deep circumflex iliac artery perforator flap (DCIAP), one iliac crest bone flap, and one femoral condyle chimeric cutaneous-osteoperichondrial flap were used in 15 patients. Eight flaps (one ALT, six SCIP, and one DCIAP) were harvested in a thin fashion; four of these eight flaps (three SCIP and one DCIAP) were elevated in a super-thin fashion. One patient was operated on with two free flaps due to a wide defect, and another patient was operated on with a secondary free flap to salvage the total loss of the first flap. All venous anastomoses and 11 arterial anastomoses were performed in an end-to-end configuration, whereas 5 arterial anastomoses were performed in an end-to-side fashion. One arterial anastomosis was performed in a ‘T’ shape in order to restore distal vascularization to the affected limb. One vein graft was used for a vein defect. Sixteen of the 17 flaps (94.1%) fully survived; however, one thin SCIP flap failed due to congestion. One end-to-side arterial anastomosis was revised and converted to an end-to-end fashion because of kinking and subsequent thrombosis. Another revision was conducted to salvage venous compression due to a hematoma without revising the anastomosis. In total, 37 anastomoses (19 veins, 18 arteries) were performed in 17 free flap transfers. Anastomoses were technically successful in 33 of 35 cases, representing an anastomosis success rate of 94.2%; double artery repair for flow-through reconstruction and the use of a vein graft were counted as double anastomoses, yet double vein repair in one case was excluded. The mean diameter of the anastomotic site was 1.8 mm (1.2-2.4 mm) for arterial anastomoses and 1.7 mm (1.1-2.6 mm) for venous anastomoses in free flap transfers. The mean time needed to complete a single arterial anastomosis was 551.9 seconds (9.1 minutes, range: 385-730 s), whereas 594.2 seconds (9.9 minutes, range: 435-745 s) were needed to complete a single venous anastomosis.
A total of 18 replantations were performed on 16 patients ( ). The patients’ ages ranged from 4 to 69 years (mean 35, median 33). Four patients were in the pediatric group (4-17 years, mean 10.75, median 11.5). All of the patients were male, and the etiology was trauma in all cases. Four patients had clean-cut injuries, while the remaining patients had different types of crush-avulsion injuries. Fourteen patients underwent surgery for digital amputation, one patient for near-total ear amputation, and one patient for toe amputation. A total of 5 vein grafts were used, two for vein defects and three for artery defects. Two of the 18 replantations failed, while the remaining 16 cases resulted in success without any anastomosis revision, representing an 88.8% success rate. The mean diameter of the vessels was 0.75 mm (0.60-1.2 mm) for arterial anastomoses and 0.70 mm (0.5-1.1 mm) for venous anastomoses in replantation cases. The mean time needed to complete a single arterial anastomosis was 426.4 seconds (7.1 minutes, range: 385-540 seconds), whereas 465.6 seconds (7.7 minutes, range: 420-615 seconds) were required to complete a single venous anastomosis. In total, 67 anastomoses (39 veins, 28 arteries) were performed in 18 replantations, including vein grafts, multi-vein repairs, and double artery repairs. Excluding double vein and artery repairs but counting the vital vein grafts as two anastomoses, the known successful patency rate was 39/41 (95.1%) ( ).
Our experimental and clinical results clearly show that the combination of these two techniques is safe, fast, and reduces the need for assistance. Our experimental study demonstrates an extremely high success rate (95-100%), which is similar to the literature (75-100%) and also comparable to the author’s experimental study using the conventional technique (96%). Our clinical study reveals a 94.2% success rate in free flap surgery, which is also in line with the literature (91-99%). A large series of replantations were reported by Fufa et al., with a digit survival rate of 57% in 121 digits, where 59% of these were due to sharp cuts. Several reported outcomes were published outside of the U.S., with higher success rates reaching over 80%. Although different types of mechanisms of injury, amputation levels, variable ischemia times, patient comorbidities, and numbers of patients make it difficult to objectively compare the outcomes between studies, we believe that our success rate (83.3%) is comparable to the literature and could be primarily attributed to the safety of the anastomosis technique. The reported time advantage in our experimental study was 32% (13 minutes vs. 8.7 minutes) when compared to the simple interrupted suture technique with conventional knot tying. We performed the anastomoses on 0.60 mm arteries. We believe that the time advantage would be more significant when projected to anastomoses on vessels greater than 2.0 mm in diameter, which represents the general diameter of vessels used in commonly preferred free flaps. Not surprisingly, our clinical study (for free flap transfers) proved to be faster than conventional anastomosis (9.1-9.9 minutes vs. 16.4 minutes) and similar to coupler anastomosis when compared with the literature. Quick and safe microvascular anastomosis increases success in microsurgery, reduces ischemia time, and alleviates the surgeon’s frustration when performing the most critical part of the procedure, as we know that the major cause of failure in free flap transfers is technical errors in microvascular anastomoses. Among the described techniques for microvascular anastomosis, the continuous suture technique remains one of the fastest options while providing high patency rates. However, Schlechter and Guyuron reported a 45% reduction in blood flow rate with the continuous suture group when compared to the interrupted suture group. Therefore, microsurgeons should be careful to provide even tension throughout the suture line to reduce the risk of constriction at the anastomosis site. In addition, the whole suture line needs to be taken down to correct small errors encountered at the end of the anastomosis. Because of this, the continuous suture technique is rarely used in microsurgery and is mostly utilized in large vessels by cardiovascular surgeons. To reduce the total anastomosis time and to see the lumen clearly at all times, as in the continuous suture technique without being concerned with the related disadvantages, we prefer to use the open-loop suture technique – a continuous suture with the simple interrupted tying concept. Because the sutures are tied separately in this technique, similar to the conventional simple interrupted suture, a reduction in blood flow is not encountered as in the conventional continuous technique. The conventional simple interrupted suture technique has certain disadvantages. This technique creates less and less space between sutures with each suture placement. Inserting micro forceps into a tight lumen space between sutures becomes more difficult and riskier with the last one or two sutures. In this situation, assistance is needed to retract a suture end, along with irrigation, to show the lumen clearly to the microsurgeon. In this situation, if the irrigation is not well-performed, suture placements are based on ‘feel’. One can feel the tactile sensation of the muscle layer without actually seeing it. However, an unnoticed small piece of adventitia or foreign body might get in the anastomosis site. Surgeon fatigue, surgeon or assistant inexperience, a deep and narrow surgical field, or a bloody environment may ease an inadvertent catch of the back wall or adventitia, which results in anastomosis failure. On the other hand, in the open-loop technique, there is enough space between sutures because the sutures are tied last. This is why the surgeon can easily use the tip of the micro forceps to elevate the edge of the vessel. This elevation not only enables one to see the lumen clearly at all times instead of relying on ‘feel’, but also helps to evert the vessel edges while avoiding adventitia and muscle layer inversion into the vessel lumen. Moreover, finding readily available assistance is not possible at all times, and this technique avoids the requirement of an assistant to irrigate the lumen or retract the suture in most situations. Therefore, this technique becomes especially useful when there is no one to assist. Another disadvantage of the conventional technique arises when there is a tendency for intimal separation, as in atherosclerotic vessels. In these vessels, using traction in the sutures without micro forceps support beneath the intima may cause advanced separation. The wide space in the lumen, thanks to the open-loop technique, enables the surgeon to easily support the intima with micro forceps. The authors do not prefer traction techniques unless the position of the vessels is exceptionally difficult to perform a microsurgical anastomosis. The airborne suture tying technique significantly speeds up the anastomosis by preventing the suture ends from sticking to the surrounding tissue. This is especially useful in preventing the suture end from adhering to adjacent tissues or materials in a moist or bloody environment. Chen et al. advocate that, with experience, incorporating the airborne knot tying into interrupted suturing could potentially save 20% of anastomosis time. In our experimental study, the reduction of anastomosis time by 32% could be partially explained by the airborne technique and partially by the open-loop technique. Venous anastomosis is considered to be more challenging and can often be technically demanding because of the thin vessel wall and collapse of the lumen during suture placement. Coupling anastomotic devices were introduced to the field of microsurgery to overcome the difficulties experienced with venous anastomoses, as well as to speed up the anastomosis. Patency rates were reported to be similar, whereas significantly decreased total anastomotic time was observed when coupling anastomotic devices were compared to hand-sewn anastomoses (9.5 minutes vs. 21 minutes, respectively), specifically when the simple interrupted suture technique is used. In the clinical part of our study, 9.1-9.9 minutes were needed for a single anastomosis in free flap transfers and 7.1-7.7 minutes in replantation cases. These results are comparable to the reported results in the coupling devices’ literature. Despite the time benefit of coupler devices, this technique has several disadvantages. The coupler device is far more suitable for venous anastomosis rather than arterial anastomosis due to the pliability of the vessel wall to be everted over the pins of the device. Moreover, it is not suitable for vessels with small diameters and end-to-side anastomoses. It is not readily usable for many centers, and assistance is required during anastomoses. The high cost and the difficulty of its application when size discrepancy is encountered limit its wide acceptance, and hand-sewn anastomosis is still considered the gold standard technique to execute a microvascular anastomosis. These techniques have a number of disadvantages, such as a steep learning curve. However, using the technical tips mentioned in the material method section, the learning curve to master the open-loop suture technique with airborne tying is not too steep, and we encourage microsurgeons to adopt this in their routine practice. Chen and Chiu advocate the use of a modification of the continuous suturing, spiral interrupted suture technique, by beginning knot tying and cutting all the loops at once. We do not prefer this because the thread ends may stick to surrounding tissue or adventitia and this may complicate catching the threads and cause time loss. Nevertheless, there may be numerous modifications of these techniques and surgeons may develop their own methods with practice. For starters, the last two or three sutures can be completed using the open-loop technique for better visualization of the lumen, as well as for familiarizing oneself with the new technique. Another technical disadvantage is that there are conditions in which this combination is difficult to perform. For example, in liver transplantations, and in cases where the anastomosis site is deep, the stump is short or there is not enough room for the vessel approximator, the posterior wall first technique can be used for the posterior wall, and then the anterior wall can be completed with the open-loop technique. Our study includes a number of limitations and strengths. Firstly, the limited sample size in the clinical study and low failure rates make it difficult to come to a significant conclusion in terms of the safety of anastomoses. Secondly, there is no control group in the clinical part of the study, as both authors prefer these techniques routinely as their first choice. Thirdly, conducting a clinical anastomosis study is extremely challenging due to numerous variables that may affect patency, such as different patient characteristics, etiology of the defect, nature of trauma, recipient sites, flap types and vessel diameters, surgeon’s experience and fatigue, and so on. However, our study diminishes these variables by incorporating an experimental study performed by a single surgeon, and a clinical study performed by another single surgeon, both of whom had already completed the learning curves in these techniques.
The open-loop suture technique with airborne knot tying allows the surgeon to complete a microvascular anastomosis in a shorter time with minimal assistance when compared to the simple interrupted suture technique. Continuous visualization of the vessel lumen comprises a low risk of inadvertent catch of the back wall or adventitia during the anastomosis. The proposed technique in our study can be the technique of choice especially for less experienced microsurgeons, in order to decrease technical failure, or for experienced microsurgeons to speed up the anastomosis with minimal assistance under difficult conditions.
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Analysis of person-hours required for proton beam therapy for pediatric tumors
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902c1377-5417-4b24-8ae7-685c36a3e658
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10214988
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Pediatrics[mh]
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Treatment for pediatric tumors is based on a multidisciplinary approach that combines surgery, chemotherapy and radiation therapy . In recent years, proton beam therapy (PBT) has been recommended for pediatric tumors to reduce late toxicities . However, radiation therapy for pediatric tumors often requires sedation and preparation, and this places a large burden on medical sites . Sedation for patients under 4 years is almost always needed, and therefore, the actual treatment time tends to be longer. In this report, we examined the person-hours required for PBT for adult and pediatric cases to obtain an accurate evaluation of the burden of PBT for pediatric tumors. The subjects were 32 pediatric patients who received PBT at our hospital from January 2010 to April 2011. Data for these patients were compared with those for 90 adult patients who received PBT from January 2022 to November 2022. Written informed consent was obtained in all cases, and the study was approved by the hospital ethics committee (H21–388, Tsukuba Clinical Research & Development Organization). For pediatric cases, the time from entering the irradiation room to leaving the room was measured for each treatment, and the number of staff (radiotherapy technicians and nurses) involved in the treatment was also examined. For adult cases, the number of staff involved in PBT is fixed at three, so only the time from entering to leaving the treatment room was measured. Pediatric patients were classified into sedation and non-sedation cases. Adult patients were classified into three groups based on irradiation from two directions without or with respiratory synchronization, and patch irradiation . Treatment person-hours were calculated as follows: (time from entering to leaving the treatment room) × (number of required personnel). Of the 32 pediatric patients in the study, 12 were treated while under sedation. Of the 90 adult patients, 30 patients were treated with and without respiratory synchronization and with patch irradiation. The mean ages of the pediatric patients were 3 (range 1–5) years for those treated with sedation and 5 (3–7) years for those treated without sedation. The mean treatment times (from entering to leaving the treatment room) were 20.7 min (pediatric cases without sedation), 30.1 min (pediatric cases with sedation), 11.5 min (without respiratory synchronization in adults), 15.6 min (with respiratory synchronization in adults) and 23.7 min (patch irradiation in adults). The mean numbers of personnel were 3.08 and 3.77 for pediatric cases without and with sedation, respectively, and the number of personnel for adult cases was fixed at 3. Based on these results, the treatment person-hours were 64.5 and 120.9 min for pediatric patients without and with sedation, respectively, and 34.3, 46.7 and 71.1 min for adult patients without and with respiratory synchronization, and with patch field irradiation, respectively . In pediatric patients, preparation procedures were performed five times on average (range 0–25) before the start of and during PBT. For sedated cases compared with non-sedated cases, the heart rate and oxygen saturation need to be monitored during the irradiation, and the final adjustments of sedation are also required after the patient has been moved to the treatment bed. It usually takes an additional 5–10 min to perform both steps. The preparation person-hours calculated as [(all preparation time) × (required personnel)]/(irradiation time) were 14.3 min for cases without sedation and 20.5 for those with sedation. The total person-hours, including the time for preparation, are shown in . In the clinical setting, it is also necessary for a pediatrician and a nurse to accompany a sedated patient during movement from the pediatric ward to the PBT facility, but this is not included in the analysis. PBT has an excellent dose concentration due to its focused energy peak and, therefore, is widely used for various tumors . For pediatric cases and adolescent and young adult patients, PBT is favored over photon radiotherapy due to reduced future adverse effects and secondary cancer . However, treatment for pediatric patients requires more time and effort for sedation and alignment compared with that for adult patients, which is a major negative point in terms of economic efficiency. In this study, the treatment person-hours for PBT for pediatric cases without sedation were 1.9, 1.4 and 0.9 times greater than those for adult patients irradiated without and with breathing synchronization, and with patch irradiation, respectively, and 3.5, 2.6 and 1.7 times greater for pediatric cases with sedation compared with the respective adult cases. Given that patch irradiation is a rare and non-standard method, the treatment person-hours for PBT for a pediatric tumor are generally 1.4–3.5 times greater than those for adult patients. We note that the study period differed between pediatric (2010–11) and adult (2022) cases, but the treatment equipment, irradiation technique and number of medical staff were similar. Various preparation procedures are used for irradiation with PBT for pediatric tumors . With the inclusion of preparation person-hours, treatment of a pediatric case without sedation took about twice as much time as treatment of adult patients (total person-hours: 78.8 vs 34.4–46.7 min) and treatment of a pediatric case with sedation took three to four times more than that for adult patients (141.4 vs 34.3–46.7 min). In addition, one pediatrician and one nurse needed to make a round trip from the ward to the treatment room for sedated patients (about 20–30 min per irradiation). The time required for this activity was not considered in the analysis; therefore, more person-hours are actually used for pediatric patients who need sedation. Given these transportation person-hours, PBT for pediatric cases in clinical practice is at least two to four times more labor-intensive than that for typical adult cases. However, this treatment is particularly effective in these cases, and further implementation of PBT requires increased support from facilities. In conclusion, the results of this study indicate that PBT for pediatric cases is much more labor-intensive than for adult cases. None declared. This work was supported by the University of Tsukuba. Research data are stored in an institutional repository and will be shared upon request to the corresponding author.
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Development and internal validation of an instrument to measure the motivation of residents for family medicine
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9a51f217-e7df-4107-aaa0-f5e71859c1e0
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10215013
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Family Medicine[mh]
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For several decades, the motivation of medical school graduates to specialise in family medicine has been decreasing and in many countries smaller than the demand for general practitioners . Moreover, residents in family medicine must be motivated for the profession, which increases the chance that they will finish residency, work in family medicine with pleasure for a long time and continue to work on developing their professional skills throughout their careers . A commonly used definition of motivation is ‘Powering people to achieve high levels of performance and overcoming barriers to change’ . Motivation is a feeling that moves people to do something and is related to purposefulness and success . The self-determination theory (SDT) argues that motivation is influenced by three psychological needs: autonomy, relatedness and competence. SDT differentiates among more or less autonomous motivation, intrinsic, extrinsic and amotivation . Intrinsic motivation reflects engagement in an activity due to inherent interest. It is an autonomous type of motivation. Extrinsic motivation refers to engagement in an activity to obtain outcomes, like to gain rewards or social approval, avoid punishment or comply with external norms. They are considered controlling types of motivation. Motivational regulations have different cognitive, affective and behavioural consequences . There is a direct relevance from educational settings to intrinsic and extrinsic motivation . As Kusurkar et al. describe in their review , motivation in medical education can be both a dependent and an independent variable. In the function of dependent variable, motivation can be influenced by changes in the curriculum or the learning environment in clinical practice. Motivation as an independent variable can influence students’ learning behaviour and study success . Motivation is a major determinant of the quality of learning, the learning strategies students use, their persistence and performance . The hypothesis is that motivated students try harder during the lesson, pay more attention and persist more to learn the skills than the less intrinsically motivated students. It confirms that autonomous forms of motivation positively influence performance . Several validated questionnaires measure the strength of motivation of medical students . This makes it possible, for example, to measure changes in motivation over time or to measure correlations between motivation and other factors, such as empathy or personality traits . Many studies have investigated factors and motivations impacting the decision to choose specific medical specialties. Motivations may include lifestyle choices, the possibility of private practice, interest in particular diseases, motivation for research or teaching or to gain a higher income. Students’ speciality preferences are influenced mainly by interaction with patients and desire to work in a challenging speciality while less common reasons are high demand for specific health services, the income potential, the workload, prestige or the duration of the residency . Studies on the motivation for a specialisation generally aim to inventory the students’ or residents’ reasons for a particular choice. These are usually self-designed questionnaires, sometimes supplemented with interviews . Due to its primarily qualitative nature, these questionnaires cannot be used to measure the strength of motivation. Goal of the current study therefore is the development and internal validation of an instrument to measure the motivation of residents for family medicine: STRength mOtivatioN General practitioner (STRONG).
Initial instrument For the development of the STRONG questionnaire, we have chosen to use an existing instrument as a starting point, namely the SMMS: Strength of Motivation for Medical School . This questionnaire is developed to measure the strength of motivation of medical students for medical school. This instrument based on the Self-Determination Theory by Ryan and Deci , has shown good psychometric properties. The SMMS-questionnaire consists of 15 items scored on a five-point Likert scale, from ‘strongly disagree’ tot ‘strongly agree’. The higher the score, the stronger the motivation. This instrument consists of three sub-scales: ‘willingness to sacrifice’, ‘readiness to start’ and ‘persistence’. The internal consistency of the three subscales, with each five items, measured by Cronbach’s alpha, ranges from 0.55 to 0.67 . Development STRONG To stay as close as possible to the original questionnaire, we used the Dutch version of the SMMS. We adapted the 15 items to make them suitable for family medicine residency and added a 16th item. This new questionnaire was translated back and forth from Dutch into German and English by native speakers. The items were then critically reviewed in two rounds by general practitioners involved in residency training ( n = 4) and family medicine residents ( n = 3). Based on the comments from these panels, the items were slightly edited. The items of the SMMS and the STRONG that we used in this validation study are listed in . Internal validation The STRONG questionnaire was sent electronically to 943 family medicine residents in Bavaria, Germany, in December 2020. A reminder was sent two weeks after the original invitation. For the statistical analyses, the software programme SPSS version 27 was used. An exploratory factor analysis for the STRONG item scores was carried out. The items were analysed for grouping into subscales by using principal component analysis. Cronbach’s alpha for internal consistency was determined for calculating the reliability of the subscales. Ethical approval Approval for the study was obtained from the Ethics Committee of the Faculty of Medicine of the Technical University of Munich (approval no. 627/19S). The survey was anonymous and voluntary, and all participants gave written consent in accordance with the Declaration of Helsinki. All residents received information on survey’s nature, purpose and procedure and their right to withhold or revoke their consent at any time.
For the development of the STRONG questionnaire, we have chosen to use an existing instrument as a starting point, namely the SMMS: Strength of Motivation for Medical School . This questionnaire is developed to measure the strength of motivation of medical students for medical school. This instrument based on the Self-Determination Theory by Ryan and Deci , has shown good psychometric properties. The SMMS-questionnaire consists of 15 items scored on a five-point Likert scale, from ‘strongly disagree’ tot ‘strongly agree’. The higher the score, the stronger the motivation. This instrument consists of three sub-scales: ‘willingness to sacrifice’, ‘readiness to start’ and ‘persistence’. The internal consistency of the three subscales, with each five items, measured by Cronbach’s alpha, ranges from 0.55 to 0.67 .
To stay as close as possible to the original questionnaire, we used the Dutch version of the SMMS. We adapted the 15 items to make them suitable for family medicine residency and added a 16th item. This new questionnaire was translated back and forth from Dutch into German and English by native speakers. The items were then critically reviewed in two rounds by general practitioners involved in residency training ( n = 4) and family medicine residents ( n = 3). Based on the comments from these panels, the items were slightly edited. The items of the SMMS and the STRONG that we used in this validation study are listed in .
The STRONG questionnaire was sent electronically to 943 family medicine residents in Bavaria, Germany, in December 2020. A reminder was sent two weeks after the original invitation. For the statistical analyses, the software programme SPSS version 27 was used. An exploratory factor analysis for the STRONG item scores was carried out. The items were analysed for grouping into subscales by using principal component analysis. Cronbach’s alpha for internal consistency was determined for calculating the reliability of the subscales.
Approval for the study was obtained from the Ethics Committee of the Faculty of Medicine of the Technical University of Munich (approval no. 627/19S). The survey was anonymous and voluntary, and all participants gave written consent in accordance with the Declaration of Helsinki. All residents received information on survey’s nature, purpose and procedure and their right to withhold or revoke their consent at any time.
Of the 943 family medicine residents, 275 completed the questionnaire making a response rate of 29.1%. The gender distribution was 83.3% females and 16.7% males. In the entire research group, 78.3% are women meaning that women are slightly over-represented among the respondents. The average age of the responders was 36.7 years, corresponding to the average age in the whole study group (36.8). The value of the KMO (Kaiser, Meyer und Olkin) test was .79 and the Bartlett Test resulted in a value of < 0.001, indicating that the data was suitable for a factor analysis . The factor analysis with Promax rotation resulted in two factors (see screeplot – ) explaining 39.6% of the variance . The questionnaire subscales could be labelled as: ‘Willingness to sacrifice’ (Subscale1) and ‘Persuasion’ (Subscale 2). Subscale 1: Willingness to sacrifice This subscale measures to what extent residents are prepared to make sacrifices to become general practitioners. Eight items based on their factor loadings (> 0.40) belonged to this subscale – see . This subscale explained 25.2% of the variance. Although item 2 had a factor loading of only 0.32, we checked the effect of adding this factor on the internal consistency of the subscale. The Cronbach’s alpha of this subscale was 0.82 and decreased to 0.81 when item 2 was added, so we decided to exclude the item from the subscale. Subscale 2: Persuasion The subscale ‘persuasion’ measures how convinced the residents are of their choice for family medicine. This subscale explained 14.4% of the variance. Four items belonged to this subscale based on the factor loadings of > 0.40 (see ). Although item 4 had a factor loading of only 0.28, we decided to include it into the subscale because it improved the internal consistency of the subscale. Cronbach’s alpha of the subscale was 0.61, which is acceptable. There were no cross loadings of items in one subscale onto the other subscale with a value of > 0.40. The Cronbach’s alpha for the full scale, with 13 items was 0.73, which is good.
This subscale measures to what extent residents are prepared to make sacrifices to become general practitioners. Eight items based on their factor loadings (> 0.40) belonged to this subscale – see . This subscale explained 25.2% of the variance. Although item 2 had a factor loading of only 0.32, we checked the effect of adding this factor on the internal consistency of the subscale. The Cronbach’s alpha of this subscale was 0.82 and decreased to 0.81 when item 2 was added, so we decided to exclude the item from the subscale.
The subscale ‘persuasion’ measures how convinced the residents are of their choice for family medicine. This subscale explained 14.4% of the variance. Four items belonged to this subscale based on the factor loadings of > 0.40 (see ). Although item 4 had a factor loading of only 0.28, we decided to include it into the subscale because it improved the internal consistency of the subscale. Cronbach’s alpha of the subscale was 0.61, which is acceptable. There were no cross loadings of items in one subscale onto the other subscale with a value of > 0.40. The Cronbach’s alpha for the full scale, with 13 items was 0.73, which is good.
Main findings As far as we know, the STRONG-questionnaire, based on the Self-Determination Theory by Ryan and Deci , is the only existing instrument for measuring the strength of motivation of residents for family medicine. Based on the internal validation described in this paper, this instrument appears to have good reliability and internal validity, assuming a two-factor structure. The reliability for the ‘willingness to sacrifice’ subscale and ‘persuasion’ subscale are very good (0.82) and acceptable (0.61), respectively. The reliability of the total instrument with 13 items is 0.73. Comparison SMMS – STRONG The SMMS, on which the STRONG is based, has three subscales . The questions in the ‘Willingness to sacrifice’ subscale largely overlap with the modified questions on scale 1 in our instrument. Therefore, we have given it the same name. The other questions fit our second subscale. We cannot confidently say why we found two rather than three subscales. The most likely explanation is that it has to do with the different study group (students versus residents) and different content (medicine in general versus family medicine). Implications of the internal validation Our study findings mean that the STRONG instrument may be used to measure the strength of the motivation of family medicine residents at different moments during residency: by regularly evaluating the strength of motivation during residency, factors which influence motivation positively or negatively can be identified . This can be done, for example, by measuring the motivation of cohorts of residents over a longer period and seeing how motivation develops. If an apparent increase or decrease can be seen at certain moments, it will be worthwhile to see what possible factors may have contributed. This is especially relevant because in addition to the primary motivation, multiple aspects such as work environment and professional development opportunities, can impact motivation for family medicine . Furthermore, insights into the strength of motivation for the profession are also important later on, as motivation affects the extent to which general practitioners continue to take courses throughout their careers, which increases their employability . To gain insight into the usefulness of this instrument, for example, whether low motivation strengths lead to a higher risk of dropping out, the instrument will have to be used over a longer period. Other possible applications for this tool include calculating the correlation with other factors, such as professional identity formation regarding family medicine or personality traits . We do not want to promote using the instrument as a selection tool for family medicine residency or in other situations that may encourage socially desirable responses. The questionnaire has not been validated for selection purposes. Moreover, this instrument may also be used during medical school, for example, for students considering taking part in a special track that prepares them for family medicine . The validation of this instrument for this group could be done in a follow-up study. As described earlier, motivation can be more or less intrinsic or extrinsic, with intrinsic and autonomous forms of motivation in particular positively affecting performance and perseverance . The SMMS, on which our instrument is based, appears to correlate especially positively with intrinsic motivation . A relevant follow-up study is to examine if the same is true for the STRONG. Strengths and limitations This study has several limitations. The most significant limitation is the moderate response rate. Because of that, we cannot exclude a selection bias, meaning that more motivated residents may be more inclined to respond. The number of female respondents is slightly higher than the percentage of women among the Bavarian family medicine residents but as the difference is small, it does not affect generalisability. Besides, the sample of this internal validation study only consisted of German residents. Although there are no indications that this target group differs greatly from residents in family medicine in other countries, it is recommended that the validation study be repeated in several countries and different contexts (for example, with residents in other disciplines) for external validation, supplemented by applying the tool in different contexts to test for feasibility.
As far as we know, the STRONG-questionnaire, based on the Self-Determination Theory by Ryan and Deci , is the only existing instrument for measuring the strength of motivation of residents for family medicine. Based on the internal validation described in this paper, this instrument appears to have good reliability and internal validity, assuming a two-factor structure. The reliability for the ‘willingness to sacrifice’ subscale and ‘persuasion’ subscale are very good (0.82) and acceptable (0.61), respectively. The reliability of the total instrument with 13 items is 0.73.
The SMMS, on which the STRONG is based, has three subscales . The questions in the ‘Willingness to sacrifice’ subscale largely overlap with the modified questions on scale 1 in our instrument. Therefore, we have given it the same name. The other questions fit our second subscale. We cannot confidently say why we found two rather than three subscales. The most likely explanation is that it has to do with the different study group (students versus residents) and different content (medicine in general versus family medicine).
Our study findings mean that the STRONG instrument may be used to measure the strength of the motivation of family medicine residents at different moments during residency: by regularly evaluating the strength of motivation during residency, factors which influence motivation positively or negatively can be identified . This can be done, for example, by measuring the motivation of cohorts of residents over a longer period and seeing how motivation develops. If an apparent increase or decrease can be seen at certain moments, it will be worthwhile to see what possible factors may have contributed. This is especially relevant because in addition to the primary motivation, multiple aspects such as work environment and professional development opportunities, can impact motivation for family medicine . Furthermore, insights into the strength of motivation for the profession are also important later on, as motivation affects the extent to which general practitioners continue to take courses throughout their careers, which increases their employability . To gain insight into the usefulness of this instrument, for example, whether low motivation strengths lead to a higher risk of dropping out, the instrument will have to be used over a longer period. Other possible applications for this tool include calculating the correlation with other factors, such as professional identity formation regarding family medicine or personality traits . We do not want to promote using the instrument as a selection tool for family medicine residency or in other situations that may encourage socially desirable responses. The questionnaire has not been validated for selection purposes. Moreover, this instrument may also be used during medical school, for example, for students considering taking part in a special track that prepares them for family medicine . The validation of this instrument for this group could be done in a follow-up study. As described earlier, motivation can be more or less intrinsic or extrinsic, with intrinsic and autonomous forms of motivation in particular positively affecting performance and perseverance . The SMMS, on which our instrument is based, appears to correlate especially positively with intrinsic motivation . A relevant follow-up study is to examine if the same is true for the STRONG.
This study has several limitations. The most significant limitation is the moderate response rate. Because of that, we cannot exclude a selection bias, meaning that more motivated residents may be more inclined to respond. The number of female respondents is slightly higher than the percentage of women among the Bavarian family medicine residents but as the difference is small, it does not affect generalisability. Besides, the sample of this internal validation study only consisted of German residents. Although there are no indications that this target group differs greatly from residents in family medicine in other countries, it is recommended that the validation study be repeated in several countries and different contexts (for example, with residents in other disciplines) for external validation, supplemented by applying the tool in different contexts to test for feasibility.
Based on the internal validation, the STRONG Instrument appears to have good reliability and internal validity, assuming a two-factor structure. This may therefore be a useful instrument for measuring the strength of the motivation of (future) family medicine residents. Insight into the motivation of family medicine residents may increase the likelihood that they will complete their training and stay in family medicine for a long time.
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Creation of 21st century anatomy facilities: designing facilities for integrated preclinical education in the Middle East
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0b8386d8-f2aa-4362-b0cb-aa9da3d0c4bd
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10215063
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Anatomy[mh]
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Khalifa University of Science and Technology (KU), a public research university located in Abu Dhabi, the capital of the United Arab Emirates, was founded in 2007 to nurture a knowledge-based economy. Initially encompassing the College of Engineering and the College of Arts and Sciences, it was decided in 2018 to establish a College of Medicine and Health Sciences (CMHS) to address the growing demand in the country of well-trained physicians and allied health workers. KU opted to create a four-year postgraduate Medical Degree (MD) program modeled after the US American system. As such, the CMHS accepted 30 students in 2019 as its inaugural student cohort to graduate in 2023. Subsequently, they accepted 34, 48, and 44 students in the following years. Part of the planning and development efforts was the creation of state-of-the-art anatomy laboratories providing appropriate facilities for a growing student cohort to eventually accommodate between 100 and 120 students per graduating year, as well as focus on interactive student-centered teaching of medical and Allied Health Science students, and for organizing Continuing Medical Education (CME) activities. Another important objective was to provide facilities for anatomical, biomedical, radiological, orthopedic, and surgical research. Education in anatomy must be appropriate to Middle Eastern culture without compromising the training of skilled medical doctors. When establishing our new Medical College, it was not feasible to establish a body donation program, and other sustainable cadaver resources had to be investigated. Moreover, establishing a body donor program is very costly and requires at least five years before universities can avail themselves of cadavers. Hence, we had to create an anatomy laboratory that reduces the number of outsourced body donations to a minimum and that can accommodate and implement current best practices in anatomy education. Estai and Bunt (2016) summarized, in a critical review, that the current best practices to teach anatomy in a medical curriculum is to combine various teaching resources, e.g., plastic models, plastinated human specimens, computer-assisted learning modalities, dissection, prosected specimens, as well as ultrasound, radiographs, CT and MRI images . This solidified the comments by Kerby et al. who state that currently there is no single teaching methodology that satisfies all the learning outcomes of a medical curriculum. Therefore, the creation of our newly created Anatomy Facilities aimed to address all these aspects of modern medical education. This included the concepts of integration of anatomy with radiology, ultrasound imaging and clinical skills, the possibility of small group interactive self-directed studies, access to a variety of educational technologies, and exposure to dissection and prosected specimens. In recent years, the methodology of anatomy education has been transformed by new technologies, such as plastination , computer-assisted learning tools, e.g., the Anatomage® tables , CAE Vimedix® ultrasound simulator mannequins , Complete Anatomy® program , as well as the creation of affordable, high-quality plastic models . At KU, anatomy is taught in an integrated system-based fashion. During period one of the curriculum (Foundations of Medicine), a four-week Introductory Anatomy Course, covering the basics from the microscopic to the organ system levels, is taught in the third month of the first year. This follows a course on genetics, biochemistry, and cellular biology, during which students are introduced to fundamental concepts of histology. During this introductory course, the basics of gross anatomy are taught in parallel with imaging technologies and radiological anatomy. In addition, case-based learning sessions introduce the students to clinical scenarios, during which they apply their knowledge in anatomy and clinical skills to solve diagnostic and therapeutic problems. Out of the 56 teaching hours of the first-year introductory course, there are 36 interactive lectures, using TurningPoint (©2021, Turning Technologies®, Youngstown, Ohio, USA), covering basics of embryology (six hours), histology (four hours), radiology (six hours) and gross anatomy (20 h). During these lectures, using Turning Point and other audience response systems, students are regularly presented questions (about 2–4 times per one lecture hour), and subsequently the answers are discussed, and misconceptions addressed. In addition, we also use an interactive approach in the dry labs (see below). The interactive lectures are complemented by twelve hours in the laboratories, five hours of case-based-learning and three hours of tutorial exercises. During the twelve-month-long second period of the curriculum (organ-systems), the anatomy of the individual organ systems is taught in greater detail in the context of clinical case scenarios, integrated with radiological anatomy, pathophysiology, therapeutics, and clinical skills. Depending on the individual course, 15–50% of the curricular activities of period two courses cover anatomy. Since plastic models are shown to be efficient supplements in teaching anatomy , our first period medical students start their gross anatomy laboratory sessions with plastic models (©2021, SOMSO®; Marcus Sommer Modelle GmbH, Coburg, Germany). In a study comparing learning outcomes of students using plastic models with students studying from cadaveric prosection to learn musculoskeletal anatomy of the upper limb, no significant difference in students’ performance was observed between the two groups . This is supplemented by the Complete Anatomy® program (©2021, 3D4Medical®, Elsevier, Dublin, Ireland), which is made available to all students at the beginning of their studies. Plastinated specimens are also employed during the laboratory sessions to give students a more realistic impression of anatomical relationships. In parallel, students also get accustomed to the Anatomage® virtual dissection table (©2021, Anatomage Inc. Santa Clara, CA, USA), which is used during the laboratory sessions as an additional resource. During the second half of the Introductory Anatomy Course, they are also introduced to prosected specimens of the thorax and abdomen. During the integrated organ system courses of period two, all these teaching resources are employed during laboratory sessions, which are based on clinical case scenarios and related problems that the students must solve in study groups. These approaches are focused on active and peer-discussion learning. Additionally, ultrasound modalities and the Sectra® table (©2021, Sectra AB, Linköping, Sweden) are utilized as additional educational tools to teach anatomy in a clinical context for contextualized learning. During period four of the curriculum (Advanced Clinical Rotations), motivated students can choose to participate in an “anatomy dissection elective”, during which a group of four to six students performs dissections on embalmed human cadavers. The prosected specimens prepared during this course are utilized in period one and two courses. The requirements for the construction of our anatomy facilities were therefore to create educational spaces that promote teacher-learner interaction, peer-assisted learning, and self-directed studies. The motivation of this project stems from the need to address specific problems when establishing anatomy facilities that allow for a combination of multiple pedagogical resources. This manuscript is beneficial for providing insight into processes and considerations for establishing Anatomical Facilities aligned with current evidence-based research.
During our literature search, we were guided by the “Preferred Reporting Items for Systematic reviews and Meta-Analyses” (PRISMA) statement . Using the PubMed, Scopus and EMBASE databases from January 1, 1990, to October 31, 2022, we included the search terms “anatomy”, “laboratory”, “medical education”, “preclinical education” “evidence-based anatomy education”. Subsequently, further searches were performed, including key words “plastination”, “3D models”, “Anatomage”, “Sectra”, “CAE Vimedix”, “dissection”, “prosection”, “ultrasound”, “sonography”, “augmented reality” and “virtual reality”. Citations within literature were also reviewed for relevant articles. Included were publications written in English, German, French and Spanish. In 2015, Brenner and colleagues proposed six techniques for anatomy education, which included in-person lectures, cadaver dissection, inspection of prosected material, models, living and radiological anatomy teaching, and computer-based learning (VR, AR, and 3D) . Suggestions for optimal modern curricula were also established by the Education Committee of the Anatomical Society of Great Britain and Ireland (ASGBI) and published in 2007 , which emphasize that maximum learning can be achieved using the following: (1) dissection/prosection, (2) multimedia, (3) practical procedures, (4) surface and clinical anatomy, and (5) radiological imaging. In this section we followed the systematic approach of a type of “needs analysis” by constructing a list of instructional methods and their effectiveness, based on the above prescribed “best teaching practices”, and from the authors experience (Table ). This represented the educational “needs” for delivering quality anatomy education. Following this, a list of facilities required to achieve these best practices (needs) was identified and was purposed with setting the foundation for the construction of a compliant facility in which to teach anatomy to modern medical students in the UAE (Table ). Student survey To assess student satisfaction with the current facilities and resources, period one and period two students (79 participants altogether) were given a survey (Institutional Review Board Protocol #H21-041). The survey, which adhered to the “Strengthening the Reporting of Observational studies in Epidemiology” (STROBE) guidelines , consisted of seven questions formulated to assess how the students benefited from the instructional methods mentioned in Table . These questions were composed using guidelines set out by Nemoto and Beglar in combination with procedures used by Spooren et al. . Table depicts how these questions were formulated to align with requirements to assess student satisfaction perceived by “best practice” instructional methods. For each item, students were asked their agreement with a statement on a 5-point Likert scale (1 = strongly disagree; 2 = disagree; 3 = neither agree nor disagree; 4 = agree; 5 = strongly agree). In addition, students were asked to provide open comments related to their experiences. Cronbach’s coefficient (α) was used to calculate the internal consistency coefficients of the items included in the survey .
To assess student satisfaction with the current facilities and resources, period one and period two students (79 participants altogether) were given a survey (Institutional Review Board Protocol #H21-041). The survey, which adhered to the “Strengthening the Reporting of Observational studies in Epidemiology” (STROBE) guidelines , consisted of seven questions formulated to assess how the students benefited from the instructional methods mentioned in Table . These questions were composed using guidelines set out by Nemoto and Beglar in combination with procedures used by Spooren et al. . Table depicts how these questions were formulated to align with requirements to assess student satisfaction perceived by “best practice” instructional methods. For each item, students were asked their agreement with a statement on a 5-point Likert scale (1 = strongly disagree; 2 = disagree; 3 = neither agree nor disagree; 4 = agree; 5 = strongly agree). In addition, students were asked to provide open comments related to their experiences. Cronbach’s coefficient (α) was used to calculate the internal consistency coefficients of the items included in the survey .
Based on the criteria set out in the material and methods, i.e., prosection, plastinated specimen, cadaver-based dissection, medical imaging, living anatomy and multimedia, the design of the facilities in the Department of Anatomy was aligned to meet with these expectations. The following facilities (Fig. ) were planned and constructed to align with the requirements to achieve the “best practice” instructional methods (Table ). Each of the facilities and how they integrate with the teaching approaches is detailed in Table . Dry laboratories Each of the three Dry Laboratories caters for 24 students and one lab instructor. In addition, audio-visual support is continually provided by the KU IT support office. The Dry Laboratories are designed and fully equipped to deliver diverse teaching methodologies, with the ultimate focus on student-centered learning and facilitation. This includes five CTOUCH® 85” Laser Nova UHD (©2021, CTOUCH, Eindhoven, Netherlands) interactive screens that broaden the capabilities of teaching and presenting content to both small and large student groups during dedicated laboratory sessions (Fig. .a.). Students collectively work in four groups of five to six students on a clinical scenario or a functional anatomical problem. Subsequently, the results of their discussions are presented on their respective screens and discussed with the other participating groups. The laboratories are centrally controlled with the Creston® application (©2021, Crestron Electronics, Inc., Rockleigh, New Jersey), allowing the venue to function both as a local, in-person presentation site, as well as a distance, off-campus learning site due to the live streaming capabilities captured with fixed and mobile Q-SYS (©2023, QSC, LLC, Costa Mesa, California, USA) PTZ-IP conference cameras and integrated Extron (©2023, Extron Electronics, Anaheim, California, USA) SMP 300 Series sound solution for capturing and distributing AV from handheld Shure (©2023, Shure, Niles, Illinois, USA) microphones and Shure ceiling array microphones. With this application, the instructor can project content from several resources within the venue, such as from the main podium, a secondary input device, a Clickshare® (©2023, Barco, Kortrijk, Belgium) device, and an Anatomage® table, onto the five interactive screens. The system also has the capability to mirror the content of one of the five CTOUCH® interactive screens to any or all the available screens. This sharing capability is also possible across the three Dry Laboratories, allowing content presented in one laboratory to be shared with and projected into the other two laboratories simultaneously. Situated within each Dry Laboratory is an Anatomage® table, which provides students with a virtual dissection tool for anatomy education and aids them with three-dimensional visualization of anatomical relations. The Anatomage® table can be orientated both in a horizontal or vertical position, providing a different perspective for the students to interact with the table (Fig. .b.). The Anatomage® table not only provides a virtual component for dissection and prosected specimens, but also enables the students to visually correlate the imaging modalities (MRI, CT scan) with the virtual specimens, thereby linking anatomy with radiology and clinical applications. The Dry Laboratories also house a wide variety of plastic SOMSO® anatomical models (Fig. .c.). The large collection of SOMSO® models has been meticulously selected to accommodate and represent all the relevant anatomy taught across the courses offered in the integrated medical curriculum. A large proportion of the models has been selected based on their ability to be disassembled and reassembled (Fig. .d), a unique feature that allows students to study and appreciate the complex nature and the relations of the human body (Fig. .e) in a systematic and fun way. The SOMSO models were thoroughly compared to other providers of models with the same technical specifications and were, in most cases, selected above their counterparts for their anatomical accuracy and build quality. Additionally, sections of the laboratory walls are painted with high gloss paint, which can be used as a whiteboard (Fig. .a.), thus aiding in the process of active learning and giving the opportunity for faculty and students to illustrate complex anatomical concepts in a visual manner, while discussing the content at hand. The Complete Anatomy® program is also available for our students and was selected as the preferred application, following a comparison with several similar applications, for its user-friendly interface, quality 3D renders, and anatomical accuracy. Imaging center The Imaging Center, located opposite the Dry Laboratories, has 24 student stations and one instructor station, each setup with Microsoft Surface Studio 2® (©2023, Microsoft Corporation, Redmond, Washington, USA). This unique setup allows faculty and students to view medical images on high quality screens with various DICOM viewers. The instructor can project the content from the instructor screen to all 24 student stations, as well as to the two CTOUCH® 85” Laser Nova UHD interactive displays. This ability to live share allows the instructor to teach, explain and demonstrate fine anatomical features and abnormalities on radiographs, CTs, or MRIs from a central point. This feature has been most useful during the social distance restrictions brought on by the COVID-19 pandemic. The Imaging Center also houses a Sectra® table, which is a multi-touch display workstation that enables faculty and students to access the Sectra Education Portal®. This permits engaging group lessons and lectures within the Imaging Center. The Sectra Education Portal® allows high-quality DICOM cases, curated by collaborating universities, to be viewed and permits tactile alterations to accommodate for the requirements of individual modules taught in the medical school. In addition to the Sectra Education Portal®, the Sectra® table includes VH Dissector Pro (©2023, Touch of Life Technologies Inc, Aurora, Colorado, USA) that offers high quality 3D reconstructed images, together with cross sectional views to easily disassemble and identify anatomical structures. With the increasing use of and push to incorporate more ultrasound teaching into medical curricula, the Imaging Center is supplemented with two CAE Vimedix® ultrasound simulator mannequins. One male mannequin for transthoracic, transesophageal echocardiography (©2023, CAE Vimedix Cardiac, CAE Healtcare®, Montreal, Canada) and a second female OB-GYN mannequin (©2023, CAE Vimedix OB-GYN, CAE Healtcare®, Montreal, Canada) to perform obstetric and gynecological transabdominal and endovaginal ultrasound examinations. Accompanying these mannequins is a comprehensive user interface that enables the instructor to present both imaging representative of normal anatomy and of pathologies of multiple diseases to support specific course objectives. The CAE Vimedix® can communicate with a Microsoft Hololens 2® (©2023, Microsoft Corporation, Redmond, Washington, USA), which allows for the 3D content to be projected onto the mannequin with augmented reality (AR) images. To fully expand and integrate this area with the rest of the laboratory spaces, the Imaging Center has the same presentation and sharing abilities as the Dry Laboratories, with two CTOUCH® 85” Laser Nova UHD interactive screens. In addition, 20 Philips Lumify® (©2023, Koninklijke Philips N.V., Amsterdam, Netherlands) ultrasound devices are regularly available to students to experience the complexity of explorative anatomy on themselves and standardized patients. Instructional studio The Instructional Studio was designed and equipped to fulfill two main functions. Firstly, to serve as an integrated teaching facility to medical students and secondly, to serve as a sophisticated venue to run CME courses to external stakeholders. The studio is fitted with standard STARLED5 NX (©2021, ACEM Spa, Argelato, Bologna, Italy) surgical lighting, VarioView 32 (Ondal Medical Systems GmbH, Hünfeld, Germany) adjustable screens, and AXCam.FHD (©2021, ACEM Spa, Argelato, Bologna, Italy) cameras at each of its eight student and two instructional stations. This intuitive setup allows for the integrative Xe® system (©2018, TechLab Works s.r.l, Blacksburg, VA, USA) software control system to remotely control the content projected on any of the station screens, including the two QM85F Samsung® Color Display Unit (©2021, Samsung, Suwon-si, South Korea) screens and two REALiS 4K501ST Pro AV (©2021, Canon Ota City, Tokyo, Japan) 4 K projectors situated on either side of the venue. In addition to the video routing function, the content visible on the respective camera can be recorded and streamed. The two instructional stations are equipped with individualized ventilation to prevent evaporation of fumes into the laboratory when dissection of embalmed specimens is conducted. These instructional stations are deliberately mobile with Mopec® Hydraulic Lift Dissection Tables (©2021, Mopec, Madison Heights, MI, USA) to allow space and mobility for the dissectors while dissecting (Fig. .a.). Along the walls of the Instructional Studio, there are both plastic SOMSO® models and von Hagens Plastination® (©2023, Gubener Plastinate GmbH, Guben, Germany) specimens stored in easily accessible and protective cabinets, to aid in teaching during sessions scheduled in the venue (Fig. .b.). Currently KU houses one of the largest collections of von Hagens Plastination® specimens outside Gubben, Germany, with more than 160 specimens. This collection includes various stages of dissection of six full bodies, 20 head and neck specimens, ten brains, nine pelvis and perineum dissections, four thorax specimens, 13 upper limbs, 21 lower limbs, and 46 organs relating to the cardiovascular, respiratory, gastrointestinal, and urinary systems. In addition, 29 pathology specimens are available, which range from pathologies such as liver cirrhosis to examples of smokers’ lungs. Moreover, the collection includes two fully disarticulated skeletons, and two complete central and peripheral nervous systems. Preparation room Adjacent to the Instructional Studio is the Preparation Room, which has a right-handed L-shaped elevating autopsy table with an integral wing dissection table (CE650, Mopec, Madison Heights, MI, USA) with standard STARLED5 NX® surgical lighting, VarioView 32® adjustable screens, and AXCam.FHD® camera capabilities linked to the Instructional Studio display system via the Xe® system. This allows sensitive dissections to be conducted and projected to students not physically present within the room. Again, these specific and tailored setup mechanisms have allowed anatomy teaching to continue amidst the restrictions imposed by the COVID-19 pandemic. This room was also designed to allow for preparing prosected specimens from donated cadavers; it has the storage capacity to keep these prosected specimens until needed. The Preparation Room also contains, to complement the histology capabilities, four ultra-cold surface units (histology freeze plates). These units can freeze histology samples quickly, thereby increasing curing process of tissue blocks, allowing dissection of frozen tissues, and reducing the overall tissue processing time by around 40%. Histology freeze plates are a necessary component for microtome sectioning in the absence of cryostat. Accompanying the dissection table and cold surface units, are two Mopec® Maestro Grossing Station® (©2021, Mopec, Madison Heights, MI, USA) and one 3DHistech Pannoramic Midi II® (©2021, 3DHISTECH Ltd., Budapest, Hungary) histological scanner. The equipment is connected to Mopec®’s PathCam Gross Imaging System (©2021, Mopec, Madison Heights, MI, USA) to simplify tagging specimens and histological microscopy. As each of the two grossing stations is equipped with a camera and accompanying software to identify barcodes, this works in harmony with the 3DHistech Pannoramic Midi II®, which has the same capabilities. The software SlideCenter® (©2021, 3DHISTECH Ltd., Budapest, Hungary) provided by 3DHistech® permits specimens to be examined remotely while a laboratory technician works on the cadaveric samples within the laboratory. Webinar room The Webinar Room comfortably seats 18 participants to engage in the exchange of ideas between faculty, staff, students, and external stakeholders. It provides multiple avenues to interact and involve various members of any discussion with a CTOUCH® 85” Laser Nova UHD interactive screen and an Epson® EB-710Ui Ultra Short Throw Projector (©2021, Seiko Epson Corporation, Suwa, Nagano, Japan) connected to an Epson® H599 LCU Projector Touch Unit (©2021, Seiko Epson Corporation, Suwa, Nagano, Japan) on either side of the venue. In addition, scattered around the oval-shaped conference table are four cubbies, which present the opportunity for any participant to project the content to any available displays, either with a HDMI, VGA, Ethernet, or a USB connection utilizing the Clickshare® functionality. Together with the Creston® integration, this room can stream the proceedings over the internet or project videos and audio to any of the Dry Laboratories and to the Imaging Center with the help of fixed and mobile Q-SYS PTZ-IP conference cameras and integrated Extron SMP 300 Series sound solution for capturing and distributing AV from handheld Shure microphones and Shure ceiling array microphones, establishing the interconnected ecosystem to relay content to any desired location. Student survey: results Student reactions towards the anatomy modalities (Table ) were positive as the response median was 5 (strongly agree) for six of the questions, which included: “I believe that the laboratory session fostered my understanding of the course content”, “Plastic models are appropriate and very useful tools during the initial anatomy teaching as a first approach to understanding the structure of the human body.”, “The use of Complete Anatomy as part of a combination of teaching tools positively influenced my acquisition of gross anatomical knowledge.”, “I am more satisfied with a combination of plastic models, plastinated specimens, prosected specimens, and computer assisted technology than with only one of these teaching modalities alone.”, “I prefer cadaveric prosection and dissection, compared to plastic, plastinated and computer models, when asked how to best understand anatomical relations and to gain anatomical knowledge in a clinical context.”, “Integration with clinical cases helped me understand the importance of anatomy.” For just one question, the median was 4 (agree) out of 5: “Combination of prosected and plastinated specimens, together with the Complete Anatomy program, fosters my anatomy knowledge better than each of these teaching methods alone.” The survey consisted of 7 items and the value for Cronbach’s Alpha for the survey was α = 0.693, which is the minimally acceptable value.
Each of the three Dry Laboratories caters for 24 students and one lab instructor. In addition, audio-visual support is continually provided by the KU IT support office. The Dry Laboratories are designed and fully equipped to deliver diverse teaching methodologies, with the ultimate focus on student-centered learning and facilitation. This includes five CTOUCH® 85” Laser Nova UHD (©2021, CTOUCH, Eindhoven, Netherlands) interactive screens that broaden the capabilities of teaching and presenting content to both small and large student groups during dedicated laboratory sessions (Fig. .a.). Students collectively work in four groups of five to six students on a clinical scenario or a functional anatomical problem. Subsequently, the results of their discussions are presented on their respective screens and discussed with the other participating groups. The laboratories are centrally controlled with the Creston® application (©2021, Crestron Electronics, Inc., Rockleigh, New Jersey), allowing the venue to function both as a local, in-person presentation site, as well as a distance, off-campus learning site due to the live streaming capabilities captured with fixed and mobile Q-SYS (©2023, QSC, LLC, Costa Mesa, California, USA) PTZ-IP conference cameras and integrated Extron (©2023, Extron Electronics, Anaheim, California, USA) SMP 300 Series sound solution for capturing and distributing AV from handheld Shure (©2023, Shure, Niles, Illinois, USA) microphones and Shure ceiling array microphones. With this application, the instructor can project content from several resources within the venue, such as from the main podium, a secondary input device, a Clickshare® (©2023, Barco, Kortrijk, Belgium) device, and an Anatomage® table, onto the five interactive screens. The system also has the capability to mirror the content of one of the five CTOUCH® interactive screens to any or all the available screens. This sharing capability is also possible across the three Dry Laboratories, allowing content presented in one laboratory to be shared with and projected into the other two laboratories simultaneously. Situated within each Dry Laboratory is an Anatomage® table, which provides students with a virtual dissection tool for anatomy education and aids them with three-dimensional visualization of anatomical relations. The Anatomage® table can be orientated both in a horizontal or vertical position, providing a different perspective for the students to interact with the table (Fig. .b.). The Anatomage® table not only provides a virtual component for dissection and prosected specimens, but also enables the students to visually correlate the imaging modalities (MRI, CT scan) with the virtual specimens, thereby linking anatomy with radiology and clinical applications. The Dry Laboratories also house a wide variety of plastic SOMSO® anatomical models (Fig. .c.). The large collection of SOMSO® models has been meticulously selected to accommodate and represent all the relevant anatomy taught across the courses offered in the integrated medical curriculum. A large proportion of the models has been selected based on their ability to be disassembled and reassembled (Fig. .d), a unique feature that allows students to study and appreciate the complex nature and the relations of the human body (Fig. .e) in a systematic and fun way. The SOMSO models were thoroughly compared to other providers of models with the same technical specifications and were, in most cases, selected above their counterparts for their anatomical accuracy and build quality. Additionally, sections of the laboratory walls are painted with high gloss paint, which can be used as a whiteboard (Fig. .a.), thus aiding in the process of active learning and giving the opportunity for faculty and students to illustrate complex anatomical concepts in a visual manner, while discussing the content at hand. The Complete Anatomy® program is also available for our students and was selected as the preferred application, following a comparison with several similar applications, for its user-friendly interface, quality 3D renders, and anatomical accuracy.
The Imaging Center, located opposite the Dry Laboratories, has 24 student stations and one instructor station, each setup with Microsoft Surface Studio 2® (©2023, Microsoft Corporation, Redmond, Washington, USA). This unique setup allows faculty and students to view medical images on high quality screens with various DICOM viewers. The instructor can project the content from the instructor screen to all 24 student stations, as well as to the two CTOUCH® 85” Laser Nova UHD interactive displays. This ability to live share allows the instructor to teach, explain and demonstrate fine anatomical features and abnormalities on radiographs, CTs, or MRIs from a central point. This feature has been most useful during the social distance restrictions brought on by the COVID-19 pandemic. The Imaging Center also houses a Sectra® table, which is a multi-touch display workstation that enables faculty and students to access the Sectra Education Portal®. This permits engaging group lessons and lectures within the Imaging Center. The Sectra Education Portal® allows high-quality DICOM cases, curated by collaborating universities, to be viewed and permits tactile alterations to accommodate for the requirements of individual modules taught in the medical school. In addition to the Sectra Education Portal®, the Sectra® table includes VH Dissector Pro (©2023, Touch of Life Technologies Inc, Aurora, Colorado, USA) that offers high quality 3D reconstructed images, together with cross sectional views to easily disassemble and identify anatomical structures. With the increasing use of and push to incorporate more ultrasound teaching into medical curricula, the Imaging Center is supplemented with two CAE Vimedix® ultrasound simulator mannequins. One male mannequin for transthoracic, transesophageal echocardiography (©2023, CAE Vimedix Cardiac, CAE Healtcare®, Montreal, Canada) and a second female OB-GYN mannequin (©2023, CAE Vimedix OB-GYN, CAE Healtcare®, Montreal, Canada) to perform obstetric and gynecological transabdominal and endovaginal ultrasound examinations. Accompanying these mannequins is a comprehensive user interface that enables the instructor to present both imaging representative of normal anatomy and of pathologies of multiple diseases to support specific course objectives. The CAE Vimedix® can communicate with a Microsoft Hololens 2® (©2023, Microsoft Corporation, Redmond, Washington, USA), which allows for the 3D content to be projected onto the mannequin with augmented reality (AR) images. To fully expand and integrate this area with the rest of the laboratory spaces, the Imaging Center has the same presentation and sharing abilities as the Dry Laboratories, with two CTOUCH® 85” Laser Nova UHD interactive screens. In addition, 20 Philips Lumify® (©2023, Koninklijke Philips N.V., Amsterdam, Netherlands) ultrasound devices are regularly available to students to experience the complexity of explorative anatomy on themselves and standardized patients.
The Instructional Studio was designed and equipped to fulfill two main functions. Firstly, to serve as an integrated teaching facility to medical students and secondly, to serve as a sophisticated venue to run CME courses to external stakeholders. The studio is fitted with standard STARLED5 NX (©2021, ACEM Spa, Argelato, Bologna, Italy) surgical lighting, VarioView 32 (Ondal Medical Systems GmbH, Hünfeld, Germany) adjustable screens, and AXCam.FHD (©2021, ACEM Spa, Argelato, Bologna, Italy) cameras at each of its eight student and two instructional stations. This intuitive setup allows for the integrative Xe® system (©2018, TechLab Works s.r.l, Blacksburg, VA, USA) software control system to remotely control the content projected on any of the station screens, including the two QM85F Samsung® Color Display Unit (©2021, Samsung, Suwon-si, South Korea) screens and two REALiS 4K501ST Pro AV (©2021, Canon Ota City, Tokyo, Japan) 4 K projectors situated on either side of the venue. In addition to the video routing function, the content visible on the respective camera can be recorded and streamed. The two instructional stations are equipped with individualized ventilation to prevent evaporation of fumes into the laboratory when dissection of embalmed specimens is conducted. These instructional stations are deliberately mobile with Mopec® Hydraulic Lift Dissection Tables (©2021, Mopec, Madison Heights, MI, USA) to allow space and mobility for the dissectors while dissecting (Fig. .a.). Along the walls of the Instructional Studio, there are both plastic SOMSO® models and von Hagens Plastination® (©2023, Gubener Plastinate GmbH, Guben, Germany) specimens stored in easily accessible and protective cabinets, to aid in teaching during sessions scheduled in the venue (Fig. .b.). Currently KU houses one of the largest collections of von Hagens Plastination® specimens outside Gubben, Germany, with more than 160 specimens. This collection includes various stages of dissection of six full bodies, 20 head and neck specimens, ten brains, nine pelvis and perineum dissections, four thorax specimens, 13 upper limbs, 21 lower limbs, and 46 organs relating to the cardiovascular, respiratory, gastrointestinal, and urinary systems. In addition, 29 pathology specimens are available, which range from pathologies such as liver cirrhosis to examples of smokers’ lungs. Moreover, the collection includes two fully disarticulated skeletons, and two complete central and peripheral nervous systems.
Adjacent to the Instructional Studio is the Preparation Room, which has a right-handed L-shaped elevating autopsy table with an integral wing dissection table (CE650, Mopec, Madison Heights, MI, USA) with standard STARLED5 NX® surgical lighting, VarioView 32® adjustable screens, and AXCam.FHD® camera capabilities linked to the Instructional Studio display system via the Xe® system. This allows sensitive dissections to be conducted and projected to students not physically present within the room. Again, these specific and tailored setup mechanisms have allowed anatomy teaching to continue amidst the restrictions imposed by the COVID-19 pandemic. This room was also designed to allow for preparing prosected specimens from donated cadavers; it has the storage capacity to keep these prosected specimens until needed. The Preparation Room also contains, to complement the histology capabilities, four ultra-cold surface units (histology freeze plates). These units can freeze histology samples quickly, thereby increasing curing process of tissue blocks, allowing dissection of frozen tissues, and reducing the overall tissue processing time by around 40%. Histology freeze plates are a necessary component for microtome sectioning in the absence of cryostat. Accompanying the dissection table and cold surface units, are two Mopec® Maestro Grossing Station® (©2021, Mopec, Madison Heights, MI, USA) and one 3DHistech Pannoramic Midi II® (©2021, 3DHISTECH Ltd., Budapest, Hungary) histological scanner. The equipment is connected to Mopec®’s PathCam Gross Imaging System (©2021, Mopec, Madison Heights, MI, USA) to simplify tagging specimens and histological microscopy. As each of the two grossing stations is equipped with a camera and accompanying software to identify barcodes, this works in harmony with the 3DHistech Pannoramic Midi II®, which has the same capabilities. The software SlideCenter® (©2021, 3DHISTECH Ltd., Budapest, Hungary) provided by 3DHistech® permits specimens to be examined remotely while a laboratory technician works on the cadaveric samples within the laboratory.
The Webinar Room comfortably seats 18 participants to engage in the exchange of ideas between faculty, staff, students, and external stakeholders. It provides multiple avenues to interact and involve various members of any discussion with a CTOUCH® 85” Laser Nova UHD interactive screen and an Epson® EB-710Ui Ultra Short Throw Projector (©2021, Seiko Epson Corporation, Suwa, Nagano, Japan) connected to an Epson® H599 LCU Projector Touch Unit (©2021, Seiko Epson Corporation, Suwa, Nagano, Japan) on either side of the venue. In addition, scattered around the oval-shaped conference table are four cubbies, which present the opportunity for any participant to project the content to any available displays, either with a HDMI, VGA, Ethernet, or a USB connection utilizing the Clickshare® functionality. Together with the Creston® integration, this room can stream the proceedings over the internet or project videos and audio to any of the Dry Laboratories and to the Imaging Center with the help of fixed and mobile Q-SYS PTZ-IP conference cameras and integrated Extron SMP 300 Series sound solution for capturing and distributing AV from handheld Shure microphones and Shure ceiling array microphones, establishing the interconnected ecosystem to relay content to any desired location.
Student reactions towards the anatomy modalities (Table ) were positive as the response median was 5 (strongly agree) for six of the questions, which included: “I believe that the laboratory session fostered my understanding of the course content”, “Plastic models are appropriate and very useful tools during the initial anatomy teaching as a first approach to understanding the structure of the human body.”, “The use of Complete Anatomy as part of a combination of teaching tools positively influenced my acquisition of gross anatomical knowledge.”, “I am more satisfied with a combination of plastic models, plastinated specimens, prosected specimens, and computer assisted technology than with only one of these teaching modalities alone.”, “I prefer cadaveric prosection and dissection, compared to plastic, plastinated and computer models, when asked how to best understand anatomical relations and to gain anatomical knowledge in a clinical context.”, “Integration with clinical cases helped me understand the importance of anatomy.” For just one question, the median was 4 (agree) out of 5: “Combination of prosected and plastinated specimens, together with the Complete Anatomy program, fosters my anatomy knowledge better than each of these teaching methods alone.” The survey consisted of 7 items and the value for Cronbach’s Alpha for the survey was α = 0.693, which is the minimally acceptable value.
This paper provides a clear guideline of how best practices for modern anatomy education listed in the Methods section (Tables and ) were meticulously considered and implemented with the design and layout of our newly created Anatomy Facilities. The design and implementation of these facilities and resources had to create a learning environment promoting student-centered learning, peer discussions and group interactions. The outcome was positively received by our students when factors such as using cadaver-based dissection, use of plastic and/or plastinated models, integrated imaging techniques, and multimedia supplementation were considered. Research has shown that plastic models are a useful tool to teach anatomy, because they are easily accessible, relatively affordable, and very effective in promoting learners’ level of gross anatomical knowledge . They allow students to obtain a notion of the three-dimensional arrangements of anatomical structures by rotating the organs in their hands and often by opening them up to investigate their interior. They have been shown to be particularly useful in teaching spatial brain anatomy . According to our survey, plastic models are appropriate and very useful tools during the initial anatomy teaching as a first approach to understanding the structure of the human body. The advantages and disadvantages of dissection , the “systematic exploration of a preserved human cadaver by sequential division of tissue layers and the liberation of certain structures … with the aim of supporting the learning of gross anatomy” have been discussed in detail . Essentially, the emotional impact of dissection, health and safety issues, practicalities, and the cost of using cadavers have been mentioned as disadvantages. Potential advantages are better knowledge acquisition and integration, appreciation of three-dimensional relationships and anatomical variability, peer-group learning, tactile experience, promotion of professionalism through direct encounter with the cadaver, and development of manual skills required for many medical specialties . A systematic review of the relevant literature dealing with objective data evaluating the cognitive learning outcomes of cadaver dissection compared to other teaching approaches concluded that there is a slight advantage of dissection over prosection . Generally, the term “dissection” is employed when students are actively dissecting, whereas “prosection” refers to the study of cadaver specimens prepared by others . We developed and offered a “Clinically Orientated Anatomy Dissection” course as an elective in 2022, in which eight Period 4 students enrolled and successfully participated. We believe that the quality of dissection performed by these students was well above standard compared to first year dissections seen in other medical curricula, as these students were more aware of the clinical significance and focused on preserving anatomical structures rather than conducting exploratory dissection. Additionally, we provide the opportunity for Period 1, 2, and 3 students to dissect weekly during a student interest group activity. Over the past 3 years, the average student attendance for this student interest group activity ranged between 10 and 18 students, depending on their curricular responsibilities, bringing the number of students participating in dissection to about a third of our student cohort. According to our own survey, which is supported by the literature , students generally prefer cadaveric prosection and dissection, compared to plastic, plastinated and computer models, when asked how to best understand anatomical relations and to gain anatomical knowledge in a clinical context. For the reasons listed above, we decided to include prosected specimens in our anatomy teaching and to allow motivated students to perform cadaveric dissection, but to reduce the number of body donations to a minimum and to supplement dissection with other teaching modalities. Many of the advantages of cadaver prosected specimens, (e.g., tactile experience, three dimensionality and exact representation of the human anatomy), without the disadvantages, (e.g., problems in procurement, difficulties in preservation, large infrastructural requirements for storage, lack of resistance to mechanical strain, exposure to fixatives, smell, and easy destruction), can be achieved by plastination . This method of preservation of organic material produces long-lasting anatomical specimens of body parts or the entire body . During the process of plastination, the body fluids of dissected organs, organ systems or entire bodies of body donors are substituted with acetone, and subsequently, acetone is replaced by a polymer, resulting in clean, dry, odorless, non-toxic, touchable, durable and authentic specimens representing the exact anatomy of the body donor that can be easily transported . Moreover, thin plastinated organ slices impart a sound knowledge of cross-sectional anatomy, which is invaluable for the understanding of radiological images . Similar sentiment is held by our students with comments in the survey suggesting “… Plastinates models gave a good idea of the reality and in visualizing …” and “I would like to use more the plastinated models because they are a more real representation of anatomy…”. The initial purchase is relatively costly, but due to the durability of the specimens lasting over decades , the investment pays off in a relatively short period. KU sourced its plastinated specimens solely from von Hagens Plastination® after an ethical sourcing inquiry. The authors personally visited the Plastination facilities and went through the records to ensure that plastinated specimens of interest were donated with the highest of ethical standards, including written consent. In addition, the tenet of body donation from von Hagens Plastination® is stipulated as follows: “Body donation for Plastination to the Institute for Plastination is not a contract, but a declaration of intent. The donor declares during their lifetime that their body should not be buried after death, but rather transferred to the Institute for Plastination. The consent form and a 30-page brochure provide detailed information so that each donor is explicitly informed about the future use of their body, including the production of teaching specimens for sale. The Institute for Plastination also organizes regular meetings for body donors. To ensure that the decision to donate one’s body to the Institute for Plastination is undertaken with free will there is no financial compensation.” Given the time-constraints of modern anatomy curricula, the use of plastinated specimens also offers the opportunity to utilize teaching time more efficiently. During a standard three-hour dissection session, half the time is spent cleaning areas of interest (i.e., removing fat and fascia), during which no anatomy is taught. By using plastinated specimens, the relationships of anatomical structures are maintained and to some degree even clearer than those often seen in cadavers . Specific areas that, due to time restraints, are not often seen in the dissection hall may be perfectly visible on the plastinated specimens due to their high quality of dissection. This environment allows students to appreciate the accurate anatomy and, importantly, the anatomical relations and integrate these with carefully prepared clinical scenarios and case-based discussions. For this reason, we acquired a large selection of plastinated specimens representing almost the entirety of gross anatomy. However, we did not completely forego dissection, since, dissection is known to develop manual skills, empathy and teamwork . Moreover, in plastinated specimens, the mobility of the individual structures is lost so that students are unable to remove organs to fully appreciate their neighboring relations. Several studies indicate that the use of computer -based technologies as part of a combination of teaching tools positively influences students’ acquisition of gross anatomical knowledge . This is corroborated by comments in our survey, such as “…The Complete Anatomy was also very useful, but I would have liked to have more of it integrated in the labs.” and “…Complete Anatomy live in the class it would have been very helpful”. It has been reported that students who employed web-based computer-aided teaching programs performed significantly better in examinations than learners who had never accessed the online content . A comparison of the learning outcomes of students taught anatomy using either the Anatomage ® table or dissection did not show any significant difference in student performance, but students were more enthusiastic about learning anatomy on the Anatomage® table and believed that they had learned more . In a cross-sectional study comparing the students’ view on anatomy teaching with either the Anatomage® table or plastinated specimens alone or with a combination of both , students were significantly more satisfied with a combination of both teaching strategies, compared to the Anatomage® table or plastinated specimens alone. Stanford et al. (1994) came to a similar conclusion, when evaluating learning outcomes after teaching cardiac anatomy by a combination of dissection and an interactive computer-assisted anatomy instruction program, which yielded better results than each of these teaching methods alone . Another group of studies, however, points to a limited value of computer-animated three-dimensional visualization of anatomical relationships, which also depends heavily on the learners’ spatial abilities . Moreover, the resolution of smaller anatomical structures, e.g., arteries and nerves, is generally rather low so that computer-based anatomy teaching benefits from additional exposure to plastinated and prosected specimens . In addition, if used as a stand-alone educational tool, “Anatomical models outperform their computer-based counterparts for anatomy learning” . Another important tool in our anatomy education is Imaging-based instruction, using ultrasound as well as 3D computerized MRI and CT images to teach anatomy. We have incorporated ultrasonography into our curriculum at a relatively early stage of student training, because it is a non-invasive and relatively inexpensive way to demonstrate anatomical and pathological structures inside the body, thereby linking basic and clinical education and allowing to learn anatomy in a clinical context. During an introductory session, students are instructed in ultrasound technology, how to handle the ultrasound probes, and how to interpret the sonographic images. The students are then allowed to use the CAE Vimedix® trainers. This way, they can locate healthy organs, their spatial relationships, and pathological changes on the mannequins, thereby mimicking the situation in the living. In addition, they also have access to portable ultrasound devices, which allow them to scan standardized patients. Moreover, the Sectra table, which is also located in the Imaging Center, provides the students with the option to generate a three-dimensional notion of anatomy, based on a variety of normal and pathological CT and MRI images in DICOM format. Patient images can be uploaded to the Sectra educational portal and can then be discussed with the students either in class using the Sectra table or online through the Sectra streaming capabilities. For the reasons described above, we decided to employ a combination of educational tools to teach anatomy in our laboratories, including plastic models, plastinated specimens, computer-assisted learning tools, prosected specimens, and imaging modalities. Given students’ different learning styles and the varying preferences for teaching approaches, the wide range of educational tools available at KU’s Department of Anatomy and Cellular Biology is highly appreciated by our students, as illustrated by the results of our survey. Moreover, in times of the COVID-19 epidemics, our broad variety of educational tools was extremely helpful in converting our anatomy laboratory education to online teaching sessions. For example, we have been able to use the Complete Anatomy® program for interactive guided visualization of three-dimensional anatomical relationships. The images and the functionality of the Anatomage® table with the assistance of streaming applications, e.g., Zoom® (©2021, Zoom Video Communications, San Jose, California, USA) or Microsoft Teams® (©2021 Microsoft Corporation, Redmond, Washington, USA), can be delivered online to the student audience. The cameras located within the Dry Laboratories, Instructional Studio, Preparation Room and Imaging Center as well as the associated streaming capabilities allow us to record and broadcast explanations of plastic models, plastinated specimens and radiological images to the students from these venues. In addition, real-time dissections can be made available to the learners. The CMHS allows the Department of Anatomy and Cellular Biology to continuously pursue novel and developing technologies and programs. We continue to assess new teaching modalities together with the Office of Medical Education, the Office of Academic Affairs, and the student cohort to evaluate if they are more advantageous than our current teaching aids. As part of this initiative, technologies such as Hololens 2 together with programs such as HoloHuman (©2022, GigXR Inc, California, USA) and HoloPatient (©2022, GigXR Inc, California, USA and ©2022, 3D4Medical®, Elsevier, Dublin, Ireland) to conduct possible case-based learning sessions are being explored. Replacement devices, such as EchoNous-KOSMOS handheld ultrasound devices (©2022, EchoNous Inc, Redmond, Washington, USA), which are driven by Artificial Intelligence (AI) to detect anatomical structures and aid in probe placement, are currently being evaluated to expand ultrasound in our anatomical teachings. We are actively looking into the option of introducing 3D printing into our curriculum and are assessing the possibilities of incorporating virtual reality, e.g., EON reality (©2022, EON Reality, Irvine, California, USA) into our anatomy teaching, according to the KU Metaverse initiative. The limitations of our study lie in the fact that we are a newly established Medical College and therefore can only determine student satisfaction on a small student sample. We do not yet have the capability of performing a longitudinal study. Moreover, we realize that not all institutions may have the financial support available to implement new or upgrade existing facilities.
After three years of planning and construction, our anatomy facilities are now fully functional. The Medical Students of KU are highly appreciative of the large variety of teaching modalities available, and their performance in standardized anatomical exams exceeded our expectations. These results are of interest to faculty tasked to create modern anatomy teaching facilities based on a combination of prosection, plastinated specimen, cadaver-based dissection, medical imaging, living anatomy and multimedia.
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a611bb0b-266f-470a-a8b0-37157d81bab7
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Microbiology[mh]
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First Pediatric Exercise Oncology Congress (PEOC)
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ea18827a-9622-41a6-afe8-bc447bdcaa3d
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10217259
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Internal Medicine[mh]
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Exercise interventions and physical activity recommendations Effects of physical activity on patient- and health-related outcomes Implementation of physical activity—strategies, models, considerations Novel methods to improve physical activity and reduce late effects Impacts of physical activity on cancer treatment response/tolerance Other topics in pediatric exercise oncology (e.g., COVID-19) Website: https://www.pediatric-exercise-oncology-congress.org accessed on 1 March 2022
2.1. Exercise Programs during Treatment for Hemat-Oncological Disease: Why Are Supervised Interventions Necessary Early after Diagnosis? Sabine Kesting 1,2 and Kinderklinik München Schwabing 1 TUM School of Medicine, Department of Paediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany 2 Institute of Preventive Paediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany The adverse effects of initial anti-cancer treatment already emerge early after diagnosis and contribute to increased physical inactivity in combination with the clinical environment and psychological burden. These circumstances can instantly result in reduced physical function, impaired activities of daily living and negatively influence health-related quality of life. Thus, the question is raised: how early should we start applying exercise interventions during acute treatment and which aspects should be considered? Experiences from a clinical perspective show a sudden interruption of normal physical activity habits immediately after hospitalization due to disease-related impairments, general uncertainty and necessary clinical diagnostics. The approaches to implement exercise programs during this sensitive phase of treatment comprise qualified and experienced staff, individual adaption of content and intensity levels, motivating and empathetic communication, creativity and flexibility. The primary objectives are the stabilization of physical activity and fitness to reduce the risk of impairments and accustoming to the new level of physical activity and possibilities to be physically active. Despite the known challenges and difficult circumstances, exercise interventions should be applied shortly after diagnosis considering the burdensome situation and the patient’s clinical status. Moreover, key components of planning and implementing exercise early are regular communication, providing information, including the patient’s wishes and concerns, to build trust and maintain physical activity levels throughout treatment. Open research questions include the effects of medication on physical function early during treatment, e.g., initial steroid application, and suitable interventions. 2.2. Cardiorespiratory Fitness in Patients and Survivors Maxime Caru Department of Pediatrics, Division of Hematology and Oncology, Penn State College of Medicine, Hershey, PA, USA Children’s and adolescents’ exposure to chemotherapeutic agents causes several long-term adverse effects affecting their cardiac health due to cardiotoxicity, physical function, and ultimately their quality of life. Physical activity is an effective strategy to prevent long-term adverse effects whether during or after treatments in pediatric oncology. Nevertheless, very few pediatric patients diagnosed with cancer meet the physical activity guidelines. Hence, less than one in two patients are doing 60 min or more of moderate-to-vigorous-intensity physical activity each day. Considering that they are physically inactive and exposed to chemotherapy-related cardiotoxicity, this has the consequence of reducing their cardiorespiratory fitness (aerobic capacity). This invited talk gives an overview of the cardiorespiratory fitness state of children and adolescents diagnosed with cancer and its long-term impact on their survivorship. In the end, the audience should be able to understand the mechanism behind the decrease in childhood cancer survivors’ cardiorespiratory fitness, identify what is needed next, and recognize clinical perspectives for this unique population. 2.3. Training to Support Physical Activity Delivery in Pediatric Oncology Amanda Wurz School of Kinesiology, University of the Fraser Valley, Abbotsford, BC, Canada Qualified exercise professionals (QEPs) play a vital role in physical activity delivery, yet there are no agreed-upon training requirements to ensure the competence of PQEPs within pediatric exercise oncology. This talk will discuss this gap, provide a case example of a QEP training protocol, and offer an opportunity for critical dialogue. Ultimately, it is hoped this talk will be a first step towards creating standardized QEP training requisites in this field. 2.4. The Burden of Cardiovascular Disease in Childhood Cancer Survivors, Its Prevention and Management Christina Schindera No abstract available. 2.5. Psychosocial Empowerment through Peer Involvement in Exercise Programs Martin Kaj Friedh No abstract available. Abstract Oral Presentations 2.6. A Bout of High-Intensity Interval Training (HIIT) in Children and Adolescents during Acute Cancer Treatment—A Feasibility Study (NOTE: Young Investigator Award Winner) Peter Weeber 1 , Sabine Kesting 2,3 , Martin Schönfelder 1 , Henning Wackerhage 1 and Irene von Luettichau 2 1 Exercise Biology, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 2 Kinderklinik München Schwabing, TUM School of Medicine, Department of Paediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany 3 Institute of Preventive Paediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany Background: Low-and moderate-intensity exercise is safe and feasible during childhood cancer treatment. However, is this also true for a bout of high-intensity interval training (HIIT)? The aim of this study is to find out whether HIIT is feasible in childhood cancer patients and to measure the cardiovascular and metabolic response to HIIT. Methods: Children with lymphoma, leukaemia, rhabdomyosarcoma, nephroblastoma, and synovial sarcoma performed ten 15 s high-intensity intervals (>90% of estimated HRmax) with 1 min active recovery on a bicycle ergometer in between. In addition to assessing the safety and feasibility of HIIT, we also recorded the perceived exertion rate, heart rate, lactate, and adrenaline concentrations. Results: A total of 11 patients at the age of 13.9 ± 3.6 years ( n = 7 female) completed a bout of HIIT without serious adverse events. The patients reached a BORG value maxima of 16 ± 1.2 during exercise and heart rate increased from 78 ± 17 bpm at rest to 178 ± 12 bpm after exercise (90 ± 6% of the predicted maximal heart rate). The power-to-weight average ratio during exercise was 2 ± 0.5 W/kg. Blood lactate concentrations increased from 1.09 ± 0.50 mmol/L at rest to 5.05 ± 1.88 mmol/L post-exercise. Adrenaline levels increased only marginally post-exercise. No adverse events were observed. Conclusions: Our study shows that HIIT is feasible for a small number of physically fit childhood cancer patients but not for patients with a low fitness level or complications. 2.7. Maximal Cardiac Output and Cardiorespiratory Fitness in Young Cancer Survivors Marcella Burghard 1,2 and Tim Takken 1,2 1 Child development and Exercise Centre, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, The Netherlands 2 Sports and Exercise Center, Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Background : It is known that cardiorespiratory fitness (CRF), measured as oxygen uptake relative to body weight (VO 2 peak/kg), is often decreased in both pediatric and adult cancer survivors. Research is unambiguous regarding a lower cardiac output as a possible cause for this impairment. Our aim was to assess if, next to CRF, there are differences in maximal cardiac output index (COI) response, including stroke volume index (SVI) and heart rate (HR) at maximal exercise, compared to healthy controls. Methods : A total of 30 young cancer survivors (mean age 14.4 ± 4.1 years) and 16 healthy controls (mean age 14.7 ± 4.5 years) performed a cardiopulmonary exercise test to measure CRF and other CPET-derived variables. COI and SVI were measured using thorax impedance measurements (Physioflow ® ). Results : CRF (VO 2 peak/kg in percentage predicted) was significantly lower ( p = 0.000) in the young cancer survivors (71.5; 63.5–82.6%) compared to the healthy controls (90.0; 84.0–95.0%). COI ( p = 0.170) and peak HR ( p = 0.914) did not differ significantly between groups, however SVI was significantly lower ( p = 0.013) in the cancer survivor group (52.2 ± 12.8 mL/m 2 ) compared to the healthy control group (61.6 ± 9.0 mL/m 2 ). Conclusions : CRF is decreased after cancer treatment in young cancer survivors. A decreased cardiac stroke volume might be one of the limiting factors for CRF. 2.8. Implementation of an Exercise Program for Advanced Pediatric Cancer Patients Ronja Beller, Gabriele Gauß, Dirk Reinhardt and Miriam Götte Department of Pediatric Hematology/Oncology, Center for Child and Adolescent Medicine, Clinic for Pediatrics III, West German Cancer Centre, University Hospital Essen, Essen, Germany. Background: Children and adolescents should have access to exercise programs throughout cancer trajectory, including during palliative care. There is only very limited experience and data available for this phase of life. An exercise project has been conducted at the University Hospital Essen for advanced cancer patients who participated in supervised exercise programs during neoadjuvant and/or adjuvant treatment, and have bonded with these programs and their exercise professionals. Methods and Results: Inclusion criteria are diagnosis of advanced cancer, age >3 years, previous participation in the hospital’s exercise program, and residence in the surrounding area of the hospital (<60 min car drive). Inclusion is discussed with the Specialized Outpatient Palliative Care Team of the clinic to consider the health status and family peculiarities. After deciding on the feasibility and gaining the acceptance of the families, the assigned exercise physiologist offered sessions once a week for 30–90 min. Sessions were usually scheduled at the patients’ homes, but also in in- and out-patient clinics if patients had appointments or complications. The contents were a combination of strength, endurance, coordination, body awareness, and mobility training that were resource-oriented, related to the child’s interests, and adapted to their respective daily condition. Data monitoring was conducted every four to six weeks. Conclusions: This concept takes into account the individual possibilities, goals, and interests of those directly affected. Since the project is not yet part of standard care, it should be evaluated and expanded. Pediatric advanced cancer patients should have the opportunity to participate in exercise programs, and benefit from physical activity support. 2.9. Implementation of a Pediatric Exercise Oncology Program in Brazil: Maple Kids Experience Alice Aparecida Rodrigues Ferreira Francisco 1,2 Jader Brito Ramos dos Santos 1 , Otávio Augusto Soares Machado 1,3 and Karen W. Wonders 1,2 1 Maple Tree Cancer Alliance Brazil, Sorocaba, Brazil 2 Maple Tree Cancer Alliance, Dayton, OH, USA 3 School of Physical Education from YMCA, Sorocaba, Brazil Background : Research has already shown that physical activity (PA) during childhood means a higher probability of maintaining active lifestyles. Therefore, it diminishes the severity of the adverse effects of cancer treatment and is a safe tool. Maple Tree Cancer Alliance (MTCA) is a non-profit organization helping cancer patients through exercise rehabilitation since 2011. Founded by Dr. Karen Wonders, in 2019 MTCA started serving patients in Brazil. Maple Kids (MK) started in 2021 in Brazil at the Childhood Cancer Research and Assistance Group Hospital (GPACI), a center attending 48 cities in the Sorocaba region. Methods : MK is an exercise program for pediatric cancer patients, to improve quality of life and overall health. We also include family participation, inspiring a healthy lifestyle for all. The main program goals are: to improve cardiac and pulmonary capacity, boost immune system, reduce fatigue, and peripheral neuropathy. Its secondary goals are to reduce psychological and cognitive problems, and to reduce second tumors and chronic diseases. Results : Maple Kids starts in January 2022, and results will soon be posted. Conclusions : Some children face debilitating physical and cognitive problems as side effects of cancer treatments. Cancer can mean not running or jumping, and feeling uncomfortable during PA. There is a lack of accessible and specialized programs, of staff prepared to support cancer patients, and a lot of misinformation from family and health professionals, which means that 62% of adult survivors have at least one chronic health condition. PA for pediatric cancer patients and survivors means better physical and psychosocial health. 2.10. Adoption of an Active Lifestyle Post Cancer Treatment: Development and Efficiency of a Behavioural, Web-Based Intervention Helena Koine 1 , Isabell Hamm 1 , Anja Chevalier 2 and Anna Vogelsang 3,4 1 Institute of Psychology, German Sport University Cologne, Cologne, Germany 2 Department of eHealth and Sports Analytics, Ruhr-University Bochum, Bochum, Germany 3 Department of Sport Economics and Sport Management, German Sport University Cologne, Cologne, Germany 4 Faculty of Humanities, MSH Medical School Hamburg, Hamburg, Germany Background: Adolescents and young adult (AYA) cancer survivors face an increased risk of physical, psychological and psychosocial late effects, long-term morbidity, and premature death. Despite the well-documented effectiveness of PA in mitigating treatment-related late effects, most adolescent and young adult (AYA) cancer survivors face various physical, behavioural, psychological, and educational barriers, which hinder them in adopting a physically active lifestyle. The current study takes survivors’ barriers into account and examines the effects of a four week online intervention on physical activity and social cognitive predictors of physical activity in AYA cancer survivors compared to a waiting control group. It is hypothesized that the physical activity levels and social cognitive predictors increase in the intervention as opposed to the control group. Methods: A randomized controlled trial with repeated measures including an intervention ( n = 30) and awaiting control group ( n = 31) was performed. The four week online intervention was developed based on the Health Action Process Approach and the Self-Concordance Model and included a baseline and post-test. Results: Significant interaction effects between time and treatment revealed greater physical activity and improved volitional and generic social cognitive predictors of physical activity in the intervention group. No significant interaction effects were found in motivational social cognitive variables. Conclusions: The results revealed the potential of a behavioural online intervention in fostering an active lifestyle in AYA cancer survivors. Given the high number of sedentary AYA and also childhood cancer survivors, the efficacy and suitability of behaviour, online lifestyle interventions for this target group will be discussed. 2.11. Moving the International Pediatric Oncology Exercise Guidelines (iPOEG) Forward: Where Do We Go from Here? Emma McLaughlin 1 , Amanda Wurz 1,2 , Colleen Cuthbert 3,4,5 and Nicole Culos-Reed 1,5,6 1 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 2 School of Kinesiology, University of the Fraser Valley, Chilliwack, BC, Canada 3 Faculty of Nursing, University of Calgary, Calgary, AB, Canada 4 Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 5 Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 6 Department of Psychosocial Resources, Tom Baker Cancer Centre, Cancer Care, Alberta Health Services, Calgary, AB, Canada Background: Following the publication of the international Pediatric Oncology Exercise Guidelines (iPOEG) in 2021, resources to support end users were developed. Collectively, these resources are referred to as the iPOEG Toolkit. To better understand the value of the iPOEG Toolkit, its dissemination, implementation, and effectiveness will be tracked and explored. Methods: A research program informed by the Knowledge to Action framework is underway. A mixed-methods approach is being used, and the RE-AIM framework is guiding the evaluation of its reach (e.g., the number of iPOEG Toolkit downloads), effectiveness (e.g., the changed physical activity levels and patient-reported outcomes among end users), adoption (e.g., the number of organizations using the Toolkit), implementation (e.g., the use of and modifications made to the iPOEG Toolkit), and maintenance (e.g., the tracking of its continued use and impact, including markers of effectiveness, over time). Data will be collected via tracking metrics (e.g., webpage views, downloads), surveys, and interviews with relevant end users. Quantitative data will be analyzed descriptively and using regression analyses as appropriate, and qualitative data will be examined using content analysis and reflective thematic analysis. Results: The iPOEG Toolkits are currently being disseminated via the Health and Wellness Lab website, emails, and social media, and data will be gathered up until 2025. Discussion: The findings from this research program will highlight if and how the iPOEG Toolkit is being used, and offer critical guidance to optimize the iPOEG Toolkit. It is hoped that this work will support the spreading of the message that it is time for children and adolescents with cancer to ‘move more’. 2.12. Effect of Physical Activity on Psychosocial Health among Adult Survivors of Childhood Cancer—The SURfit Study Wei H. Deng 1,2 , Simeon J. Zürcher 3 , Christina Schindera 4 , Ruedi Jung 5 , Helge Hebestreit 6 , Iris Bänteli 7 , Katja Bologna 8 , Nicolas von der Weid 4,† , Susi Kriemler 5,† and Corina S Rueegg 1,† 1 Oslo Centre for Biostatistics and Epidemiology, Oslo University Hospital, Oslo, Norway 2 Oslo Centre for Biostatistics and Epidemiology, Department of Biostatistics, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 3 Center for Psychiatric Rehabilitation, Universitäre Psychiatrische Dienste Bern (UPD) and University Hospital of Psychiatry and Psychotherapy, University of Bern, Bern, Switzerland 4 Department of Pediatric Hematology and Oncology, University Children’s Hospital Basel (UKBB) and University of Basel, Basel, Switzerland 5 Epidemiology, Biostatistics and Prevention Institute, University of Zurich, Zurich, Switzerland 6 Pediatric Department, University Hospital, Julius-Maximilians University, Würzburg, Germany 7 Department of Psychosomatic Medicine, University Hospital and University of Basel, Basel, Switzerland 8 Departement of Internal Medicine, Kantonsspital St. Gallen, St. Gallen, Switzerland † These authors contributed equally to this work. Backgound: Childhood cancer survivors (CCSs) are at elevated risk of experiencing fatigue, depression, and reduced quality of life. Low physical activity (PA) levels may worsen these. Very few randomized controlled trials have investigated the effect of PA on psychosocial health among CCSs. We investigated the effect of a one year individualized exercise intervention on fatigue, mental health, and health-related quality of life (HRQoL) in adult CCSs. Methods: We randomized 151 CCSs aged ≥16 years, <16 at diagnosis, and ≥5 years since diagnosis, identified through the Swiss Childhood Cancer Registry. The intervention participants received personalized exercise counselling to increase intense PA by ≥2.5 h/week for one year. Controls maintained usual PA levels. We assessed the severity of fatigue, psychological distress, and physical and mental HRQoL at baseline, 3, 6, and 12 months. Outcomes were transformed into T-scores (mean = 50, standard deviation (SD) = 10). We used generalized linear mixed-effects models with intention-to-treat (ITT, primary), as well as per protocol allocations with compliance based on self-reported PA or a cardiopulmonary fitness test. Results: The mean baseline outcomes ranged from T-score 49.8 to 52.2, of the 133 (88%) who completed the trial. ITT analyses found significantly lower severity of fatigue by T-score −3.56 (95% confidence interval (CI) −5.69 to −1.43, p = 0.001) in the intervention group compared to controls at 12 months. The physical component of HRQoL was significantly better than controls in the intervention group for per protocol analyses by 3.06 (95%CI 0.99 to 5.14, p = 0.004) and 3.54 (1.13 to 5.96, p = 0.004). Conclusions: Individualized exercise interventions may improve fatigue and physical HRQoL among adult long-term survivors of childhood cancer. This is despite no effects on psychological distress. 2.13. Exercise Tolerance and Physical Activity Short after Intensive Treatment in Patients with Childhood Cancer Miek Hornikx 1 , Anne Uyttebroeck 2 , Deveny Vanrusselt 2 , Charlotte Sleurs 2 , Marc Gewillig 3 and Sabine Verschueren 1 1 Department of Rehabilitation Sciences, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium 2 Department of Oncology, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium 3 Department of Cardiovascular Sciences, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium Background: Patients with childhood cancer are confronted with exercise intolerance (EI (VO 2 peak < 85% predicted)) after treatment, with a detrimental effect on quality of life and mortality. Knowledge about the limiting factor(s) for this EI and its relation to physical activity (PA) is essential in order to prescribe individually tailored rehabilitation and to stimulate physical and social reintegration. Methods: A total of 41 patients with childhood cancer (13 ± 3 years; 71% boys), diagnosed with leukemia/lymphoma (61%), a solid tumor (32%) or brain tumor (7%), and who had recently finalized their oncology-related treatment, were included in the study. Patients performed a maximal symptom-limited cardiopulmonary exercise test on a treadmill (4.8 km/h; +2% elevation/min). PA was recorded with a 3-axial accelerometer (Dynaport MoveMonitor, McRoberts, The Hague), that patients wore for 7 consecutive days. Active time (standing and walking), sedentary time, and steps were withheld. Results: Exercise tolerance (VO 2 peak: 29.7 ± 7.8 mL/min/kg (67 ± 16% predicted)) was markedly reduced in patients with childhood cancer compared to healthy peers. The majority of patients were peripherally limited (83%). A cardiac limitation was present in 71% of patients and was predominantly due to a reduced oxygen pulse (97%). Hyperventilation (32%) and a ventilatory limitation (12%) were less prevalent. The PA data of 13 patients were available (Active time: 178 ± 67 min/day; sedentary time: 515 ± 113 min/day; steps: 6411 [4458–6838]). Conclusions: Exercise tolerance is markedly reduced in patients with childhood cancer shortly after intensive treatment and this is mainly caused by the deconditioning of peripheral muscles and a reduced oxygen pulse. Further research is necessary to study the link with physical activity. 2.14. Implementation of a Low-Cost, High Impact Pediatric Exercise Oncology Program in Tanzania Manuel Ester 1,2 and Patricia Scanlan 2,3 1 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 2 Tumaini La Maisha, Tanzania 3 Department of Pediatric Oncology, Muhimbili National Hospital, Tanzania Background : Despite the physical and psychosocial benefits of exercise for children and adolescents living with and beyond cancer, few exercise oncology programs have been successfully implemented for this population. Implementation barriers herein include cost as well as a lack of qualified exercise professionals and appropriate facilities, which may be exacerbated in resource-limited countries. The purpose of this implementation project was to overcome these barriers and implement a sustainable exercise oncology program with Tumaini La Maisha (TLM) in Tanzania. Methods : Interdisciplinary stakeholder discussions were held between nurses, oncologists (PS), caregivers, TLM teachers, and the visiting exercise professional (ME) in order to provide a broad range of perspectives and agree upon a feasible exercise program for implementation at Muhimbili National Hospital. Cost barriers were addressed by using volunteers and paid TLM staff for program delivery, with the exercise professional educating staff on exercise principles. To address facility barriers, a public sports field served as the exercise space. Results : Discussions led to the development and implementation of a 30 min teacher-led, group- and family-focused, play-based exercise program. The program was delivered daily for 3 months, with high attendance and satisfaction from parents and children. Three TLM teachers were trained for long-term program delivery. Newly identified barriers included sun exposure risks (addressed via indoor exercise) and a lack of cultural relevancy (addressed by integrating traditional dance within the program). Conclusions : The involvement of stakeholders in the implementation planning and consideration of resource limitations led to the successful implementation and maintenance of an exercise oncology program in a resource-limited setting. 2.15. Network ActiveOncoKids as a National Implementation Approach for Exercise as Usual Care in Pediatric and Adolescent Oncology Miriam Götte 1 , Regine Söntgerath 2 , Gabriele Gauß 1 , Joachim Wiskemann 3 , Mirko Buždon 4 and Sabine Kesting 5,6 1 Department of Pediatric Hematology/Oncology, Clinic for Pediatrics III, West German Cancer Centre, University Hospital Essen, Essen, Germany 2 Department of Pediatric Oncology, Hematology and Hemostaseology, University Hospital Leipzig, Leipzig, Germany 3 Working Group Exercise Oncology, Division of Medical Oncology, University Clinic Heidelberg and National Center for Tumor Diseases (NCT), Heidelberg, Germany 4 Institute of Sports Medicine, Hannover Medical School (MHH), Hannover, Germany 5 Institute of Preventive Pediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 6 Kinderklinik München Schwabing, TUM School of Medicine; Department of Pediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany Background: Cancer and the acute and late effects of its treatment are associated with a decline of physical activity behavior in childhood cancer patients and survivors. Children have a legal right to exercise as well as to the active participation in physical activities, and to the positive effects of exercise-related benefits. Methods: Network ActiveOncoKids (NAOK) is a Germany-wide initiative with the main goal of enabling children, adolescents, and young adults with exercise opportunities during and after cancer treatment. The network uses and bundles the knowledge, expertise, and experience of decentralized clinical sites and working groups on the topic of “Exercise and Sport in Pediatric Oncology”. For this purpose, NAOK was founded in 2012, striving for networking and structuring. It is managed and accompanied by an overarching coordination, a steering group, and an advisory board. Its main aims are A) physical activity support for patients and families, B) policy change to establish structures and guidelines, and C) the generating of evidence through scientific projects. Results: NAOK brings together 33 pediatric oncology treatment centers that offer exercise interventions either during acute treatment and/or in follow-up care. The main focus of the last 2.5 years was to support children, adolescents, and young adults through individualized counselling, to help in implementing exercise programs at pediatric oncology centers, and to adapt and change medical and care structures (e.g., via guidelines and educational lessons). Conclusions: NAOK is an interdisciplinary network that supports the implementation of pediatric exercise oncology in usual care. In the past few years, great progress has been achieved in the areas of exercise implementation, structural organization, and communication, which might serve as a model for other countries. 2.16. Reh-PLAY Project: The Italian Rehabilitation Website for Children and Adolescents Affected by Oncoematological Diseases in the Pandemic COVID-19 Context Lucia Longo 1 , Annalisa Cornelli 2 , Riccardo Ruisi 3 , Francesca Rossi 4 , Filippo Gatti 5 , Federica Ricci 4 , Daniele Bertin 4 , Giulia Zucchetti 4 and Franca Fagioli 4,6 1 Associazione Unione Genitori Italiani Contro il Tumore dei Bambini ODV, Torino, Italy 2 Ospedale Papa Giovanni XXIII, Associazione ConGiulia ONLUS, Bergamo, Italy 3 Istituto Ortopedico Rizzoli, Bologna, Italy 4 A.O.U. Città Della Salute e Della Scienza, Presidio Ospedale Infantile Regina Margherita, Torino, Italy 5 Università degli Studi di Torino, Corso di Laurea in Terapia della Neuro e Psicomotricità dell’Età Evolutiva 6 Università degli Studi di Torino, Torino, Italy Background: With the onset of the COVID-19 pandemic in Italy and its related restrictions, the National Orders Federation of Technical, Rehabilitation and Prevention Health Professions, recommended remote rehabilitation interventions. Therefore, in April 2020, the rehabilitation working group of the Italian Hematology and Oncology Association (AIEOP) created a website for telerehabilitation within the “#IoMiMuovoACasa” (#IDoHomeTraining) pilot project. According to the preliminary safety and efficacy results, and users’ opinions, the need for a more structured website arose. The Reh-PLAY project aims to create a new website to support remote individualized rehabilitation interventions, in addition to the direct treatment, for the prevention/rehabilitation of motor difficulties related to the disease and/or to treatment side effects. Methods: The website contents are written according to the AIEOP Consensus Conference on Rehabilitation in Pediatric Oncohematology. Exercise videos created by physiotherapists are based on their expertise and are smartly accessible by the professionals who can create personalized exercise programs, available in a patient’s personal area. Results: In one month, twenty patients used the website. The Reh-PLAY website is smartly accessible for families, patients, and physiotherapists from the AIEOP website. Professionals can select texts/infographics about prevention and rehabilitation, and access 250 exercise videos, divided by type, age, and intensity. Patients can download information brochures, adhesion diaries, patients reporting outcome measures, and a questionnaire to improve the service. Conclusions : Growing evidence in Pediatric Oncohematology identifies rehabilitation as a fundamental aspect of treatment. The Reh-PLAY website improves continuity in rehabilitation management, supporting families and patients with telerehabilitation, and facilitating the dissemination of knowledge between professionals. 2.17. Lessons Learned from Innovating the Pediatric Cancer Patients and Survivors Engaging in Exercise for Recovery (PEER) Program to an Online Modality Due to COVID-19 Shaelene Standing 1 , Rachel McInnes 2 , S. Nicole Culos-Reed 3 , Mackenzie Murawsky 2,4 , Gregory M. T. Guilcher 5,6 , Fiona Schulte 5,6 and Carolina Chamorro Vina 2,3 1 Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2 Kids Cancer Care Foundation of Alberta, Calgary, AB, Canada 3 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 4 Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada 5 Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 6 Section of Oncology/Transplant, Alberta Children’s Hospital, Calgary, AB, Canada Background: The focus of the PEER program is to increase physical literacy, fitness, and to promote socialization for children affected by cancer and their siblings. In response to the COVID-19 pandemic, the program transitioned to an online format. The sessions, for children (6–11 years and 12–17 years), ran 1/week. Sport kits were provided to participants. Methods: After 12 months of running the program, which was attended regularly by 17 families (16 kids and 9 teens), we sought participant feedback through anonymous surveys. A total of 12 parent and 10 participant responses were collected outlining information about motivators for and barriers to online programming, safety, and satisfaction. Results: The families participated in the program mainly to (1) improve fitness and (2) increase peer connections. Of these, 100% felt safe, 95% agreed that sport kits increased engagement, and most agreed that sport kits also helped kids increase their activity level after participation in the PEER sessions. Furthermore, 86% felt that 45–60 min was a good session duration and that participation in the program increased their child’s fitness level. Conclusions: This program was (i) engaging, (ii) safe, and (iii) accessible for children affected by cancer who are medically vulnerable and/or remotely located. We suggest a program length of 45–60 min based on the age group. Exercises should have a greater focus on physical literacy and fundamental actions for younger age groups and should be more fitness-based for teens. Fun and the inclusion of socialization are key elements in increasing enjoyment, adherence, and community belongingness. The provision of a sport kit is recommended. Individualized adaptations are necessary for program success. 2.18. Promoting Positive Physical Activity Behaviours in Children and Adolescents Undergoing Acute Cancer Treatment: Feasibility of the CanMOVE Intervention Sarah L. Grimshaw 1,2 , Nicholas F. Taylor 1 , Rachel Conyers 2 and Nora Shields 1 1 La Trobe University, School of Allied Health, Human Services and Sport, Bundoora, Melbourne, VIC 3086, Australia 2 Murdoch Children’s Research Institute, Parkville, Melbourne, VIC 3052, Australia Background: Supporting children/adolescents with cancer to be more physically active has the potential to improve short- and long-term outcomes. The objective of this study was to assess the feasibility of CanMOVE, a complex, theoretically informed, behaviour change intervention to promote participation in physical activity for children/adolescents undergoing acute cancer treatment. Method: A feasibility study utilising single-group, repeated measures, and a mixed methods design was completed. Participants completed the 10 week CanMOVE intervention, which involved structured support from a physiotherapist and the provision of a Fitbit (child and parent). Feasibility domains of demand, acceptability, implementation, practicality, limited efficacy, and integration were evaluated. Objective assessments of physical activity, physical function, and health-related quality of life were completed. Qualitative data were collected via semi-structured interviews with participants (parents and children/adolescents) and focus groups with clinical staff. Results: Twenty families completed CanMOVE, including children/adolescents (median age 12 years, range 5–16) with a mix of cancer diagnoses. There was a high demand for CanMOVE with a 95% enrolment rate. CanMOVE was acceptable from both participant and staff perspectives. All feasibility thresholds set for implementation were met. There were no serious adverse events. Under limited efficacy, data indicate that CanMOVE shows promise in influencing child/adolescent physical activity behaviour. Positive impacts were also seen in parent and staff behaviour towards physical activity promotion. The pre/post physical function and HRQOL assessments showed positive trends. Conclusions: CanMOVE is feasible and safe to implement in the paediatric oncology setting. CanMOVE shows potential in influencing the behaviour of children/adolescents and of the people in their social and professional support networks. These findings can be used to inform services in the paediatric cancer setting to ensure physical activity promotion is a considered and prioritised aspect of clinical care. Abstract Poster Presentations 2.19. Maximal Activity; Health Care Program for Children with Cancer during Hospitalization F V. Engels, D Agterberg, W. P. Bekkering and Patrick van der Torre Department of Sports and Exercise Center, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, The Netherlands Background: International studies and organizations, such as the World Health Organisation, have identified the health risks across lifespan that are associated with physical inactivity. Childhood cancer and its treatment have considerable impact on a child’s physical and mental wellbeing and often lead to reduced physical activity levels and sedentary behaviour. The combination of long-term chemotherapy, surgery and/or radiotherapy as administered in children with cancer especially impairs physical activity and fitness, both during and after therapy. Physical activities are important for the development of children and increasing evidence suggests the beneficial effects of physical activity promotion during cancer treatment as well. Therefore, ways to promote physical activity and exercise are becoming an important part of children’s cancer treatment. By means of our “Maximal Activity” program in the Princess Máxima Center for Pediatric Oncology, we want to encourage our patients to get out of bed and be as active as possible, both during and after treatment. Methods: A qualitative survey of 30 individuals (children, families and health care professionals) was used to ask about the experiences of the developmentally appropriate care program, of which ‘Maximal Activity’ is a part. Results: An important point that emerged was that the facilities and environment invite physical activity. The program was named by many as valuable. Trust and confidence in physical activity increased during hospitalization. Other results still need to be analysed and will be presented at the congress. Conclusions: Maximal Activity is a health program that contributes to a stimulating environment for physical activity and is appreciated by all stakeholders. 2.20. Assessing the Physical Activity Behaviour of Parents Whose Children Have Cancer before and during Intensive Cancer Treatment Carolin Ohnmacht and Alexander Puzik Department of Pediatric Hematology and Oncology, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Mathildenstraße 1, D-79106 Freiburg, Germany Background: Regular physical activity (PA) is essential for biopsychosocial health, while reduced PA during childhood cancer treatment leads to increased late effects in affected children. Moreover, cancer and its treatment determine the everyday lives of families and parents who spend plenty of time with their children in hospital. Thus, it can be assumed that the parents’ PA is significantly affected during cancer treatment. Meanwhile, parents’ PA-behaviour has a pronounced influence on their children’s behaviour. The aim of this project is to investigate the parents’ PA-behaviour before and during their children’s cancer treatment. Methods: The PA and sedentary behaviour of the parents were assessed before and during their children’s intensive oncological therapy in a cross-sectional design using the International Physical Activity Questionnaire (IPAQ-SF). Results: A total of 40 parents of children with cancer took part in this survey. They were interviewed no earlier than their children’s second inpatient stay. The parents’ PA levels before diagnosis were in line with the reference values for healthy adults in Germany. During the children’s treatment, all dimensions of the parents’ daily PA, and the number of minutes of PA per week, decreased significantly ( p < 0.001). The greatest reduction in PA was identified during inpatient stays, with a significant increase in sitting time ( p < 0.001). Conclusions: This is the first study to show that the PA of parents whose children have cancer decreases significantly during cancer treatment. As parents’ PA behaviour significantly affects that of their children, even after completion of cancer treatment, future exercise programs in pediatric oncology should include parents in order to reduce inactivity-related late effects. 2.21. Get Strong to Fight Childhood Cancer: An Exercise Intervention for Children and Adolescents Undergoing Anti-Cancer Treatment (FORTEe) Sandra Stöessel 1 , Elias Dreismickenbecker 1 , Marie A. Neu 1 , Lisa Ploch 1 , Norbert Paul 1 , Christian Ruckes 1 , Adriana Balduzzi 2 , Francesca Lanfranconi 2 , Peter Wright 3 , Stan Windsor 3 , Joachim Wiskemann 4 , Inaam El-Rajab 4 , Veronika Picmanova 5 , Céline Gravot 5 , Rodolf Mongondry 6 , Wilhelm Bloch 7 , Katie Rizvi 8 and on behalf of Youth Cancer Europe, Martin K. Fridh 9 , Alejandro Lucia 10 , Carmen Fiuza-Luces 10 , Miriam Götte 11 and on behalf of Network ActiveOncoKids, Filippo Spreafico 12 , Barbara Konda 13 , Lidija Kitanovski 14 , Lena Werthmann 15 , Tobias Baader 16 and Jorg Faber 1 and on behalf of the FORTEe Consortium 1 University Medical Center of the Johannes Gutenberg, University Mainz (DE) 2 Fondazione Monza e Brianza per Il Bambino e La Sua Mamma (IT) 3 Oxford Brookes University (UK) 4 Heidelberg University Hospital (DE) 5 Concentris Research Management GmbH (DE) 6 Centre de Lutte Contre le Cancer Léon Bérard (FR) 7 German Sport University Cologne (DE) 8 Youth Cancer Europe (RO) 9 Department of Pediatric and Adolescent Medicine, Copenhagen University Hospital, Copenhagen, Denmark (DK) 10 Universidad Europea de Madrid (ES) 11 Essen University Hospital (DE) 12 Fondazione IRCCS Istituto Nazionale dei Tumori (IT) 13 Forma 3D Ltd. (SI) 14 University Medical Center Ljubljana, Division of Pediatrics, Department of Haematooncology (SI) 15 Nurogames GmbH (DE) 16 Pixformance Sports GmbH (DE) Background: Cancer is the leading cause of death by non-communicable diseases in children in Europe. During cancer treatment, patients’ morbidity is increased due to physical inactivity and cancer-related fatigue. Precision-based exercise training programs in children and adolescents attending the intensive phases of cancer treatment is an increasingly promising therapy. However, strong evidence for exercise efficiency is lacking in paediatric oncology and, thus, precision exercise training is not part of standard care and does not reach the majority of patients. Methods: The FORTEe project is structured in seven work packages and intends to evaluate a personalised and standardised exercise intervention in 450 children, adolescents and young adults undergoing cancer treatment in 9 centres across Europe. The randomised, controlled FORTEe trial aims to generate high evidence for an innovative, patient-centred exercise treatment. Supervised exercise training intends to impact the efficiency of systems involved in the oxidative metabolism chain, including the skeletal muscle. The tailored training is also focused on strength in order to counteract muscular atrophy. Within the project, digital and innovative technologies (a FORTEe app, an augmented reality program and an interactive digital training) will be developed and applied to make the exercise training more attractive, age-adapted and inspirational. FORTEe will stimulate multidisciplinary research by involving paediatricians and exercise scientist in order to provide more inclusive access to paediatric exercise oncology. Conclusions: Progressing beyond the current state-of-the-art standard, FORTEe has the ambition of implementing paediatric exercise oncology as an evidence-based standard in clinical care for all childhood cancer patients worldwide. 2.22. Therapeutic Exercise in Children Diagnosed with Solid Tumors: A Scoping Review Brooke E. Kohler, 1 Emmah Baque 1,2 , Carolina X. Sandler 3,4,5 and Stewart G. Trost 1,6 1 Faculty of Health, Queensland Centre for Children’s Health Research, Queensland University of Technology (QUT) Brisbane, Brisbane, QLD, Australia 2 School of Health Sciences and Social Work, Griffith University, Nathan, QLD, Australia 3 UNSW Fatigue Research Program, Kirby Institute, University of New South Wales, Sydney, NSW, Australia 4 School of Sport and Exercise Science, School of Health Sciences, Western Sydney University, Sydney, NSW, Australia 5 Menzies Health Institute Queensland, Griffith University, Griffith, QLD, Australia 6 School of Human Movement and Nutrition Sciences, University of Queensland, Brisbane, QLD, Australia Background: Increasing survival rates for children with solid tumors present an ongoing challenge regarding how to maximize the quality of survivorship and effectively manage the short- and long-term complications of the disease and its treatment. To gain a better understanding of the research pertaining to therapeutic exercise programs, we conducted a scoping review of exercise training studies conducted in children diagnosed with solid tumors. Method: A systematic literature search was performed across four electronic databases. Articles were selected for full-text review if they included participants diagnosed with a solid tumor, if at least 50% of the participants were aged ≤21 years, if they evaluated an exercise program ≥ 2-weeks in duration, and if they were published in an English, peer-reviewed journal. Results: Of the 6648 citations identified, 14 articles met the inclusion criteria. Only three studies were conducted as randomized controlled trials. The duration of the exercise programs ranged from 3 to 40 weeks and their frequency ranged from 3 to 11 sessions per week. Exercise session duration ranged from 15 to 180 min, with most articles reporting 30–90-min sessions. A range of exercise modalities were employed, including ergometry, resistance training, yoga, and active videogaming. Adherence ranged from 77% to 100%, with no adverse events reported. Exercise training resulted in improvements in cardiorespiratory fitness, functional strength, physical activity, and quality of life. Conclusions: A small number of mostly low-quality studies have evaluated therapeutic exercise in pediatric survivors of solid tumors. Although limited, the extent of research supports the feasibility and safety of therapeutic exercise and suggests that therapeutic exercise may be potentially effective in improving a range of health outcomes. 2.23. Preventing Sensory and Motor Dysfunctions in Children Receiving Neurotoxic Chemotherapy—Overview of the Current Therapy Options and Study Protocol of the PrepAIR Randomized Controlled Multi-Center Trial Clémentine Bischoff 1 , Nicolas Von der Weid 2 , Oliver Faude 1 , Fiona Streckmann 1,3 and on behalf of the PrepAIR Study Group † 1 Department of Sport, Exercise and Health, University of Basel, Grosse Allee 6, 4052 Basel, Switzerland 2 Department of Pediatric Oncology and Hematology, University Children’s Hospital Basel, University of Basel, Spitalstrasse 33, 4056 Basel, Switzerland 3 Department of Oncology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland † PrepAIR Study Group: Katrin Scheinemann (KS Aarau), Uta Tacke (UKBB), Jochen Rössler (Insel Bern), Pablo Hernaiz Driever (Charité Berlin), Miriam van Buiren & Alexander Puzik (Uniklinik Freiburg, Jeanette Greiner (KISPI St. Gallen), Aline Rehm & Patrizia Brunner (Universität Basel), Valentin Benzig & Regula Everts (Universität Bern), Anja Wehrle (Universität Freiburg), Claudia Bucher (KS Aarau), Sandra Frauchiger (Insel Bern), Carolin Ohnmacht (Uniklinik Freiburg), Martina Sigg (KISPI St. Gallen). Background: Modern therapy improved survival for children with cancer. However, the treatment has unintended consequences. Depending on neurotoxic agents, 52–100% of children develop a chemotherapy-induced peripheral neuropathy (CIPN). The severe symptoms such as loss of sensation, numbness, pain, absent reflexes and loss of balance control, not only delay motor development milestones such as walking, running, jumping, or climbing, diminishing children’s quality of life and affecting their social reintegration, but are also of high clinical relevance. Streckmann et al. confirmed in their meta-analysis that recovery is poor, but there is a clear benefit for adults in favor of sensorimotor training (SMT) to target the symptoms of CIPN. Methods : In our RCT, we will recruit N = 131 children from 6 centers (Switzerland and Germany). Immediately after being scheduled to receive neurotoxic chemotherapy, the intervention group will perform a standardized, age-adjusted, specific playful SMT program twice a week for the duration of their medical therapy, while the control group will receive treatment as usual. Results : For the intervention, we created a training manual which will finally lead to a simple therapy option for children suffering from CIPN. The manual provides a basis for playful SMT based on a modular system, which leaves the therapists and the children room for scope and still respects all the training modalities within the background of “specific exercise is medicine”. Conclusions: We hypothesize that children in the intervention group will develop fewer symptoms of CIPN and will be able to maintain their motor and sensory functions for an age-appropriate motor development. 2.24. Impact of the COVID-19 Pandemic on the Availability of Exercise Programs in Pediatric Oncology: A Survey of Providers in Germany Dominik Gaser 1,2 , Christiane Peters 2 , Renate Oberhoffer-Fritz 2 , Miriam Götte 3 , Gabriele Gauß 3 , Irene Teichert-von Lüttichau 1 and Sabine Kesting 1,2 1 Department of Pediatrics and Children’s Cancer Research Centre, Children’s Hospital München Schwabing, TUM School of Medicine, Technical University of Munich, Munich, Germany 2 Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 3 Department of Hematology and Oncology, Clinic of Pediatrics III, West German Cancer Centre Essen, University Hospital, Essen, Germany Background: An increasing amount of in-hospital and out-patient physical activity offers facilitate access to professional exercise programs for children and adolescents during acute anticancer treatment and surveillance. The COVID-19 pandemic has presented major challenges to hospitals and rehabilitation facilities. An online survey among providers in Germany investigates the impact of the pandemic on individual sectors of exercise programs in pediatric oncology. Methods: From 19 January until 9 February 2022, all German clinics and institutions with an exercise program for pediatric cancer patients and/or survivors are invited to participate in an online survey. Methodology and recruitment is conducted in cooperation with Network ActiveOncoKids. Limitations, challenges and measures for adapting offers in the context of the individual pandemic waves, are collected. In addition, challenges for the implementation of scientific studies are analyzed. Results: Thirty-three sites have been requested to participate in the survey. We assume that exercise professionals and scientists have used the pandemic-related challenges to modify the existing concepts of exercise promotion and adapt them to the specific local conditions. Conclusions: We expect new ideas and approaches for the realization of exercise programs under pandemic conditions. The extension of digital offers may improve the access for children and adolescents to exercise programs in pediatric oncology in the long term and, therefore, could potentially be adopted into standard exercise care. 2.25. A Review to Map the Evidence of Physical Activity Interventions in Post-Treatment Adolescent and Young Adult Cancer Survivors Maxime Caru 1,2 , Ariane Levesque 3 , Pooja Rao 1 , Smita Dandekar 1 , Christopher Terry 4 , Valerie Brown 1 , Lisa McGregor 1 and Kathryn Schmitz 2 1 Department of Pediatric Hematology and Oncology, Penn State College of Medicine, Hershey, PA, USA 2 Department of Public Health Sciences, Penn State College of Medicine, Hershey, PA, USA 3 Department of Psychology, University of Montreal, Montreal, QC, Canada 4 Department of Medical Oncology, Sidney Kimmel Cancer Center—Thomas Jefferson University, Philadelphia, PA, USA Background: AYA cancer survivors are a population that have unmet needs, and researchers believe that physical activity (PA) interventions can address many of these needs. This review describes and synthesizes previously reported data in order to map the current evidence as well as to identify gaps in knowledge and future research needs. Methods: A search of the literature was conducted in PubMed, CINAHL, EMBASE, Web of Science and Cochrane Library. This review followed the PRISMA-ScR statement. We included all original studies investigating PA interventions in post-treatment AYA cancer survivors who were between 15 and 39 years old at the time of their initial cancer diagnosis. Results: A total of 8 studies were included in this review and showed that PA interventions were feasible and acceptable in AYA cancer survivors. Overall, PA interventions were individualized and mainly aerobic in nature. Studies examining the effects of PA interventions on AYA cancer survivors’ health evaluated physical and mental health outcomes, including, but not limited to, physical functioning, functional capacity, occupational performance, health-related quality of life, physical capacities, fitness, mood, and self-perception after cancer treatments. Conclusions: Our review maps the current evidence of PA interventions and highlights the paucity of data in this area of investigation, obviating how much work remains to be carried out to demonstrate the potential benefits of PA on AYA cancer survivors’ health outcomes. Future studies should consider the development PA interventions that address patients’ clinical needs, in addition to providing a detailed description of PA interventions. 2.26. Sensorimotor Training—Therapeutic Potentials and a Child-Specific Training Concept for Pediatric Cancer Patients Sarah Otten 1 , Clémentine Bischoff 2 , Julia Däggelmann 1 , Fiona Streckmann 2,3 , Wilhelm Bloch 1 and Vanessa Oschwald 1 1 Institute for Cardiovascular Research and Sports Medicine, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany 2 Department of Sport, Exercise and Health, University of Basel, Birsstr. 320B, 4052 Basel, Switzerland 3 Department of Oncology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland Background: A sensorimotor training (SMT), mostly applied as balance training on different surfaces and in different positions, has the potential to contribute to the nervous system’s plasticity and to improve lower extremity impairments. In recent years, SMT has not only been conducted in rehabilitation, injury and fall prevention, but has also been successfully applied in adult oncology. In this context, studies showed improvements in common lower extremity impairments, such as impaired balance control, sensory and motor symptoms. While SMT or associated training modalities have been investigated in different pediatric patient collectives, they have rarely been conducted in pediatric oncology, though the outlined effects are promising for these patients. Methods: In order to identify therapeutic potentials of SMT for pediatric oncology, current data on SMT in different pediatric patient collectives are reviewed. Furthermore, to gather preliminary insights on a playful and child-specific SMT in pediatric oncology, first pilot studies during and after inpatient medical treatment in this field are conducted. Results: SMT resp.-related training modalities with various pediatric patients demonstrate potential effects on lower extremity parameters. The preliminary results in pediatric oncology indicate that a child-specific and playful SMT for children after cancer treatment is feasible. Motivating sensorimotor exercises are identified. Further study results on SMT performed during acute pediatric oncological therapy will be available and presented at the congress. Conclusions: The reviewed interventions in pediatric collectives, and preliminary study results in pediatric oncology in particular, suggest that SMT might be a promising and targeted training modality supplementing exercise therapy for children with cancer. 2.27. Dancing Can Improve Perceived Psychosocial Wellbeing and Coping during in-Patient Admissions on a Paediatric Cancer Ward Rachael Keating 1 , Eilis O’Shea 2 and Susan Hurley 3 1 Physiotherapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland 2 Occupational Therapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland 3 Music Therapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland Background: The “Tuesday Boogie” is a quality improvement initiative which was developed to promote physical activity and enhance psychosocial wellbeing amongst in-patients, parents and staff on an in-patient paediatric cancer ward. It involves children, parents and staff engaging in a 15 min group dancing session and runs on a weekly basis. We conducted a service evaluation to assess the feasibility of the dance-based intervention and whether it influenced the psychosocial wellbeing of participants. Methods: Child, parent and staff questionnaires were designed by the research team to evaluate the intervention. Both quantitative (Likert, multiple choice, dichotomous) and qualitative open format questions were included. Data were collected on a voluntary, anonymous basis from June to July 2021. Results: In total, 39 questionnaires were completed ( n = 4 child, n = 9 parent, n = 26 staff). A total of 97% of respondents had taken part in the intervention with 100% reporting the intervention was worthwhile. Of the respondents, 95% felt taking part improved their mood and 92% reported it helped them to cope with being in hospital. Furthermore, 77% of respondents felt less worry or stress during the intervention. The following qualitative data were collected: C1 “It is nice to be happy and dance instead of worrying”, P3 “Changes the energy…real mood shifter and connector”, P9 “M gets a chance to have fun and to interact with other kids which is so rare these days”, S22 “Shows patients that not everything that happens in hospital is scary”. Conclusions: A dance-based intervention for children, parents and staff on an in-patient paediatric cancer ward is feasible and has numerous psychosocial benefits. 2.28. The Potential of Ambulatory Assessment for Exercise Oncology Anna Vogelsang 1,2 , Corinna Meyer-Schwickerath 3,4 , Joachim Wiskemann 3,4 and Markus Reichert 1,5,6 1 Department of eHealth and Sports Analytics, Faculty of Sport Science, Ruhr-University Bochum, Bochum, Germany 2 Faculty of Humanities, MSH Medical School Hamburg, Hamburg, Germany 3 University of Heidelberg, Institute of Sports and Sports Sciences, Heidelberg, Germany 4 Department of Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany 5 Mental mHealth Lab, Department of Sport and Sport Science, Karlsruhe Institute of Technology, Karlsruhe, Germany 6 Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany Background: The growing population of childhood cancer survivors is at risk of developing late chronic conditions and premature mortality. Physical activity (PA) can mitigate physical and psychological late effects, but only if engaged in regularly over sustained time periods. PA sustainment, however, remains challenging due to daily and within-daily fluctuations in how survivors feel, with whom they interact and in which environments they live. Thus far, exercise psychology has mainly focused on macro-temporal processes unfolding over weeks and months. To promote sustainable PA engagement, micro-temporal processes (e.g., unfolding over minutes, hours, days) need to be studied. Therefore, this review introduces ambulatory assessment as a method to gain insights into dynamic behavioural processes as they unfold in survivors’ everyday lives. Methods: Ambulatory assessment (AA) describes a group of computer- or smartphone-assisted methods to study behavioural, biological, and psychological processes in survivors’ everyday lives near real time. In this review, we discuss the characteristics of AA and expand on its promise for researching micro-temporal behavioural processes and on its potential for exercise oncology, such as the prediction of critical phases of PA relapse. This is exemplified in ongoing projects, and we expand on future avenues where ambulatory interventions might benefit survivors’ tailored care. Conclusions: Insights into dynamic behavioural processes as they unfold in everyday life may critically add to our understanding of sustainable PA and the development of individualised treatment where and when it is needed. Avenues for research, prevention and treatment will be discussed as well as the acceptability, compliance and ethical issues of AA. 2.29. Factors Related to Rehabilitation Adherence in Childhood Cancer: A Review Lynn R. Tanner 1,2 and Erica N. Schorr 1 1 School of Nursing, University of Minnesota, Minneapolis, MN, USA 2 Physical Medicine & Rehabilitation, Children’s Minnesota, MN, USA Background: Children and adolescents with cancer receive rehabilitation interventions for the functional impacts of the disease and its corresponding treatment. Adherence to these interventions varies greatly. The purpose of this review was to identify the factors related to adherence in rehabilitation. Methods: A systematic review was conducted using Ovid Medline and CINAHL databases with search terms related to pediatric cancer, rehabilitation, and adherence. Study eligibility criteria included the following: English language, participants receiving a physical therapy, occupational therapy, speech-language pathology, cognitive or exercise intervention or service, mean age of ≤18 years old, and measurement of factors related to adherence. The PRISMA 2020 statement for reporting systematic reviews guided data synthesis. The study quality was assessed using the Joanna Briggs Institute Critical Appraisal Tools. Results: The review included 13 studies providing rehabilitation interventions (exercise, yoga, walking, cognitive training, and vibration plate) to 283 children aged 3–19 years with adherence levels of 61–91% measured by session attendance. The majority (85%) of the studies comprised exercise interventions with 69% of the interventions being multifaceted in nature including aerobic, strengthening, and flexibility components. The factors related to adherence fell into three categories: (1) organizational; (2) condition-related; and (3) personal. Common barriers included fatigue, illness, time, family scheduling, and motivation. Facilitators included peer or caregiver support and supervision. Conclusions: The existing literature on rehabilitation adherence focuses mostly on exercise interventions delivered by a multitude of health care professionals. More research is needed to improve understanding of the factors related to adherence to rehabilitation interventions and services in survivors of childhood cancer.
Sabine Kesting 1,2 and Kinderklinik München Schwabing 1 TUM School of Medicine, Department of Paediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany 2 Institute of Preventive Paediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany The adverse effects of initial anti-cancer treatment already emerge early after diagnosis and contribute to increased physical inactivity in combination with the clinical environment and psychological burden. These circumstances can instantly result in reduced physical function, impaired activities of daily living and negatively influence health-related quality of life. Thus, the question is raised: how early should we start applying exercise interventions during acute treatment and which aspects should be considered? Experiences from a clinical perspective show a sudden interruption of normal physical activity habits immediately after hospitalization due to disease-related impairments, general uncertainty and necessary clinical diagnostics. The approaches to implement exercise programs during this sensitive phase of treatment comprise qualified and experienced staff, individual adaption of content and intensity levels, motivating and empathetic communication, creativity and flexibility. The primary objectives are the stabilization of physical activity and fitness to reduce the risk of impairments and accustoming to the new level of physical activity and possibilities to be physically active. Despite the known challenges and difficult circumstances, exercise interventions should be applied shortly after diagnosis considering the burdensome situation and the patient’s clinical status. Moreover, key components of planning and implementing exercise early are regular communication, providing information, including the patient’s wishes and concerns, to build trust and maintain physical activity levels throughout treatment. Open research questions include the effects of medication on physical function early during treatment, e.g., initial steroid application, and suitable interventions.
Maxime Caru Department of Pediatrics, Division of Hematology and Oncology, Penn State College of Medicine, Hershey, PA, USA Children’s and adolescents’ exposure to chemotherapeutic agents causes several long-term adverse effects affecting their cardiac health due to cardiotoxicity, physical function, and ultimately their quality of life. Physical activity is an effective strategy to prevent long-term adverse effects whether during or after treatments in pediatric oncology. Nevertheless, very few pediatric patients diagnosed with cancer meet the physical activity guidelines. Hence, less than one in two patients are doing 60 min or more of moderate-to-vigorous-intensity physical activity each day. Considering that they are physically inactive and exposed to chemotherapy-related cardiotoxicity, this has the consequence of reducing their cardiorespiratory fitness (aerobic capacity). This invited talk gives an overview of the cardiorespiratory fitness state of children and adolescents diagnosed with cancer and its long-term impact on their survivorship. In the end, the audience should be able to understand the mechanism behind the decrease in childhood cancer survivors’ cardiorespiratory fitness, identify what is needed next, and recognize clinical perspectives for this unique population.
Amanda Wurz School of Kinesiology, University of the Fraser Valley, Abbotsford, BC, Canada Qualified exercise professionals (QEPs) play a vital role in physical activity delivery, yet there are no agreed-upon training requirements to ensure the competence of PQEPs within pediatric exercise oncology. This talk will discuss this gap, provide a case example of a QEP training protocol, and offer an opportunity for critical dialogue. Ultimately, it is hoped this talk will be a first step towards creating standardized QEP training requisites in this field.
Christina Schindera No abstract available.
Martin Kaj Friedh No abstract available. Abstract Oral Presentations
Peter Weeber 1 , Sabine Kesting 2,3 , Martin Schönfelder 1 , Henning Wackerhage 1 and Irene von Luettichau 2 1 Exercise Biology, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 2 Kinderklinik München Schwabing, TUM School of Medicine, Department of Paediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany 3 Institute of Preventive Paediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany Background: Low-and moderate-intensity exercise is safe and feasible during childhood cancer treatment. However, is this also true for a bout of high-intensity interval training (HIIT)? The aim of this study is to find out whether HIIT is feasible in childhood cancer patients and to measure the cardiovascular and metabolic response to HIIT. Methods: Children with lymphoma, leukaemia, rhabdomyosarcoma, nephroblastoma, and synovial sarcoma performed ten 15 s high-intensity intervals (>90% of estimated HRmax) with 1 min active recovery on a bicycle ergometer in between. In addition to assessing the safety and feasibility of HIIT, we also recorded the perceived exertion rate, heart rate, lactate, and adrenaline concentrations. Results: A total of 11 patients at the age of 13.9 ± 3.6 years ( n = 7 female) completed a bout of HIIT without serious adverse events. The patients reached a BORG value maxima of 16 ± 1.2 during exercise and heart rate increased from 78 ± 17 bpm at rest to 178 ± 12 bpm after exercise (90 ± 6% of the predicted maximal heart rate). The power-to-weight average ratio during exercise was 2 ± 0.5 W/kg. Blood lactate concentrations increased from 1.09 ± 0.50 mmol/L at rest to 5.05 ± 1.88 mmol/L post-exercise. Adrenaline levels increased only marginally post-exercise. No adverse events were observed. Conclusions: Our study shows that HIIT is feasible for a small number of physically fit childhood cancer patients but not for patients with a low fitness level or complications.
Marcella Burghard 1,2 and Tim Takken 1,2 1 Child development and Exercise Centre, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, Utrecht, The Netherlands 2 Sports and Exercise Center, Princess Máxima Center for Pediatric Oncology, Utrecht, The Netherlands Background : It is known that cardiorespiratory fitness (CRF), measured as oxygen uptake relative to body weight (VO 2 peak/kg), is often decreased in both pediatric and adult cancer survivors. Research is unambiguous regarding a lower cardiac output as a possible cause for this impairment. Our aim was to assess if, next to CRF, there are differences in maximal cardiac output index (COI) response, including stroke volume index (SVI) and heart rate (HR) at maximal exercise, compared to healthy controls. Methods : A total of 30 young cancer survivors (mean age 14.4 ± 4.1 years) and 16 healthy controls (mean age 14.7 ± 4.5 years) performed a cardiopulmonary exercise test to measure CRF and other CPET-derived variables. COI and SVI were measured using thorax impedance measurements (Physioflow ® ). Results : CRF (VO 2 peak/kg in percentage predicted) was significantly lower ( p = 0.000) in the young cancer survivors (71.5; 63.5–82.6%) compared to the healthy controls (90.0; 84.0–95.0%). COI ( p = 0.170) and peak HR ( p = 0.914) did not differ significantly between groups, however SVI was significantly lower ( p = 0.013) in the cancer survivor group (52.2 ± 12.8 mL/m 2 ) compared to the healthy control group (61.6 ± 9.0 mL/m 2 ). Conclusions : CRF is decreased after cancer treatment in young cancer survivors. A decreased cardiac stroke volume might be one of the limiting factors for CRF.
Ronja Beller, Gabriele Gauß, Dirk Reinhardt and Miriam Götte Department of Pediatric Hematology/Oncology, Center for Child and Adolescent Medicine, Clinic for Pediatrics III, West German Cancer Centre, University Hospital Essen, Essen, Germany. Background: Children and adolescents should have access to exercise programs throughout cancer trajectory, including during palliative care. There is only very limited experience and data available for this phase of life. An exercise project has been conducted at the University Hospital Essen for advanced cancer patients who participated in supervised exercise programs during neoadjuvant and/or adjuvant treatment, and have bonded with these programs and their exercise professionals. Methods and Results: Inclusion criteria are diagnosis of advanced cancer, age >3 years, previous participation in the hospital’s exercise program, and residence in the surrounding area of the hospital (<60 min car drive). Inclusion is discussed with the Specialized Outpatient Palliative Care Team of the clinic to consider the health status and family peculiarities. After deciding on the feasibility and gaining the acceptance of the families, the assigned exercise physiologist offered sessions once a week for 30–90 min. Sessions were usually scheduled at the patients’ homes, but also in in- and out-patient clinics if patients had appointments or complications. The contents were a combination of strength, endurance, coordination, body awareness, and mobility training that were resource-oriented, related to the child’s interests, and adapted to their respective daily condition. Data monitoring was conducted every four to six weeks. Conclusions: This concept takes into account the individual possibilities, goals, and interests of those directly affected. Since the project is not yet part of standard care, it should be evaluated and expanded. Pediatric advanced cancer patients should have the opportunity to participate in exercise programs, and benefit from physical activity support.
Alice Aparecida Rodrigues Ferreira Francisco 1,2 Jader Brito Ramos dos Santos 1 , Otávio Augusto Soares Machado 1,3 and Karen W. Wonders 1,2 1 Maple Tree Cancer Alliance Brazil, Sorocaba, Brazil 2 Maple Tree Cancer Alliance, Dayton, OH, USA 3 School of Physical Education from YMCA, Sorocaba, Brazil Background : Research has already shown that physical activity (PA) during childhood means a higher probability of maintaining active lifestyles. Therefore, it diminishes the severity of the adverse effects of cancer treatment and is a safe tool. Maple Tree Cancer Alliance (MTCA) is a non-profit organization helping cancer patients through exercise rehabilitation since 2011. Founded by Dr. Karen Wonders, in 2019 MTCA started serving patients in Brazil. Maple Kids (MK) started in 2021 in Brazil at the Childhood Cancer Research and Assistance Group Hospital (GPACI), a center attending 48 cities in the Sorocaba region. Methods : MK is an exercise program for pediatric cancer patients, to improve quality of life and overall health. We also include family participation, inspiring a healthy lifestyle for all. The main program goals are: to improve cardiac and pulmonary capacity, boost immune system, reduce fatigue, and peripheral neuropathy. Its secondary goals are to reduce psychological and cognitive problems, and to reduce second tumors and chronic diseases. Results : Maple Kids starts in January 2022, and results will soon be posted. Conclusions : Some children face debilitating physical and cognitive problems as side effects of cancer treatments. Cancer can mean not running or jumping, and feeling uncomfortable during PA. There is a lack of accessible and specialized programs, of staff prepared to support cancer patients, and a lot of misinformation from family and health professionals, which means that 62% of adult survivors have at least one chronic health condition. PA for pediatric cancer patients and survivors means better physical and psychosocial health.
Helena Koine 1 , Isabell Hamm 1 , Anja Chevalier 2 and Anna Vogelsang 3,4 1 Institute of Psychology, German Sport University Cologne, Cologne, Germany 2 Department of eHealth and Sports Analytics, Ruhr-University Bochum, Bochum, Germany 3 Department of Sport Economics and Sport Management, German Sport University Cologne, Cologne, Germany 4 Faculty of Humanities, MSH Medical School Hamburg, Hamburg, Germany Background: Adolescents and young adult (AYA) cancer survivors face an increased risk of physical, psychological and psychosocial late effects, long-term morbidity, and premature death. Despite the well-documented effectiveness of PA in mitigating treatment-related late effects, most adolescent and young adult (AYA) cancer survivors face various physical, behavioural, psychological, and educational barriers, which hinder them in adopting a physically active lifestyle. The current study takes survivors’ barriers into account and examines the effects of a four week online intervention on physical activity and social cognitive predictors of physical activity in AYA cancer survivors compared to a waiting control group. It is hypothesized that the physical activity levels and social cognitive predictors increase in the intervention as opposed to the control group. Methods: A randomized controlled trial with repeated measures including an intervention ( n = 30) and awaiting control group ( n = 31) was performed. The four week online intervention was developed based on the Health Action Process Approach and the Self-Concordance Model and included a baseline and post-test. Results: Significant interaction effects between time and treatment revealed greater physical activity and improved volitional and generic social cognitive predictors of physical activity in the intervention group. No significant interaction effects were found in motivational social cognitive variables. Conclusions: The results revealed the potential of a behavioural online intervention in fostering an active lifestyle in AYA cancer survivors. Given the high number of sedentary AYA and also childhood cancer survivors, the efficacy and suitability of behaviour, online lifestyle interventions for this target group will be discussed.
Emma McLaughlin 1 , Amanda Wurz 1,2 , Colleen Cuthbert 3,4,5 and Nicole Culos-Reed 1,5,6 1 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 2 School of Kinesiology, University of the Fraser Valley, Chilliwack, BC, Canada 3 Faculty of Nursing, University of Calgary, Calgary, AB, Canada 4 Department of Community Health Sciences, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 5 Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 6 Department of Psychosocial Resources, Tom Baker Cancer Centre, Cancer Care, Alberta Health Services, Calgary, AB, Canada Background: Following the publication of the international Pediatric Oncology Exercise Guidelines (iPOEG) in 2021, resources to support end users were developed. Collectively, these resources are referred to as the iPOEG Toolkit. To better understand the value of the iPOEG Toolkit, its dissemination, implementation, and effectiveness will be tracked and explored. Methods: A research program informed by the Knowledge to Action framework is underway. A mixed-methods approach is being used, and the RE-AIM framework is guiding the evaluation of its reach (e.g., the number of iPOEG Toolkit downloads), effectiveness (e.g., the changed physical activity levels and patient-reported outcomes among end users), adoption (e.g., the number of organizations using the Toolkit), implementation (e.g., the use of and modifications made to the iPOEG Toolkit), and maintenance (e.g., the tracking of its continued use and impact, including markers of effectiveness, over time). Data will be collected via tracking metrics (e.g., webpage views, downloads), surveys, and interviews with relevant end users. Quantitative data will be analyzed descriptively and using regression analyses as appropriate, and qualitative data will be examined using content analysis and reflective thematic analysis. Results: The iPOEG Toolkits are currently being disseminated via the Health and Wellness Lab website, emails, and social media, and data will be gathered up until 2025. Discussion: The findings from this research program will highlight if and how the iPOEG Toolkit is being used, and offer critical guidance to optimize the iPOEG Toolkit. It is hoped that this work will support the spreading of the message that it is time for children and adolescents with cancer to ‘move more’.
Wei H. Deng 1,2 , Simeon J. Zürcher 3 , Christina Schindera 4 , Ruedi Jung 5 , Helge Hebestreit 6 , Iris Bänteli 7 , Katja Bologna 8 , Nicolas von der Weid 4,† , Susi Kriemler 5,† and Corina S Rueegg 1,† 1 Oslo Centre for Biostatistics and Epidemiology, Oslo University Hospital, Oslo, Norway 2 Oslo Centre for Biostatistics and Epidemiology, Department of Biostatistics, Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway 3 Center for Psychiatric Rehabilitation, Universitäre Psychiatrische Dienste Bern (UPD) and University Hospital of Psychiatry and Psychotherapy, University of Bern, Bern, Switzerland 4 Department of Pediatric Hematology and Oncology, University Children’s Hospital Basel (UKBB) and University of Basel, Basel, Switzerland 5 Epidemiology, Biostatistics and Prevention Institute, University of Zurich, Zurich, Switzerland 6 Pediatric Department, University Hospital, Julius-Maximilians University, Würzburg, Germany 7 Department of Psychosomatic Medicine, University Hospital and University of Basel, Basel, Switzerland 8 Departement of Internal Medicine, Kantonsspital St. Gallen, St. Gallen, Switzerland † These authors contributed equally to this work. Backgound: Childhood cancer survivors (CCSs) are at elevated risk of experiencing fatigue, depression, and reduced quality of life. Low physical activity (PA) levels may worsen these. Very few randomized controlled trials have investigated the effect of PA on psychosocial health among CCSs. We investigated the effect of a one year individualized exercise intervention on fatigue, mental health, and health-related quality of life (HRQoL) in adult CCSs. Methods: We randomized 151 CCSs aged ≥16 years, <16 at diagnosis, and ≥5 years since diagnosis, identified through the Swiss Childhood Cancer Registry. The intervention participants received personalized exercise counselling to increase intense PA by ≥2.5 h/week for one year. Controls maintained usual PA levels. We assessed the severity of fatigue, psychological distress, and physical and mental HRQoL at baseline, 3, 6, and 12 months. Outcomes were transformed into T-scores (mean = 50, standard deviation (SD) = 10). We used generalized linear mixed-effects models with intention-to-treat (ITT, primary), as well as per protocol allocations with compliance based on self-reported PA or a cardiopulmonary fitness test. Results: The mean baseline outcomes ranged from T-score 49.8 to 52.2, of the 133 (88%) who completed the trial. ITT analyses found significantly lower severity of fatigue by T-score −3.56 (95% confidence interval (CI) −5.69 to −1.43, p = 0.001) in the intervention group compared to controls at 12 months. The physical component of HRQoL was significantly better than controls in the intervention group for per protocol analyses by 3.06 (95%CI 0.99 to 5.14, p = 0.004) and 3.54 (1.13 to 5.96, p = 0.004). Conclusions: Individualized exercise interventions may improve fatigue and physical HRQoL among adult long-term survivors of childhood cancer. This is despite no effects on psychological distress.
Miek Hornikx 1 , Anne Uyttebroeck 2 , Deveny Vanrusselt 2 , Charlotte Sleurs 2 , Marc Gewillig 3 and Sabine Verschueren 1 1 Department of Rehabilitation Sciences, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium 2 Department of Oncology, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium 3 Department of Cardiovascular Sciences, KU Leuven-University of Leuven, University Hospitals Leuven, Leuven, Belgium Background: Patients with childhood cancer are confronted with exercise intolerance (EI (VO 2 peak < 85% predicted)) after treatment, with a detrimental effect on quality of life and mortality. Knowledge about the limiting factor(s) for this EI and its relation to physical activity (PA) is essential in order to prescribe individually tailored rehabilitation and to stimulate physical and social reintegration. Methods: A total of 41 patients with childhood cancer (13 ± 3 years; 71% boys), diagnosed with leukemia/lymphoma (61%), a solid tumor (32%) or brain tumor (7%), and who had recently finalized their oncology-related treatment, were included in the study. Patients performed a maximal symptom-limited cardiopulmonary exercise test on a treadmill (4.8 km/h; +2% elevation/min). PA was recorded with a 3-axial accelerometer (Dynaport MoveMonitor, McRoberts, The Hague), that patients wore for 7 consecutive days. Active time (standing and walking), sedentary time, and steps were withheld. Results: Exercise tolerance (VO 2 peak: 29.7 ± 7.8 mL/min/kg (67 ± 16% predicted)) was markedly reduced in patients with childhood cancer compared to healthy peers. The majority of patients were peripherally limited (83%). A cardiac limitation was present in 71% of patients and was predominantly due to a reduced oxygen pulse (97%). Hyperventilation (32%) and a ventilatory limitation (12%) were less prevalent. The PA data of 13 patients were available (Active time: 178 ± 67 min/day; sedentary time: 515 ± 113 min/day; steps: 6411 [4458–6838]). Conclusions: Exercise tolerance is markedly reduced in patients with childhood cancer shortly after intensive treatment and this is mainly caused by the deconditioning of peripheral muscles and a reduced oxygen pulse. Further research is necessary to study the link with physical activity.
Manuel Ester 1,2 and Patricia Scanlan 2,3 1 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 2 Tumaini La Maisha, Tanzania 3 Department of Pediatric Oncology, Muhimbili National Hospital, Tanzania Background : Despite the physical and psychosocial benefits of exercise for children and adolescents living with and beyond cancer, few exercise oncology programs have been successfully implemented for this population. Implementation barriers herein include cost as well as a lack of qualified exercise professionals and appropriate facilities, which may be exacerbated in resource-limited countries. The purpose of this implementation project was to overcome these barriers and implement a sustainable exercise oncology program with Tumaini La Maisha (TLM) in Tanzania. Methods : Interdisciplinary stakeholder discussions were held between nurses, oncologists (PS), caregivers, TLM teachers, and the visiting exercise professional (ME) in order to provide a broad range of perspectives and agree upon a feasible exercise program for implementation at Muhimbili National Hospital. Cost barriers were addressed by using volunteers and paid TLM staff for program delivery, with the exercise professional educating staff on exercise principles. To address facility barriers, a public sports field served as the exercise space. Results : Discussions led to the development and implementation of a 30 min teacher-led, group- and family-focused, play-based exercise program. The program was delivered daily for 3 months, with high attendance and satisfaction from parents and children. Three TLM teachers were trained for long-term program delivery. Newly identified barriers included sun exposure risks (addressed via indoor exercise) and a lack of cultural relevancy (addressed by integrating traditional dance within the program). Conclusions : The involvement of stakeholders in the implementation planning and consideration of resource limitations led to the successful implementation and maintenance of an exercise oncology program in a resource-limited setting.
Miriam Götte 1 , Regine Söntgerath 2 , Gabriele Gauß 1 , Joachim Wiskemann 3 , Mirko Buždon 4 and Sabine Kesting 5,6 1 Department of Pediatric Hematology/Oncology, Clinic for Pediatrics III, West German Cancer Centre, University Hospital Essen, Essen, Germany 2 Department of Pediatric Oncology, Hematology and Hemostaseology, University Hospital Leipzig, Leipzig, Germany 3 Working Group Exercise Oncology, Division of Medical Oncology, University Clinic Heidelberg and National Center for Tumor Diseases (NCT), Heidelberg, Germany 4 Institute of Sports Medicine, Hannover Medical School (MHH), Hannover, Germany 5 Institute of Preventive Pediatrics, Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 6 Kinderklinik München Schwabing, TUM School of Medicine; Department of Pediatrics and Children’s Cancer Research Center, Technical University of Munich, Munich, Germany Background: Cancer and the acute and late effects of its treatment are associated with a decline of physical activity behavior in childhood cancer patients and survivors. Children have a legal right to exercise as well as to the active participation in physical activities, and to the positive effects of exercise-related benefits. Methods: Network ActiveOncoKids (NAOK) is a Germany-wide initiative with the main goal of enabling children, adolescents, and young adults with exercise opportunities during and after cancer treatment. The network uses and bundles the knowledge, expertise, and experience of decentralized clinical sites and working groups on the topic of “Exercise and Sport in Pediatric Oncology”. For this purpose, NAOK was founded in 2012, striving for networking and structuring. It is managed and accompanied by an overarching coordination, a steering group, and an advisory board. Its main aims are A) physical activity support for patients and families, B) policy change to establish structures and guidelines, and C) the generating of evidence through scientific projects. Results: NAOK brings together 33 pediatric oncology treatment centers that offer exercise interventions either during acute treatment and/or in follow-up care. The main focus of the last 2.5 years was to support children, adolescents, and young adults through individualized counselling, to help in implementing exercise programs at pediatric oncology centers, and to adapt and change medical and care structures (e.g., via guidelines and educational lessons). Conclusions: NAOK is an interdisciplinary network that supports the implementation of pediatric exercise oncology in usual care. In the past few years, great progress has been achieved in the areas of exercise implementation, structural organization, and communication, which might serve as a model for other countries.
Lucia Longo 1 , Annalisa Cornelli 2 , Riccardo Ruisi 3 , Francesca Rossi 4 , Filippo Gatti 5 , Federica Ricci 4 , Daniele Bertin 4 , Giulia Zucchetti 4 and Franca Fagioli 4,6 1 Associazione Unione Genitori Italiani Contro il Tumore dei Bambini ODV, Torino, Italy 2 Ospedale Papa Giovanni XXIII, Associazione ConGiulia ONLUS, Bergamo, Italy 3 Istituto Ortopedico Rizzoli, Bologna, Italy 4 A.O.U. Città Della Salute e Della Scienza, Presidio Ospedale Infantile Regina Margherita, Torino, Italy 5 Università degli Studi di Torino, Corso di Laurea in Terapia della Neuro e Psicomotricità dell’Età Evolutiva 6 Università degli Studi di Torino, Torino, Italy Background: With the onset of the COVID-19 pandemic in Italy and its related restrictions, the National Orders Federation of Technical, Rehabilitation and Prevention Health Professions, recommended remote rehabilitation interventions. Therefore, in April 2020, the rehabilitation working group of the Italian Hematology and Oncology Association (AIEOP) created a website for telerehabilitation within the “#IoMiMuovoACasa” (#IDoHomeTraining) pilot project. According to the preliminary safety and efficacy results, and users’ opinions, the need for a more structured website arose. The Reh-PLAY project aims to create a new website to support remote individualized rehabilitation interventions, in addition to the direct treatment, for the prevention/rehabilitation of motor difficulties related to the disease and/or to treatment side effects. Methods: The website contents are written according to the AIEOP Consensus Conference on Rehabilitation in Pediatric Oncohematology. Exercise videos created by physiotherapists are based on their expertise and are smartly accessible by the professionals who can create personalized exercise programs, available in a patient’s personal area. Results: In one month, twenty patients used the website. The Reh-PLAY website is smartly accessible for families, patients, and physiotherapists from the AIEOP website. Professionals can select texts/infographics about prevention and rehabilitation, and access 250 exercise videos, divided by type, age, and intensity. Patients can download information brochures, adhesion diaries, patients reporting outcome measures, and a questionnaire to improve the service. Conclusions : Growing evidence in Pediatric Oncohematology identifies rehabilitation as a fundamental aspect of treatment. The Reh-PLAY website improves continuity in rehabilitation management, supporting families and patients with telerehabilitation, and facilitating the dissemination of knowledge between professionals.
Shaelene Standing 1 , Rachel McInnes 2 , S. Nicole Culos-Reed 3 , Mackenzie Murawsky 2,4 , Gregory M. T. Guilcher 5,6 , Fiona Schulte 5,6 and Carolina Chamorro Vina 2,3 1 Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 2 Kids Cancer Care Foundation of Alberta, Calgary, AB, Canada 3 Faculty of Kinesiology, University of Calgary, Calgary, AB, Canada 4 Faculty of Health Sciences, University of Ottawa, Ottawa, ON, Canada 5 Department of Oncology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada 6 Section of Oncology/Transplant, Alberta Children’s Hospital, Calgary, AB, Canada Background: The focus of the PEER program is to increase physical literacy, fitness, and to promote socialization for children affected by cancer and their siblings. In response to the COVID-19 pandemic, the program transitioned to an online format. The sessions, for children (6–11 years and 12–17 years), ran 1/week. Sport kits were provided to participants. Methods: After 12 months of running the program, which was attended regularly by 17 families (16 kids and 9 teens), we sought participant feedback through anonymous surveys. A total of 12 parent and 10 participant responses were collected outlining information about motivators for and barriers to online programming, safety, and satisfaction. Results: The families participated in the program mainly to (1) improve fitness and (2) increase peer connections. Of these, 100% felt safe, 95% agreed that sport kits increased engagement, and most agreed that sport kits also helped kids increase their activity level after participation in the PEER sessions. Furthermore, 86% felt that 45–60 min was a good session duration and that participation in the program increased their child’s fitness level. Conclusions: This program was (i) engaging, (ii) safe, and (iii) accessible for children affected by cancer who are medically vulnerable and/or remotely located. We suggest a program length of 45–60 min based on the age group. Exercises should have a greater focus on physical literacy and fundamental actions for younger age groups and should be more fitness-based for teens. Fun and the inclusion of socialization are key elements in increasing enjoyment, adherence, and community belongingness. The provision of a sport kit is recommended. Individualized adaptations are necessary for program success.
Sarah L. Grimshaw 1,2 , Nicholas F. Taylor 1 , Rachel Conyers 2 and Nora Shields 1 1 La Trobe University, School of Allied Health, Human Services and Sport, Bundoora, Melbourne, VIC 3086, Australia 2 Murdoch Children’s Research Institute, Parkville, Melbourne, VIC 3052, Australia Background: Supporting children/adolescents with cancer to be more physically active has the potential to improve short- and long-term outcomes. The objective of this study was to assess the feasibility of CanMOVE, a complex, theoretically informed, behaviour change intervention to promote participation in physical activity for children/adolescents undergoing acute cancer treatment. Method: A feasibility study utilising single-group, repeated measures, and a mixed methods design was completed. Participants completed the 10 week CanMOVE intervention, which involved structured support from a physiotherapist and the provision of a Fitbit (child and parent). Feasibility domains of demand, acceptability, implementation, practicality, limited efficacy, and integration were evaluated. Objective assessments of physical activity, physical function, and health-related quality of life were completed. Qualitative data were collected via semi-structured interviews with participants (parents and children/adolescents) and focus groups with clinical staff. Results: Twenty families completed CanMOVE, including children/adolescents (median age 12 years, range 5–16) with a mix of cancer diagnoses. There was a high demand for CanMOVE with a 95% enrolment rate. CanMOVE was acceptable from both participant and staff perspectives. All feasibility thresholds set for implementation were met. There were no serious adverse events. Under limited efficacy, data indicate that CanMOVE shows promise in influencing child/adolescent physical activity behaviour. Positive impacts were also seen in parent and staff behaviour towards physical activity promotion. The pre/post physical function and HRQOL assessments showed positive trends. Conclusions: CanMOVE is feasible and safe to implement in the paediatric oncology setting. CanMOVE shows potential in influencing the behaviour of children/adolescents and of the people in their social and professional support networks. These findings can be used to inform services in the paediatric cancer setting to ensure physical activity promotion is a considered and prioritised aspect of clinical care. Abstract Poster Presentations
F V. Engels, D Agterberg, W. P. Bekkering and Patrick van der Torre Department of Sports and Exercise Center, Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS Utrecht, The Netherlands Background: International studies and organizations, such as the World Health Organisation, have identified the health risks across lifespan that are associated with physical inactivity. Childhood cancer and its treatment have considerable impact on a child’s physical and mental wellbeing and often lead to reduced physical activity levels and sedentary behaviour. The combination of long-term chemotherapy, surgery and/or radiotherapy as administered in children with cancer especially impairs physical activity and fitness, both during and after therapy. Physical activities are important for the development of children and increasing evidence suggests the beneficial effects of physical activity promotion during cancer treatment as well. Therefore, ways to promote physical activity and exercise are becoming an important part of children’s cancer treatment. By means of our “Maximal Activity” program in the Princess Máxima Center for Pediatric Oncology, we want to encourage our patients to get out of bed and be as active as possible, both during and after treatment. Methods: A qualitative survey of 30 individuals (children, families and health care professionals) was used to ask about the experiences of the developmentally appropriate care program, of which ‘Maximal Activity’ is a part. Results: An important point that emerged was that the facilities and environment invite physical activity. The program was named by many as valuable. Trust and confidence in physical activity increased during hospitalization. Other results still need to be analysed and will be presented at the congress. Conclusions: Maximal Activity is a health program that contributes to a stimulating environment for physical activity and is appreciated by all stakeholders.
Carolin Ohnmacht and Alexander Puzik Department of Pediatric Hematology and Oncology, Medical Center—University of Freiburg, Faculty of Medicine, University of Freiburg, Mathildenstraße 1, D-79106 Freiburg, Germany Background: Regular physical activity (PA) is essential for biopsychosocial health, while reduced PA during childhood cancer treatment leads to increased late effects in affected children. Moreover, cancer and its treatment determine the everyday lives of families and parents who spend plenty of time with their children in hospital. Thus, it can be assumed that the parents’ PA is significantly affected during cancer treatment. Meanwhile, parents’ PA-behaviour has a pronounced influence on their children’s behaviour. The aim of this project is to investigate the parents’ PA-behaviour before and during their children’s cancer treatment. Methods: The PA and sedentary behaviour of the parents were assessed before and during their children’s intensive oncological therapy in a cross-sectional design using the International Physical Activity Questionnaire (IPAQ-SF). Results: A total of 40 parents of children with cancer took part in this survey. They were interviewed no earlier than their children’s second inpatient stay. The parents’ PA levels before diagnosis were in line with the reference values for healthy adults in Germany. During the children’s treatment, all dimensions of the parents’ daily PA, and the number of minutes of PA per week, decreased significantly ( p < 0.001). The greatest reduction in PA was identified during inpatient stays, with a significant increase in sitting time ( p < 0.001). Conclusions: This is the first study to show that the PA of parents whose children have cancer decreases significantly during cancer treatment. As parents’ PA behaviour significantly affects that of their children, even after completion of cancer treatment, future exercise programs in pediatric oncology should include parents in order to reduce inactivity-related late effects.
Sandra Stöessel 1 , Elias Dreismickenbecker 1 , Marie A. Neu 1 , Lisa Ploch 1 , Norbert Paul 1 , Christian Ruckes 1 , Adriana Balduzzi 2 , Francesca Lanfranconi 2 , Peter Wright 3 , Stan Windsor 3 , Joachim Wiskemann 4 , Inaam El-Rajab 4 , Veronika Picmanova 5 , Céline Gravot 5 , Rodolf Mongondry 6 , Wilhelm Bloch 7 , Katie Rizvi 8 and on behalf of Youth Cancer Europe, Martin K. Fridh 9 , Alejandro Lucia 10 , Carmen Fiuza-Luces 10 , Miriam Götte 11 and on behalf of Network ActiveOncoKids, Filippo Spreafico 12 , Barbara Konda 13 , Lidija Kitanovski 14 , Lena Werthmann 15 , Tobias Baader 16 and Jorg Faber 1 and on behalf of the FORTEe Consortium 1 University Medical Center of the Johannes Gutenberg, University Mainz (DE) 2 Fondazione Monza e Brianza per Il Bambino e La Sua Mamma (IT) 3 Oxford Brookes University (UK) 4 Heidelberg University Hospital (DE) 5 Concentris Research Management GmbH (DE) 6 Centre de Lutte Contre le Cancer Léon Bérard (FR) 7 German Sport University Cologne (DE) 8 Youth Cancer Europe (RO) 9 Department of Pediatric and Adolescent Medicine, Copenhagen University Hospital, Copenhagen, Denmark (DK) 10 Universidad Europea de Madrid (ES) 11 Essen University Hospital (DE) 12 Fondazione IRCCS Istituto Nazionale dei Tumori (IT) 13 Forma 3D Ltd. (SI) 14 University Medical Center Ljubljana, Division of Pediatrics, Department of Haematooncology (SI) 15 Nurogames GmbH (DE) 16 Pixformance Sports GmbH (DE) Background: Cancer is the leading cause of death by non-communicable diseases in children in Europe. During cancer treatment, patients’ morbidity is increased due to physical inactivity and cancer-related fatigue. Precision-based exercise training programs in children and adolescents attending the intensive phases of cancer treatment is an increasingly promising therapy. However, strong evidence for exercise efficiency is lacking in paediatric oncology and, thus, precision exercise training is not part of standard care and does not reach the majority of patients. Methods: The FORTEe project is structured in seven work packages and intends to evaluate a personalised and standardised exercise intervention in 450 children, adolescents and young adults undergoing cancer treatment in 9 centres across Europe. The randomised, controlled FORTEe trial aims to generate high evidence for an innovative, patient-centred exercise treatment. Supervised exercise training intends to impact the efficiency of systems involved in the oxidative metabolism chain, including the skeletal muscle. The tailored training is also focused on strength in order to counteract muscular atrophy. Within the project, digital and innovative technologies (a FORTEe app, an augmented reality program and an interactive digital training) will be developed and applied to make the exercise training more attractive, age-adapted and inspirational. FORTEe will stimulate multidisciplinary research by involving paediatricians and exercise scientist in order to provide more inclusive access to paediatric exercise oncology. Conclusions: Progressing beyond the current state-of-the-art standard, FORTEe has the ambition of implementing paediatric exercise oncology as an evidence-based standard in clinical care for all childhood cancer patients worldwide.
Brooke E. Kohler, 1 Emmah Baque 1,2 , Carolina X. Sandler 3,4,5 and Stewart G. Trost 1,6 1 Faculty of Health, Queensland Centre for Children’s Health Research, Queensland University of Technology (QUT) Brisbane, Brisbane, QLD, Australia 2 School of Health Sciences and Social Work, Griffith University, Nathan, QLD, Australia 3 UNSW Fatigue Research Program, Kirby Institute, University of New South Wales, Sydney, NSW, Australia 4 School of Sport and Exercise Science, School of Health Sciences, Western Sydney University, Sydney, NSW, Australia 5 Menzies Health Institute Queensland, Griffith University, Griffith, QLD, Australia 6 School of Human Movement and Nutrition Sciences, University of Queensland, Brisbane, QLD, Australia Background: Increasing survival rates for children with solid tumors present an ongoing challenge regarding how to maximize the quality of survivorship and effectively manage the short- and long-term complications of the disease and its treatment. To gain a better understanding of the research pertaining to therapeutic exercise programs, we conducted a scoping review of exercise training studies conducted in children diagnosed with solid tumors. Method: A systematic literature search was performed across four electronic databases. Articles were selected for full-text review if they included participants diagnosed with a solid tumor, if at least 50% of the participants were aged ≤21 years, if they evaluated an exercise program ≥ 2-weeks in duration, and if they were published in an English, peer-reviewed journal. Results: Of the 6648 citations identified, 14 articles met the inclusion criteria. Only three studies were conducted as randomized controlled trials. The duration of the exercise programs ranged from 3 to 40 weeks and their frequency ranged from 3 to 11 sessions per week. Exercise session duration ranged from 15 to 180 min, with most articles reporting 30–90-min sessions. A range of exercise modalities were employed, including ergometry, resistance training, yoga, and active videogaming. Adherence ranged from 77% to 100%, with no adverse events reported. Exercise training resulted in improvements in cardiorespiratory fitness, functional strength, physical activity, and quality of life. Conclusions: A small number of mostly low-quality studies have evaluated therapeutic exercise in pediatric survivors of solid tumors. Although limited, the extent of research supports the feasibility and safety of therapeutic exercise and suggests that therapeutic exercise may be potentially effective in improving a range of health outcomes.
Clémentine Bischoff 1 , Nicolas Von der Weid 2 , Oliver Faude 1 , Fiona Streckmann 1,3 and on behalf of the PrepAIR Study Group † 1 Department of Sport, Exercise and Health, University of Basel, Grosse Allee 6, 4052 Basel, Switzerland 2 Department of Pediatric Oncology and Hematology, University Children’s Hospital Basel, University of Basel, Spitalstrasse 33, 4056 Basel, Switzerland 3 Department of Oncology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland † PrepAIR Study Group: Katrin Scheinemann (KS Aarau), Uta Tacke (UKBB), Jochen Rössler (Insel Bern), Pablo Hernaiz Driever (Charité Berlin), Miriam van Buiren & Alexander Puzik (Uniklinik Freiburg, Jeanette Greiner (KISPI St. Gallen), Aline Rehm & Patrizia Brunner (Universität Basel), Valentin Benzig & Regula Everts (Universität Bern), Anja Wehrle (Universität Freiburg), Claudia Bucher (KS Aarau), Sandra Frauchiger (Insel Bern), Carolin Ohnmacht (Uniklinik Freiburg), Martina Sigg (KISPI St. Gallen). Background: Modern therapy improved survival for children with cancer. However, the treatment has unintended consequences. Depending on neurotoxic agents, 52–100% of children develop a chemotherapy-induced peripheral neuropathy (CIPN). The severe symptoms such as loss of sensation, numbness, pain, absent reflexes and loss of balance control, not only delay motor development milestones such as walking, running, jumping, or climbing, diminishing children’s quality of life and affecting their social reintegration, but are also of high clinical relevance. Streckmann et al. confirmed in their meta-analysis that recovery is poor, but there is a clear benefit for adults in favor of sensorimotor training (SMT) to target the symptoms of CIPN. Methods : In our RCT, we will recruit N = 131 children from 6 centers (Switzerland and Germany). Immediately after being scheduled to receive neurotoxic chemotherapy, the intervention group will perform a standardized, age-adjusted, specific playful SMT program twice a week for the duration of their medical therapy, while the control group will receive treatment as usual. Results : For the intervention, we created a training manual which will finally lead to a simple therapy option for children suffering from CIPN. The manual provides a basis for playful SMT based on a modular system, which leaves the therapists and the children room for scope and still respects all the training modalities within the background of “specific exercise is medicine”. Conclusions: We hypothesize that children in the intervention group will develop fewer symptoms of CIPN and will be able to maintain their motor and sensory functions for an age-appropriate motor development.
Dominik Gaser 1,2 , Christiane Peters 2 , Renate Oberhoffer-Fritz 2 , Miriam Götte 3 , Gabriele Gauß 3 , Irene Teichert-von Lüttichau 1 and Sabine Kesting 1,2 1 Department of Pediatrics and Children’s Cancer Research Centre, Children’s Hospital München Schwabing, TUM School of Medicine, Technical University of Munich, Munich, Germany 2 Department of Sport and Health Sciences, Technical University of Munich, Munich, Germany 3 Department of Hematology and Oncology, Clinic of Pediatrics III, West German Cancer Centre Essen, University Hospital, Essen, Germany Background: An increasing amount of in-hospital and out-patient physical activity offers facilitate access to professional exercise programs for children and adolescents during acute anticancer treatment and surveillance. The COVID-19 pandemic has presented major challenges to hospitals and rehabilitation facilities. An online survey among providers in Germany investigates the impact of the pandemic on individual sectors of exercise programs in pediatric oncology. Methods: From 19 January until 9 February 2022, all German clinics and institutions with an exercise program for pediatric cancer patients and/or survivors are invited to participate in an online survey. Methodology and recruitment is conducted in cooperation with Network ActiveOncoKids. Limitations, challenges and measures for adapting offers in the context of the individual pandemic waves, are collected. In addition, challenges for the implementation of scientific studies are analyzed. Results: Thirty-three sites have been requested to participate in the survey. We assume that exercise professionals and scientists have used the pandemic-related challenges to modify the existing concepts of exercise promotion and adapt them to the specific local conditions. Conclusions: We expect new ideas and approaches for the realization of exercise programs under pandemic conditions. The extension of digital offers may improve the access for children and adolescents to exercise programs in pediatric oncology in the long term and, therefore, could potentially be adopted into standard exercise care.
Maxime Caru 1,2 , Ariane Levesque 3 , Pooja Rao 1 , Smita Dandekar 1 , Christopher Terry 4 , Valerie Brown 1 , Lisa McGregor 1 and Kathryn Schmitz 2 1 Department of Pediatric Hematology and Oncology, Penn State College of Medicine, Hershey, PA, USA 2 Department of Public Health Sciences, Penn State College of Medicine, Hershey, PA, USA 3 Department of Psychology, University of Montreal, Montreal, QC, Canada 4 Department of Medical Oncology, Sidney Kimmel Cancer Center—Thomas Jefferson University, Philadelphia, PA, USA Background: AYA cancer survivors are a population that have unmet needs, and researchers believe that physical activity (PA) interventions can address many of these needs. This review describes and synthesizes previously reported data in order to map the current evidence as well as to identify gaps in knowledge and future research needs. Methods: A search of the literature was conducted in PubMed, CINAHL, EMBASE, Web of Science and Cochrane Library. This review followed the PRISMA-ScR statement. We included all original studies investigating PA interventions in post-treatment AYA cancer survivors who were between 15 and 39 years old at the time of their initial cancer diagnosis. Results: A total of 8 studies were included in this review and showed that PA interventions were feasible and acceptable in AYA cancer survivors. Overall, PA interventions were individualized and mainly aerobic in nature. Studies examining the effects of PA interventions on AYA cancer survivors’ health evaluated physical and mental health outcomes, including, but not limited to, physical functioning, functional capacity, occupational performance, health-related quality of life, physical capacities, fitness, mood, and self-perception after cancer treatments. Conclusions: Our review maps the current evidence of PA interventions and highlights the paucity of data in this area of investigation, obviating how much work remains to be carried out to demonstrate the potential benefits of PA on AYA cancer survivors’ health outcomes. Future studies should consider the development PA interventions that address patients’ clinical needs, in addition to providing a detailed description of PA interventions.
Sarah Otten 1 , Clémentine Bischoff 2 , Julia Däggelmann 1 , Fiona Streckmann 2,3 , Wilhelm Bloch 1 and Vanessa Oschwald 1 1 Institute for Cardiovascular Research and Sports Medicine, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany 2 Department of Sport, Exercise and Health, University of Basel, Birsstr. 320B, 4052 Basel, Switzerland 3 Department of Oncology, University Hospital Basel, Petersgraben 4, 4031 Basel, Switzerland Background: A sensorimotor training (SMT), mostly applied as balance training on different surfaces and in different positions, has the potential to contribute to the nervous system’s plasticity and to improve lower extremity impairments. In recent years, SMT has not only been conducted in rehabilitation, injury and fall prevention, but has also been successfully applied in adult oncology. In this context, studies showed improvements in common lower extremity impairments, such as impaired balance control, sensory and motor symptoms. While SMT or associated training modalities have been investigated in different pediatric patient collectives, they have rarely been conducted in pediatric oncology, though the outlined effects are promising for these patients. Methods: In order to identify therapeutic potentials of SMT for pediatric oncology, current data on SMT in different pediatric patient collectives are reviewed. Furthermore, to gather preliminary insights on a playful and child-specific SMT in pediatric oncology, first pilot studies during and after inpatient medical treatment in this field are conducted. Results: SMT resp.-related training modalities with various pediatric patients demonstrate potential effects on lower extremity parameters. The preliminary results in pediatric oncology indicate that a child-specific and playful SMT for children after cancer treatment is feasible. Motivating sensorimotor exercises are identified. Further study results on SMT performed during acute pediatric oncological therapy will be available and presented at the congress. Conclusions: The reviewed interventions in pediatric collectives, and preliminary study results in pediatric oncology in particular, suggest that SMT might be a promising and targeted training modality supplementing exercise therapy for children with cancer.
Rachael Keating 1 , Eilis O’Shea 2 and Susan Hurley 3 1 Physiotherapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland 2 Occupational Therapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland 3 Music Therapy Department, Children’s Health Ireland at Crumlin, Dublin, Ireland Background: The “Tuesday Boogie” is a quality improvement initiative which was developed to promote physical activity and enhance psychosocial wellbeing amongst in-patients, parents and staff on an in-patient paediatric cancer ward. It involves children, parents and staff engaging in a 15 min group dancing session and runs on a weekly basis. We conducted a service evaluation to assess the feasibility of the dance-based intervention and whether it influenced the psychosocial wellbeing of participants. Methods: Child, parent and staff questionnaires were designed by the research team to evaluate the intervention. Both quantitative (Likert, multiple choice, dichotomous) and qualitative open format questions were included. Data were collected on a voluntary, anonymous basis from June to July 2021. Results: In total, 39 questionnaires were completed ( n = 4 child, n = 9 parent, n = 26 staff). A total of 97% of respondents had taken part in the intervention with 100% reporting the intervention was worthwhile. Of the respondents, 95% felt taking part improved their mood and 92% reported it helped them to cope with being in hospital. Furthermore, 77% of respondents felt less worry or stress during the intervention. The following qualitative data were collected: C1 “It is nice to be happy and dance instead of worrying”, P3 “Changes the energy…real mood shifter and connector”, P9 “M gets a chance to have fun and to interact with other kids which is so rare these days”, S22 “Shows patients that not everything that happens in hospital is scary”. Conclusions: A dance-based intervention for children, parents and staff on an in-patient paediatric cancer ward is feasible and has numerous psychosocial benefits.
Anna Vogelsang 1,2 , Corinna Meyer-Schwickerath 3,4 , Joachim Wiskemann 3,4 and Markus Reichert 1,5,6 1 Department of eHealth and Sports Analytics, Faculty of Sport Science, Ruhr-University Bochum, Bochum, Germany 2 Faculty of Humanities, MSH Medical School Hamburg, Hamburg, Germany 3 University of Heidelberg, Institute of Sports and Sports Sciences, Heidelberg, Germany 4 Department of Medical Oncology, National Center for Tumor Diseases (NCT), Heidelberg, Germany 5 Mental mHealth Lab, Department of Sport and Sport Science, Karlsruhe Institute of Technology, Karlsruhe, Germany 6 Department of Psychiatry and Psychotherapy, Central Institute of Mental Health, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany Background: The growing population of childhood cancer survivors is at risk of developing late chronic conditions and premature mortality. Physical activity (PA) can mitigate physical and psychological late effects, but only if engaged in regularly over sustained time periods. PA sustainment, however, remains challenging due to daily and within-daily fluctuations in how survivors feel, with whom they interact and in which environments they live. Thus far, exercise psychology has mainly focused on macro-temporal processes unfolding over weeks and months. To promote sustainable PA engagement, micro-temporal processes (e.g., unfolding over minutes, hours, days) need to be studied. Therefore, this review introduces ambulatory assessment as a method to gain insights into dynamic behavioural processes as they unfold in survivors’ everyday lives. Methods: Ambulatory assessment (AA) describes a group of computer- or smartphone-assisted methods to study behavioural, biological, and psychological processes in survivors’ everyday lives near real time. In this review, we discuss the characteristics of AA and expand on its promise for researching micro-temporal behavioural processes and on its potential for exercise oncology, such as the prediction of critical phases of PA relapse. This is exemplified in ongoing projects, and we expand on future avenues where ambulatory interventions might benefit survivors’ tailored care. Conclusions: Insights into dynamic behavioural processes as they unfold in everyday life may critically add to our understanding of sustainable PA and the development of individualised treatment where and when it is needed. Avenues for research, prevention and treatment will be discussed as well as the acceptability, compliance and ethical issues of AA.
Lynn R. Tanner 1,2 and Erica N. Schorr 1 1 School of Nursing, University of Minnesota, Minneapolis, MN, USA 2 Physical Medicine & Rehabilitation, Children’s Minnesota, MN, USA Background: Children and adolescents with cancer receive rehabilitation interventions for the functional impacts of the disease and its corresponding treatment. Adherence to these interventions varies greatly. The purpose of this review was to identify the factors related to adherence in rehabilitation. Methods: A systematic review was conducted using Ovid Medline and CINAHL databases with search terms related to pediatric cancer, rehabilitation, and adherence. Study eligibility criteria included the following: English language, participants receiving a physical therapy, occupational therapy, speech-language pathology, cognitive or exercise intervention or service, mean age of ≤18 years old, and measurement of factors related to adherence. The PRISMA 2020 statement for reporting systematic reviews guided data synthesis. The study quality was assessed using the Joanna Briggs Institute Critical Appraisal Tools. Results: The review included 13 studies providing rehabilitation interventions (exercise, yoga, walking, cognitive training, and vibration plate) to 283 children aged 3–19 years with adherence levels of 61–91% measured by session attendance. The majority (85%) of the studies comprised exercise interventions with 69% of the interventions being multifaceted in nature including aerobic, strengthening, and flexibility components. The factors related to adherence fell into three categories: (1) organizational; (2) condition-related; and (3) personal. Common barriers included fatigue, illness, time, family scheduling, and motivation. Facilitators included peer or caregiver support and supervision. Conclusions: The existing literature on rehabilitation adherence focuses mostly on exercise interventions delivered by a multitude of health care professionals. More research is needed to improve understanding of the factors related to adherence to rehabilitation interventions and services in survivors of childhood cancer.
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The Role of Telemedicine for Psychological Support for Oncological Patients Who Have Received Radiotherapy
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812dc1f0-44b5-4cfd-988e-2b721db86d5f
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10217284
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Internal Medicine[mh]
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Receiving a cancer diagnosis leads to the development of several psychological dynamics: fear of dying, uncertainty, loss of control, change in interpersonal relationships and change in self-image. For these patients, the psychological structure often represents “baggage” during cancer treatments. Surgery, chemotherapy and radiotherapy (RT) are the main treatment weapons in the armamentarium of the oncological battle. These therapies could be stressful for patients (fear of oncological pathway, fear of side effects, fear of pain, fear of death); however, they have a “life-saving” purpose. In the present context, during the last few years, SARS-CoV-2 infection has had a significant impact on everyone’s daily life: higher levels of perceived stress were associated with the fear of infection in the whole population, as suggested by recent studies . Furthermore, the lives of oncological patients received additional stress due to the COVID-19 pandemic . In fact, they experienced additional conflicting emotional moods due to the needs of receiving life-saving treatments and the necessity to “stay at home” for fear of exposing themselves to the contagion risk . Moreover, the loneliness that affects all oncological patients added to the social distancing procedures/restrictions in access to care may increase patient fears and concerns about receiving cancer treatments and disease recurrence . In the management of the relationship between stressful events and disease, the possibility of receiving dedicated support, both in the intimate and extended environment, is a relevant aspect. However, as the pattern and duration of stressors might vary, the responses of each individual could also be significantly variable. They depend on cognitive, neurobiological and emotional processes, such as the style of attachment or the coping performed in response to stress . The differences in the latter responses among patients contribute to higher or lower rates and severity of psychological and/or physical symptoms: for the cancer population, depression occurs in 9–30% and anxiety in 9–50% . During the COVID-19 pandemic, a high level of stress, about 30%, was reported in the general population, while in the cancer population, it is slightly increased . In addition, the Impact of Event Scale-revised (IES-R) total scores showed a strong state of stress: these findings indicate that during this pandemic, oncological patients experienced a clinically meaningful level of stress that could be comparable to post-traumatic stress disorder . In particular, as noted in a recent systematic review, the prevalence rates for COVID-19-related anxiety and depression were 31.9% and 33.7%, respectively, while in the oncological population, they occurred up to 31% and 36%, respectively . For these reasons, the role of psychological support for cancer patients is emerging as a priority. RT provides significant survival benefit for patients affected by cancer , and the benefit of psychological support for oncological patients has been demonstrated both during radiation therapy and during follow-up. Due to the COVID-19 pandemic, the use of telemedicine (video or telephone visits), in order to avoid the unnecessary exposure risks in an ambulatory hospital, experienced unexpected growth. For oncological patients, psychological meetings could be difficult to organize due to the various disease commitments (chemotherapy appointments, medical visits, diagnostic procedures, and so on). Based on COVID-19 experiences, the expansion of telemedicine for psychological assessment could endure due to the potential benefit for both oncological patients and the healthcare system . Telemedicine may provide consistent advantages over in-person care, especially in chronic patients such as oncological ones, which require frequent psychological assessment and support. Surely, if patients do not have basic technology skills and do not feel comfortable using technologies (phone, computer, and so on), it is difficult to receive any benefit from telemedicine. This may be challenging for some older patients (due to inexperience/lack of access to technology) and for patients with cognitive disability . Many studies pre- and post-COVID-19 showed the benefit of telemedicine in cancer patients, but few data are reported on psychological tele-visits, especially in patients who underwent radiation treatment . In our radiation department, all patients received psycho-oncological support during RT and during follow-up. Based on the latter, the aim of this retrospective analysis was to evaluate the role of tele-visits and in-person psychological support for cancer patients after RT, and to report a descriptive analysis pointing out the needs of psychosocial intervention in a radiation department during radiation treatment.
From June 2019 to June 2022, according to our institutional care management, all patients receiving radiation therapy were prospectively enrolled to receive charge-free assessment of their cognitive, emotional and physical states and psycho-oncological support during treatment. For the whole population who accepted the psychological support during RT, a descriptive analysis was reported. For all patients who accepted to be followed up by a psycho-oncologist, tele-consultations (video-call or telephone) (TC group) or on-site psychological visits (OS Group) were proposed. Inclusion in the TC group or OS group was based on patients’ preferences. Inclusion criteria were as follows: patients aged more than 18 years old; diagnosis of cancer; treated with a long course of RT (more than 5 fractions); signed consent form; and psychological assessment and evaluation during radiation therapy and during follow-up (minimum 2 meetings after RT). Exclusion criteria were as follows: no signed consent form; no evaluation during follow-up; age less than 18 or more than 80 years old. The aims of this analysis were to describe (in terms of anxiety, depression, coping and post-traumatic stress disorder) the whole population, who received psychological support during radiation therapy, and to evaluate the differences in terms of psychological results between tele-visits or in-person visits for the sub-population who were followed up after RT. For those who adhered to the psycho-oncological support program, the first interview (pre-RT/baseline) and all subsequent psychological meetings were structured as follows by a psycho-oncologist: A descriptive collection of personality features, psychosocial elements and cognitive and emotional processing capacity regarding the event, paying attention to the patient’s history. An evaluation of anxiety and depression with Hospital Anxiety Depression Scale (HADS) , which is a four-point scale (from 0 to 3) consisting of 14 questions to which the patients had to respond, referring to their symptoms during the previous week. The HADS scale is divided into 2 subscales which investigate anxiety (HADS-A) and depression (HADS-D), respectively. Seven of the 14 elements of the HADS scale correspond to the HADS-A subscale and the remaining 7 to the HADS-D subscale, ranging from 0 (no discomfort) to 21 (extreme discomfort). Scores of 8 or more in both subscales indicate the presence of a disorder. An evaluation of distress using the Distress Thermometer (DT) . It is a single-item instrument, consisting of a visual analog scale and represented by a thermometer ranging from 0 (no discomfort) to 10 (extreme discomfort). A score of 4 or more indicates the presence of a disorder. An assessment of the varying coping strategies used by patients in response to stress by the Brief COPE (BC) . It is an instrument composed of 14 scales for a total of 28 items (positive restructuring, distracting attention, expression, use of instrumental support, operational coping, denial, religion, humor, behavioral disengagement, use of emotional support, substance use, acceptance, planning, self-accusation). An evaluation of post-traumatic stress symptoms using the Impact of Event Scale-revised (IES-R) . The IES-R is a 22-item self-report measure (for DSM-IV ) that assesses subjective distress caused by traumatic events. It is a revised version of the older version; the IES-R contains 7 additional items related to the hyperarousal symptoms of PTSD (post-traumatic stress disorder), which were not included in the original IES. Respondents are asked to identify a specific stressful life event and then indicate how much they were distressed or bothered during the previous seven days by each “difficulty” listed. Items are rated on a 5-point scale ranging from 0 (“not at all”) to 4 (“extremely”). The IES-R yields a total score (ranging from 0 to 88); significant symptoms were defined by a score of more than 33. During radiation therapy and follow-up, easy implementations, including mindfulness-based stress reduction techniques, were performed to manage stress and pain perception, to reduce physical symptoms and to improve mood and sleep quality . These psycho-therapeutic approaches were developed in individual sessions and/or in small group sessions (only during RT), based on the inclination of the patient . Statistical Analysis A statistical analysis was carried out to detect differences between study groups, using a Mann–Whitney test for quantitative variables to evaluate differences in median, while t-tests or Chi-square tests were used for qualitative variables to test differences in proportion. A p-value equal or less than 0.05 was considered statistically significant.
A statistical analysis was carried out to detect differences between study groups, using a Mann–Whitney test for quantitative variables to evaluate differences in median, while t-tests or Chi-square tests were used for qualitative variables to test differences in proportion. A p-value equal or less than 0.05 was considered statistically significant.
3.1. Overall Population Who Received Psychological Assessment during RT From July 2019 to June 2022, 2730 oncological patients underwent RT in our radiation oncology department. Of these, 2290 (84%) agreed to participate in a psycho-oncological support program, receiving at least a first evaluation. The median age was 60 years (range 20–78). There were 1671 females (73%) and 619 males (27%). In terms of anxiety, depression and distress, for the entire study population during RT, the median HADS-A was 8 (range 1–19), the median HADS-D was 5 (range 1–18) and the median DT was 5 (range 0–10). Excluding patients treated with a short course of RT (1–5 fractions) who received only one psychological evaluation, the remaining 1145 cases were evaluated during RT with structured psycho-oncological interviews for a median of 3 sessions (range 2–5). For the latter population, the median RT fractions was 25 (range 15–33) and the most common histologies were breast and gynecological cancer. During their first psycho-oncological interview, all the 1145 patients experienced the assessment of anxiety, depression and distress levels, reporting the following results: concerning the HADS-A scale, 50% of cases (574 patients) reported a pathological score ≥8; concerning the HADS-D scale, 30% of cases (340 patients) reported a pathological score ≥8; concerning the DT scale, 60% (687 patients) reported a pathological score ≥4. All patients completed their radiation treatment without delays, reporting a reduction in RT anxiety during the interviews and a “feeling of welcome” at the end of RT. Population Followed after RT: Tele-Visit Versus in-Person Evaluation From July 2019 to June 2022, only 82 patients out 1145 (7.2%) were followed up after RT: 30 patients with on-site consult (Group OS) and 52 patients with tele-consult (Group TC). The main clinical and physiological characteristics are reported in . Sixty-one patients (74.4%) were female and 21 (25.6%) were male; the median age was 50 years old (range 18–75 years old). The breast cancer (39 cases, 47.5%), gynecological cancer (8 cases, 9.7%), brain tumor (12 cases, 14.6%) and prostate cancer (6 cases, 7.3%) were the most represented histopathologies that affected patients. During RT, all patients received a median of 4 psychological visits, while during follow-up, they received a median of 8 meetings (1 or 2 meeting per week, range 4–28), without significant difference between the two groups. For the whole population of 82 patients, from baseline to the last follow-up, a significant improvement in terms of HADS-A, global HADS and BC was shown ( p 0.04; p 0.05; p 0.0008, respectively). shows the differences in terms of each scale (and relative pathological value) between baseline and last follow-up. Regarding Group-OS, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 9 (range 4–16) versus 8 (4–14); regarding the HADS-D scale, a score of 6 (range 2–14) versus 5.5 (2–11); regarding the DT scale, 20 patients (66.7%) versus 19 patients (63.3%) reported a pathological score ≥ 4; regarding the BC scale, 10 patients (33.3%) versus 3 patients (10%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 14–83) versus 43 (range 11–88). Regarding Group-TC, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 8.5 (range 3–16) versus 7.7 (3–12); regarding the HADS-D scale, a score of 7 (range 2–14) versus 6 (2–12); regarding the DT scale, 32 patients (61.5%) versus 31 patients (59.6%) reported a pathological score ≥ 4; regarding the BC scale, 18 patients (34.6%) versus 8 patients (15.4%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 12–88) versus 46.5 (range 11–80). In general, with respect to baseline, statistically significant differences were observed between the two groups in terms of anxiety (HADS, HADS-A: p 0.00002 and 0.03) in favor of on-site visits. In each group, a statistical improvement was observed in BC ( p 0.01), score ≥ 4. shows, for each scale used, the percentage of patients with pathological score at baseline and at last follow-up for each group (tele-consult and on-site).
From July 2019 to June 2022, 2730 oncological patients underwent RT in our radiation oncology department. Of these, 2290 (84%) agreed to participate in a psycho-oncological support program, receiving at least a first evaluation. The median age was 60 years (range 20–78). There were 1671 females (73%) and 619 males (27%). In terms of anxiety, depression and distress, for the entire study population during RT, the median HADS-A was 8 (range 1–19), the median HADS-D was 5 (range 1–18) and the median DT was 5 (range 0–10). Excluding patients treated with a short course of RT (1–5 fractions) who received only one psychological evaluation, the remaining 1145 cases were evaluated during RT with structured psycho-oncological interviews for a median of 3 sessions (range 2–5). For the latter population, the median RT fractions was 25 (range 15–33) and the most common histologies were breast and gynecological cancer. During their first psycho-oncological interview, all the 1145 patients experienced the assessment of anxiety, depression and distress levels, reporting the following results: concerning the HADS-A scale, 50% of cases (574 patients) reported a pathological score ≥8; concerning the HADS-D scale, 30% of cases (340 patients) reported a pathological score ≥8; concerning the DT scale, 60% (687 patients) reported a pathological score ≥4. All patients completed their radiation treatment without delays, reporting a reduction in RT anxiety during the interviews and a “feeling of welcome” at the end of RT. Population Followed after RT: Tele-Visit Versus in-Person Evaluation From July 2019 to June 2022, only 82 patients out 1145 (7.2%) were followed up after RT: 30 patients with on-site consult (Group OS) and 52 patients with tele-consult (Group TC). The main clinical and physiological characteristics are reported in . Sixty-one patients (74.4%) were female and 21 (25.6%) were male; the median age was 50 years old (range 18–75 years old). The breast cancer (39 cases, 47.5%), gynecological cancer (8 cases, 9.7%), brain tumor (12 cases, 14.6%) and prostate cancer (6 cases, 7.3%) were the most represented histopathologies that affected patients. During RT, all patients received a median of 4 psychological visits, while during follow-up, they received a median of 8 meetings (1 or 2 meeting per week, range 4–28), without significant difference between the two groups. For the whole population of 82 patients, from baseline to the last follow-up, a significant improvement in terms of HADS-A, global HADS and BC was shown ( p 0.04; p 0.05; p 0.0008, respectively). shows the differences in terms of each scale (and relative pathological value) between baseline and last follow-up. Regarding Group-OS, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 9 (range 4–16) versus 8 (4–14); regarding the HADS-D scale, a score of 6 (range 2–14) versus 5.5 (2–11); regarding the DT scale, 20 patients (66.7%) versus 19 patients (63.3%) reported a pathological score ≥ 4; regarding the BC scale, 10 patients (33.3%) versus 3 patients (10%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 14–83) versus 43 (range 11–88). Regarding Group-TC, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 8.5 (range 3–16) versus 7.7 (3–12); regarding the HADS-D scale, a score of 7 (range 2–14) versus 6 (2–12); regarding the DT scale, 32 patients (61.5%) versus 31 patients (59.6%) reported a pathological score ≥ 4; regarding the BC scale, 18 patients (34.6%) versus 8 patients (15.4%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 12–88) versus 46.5 (range 11–80). In general, with respect to baseline, statistically significant differences were observed between the two groups in terms of anxiety (HADS, HADS-A: p 0.00002 and 0.03) in favor of on-site visits. In each group, a statistical improvement was observed in BC ( p 0.01), score ≥ 4. shows, for each scale used, the percentage of patients with pathological score at baseline and at last follow-up for each group (tele-consult and on-site).
From July 2019 to June 2022, only 82 patients out 1145 (7.2%) were followed up after RT: 30 patients with on-site consult (Group OS) and 52 patients with tele-consult (Group TC). The main clinical and physiological characteristics are reported in . Sixty-one patients (74.4%) were female and 21 (25.6%) were male; the median age was 50 years old (range 18–75 years old). The breast cancer (39 cases, 47.5%), gynecological cancer (8 cases, 9.7%), brain tumor (12 cases, 14.6%) and prostate cancer (6 cases, 7.3%) were the most represented histopathologies that affected patients. During RT, all patients received a median of 4 psychological visits, while during follow-up, they received a median of 8 meetings (1 or 2 meeting per week, range 4–28), without significant difference between the two groups. For the whole population of 82 patients, from baseline to the last follow-up, a significant improvement in terms of HADS-A, global HADS and BC was shown ( p 0.04; p 0.05; p 0.0008, respectively). shows the differences in terms of each scale (and relative pathological value) between baseline and last follow-up. Regarding Group-OS, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 9 (range 4–16) versus 8 (4–14); regarding the HADS-D scale, a score of 6 (range 2–14) versus 5.5 (2–11); regarding the DT scale, 20 patients (66.7%) versus 19 patients (63.3%) reported a pathological score ≥ 4; regarding the BC scale, 10 patients (33.3%) versus 3 patients (10%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 14–83) versus 43 (range 11–88). Regarding Group-TC, the following ratings of anxiety, depression and distress were reported, comparing baseline to last follow-up evaluation, respectively: regarding the HADS-A scale, a score of 8.5 (range 3–16) versus 7.7 (3–12); regarding the HADS-D scale, a score of 7 (range 2–14) versus 6 (2–12); regarding the DT scale, 32 patients (61.5%) versus 31 patients (59.6%) reported a pathological score ≥ 4; regarding the BC scale, 18 patients (34.6%) versus 8 patients (15.4%) reported an avoidant behavior; regarding the IES-R scale, a score of 51 (range 12–88) versus 46.5 (range 11–80). In general, with respect to baseline, statistically significant differences were observed between the two groups in terms of anxiety (HADS, HADS-A: p 0.00002 and 0.03) in favor of on-site visits. In each group, a statistical improvement was observed in BC ( p 0.01), score ≥ 4. shows, for each scale used, the percentage of patients with pathological score at baseline and at last follow-up for each group (tele-consult and on-site).
The aims of the present analysis were to describe the role of psychological support during radiation therapy in oncological patients, and to evaluate the difference in terms of the emotional states of oncological patients who were followed up by tele-consults or in-person visits, to define the role of telemedicine in this setting. In order to reduce the number of on-site visits, telemedicine has grown during the pandemic, but few studies in the scientific literature have assessed its employment . Telemedicine can improve the burden on the healthcare system by reducing appointments, especially with regard to patients with physical disabilities and dependency on caregivers for better quality of life. Moreover, at the base of the success of telemedicine, there is the need for patients to be able and willing to use technology. A recent evaluation on 15 elderly patients with early breast cancer showed that 13 participants reported a preference for a hybrid care model: telemedicine care plus on-site visits, rather than in-person care alone . The in-person appointment is preferred for 2 reasons: the comfort with familiar face-to-face interactions, and to have a physical exam. In this study, patients preferred in-person appointments during the post-primary treatment phase, while they preferred telemedicine when relationships were well-established and the patients were in follow-up. To our knowledge, in fact, no substantial data evaluating the changes in emotional states of cancer patients treated with radiation therapy are reported. Receiving a cancer diagnosis is associated with higher levels of perceived stress; therefore, at the beginning of RT treatment, psychological support was offered to all patients. In analyzing how many patients adhered to our therapeutic protocol, it emerged that a significant increase in psychological requests were reported during the COVID-19 pandemic in 2020. Particularly, with respect to the July to December 2019 period (pre-COVID), there was a significant increase in patients requesting support (13 patients per month pre COVID-19 period vs. 60 patients per month post-COVID period; p < 0.0001). Regarding anxiety and depression for all populations of the study, based on two scales, the data showed that anxiety was present in roughly 50% of patients with a median HADS-A of 8, while only about 30% of patients suffered depression (median HADS-D of 5). These data are in line with other publications . Moreover, some data in the literature showed that the word “radiation” is often associated with a negative connotation based on media information or a secondhand experience. This produces a high grade of anxiety in patients who are going to receive RT. Gillan et al. conducted a multiphase survey to define the perceptions of radiation in the general population (phase I) and, in phase II, to define the perceptions of RT in patients who received RT . The latter study assessed that in the phase I survey, the public perceptions of radiation were usually negative. In phase II of this analysis, toxic events during and after RT were reported as concerns, such as misperceptions about infertily due to radiation and becoming radioactive. In conclusion, in Canada, many patients refused RT only for fear or misinformation about risks, or they accepted it, but with a high grade of anxiety. For this reason, the psychological support during active therapy, especially RT, was useful. In fact, all patients completed their treatment without delays, reporting a reduction in RT anxiety during the interviews and a “feeling of welcome” at the end of treatment. Regarding the aim of establishing the difference between on-site and telemedicine, 82 patients accepted our psychological support by a psycho-oncologist after RT: 52 out 82 (63.4%) wanted to be followed by a tele-visit (video call or telephone). As reported by Buse et al., and also for our population, COVID-19, physical disability, and transportation burdens were the most common factors for tele-visit preference. The median number of visits during follow-up was 8 (range 4–28), with no difference between the two groups. Anxiety and depression have expression throughout the body. During the psychological interview of all patients, it was possible to reduce the levels of distress and somatic disorders, including fatigue, sleep disorders, gastritis, cognitive ruminations and risk behaviors. Cognitive-behavioral techniques for processing destabilizing events, from diagnosis to related treatments, and relaxation techniques for the management of anxiety and pain, were used to obtain anxiety reduction. Our data showed that the psychological support during and after RT treatment reported an improvement in anxiety and depression, by activating a propositive behavior (BC) for a better quality of life perception. In terms of depression and post-traumatic stress, no improvements were documented. This is probably due to the emotional state of oncological patients after treatment and during follow-up, characterized by the fear of disease recurrence or metastases and risk of death. These results are also evident in each group, in-person evaluation and tele-consult. In the present analysis, patients followed by tele-visits reported comparable quantitative results in terms of depression and post-traumatic stress (DT and IES-R) with respect to the in-person visits group, with the only difference being in terms of anxiety ( p 0.002). In fact, patients who were followed up with the on-site approach reported a better decrease in the anxiety score. Three main reasons could be hypothesized: First of all, in the TC group, a predominance of poor-prognosis diseases was present (brain tumor, metastatic tumor and head and neck tumor), with a high incidence of recurrence post-treatment and a subsequent higher anxiety score. In the OS group, most of the patients had breast cancer (young patients with good prognosis); the high level of anxiety was also reduced during follow-up because no recurrence was reported. For phycologists, the evaluation of expression throughout the body is more difficult to evaluate during tele-consults; thus, the psychological strategy could be longer and less precise. The present data are in line with a recent Cochrane publication. The predominance of telephone-delivered interventions for psychological symptoms is unsurprising. Telephone counseling has been shown to be effective in reducing psychological symptoms, including depression and anxiety, in patient populations other than those affected by cancer. Further, this review has demonstrated that telephone-delivered interventions are being developed for managing a range of physical as well as psychological cancer-related symptoms . Surely, our data have some limitations, including small sample size, no randomization, short follow-up, and so on, but are interesting both for underlining the important role of psycho-oncology for oncological patients and the importance of following up with patients by telemedicine.
As reported in the literature, the data reveal the importance of psychological support for oncological patients . For psychological follow-up, telephone interventions, or tele-visits, are convenient for patients, their families and healthcare workers, as they reduce distress and anxiety and activate a propositive behavior. However, considering the absence of solid data, rigorous research on this topic with more homogeneous populations (according to disease status, age, psychological attitude, etc.) and therapeutic approaches, in a greater sample size, are necessary.
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Radiomics and Radiogenomics in Pelvic Oncology: Current Applications and Future Directions
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d8991424-4811-429e-bec8-9799cc422119
|
10217419
|
Internal Medicine[mh]
|
Radiomics refers to the conversion of medical imaging into high-throughput, quantifiable data in order to analyse disease patterns, guide prognosis and aid in decision making . Since its inception in 2012, data have been extracted and analysed from a variety of imaging modalities, such as computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) scans, in an attempt to determine prognosis and to predict patient outcomes, particularly in the field of surgical oncology . The process can be broadly broken down into three primary steps: the use of automated or semi-automated methods to identify the volumes of interest in a tumour, the generation of multiple quantitative features from raw imaging data regarding the region of interest (ROI), and finally the development of models that predict tumour characteristics and guide decision making . Radiogenomics is an extension of radiomics that combines conventional radiomics techniques with molecular analysis in the form of genomic and transcriptomic data . This particular field of medicine aims to offset the high costs and labour intensity associated with traditional genetic testing via the development of imaging surrogates that may serve as an alternative method for identifying which patients carry specific oncogenes . In short, parameters derived from advanced image processing and analysis can be used to identify specific phenotypic and genotypic characteristics of the tumour, without the need for costly genetic testing . The ultimate goal of radiogenomics is to offset the high costs, workload and invasiveness associated with traditional genetic testing by developing imaging surrogates that have the potential to serve as alternative methods for identifying oncogene status . Its role has been assessed in several fields of oncology, including glioblastoma, breast cancer, renal cell carcinoma and colorectal cancer . While several studies have demonstrated the feasibility of constructing a radiogenomic signature, most lack prospective validation with an external cohort . Artificial intelligence (AI) is increasingly employed in medicine due to its ability to perform higher cognitive functions, such as problem solving and decision making . A subfield within AI, machine learning facilitates the ability to search for data and recognize patterns, allowing the accurate prediction of results or outcomes . This is particularly useful for identifying subtle patterns in large datasets that are often imperceptible to individual review . The incorporation of machine learning with other new fields of research, such as radiomics and radiogenomics, has the potential to revolutionise precision medicine by predicting treatment response, prognosis and patient outcomes . This review aims to shed light on the current implementation of radiomics and radiogenomics in the field of pelvic oncology, paying particular attention to individual subspecialty use and implications for the future. We provide a sample of the current literature within this field, highlighting the strengths, weaknesses and gaps in knowledge that may guide researchers in their future endeavours. Included studies deemed interesting and representative of the literature were selected after relevant articles were searched in the National Institutes of Health (NIH) PubMed database. Broad applications are highlighted in .
2.1. Colorectal (i) Prediction of response to neoadjuvant chemotherapy Several studies have investigated the application of radiomics and radiogenomics in colorectal surgery . Nakanishi et al. developed a radiomics-based model for predicting lateral pelvic lymph node metastases (LPLNM) following neoadjuvant (chemo)radiotherapy (nCRT) for advanced low rectal cancers . A retrospective study of 247 patients demonstrated that a radiomics-based prediction model was superior to the gross measurement of LPLN short-axis diameter in the prediction of LPLNM post-neoadjuvant (chemo)radiotherapy for rectal cancer . This study demonstrated how a radiomics-based prediction model can be applied to avoid unnecessary exposure of patients to high morbidity procedures such as LPLN dissection in those patients deemed ‘low risk’ by the model. Recent studies have investigated the feasibility of radiomics nomograms in predicting a pathological complete response (pCR) . Liu et al. developed a radiomics-based model using pre- and post-nCRT T2 and diffusion-weighted images (DWI) in combination with tumour length, with the ability to predict pCR with a diagnostic accuracy of 94% . Similarly, Wang et al. extracted radiomic features from 183 pre-operative mpMRI scans to develop a model capable of predicting the response to nCRT . Their nomogram, which included MRI T-stage, circumferential resection margin and apparent diffusion coefficient values, predicted a good response to nCRT with a specificity of 88% and a sensitivity of 71% . (ii) Prediction of mutation status The role of fluorodeoxyglucose ( 18 F-FDG) PET in the assessment of KRAS mutations in colorectal cancer is another topic of research in colorectal radiogenomics . This radiotracer detects areas of abnormal glucose metabolism and serves to evaluate metabolic and tumour activity . Several studies have demonstrated a higher standardised uptake value (SUV) of this radiotracer in patients with KRAS mutations, with a reported diagnostic accuracy of up to 75% . MRI remains the gold standard imaging modality for rectal cancer due to its ability to assess extramural spread, accurately stage and detect local recurrence of the disease . Studies have demonstrated that rectal tumours with KRAS mutations are more likely to exhibit a longer axial length as well as a greater axial: longitudinal ratio on pre-treatment MRI . Since genomic analysis is now essential to guide therapeutic decisions in colorectal cancer, the development of radiogenomic models capable of predicting the involvement of various genetic mutations will allow for targeted therapy and improved patient outcomes . Radiogenomic prediction of specific genetic aberrations has the potential to serve as a non-invasive alternative to conventional genetic testing in the future . Further studies should focus on applying this novel technology to other genetic mutations as well as validating the findings of existing studies. (iii) Prediction of oncological outcomes Exenterative surgery can be associated with major complications . Despite advancements in surgical techniques and our understanding of advanced and recurrent pelvic malignancies, the rate of recurrence after pelvic exenteration remains unacceptably high . Radiomics and radiogenomics offer the potential to offset the risk of recurrence in this cohort of patients by accurately predicting which patients are likely to experience recurrence and subsequently avoid exenterative surgery and its associated morbidity . Badic et al. aimed to use radiomics to assess the value of contrast-enhanced CT scans as predictors of recurrence in patients with stage II and III colorectal cancer . The authors used three separate machine learning models to predict disease-free survival (DFS) in this cohort of patients. A signature was developed based on clinical, histopathological and radiomic characteristics, and a predictor of recurrence showed value when compared with the traditional staging . Similarly, Jayaprakasam et al. demonstrated the capabilities of MRI radiomics in predicting tumour recurrence and response to neoadjuvant chemotherapy in patients with locally advanced rectal cancer . Their study exploited the hypothesis that the tumour and mesorectal fat interaction results in microscopic changes to adipocytes and subtle changes on MRI that are invisible to the naked eye . In terms of predicting response, eleven radiomics features differed significantly between complete and non-complete responders. The final predictive model obtained a diagnostic accuracy of 83.9% and an area under the curve (AUC) of 0.89. Univariate analysis revealed 36 radiomics features that were significant in predicting local recurrence. The final predictive model obtained a diagnostic accuracy of 78.3% and an AUC of 0.79 . These studies demonstrated the feasibility and efficacy of employing radiomics models in pelvic malignancies to predict various oncological outcomes such as response to chemotherapy and risk of recurrence, with relatively strong accuracy. 2.2. Urological (i) Prostate cancer; prediction of oncological outcomes Recent years have seen a rapid increase in the number of publications applying radiomics and radiogenomics to genitourinary cancers . In the realm of prostate cancer, Bourbonne et al. developed a multiparametric MRI (mpMRI)-based radiomic model to predict the risk of biochemical recurrence (BCR) and BCR-free survival post-radical prostatectomy . The authors used a size-zone emphasis from apparent diffusion coefficient (ADC) maps extracted from 107 pre-therapeutic diffusion-weighted images, and demonstrated a balanced accuracy of 78% in predicting BCR post-radical prostatectomy . This model was externally validated and may be used to stratify patients post-operatively by the risk of recurrence and tailor post-operative management accordingly. External validation is a labour-intensive but valuable component of radiomic nomogram construction, verifying reproducibility across other centres. The Miami MAST trial ( ClinicalTrials.gov : NCT02242773), currently ongoing, aims to extract data from mpMRI-guided MRI/ultrasound (US) fusion biopsies in order to identify high-grade tumours early on in the investigations . This study is estimated to be completed in 2024 and will aid physicians in their decision to commence radical treatment earlier in high-risk diseases. The current literature on radiogenomics in prostate cancer is limited, particularly regarding the detection of specific biomarkers that may be used to guide prognosis and treatment decisions . The three current commercially approved genomic panels performed on prostate biopsy cores (Genomic Health’s Oncotype Dx test ®® , Myriad’s Prolaris test ®® and Genome Dx’s Decipher test ®® ) will provide a good basis for future research into prostate cancer radiogenomics . (ii) Bladder cancer; prediction of oncological outcomes Several studies have investigated the role of radiomics and radiogenomics in predicting clinical outcomes in bladder cancer . Lin et al. sequestered RNA sequencing data, radiomics features and clinical parameters of 62 patients with transitional cell carcinoma (TCC) of the bladder to create an integrative nomogram capable of stratifying patients into low- and high-risk groups and subsequently predicting progression-free interval (PFI) with excellent accuracy . CD8A is a novel protective gene in bladder cancer and a marker of immunotherapeutic response and immune cell infiltration . Low expression is associated with immunotherapeutic failure and poor oncological outcomes . Zheng et al. developed a radiomics signature using pre-operative radiomics features and RNA-sequencing data of 111 bladder tumour samples to predict CD8A expression and subsequent response to immunotherapy . Receiver operating characteristic (ROC) curves revealed that the nomogram had good performance in survival prediction with 1-, 3- and 5-year area AUC of 0.679, 0.722 and 0.722, respectively. This study demonstrated the ability of a radiomics signature based on nine MRI-derived radiomics features to predict the prognosis and immunotherapeutic susceptibility in patients with bladder cancer . While studies aiming to predict muscle invasiveness of bladder cancer are bountiful, those aiming to construct nomograms capable of predicting oncological outcomes are scarce . Future research should focus on predicting the response to neoadjuvant therapy and the long-term oncological outcomes. 2.3. Gynaecological (i) Ovarian cancer; prediction of BRCA status Despite recent progress in chemotherapeutic and surgical approaches, the high morbidity and mortality associated with many gynaecological malignancies necessitate an improved understanding of how these tumours behave from a radiological and genetic perspective . Few studies have described the use of radiomics and radiogenomics in gynae-oncology . Nero et al. developed an automated machine learning pipeline model in order to identify gBRCA1/2 status based on ultrasound images of healthy ovaries, with encouraging performance . With a positive predictive value (PPV) of 0.87, only 13/100 women will be exposed to unnecessary genetic testing. Despite this, the model describes a negative predictive value (NPV) of 0.67, implying that 27/100 women carrying the gene would be missed; thus, it is a major limitation of the approach . (ii) Endometrial cancer; prediction of oncological outcomes Hoivik et al. integrated MRI radiomic features with histologic, transcriptomic and molecular biomarkers in their study of 866 patients with endometrial cancer in an attempt to identify those with aggressive tumour features . In this study, a fully automated machine learning-based tumour segmentation algorithm reproduced the same radiomic prognostic groups as manual whole-volume tumour radiomic profiling by radiologists . The authors identified an 11-gene high-risk signature associated with poor survival that will aid in prognosis and guide treatment decisions in patients with endometrial carcinoma. Further research is necessary to bring radiomics-based research within gynae-oncology to the same standard as currently available in the fields of colorectal and urology. 2.4. Sarcoma The use of artificial intelligence in soft-tissue sarcomas is a relatively novel concept that aims to serve as a non-invasive method of providing information regarding the diagnosis and prognosis of tumours . The employment of radiomic texture analysis in this field has resulted in the development of radiomics MRI-based models that can distinguish histotypes, determine grades and predict response and overall survival . Using T1 and fat-suppressed-T2 weighted imaging, Wang et al. identified specific radiomic features that were significantly correlated with malignant soft-tissue lesions and subsequently constructed a radiomics nomogram with superior predictive performance than that of the clinical model based on the experience of radiologists . Peeken et al. compared the value of MRI-based radiomics with expert-derived clinical profiling for the prediction of overall survival (OS) . A total of 105 radiomic features were extracted from the images of 108 patients and subjected to three separate machine learning techniques to predict OS. The findings were compared to the semantic imaging features determined by radiologists. T2-weighted sequence and T1-weighted fat-saturated sequence radiomic models were superior to semantic imaging features in determining the prognosis of soft-tissue sarcomas . To our knowledge, no studies investigating the use of radiomics or radiogenomics in the diagnosis and prognosis of intra-abdominal sarcomatous disease have been published. Future studies should focus on filling this void, paying particular focus to the prediction of oncological outcomes in patients with this aggressive disease.
(i) Prediction of response to neoadjuvant chemotherapy Several studies have investigated the application of radiomics and radiogenomics in colorectal surgery . Nakanishi et al. developed a radiomics-based model for predicting lateral pelvic lymph node metastases (LPLNM) following neoadjuvant (chemo)radiotherapy (nCRT) for advanced low rectal cancers . A retrospective study of 247 patients demonstrated that a radiomics-based prediction model was superior to the gross measurement of LPLN short-axis diameter in the prediction of LPLNM post-neoadjuvant (chemo)radiotherapy for rectal cancer . This study demonstrated how a radiomics-based prediction model can be applied to avoid unnecessary exposure of patients to high morbidity procedures such as LPLN dissection in those patients deemed ‘low risk’ by the model. Recent studies have investigated the feasibility of radiomics nomograms in predicting a pathological complete response (pCR) . Liu et al. developed a radiomics-based model using pre- and post-nCRT T2 and diffusion-weighted images (DWI) in combination with tumour length, with the ability to predict pCR with a diagnostic accuracy of 94% . Similarly, Wang et al. extracted radiomic features from 183 pre-operative mpMRI scans to develop a model capable of predicting the response to nCRT . Their nomogram, which included MRI T-stage, circumferential resection margin and apparent diffusion coefficient values, predicted a good response to nCRT with a specificity of 88% and a sensitivity of 71% . (ii) Prediction of mutation status The role of fluorodeoxyglucose ( 18 F-FDG) PET in the assessment of KRAS mutations in colorectal cancer is another topic of research in colorectal radiogenomics . This radiotracer detects areas of abnormal glucose metabolism and serves to evaluate metabolic and tumour activity . Several studies have demonstrated a higher standardised uptake value (SUV) of this radiotracer in patients with KRAS mutations, with a reported diagnostic accuracy of up to 75% . MRI remains the gold standard imaging modality for rectal cancer due to its ability to assess extramural spread, accurately stage and detect local recurrence of the disease . Studies have demonstrated that rectal tumours with KRAS mutations are more likely to exhibit a longer axial length as well as a greater axial: longitudinal ratio on pre-treatment MRI . Since genomic analysis is now essential to guide therapeutic decisions in colorectal cancer, the development of radiogenomic models capable of predicting the involvement of various genetic mutations will allow for targeted therapy and improved patient outcomes . Radiogenomic prediction of specific genetic aberrations has the potential to serve as a non-invasive alternative to conventional genetic testing in the future . Further studies should focus on applying this novel technology to other genetic mutations as well as validating the findings of existing studies. (iii) Prediction of oncological outcomes Exenterative surgery can be associated with major complications . Despite advancements in surgical techniques and our understanding of advanced and recurrent pelvic malignancies, the rate of recurrence after pelvic exenteration remains unacceptably high . Radiomics and radiogenomics offer the potential to offset the risk of recurrence in this cohort of patients by accurately predicting which patients are likely to experience recurrence and subsequently avoid exenterative surgery and its associated morbidity . Badic et al. aimed to use radiomics to assess the value of contrast-enhanced CT scans as predictors of recurrence in patients with stage II and III colorectal cancer . The authors used three separate machine learning models to predict disease-free survival (DFS) in this cohort of patients. A signature was developed based on clinical, histopathological and radiomic characteristics, and a predictor of recurrence showed value when compared with the traditional staging . Similarly, Jayaprakasam et al. demonstrated the capabilities of MRI radiomics in predicting tumour recurrence and response to neoadjuvant chemotherapy in patients with locally advanced rectal cancer . Their study exploited the hypothesis that the tumour and mesorectal fat interaction results in microscopic changes to adipocytes and subtle changes on MRI that are invisible to the naked eye . In terms of predicting response, eleven radiomics features differed significantly between complete and non-complete responders. The final predictive model obtained a diagnostic accuracy of 83.9% and an area under the curve (AUC) of 0.89. Univariate analysis revealed 36 radiomics features that were significant in predicting local recurrence. The final predictive model obtained a diagnostic accuracy of 78.3% and an AUC of 0.79 . These studies demonstrated the feasibility and efficacy of employing radiomics models in pelvic malignancies to predict various oncological outcomes such as response to chemotherapy and risk of recurrence, with relatively strong accuracy.
(i) Prostate cancer; prediction of oncological outcomes Recent years have seen a rapid increase in the number of publications applying radiomics and radiogenomics to genitourinary cancers . In the realm of prostate cancer, Bourbonne et al. developed a multiparametric MRI (mpMRI)-based radiomic model to predict the risk of biochemical recurrence (BCR) and BCR-free survival post-radical prostatectomy . The authors used a size-zone emphasis from apparent diffusion coefficient (ADC) maps extracted from 107 pre-therapeutic diffusion-weighted images, and demonstrated a balanced accuracy of 78% in predicting BCR post-radical prostatectomy . This model was externally validated and may be used to stratify patients post-operatively by the risk of recurrence and tailor post-operative management accordingly. External validation is a labour-intensive but valuable component of radiomic nomogram construction, verifying reproducibility across other centres. The Miami MAST trial ( ClinicalTrials.gov : NCT02242773), currently ongoing, aims to extract data from mpMRI-guided MRI/ultrasound (US) fusion biopsies in order to identify high-grade tumours early on in the investigations . This study is estimated to be completed in 2024 and will aid physicians in their decision to commence radical treatment earlier in high-risk diseases. The current literature on radiogenomics in prostate cancer is limited, particularly regarding the detection of specific biomarkers that may be used to guide prognosis and treatment decisions . The three current commercially approved genomic panels performed on prostate biopsy cores (Genomic Health’s Oncotype Dx test ®® , Myriad’s Prolaris test ®® and Genome Dx’s Decipher test ®® ) will provide a good basis for future research into prostate cancer radiogenomics . (ii) Bladder cancer; prediction of oncological outcomes Several studies have investigated the role of radiomics and radiogenomics in predicting clinical outcomes in bladder cancer . Lin et al. sequestered RNA sequencing data, radiomics features and clinical parameters of 62 patients with transitional cell carcinoma (TCC) of the bladder to create an integrative nomogram capable of stratifying patients into low- and high-risk groups and subsequently predicting progression-free interval (PFI) with excellent accuracy . CD8A is a novel protective gene in bladder cancer and a marker of immunotherapeutic response and immune cell infiltration . Low expression is associated with immunotherapeutic failure and poor oncological outcomes . Zheng et al. developed a radiomics signature using pre-operative radiomics features and RNA-sequencing data of 111 bladder tumour samples to predict CD8A expression and subsequent response to immunotherapy . Receiver operating characteristic (ROC) curves revealed that the nomogram had good performance in survival prediction with 1-, 3- and 5-year area AUC of 0.679, 0.722 and 0.722, respectively. This study demonstrated the ability of a radiomics signature based on nine MRI-derived radiomics features to predict the prognosis and immunotherapeutic susceptibility in patients with bladder cancer . While studies aiming to predict muscle invasiveness of bladder cancer are bountiful, those aiming to construct nomograms capable of predicting oncological outcomes are scarce . Future research should focus on predicting the response to neoadjuvant therapy and the long-term oncological outcomes.
(i) Ovarian cancer; prediction of BRCA status Despite recent progress in chemotherapeutic and surgical approaches, the high morbidity and mortality associated with many gynaecological malignancies necessitate an improved understanding of how these tumours behave from a radiological and genetic perspective . Few studies have described the use of radiomics and radiogenomics in gynae-oncology . Nero et al. developed an automated machine learning pipeline model in order to identify gBRCA1/2 status based on ultrasound images of healthy ovaries, with encouraging performance . With a positive predictive value (PPV) of 0.87, only 13/100 women will be exposed to unnecessary genetic testing. Despite this, the model describes a negative predictive value (NPV) of 0.67, implying that 27/100 women carrying the gene would be missed; thus, it is a major limitation of the approach . (ii) Endometrial cancer; prediction of oncological outcomes Hoivik et al. integrated MRI radiomic features with histologic, transcriptomic and molecular biomarkers in their study of 866 patients with endometrial cancer in an attempt to identify those with aggressive tumour features . In this study, a fully automated machine learning-based tumour segmentation algorithm reproduced the same radiomic prognostic groups as manual whole-volume tumour radiomic profiling by radiologists . The authors identified an 11-gene high-risk signature associated with poor survival that will aid in prognosis and guide treatment decisions in patients with endometrial carcinoma. Further research is necessary to bring radiomics-based research within gynae-oncology to the same standard as currently available in the fields of colorectal and urology.
The use of artificial intelligence in soft-tissue sarcomas is a relatively novel concept that aims to serve as a non-invasive method of providing information regarding the diagnosis and prognosis of tumours . The employment of radiomic texture analysis in this field has resulted in the development of radiomics MRI-based models that can distinguish histotypes, determine grades and predict response and overall survival . Using T1 and fat-suppressed-T2 weighted imaging, Wang et al. identified specific radiomic features that were significantly correlated with malignant soft-tissue lesions and subsequently constructed a radiomics nomogram with superior predictive performance than that of the clinical model based on the experience of radiologists . Peeken et al. compared the value of MRI-based radiomics with expert-derived clinical profiling for the prediction of overall survival (OS) . A total of 105 radiomic features were extracted from the images of 108 patients and subjected to three separate machine learning techniques to predict OS. The findings were compared to the semantic imaging features determined by radiologists. T2-weighted sequence and T1-weighted fat-saturated sequence radiomic models were superior to semantic imaging features in determining the prognosis of soft-tissue sarcomas . To our knowledge, no studies investigating the use of radiomics or radiogenomics in the diagnosis and prognosis of intra-abdominal sarcomatous disease have been published. Future studies should focus on filling this void, paying particular focus to the prediction of oncological outcomes in patients with this aggressive disease.
Despite significant potential in the field of pelvic oncology, current evidence is limited by variability in feature extraction and a lack of reproducibility . Model performance is sensitive to many intrinsic variables, including heterogeneous image acquisition parameters, segmentations and feature extraction software, as well as small and mixed patient cohorts . As research in this field advances, more open-source databases and software packages are being made available in an attempt to standardise models and accelerate the development and external validation of these signatures . Future studies should focus on standardising the imaging protocols and radiomic techniques . Standards for radiomic features need to be set in order to allow comparability between studies and reproducibility for new studies. Similarly, the stability and reproducibility of radiomic models for predicting prognosis must be externally assessed prior to their application in the clinical setting . Internal validation may not be sufficient to extrapolate performance in an external setting due to relatively small datasets, as seen in most of the current studies, and external datasets should thus be validated in a large multicentre setting prior to implementation in clinical practice . Multiple studies have attempted to identify reproducible radiomics features to improve the repeatability and application of radiomics and radiogenomics. Traverso et al. performed a systematic review to identify radiomics features that were repeatable and reproducible . The authors found that first-order features (histogram-based), in particular entropy, had higher reproducibility than shape metrics and textural features. The Image Biomarker Standardisation Initiative (IBSI), published in 2020, aimed to standardise a set of radiomics features . Over three phases, the authors achieved good to excellent reproducibility for 167 different radiomics features utilising CT, MRI and FDG-PET in 51 patients with soft-tissue sarcoma. Similarly, Pfaehler et al. developed a checklist aiming to simplify and improve the reporting of radiomic signatures, with the goal of eventually guaranteeing full replication and validation of these studies . Further systematic reviews focusing on the repeatability and reproducibility of radiomics features are necessary to further improve the standardisation of results from radiomics studies .
The use of radiomics and radiogenomics for predicting correlations with genetic or transcriptomic abnormalities of tumours and subsequently determining prognosis and guiding treatment decisions needs much larger data studies to become a validated tool . Future studies should focus on addressing the limitations outlined previously and evaluating the datasets in a multicentre, prospective setting. There is a paucity of evidence in the literature surrounding the use of radiomics and radiogenomics to identify patients at high risk of recurrence of locally advanced or locally recurrent pelvic cancers. The development of a radiomics nomogram aimed at predicting disease recurrence in this cohort of patients may offer surgeons the ability to avoid highly morbid procedures, such as exenteration in patients who are likely to recur, or at least counsel patients better regarding expected outcomes . Quantification of circulating tumour DNA (ctDNA) and cell-free DNA (cfDNA) represents a novel area of research in cancer detection and evaluation of disease burden, with the potential to revolutionise our assessment of therapeutic responses and our understanding of personalised medicine as a whole . Current applications include the detection of microscopic residual disease following radiotherapy, an alternative non-invasive method of genotyping, as well as early detection of tumour recurrence . Research on ctDNA in the field of radiomics and radiogenomics is scarce . Lafata et al. aimed to create patient-specific radiogenomic expression patterns to guide prognosis by combining CT radiomics, next-generation sequencing of ctDNA and serum cfDNA in patients with locally advanced lung cancer receiving chemoradiotherapy . Two distinct radiomic signatures were identified prior to treatment, which were subsequently associated with the presence of TP53 mutations within ctDNA and changes in cfDNA two weeks post-chemoradiation. The authors found that heterogeneous and low-attenuating disease, without a detectable ctDNA TP53 mutation, was linked to an early surge in post-treatment cfDNA concentration and improved overall survival . These findings highlight the feasibility and efficacy of this approach in predicting treatment response and guiding prognosis. Further studies, ideally larger randomised clinical studies, are necessary to validate these findings. Perhaps one of the greatest obstacles to radiomic-based research is feature reliability . Heterogeneity and uncertainty can arise from many areas within a complex workflow, ultimately impeding feature reproducibility, stability and validity . In their review of 481 radiomics studies, Xue et al. attempted to define reliability using intraclass correlation coefficient (ICC) expression . ICC is a reliability index widely adopted in the medical literature, which can be applied to any radiomic feature that has continuous values . The authors conclude by offering several suggestions for researchers carrying out radiomics-based research to mitigate the pitfalls identified in their analysis of 481 manuscripts. Koo et al. also provide clinical researchers with a practical step-by-step guideline to select the correct form of ICC and to avoid selecting inappropriate ICC forms that may result in misleading interpretations .
Despite a rapid increase in publications investigating the use of radiomics and radiogenomics in pelvic oncology, current evidence is limited by poor reproducibility and small datasets . In the era of personalised medicine, this novel field of research has significant potential, particularly for predicting prognosis and guiding therapeutic decisions . Future research may provide fundamental data on how we treat this cohort of patients, with the aim of reducing the exposure of high-risk patients to highly morbid procedures. While many of the studies discussed in our narrative review are small, single-centre projects, they provide the foundation necessary and proof of efficacy required for the production of further high-impact studies in the future. Large-scale, multi-institutional studies with external validation of models are required to ultimately change clinical practice.
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Direct and Secondary Transfer of Touch DNA on a Credit Card: Evidence Evaluation Given Activity Level Propositions and Application of Bayesian Networks
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095f9290-7907-4d08-ad86-e023111f7be1
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10217942
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Forensic Medicine[mh]
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Forensic genetics has been considered the gold standard for investigative and judiciary purposes to assess whose DNA has been recovered from a crime scene and to aid the Court in identifying the individuals who may have taken part in the dynamic of the criminal event . However, the identification of the donor of a biological trace in and of itself is not sufficient anymore. Forensic experts are increasingly required to aid in the understanding of the relevance of said biological trace for the criminal case in question. Questions concerning the timing and mechanisms of deposition, and the effect that other variables, such as recovery and persistence, contamination, and presence of background DNA, may have on findings are ever more common. This is particularly pertinent in the case of analysis and evaluation of trace DNA, meaning scarce quantities of DNA, usually a mixture, not attributed to a specific body fluid . Since hands are the most prominent means of interaction with objects and surroundings (not necessarily in a criminal offence), these considerations also apply to touch DNA, meaning biological material released from the skin, derived from shed keratinocytes, nucleated cells from other body parts, secretions, and cell-free DNA [ , , , , , , , ]. The last is particularly informative and yields a higher amount of DNA than other components; however, its origin is not yet clear [ , , ]. The amount of touch DNA that individuals may deposit, known as shedder status, varies greatly due to intra- and inter-individual variables. Body location, age, sex, skin conditions, activity performed, type and length of contact, and the surface of contact are all factors that increase variation in terms of shedding propensity [ , , , , , , , , ]. Thanks to the increase in sensitivity of the current forensic genetics techniques, it is now possible to recover and obtain better DNA profiles from minute quantities of biological material, such as trace and touch DNA. Despite the obvious advantage of being able to infer more robust results and relevant information from limited amounts of DNA, this improvement led to the generation of more complex DNA profiles, often multi-contributors, with increased detection of background DNA [ , , , , , , , , ], contamination events, and DNA profiles not resulting from a direct transfer. In particular, DNA transfer, especially secondary or higher level through one or more intermediate vectors, is a well-recognised phenomenon that further complicates DNA analysis results’ interpretation [ , , , , , , , , , , , ]. Specifically, in a judiciary setting, such considerations moved the evaluation of the DNA evidence to a further step in the hierarchy of propositions. With hierarchy of propositions, we define a set of broad categories at which the evidence can be evaluated depending on the characteristics that are taken into consideration, the information available, and the propositions that are being formulated . In particular, the evaluative framework was moved from a (sub)source level, where the question “Whose DNA is this?” is answered , to the activity level, where the source of the DNA is not disputed, but the mechanism of transfer and the influence of other case-specific factors are [ , , , , ]. Assuming that the suspect is the origin of a biological trace, it is essential to explore how the DNA of the suspect got to the crime scene, either via a criminal activity, secondary or higher degree transfer, or a legitimate activity . For this kind of estimation, the experts assess the strength of the evidence given competing alternative hypotheses (Hp and Hd) regarding the activity being disputed. Possible mechanisms and instances of DNA transfer and their relative likelihood have to be presented, in light of the Influence that other case-specific factors may have. Therefore, it’s important to properly evaluate the case circumstances and to assess if a trace, given its qualitative and quantitative features, could be the result of a primary or further level of transfer [ , , , , , , , , ]. This approach is termed evaluative reporting, and it has to be objective and based on case-relevant information [ , , ]. It is recommended that the expert opinion is informed by empirical and experimental data, present in the literature or produced in-house. Given the high number of case-specific relevant variables and of data regarding the activities that may have led to the deposition and transfer of DNA, the experts are increasingly relying on the use of Bayesian Networks (BN) [ , , , , , ]. BNs are graphical representations of the case that include all the relevant variables and display their dependency relationships and interactions. Taking into consideration all the important and pertinent elements to the case, BNs allow making complex statistical evaluations of the DNA analysis results, based on every possible combination of events and on which disputed activity is assumed as true (according to either Hp or Hd) [ , , , , , , ]. As mentioned above, Bayesian Network construction and more generally DNA evidence evaluation at the activity level have to be informed by experimental data. When probabilistic observations on relevant case-specific variables are not available from previously published, peer-reviewed studies, the expert must either adapt existing data, if not specific, or produce in-house results from ad hoc experiments. The present study is an example of a touch DNA evidence evaluation given activity level propositions regarding the direct manipulation of a credit card and, albeit this assessment was not required for judiciary purposes, the case scenario which prompted such an investigation.
2.1. Case Circumstances For a case of alleged fraudulent use of a credit card, our laboratory was commissioned to perform DNA analysis and evaluation only at the sub-source level. However, the case circumstances prompted the experimental reproduction of the case and the investigation of touch DNA transfer mechanisms and trace evaluation given activity level propositions. The credit card owner (O) works in an office shared with another individual. While on sick leave, she noticed unauthorised withdrawals from her bank account. The card linked to said bank account was left in her private locker in the office while she was on leave. Given circumstantial evidence, the person suspected of having unduly used the card was her co-worker (POI). POI stated that he never used O’s card and that he never handled it. He stated, however, that O was known for not storing the card in a personal and safe place. She often left it on her and other co-workers’ desks, including POI’s. When getting back to the office, O did not touch the card again, only checking for its presence in her locker. It is assumed that no one else touched the card since it got back into the owner’s possession. The DNA traces found on O’s card turned out to be compatible with a mixture of O, POI, and an unknown individual, to which POI was the second most prominent contributor. Relative mixture proportions were calculated with EuroForMix v. 4.0.1 (quantitative LR model) and returned values of 0.5 for O, 0.4 for POI, and 0.1 for the unknown individual. We decided it was worthwhile replicating and studying the event, to investigate the differences (if existing) in DNA traces’ characteristics for primary deposition and secondary transfer of touch DNA on a credit card, a non-porous plastic support. Particularly for the secondary transfer, additional interest arose from the social setting, meaning that the individuals shared the same working space, thus possibly increasing the complexity of the results interpretation [ , , ]. The study was approved by the Ethics Committee of the University of Perugia (protocol code 92184). To mirror the co-working conditions of this scenario, laboratory staff volunteered for the study and signed informed consent to the analysis of the deposited DNA traces and to the use of their genotypes, present in the exclusion database of the laboratory, for reference purposes. A total of four participants were included in the study. However, to minimize the possibility of contamination events, the sample collection and laboratory processing phase were carried out by personnel who did not participate in the study, and, during this process, participants were not allowed in the laboratory spaces, after their careful sterilization. The experimental set-up of the study consisted of four phases. 2.2. Phase I—Shedder Status Since shedder status influences transfer, persistence, and recovery of DNA, phase I of the experiment consisted of the shedder status assessment for all participants (coded from A to D). Participants were first asked to wash their hands with antibacterial soap and then dry them with a sterile, personal towel. Immediately after handwashing, participants were asked to hold, with both hands and for 30 s, a 15 mL falcon tube that had been previously sterilized with UV lights for 1 h. The falcon tubes were immediately sampled and labelled with the code of the participant and the time point of the sampling, t 0. The volunteers were asked to repeat the experiment at three additional time points after handwashing, 1 h, 2 h, and 3 h, respectively. These samples were labelled with the code of the participant and the relative time point, meaning t 1, t 2, and t 3. During the time elapsed between the handwashing and the experiment at t 3, participants were not given any particular imposition but were instructed to carry out routine daily activities; however, hand washing was not allowed. Four shedding samples per participant were collected, for a total of 16 samples. The focus of this phase was not strictly the investigation of shedding status variability among individuals. It was, rather, the informed pairing of participants in phases II and III, to obtain variability in the pairings while also maintaining even differences among couples. The aim is to preserve similar experimental conditions with different individuals. Additionally, information on the participants’ shedder status was collected to better interpret phases II and III results. 2.3. Phase II—Direct Transfer Pairings of participants were established based on the results of phase I. In phase II, the direct transfer scenario was replicated. Participants were asked to provide one or more frequently used credit cards, for a total of 11 cards. The card, whose owner was indicated with the code “O”, was then manipulated, without gloves, by the counterpart in the pairing, labelled as “POI”. The card was handled for 30 s by POI, who then simulated an ATM cash withdrawal. To replicate realistic case-work conditions, the cards were not sterilized, nor were the participants asked to wash their hands. The cards were then immediately sampled, and the samples were assigned the code from D1 to D11 (“D” standing for Direct transfer). 2.4. Phase III—Secondary Transfer After the sampling in phase II, the cards were sterilised by means of UV lights, 30 min per side, to remove any possible DNA trace from POI that may have persisted on the cards. The owners were then asked to use them as they regularly do for a month. After this period, phase III was carried out: the pairings from phase II were maintained and the same number of cards was used (11 cards). In this phase, the secondary transfer scenario was investigated, meaning that the owner’s (O) card was placed and moved around the surface of POI’s desk, applying slight pressure, for 30 s. To conform to realistic experimental conditions, the desks’ surfaces and the cards were not sterilized. However, the desks are personal to the participants and before the card was placed upon them, the POIs touched the surfaces with their bare hands and forearms, to replicate a “uniform” use of the desk among participants. The cards were immediately sampled, and the samples were given codes ranging from S1 to S11 (“S” standing for Secondary transfer). 2.5. Phase IV—Laboratory Processing and Statistical Analysis Through all the experimental phases, the items (falcon tubes and cards) were sampled by using the tape lifting technique. Despite a more difficult interpretation of results, this recovery method has yielded a higher amount of DNA than other techniques [ , , , ]. All samples were extracted employing the phenol–chloroform organic method , and the pellet of each sample was resuspended in 30 μL of sterile water. The DNA extracts were quantified using the PowerQuant ® System (Promega, Madison, WI, USA) . As described in , quantification results were quite low; therefore, samples were later amplified undiluted as is, and no DNA concentration threshold was applied to further process the samples, given the explorative approach of this study. PCR amplification was carried out using the PowerPlex ® ESX17 Fast kit (Promega, Madison, WI, USA), as per the manufacturer’s instructions ; given the nature of the study, no modifications for low template DNA amplification were applied. Capillary electrophoresis followed by means of the Applied Biosystems TM SeqStudio TM Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with the following conditions: injection time, 7 s; injection voltage, 1200 V; run time, 1440 s; run voltage, 9000 V. Results were analysed using the software GeneMapper ® ID-X v 1.6 (Thermo Fisher Scientific, Waltham, MA, USA). Analytical threshold for alleles (AT) was set at 50 RFU. As described in , further statistical analyses and calculation of LR at the sub-source level (LRϕ) were performed using EuroForMix v 4.0.1. Reference profiles of the participants were already available in the laboratory exclusion database. The LR at activity level (LRα) was calculated manually and results were confirmed by using the software Hugin Expert. The plots in this paper were created by using the R package ggplot2.
For a case of alleged fraudulent use of a credit card, our laboratory was commissioned to perform DNA analysis and evaluation only at the sub-source level. However, the case circumstances prompted the experimental reproduction of the case and the investigation of touch DNA transfer mechanisms and trace evaluation given activity level propositions. The credit card owner (O) works in an office shared with another individual. While on sick leave, she noticed unauthorised withdrawals from her bank account. The card linked to said bank account was left in her private locker in the office while she was on leave. Given circumstantial evidence, the person suspected of having unduly used the card was her co-worker (POI). POI stated that he never used O’s card and that he never handled it. He stated, however, that O was known for not storing the card in a personal and safe place. She often left it on her and other co-workers’ desks, including POI’s. When getting back to the office, O did not touch the card again, only checking for its presence in her locker. It is assumed that no one else touched the card since it got back into the owner’s possession. The DNA traces found on O’s card turned out to be compatible with a mixture of O, POI, and an unknown individual, to which POI was the second most prominent contributor. Relative mixture proportions were calculated with EuroForMix v. 4.0.1 (quantitative LR model) and returned values of 0.5 for O, 0.4 for POI, and 0.1 for the unknown individual. We decided it was worthwhile replicating and studying the event, to investigate the differences (if existing) in DNA traces’ characteristics for primary deposition and secondary transfer of touch DNA on a credit card, a non-porous plastic support. Particularly for the secondary transfer, additional interest arose from the social setting, meaning that the individuals shared the same working space, thus possibly increasing the complexity of the results interpretation [ , , ]. The study was approved by the Ethics Committee of the University of Perugia (protocol code 92184). To mirror the co-working conditions of this scenario, laboratory staff volunteered for the study and signed informed consent to the analysis of the deposited DNA traces and to the use of their genotypes, present in the exclusion database of the laboratory, for reference purposes. A total of four participants were included in the study. However, to minimize the possibility of contamination events, the sample collection and laboratory processing phase were carried out by personnel who did not participate in the study, and, during this process, participants were not allowed in the laboratory spaces, after their careful sterilization. The experimental set-up of the study consisted of four phases.
Since shedder status influences transfer, persistence, and recovery of DNA, phase I of the experiment consisted of the shedder status assessment for all participants (coded from A to D). Participants were first asked to wash their hands with antibacterial soap and then dry them with a sterile, personal towel. Immediately after handwashing, participants were asked to hold, with both hands and for 30 s, a 15 mL falcon tube that had been previously sterilized with UV lights for 1 h. The falcon tubes were immediately sampled and labelled with the code of the participant and the time point of the sampling, t 0. The volunteers were asked to repeat the experiment at three additional time points after handwashing, 1 h, 2 h, and 3 h, respectively. These samples were labelled with the code of the participant and the relative time point, meaning t 1, t 2, and t 3. During the time elapsed between the handwashing and the experiment at t 3, participants were not given any particular imposition but were instructed to carry out routine daily activities; however, hand washing was not allowed. Four shedding samples per participant were collected, for a total of 16 samples. The focus of this phase was not strictly the investigation of shedding status variability among individuals. It was, rather, the informed pairing of participants in phases II and III, to obtain variability in the pairings while also maintaining even differences among couples. The aim is to preserve similar experimental conditions with different individuals. Additionally, information on the participants’ shedder status was collected to better interpret phases II and III results.
Pairings of participants were established based on the results of phase I. In phase II, the direct transfer scenario was replicated. Participants were asked to provide one or more frequently used credit cards, for a total of 11 cards. The card, whose owner was indicated with the code “O”, was then manipulated, without gloves, by the counterpart in the pairing, labelled as “POI”. The card was handled for 30 s by POI, who then simulated an ATM cash withdrawal. To replicate realistic case-work conditions, the cards were not sterilized, nor were the participants asked to wash their hands. The cards were then immediately sampled, and the samples were assigned the code from D1 to D11 (“D” standing for Direct transfer).
After the sampling in phase II, the cards were sterilised by means of UV lights, 30 min per side, to remove any possible DNA trace from POI that may have persisted on the cards. The owners were then asked to use them as they regularly do for a month. After this period, phase III was carried out: the pairings from phase II were maintained and the same number of cards was used (11 cards). In this phase, the secondary transfer scenario was investigated, meaning that the owner’s (O) card was placed and moved around the surface of POI’s desk, applying slight pressure, for 30 s. To conform to realistic experimental conditions, the desks’ surfaces and the cards were not sterilized. However, the desks are personal to the participants and before the card was placed upon them, the POIs touched the surfaces with their bare hands and forearms, to replicate a “uniform” use of the desk among participants. The cards were immediately sampled, and the samples were given codes ranging from S1 to S11 (“S” standing for Secondary transfer).
Through all the experimental phases, the items (falcon tubes and cards) were sampled by using the tape lifting technique. Despite a more difficult interpretation of results, this recovery method has yielded a higher amount of DNA than other techniques [ , , , ]. All samples were extracted employing the phenol–chloroform organic method , and the pellet of each sample was resuspended in 30 μL of sterile water. The DNA extracts were quantified using the PowerQuant ® System (Promega, Madison, WI, USA) . As described in , quantification results were quite low; therefore, samples were later amplified undiluted as is, and no DNA concentration threshold was applied to further process the samples, given the explorative approach of this study. PCR amplification was carried out using the PowerPlex ® ESX17 Fast kit (Promega, Madison, WI, USA), as per the manufacturer’s instructions ; given the nature of the study, no modifications for low template DNA amplification were applied. Capillary electrophoresis followed by means of the Applied Biosystems TM SeqStudio TM Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) with the following conditions: injection time, 7 s; injection voltage, 1200 V; run time, 1440 s; run voltage, 9000 V. Results were analysed using the software GeneMapper ® ID-X v 1.6 (Thermo Fisher Scientific, Waltham, MA, USA). Analytical threshold for alleles (AT) was set at 50 RFU. As described in , further statistical analyses and calculation of LR at the sub-source level (LRϕ) were performed using EuroForMix v 4.0.1. Reference profiles of the participants were already available in the laboratory exclusion database. The LR at activity level (LRα) was calculated manually and results were confirmed by using the software Hugin Expert. The plots in this paper were created by using the R package ggplot2.
3.1. Phase I—Shedder Status All data regarding the results of phase I are reported in ; DNA quantity and profile completeness of the donor of the trace were investigated. The correlation between profile completeness for each participant and time elapsed from hand washing is plotted in . Samples showed a concentration ranging from 0.0005 to 0.0270 ng/μL, for a total of 0.015 to 0.810 ng in 30 μL. As expected, the samples collected at t 0 yielded the lowest DNA amounts. For the following time points an increase in DNA amounts, consistent among participants, was observed. These results are mirrored by the value of profile completeness, calculated on the proportion of alleles present in the sample that match the profile of the participant. The overall lower results are reported for participant B; however, it is possible to observe only slight differences with individuals A and D. Only participant C shows a distinct trend, both in terms of quantitation and profile completeness, yielding the highest values. Participant D sheds, on average, twice as much DNA as participant B ( ). Taking into consideration previously published works and our results, shedder categories were established based on profile completeness, keeping in mind quantitation results. Additionally, given our observation that 3 out of 4 individuals displayed intermediate values, we established three shedding categories: Poor shedder (profile completeness degree between 0 and 0.3), Intermediate shedder (profile completeness degree between 0.31 and 0.60), and Good shedder (profile completeness degree between 0.61 and 1). Participant C fell into the Good shedder category and participants A and D were in the Intermediate. Given the thresholds we established, participant B was classified as a Poor shedder; however, as mentioned above, the results obtained did not differ much from those observed for participants A and D ( , ). Based on these results, couples were created pairing Poor with Intermediate shedders and Intermediate shedders with Good shedders, to create more homogenous results while maintaining a margin of variability. 3.2. Phases II and III—Direct and Secondary Transfer Results were initially evaluated taking into consideration quantitation data and O/POI’s profile completeness. Results are reported in and the differences observed between direct and secondary transfer are shown in , along with their mean values. DNA traces from the primary transfer scenario show a DNA quantity ranging from 0.345 ng to 2.343 ng (in 30 μL), with an average of 1.6 ng. These results are around double the values of DNA quantities retrieved in the secondary transfer scenario (from 0.171 to 1.7 ng, with an average value of 0.657 ng in 30 μL) and they are consistent with previous findings [ , , , ] ( A,B). Profile completeness was calculated with a double approach. For both O and POI, it was initially calculated as the number of unique alleles (alleles not shared by O and POI) present in the trace divided by the number of possible unique alleles (of either O or POI) [ , , , , ] ( D,E). Profile completeness was then calculated based on all the possible alleles that could be observed in the reference profiles, including the alleles shared by O and POI ( F,G). For both calculation methods, the profile completeness of the owner (O) is consistent between primary and secondary transfer events, as expected with consistent use of the card in both instances. Instead, the profile completeness of POI drops from a mean value of 0.90 in the primary transfer to 0.68 in the secondary transfer event for the “unique alleles” method, while dropping from a value of 0.91 to 0.72 for the “all alleles” calculation approach. Further statistical analysis was performed by using EuroForMix v 4.0.1 , results are reported in and . For each sample, the Likelihood Ratio at sub-source level (LRϕ) was calculated using the continuous (quantitative) model with the following parameters: AT set at 50 RFU; FST correction set at 0; PrC set at 0.05; lambda set at 0.01. Model options for degradation, forward and backward stutters, and number of contributors were chosen for each DNA trace based on the optimal adjLoglik value recommended by EuroForMix’s “automatic model search option”. As further discussed in , a discriminatory value of 10 6 for LR at sub-source level was taken into consideration. In 82% (9 out of 11) of primary transfer samples, an LRϕ greater than 10 6 was obtained, thus sufficient to agree to the identity of POI as a contributor to the trace. In the secondary transfer scenario, only 36% of the samples (4 out of 11) yielded a log10LRϕ greater than 6 ( ). As shown in C, mean values of log10LRϕ vary greatly between transfer mechanisms, dropping from a value of 14.93 for direct transfer to 4.62 for secondary transfer. In this case, a low LR (log10LRϕ < 6) corresponded to a very low DNA quantity yield. Mixture proportion values for O, POI, and unknown (for samples with three contributors) were calculated by EuroForMix under Hp and are reported in and H,I. O and POI’s contribution to the trace in terms of DNA quantity (ng) was calculated by multiplying O and POI’s relative mixture proportion with the DNA quantity (ng in 30 μL) of the trace ( , J,K). The owner’s contribution is expected to be quantitatively the same for both direct and secondary transfer; however, a slight decrease in DNA amounts is observed. O’s contribution drops from a mean value of 0.478 ng in direct transfer to 0.314 ng. A possible explanation is discussed in . As can be observed ( , I,K), in the direct transfer simulations, the last user (POI) contributes generally more to the make-up of the trace. In terms of mixture proportion, POI’s contribution drops slightly, from a mean of 0.5 in the primary transfer to a value of 0.39 in the secondary transfer ( I). At the same time, the mean value of DNA quantity (ng) deposited by POI in the direct transfer (0.846 ng) is around four times greater than the mean value for the secondary transfer experiment (0.201 ng), given that POI’s presence is the result of an indirect contact through a surface ( K). The correlation between contribution and DNA amount is to be expected since the mixture proportion is calculated based on the observed alleles matching the individual’s reference genotype and their respective peak height (in RFU), which is, in turn, proportional to the trace’s DNA amount deposited by the individual . However, for the secondary transfer scenario, the decrease of POI’s contribution in terms of mixture proportion lead to an increase in O’s mixture proportion value, albeit corresponding to a slightly lower DNA yield. Additionally, the results of O and POI’s contributions to the trace are broadly reflective of the results observed for the profile completeness values. Ultimately, POI was a major contributor, both in terms of mixture proportion and DNA amount contributed to the trace, in 64% (7 out of 11) of the primary transfer samples and only in 36% (4 out of 11) of the secondary transfer events. Student’s two-way t -tests were used to compare variables expected to differ between the mode of transfer (direct and secondary): DNA quantity (ng in 30 μL), log10LRϕ, and parameters referring to POI’s contribution to the trace, meaning profile completeness, mixture proportion, and DNA amount contribution. Two variables, log10LRϕ ( p -value of 0.0019) and POI contribution in terms of DNA amount ( p -value of 0.0057), were chosen as the criteria for the calculation of direct and secondary transfer mechanisms’ probabilities, as will be discussed in . 3.3. Background DNA Background DNA is defined as the presence of alleles, not observed in the prevalent DNA, due to unknown sources . As can be observed in , 19 out of 22 total samples presented 3 contributors, therefore we estimated that background DNA was present in 86.4% of the samples. The third, unknown contributor had a higher relative contribution to the mixture in the secondary transfer rather than in the direct transfer. In one sample (S8), the third contributors’ value was higher than POI’s contribution, approximating the value calculated for the owner. In our case, given that the objects involved in the study (cards and personal desks) were not sterilized, nor had the participants washed their hands before handling the cards, it is not possible to determine the source of background DNA. Therefore, for our purposes, only a qualitative assessment of the presence or absence of background DNA is performed. This assessment was, however, possible for samples from phase I: 5 out of 16 samples presented a second contributor. In this case, the non-prevalent DNA must have originated from the hands of the participant . This data, albeit informative, was not included in the probabilistic calculations described below. 3.4. Constructing a Bayesian Network A Bayesian Network (BN) is a graphical representation of complex probability calculations and is becoming a commonly used tool in evaluating the strength of the evidence under different information and assumptions [ , , , , , , ]. BNs are highly customisable and can be constructed reflecting case-specific scenarios: in this study, DNA results are evaluated under competing propositions regarding DNA deposition activities. Therefore, based on the circumstances of the case in the study, the relevant information was identified as follows: O and POI share the office workspace; O often left her credit card on POI and other co-workers’ desks; If POI did not use the card, someone else did (evidence given by the bank withdrawal). In addition, it is assumed, by both the prosecution and the defence, that POI is the origin of the DNA retrieved from the credit card. Since what is disputed is not the identification of POI as a donor to the DNA trace but how the DNA got on the card, the evidence is evaluated given the following alternative propositions: Hp: POI used O’s card and made the cash withdrawals (primary transfer); Hd: POI did not use the card, an unknown individual did, and the presence of POI as a contributor to the trace is due to coworking (secondary transfer). Hd supports legitimate reasons for POI’s DNA to be on the card through previous innocent transfer. Starting from these two alternative competing propositions, the Bayesian Network (BN) was constructed according to the guidelines provided by various works [ , , , , , , ]. The construction of the BN was case-specific, meaning that it was tailored to the case-relevant information and the mechanisms of transfer we investigated. Its design is shown in . The first node, defined as the main proposition node , reflects the hypothesis of the prosecution and defence, which are stated above. The nodes that directly depart from the main proposition node are known as activity nodes and they describe the activity that is being disputed, in our case the mechanism of transfer that could have led to POI’s DNA transfer to the card: POI and O share the workspace; POI used the card (without gloves); An unknown alternative offender (AO) used the card (without gloves). Below activity nodes, finding nodes are placed and they describe the DNA transfer events, meaning the probability of recovering DNA with certain defined characteristics. As suggested , accumulation nodes are added, to highlight the possible observations on DNA transfer: POI’s DNA is on the card; Unknown DNA is on the card. Additionally, given that background DNA is a variable taken into consideration in our study, a root node considering the probability of recovering background DNA is added and it has a parental relationship with the finding node “Unknown DNA on the card”. When DNA from an unknown source is recovered, the presence of background DNA has to be included, because said unknown profile may be attributable to background DNA or an unknown, alternative offender (AO) . Lastly, a result node is added. This node displays the possible outcomes and their posterior probability values, depending on which proposition is considered true (either Hp or Hd). When constructing the BN with software, an additional node, termed function node, can be added to calculate the ratio of said probabilities (and thus directly calculate the LRαs). Since the BN shown in was not constructed with software, a function node is not reported. The probabilities underlying each node are shown in . The probabilities of the sub-activity, accumulation, and results nodes are Boolean, meaning they either have a value Pr = 0 or Pr = 1, depending on the parental proposition considered to be true, and thus on the disputed activity. The only activity that is not being disputed and is accepted as true by both the prosecution and defence is the sharing of the workspace (thus the corresponding sub-activity node has Pr = 1 under both propositions) . The probability of transfer events, either primary or secondary, and background DNA, are inferred from our observations and will inform the respective finding nodes and the background DNA root node. As of today, there is no general agreement on which DNA mixture attribute is the most suitable for comparing results from different transfer scenarios [ , , , , ]. Therefore, to inform our probability values, POI’s relative contribution in ng to the trace ( ) was used as a quality indicator for the DNA trace’s properties we were investigating. However, since our study is explorative in nature, only a qualitative and discrete assessment was performed, meaning that only the presence or absence of the POI as a major contributor to the trace was taken into account. This reasoning is based both on our observations and on previous works according to which the order of subjects handling an object could at times be inferred by the most prominent contributor [ , , , , ]. The probabilities, and their values, that will inform our network are the following: t = the probability that a DNA amount, leading to a reportable major contribution from POI, will be transferred from POI and recovered on the card, given that POI used the card. t’ = the probability that a DNA amount, leading to a reportable major contribution from an unknown, alternative offender (AO), will be transferred from UNK and recovered on the card, given that UNK used the card. s = the probability that a DNA amount, leading to a reportable major contribution from POI, will be transferred and recovered on O’s card, given that O and POI share the same workspace, and O left the card on POI’s desk. b = probability of observing background DNA on the card. To infer the probability of DNA transfer for both transfer mechanisms, two criteria based on the chosen discriminatory parameters (log10LRϕ and POI’s DNA amount) were applied. In particular, the formula “reportable major profile” verbally translates the probability of observing DNA traces with both a log10LRϕ > 6 and POI as a major contributor. For personal identification purposes, an “extremely strong support” in favour of the inclusion of an individual as a donor of a trace requires an LRϕ greater than 10 6 [ , , ]. Therefore, the observation of a reportable profile (log10LRϕ > 6) has been labelled as the 1st level criterion. The 2nd level criterion is POI being the major contributor in terms of DNA amounts. t and t’ are the probabilities relative to the primary transfer scenario, and they were extrapolated by determining what was the proportion of primary transfer events that presented a major reportable contribution from POI to the trace. 1st Level: probability of log10LRϕ > 6 = 9/11 = 0.818; 2nd Level: probability of POI being a major contributor (ng) = 7/9 = 0.778. The probability of log10LRϕ > 6 and POI being a major contributor = 0.818 × 0.778 = 0.636. s is the probability of observing POI as the reportable major contributor in the secondary transfer scenario. 1st Level: probability of log10LRϕ > 6 = 4/11 = 0.364; 2nd Level: probability of POI being a major contributor (ng) = 2/4 = 0.5. The probability of log10LRϕ > 6 and POI being a major contributor = 0.364 × 0.5 = 0.182. As for the background DNA probability, it is calculated as the number of events in which a third, unknown contributor, other than POI and O, was present in both direct and secondary transfer events, and this value is b = 19/22 = 0.864. To evaluate the strength of evidence depending on activity level propositions, LRα was manually calculated under the four different possible outcomes of the DNA analysis, according to the formulae reported by Gill et al. in the Supplementary material . The results are listed in . As can be observed, when POI only is recovered as the contributor to the trace, other than O (who is always assumed to be present), the prosecution’s proposition (that POI used the card) is ten times more likely than the defence proposition. When both POI and an unknown individual are observed, so the trace has three contributors, the LRα is slightly lower but still favours the prosecution. As can be expected, when no DNA other than that of the owner is retrieved, the LRα has a neutral value of 1 and when only unknown DNA is observed (and not POI’s), the LRα value favours the defence hypothesis. Our results were then tested by creating the BN in the software Hugin Expert .
All data regarding the results of phase I are reported in ; DNA quantity and profile completeness of the donor of the trace were investigated. The correlation between profile completeness for each participant and time elapsed from hand washing is plotted in . Samples showed a concentration ranging from 0.0005 to 0.0270 ng/μL, for a total of 0.015 to 0.810 ng in 30 μL. As expected, the samples collected at t 0 yielded the lowest DNA amounts. For the following time points an increase in DNA amounts, consistent among participants, was observed. These results are mirrored by the value of profile completeness, calculated on the proportion of alleles present in the sample that match the profile of the participant. The overall lower results are reported for participant B; however, it is possible to observe only slight differences with individuals A and D. Only participant C shows a distinct trend, both in terms of quantitation and profile completeness, yielding the highest values. Participant D sheds, on average, twice as much DNA as participant B ( ). Taking into consideration previously published works and our results, shedder categories were established based on profile completeness, keeping in mind quantitation results. Additionally, given our observation that 3 out of 4 individuals displayed intermediate values, we established three shedding categories: Poor shedder (profile completeness degree between 0 and 0.3), Intermediate shedder (profile completeness degree between 0.31 and 0.60), and Good shedder (profile completeness degree between 0.61 and 1). Participant C fell into the Good shedder category and participants A and D were in the Intermediate. Given the thresholds we established, participant B was classified as a Poor shedder; however, as mentioned above, the results obtained did not differ much from those observed for participants A and D ( , ). Based on these results, couples were created pairing Poor with Intermediate shedders and Intermediate shedders with Good shedders, to create more homogenous results while maintaining a margin of variability.
Results were initially evaluated taking into consideration quantitation data and O/POI’s profile completeness. Results are reported in and the differences observed between direct and secondary transfer are shown in , along with their mean values. DNA traces from the primary transfer scenario show a DNA quantity ranging from 0.345 ng to 2.343 ng (in 30 μL), with an average of 1.6 ng. These results are around double the values of DNA quantities retrieved in the secondary transfer scenario (from 0.171 to 1.7 ng, with an average value of 0.657 ng in 30 μL) and they are consistent with previous findings [ , , , ] ( A,B). Profile completeness was calculated with a double approach. For both O and POI, it was initially calculated as the number of unique alleles (alleles not shared by O and POI) present in the trace divided by the number of possible unique alleles (of either O or POI) [ , , , , ] ( D,E). Profile completeness was then calculated based on all the possible alleles that could be observed in the reference profiles, including the alleles shared by O and POI ( F,G). For both calculation methods, the profile completeness of the owner (O) is consistent between primary and secondary transfer events, as expected with consistent use of the card in both instances. Instead, the profile completeness of POI drops from a mean value of 0.90 in the primary transfer to 0.68 in the secondary transfer event for the “unique alleles” method, while dropping from a value of 0.91 to 0.72 for the “all alleles” calculation approach. Further statistical analysis was performed by using EuroForMix v 4.0.1 , results are reported in and . For each sample, the Likelihood Ratio at sub-source level (LRϕ) was calculated using the continuous (quantitative) model with the following parameters: AT set at 50 RFU; FST correction set at 0; PrC set at 0.05; lambda set at 0.01. Model options for degradation, forward and backward stutters, and number of contributors were chosen for each DNA trace based on the optimal adjLoglik value recommended by EuroForMix’s “automatic model search option”. As further discussed in , a discriminatory value of 10 6 for LR at sub-source level was taken into consideration. In 82% (9 out of 11) of primary transfer samples, an LRϕ greater than 10 6 was obtained, thus sufficient to agree to the identity of POI as a contributor to the trace. In the secondary transfer scenario, only 36% of the samples (4 out of 11) yielded a log10LRϕ greater than 6 ( ). As shown in C, mean values of log10LRϕ vary greatly between transfer mechanisms, dropping from a value of 14.93 for direct transfer to 4.62 for secondary transfer. In this case, a low LR (log10LRϕ < 6) corresponded to a very low DNA quantity yield. Mixture proportion values for O, POI, and unknown (for samples with three contributors) were calculated by EuroForMix under Hp and are reported in and H,I. O and POI’s contribution to the trace in terms of DNA quantity (ng) was calculated by multiplying O and POI’s relative mixture proportion with the DNA quantity (ng in 30 μL) of the trace ( , J,K). The owner’s contribution is expected to be quantitatively the same for both direct and secondary transfer; however, a slight decrease in DNA amounts is observed. O’s contribution drops from a mean value of 0.478 ng in direct transfer to 0.314 ng. A possible explanation is discussed in . As can be observed ( , I,K), in the direct transfer simulations, the last user (POI) contributes generally more to the make-up of the trace. In terms of mixture proportion, POI’s contribution drops slightly, from a mean of 0.5 in the primary transfer to a value of 0.39 in the secondary transfer ( I). At the same time, the mean value of DNA quantity (ng) deposited by POI in the direct transfer (0.846 ng) is around four times greater than the mean value for the secondary transfer experiment (0.201 ng), given that POI’s presence is the result of an indirect contact through a surface ( K). The correlation between contribution and DNA amount is to be expected since the mixture proportion is calculated based on the observed alleles matching the individual’s reference genotype and their respective peak height (in RFU), which is, in turn, proportional to the trace’s DNA amount deposited by the individual . However, for the secondary transfer scenario, the decrease of POI’s contribution in terms of mixture proportion lead to an increase in O’s mixture proportion value, albeit corresponding to a slightly lower DNA yield. Additionally, the results of O and POI’s contributions to the trace are broadly reflective of the results observed for the profile completeness values. Ultimately, POI was a major contributor, both in terms of mixture proportion and DNA amount contributed to the trace, in 64% (7 out of 11) of the primary transfer samples and only in 36% (4 out of 11) of the secondary transfer events. Student’s two-way t -tests were used to compare variables expected to differ between the mode of transfer (direct and secondary): DNA quantity (ng in 30 μL), log10LRϕ, and parameters referring to POI’s contribution to the trace, meaning profile completeness, mixture proportion, and DNA amount contribution. Two variables, log10LRϕ ( p -value of 0.0019) and POI contribution in terms of DNA amount ( p -value of 0.0057), were chosen as the criteria for the calculation of direct and secondary transfer mechanisms’ probabilities, as will be discussed in .
Background DNA is defined as the presence of alleles, not observed in the prevalent DNA, due to unknown sources . As can be observed in , 19 out of 22 total samples presented 3 contributors, therefore we estimated that background DNA was present in 86.4% of the samples. The third, unknown contributor had a higher relative contribution to the mixture in the secondary transfer rather than in the direct transfer. In one sample (S8), the third contributors’ value was higher than POI’s contribution, approximating the value calculated for the owner. In our case, given that the objects involved in the study (cards and personal desks) were not sterilized, nor had the participants washed their hands before handling the cards, it is not possible to determine the source of background DNA. Therefore, for our purposes, only a qualitative assessment of the presence or absence of background DNA is performed. This assessment was, however, possible for samples from phase I: 5 out of 16 samples presented a second contributor. In this case, the non-prevalent DNA must have originated from the hands of the participant . This data, albeit informative, was not included in the probabilistic calculations described below.
A Bayesian Network (BN) is a graphical representation of complex probability calculations and is becoming a commonly used tool in evaluating the strength of the evidence under different information and assumptions [ , , , , , , ]. BNs are highly customisable and can be constructed reflecting case-specific scenarios: in this study, DNA results are evaluated under competing propositions regarding DNA deposition activities. Therefore, based on the circumstances of the case in the study, the relevant information was identified as follows: O and POI share the office workspace; O often left her credit card on POI and other co-workers’ desks; If POI did not use the card, someone else did (evidence given by the bank withdrawal). In addition, it is assumed, by both the prosecution and the defence, that POI is the origin of the DNA retrieved from the credit card. Since what is disputed is not the identification of POI as a donor to the DNA trace but how the DNA got on the card, the evidence is evaluated given the following alternative propositions: Hp: POI used O’s card and made the cash withdrawals (primary transfer); Hd: POI did not use the card, an unknown individual did, and the presence of POI as a contributor to the trace is due to coworking (secondary transfer). Hd supports legitimate reasons for POI’s DNA to be on the card through previous innocent transfer. Starting from these two alternative competing propositions, the Bayesian Network (BN) was constructed according to the guidelines provided by various works [ , , , , , , ]. The construction of the BN was case-specific, meaning that it was tailored to the case-relevant information and the mechanisms of transfer we investigated. Its design is shown in . The first node, defined as the main proposition node , reflects the hypothesis of the prosecution and defence, which are stated above. The nodes that directly depart from the main proposition node are known as activity nodes and they describe the activity that is being disputed, in our case the mechanism of transfer that could have led to POI’s DNA transfer to the card: POI and O share the workspace; POI used the card (without gloves); An unknown alternative offender (AO) used the card (without gloves). Below activity nodes, finding nodes are placed and they describe the DNA transfer events, meaning the probability of recovering DNA with certain defined characteristics. As suggested , accumulation nodes are added, to highlight the possible observations on DNA transfer: POI’s DNA is on the card; Unknown DNA is on the card. Additionally, given that background DNA is a variable taken into consideration in our study, a root node considering the probability of recovering background DNA is added and it has a parental relationship with the finding node “Unknown DNA on the card”. When DNA from an unknown source is recovered, the presence of background DNA has to be included, because said unknown profile may be attributable to background DNA or an unknown, alternative offender (AO) . Lastly, a result node is added. This node displays the possible outcomes and their posterior probability values, depending on which proposition is considered true (either Hp or Hd). When constructing the BN with software, an additional node, termed function node, can be added to calculate the ratio of said probabilities (and thus directly calculate the LRαs). Since the BN shown in was not constructed with software, a function node is not reported. The probabilities underlying each node are shown in . The probabilities of the sub-activity, accumulation, and results nodes are Boolean, meaning they either have a value Pr = 0 or Pr = 1, depending on the parental proposition considered to be true, and thus on the disputed activity. The only activity that is not being disputed and is accepted as true by both the prosecution and defence is the sharing of the workspace (thus the corresponding sub-activity node has Pr = 1 under both propositions) . The probability of transfer events, either primary or secondary, and background DNA, are inferred from our observations and will inform the respective finding nodes and the background DNA root node. As of today, there is no general agreement on which DNA mixture attribute is the most suitable for comparing results from different transfer scenarios [ , , , , ]. Therefore, to inform our probability values, POI’s relative contribution in ng to the trace ( ) was used as a quality indicator for the DNA trace’s properties we were investigating. However, since our study is explorative in nature, only a qualitative and discrete assessment was performed, meaning that only the presence or absence of the POI as a major contributor to the trace was taken into account. This reasoning is based both on our observations and on previous works according to which the order of subjects handling an object could at times be inferred by the most prominent contributor [ , , , , ]. The probabilities, and their values, that will inform our network are the following: t = the probability that a DNA amount, leading to a reportable major contribution from POI, will be transferred from POI and recovered on the card, given that POI used the card. t’ = the probability that a DNA amount, leading to a reportable major contribution from an unknown, alternative offender (AO), will be transferred from UNK and recovered on the card, given that UNK used the card. s = the probability that a DNA amount, leading to a reportable major contribution from POI, will be transferred and recovered on O’s card, given that O and POI share the same workspace, and O left the card on POI’s desk. b = probability of observing background DNA on the card. To infer the probability of DNA transfer for both transfer mechanisms, two criteria based on the chosen discriminatory parameters (log10LRϕ and POI’s DNA amount) were applied. In particular, the formula “reportable major profile” verbally translates the probability of observing DNA traces with both a log10LRϕ > 6 and POI as a major contributor. For personal identification purposes, an “extremely strong support” in favour of the inclusion of an individual as a donor of a trace requires an LRϕ greater than 10 6 [ , , ]. Therefore, the observation of a reportable profile (log10LRϕ > 6) has been labelled as the 1st level criterion. The 2nd level criterion is POI being the major contributor in terms of DNA amounts. t and t’ are the probabilities relative to the primary transfer scenario, and they were extrapolated by determining what was the proportion of primary transfer events that presented a major reportable contribution from POI to the trace. 1st Level: probability of log10LRϕ > 6 = 9/11 = 0.818; 2nd Level: probability of POI being a major contributor (ng) = 7/9 = 0.778. The probability of log10LRϕ > 6 and POI being a major contributor = 0.818 × 0.778 = 0.636. s is the probability of observing POI as the reportable major contributor in the secondary transfer scenario. 1st Level: probability of log10LRϕ > 6 = 4/11 = 0.364; 2nd Level: probability of POI being a major contributor (ng) = 2/4 = 0.5. The probability of log10LRϕ > 6 and POI being a major contributor = 0.364 × 0.5 = 0.182. As for the background DNA probability, it is calculated as the number of events in which a third, unknown contributor, other than POI and O, was present in both direct and secondary transfer events, and this value is b = 19/22 = 0.864. To evaluate the strength of evidence depending on activity level propositions, LRα was manually calculated under the four different possible outcomes of the DNA analysis, according to the formulae reported by Gill et al. in the Supplementary material . The results are listed in . As can be observed, when POI only is recovered as the contributor to the trace, other than O (who is always assumed to be present), the prosecution’s proposition (that POI used the card) is ten times more likely than the defence proposition. When both POI and an unknown individual are observed, so the trace has three contributors, the LRα is slightly lower but still favours the prosecution. As can be expected, when no DNA other than that of the owner is retrieved, the LRα has a neutral value of 1 and when only unknown DNA is observed (and not POI’s), the LRα value favours the defence hypothesis. Our results were then tested by creating the BN in the software Hugin Expert .
4.1. Phase I–Shedder Status As reported in previous studies, great inter- and intra-individual variations exist in the amount of DNA deposited through touch [ , , , , , ]. Additionally, other features that influence the deposition of touch DNA are the length of contact, the type of item and the nature of the surface, the presence of moisture on the surface or in the sample, etc. [ , , ]. In terms of DNA quantities deposited on our surface of choice for the assessment of shedder status (the plastic tube), our results are comparable with those reported in the literature based on the same material, nature of the contact, and length of contact [ , , , ]. In particular, they were in line with the results obtained by Fonneløp et al. , who performed the same kind of assessment (with falcon tubes). As far as the participant’s individual characteristics, age and skin conditions seem to be factors determining the shedding propensity [ , , , , , , , , ]; however, participants in the study fell in the age range between 25 and 65 years of age and had no known skin conditions, thus minimizing interindividual variability. To maintain realistic casework conditions, participants were allowed to perform routine daily activities, which, of course, varied among them. Keeping this in mind, our experiment allowed for the broad classification of participants into shedding categories that are not dependent on strict laboratory experimental conditions. It has to be considered that the shedding propensity of an individual may vary depending on activities performed and intra-individual variability [ , , , , , , ]. For the same reason, the profile completeness at time t 0 after handwashing was included in the calculations for shedding status assessment. Handwashing reduces the quantity of retrievable DNA (whose quantity then increases over time) ; however, we considered this value important for a general assessment of shedding tendency, given different activities that an individual can perform before committing a criminal offence (or generally touching something). Additionally, despite our observations being too few, none of the participants showed an extremely low shedding value. We observed that 75% of participants (3 out of 4) appeared to belong to a more intermediate shedding range, thus informing our belief that a binary categorization system (Poor/High shedder) may be too simplistic, while also considering that individuals may fall at different points of a continuous distribution range [ , , , , ]. Therefore, an Intermediate shedder category was added, even though we are well aware that this categorization has to be informed by more data. 4.2. Phase II—Direct and Secondary Transfer This study aimed to investigate qualitative and quantitative characteristics of DNA traces given different transfer mechanisms, so as to verify whether, based on said attributes of a DNA mixture, it is possible to discriminate the mode of transfer . In terms of qualitative observations, both evaluation approaches for O and POI’s profile completeness showed high mean values in the direct and secondary transfer alike; however, some differences are present. O’s profile completeness remains more or less constant for both direct and secondary transfer ( , D,F), since O used the card as per their usual habits prior to both experiments. Conversely, the degree of POI’s profile completeness, for both methods of calculation, differs between transfer mechanisms. POI’s profile completeness ( , E,G) in the direct transfer samples is higher than in the secondary ones. Given the secondary transfer of DNA by means of an intermediate surface, a lower quality of POI’s profile is to be expected. However, these results are to be interpreted considering that POI’s DNA was freshly deposited on the cards right before their sampling. In terms of DNA quantity deposited and retrieved from credit cards, in both direct and secondary transfer, our results are generally comparable with those reported in other studies, albeit slightly lower [ , , , , , , ]. However, the limited surface and length of use of a credit card have to be taken into account. Differences in DNA quantities can be observed between primary and secondary transfer: secondary transfer touch DNA samples show roughly half the DNA quantities as compared to direct transfer samples. Results are consistent with previous studies regarding touch DNA [ , , , ] also considering that, as common sense suggests, the cards directly handled, without gloves, by the POI will yield a higher DNA amount than the cards of the secondary transfer scenario. When considering the secondary transfer scenario, we expected a reasonably good outcome. Our expectations were based on the fact that secondary touch DNA transfer by means of an object (the desk surface) resulted in a greater amount of DNA being transferred when the trace was fresh, the surfaces involved were hard and non-porous (such as plastic), and slight pressure and friction was applied [ , , , , , , ]. This could explain why, in 50% of secondary transfer DNA traces that returned an LRϕ > 10 6 , POI was the major contributor. Breaking down the observed DNA quantities by contributor, the owner (O) is expected to always be present in the trace in the same quantity and quality, ideally due to the same frequency of use of the card prior to transfer experiments. However, despite presenting roughly the same profile completeness values both in the direct and secondary transfer, O contributes to the DNA trace with a slightly lower DNA amount in the secondary transfer scenario ( J). This could be due to the sterilization of the cards after the first experiment and to the fact that only one month passed between the first (direct transfer) and second experiment (secondary transfer). This period of time was initially considered to be sufficient for O to deposit DNA amounts reflective of frequent and extensive use of the card. However, in a future study, it would be useful to investigate how a longer time interval between experiments could change O’s DNA amounts deposited on the card. Additionally, O has a higher mixture proportion in the secondary transfer rather than in the primary one ( H), realistically due to POI’s decrease in contribution in terms of mixture proportion. In fact, for the direct transfer scenario, POI is generally a major contributor, both in mixture proportion and DNA amounts deposited, as well as showing a higher profile completeness degree ( E,G,I,K, “D” transfer). All of these three parameters drop in value for the secondary transfer ( E,G,I,K, “S” transfer). This difference between O and POI’s characteristics is reasonable given that, when directly handling the card, POI leaves fresher DNA and in higher quantities in relation to the DNA traces previously deposited, whose quantity and quality may have reduced over time. Additionally, this is in line with previous studies, where the last individual to handle an object was generally retrieved as the most prominent contributor [ , , , , ]. 4.3. Background DNA As could be expected, the probability of recovering DNA originating from unknown sources is quite high. Our results are comparable with those described in the literature, which reports the presence of background DNA in more than 60% of the samples [ , , , , , , , ]. These observations highlight the importance of carrying out experiments that are as close to real life as possible since non-prevalent DNA is present virtually everywhere . This consideration is particularly relevant for substrates and items that could be frequently subjected to foreign DNA transfer. Credit cards are often handled by non-owners, such as shop clerks, or may be used in an ATM withdrawal where other individuals’ cards are inserted thus leaving behind their DNA, that could be subsequently picked up by other cards. Therefore, the detection of unknown DNA from such an item is highly probable. 4.4. General Considerations Despite the good preliminary results obtained, the LRα values show moderate to low statistical support in favour of Hp. In particular, the LRαs obtained are similar to results obtained in other touch DNA studies [ , , , , ]. For the discussion of the results, the following considerations have to be kept in mind: the owner’s contribution is always assumed; both the prosecution and the defence agree on the possibility of POI’s DNA being transferred due to co-working with O; the card was handled bare-handed; if POI did not use the card, someone else (AO) did, given the evidence of cash withdrawal. For the “POI only” and “POI and unknown” outcomes, the LRα values indicate that the observed evidence is, respectively, 10.6 and 3.5 times more likely if the prosecution’s hypothesis on the mode of transfer (direct deposition) was true than if Hd was true (secondary transfer). The LRα for the “POI and unknown” scenario still favours Hp; however, it yields a lower LRα than the “POI only” outcome. In fact, the presence of a third, unknown contributor strengthens the defence’s hypothesis of an unknown individual (AO) being the offender and POI’s DNA on the card resulting from a secondary transfer due to co-working. As expected, the “Unknown only” outcome is in favour of Hd, albeit with low statistical support, given the fact that the probability of recovering background DNA is quite high ( b = 0.864) and that the origin of the unknown DNA found on the card is not known (from AO or background DNA). The “No DNA” scenario, meaning that only DNA from the owner is recovered, is not informative. The result is explained by the fact that the absence of DNA does not exclude that POI handled the card. Given that neither POI’s nor an unknown donor’s profiles have been observed, POI and AO have the same probability of leaving a detectable DNA amount leading to a reportable major contribution ( t and t’ , respectively) as much as they have the same probability of leaving no detectable DNA traces (1− t and 1− t’ , respectively). In fact, in some instances “masked touching”, meaning touching an object without leaving detectable DNA, has been observed [ , , , , , ]. The present study offers the investigative and preliminary basis for the set-up of another, more in-depth study; therefore, performing a number of subsequent steps would be appropriate. First, it would be advisable to increase the number of individuals taking part in the study. As already mentioned, the small dataset here investigated poses some limitations. Individuals’ shedding tendency should be further investigated in a larger population: more information regarding intra- and inter-individual variation has to be collected to support our preliminary classification of shedding status into three classes, while also taking into consideration variability due to performed activities. The supplementary data thus obtained on shedding status would be of useful implementation to the Bayesian Network here constructed. Second, the number of experiments carried out for both primary and secondary transfer should be implemented. This can be achieved by increasing the number of individuals investigated, creating pairings that are higher in number and more varied in terms of shedding status differences between participants. This would allow us to obtain more information on POI’s prevalence in the DNA mixtures and on other qualitative and quantitative DNA profiles’ characteristics. Additionally, statistical simulations would allow increasing the number of samples based on the effective observations, thus increasing the number of statistically relevant events per DNA transfer mechanism. Third, sensitivity testing should be carried out, to analyse how the LRα values would change depending on plausible ranges of probabilities for primary and secondary transfer, conditioned upon different values of POI’s relative contribution, not only based on presence/absence calculations . For example, Fonneløp and colleagues showed how a continuous quantitative evaluation of the chosen DNA attribute and sensitivity testing increased the value of the evidence. Fourth, DNA persistence and recovery on credit cards should be investigated. To maximize DNA recovery rates, we decided to collect the samples right after the transfer event; however, it is commonly known that DNA trace quantities decrease as the time elapsed from the deposition of the traces increases [ , , ]. Lastly, the complexity of the Bayesian Network could be improved by implementing other nodes regarding, for example, information on shedder status, DNA persistence and recovery (thus comparing different recovery techniques), and extraction efficiency.
As reported in previous studies, great inter- and intra-individual variations exist in the amount of DNA deposited through touch [ , , , , , ]. Additionally, other features that influence the deposition of touch DNA are the length of contact, the type of item and the nature of the surface, the presence of moisture on the surface or in the sample, etc. [ , , ]. In terms of DNA quantities deposited on our surface of choice for the assessment of shedder status (the plastic tube), our results are comparable with those reported in the literature based on the same material, nature of the contact, and length of contact [ , , , ]. In particular, they were in line with the results obtained by Fonneløp et al. , who performed the same kind of assessment (with falcon tubes). As far as the participant’s individual characteristics, age and skin conditions seem to be factors determining the shedding propensity [ , , , , , , , , ]; however, participants in the study fell in the age range between 25 and 65 years of age and had no known skin conditions, thus minimizing interindividual variability. To maintain realistic casework conditions, participants were allowed to perform routine daily activities, which, of course, varied among them. Keeping this in mind, our experiment allowed for the broad classification of participants into shedding categories that are not dependent on strict laboratory experimental conditions. It has to be considered that the shedding propensity of an individual may vary depending on activities performed and intra-individual variability [ , , , , , , ]. For the same reason, the profile completeness at time t 0 after handwashing was included in the calculations for shedding status assessment. Handwashing reduces the quantity of retrievable DNA (whose quantity then increases over time) ; however, we considered this value important for a general assessment of shedding tendency, given different activities that an individual can perform before committing a criminal offence (or generally touching something). Additionally, despite our observations being too few, none of the participants showed an extremely low shedding value. We observed that 75% of participants (3 out of 4) appeared to belong to a more intermediate shedding range, thus informing our belief that a binary categorization system (Poor/High shedder) may be too simplistic, while also considering that individuals may fall at different points of a continuous distribution range [ , , , , ]. Therefore, an Intermediate shedder category was added, even though we are well aware that this categorization has to be informed by more data.
This study aimed to investigate qualitative and quantitative characteristics of DNA traces given different transfer mechanisms, so as to verify whether, based on said attributes of a DNA mixture, it is possible to discriminate the mode of transfer . In terms of qualitative observations, both evaluation approaches for O and POI’s profile completeness showed high mean values in the direct and secondary transfer alike; however, some differences are present. O’s profile completeness remains more or less constant for both direct and secondary transfer ( , D,F), since O used the card as per their usual habits prior to both experiments. Conversely, the degree of POI’s profile completeness, for both methods of calculation, differs between transfer mechanisms. POI’s profile completeness ( , E,G) in the direct transfer samples is higher than in the secondary ones. Given the secondary transfer of DNA by means of an intermediate surface, a lower quality of POI’s profile is to be expected. However, these results are to be interpreted considering that POI’s DNA was freshly deposited on the cards right before their sampling. In terms of DNA quantity deposited and retrieved from credit cards, in both direct and secondary transfer, our results are generally comparable with those reported in other studies, albeit slightly lower [ , , , , , , ]. However, the limited surface and length of use of a credit card have to be taken into account. Differences in DNA quantities can be observed between primary and secondary transfer: secondary transfer touch DNA samples show roughly half the DNA quantities as compared to direct transfer samples. Results are consistent with previous studies regarding touch DNA [ , , , ] also considering that, as common sense suggests, the cards directly handled, without gloves, by the POI will yield a higher DNA amount than the cards of the secondary transfer scenario. When considering the secondary transfer scenario, we expected a reasonably good outcome. Our expectations were based on the fact that secondary touch DNA transfer by means of an object (the desk surface) resulted in a greater amount of DNA being transferred when the trace was fresh, the surfaces involved were hard and non-porous (such as plastic), and slight pressure and friction was applied [ , , , , , , ]. This could explain why, in 50% of secondary transfer DNA traces that returned an LRϕ > 10 6 , POI was the major contributor. Breaking down the observed DNA quantities by contributor, the owner (O) is expected to always be present in the trace in the same quantity and quality, ideally due to the same frequency of use of the card prior to transfer experiments. However, despite presenting roughly the same profile completeness values both in the direct and secondary transfer, O contributes to the DNA trace with a slightly lower DNA amount in the secondary transfer scenario ( J). This could be due to the sterilization of the cards after the first experiment and to the fact that only one month passed between the first (direct transfer) and second experiment (secondary transfer). This period of time was initially considered to be sufficient for O to deposit DNA amounts reflective of frequent and extensive use of the card. However, in a future study, it would be useful to investigate how a longer time interval between experiments could change O’s DNA amounts deposited on the card. Additionally, O has a higher mixture proportion in the secondary transfer rather than in the primary one ( H), realistically due to POI’s decrease in contribution in terms of mixture proportion. In fact, for the direct transfer scenario, POI is generally a major contributor, both in mixture proportion and DNA amounts deposited, as well as showing a higher profile completeness degree ( E,G,I,K, “D” transfer). All of these three parameters drop in value for the secondary transfer ( E,G,I,K, “S” transfer). This difference between O and POI’s characteristics is reasonable given that, when directly handling the card, POI leaves fresher DNA and in higher quantities in relation to the DNA traces previously deposited, whose quantity and quality may have reduced over time. Additionally, this is in line with previous studies, where the last individual to handle an object was generally retrieved as the most prominent contributor [ , , , , ].
As could be expected, the probability of recovering DNA originating from unknown sources is quite high. Our results are comparable with those described in the literature, which reports the presence of background DNA in more than 60% of the samples [ , , , , , , , ]. These observations highlight the importance of carrying out experiments that are as close to real life as possible since non-prevalent DNA is present virtually everywhere . This consideration is particularly relevant for substrates and items that could be frequently subjected to foreign DNA transfer. Credit cards are often handled by non-owners, such as shop clerks, or may be used in an ATM withdrawal where other individuals’ cards are inserted thus leaving behind their DNA, that could be subsequently picked up by other cards. Therefore, the detection of unknown DNA from such an item is highly probable.
Despite the good preliminary results obtained, the LRα values show moderate to low statistical support in favour of Hp. In particular, the LRαs obtained are similar to results obtained in other touch DNA studies [ , , , , ]. For the discussion of the results, the following considerations have to be kept in mind: the owner’s contribution is always assumed; both the prosecution and the defence agree on the possibility of POI’s DNA being transferred due to co-working with O; the card was handled bare-handed; if POI did not use the card, someone else (AO) did, given the evidence of cash withdrawal. For the “POI only” and “POI and unknown” outcomes, the LRα values indicate that the observed evidence is, respectively, 10.6 and 3.5 times more likely if the prosecution’s hypothesis on the mode of transfer (direct deposition) was true than if Hd was true (secondary transfer). The LRα for the “POI and unknown” scenario still favours Hp; however, it yields a lower LRα than the “POI only” outcome. In fact, the presence of a third, unknown contributor strengthens the defence’s hypothesis of an unknown individual (AO) being the offender and POI’s DNA on the card resulting from a secondary transfer due to co-working. As expected, the “Unknown only” outcome is in favour of Hd, albeit with low statistical support, given the fact that the probability of recovering background DNA is quite high ( b = 0.864) and that the origin of the unknown DNA found on the card is not known (from AO or background DNA). The “No DNA” scenario, meaning that only DNA from the owner is recovered, is not informative. The result is explained by the fact that the absence of DNA does not exclude that POI handled the card. Given that neither POI’s nor an unknown donor’s profiles have been observed, POI and AO have the same probability of leaving a detectable DNA amount leading to a reportable major contribution ( t and t’ , respectively) as much as they have the same probability of leaving no detectable DNA traces (1− t and 1− t’ , respectively). In fact, in some instances “masked touching”, meaning touching an object without leaving detectable DNA, has been observed [ , , , , , ]. The present study offers the investigative and preliminary basis for the set-up of another, more in-depth study; therefore, performing a number of subsequent steps would be appropriate. First, it would be advisable to increase the number of individuals taking part in the study. As already mentioned, the small dataset here investigated poses some limitations. Individuals’ shedding tendency should be further investigated in a larger population: more information regarding intra- and inter-individual variation has to be collected to support our preliminary classification of shedding status into three classes, while also taking into consideration variability due to performed activities. The supplementary data thus obtained on shedding status would be of useful implementation to the Bayesian Network here constructed. Second, the number of experiments carried out for both primary and secondary transfer should be implemented. This can be achieved by increasing the number of individuals investigated, creating pairings that are higher in number and more varied in terms of shedding status differences between participants. This would allow us to obtain more information on POI’s prevalence in the DNA mixtures and on other qualitative and quantitative DNA profiles’ characteristics. Additionally, statistical simulations would allow increasing the number of samples based on the effective observations, thus increasing the number of statistically relevant events per DNA transfer mechanism. Third, sensitivity testing should be carried out, to analyse how the LRα values would change depending on plausible ranges of probabilities for primary and secondary transfer, conditioned upon different values of POI’s relative contribution, not only based on presence/absence calculations . For example, Fonneløp and colleagues showed how a continuous quantitative evaluation of the chosen DNA attribute and sensitivity testing increased the value of the evidence. Fourth, DNA persistence and recovery on credit cards should be investigated. To maximize DNA recovery rates, we decided to collect the samples right after the transfer event; however, it is commonly known that DNA trace quantities decrease as the time elapsed from the deposition of the traces increases [ , , ]. Lastly, the complexity of the Bayesian Network could be improved by implementing other nodes regarding, for example, information on shedder status, DNA persistence and recovery (thus comparing different recovery techniques), and extraction efficiency.
This study aimed to investigate qualitative and quantitative characteristics of DNA traces given different transfer mechanisms, so as to verify whether, based on said attributes of a DNA mixture, it is possible to discriminate the mode of transfer. Based on a real-life casework scenario, direct and secondary DNA transfer experiments were set up, after investigating the DNA shedding propensity of the participants. Qualitative and quantitative differences were observed between the traces produced during the primary transfer scenario and the secondary transfer scenario. The DNA trace attribute herein taken into consideration is the relative mixture contribution in ng of POI and discrete observations (presence/absence of POI as a major contributor) were used to inform the probabilities of primary and secondary transfer events. Given the realistic experimental conditions, the objects utilized in the study (cards and desktops) were not sterilized and participants were not asked to wash their hands before handling the cards. Therefore, the majority of the samples showed the presence of a third, unknown contributor. This observation was used to infer the probability of observing background DNA. Based on the relevant case-specific information and the data collected, a Bayesian Network was constructed. Likelihood Ratios at activity level (LRα) were calculated for each of the possible outcomes resulting from the DNA analysis. In instances where only POI and POI plus an unknown individual are retrieved, the values obtained show moderate to low support in favour of the prosecution proposition. This means that observing POI as a contributor to the trace and their relative contribution higher than the owner’s, it is more likely if POI used the card than if POI’s DNA was indirectly transferred due to co-working with the card’s owner. These results are promising in terms of discriminating between traces resulting from a primary and secondary transfer, especially in a case that involves touch DNA with case circumstances that, to our knowledge, have not been previously investigated. A more in-depth analysis of the qualitative and quantitative characteristics of a touch DNA trace, of individual shedder status, and of the sensitivity should be the natural next steps in the development of this study.
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Evaluation and Verification of a microRNA Panel Using Quadratic Discriminant Analysis for the Classification of Human Body Fluids in DNA Extracts
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069af578-1c7d-4c42-a44b-bc371690265d
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10218048
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Forensic Medicine[mh]
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DNA evidence is a valuable forensic tool that can place persons involved in a crime at the scene or tie them to evidence. However, body fluid identification is still important for corroborating testimony and lending additional information about the specific events of an alleged crime, especially in violent crimes, such as homicide or sexual assault. These limitations indicate a need for more accurate body fluid identification assays which can accurately identify all forensically relevant body fluids and do not contribute to unnecessary sample consumption. MicroRNAs are small, non-coding RNAs that range from 21 to 24 nucleotides long, and because of this, are less susceptible to degradation as compared to longer mRNAs [ , , , , , ]. Many miRNAs are differentially expressed in body fluids, and the expression levels of these diagnostic markers can be evaluated to identify forensically relevant body fluids [ , , , , , , , , , , , , , , ]. One challenge of traditional RNA assays is the need for a separate RNA extraction, as it unnecessarily consumes the sample . However, miRNAs can be co-extracted with DNA using several commonly used DNA extraction methods without the need for an extra DNase treatment step, which cuts down on time and sample consumption [ , , , , ]. Seashols-Williams et al. previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs in RNA extracts that classified venous and menstrual secretions, feces, urine, saliva, semen, and vaginal secretions through analysis of differential expression. This panel of miRNAs includes a pair of endogenous reference markers that provide normalization of miRNA expression without evaluation of the RNA quality or known input quantity. In a subsequent report, the expression levels of these markers were validated using blood, semen, saliva, urine, vaginal fluid, menstrual secretions, and perspiration. The ability of this panel to identify body fluids was tested using a quadratic discriminate analysis (QDA) model, which was created using the statistical software R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria), and consisted of a ten-fold cross validation of the model. This model used the normalized expression levels obtained from experimental samples to predict the presence of body fluids . The model correctly classified 93.3% of samples when tested in blood, menstrual secretions, feces, urine, saliva, semen, and vaginal fluid. Following these results, the expression levels were investigated within a donor over a biological cycle in RNA extracts. The correct classification rates in blood, feces, urine, and vaginal fluid were comparable to that of the population studies; however, the classification rates of saliva, semen, and menstrual secretions were lower. To create a more easily implemented assay for forensic DNA laboratories, the presence of miRNA retention and expression in DNA extracts was assessed and found to be only slightly lower than in RNA extracts . Based on those results, we were interested in assessing the ability of our body fluid classification model to identify body fluids using miRNAs retained in DNA extracts.
2.1. Validation of miRNA Panel for Body Fluid Identification in DNA Extracts The following sample collection, DNA isolation and RT-qPCR analysis methods apply to all analyses unless stated otherwise. 2.1.1. Sample Collection Blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions were collected between 2016–2020 using informed consent in accordance with the approved Institutional Review Board Human Subjects Research Protocol (HM200009027). Menstrual, fecal, and vaginal samples were collected on sterile cotton swabs by the donors and returned in swab boxes. Blood was deposited onto a sterile cotton swab after sterilizing and pricking the donor’s finger with a Unistik ® 3 Normal lancet (Owen Mumford Ltd., Woodstock, UK), and saliva was collected by rolling a sterile cotton swab along the inside of the donor’s cheek. Urine and semen were deposited into sterile collection cups supplied to the donor, which were returned on ice within 24 h before aliquoting each onto sterile cotton swabs (50 µL of semen and 100 µL of urine). All swabs were dried and stored in swab boxes at room temperature until treatment and/or DNA extraction. 2.1.2. DNA Isolation DNA was isolated from whole swabs using the QIAgen DNA Investigator Kit on the QIAcube (Qiagen, Valencia, CA, USA) and the previously validated manufacturer’s protocol for forensic casework samples. No modifications such as the addition of glycogen were used in an effort to mimic a forensic workflow as closely as possible. Final elution volumes were as follows: 30 μL for saliva, blood, menstrual secretions, semen, and vaginal fluid, 50 μL for feces, and 20 μL for urine. Reagent blanks were included with each batch of DNA extractions, and extracts were stored at −80 °C until further use. DNase treatment was not performed on the sample extracts, as primer evaluation and previous work has demonstrated no significant difference on miRNA detection levels , indicating that genomic DNA does not interfere with miRNA detection. Reverse Transcription and qPCR controls were analyzed to verify lack of interference in miRNA amplification by genomic DNA. DNA extractions and subsequent cDNA synthesis were performed in a dedicated workspace for RNA, following strict procedures to help limit the effect of contamination, including physical isolation from post-PCR laboratories, use of personal protective equipment, and use of molecular biology grade reagents and consumables. miRNA isolation efficiency was measured through RT-qPCR analysis, as our previous work has shown that UV spectrophotometry and other methods cannot precisely predict miRNA concentrations in the low concentrations observed in biological fluids [ , , , ]. 2.1.3. Sample Treatment for Compromised Analysis Blood, semen, saliva, and urine were collected from three different donors. Blood was collected into a Vacutainer ® containing EDTA (Beckton, Dickinson & Company, Franklin Lakes, NJ, USA) and inverted for 15 s before 50 μL was deposited onto a sterile cotton swab. Urine, semen, and saliva were collected into a sterile collection cup, and 50 μL (semen, saliva) or 100 μL (urine) were deposited onto sterile cotton swabs. The swabs were dried at room temperature for 24–48 h and then stored at −20 °C until treatment, which was performed within 72 h of drying. For heat-compromised samples, swabs were exposed to either 55 °C or 95 °C for 0.5, 1, 2, 4, or 24 h. For the chemically treated samples, 100 μL of either 1:10 (87 nM) or full-strength (870 mM) sodium hypochlorite, dish soap (Dawn Ultra Dishwashing Liquid, Proctor & Gamble, Cincinnati, OH), or glacial acetic acid (pH 2.5, 17.4 M) were deposited onto the prepared swabs and dried for 72 h. UV-treated samples were exposed to 4 h of 302 nm light at room temperature using the UVP high-performance ultra-violet transilluminator (UVP, Upland, CA, USA). This wavelength was chosen as it is in the middle of the ultraviolet range and has been demonstrated to induce the most significant DNA damage . After treatment, all swabs were stored at −20 °C until DNA isolation. Dried blood, semen, saliva, and urine were also tested in an environmental chamber for simulated outdoor conditions. Samples were deposited onto a cotton swatch from Layne et al. using a single donor for each body fluid. The samples were exposed to treatment in a Q-sun Ce-3 Environmental Chamber (Q-Lab Corporation, Westlake, OH, USA) at the Federal Bureau of Investigation Research Laboratory. The Environmental chamber controlled for temperature, humidity, and a 24 h light/dark cycle to imitate a summer day in Virginia ( ). The samples were removed from the chamber at 48 h intervals up to 14 days and stored at −80 °C until punches were taken. Using a biopsy punch, 4 mm punches were taken from the remaining cotton swatches and stored at −80 °C until DNA isolation (5 years). 2.1.4. RT-qPCR Reverse transcription was performed on the Proflex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the qScript™ microRNA Quantification System (Quanta Biosciences, Gaithersburg, MD, USA) following the previously reported protocol . qPCR primers for all miRNA target sequences were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA) ( ), and qPCR was performed according to the protocol in quarter volume reactions: 6.25 µL of 2X PerfeCTa ® SYBR Green SuperMix, 0.25 µL of PerfeCTa ® Universal Primer (UP: 5′-ATGGCGGTAAGTCCAGATACG-3′)(Quanta Biosciences) and IDT MicroRNA Primer Assay (2.5 µM), 3.75 µL of nuclease-free water, and 2 µL of cDNA reaction for a total reaction volume of 12.5 µL. Thermal cycling parameters on the QuantStudio™ 6 Flex Real-Time PCR Instrument (Thermo Fisher Scientific) were set at: 95 °C for 2 min, 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 70 °C for 34 s. Raw data were analyzed at a threshold of 0.015 within QuantStudio™ Real-Time PCR software v1.3 (Thermo Fisher Scientific) and exported into Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Differential expression or delta quantification cycle (ΔCq) values were calculated by subtracting the average Cq of let-7g and let-7i from the Cq value of the target miRNA (ΔCq = Cq( miRNA target ) − Cq( avg let-7g & let-7i )). All experiments were performed and analyzed according to Minimum Information for Publication of Quantitative Real-Time PR Experiments (MIQE) guidelines . Each miRNA target was amplified in duplicate technical replicates for each sample with no template controls (NTCs) and negative reverse transcription (RT) controls (non-transcribed RNA extract—to eliminate genomic DNA contamination as a variable) on each plate. 2.2. Statistical Analyses Initial statistical analyses of the raw and ΔCq data were performed in JMP ® v14.2.0 (SAS Institute, Cary, NC, USA). Normal distribution and equal variance were confirmed for all sample sets using quantile–quantile plots and Levine’s test, respectively. In multi-group comparisons, a one-way ANOVA test was performed with Tukey’s HSD pairwise comparison. A significance level of α = 0.05 was used to determine which tests are declared statistically significant. Subsequent statistical prediction modeling was performed in R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). For predictive analysis, Quadratic Discriminant Analysis (qda in R) was used. A 10-fold cross validation procedure was performed to ensure that each observation was in both training and validation sets by splitting the data into ten non-overlapping groups, corresponding to 10% of the samples. Each group was in turn used as a test set while the remaining (i.e., 90% of the data) were used to train the model. Once sample collection was complete, the population dataset consisted of 355 samples: 51 blood, 53 menstrual secretions, 50 feces, 46 urine, 53 saliva, 52 semen, and 50 vaginal secretions ( ). For some of the markers and body fluid combinations, there were some samples in which not all miRNAs were tested; thus, across the total dataset, there were 252 fully observed values. To address the issues associated with these missing values, a single imputation using the conditional multivariate mean was employed. This allowed for the imputed value to depend on all the observed values for the subject. Multiple imputation was avoided as the goal of the project is correct classification, and multiple imputation would not allow for easy model validation. To improve the ability of the classifiers, an “Other” category was created by fitting a multivariate normal distribution to the entire dataset and drawing samples outside the 3.5-standard-deviation ellipsoid. Without this other category, any future extreme observations will be classified as the body fluid closest even though it is truly an extrapolation. This Other category allows for only those body fluids with measurements in the range observed to be classified as a body fluid. All measurements outside the observed measurement range will be classified as Other. After development of the QDA model and cross-validation, classification testing was set at 50% confidence for body fluid classification. 2.3. Variation within Individuals over Time To evaluate variation in differential expression within an individual over time or within a biological cycle, three volunteers for each body fluid donated multiple samples according to the time conditions listed in . Sample collection, DNA extraction, and RT-qPCR analysis were performed as described above.
The following sample collection, DNA isolation and RT-qPCR analysis methods apply to all analyses unless stated otherwise. 2.1.1. Sample Collection Blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions were collected between 2016–2020 using informed consent in accordance with the approved Institutional Review Board Human Subjects Research Protocol (HM200009027). Menstrual, fecal, and vaginal samples were collected on sterile cotton swabs by the donors and returned in swab boxes. Blood was deposited onto a sterile cotton swab after sterilizing and pricking the donor’s finger with a Unistik ® 3 Normal lancet (Owen Mumford Ltd., Woodstock, UK), and saliva was collected by rolling a sterile cotton swab along the inside of the donor’s cheek. Urine and semen were deposited into sterile collection cups supplied to the donor, which were returned on ice within 24 h before aliquoting each onto sterile cotton swabs (50 µL of semen and 100 µL of urine). All swabs were dried and stored in swab boxes at room temperature until treatment and/or DNA extraction. 2.1.2. DNA Isolation DNA was isolated from whole swabs using the QIAgen DNA Investigator Kit on the QIAcube (Qiagen, Valencia, CA, USA) and the previously validated manufacturer’s protocol for forensic casework samples. No modifications such as the addition of glycogen were used in an effort to mimic a forensic workflow as closely as possible. Final elution volumes were as follows: 30 μL for saliva, blood, menstrual secretions, semen, and vaginal fluid, 50 μL for feces, and 20 μL for urine. Reagent blanks were included with each batch of DNA extractions, and extracts were stored at −80 °C until further use. DNase treatment was not performed on the sample extracts, as primer evaluation and previous work has demonstrated no significant difference on miRNA detection levels , indicating that genomic DNA does not interfere with miRNA detection. Reverse Transcription and qPCR controls were analyzed to verify lack of interference in miRNA amplification by genomic DNA. DNA extractions and subsequent cDNA synthesis were performed in a dedicated workspace for RNA, following strict procedures to help limit the effect of contamination, including physical isolation from post-PCR laboratories, use of personal protective equipment, and use of molecular biology grade reagents and consumables. miRNA isolation efficiency was measured through RT-qPCR analysis, as our previous work has shown that UV spectrophotometry and other methods cannot precisely predict miRNA concentrations in the low concentrations observed in biological fluids [ , , , ]. 2.1.3. Sample Treatment for Compromised Analysis Blood, semen, saliva, and urine were collected from three different donors. Blood was collected into a Vacutainer ® containing EDTA (Beckton, Dickinson & Company, Franklin Lakes, NJ, USA) and inverted for 15 s before 50 μL was deposited onto a sterile cotton swab. Urine, semen, and saliva were collected into a sterile collection cup, and 50 μL (semen, saliva) or 100 μL (urine) were deposited onto sterile cotton swabs. The swabs were dried at room temperature for 24–48 h and then stored at −20 °C until treatment, which was performed within 72 h of drying. For heat-compromised samples, swabs were exposed to either 55 °C or 95 °C for 0.5, 1, 2, 4, or 24 h. For the chemically treated samples, 100 μL of either 1:10 (87 nM) or full-strength (870 mM) sodium hypochlorite, dish soap (Dawn Ultra Dishwashing Liquid, Proctor & Gamble, Cincinnati, OH), or glacial acetic acid (pH 2.5, 17.4 M) were deposited onto the prepared swabs and dried for 72 h. UV-treated samples were exposed to 4 h of 302 nm light at room temperature using the UVP high-performance ultra-violet transilluminator (UVP, Upland, CA, USA). This wavelength was chosen as it is in the middle of the ultraviolet range and has been demonstrated to induce the most significant DNA damage . After treatment, all swabs were stored at −20 °C until DNA isolation. Dried blood, semen, saliva, and urine were also tested in an environmental chamber for simulated outdoor conditions. Samples were deposited onto a cotton swatch from Layne et al. using a single donor for each body fluid. The samples were exposed to treatment in a Q-sun Ce-3 Environmental Chamber (Q-Lab Corporation, Westlake, OH, USA) at the Federal Bureau of Investigation Research Laboratory. The Environmental chamber controlled for temperature, humidity, and a 24 h light/dark cycle to imitate a summer day in Virginia ( ). The samples were removed from the chamber at 48 h intervals up to 14 days and stored at −80 °C until punches were taken. Using a biopsy punch, 4 mm punches were taken from the remaining cotton swatches and stored at −80 °C until DNA isolation (5 years). 2.1.4. RT-qPCR Reverse transcription was performed on the Proflex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the qScript™ microRNA Quantification System (Quanta Biosciences, Gaithersburg, MD, USA) following the previously reported protocol . qPCR primers for all miRNA target sequences were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA) ( ), and qPCR was performed according to the protocol in quarter volume reactions: 6.25 µL of 2X PerfeCTa ® SYBR Green SuperMix, 0.25 µL of PerfeCTa ® Universal Primer (UP: 5′-ATGGCGGTAAGTCCAGATACG-3′)(Quanta Biosciences) and IDT MicroRNA Primer Assay (2.5 µM), 3.75 µL of nuclease-free water, and 2 µL of cDNA reaction for a total reaction volume of 12.5 µL. Thermal cycling parameters on the QuantStudio™ 6 Flex Real-Time PCR Instrument (Thermo Fisher Scientific) were set at: 95 °C for 2 min, 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 70 °C for 34 s. Raw data were analyzed at a threshold of 0.015 within QuantStudio™ Real-Time PCR software v1.3 (Thermo Fisher Scientific) and exported into Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Differential expression or delta quantification cycle (ΔCq) values were calculated by subtracting the average Cq of let-7g and let-7i from the Cq value of the target miRNA (ΔCq = Cq( miRNA target ) − Cq( avg let-7g & let-7i )). All experiments were performed and analyzed according to Minimum Information for Publication of Quantitative Real-Time PR Experiments (MIQE) guidelines . Each miRNA target was amplified in duplicate technical replicates for each sample with no template controls (NTCs) and negative reverse transcription (RT) controls (non-transcribed RNA extract—to eliminate genomic DNA contamination as a variable) on each plate.
Blood, menstrual secretions, feces, urine, saliva, semen, and vaginal secretions were collected between 2016–2020 using informed consent in accordance with the approved Institutional Review Board Human Subjects Research Protocol (HM200009027). Menstrual, fecal, and vaginal samples were collected on sterile cotton swabs by the donors and returned in swab boxes. Blood was deposited onto a sterile cotton swab after sterilizing and pricking the donor’s finger with a Unistik ® 3 Normal lancet (Owen Mumford Ltd., Woodstock, UK), and saliva was collected by rolling a sterile cotton swab along the inside of the donor’s cheek. Urine and semen were deposited into sterile collection cups supplied to the donor, which were returned on ice within 24 h before aliquoting each onto sterile cotton swabs (50 µL of semen and 100 µL of urine). All swabs were dried and stored in swab boxes at room temperature until treatment and/or DNA extraction.
DNA was isolated from whole swabs using the QIAgen DNA Investigator Kit on the QIAcube (Qiagen, Valencia, CA, USA) and the previously validated manufacturer’s protocol for forensic casework samples. No modifications such as the addition of glycogen were used in an effort to mimic a forensic workflow as closely as possible. Final elution volumes were as follows: 30 μL for saliva, blood, menstrual secretions, semen, and vaginal fluid, 50 μL for feces, and 20 μL for urine. Reagent blanks were included with each batch of DNA extractions, and extracts were stored at −80 °C until further use. DNase treatment was not performed on the sample extracts, as primer evaluation and previous work has demonstrated no significant difference on miRNA detection levels , indicating that genomic DNA does not interfere with miRNA detection. Reverse Transcription and qPCR controls were analyzed to verify lack of interference in miRNA amplification by genomic DNA. DNA extractions and subsequent cDNA synthesis were performed in a dedicated workspace for RNA, following strict procedures to help limit the effect of contamination, including physical isolation from post-PCR laboratories, use of personal protective equipment, and use of molecular biology grade reagents and consumables. miRNA isolation efficiency was measured through RT-qPCR analysis, as our previous work has shown that UV spectrophotometry and other methods cannot precisely predict miRNA concentrations in the low concentrations observed in biological fluids [ , , , ].
Blood, semen, saliva, and urine were collected from three different donors. Blood was collected into a Vacutainer ® containing EDTA (Beckton, Dickinson & Company, Franklin Lakes, NJ, USA) and inverted for 15 s before 50 μL was deposited onto a sterile cotton swab. Urine, semen, and saliva were collected into a sterile collection cup, and 50 μL (semen, saliva) or 100 μL (urine) were deposited onto sterile cotton swabs. The swabs were dried at room temperature for 24–48 h and then stored at −20 °C until treatment, which was performed within 72 h of drying. For heat-compromised samples, swabs were exposed to either 55 °C or 95 °C for 0.5, 1, 2, 4, or 24 h. For the chemically treated samples, 100 μL of either 1:10 (87 nM) or full-strength (870 mM) sodium hypochlorite, dish soap (Dawn Ultra Dishwashing Liquid, Proctor & Gamble, Cincinnati, OH), or glacial acetic acid (pH 2.5, 17.4 M) were deposited onto the prepared swabs and dried for 72 h. UV-treated samples were exposed to 4 h of 302 nm light at room temperature using the UVP high-performance ultra-violet transilluminator (UVP, Upland, CA, USA). This wavelength was chosen as it is in the middle of the ultraviolet range and has been demonstrated to induce the most significant DNA damage . After treatment, all swabs were stored at −20 °C until DNA isolation. Dried blood, semen, saliva, and urine were also tested in an environmental chamber for simulated outdoor conditions. Samples were deposited onto a cotton swatch from Layne et al. using a single donor for each body fluid. The samples were exposed to treatment in a Q-sun Ce-3 Environmental Chamber (Q-Lab Corporation, Westlake, OH, USA) at the Federal Bureau of Investigation Research Laboratory. The Environmental chamber controlled for temperature, humidity, and a 24 h light/dark cycle to imitate a summer day in Virginia ( ). The samples were removed from the chamber at 48 h intervals up to 14 days and stored at −80 °C until punches were taken. Using a biopsy punch, 4 mm punches were taken from the remaining cotton swatches and stored at −80 °C until DNA isolation (5 years).
Reverse transcription was performed on the Proflex PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using the qScript™ microRNA Quantification System (Quanta Biosciences, Gaithersburg, MD, USA) following the previously reported protocol . qPCR primers for all miRNA target sequences were purchased from IDT (Integrated DNA Technologies, Coralville, IA, USA) ( ), and qPCR was performed according to the protocol in quarter volume reactions: 6.25 µL of 2X PerfeCTa ® SYBR Green SuperMix, 0.25 µL of PerfeCTa ® Universal Primer (UP: 5′-ATGGCGGTAAGTCCAGATACG-3′)(Quanta Biosciences) and IDT MicroRNA Primer Assay (2.5 µM), 3.75 µL of nuclease-free water, and 2 µL of cDNA reaction for a total reaction volume of 12.5 µL. Thermal cycling parameters on the QuantStudio™ 6 Flex Real-Time PCR Instrument (Thermo Fisher Scientific) were set at: 95 °C for 2 min, 40 cycles of 95 °C for 5 s, 60 °C for 15 s, and 70 °C for 34 s. Raw data were analyzed at a threshold of 0.015 within QuantStudio™ Real-Time PCR software v1.3 (Thermo Fisher Scientific) and exported into Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). Differential expression or delta quantification cycle (ΔCq) values were calculated by subtracting the average Cq of let-7g and let-7i from the Cq value of the target miRNA (ΔCq = Cq( miRNA target ) − Cq( avg let-7g & let-7i )). All experiments were performed and analyzed according to Minimum Information for Publication of Quantitative Real-Time PR Experiments (MIQE) guidelines . Each miRNA target was amplified in duplicate technical replicates for each sample with no template controls (NTCs) and negative reverse transcription (RT) controls (non-transcribed RNA extract—to eliminate genomic DNA contamination as a variable) on each plate.
Initial statistical analyses of the raw and ΔCq data were performed in JMP ® v14.2.0 (SAS Institute, Cary, NC, USA). Normal distribution and equal variance were confirmed for all sample sets using quantile–quantile plots and Levine’s test, respectively. In multi-group comparisons, a one-way ANOVA test was performed with Tukey’s HSD pairwise comparison. A significance level of α = 0.05 was used to determine which tests are declared statistically significant. Subsequent statistical prediction modeling was performed in R version 4.0.2 (R Foundation for Statistical Computing, Vienna, Austria). For predictive analysis, Quadratic Discriminant Analysis (qda in R) was used. A 10-fold cross validation procedure was performed to ensure that each observation was in both training and validation sets by splitting the data into ten non-overlapping groups, corresponding to 10% of the samples. Each group was in turn used as a test set while the remaining (i.e., 90% of the data) were used to train the model. Once sample collection was complete, the population dataset consisted of 355 samples: 51 blood, 53 menstrual secretions, 50 feces, 46 urine, 53 saliva, 52 semen, and 50 vaginal secretions ( ). For some of the markers and body fluid combinations, there were some samples in which not all miRNAs were tested; thus, across the total dataset, there were 252 fully observed values. To address the issues associated with these missing values, a single imputation using the conditional multivariate mean was employed. This allowed for the imputed value to depend on all the observed values for the subject. Multiple imputation was avoided as the goal of the project is correct classification, and multiple imputation would not allow for easy model validation. To improve the ability of the classifiers, an “Other” category was created by fitting a multivariate normal distribution to the entire dataset and drawing samples outside the 3.5-standard-deviation ellipsoid. Without this other category, any future extreme observations will be classified as the body fluid closest even though it is truly an extrapolation. This Other category allows for only those body fluids with measurements in the range observed to be classified as a body fluid. All measurements outside the observed measurement range will be classified as Other. After development of the QDA model and cross-validation, classification testing was set at 50% confidence for body fluid classification.
To evaluate variation in differential expression within an individual over time or within a biological cycle, three volunteers for each body fluid donated multiple samples according to the time conditions listed in . Sample collection, DNA extraction, and RT-qPCR analysis were performed as described above.
3.1. Validation of miRNA Panel for Body Fluid Identification Initial evaluation of miRNA detection for body fluid identification in DNA extracts were modeled on our previous work in RNA extracts by testing the same panel of miRNAs (miRs 200b, 320c, 10b, and 891a relative to the average expression of lets-7g and 7i) in 50 population samples for each biological fluid (blood, semen, vaginal and menstrual secretions, saliva, feces and urine). Development, verification, and 10-fold cross validation of an analogous QDA model for DNA extracts demonstrated 88.0% overall accuracy. Identification of the biological fluids was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72–98% of the unknown samples classified correctly ( ). As expected, miRNA detection was slightly different in DNA extracts as compared to the RNA extracts previously validated, and so the predictive model was not as accurate as the identification in RNA extracts. Therefore, we identified additional markers to test and improve accuracy of the panel. We identified markers from the literature and our previous work that had the potential to discriminate body fluids in DNA extracts and evaluated them using a tiered population sample approach to conserve samples. We tested several miRNAs identified in the literature with a sample of our population extracts and identified miRs-141, 412, and 205 as being possibly discriminatory and useful to add to the panel. We found that all three miRNAs could assist in improving discrimination, particularly in feces, menstrual and vaginal secretions, saliva ( ) and urine, for an overall prediction accuracy of 92.1% in a 10x-cross-fold validation of the quantitative discriminant analysis (QDA) method ( , ). We have made this prediction model available for public use ( https://vcu-frsc-sswlab.shinyapps.io/QDA-Prediction-Analysis-wDNA/ accessed on 24 April 2023), and the code can be found at https://github.com/VCU-Forensic-Science-Williams-Lab accessed on 24 April 2023 (file name miRNA panel in DNA-7 miRs (1).R). Note that the only disadvantage of including more markers is time and wells in preparation of the qPCR plate, as preparation of the reverse transcription reaction is the same regardless of the number of markers (until the reverse transcription reaction is depleted), and downstream data analysis is also unchanged for the practitioner. 3.2. Variation within Individuals over Time miRNA detection from DNA extracts of each body fluid were measured to observe whether there was a change over the course of biological cycles or time ( ). Blood, urine, and vaginal secretions performed similarly in classification rates to the population samples ( ), while semen was classified less accurately (77.8% as compared to 90% in population samples). However, menstrual secretions were dramatically decreased in terms of their accurate classification rate, though they were mostly misclassified as vaginal secretions, and none were misclassified as blood. Saliva classification was very poor in these samples—the expression of miR-412 and miR-205 in the saliva samples after eating on the first day were significantly different from the previous donation and the donation directly following it ( p < 0.05,). The expression of miR-205 was also significantly different in the day 2 wake up donation when compared to the donation before it ( p < 0.05). The classification rate in these samples was very low due to these differences, which could be caused by differences in metabolism and stimulation of different salivary glands prior to and after eating a meal . 3.3. Detection of miRNAs in Treated Samples 3.3.1. Heat Treatment The robustness of miRNA marker detection from DNA extracts of blood, semen, saliva, and urine was tested over a period of 24 h at a temperature exposure of either 55 °C or 95 °C. MicroRNA levels in blood proved to be highly resistant to degradation over all heat treatments and showed no significant variation among markers when compared to the untreated controls ( ). These findings are similar to those of Fang et al., which also demonstrated the robustness of miRNA markers in RNA extracts from blood at elevated heat conditions , as well as the findings from Layne et al. and Mayes et al. using RNA extracts of blood . While urine demonstrated some evidence of degradation, classification rates were similar to the population samples. In contrast, semen and saliva markers were degraded over time after exposure to heat treatment, resulting in a reduced classification rate of 57.6 and 48.5%, respectively, compared to 90 and 84%, respectively, for population classification ( ). 3.3.2. Chemical and Ultraviolet Treatment The effects of chemical or UV treatment on blood, semen, saliva, and urine showed similar degrading patterns, with blood and urine classifying at a rate similar to the population samples, and semen and saliva demonstrating degradation resulting in reduced classification rates ( , ). Semen was most greatly impacted by the application of dish soap, 1:10 bleach dilution, or full-strength bleach. Markers miR200b, miR10b, and miR205 were the most significantly different from the untreated controls after these treatments in semen samples ( p < 0.05). These differences are reflected in the low correct classification rate of 66.7%. The low classification rate is supported by findings from Mayes et al., which found differing ΔCq values after laundering with a detergent . Saliva exhibited a low classification rate of 33.3% and showed greater sensitivity to UV and glacial acetic acid (GAA) treatment. Saliva also showed significant degradation after full strength bleach treatment in all markers except for miR200b. These findings differ from the results in corresponding RNA extracts in Layne et al., which found that urine was not significantly affected by these treatments . 3.3.3. Environmental Chamber Stability Exposure to controlled heat, light/dark cycle, and humidity showed the same pattern, with blood and urine classification unchanged from population samples, indicating the robustness of the markers and classification method. In contrast, neither semen nor saliva samples were correctly classified in any of the treated or untreated samples ( ). Since the untreated samples were also incorrectly classified, and showed high raw Cq values, the results for this sample set are inconclusive ( ). The age of these samples may have also been a factor, considering that they were stored at −80 °C for five years after treatment, implying that future sample age studies should be evaluated further.
Initial evaluation of miRNA detection for body fluid identification in DNA extracts were modeled on our previous work in RNA extracts by testing the same panel of miRNAs (miRs 200b, 320c, 10b, and 891a relative to the average expression of lets-7g and 7i) in 50 population samples for each biological fluid (blood, semen, vaginal and menstrual secretions, saliva, feces and urine). Development, verification, and 10-fold cross validation of an analogous QDA model for DNA extracts demonstrated 88.0% overall accuracy. Identification of the biological fluids was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72–98% of the unknown samples classified correctly ( ). As expected, miRNA detection was slightly different in DNA extracts as compared to the RNA extracts previously validated, and so the predictive model was not as accurate as the identification in RNA extracts. Therefore, we identified additional markers to test and improve accuracy of the panel. We identified markers from the literature and our previous work that had the potential to discriminate body fluids in DNA extracts and evaluated them using a tiered population sample approach to conserve samples. We tested several miRNAs identified in the literature with a sample of our population extracts and identified miRs-141, 412, and 205 as being possibly discriminatory and useful to add to the panel. We found that all three miRNAs could assist in improving discrimination, particularly in feces, menstrual and vaginal secretions, saliva ( ) and urine, for an overall prediction accuracy of 92.1% in a 10x-cross-fold validation of the quantitative discriminant analysis (QDA) method ( , ). We have made this prediction model available for public use ( https://vcu-frsc-sswlab.shinyapps.io/QDA-Prediction-Analysis-wDNA/ accessed on 24 April 2023), and the code can be found at https://github.com/VCU-Forensic-Science-Williams-Lab accessed on 24 April 2023 (file name miRNA panel in DNA-7 miRs (1).R). Note that the only disadvantage of including more markers is time and wells in preparation of the qPCR plate, as preparation of the reverse transcription reaction is the same regardless of the number of markers (until the reverse transcription reaction is depleted), and downstream data analysis is also unchanged for the practitioner.
miRNA detection from DNA extracts of each body fluid were measured to observe whether there was a change over the course of biological cycles or time ( ). Blood, urine, and vaginal secretions performed similarly in classification rates to the population samples ( ), while semen was classified less accurately (77.8% as compared to 90% in population samples). However, menstrual secretions were dramatically decreased in terms of their accurate classification rate, though they were mostly misclassified as vaginal secretions, and none were misclassified as blood. Saliva classification was very poor in these samples—the expression of miR-412 and miR-205 in the saliva samples after eating on the first day were significantly different from the previous donation and the donation directly following it ( p < 0.05,). The expression of miR-205 was also significantly different in the day 2 wake up donation when compared to the donation before it ( p < 0.05). The classification rate in these samples was very low due to these differences, which could be caused by differences in metabolism and stimulation of different salivary glands prior to and after eating a meal .
3.3.1. Heat Treatment The robustness of miRNA marker detection from DNA extracts of blood, semen, saliva, and urine was tested over a period of 24 h at a temperature exposure of either 55 °C or 95 °C. MicroRNA levels in blood proved to be highly resistant to degradation over all heat treatments and showed no significant variation among markers when compared to the untreated controls ( ). These findings are similar to those of Fang et al., which also demonstrated the robustness of miRNA markers in RNA extracts from blood at elevated heat conditions , as well as the findings from Layne et al. and Mayes et al. using RNA extracts of blood . While urine demonstrated some evidence of degradation, classification rates were similar to the population samples. In contrast, semen and saliva markers were degraded over time after exposure to heat treatment, resulting in a reduced classification rate of 57.6 and 48.5%, respectively, compared to 90 and 84%, respectively, for population classification ( ). 3.3.2. Chemical and Ultraviolet Treatment The effects of chemical or UV treatment on blood, semen, saliva, and urine showed similar degrading patterns, with blood and urine classifying at a rate similar to the population samples, and semen and saliva demonstrating degradation resulting in reduced classification rates ( , ). Semen was most greatly impacted by the application of dish soap, 1:10 bleach dilution, or full-strength bleach. Markers miR200b, miR10b, and miR205 were the most significantly different from the untreated controls after these treatments in semen samples ( p < 0.05). These differences are reflected in the low correct classification rate of 66.7%. The low classification rate is supported by findings from Mayes et al., which found differing ΔCq values after laundering with a detergent . Saliva exhibited a low classification rate of 33.3% and showed greater sensitivity to UV and glacial acetic acid (GAA) treatment. Saliva also showed significant degradation after full strength bleach treatment in all markers except for miR200b. These findings differ from the results in corresponding RNA extracts in Layne et al., which found that urine was not significantly affected by these treatments . 3.3.3. Environmental Chamber Stability Exposure to controlled heat, light/dark cycle, and humidity showed the same pattern, with blood and urine classification unchanged from population samples, indicating the robustness of the markers and classification method. In contrast, neither semen nor saliva samples were correctly classified in any of the treated or untreated samples ( ). Since the untreated samples were also incorrectly classified, and showed high raw Cq values, the results for this sample set are inconclusive ( ). The age of these samples may have also been a factor, considering that they were stored at −80 °C for five years after treatment, implying that future sample age studies should be evaluated further.
The robustness of miRNA marker detection from DNA extracts of blood, semen, saliva, and urine was tested over a period of 24 h at a temperature exposure of either 55 °C or 95 °C. MicroRNA levels in blood proved to be highly resistant to degradation over all heat treatments and showed no significant variation among markers when compared to the untreated controls ( ). These findings are similar to those of Fang et al., which also demonstrated the robustness of miRNA markers in RNA extracts from blood at elevated heat conditions , as well as the findings from Layne et al. and Mayes et al. using RNA extracts of blood . While urine demonstrated some evidence of degradation, classification rates were similar to the population samples. In contrast, semen and saliva markers were degraded over time after exposure to heat treatment, resulting in a reduced classification rate of 57.6 and 48.5%, respectively, compared to 90 and 84%, respectively, for population classification ( ).
The effects of chemical or UV treatment on blood, semen, saliva, and urine showed similar degrading patterns, with blood and urine classifying at a rate similar to the population samples, and semen and saliva demonstrating degradation resulting in reduced classification rates ( , ). Semen was most greatly impacted by the application of dish soap, 1:10 bleach dilution, or full-strength bleach. Markers miR200b, miR10b, and miR205 were the most significantly different from the untreated controls after these treatments in semen samples ( p < 0.05). These differences are reflected in the low correct classification rate of 66.7%. The low classification rate is supported by findings from Mayes et al., which found differing ΔCq values after laundering with a detergent . Saliva exhibited a low classification rate of 33.3% and showed greater sensitivity to UV and glacial acetic acid (GAA) treatment. Saliva also showed significant degradation after full strength bleach treatment in all markers except for miR200b. These findings differ from the results in corresponding RNA extracts in Layne et al., which found that urine was not significantly affected by these treatments .
Exposure to controlled heat, light/dark cycle, and humidity showed the same pattern, with blood and urine classification unchanged from population samples, indicating the robustness of the markers and classification method. In contrast, neither semen nor saliva samples were correctly classified in any of the treated or untreated samples ( ). Since the untreated samples were also incorrectly classified, and showed high raw Cq values, the results for this sample set are inconclusive ( ). The age of these samples may have also been a factor, considering that they were stored at −80 °C for five years after treatment, implying that future sample age studies should be evaluated further.
This study investigated the ability to classify body fluids in DNA extracts using a more comprehensive set of miRNA markers than previously evaluated. A total of 355 samples of DNA extracts from blood, semen, vaginal and menstrual secretions, saliva, feces and urine were tested for classification accuracy in the original and subsequently expanded panel of miRNAs, resulting in a QDA model with an overall accuracy of 92%. Further evaluation of panel performance in individuals over time and compromised samples demonstrated some limitations of the method in certain biological fluids. Heat treatments had a greater impact on both the detected miRNA quantities in semen and saliva as well as the classification accuracy compared to blood and urine. This trend continued with the chemical and UV treatments; however, the overall decrease in detectability proved to be treatment, body fluid, and marker dependent. Marker expression across a biological cycle appeared to impact correct classification rates dramatically in saliva and menstrual secretions, indicating that the addition of other more consistent markers may be necessary for reliable prediction. As these findings model those results from the same sample types in RNA extracts, our data suggest that detection and prediction in saliva and semen tends to be less robust. Additionally, our previous work in RNA extracts indicated a limitation of the prediction model for handling samples of mixed sources, which of course are often encountered, particularly in sexual assault samples . The development of a panel of miRNAs that can predict body fluids with over 90% accuracy from DNA extracts is a significant step forward. By developing a robust method that uses DNA extracts instead of RNA extracts, a significant barrier to implementation is removed—that of additional analyst time, reagent costs, and sample consumption required for a separate RNA isolation method. Much of the historical resistance to a novel body fluid identification method such as mRNA or miRNAs has been due to the additional isolation methods required; therefore, using a DNA extract for body fluid identification combined with analysis methods that utilize existing equipment in a forensic laboratory could lead to rapid, large-scale implementation into the forensic DNA analysis workflow. However, before the assay can be implemented into casework, an evaluation of different analysis methods and prediction modeling in which compromised and mixed samples can be accurately classified is important in order to address real-world sample types. It may also be beneficial to explore adding non-miRNA markers to the panel, such as a combinatorial assay using microbial DNA markers and/or methylation, which may be more accurate than miRNA markers alone given the relative strengths of each marker type. Having now demonstrated that miRNAs are detectable in DNA extracts, this is a possibility, and biomarkers can be combined into a more comprehensive assay . As more markers are added to the panel, targeted high-throughput sequencing may be considered instead of qPCR, since it would allow this assay to be performed more quickly while simultaneously evaluating many markers of different origins.
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THE IMPACT OF COVID-19 PANDEMIC ON DIAGNOSIS AND TREATMENT IN OTORHINOLARYNGOLOGY PATIENTS IN CROATIA
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a9469bc5-875a-4d68-ad40-97a4a550ab38
|
10218082
|
Otolaryngology[mh]
|
The World Health Organization declared the SARS-CoV-2 pandemic on March 11, 2020. The pandemic reached Croatia on February 25, 2020, when the first patient tested positive for SARS-CoV-2. There were, and still are, many unknowns about this virus, its contagiousness, prevention of the disease, clinical presentation, possible treatment, and outcomes, compounded by its frequent mutations and subsequent variants. Otorhinolaryngology was recognized early as one of the clinical specialties at a high risk of potential professional exposure ( - ). Clinical practice among ENT specialists has therefore changed ( , ). Early after the pandemic had started, we noticed a significant decrease in non-COVID referrals on our and other ENT departments due to several reasons (decrease in primary health care visits, fear among patients themselves, decrease in the capacity of ENT departments and hospitals in general, etc.). It was logical to anticipate that this would result in some consequences for patients. Several cases of delayed diagnosis and treatment in non-COVID patients have been documented in our department as in other medical centers worldwide ( - ). The aim of the present work was to perform an anonymous survey among Croatian ENT specialists to assess how the pandemic influenced the ENT practice in Croatia.
A questionnaire was sent to e-mail addresses of all registered otorhinolaryngologists in Croatia (300 e-mail addresses), and a reminder one month later. It was made using the QuestionPro web platform and was available for two months, from February 22 to April 22, 2021. Two types of questions were used, i.e., nominal questions and questions based on the Likert scale. Participants were asked about the most used source of information during the COVID-19 pandemic, satisfaction with available personal protective equipment (PPE), changes in the number of diagnostic and therapeutic procedures, and evidence for delay in diagnosis and/or treatment of different diseases in ENT. The complete questionnaire is reported in . We received 123 completely answered and 157 partially answered questionnaires. Only complete responses were considered for analysis. Statistical analysis was performed using Jamovi free statistical software ( ).
The majority of participants that completed the survey are employed at clinical hospitals or university hospital centers (57%) and general or regional hospitals (32%). The remaining participants are private practitioners (9%) or work only as ENT consultants at Community Health Centers (2%). At the beginning of the pandemic, the majority of physicians employed at tertiary health care institutions performed complete clinical examinations, including endoscopic procedures, with the highest available level of PPE (41%), 29% of respondents performed only urgent examinations, and 27% of respondents continued normal clinical practice relying only on patient history data (possible exposure to COVID and symptoms) ( ). Cross-tabulation of answers showed similar distributions of these different clinical practices among ENT specialists regardless of the place where they work, level of health care they provide (secondary or tertiary), and their main source of information on COVID-19. Every third ENT specialist was quarantined due to professional exposure to COVID-19 (37%), and every sixth ENT specialist acquired COVID-19 infection due to professional exposure (16%). The most common source of information used to guide clinical practice during the COVID-19 pandemic was internet (31%), followed by various ENT associations (29%), official employment institution instructions (25%), and consultation with colleagues (17%). Only 6% of participants used research papers as their primary and most used source of information. A large proportion of otorhinolaryngologists were satisfied or very satisfied (48%) or neutral (33%) regarding given instructions and PPE available at the workplace. Unsatisfied physicians were those that got most of their information about work during the pandemic from colleagues and internet. The vast majority of respondents stated significant reduction in both the number of patient referrals and their capacities ( ). Only 4% of physicians had the same number of patients and 7% of physicians did not observe decrease in the capacity to admit patients. When asked about delay in the diagnosis and treatment in particular fields in ENT practice, 44% of participants stated that there was delay in the diagnosis and treatment in patients with chronic ENT diseases due to the COVID-19 pandemic but believed that this delay did not result in severe consequences for patients ( ). However, 62% of participants answered that there were many cases of significant delay in the diagnosis and treatment of patients with head and neck cancers that would have an impact on the patient outcomes ( ). During the first months of the pandemic, 37% of otorhinolaryngologists stopped performing elective surgery in patients with aesthetic and/or functional impairment ( ). Only 6% continued performing these procedures without delay. The rest of the respondents performed these procedures, but with significant delay in scheduling.
At the beginning of the first pandemic wave in Croatia, many facts regarding the virus contagiousness, morbidity, prevention, outcomes, etc. were unknown. Consequently, a significant reduction in various diagnostic and therapeutic procedures was noted. Several months after the beginning of the pandemic, we noticed an increase in the number of patients whose conditions might have been attributable to delay in the diagnosis or treatment of the initial medical condition. Examples of this are cases of odontogenic sinusitis, massive untreated polyposis, and several cases of advanced sinonasal tumors ( ). Since the overall number of them is generally low (complications of untreated diseases, sinonasal and especially advanced sinonasal tumors), a statistically significant increase due to some extrinsic cause will be hard to demonstrate in a short time. It was the reason to perform a survey among the physicians who practice otorhinolaryngology. We think that the number of complete responses, which is nearly half of all working otorhinolaryngologists in Croatia, is representative to draw some valid conclusions. The results of this survey confirmed that many ENT specialists shared our grim view regarding reduction in patient referrals, thus compromising patient welfare in terms of diagnosing and treating various ENT medical conditions. During the pandemic, the focus of medical interest and capacities shifted to COVID patients ( ). As a result, many non-COVID patients experienced difficulties in obtaining primary and secondary health care. We are aware that, even before the pandemic, there was delay in the diagnosis of many ENT malignancies ( ). The main reason for the delay is that the symptoms of ENT tumors in the early phase are usually mild and overlapping with the symptoms of chronic inflammatory diseases. According to our tumor patient data, the duration of symptoms prior to obtaining the diagnosis is approximately two months, and in some cases several months, which is in accordance with literature data ( ). Several models aiming to reduce the time from initial symptoms to definitive treatment have been introduced in different countries. Examples are The Two-Week Rule for Head and Neck Cancers in the United Kingdom in 2000 ( ) and The Danish Fast-Track for Head and Neck Cancers in 2007 ( ). In our tertiary center, there was also an established fast-track program in which patients with head and neck cancer would be treated within 2 to 3 weeks from initial referral. The pandemic reorganization of our department did not adversely affect this interval. Although the pandemic will likely end at some point, it is still active at the time of writing this paper. Furthermore, there are reasonable fears of a similar event occurring in the future, and preparations have already begun ( ). In this context, there should not be any doubt regarding the necessity for reorganization of health care systems, particularly with the aim of improving the availability of health care at secondary and tertiary centers. We believe there is room for improvement based on several facts which are elaborated below. Our data showed that patients with protracted symptoms did not get appropriate referral in a timely manner. Although patient history and symptoms, without clinical and endoscopic examination, are insufficient for an accurate diagnosis, especially given that symptoms of early tumors overlap with other chronic and inflammatory diseases, carefully taken history (especially if taken by an experienced ENT specialist) may serve as an appropriate screening method for detecting patients in need of a prompt clinical examination. In addition, an important reason for reduction of capacities during the pandemic was very likely the need for quarantine of physicians, nurses, and patients after contact with a COVID-positive person or contracting COVID-19 infection without necessarily developing severe (or any) symptoms. This means that many of those isolated, both patients and health care workers, were not be ill, but will be unavailable for diagnostics and treatment of non-COVID patients. While their quarantine was necessary, these extensive time periods could have been used for telephone or even video-call consultations and evaluations by selected specialists. Technical requirements are already fulfilled since access to telephones and camera-enabled mobile devices is ubiquitous today. The only remaining task would be developing an organizational framework for such consultations. In conclusion, the COVID-19 pandemic brought forth many challenges for the health care system and resulted in many restrictions and their consequences. Along with the heavy disease burden of COVID-19 itself, our survey shows that many experts believe the buckling of the health care system has reflected intensely on non-COVID patients. This pandemic has shown that we must be able to adapt faster to similar situations and reduce the potential negative impact on our patients.
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Preliminary Study: DNA Transfer and Persistence on Non-Porous Surfaces Submerged in Spring Water
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745d15e4-539f-4164-a746-34968208c6e8
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10218150
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Forensic Medicine[mh]
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Submerged items can occur in a variety of forensic situations from underwater to indoor crime scenes. At indoor scenes, investigators may find items left behind in sinks, showers, toilets, and bathtubs. Even if a crime occurs elsewhere, a perpetrator may try to destroy evidence by throwing it into a river, lake, or ocean . Many investigators believe that submerged items no longer hold evidentiary value, especially for identification purposes . However, identifiable trace evidence, fingerprints, and DNA have all been successfully recovered from submerged items. After the introduction of RFLP (Restriction Fragment Length Polymorphism), DNA has become one of the most prominent forensic techniques , but research suggests that its recovery potential from aquatic conditions is dependent on numerous factors . In addition to the physical presence of water, DNA recovery on submerged items seems to be further hindered by higher salinities and stronger currents . The structure of the items appears to influence DNA recovery as well. Porous items, such as bedsheets and paper, appear to retain submerged DNA for longer times than non-porous items, such as car doors and bottles . The crevices of fibers existing in porous items may be able to protect DNA from being lost to currents . Cellular material surrounding DNA may be able to protect DNA for a given time, but the water itself is capable of hydrolyzing DNA and destroying valuable evidence . However, submerged non-porous items can still hold evidentiary value after this restrictive timeframe. Multiple studies have shown that latent fingerprints can be recovered from non-porous items [ , , ] even after a month of submersion . Similar to DNA, submerged latent fingerprint recovery appears to be further hindered by higher temperatures, higher salinities, and faster currents [ , , ]. When depositing a latent fingerprint (also referred to as a fingermark), one leaves behind natural oils and insoluble secretions which may be less miscible in water than soluble DNA. Fingerprint samples are capable of generating low levels of DNA, although the nature of that DNA (shed keratinocytes, epithelial cells, nucleated cells from other parts of the body that the hands have contacted, and cell-free DNA as examples) is still debated . Some refer to this recovery of low-level DNA as “touch DNA”, or “transfer DNA” yet the term “trace DNA” will be utilized in this study. Because fingerprints are retained on submerged non-porous evidence, it was hypothesized that the insoluble components of fingerprint secretions may protect DNA by trapping any residual epithelial cells between the secretions and the surface itself, permitting trace DNA recovery even under submerged conditions. Previous studies have highlighted the ability to obtain DNA from latent fingerprints [ , , , ]. Investigators and analysts could utilize this knowledge to determine the cost–benefit of pursuing friction ridge comparison in addition to, or possibly in lieu of, trace DNA analysis. Such knowledge could supplement research that focuses on choosing optimal samples for trace DNA analysis. For example, nucleic acid dyes, such as SYBR Green I, have been utilized to visualize low-level DNA on touched objects to evaluate the cost–benefit of pursuing trace DNA analysis . The behavior of DNA in saliva deposition slide samples was evaluated in addition to the mentioned fingerprint samples. Trace samples have an inherent unpredictability in the amount of DNA that will transfer in deposition, so saliva slide samples provided a way of being able to deposit a known amount of DNA and monitor loss over time. Therefore, saliva deposition slide samples served as controlled samples, while fingerprint slide samples were representative of mock casework samples. Because non-porous surfaces do not have traits that might aid in DNA retention, it was hypothesized that DNA quantities and the number of donor alleles recovered would decrease over longer submersion periods. Additionally, it was hypothesized that DNA quantity and the number of alleles would be negatively affected by flow conditions.
2.1. Experimental Phases The experiment was broken into four phases. In Phases 1 and 2, slides containing the saliva samples were placed into stagnant and flowing spring water, respectively. In Phases 3 and 4, slides containing the fingerprint samples were placed into stagnant and flowing spring water, respectively. Phases 1 and 2 pertain to controlled samples with known initial quantities of DNA, while Phases 3 and 4 reflect mock casework samples where the initial quantity of DNA is unknown. Additionally, two replicates of blank slide samples per submersion time were utilized for each phase. 2.2. Water Environments Two water environments were utilized: stagnant spring water and flowing spring water. All water environments were created in an indoor laboratory setting with Poland Spring ® water, as the company provides information about the levels of various chemical and biological components and physical characteristics of the water. Before experimentation, samples from the Poland Spring ® water jugs were extracted and quantified for the possible presence of background human DNA, and none was detected for the methods utilized (see below). The stagnant environment was created by filling a glass tray with approximately 1.5 L spring water. The glass tray was cleaned prior to use by spraying the tray with 20% bleach, 70% reagent alcohol, and distilled water followed by cross-linking for 1000 s with a SpectroLinker XL-1500 UV Crosslinker. While in use, the glass tray was stored under a PCR hood ( ). The flowing environment was created by filling a horizontal flume with approximately 5 L spring water. The flume was formed with 2-inch diameter polyvinyl chloride (PVC) plastic, with overall dimensions measuring 32 by 56 inches at its widest points. The flume was powered by a PULACO aquarium wave-making pump with a maximal flow rate of 50 GPH (Gallons per Hour). A few inches after the pump were deemed a “dead zone” where no samples were placed, permitting the flow to rise to a constant speed of approximately 10 cm/s. PVC was cut horizontally in the “sample zone” to allow samples to be placed into and removed from the water. Samples were oriented such that the wide surface of the slides was parallel to the direction of the current for the samples to experience a constant flow. The water recirculated through the flume over the course of the experiment. To avoid disrupting the flow when rounding the corners, the flume utilized two 45-degree PVC elbows rather than 90-degree angles. Between uses, the flume was cleaned by spraying with 20% bleach, 70% reagent alcohol, and distilled water. Due to its size, the flume was unable to be cross-linked and was stored in an active research room when in use. Bins were placed over open areas in the flume to attempt to avoid extraneous contamination ( ). 2.3. Creating and Depositing Samples Sirchie glass microscope slides were utilized as the non-porous surface. The glass slides were cross-linked on both sides for 1000 s in a SpectroLinker XL-1500 UV Crosslinker before use. Three random slides were swabbed and processed for human DNA after cross-linking. No quantity of human DNA was recovered, so it was assumed that cross-linking for 1000 s on both sides would remove any background human DNA from the slides. Sample slides were divided into three main categories. “Blank” slide samples had nothing added to the slide after cross-linking. For Phases 1 and 2, “deposition” slide samples had 5 μL fresh neat saliva added to the slide. A portion of the neat saliva (20 μL) was extracted following the QIAamp DNA Investigator Protocol: Isolation of Total DNA from Small Volumes of Blood or Saliva and quantified (yielding a concentration of approximately 6 ng/μL) before deposition to estimate the initial amount of DNA being deposited onto each sample. Two blank slide samples and five deposition samples were utilized per submersion time per water condition for these phases. For Phases 3 and 4, “fingerprint” slide samples had a sebaceous fingerprint deposited onto the slide with a force of approximately 400 g with contact lasting three seconds. Force was measured by placing the slide on a scale when depositing the fingerprint. The right thumb of one donor was utilized as the fingerprint source for all samples. Before depositing the fingerprints, the donor washed their hands for 20 s with warm soapy water and allowed their hands to air-dry for approximately 5 min. The donor then wiped their right thumb across their forehead to obtain sebaceous secretions and deposited the print onto the designated glass slide. The cleaning process was repeated between fingerprint depositions. Two blank slide samples and five fingerprint slide samples were utilized per submersion time per water condition for these phases. Due to the inherent unpredictability of touch DNA samples, the initial amount of DNA transferred from the finger to the slide was unknown. However, three fingerprint samples (right thumb) were created, extracted, quantified, and amplified for human DNA prior to submersion to attempt to establish a baseline. The quantities of these initial fingerprint samples ranged from 0.0106 to 0.0343 ng/μL with an average of 0.0224 ng/μL (σ 2 = 1.404 × 10 −4 ). One male donor aged 22–24 was utilized in this study and was the donor for both saliva and fingerprint samples. All samples were obtained according to informed consent. 2.4. Sample Submersion and Recovery For Phases 1 and 2, all blank and deposition slide samples entered the water at the same time, and a set of slides were removed at each time interval: 6 h, 12 h, 24 h, 48 h, 72 h, and 168 h (1 week). For Phases 3 and 4, all blank and fingerprint slide samples entered the water at the same time, and a set of slides were removed at each time interval: 24 h, 48 h, 72 h, and 168 h (1 week). An additional set was evaluated at 0 h to serve as an initial assessment. The experiment was carried out in an indoor lab environment at room temperature (approximately 20 °C). In the flowing environment, cleaned blank slides were inserted in place of the removed slides to avoid altering the way water flowed through the flume. To reduce the risk of introducing microbes when removing the sets, researchers utilized proper PPE and disinfected gloves with 70% reagent alcohol. As slides were removed from the water, they were placed on a sterile benchtop to air dry. When fully dry, the slides were either swabbed and extracted for DNA or stored at −80 °C until extraction. When stored at −80 °C, slides were kept in cross-linked microscope slide holder boxes. The wet-dry swab method was utilized to collect potential DNA from the slides which is a common technique for trace DNA recovery . 2.5. DNA Analysis Qiagen’s QIAamp DNA Investigator Kit was utilized to extract the DNA collected from each sample. All swabs from samples (blank slides, deposition slides, and fingerprint slides) were extracted following the QIAamp DNA Investigator Protocol: Isolation of Total DNA from Surface and Buccal Swabs. This extraction procedure included adding proteinase K and Buffer ATL to the sample, incubating at 56 °C with shaking for two hours, adding Buffer AL, incubating at 70 °C with shaking for ten minutes, adding ethanol, and transferring the lysate to a QIAamp MinElute column. Samples were washed with Buffer AW1, Buffer AW2, and ethanol before eluting from the QIAamp MinElute column utilizing 50 μL Buffer ATE. Used glass slides and swab heads were saved and stored at −80 °C in case needed for further analysis. Samples were stored in 1.5 mL Eppendorf tubes at −80 °C after extraction. The Quantifiler TM Human DNA Quantification Kit with a QuantStudio 5 Real-Time PCR System (Design and Analysis Software v1.5.1) was utilized to quantify the human DNA present in samples. The Quantifiler TM Human DNA Quantification Kit has a lower threshold of detection of approximately 16 pg/μL of human genomic DNA . The quantity of human DNA present on all samples was recorded. STR amplification was performed for both submersion conditions at every submersion time on all blank slide samples, three saliva slide deposition, and three fingerprint slide samples. Applied Biosystems’s GlobalFiler TM PCR Amplification Kit was utilized to amplify twenty-four STR loci. Capillary electrophoresis was performed with ILS600 LIZ Size Standard on an Applied Biosystems 3130xl Genetic Analyzer (16 capillary array 50 cm, POP4 polymer, 1× buffer). The injection conditions utilized were 3 kV/5 s, and the run conditions were 15 kV/1500 s. STR amplification results were interpreted with the GeneMarker HID STR Human Identity Software (version 2.4). Sample amplification data recorded included number and designation of alleles present, individual allele peak heights, and heterozygous peak height ratios. Internal controls were utilized and during all steps (extraction, quantification, and amplification) in order to monitor any contamination or error. Negative internal controls yielded no indication of contamination. 2.6. Statistical Methods T -Tests (two-tailed) were utilized to calculate p -values as estimates of statistical significance ( p < 0.05 in all instances) when comparing one condition to another (for example, comparing DNA quantities recovered at 24 h in the flowing versus stagnant water conditions). Other mathematical analyses included calculating averages and variance values (σ 2 ) or standard deviation values based on the samples for both DNA quantity and the number of alleles recovered.
The experiment was broken into four phases. In Phases 1 and 2, slides containing the saliva samples were placed into stagnant and flowing spring water, respectively. In Phases 3 and 4, slides containing the fingerprint samples were placed into stagnant and flowing spring water, respectively. Phases 1 and 2 pertain to controlled samples with known initial quantities of DNA, while Phases 3 and 4 reflect mock casework samples where the initial quantity of DNA is unknown. Additionally, two replicates of blank slide samples per submersion time were utilized for each phase.
Two water environments were utilized: stagnant spring water and flowing spring water. All water environments were created in an indoor laboratory setting with Poland Spring ® water, as the company provides information about the levels of various chemical and biological components and physical characteristics of the water. Before experimentation, samples from the Poland Spring ® water jugs were extracted and quantified for the possible presence of background human DNA, and none was detected for the methods utilized (see below). The stagnant environment was created by filling a glass tray with approximately 1.5 L spring water. The glass tray was cleaned prior to use by spraying the tray with 20% bleach, 70% reagent alcohol, and distilled water followed by cross-linking for 1000 s with a SpectroLinker XL-1500 UV Crosslinker. While in use, the glass tray was stored under a PCR hood ( ). The flowing environment was created by filling a horizontal flume with approximately 5 L spring water. The flume was formed with 2-inch diameter polyvinyl chloride (PVC) plastic, with overall dimensions measuring 32 by 56 inches at its widest points. The flume was powered by a PULACO aquarium wave-making pump with a maximal flow rate of 50 GPH (Gallons per Hour). A few inches after the pump were deemed a “dead zone” where no samples were placed, permitting the flow to rise to a constant speed of approximately 10 cm/s. PVC was cut horizontally in the “sample zone” to allow samples to be placed into and removed from the water. Samples were oriented such that the wide surface of the slides was parallel to the direction of the current for the samples to experience a constant flow. The water recirculated through the flume over the course of the experiment. To avoid disrupting the flow when rounding the corners, the flume utilized two 45-degree PVC elbows rather than 90-degree angles. Between uses, the flume was cleaned by spraying with 20% bleach, 70% reagent alcohol, and distilled water. Due to its size, the flume was unable to be cross-linked and was stored in an active research room when in use. Bins were placed over open areas in the flume to attempt to avoid extraneous contamination ( ).
Sirchie glass microscope slides were utilized as the non-porous surface. The glass slides were cross-linked on both sides for 1000 s in a SpectroLinker XL-1500 UV Crosslinker before use. Three random slides were swabbed and processed for human DNA after cross-linking. No quantity of human DNA was recovered, so it was assumed that cross-linking for 1000 s on both sides would remove any background human DNA from the slides. Sample slides were divided into three main categories. “Blank” slide samples had nothing added to the slide after cross-linking. For Phases 1 and 2, “deposition” slide samples had 5 μL fresh neat saliva added to the slide. A portion of the neat saliva (20 μL) was extracted following the QIAamp DNA Investigator Protocol: Isolation of Total DNA from Small Volumes of Blood or Saliva and quantified (yielding a concentration of approximately 6 ng/μL) before deposition to estimate the initial amount of DNA being deposited onto each sample. Two blank slide samples and five deposition samples were utilized per submersion time per water condition for these phases. For Phases 3 and 4, “fingerprint” slide samples had a sebaceous fingerprint deposited onto the slide with a force of approximately 400 g with contact lasting three seconds. Force was measured by placing the slide on a scale when depositing the fingerprint. The right thumb of one donor was utilized as the fingerprint source for all samples. Before depositing the fingerprints, the donor washed their hands for 20 s with warm soapy water and allowed their hands to air-dry for approximately 5 min. The donor then wiped their right thumb across their forehead to obtain sebaceous secretions and deposited the print onto the designated glass slide. The cleaning process was repeated between fingerprint depositions. Two blank slide samples and five fingerprint slide samples were utilized per submersion time per water condition for these phases. Due to the inherent unpredictability of touch DNA samples, the initial amount of DNA transferred from the finger to the slide was unknown. However, three fingerprint samples (right thumb) were created, extracted, quantified, and amplified for human DNA prior to submersion to attempt to establish a baseline. The quantities of these initial fingerprint samples ranged from 0.0106 to 0.0343 ng/μL with an average of 0.0224 ng/μL (σ 2 = 1.404 × 10 −4 ). One male donor aged 22–24 was utilized in this study and was the donor for both saliva and fingerprint samples. All samples were obtained according to informed consent.
For Phases 1 and 2, all blank and deposition slide samples entered the water at the same time, and a set of slides were removed at each time interval: 6 h, 12 h, 24 h, 48 h, 72 h, and 168 h (1 week). For Phases 3 and 4, all blank and fingerprint slide samples entered the water at the same time, and a set of slides were removed at each time interval: 24 h, 48 h, 72 h, and 168 h (1 week). An additional set was evaluated at 0 h to serve as an initial assessment. The experiment was carried out in an indoor lab environment at room temperature (approximately 20 °C). In the flowing environment, cleaned blank slides were inserted in place of the removed slides to avoid altering the way water flowed through the flume. To reduce the risk of introducing microbes when removing the sets, researchers utilized proper PPE and disinfected gloves with 70% reagent alcohol. As slides were removed from the water, they were placed on a sterile benchtop to air dry. When fully dry, the slides were either swabbed and extracted for DNA or stored at −80 °C until extraction. When stored at −80 °C, slides were kept in cross-linked microscope slide holder boxes. The wet-dry swab method was utilized to collect potential DNA from the slides which is a common technique for trace DNA recovery .
Qiagen’s QIAamp DNA Investigator Kit was utilized to extract the DNA collected from each sample. All swabs from samples (blank slides, deposition slides, and fingerprint slides) were extracted following the QIAamp DNA Investigator Protocol: Isolation of Total DNA from Surface and Buccal Swabs. This extraction procedure included adding proteinase K and Buffer ATL to the sample, incubating at 56 °C with shaking for two hours, adding Buffer AL, incubating at 70 °C with shaking for ten minutes, adding ethanol, and transferring the lysate to a QIAamp MinElute column. Samples were washed with Buffer AW1, Buffer AW2, and ethanol before eluting from the QIAamp MinElute column utilizing 50 μL Buffer ATE. Used glass slides and swab heads were saved and stored at −80 °C in case needed for further analysis. Samples were stored in 1.5 mL Eppendorf tubes at −80 °C after extraction. The Quantifiler TM Human DNA Quantification Kit with a QuantStudio 5 Real-Time PCR System (Design and Analysis Software v1.5.1) was utilized to quantify the human DNA present in samples. The Quantifiler TM Human DNA Quantification Kit has a lower threshold of detection of approximately 16 pg/μL of human genomic DNA . The quantity of human DNA present on all samples was recorded. STR amplification was performed for both submersion conditions at every submersion time on all blank slide samples, three saliva slide deposition, and three fingerprint slide samples. Applied Biosystems’s GlobalFiler TM PCR Amplification Kit was utilized to amplify twenty-four STR loci. Capillary electrophoresis was performed with ILS600 LIZ Size Standard on an Applied Biosystems 3130xl Genetic Analyzer (16 capillary array 50 cm, POP4 polymer, 1× buffer). The injection conditions utilized were 3 kV/5 s, and the run conditions were 15 kV/1500 s. STR amplification results were interpreted with the GeneMarker HID STR Human Identity Software (version 2.4). Sample amplification data recorded included number and designation of alleles present, individual allele peak heights, and heterozygous peak height ratios. Internal controls were utilized and during all steps (extraction, quantification, and amplification) in order to monitor any contamination or error. Negative internal controls yielded no indication of contamination.
T -Tests (two-tailed) were utilized to calculate p -values as estimates of statistical significance ( p < 0.05 in all instances) when comparing one condition to another (for example, comparing DNA quantities recovered at 24 h in the flowing versus stagnant water conditions). Other mathematical analyses included calculating averages and variance values (σ 2 ) or standard deviation values based on the samples for both DNA quantity and the number of alleles recovered.
3.1. Phases 1 and 2: DNA Quantity In general, DNA quantities of deposition slide samples in both stagnant and flowing conditions decreased over the one-week submersion period ( ). Initial deposition slide samples were created by adding neat saliva to cleaned glass slides, letting the slides air-dry, and swabbing with the wet-dry swab method. Initial DNA quantities from the slides ranged from 2.9653 to 5.1249 ng/μL with an average of 3.96142 ng/μL (σ 2 = 0.8360). When comparing the DNA quantities from deposition slide samples obtained between the stagnant versus flowing conditions for each submersion time, there was little statistical significance ( p = 0.824, p = 0.810, p = 0.016, p = 0.332, p = 0.20, and p = 0.028 for each submersion interval, respectively, significance level p < 0.05). However, at 24 h and at 1 week, DNA quantities were statistically significantly higher in the stagnant condition than in the flowing ( p = 0.016 and p = 0.028, respectively). These results may indicate that DNA behaves similarly in stagnant and flowing conditions at shorter submersion times, but with prolonged submersion, flow can negatively affect DNA recovery. When comparing the DNA quantities from deposition slide samples obtained for each submersion time versus the initial DNA quantities, DNA quantities were statistically significantly lower in both stagnant and flowing conditions for every time period (Stagnant: p = 0.0073, p = 0.0033, p = 0.0006, p = 4.3 × 10 −5 , p = 2.1 × 10 −5 , and p = 1.3 × 10 −5 ; Flowing: p = 0.0006, p = 0.0009, p = 0.006, p = 0.0001, p = 6.7 × 10 −5 , and p = 4.7 × 10 −5 ; for each submersion interval, respectively, significance level p < 0.05). Although longer submersion times negatively affected DNA recovery, this suggests that a portion of DNA is lost from samples even with shorter submersion times. In general, DNA quantities of blank slide samples in both stagnant and flowing conditions increased over the one-week submersion period ( ). A subset of three blank slides were swabbed after cross linking and before submersion. The initial DNA quantities from these blank slides were undetermined with Quantifiler TM Human, so the DNA quantities were assumed to be zero. Of note, multiple blank slide samples were of sufficient quantity that the samples were expected to pass the amplification threshold. For instance, at one week, the highest DNA quantity recovered from a blank slide sample was approximately 0.07 ng/μL, which could theoretically provide 1.05 ng DNA for amplification, therefore, meeting the target of approximately 1 ng. Statistical methods were not applied to blank samples due to the limited sample size to make any determinations of statistical significance. However, the recovery of sufficient DNA quantities on multiple (initially blank) samples over various submersion times is noteworthy, particularly in the implications for forensic DNA analysis which will be discussed further. 3.2. Phases 1 and 2: Number of Donor Alleles Recovered A reference sample from the donor was utilized to gather information about the donor’s alleles at AMEL and 17 STR loci. In total, 30 alleles of the donor were considered. For deposition slide samples, it was noted when one of the 30 donor alleles had dropped out from the electropherogram. For blank slide samples, it was noted when one of the 30 donor alleles had dropped into the electropherogram. Any foreign alleles detected were also noted and were not included in the calculation of the number of donor alleles recovered. When foreign alleles were detected, alleles consistent with the donor were also detected, and foreign alleles produced less signal in the electropherograms. There were no samples where alleles were detected without any being consistent with the donor. Foreign alleles were not common but were most prevalent in the flowing water condition which may have been due to the assembly of the horizontal flume, the inability to cross-link the horizontal flume due to its size, the storage of the horizontal flume in a shared laboratory space, or other additional factors. For the sake of the study, it was assumed that foreign alleles were not shared with donor alleles. In the stagnant condition, on average, over 70% of the donor alleles were recovered from the deposition slide samples at every submersion interval over one week ( ). In the flowing condition, on average, over 70% of the donor alleles were recovered at every submersion interval except at one week ( ). There were no statistically significant differences when comparing the number of donor alleles recovered in stagnant versus flowing conditions for all submersion intervals ( p = 0.579, p = 0.292, p = 0.169, p = 0.329, p = 0.588, and p = 0.252 for each submersion interval, respectively, significance level p < 0.05). In the stagnant condition, donor alleles were not detected on the blank slide samples until 24 h submersion. No donor alleles were detected on the subset of initial blank slide samples prior to submersion. The number of donor alleles recovered varied over the course of one week, but at one week submersion, on average, over 50% of the donor alleles were recovered from the blank slide samples in stagnant water ( ). In the flowing condition, donor alleles were detected on the blank slide samples starting at 6 h submersion. However, the average number of donor alleles recovered was below 25% for each submersion interval in flowing water ( ). Large standard deviations in the number of donor alleles recovered from blank slide samples may reflect the unpredictability in DNA transfer and persistence ( ). When comparing the number of donor alleles recovered on deposition slide samples at all submersion times to the initial 30 alleles, the number of alleles detected was statistically significantly lower than the initial at 72 h and one week submersion in stagnant ( p = 0.024 and p = 0.02, respectively, significance level p < 0.05). In flowing water, the number of donor alleles recovered at 12 h was statistically significantly lower than the initial ( p = 0.038, significance level p < 0.05), but there was no statistically significant difference at any other submersion interval. This may indicate that enough DNA to yield a sufficient STR profile may be retained on non-porous surfaces even after multiple days submersion. 3.3. Phases 3 and 4: DNA Quantity In general, DNA quantities of fingerprint slide samples in both stagnant and flowing conditions decreased over the one-week submersion period ( ). Initial DNA quantities from the fingerprint slide samples ranged from 0.0106 to 0.0343 ng/μL with an average of 0.0224 ng/μL (σ 2 = 1.404 × 10 −4 ). These initial quantities were lower than that of the deposition samples associated with Phases 1 and 2, so the performance of fingerprint slide samples was not compared to that of the deposition slide samples. When comparing the DNA quantities from fingerprint slide samples obtained between the stagnant and flowing conditions for each submersion interval, there was no statistical significance (Stagnant: p = 0.639, p = 0.485, p = 0.153, and p = 0.297; Flowing: p = UND, p = 0.423, p = UND, and p = 0.423; for each submersion interval, respectively, significance level p < 0.05). When comparing the DNA quantities from fingerprint slide samples obtained for each submersion interval versus the initial fingerprint DNA quantities, there was not a significant decline in DNA quantity until the 72 h submersion duration (Stagnant: p = 0.57, p = 0.64, p = 0.9, and 0.02; Flowing: p = 0.98, p = 0.17, p = 0.04, and p = 0.006; for each submersion interval, respectively, significance level p < 0.05). For the flowing water, fingerprint DNA quantities were statistically significantly lower at 72 h and 1 week than the initial values ( p = 0.04 and p = 0.006). For the stagnant water, fingerprint DNA quantities were statistically significantly lower at 1 week than the initial values ( p = 0.02). This indicates that fingerprint samples may experience loss of DNA at shorter submersion times in flowing water in comparison to stagnant, supporting that flow negatively affects DNA recovery. In general, DNA quantities of blank slide samples in both the stagnant and flowing conditions were similar over time. All quantities for blank slide samples were below 0.006 ng/μL and, therefore, were expected to fall below the target amplification threshold of 1 ng. 3.4. Phases 3 and 4: Number of Alleles Recovered The average number of donor alleles recovered from fingerprint slide samples differed for all submersion intervals in the stagnant condition ( ). The average percent of donor alleles recovered was 62.22% at 24 h (σ 2 = 2670.37), 47.78% at 48 h (σ 2 = 1048.15), 81.11% at 72 h (σ 2 = 114.81), and 10.00% at 1 week (σ 2 = 44.44). Variance values were highest at the first submersion time, and less variation was observed with longer submersion. High variation within the earlier submersion times may be reflective of the unpredictability of touch and low-quantity samples prior to any influence from the stagnant water. In the flowing condition, no donor alleles were recovered from fingerprint slide samples. Despite this, visible ridge detail was visible in all fingerprint samples at each submersion time in either condition ( ). No donor or foreign alleles were recovered from the blank slide samples for any of the submersion times in either water condition. Although the donor had 30 known alleles, three fingerprint slide samples were deposited and collected for DNA analysis without any submersion in water. The three samples contained 73.3%, 20%, and 20% of the 30 known donor alleles, respectively. For the purposes of Phase 3 and 4, the initial average number of alleles was considered to be 37.8% (11.34 alleles) of the 30 donor alleles. When comparing the number of donor alleles recovered on fingerprint slide samples at all submersion times to the initial average number of alleles (37.8% of the 30 donor alleles), the number of alleles detected was not statistically significantly different at any submersion time for either condition (Stagnant: p = 0.52, p = 0.718, p = 0.246, and p = 0.201 for each submersion interval, respectively; Flowing: p = 0.168 at all submersion intervals; significance level p < 0.05). However, the lack of statistically significant differences may be partially attributed to the inherent inconsistency in depositing a full DNA profile through fingerprint deposition.
In general, DNA quantities of deposition slide samples in both stagnant and flowing conditions decreased over the one-week submersion period ( ). Initial deposition slide samples were created by adding neat saliva to cleaned glass slides, letting the slides air-dry, and swabbing with the wet-dry swab method. Initial DNA quantities from the slides ranged from 2.9653 to 5.1249 ng/μL with an average of 3.96142 ng/μL (σ 2 = 0.8360). When comparing the DNA quantities from deposition slide samples obtained between the stagnant versus flowing conditions for each submersion time, there was little statistical significance ( p = 0.824, p = 0.810, p = 0.016, p = 0.332, p = 0.20, and p = 0.028 for each submersion interval, respectively, significance level p < 0.05). However, at 24 h and at 1 week, DNA quantities were statistically significantly higher in the stagnant condition than in the flowing ( p = 0.016 and p = 0.028, respectively). These results may indicate that DNA behaves similarly in stagnant and flowing conditions at shorter submersion times, but with prolonged submersion, flow can negatively affect DNA recovery. When comparing the DNA quantities from deposition slide samples obtained for each submersion time versus the initial DNA quantities, DNA quantities were statistically significantly lower in both stagnant and flowing conditions for every time period (Stagnant: p = 0.0073, p = 0.0033, p = 0.0006, p = 4.3 × 10 −5 , p = 2.1 × 10 −5 , and p = 1.3 × 10 −5 ; Flowing: p = 0.0006, p = 0.0009, p = 0.006, p = 0.0001, p = 6.7 × 10 −5 , and p = 4.7 × 10 −5 ; for each submersion interval, respectively, significance level p < 0.05). Although longer submersion times negatively affected DNA recovery, this suggests that a portion of DNA is lost from samples even with shorter submersion times. In general, DNA quantities of blank slide samples in both stagnant and flowing conditions increased over the one-week submersion period ( ). A subset of three blank slides were swabbed after cross linking and before submersion. The initial DNA quantities from these blank slides were undetermined with Quantifiler TM Human, so the DNA quantities were assumed to be zero. Of note, multiple blank slide samples were of sufficient quantity that the samples were expected to pass the amplification threshold. For instance, at one week, the highest DNA quantity recovered from a blank slide sample was approximately 0.07 ng/μL, which could theoretically provide 1.05 ng DNA for amplification, therefore, meeting the target of approximately 1 ng. Statistical methods were not applied to blank samples due to the limited sample size to make any determinations of statistical significance. However, the recovery of sufficient DNA quantities on multiple (initially blank) samples over various submersion times is noteworthy, particularly in the implications for forensic DNA analysis which will be discussed further.
A reference sample from the donor was utilized to gather information about the donor’s alleles at AMEL and 17 STR loci. In total, 30 alleles of the donor were considered. For deposition slide samples, it was noted when one of the 30 donor alleles had dropped out from the electropherogram. For blank slide samples, it was noted when one of the 30 donor alleles had dropped into the electropherogram. Any foreign alleles detected were also noted and were not included in the calculation of the number of donor alleles recovered. When foreign alleles were detected, alleles consistent with the donor were also detected, and foreign alleles produced less signal in the electropherograms. There were no samples where alleles were detected without any being consistent with the donor. Foreign alleles were not common but were most prevalent in the flowing water condition which may have been due to the assembly of the horizontal flume, the inability to cross-link the horizontal flume due to its size, the storage of the horizontal flume in a shared laboratory space, or other additional factors. For the sake of the study, it was assumed that foreign alleles were not shared with donor alleles. In the stagnant condition, on average, over 70% of the donor alleles were recovered from the deposition slide samples at every submersion interval over one week ( ). In the flowing condition, on average, over 70% of the donor alleles were recovered at every submersion interval except at one week ( ). There were no statistically significant differences when comparing the number of donor alleles recovered in stagnant versus flowing conditions for all submersion intervals ( p = 0.579, p = 0.292, p = 0.169, p = 0.329, p = 0.588, and p = 0.252 for each submersion interval, respectively, significance level p < 0.05). In the stagnant condition, donor alleles were not detected on the blank slide samples until 24 h submersion. No donor alleles were detected on the subset of initial blank slide samples prior to submersion. The number of donor alleles recovered varied over the course of one week, but at one week submersion, on average, over 50% of the donor alleles were recovered from the blank slide samples in stagnant water ( ). In the flowing condition, donor alleles were detected on the blank slide samples starting at 6 h submersion. However, the average number of donor alleles recovered was below 25% for each submersion interval in flowing water ( ). Large standard deviations in the number of donor alleles recovered from blank slide samples may reflect the unpredictability in DNA transfer and persistence ( ). When comparing the number of donor alleles recovered on deposition slide samples at all submersion times to the initial 30 alleles, the number of alleles detected was statistically significantly lower than the initial at 72 h and one week submersion in stagnant ( p = 0.024 and p = 0.02, respectively, significance level p < 0.05). In flowing water, the number of donor alleles recovered at 12 h was statistically significantly lower than the initial ( p = 0.038, significance level p < 0.05), but there was no statistically significant difference at any other submersion interval. This may indicate that enough DNA to yield a sufficient STR profile may be retained on non-porous surfaces even after multiple days submersion.
In general, DNA quantities of fingerprint slide samples in both stagnant and flowing conditions decreased over the one-week submersion period ( ). Initial DNA quantities from the fingerprint slide samples ranged from 0.0106 to 0.0343 ng/μL with an average of 0.0224 ng/μL (σ 2 = 1.404 × 10 −4 ). These initial quantities were lower than that of the deposition samples associated with Phases 1 and 2, so the performance of fingerprint slide samples was not compared to that of the deposition slide samples. When comparing the DNA quantities from fingerprint slide samples obtained between the stagnant and flowing conditions for each submersion interval, there was no statistical significance (Stagnant: p = 0.639, p = 0.485, p = 0.153, and p = 0.297; Flowing: p = UND, p = 0.423, p = UND, and p = 0.423; for each submersion interval, respectively, significance level p < 0.05). When comparing the DNA quantities from fingerprint slide samples obtained for each submersion interval versus the initial fingerprint DNA quantities, there was not a significant decline in DNA quantity until the 72 h submersion duration (Stagnant: p = 0.57, p = 0.64, p = 0.9, and 0.02; Flowing: p = 0.98, p = 0.17, p = 0.04, and p = 0.006; for each submersion interval, respectively, significance level p < 0.05). For the flowing water, fingerprint DNA quantities were statistically significantly lower at 72 h and 1 week than the initial values ( p = 0.04 and p = 0.006). For the stagnant water, fingerprint DNA quantities were statistically significantly lower at 1 week than the initial values ( p = 0.02). This indicates that fingerprint samples may experience loss of DNA at shorter submersion times in flowing water in comparison to stagnant, supporting that flow negatively affects DNA recovery. In general, DNA quantities of blank slide samples in both the stagnant and flowing conditions were similar over time. All quantities for blank slide samples were below 0.006 ng/μL and, therefore, were expected to fall below the target amplification threshold of 1 ng.
The average number of donor alleles recovered from fingerprint slide samples differed for all submersion intervals in the stagnant condition ( ). The average percent of donor alleles recovered was 62.22% at 24 h (σ 2 = 2670.37), 47.78% at 48 h (σ 2 = 1048.15), 81.11% at 72 h (σ 2 = 114.81), and 10.00% at 1 week (σ 2 = 44.44). Variance values were highest at the first submersion time, and less variation was observed with longer submersion. High variation within the earlier submersion times may be reflective of the unpredictability of touch and low-quantity samples prior to any influence from the stagnant water. In the flowing condition, no donor alleles were recovered from fingerprint slide samples. Despite this, visible ridge detail was visible in all fingerprint samples at each submersion time in either condition ( ). No donor or foreign alleles were recovered from the blank slide samples for any of the submersion times in either water condition. Although the donor had 30 known alleles, three fingerprint slide samples were deposited and collected for DNA analysis without any submersion in water. The three samples contained 73.3%, 20%, and 20% of the 30 known donor alleles, respectively. For the purposes of Phase 3 and 4, the initial average number of alleles was considered to be 37.8% (11.34 alleles) of the 30 donor alleles. When comparing the number of donor alleles recovered on fingerprint slide samples at all submersion times to the initial average number of alleles (37.8% of the 30 donor alleles), the number of alleles detected was not statistically significantly different at any submersion time for either condition (Stagnant: p = 0.52, p = 0.718, p = 0.246, and p = 0.201 for each submersion interval, respectively; Flowing: p = 0.168 at all submersion intervals; significance level p < 0.05). However, the lack of statistically significant differences may be partially attributed to the inherent inconsistency in depositing a full DNA profile through fingerprint deposition.
4.1. Revisiting Hypotheses For Phases 1 and 2, overall DNA quantity of deposition slide samples decreased over one week submersion. However, minimal allele drop-out was observed for deposition slide samples, and some instances of allele drop-in were observed for the blank slide samples. Although longer submersion times resulted in a decrease of DNA quantity, one week submersion did not significantly affect the ability to recover donor alleles from deposition slide samples. For Phases 3 and 4, overall DNA quantity of fingerprint slide samples decreased, with the decline being statistically significant only at 72 h and one week submersion ( p = 0.04 and p = 0.006, respectively, significance level p < 0.05). Multiple instances of allele drop-out were observed for fingerprint slide samples. Over one week submersion, fingerprint slide samples in flowing water experienced a decline in the number of alleles recovered while fingerprint slide samples in stagnant water did not. For Phases 1 and 2, overall DNA quantities were lower in flowing than in stagnant water, but the two submersion intervals where the difference was statistically significant were at 24 h and one week submersion ( p = 0.016 and p = 0.028, respectively, significance level p < 0.05). There was no difference in the number of donor alleles recovered between stagnant and flowing conditions. Flow did affect DNA quantities at some submersion times but did not hinder the ability to recover donor alleles from deposition samples. For Phases 3 and 4, there was no difference in the DNA quantities between stagnant and flowing conditions. The presence of fingerprint secretions possibly aided in retaining DNA to the slides even in flow conditions. The number of donor alleles recovered from fingerprint slide samples were lower in flowing than in stagnant water for all submersion times. Although flow did not affect DNA quantities of fingerprint samples, flow hindered the ability to recover donor alleles. 4.2. Limitations For this study, there were five replicates of deposition or fingerprint slide samples and two replicates of blank slide samples per submersion time for DNA quantitation purposes. Additionally, there were three replicates of deposition or fingerprint slide samples and two replicates of blank slide samples per submersion time for STR amplification purposes. Ideally, the sample size would have been increased for both DNA quantitation and STR amplification. Only one donor was utilized in this study, and the same individual donated DNA and fingerprints. Incorporating more individuals could account for variability among donors, especially with the fingerprint samples. Both limitations are due to time and budget constraints. 4.3. Implications 4.3.1. Trace DNA from Fingerprint Samples One of the main goals of the study was to evaluate the performance of fingerprint samples in terms of the retention of both fingerprint secretions and DNA. The quantity of DNA transferred from the donor’s finger to the glass surfaces was lower than anticipated at all submersion times, including the initial time when the fingerprints were not submerged. The highest quantity was collected at 48 h submersion (0.0055 ng/μL in stagnant water), yet this value was not significantly different from any of the other DNA quantities collected from fingerprints for any of the submersion times. Despite difficulty recovering DNA after one week of submersion, fingerprints recovered from both stagnant and flowing water contained visible ridge detail without the need for enhancement throughout the duration of the experiment. These findings support that of previous studies where fingerprints were able to be recovered from submerged non-porous surfaces up to 42 days of submersion, with the highest success being within a week of submersion . The results of this particular study indicate that if a fingerprint is observable on a non-porous item recovered from the water, then investigators should consider prioritizing friction ridge comparison over DNA recovery, especially if recovered from flowing environments. 4.3.2. Possibility of Transfer In designing the experiment, the goal of incorporating blank slide samples to be removed at each submersion time was to act as a control measure for detecting background DNA in the water throughout the duration of the experiment. Increasing DNA quantities of blank slide samples, particularly in Phases 1 and 2, indicated that some degree of drop in was occurring in the blank slide samples, as no human DNA was detected on blank slides prior to submersion. However, STR detection revealed alleles consistent with the donor from the deposition slide samples on the blank slides. Although foreign alleles were detected on some of the blank slide samples in the flowing condition, alleles consistent with the donor were recovered in addition to those foreign ones. Because negative internal controls did not yield any detectable DNA in either quantification or STR detection, it was assumed that any alleles present on the blank slide samples were a result of transfer rather than contamination. During the experiment, donor DNA from the deposition slide samples may have been transferred into the water before transferring onto the blank slide samples. Transfer refers to the mode by which DNA arrives on an item. DNA can arrive on an item through direct contacts, such as when saliva was directly applied to deposition slides in Phases 1 and 2 ( ). Indirect transfer can occur when DNA deposited on Surface A arrives on Surface B, such as DNA from deposition slide samples arriving on blank slide samples ( ). DNA may also undergo multiple degrees of transfer, such as secondary to tertiary to quaternary and so on . DNA transferring from deposition slide samples to the water to the blank slide samples would be considered a tertiary transfer as DNA is transferring from Surface A (deposition slide) to B (water) to C (blank slide) ( ). For Phases 1 and 2, transfer onto blank slide samples in stagnant water appeared to be most prominent at one week. Without knowledge of the sample identification, the electropherograms of deposition and blank slide samples at one week would be extremely difficult to distinguish ( and ). With how the experiment was designed, all deposition and blank slides were submerged into the same water vessel at the same moment, and then after designated submersion intervals, sets of slides were removed. Initially, it was anticipated that any DNA lost from the deposition samples to the surrounding water would be hydrolyzed and suffer in quality. Additionally, transfer from the deposition slide samples to the blank slides was not anticipated. In theory, DNA lost from deposition slide samples yet not destroyed in the water could have cross-transferred to the other deposition slide samples or transferred onto the blank slide samples. Slides in the set removed at one week could have been subjected to higher DNA concentrations in the water than the other sets. In stagnant water, the higher DNA concentrations may have eventually settled onto the blank slides, resulting in high yields of donor alleles. In flowing water, transfer onto blank slide samples was most prominent at 6 h and then relatively consistent for the remainder of the submersion times. Because samples in the flowing condition were experiencing a constant flow, any DNA that was lost to the water and transferred onto blank slide samples may have been pushed off again and continued to circulate in the horizontal flume. Transfer onto blank slide samples was not observed in Phases 3 and 4. This may be due to the fingerprint slide samples containing a lesser amount of DNA prior to submersion than in Phases 1 and 2. Additionally, components in saliva may have aided in DNA persistence in Phases 1 and 2. Another possibility is that if DNA were lost from the fingerprint samples in Phases 3 and 4, the DNA may be repelled from any remaining oils in fingerprint secretions, preventing lost DNA from redepositing and cross-transferring onto other fingerprint samples in the same water condition. Due to multiple samples being submerged in the same water together, the transfer observed is not necessarily reflective of a situation where DNA is indirectly transferred from Surface A to Surface B without cross-transfer from additional surfaces containing DNA from the same donor. DNA transfer observed in the flowing water was under recirculating conditions (similar to pools, tanks, and fountains), and free-flowing conditions (similar to rivers and streams) may have different possibilities for DNA transfer. For instance, DNA lost from Surface A in a free-flowing condition may be swept away before having the opportunity to transfer to Surface B. Under recirculating conditions, DNA lost from Surface A may have the opportunity to deposit back onto Surface A, transfer to Surface B, and remain in the water as examples. Additionally, DNA transfer through water may be affected by the volume of water, direction of current, and consistency of flow rate, in addition to other environmental factors, including salinity, pH, contaminants, UV light, physical obstacles in the environment, etc. However, the ability to recover donor DNA from the blank slide samples at least indicates the ability of DNA to transfer and persist under submerged conditions without providing a statement of probability. While perhaps not to the extent of what was observed in this study, these results indicate that investigators and analysts should at least be wary of overstating the significance of recovering submerged DNA. For instance, the increased sensitivity of forensic DNA analysis to detect low levels of DNA has, in turn, permitted the recovery of DNA not deposited by direct contact . By assuming the recovery of DNA from an item, even if a full profile, was a result of the individual directly contacting the item, an analyst can overstate the significance of detecting that DNA. 4.4. Future Possibilities Future studies could incorporate metal and plastic items of various smooth and textured surfaces to determine if any surface combinations provide the best conditions for DNA recovery. Additionally, future studies could evaluate how submersion in water in addition to other environmental factors influences DNA recovery which could provide a well-rounded view to submerged DNA behavior in natural environments. Introducing soap or detergents as variables may also be informative for submerged items recovered at indoor crime scenes. The results of an additional study could aid investigators in determining if a non-porous item recovered from water should be considered for DNA analysis, taking into account the item’s surface and environmental conditions.
For Phases 1 and 2, overall DNA quantity of deposition slide samples decreased over one week submersion. However, minimal allele drop-out was observed for deposition slide samples, and some instances of allele drop-in were observed for the blank slide samples. Although longer submersion times resulted in a decrease of DNA quantity, one week submersion did not significantly affect the ability to recover donor alleles from deposition slide samples. For Phases 3 and 4, overall DNA quantity of fingerprint slide samples decreased, with the decline being statistically significant only at 72 h and one week submersion ( p = 0.04 and p = 0.006, respectively, significance level p < 0.05). Multiple instances of allele drop-out were observed for fingerprint slide samples. Over one week submersion, fingerprint slide samples in flowing water experienced a decline in the number of alleles recovered while fingerprint slide samples in stagnant water did not. For Phases 1 and 2, overall DNA quantities were lower in flowing than in stagnant water, but the two submersion intervals where the difference was statistically significant were at 24 h and one week submersion ( p = 0.016 and p = 0.028, respectively, significance level p < 0.05). There was no difference in the number of donor alleles recovered between stagnant and flowing conditions. Flow did affect DNA quantities at some submersion times but did not hinder the ability to recover donor alleles from deposition samples. For Phases 3 and 4, there was no difference in the DNA quantities between stagnant and flowing conditions. The presence of fingerprint secretions possibly aided in retaining DNA to the slides even in flow conditions. The number of donor alleles recovered from fingerprint slide samples were lower in flowing than in stagnant water for all submersion times. Although flow did not affect DNA quantities of fingerprint samples, flow hindered the ability to recover donor alleles.
For this study, there were five replicates of deposition or fingerprint slide samples and two replicates of blank slide samples per submersion time for DNA quantitation purposes. Additionally, there were three replicates of deposition or fingerprint slide samples and two replicates of blank slide samples per submersion time for STR amplification purposes. Ideally, the sample size would have been increased for both DNA quantitation and STR amplification. Only one donor was utilized in this study, and the same individual donated DNA and fingerprints. Incorporating more individuals could account for variability among donors, especially with the fingerprint samples. Both limitations are due to time and budget constraints.
4.3.1. Trace DNA from Fingerprint Samples One of the main goals of the study was to evaluate the performance of fingerprint samples in terms of the retention of both fingerprint secretions and DNA. The quantity of DNA transferred from the donor’s finger to the glass surfaces was lower than anticipated at all submersion times, including the initial time when the fingerprints were not submerged. The highest quantity was collected at 48 h submersion (0.0055 ng/μL in stagnant water), yet this value was not significantly different from any of the other DNA quantities collected from fingerprints for any of the submersion times. Despite difficulty recovering DNA after one week of submersion, fingerprints recovered from both stagnant and flowing water contained visible ridge detail without the need for enhancement throughout the duration of the experiment. These findings support that of previous studies where fingerprints were able to be recovered from submerged non-porous surfaces up to 42 days of submersion, with the highest success being within a week of submersion . The results of this particular study indicate that if a fingerprint is observable on a non-porous item recovered from the water, then investigators should consider prioritizing friction ridge comparison over DNA recovery, especially if recovered from flowing environments. 4.3.2. Possibility of Transfer In designing the experiment, the goal of incorporating blank slide samples to be removed at each submersion time was to act as a control measure for detecting background DNA in the water throughout the duration of the experiment. Increasing DNA quantities of blank slide samples, particularly in Phases 1 and 2, indicated that some degree of drop in was occurring in the blank slide samples, as no human DNA was detected on blank slides prior to submersion. However, STR detection revealed alleles consistent with the donor from the deposition slide samples on the blank slides. Although foreign alleles were detected on some of the blank slide samples in the flowing condition, alleles consistent with the donor were recovered in addition to those foreign ones. Because negative internal controls did not yield any detectable DNA in either quantification or STR detection, it was assumed that any alleles present on the blank slide samples were a result of transfer rather than contamination. During the experiment, donor DNA from the deposition slide samples may have been transferred into the water before transferring onto the blank slide samples. Transfer refers to the mode by which DNA arrives on an item. DNA can arrive on an item through direct contacts, such as when saliva was directly applied to deposition slides in Phases 1 and 2 ( ). Indirect transfer can occur when DNA deposited on Surface A arrives on Surface B, such as DNA from deposition slide samples arriving on blank slide samples ( ). DNA may also undergo multiple degrees of transfer, such as secondary to tertiary to quaternary and so on . DNA transferring from deposition slide samples to the water to the blank slide samples would be considered a tertiary transfer as DNA is transferring from Surface A (deposition slide) to B (water) to C (blank slide) ( ). For Phases 1 and 2, transfer onto blank slide samples in stagnant water appeared to be most prominent at one week. Without knowledge of the sample identification, the electropherograms of deposition and blank slide samples at one week would be extremely difficult to distinguish ( and ). With how the experiment was designed, all deposition and blank slides were submerged into the same water vessel at the same moment, and then after designated submersion intervals, sets of slides were removed. Initially, it was anticipated that any DNA lost from the deposition samples to the surrounding water would be hydrolyzed and suffer in quality. Additionally, transfer from the deposition slide samples to the blank slides was not anticipated. In theory, DNA lost from deposition slide samples yet not destroyed in the water could have cross-transferred to the other deposition slide samples or transferred onto the blank slide samples. Slides in the set removed at one week could have been subjected to higher DNA concentrations in the water than the other sets. In stagnant water, the higher DNA concentrations may have eventually settled onto the blank slides, resulting in high yields of donor alleles. In flowing water, transfer onto blank slide samples was most prominent at 6 h and then relatively consistent for the remainder of the submersion times. Because samples in the flowing condition were experiencing a constant flow, any DNA that was lost to the water and transferred onto blank slide samples may have been pushed off again and continued to circulate in the horizontal flume. Transfer onto blank slide samples was not observed in Phases 3 and 4. This may be due to the fingerprint slide samples containing a lesser amount of DNA prior to submersion than in Phases 1 and 2. Additionally, components in saliva may have aided in DNA persistence in Phases 1 and 2. Another possibility is that if DNA were lost from the fingerprint samples in Phases 3 and 4, the DNA may be repelled from any remaining oils in fingerprint secretions, preventing lost DNA from redepositing and cross-transferring onto other fingerprint samples in the same water condition. Due to multiple samples being submerged in the same water together, the transfer observed is not necessarily reflective of a situation where DNA is indirectly transferred from Surface A to Surface B without cross-transfer from additional surfaces containing DNA from the same donor. DNA transfer observed in the flowing water was under recirculating conditions (similar to pools, tanks, and fountains), and free-flowing conditions (similar to rivers and streams) may have different possibilities for DNA transfer. For instance, DNA lost from Surface A in a free-flowing condition may be swept away before having the opportunity to transfer to Surface B. Under recirculating conditions, DNA lost from Surface A may have the opportunity to deposit back onto Surface A, transfer to Surface B, and remain in the water as examples. Additionally, DNA transfer through water may be affected by the volume of water, direction of current, and consistency of flow rate, in addition to other environmental factors, including salinity, pH, contaminants, UV light, physical obstacles in the environment, etc. However, the ability to recover donor DNA from the blank slide samples at least indicates the ability of DNA to transfer and persist under submerged conditions without providing a statement of probability. While perhaps not to the extent of what was observed in this study, these results indicate that investigators and analysts should at least be wary of overstating the significance of recovering submerged DNA. For instance, the increased sensitivity of forensic DNA analysis to detect low levels of DNA has, in turn, permitted the recovery of DNA not deposited by direct contact . By assuming the recovery of DNA from an item, even if a full profile, was a result of the individual directly contacting the item, an analyst can overstate the significance of detecting that DNA.
One of the main goals of the study was to evaluate the performance of fingerprint samples in terms of the retention of both fingerprint secretions and DNA. The quantity of DNA transferred from the donor’s finger to the glass surfaces was lower than anticipated at all submersion times, including the initial time when the fingerprints were not submerged. The highest quantity was collected at 48 h submersion (0.0055 ng/μL in stagnant water), yet this value was not significantly different from any of the other DNA quantities collected from fingerprints for any of the submersion times. Despite difficulty recovering DNA after one week of submersion, fingerprints recovered from both stagnant and flowing water contained visible ridge detail without the need for enhancement throughout the duration of the experiment. These findings support that of previous studies where fingerprints were able to be recovered from submerged non-porous surfaces up to 42 days of submersion, with the highest success being within a week of submersion . The results of this particular study indicate that if a fingerprint is observable on a non-porous item recovered from the water, then investigators should consider prioritizing friction ridge comparison over DNA recovery, especially if recovered from flowing environments.
In designing the experiment, the goal of incorporating blank slide samples to be removed at each submersion time was to act as a control measure for detecting background DNA in the water throughout the duration of the experiment. Increasing DNA quantities of blank slide samples, particularly in Phases 1 and 2, indicated that some degree of drop in was occurring in the blank slide samples, as no human DNA was detected on blank slides prior to submersion. However, STR detection revealed alleles consistent with the donor from the deposition slide samples on the blank slides. Although foreign alleles were detected on some of the blank slide samples in the flowing condition, alleles consistent with the donor were recovered in addition to those foreign ones. Because negative internal controls did not yield any detectable DNA in either quantification or STR detection, it was assumed that any alleles present on the blank slide samples were a result of transfer rather than contamination. During the experiment, donor DNA from the deposition slide samples may have been transferred into the water before transferring onto the blank slide samples. Transfer refers to the mode by which DNA arrives on an item. DNA can arrive on an item through direct contacts, such as when saliva was directly applied to deposition slides in Phases 1 and 2 ( ). Indirect transfer can occur when DNA deposited on Surface A arrives on Surface B, such as DNA from deposition slide samples arriving on blank slide samples ( ). DNA may also undergo multiple degrees of transfer, such as secondary to tertiary to quaternary and so on . DNA transferring from deposition slide samples to the water to the blank slide samples would be considered a tertiary transfer as DNA is transferring from Surface A (deposition slide) to B (water) to C (blank slide) ( ). For Phases 1 and 2, transfer onto blank slide samples in stagnant water appeared to be most prominent at one week. Without knowledge of the sample identification, the electropherograms of deposition and blank slide samples at one week would be extremely difficult to distinguish ( and ). With how the experiment was designed, all deposition and blank slides were submerged into the same water vessel at the same moment, and then after designated submersion intervals, sets of slides were removed. Initially, it was anticipated that any DNA lost from the deposition samples to the surrounding water would be hydrolyzed and suffer in quality. Additionally, transfer from the deposition slide samples to the blank slides was not anticipated. In theory, DNA lost from deposition slide samples yet not destroyed in the water could have cross-transferred to the other deposition slide samples or transferred onto the blank slide samples. Slides in the set removed at one week could have been subjected to higher DNA concentrations in the water than the other sets. In stagnant water, the higher DNA concentrations may have eventually settled onto the blank slides, resulting in high yields of donor alleles. In flowing water, transfer onto blank slide samples was most prominent at 6 h and then relatively consistent for the remainder of the submersion times. Because samples in the flowing condition were experiencing a constant flow, any DNA that was lost to the water and transferred onto blank slide samples may have been pushed off again and continued to circulate in the horizontal flume. Transfer onto blank slide samples was not observed in Phases 3 and 4. This may be due to the fingerprint slide samples containing a lesser amount of DNA prior to submersion than in Phases 1 and 2. Additionally, components in saliva may have aided in DNA persistence in Phases 1 and 2. Another possibility is that if DNA were lost from the fingerprint samples in Phases 3 and 4, the DNA may be repelled from any remaining oils in fingerprint secretions, preventing lost DNA from redepositing and cross-transferring onto other fingerprint samples in the same water condition. Due to multiple samples being submerged in the same water together, the transfer observed is not necessarily reflective of a situation where DNA is indirectly transferred from Surface A to Surface B without cross-transfer from additional surfaces containing DNA from the same donor. DNA transfer observed in the flowing water was under recirculating conditions (similar to pools, tanks, and fountains), and free-flowing conditions (similar to rivers and streams) may have different possibilities for DNA transfer. For instance, DNA lost from Surface A in a free-flowing condition may be swept away before having the opportunity to transfer to Surface B. Under recirculating conditions, DNA lost from Surface A may have the opportunity to deposit back onto Surface A, transfer to Surface B, and remain in the water as examples. Additionally, DNA transfer through water may be affected by the volume of water, direction of current, and consistency of flow rate, in addition to other environmental factors, including salinity, pH, contaminants, UV light, physical obstacles in the environment, etc. However, the ability to recover donor DNA from the blank slide samples at least indicates the ability of DNA to transfer and persist under submerged conditions without providing a statement of probability. While perhaps not to the extent of what was observed in this study, these results indicate that investigators and analysts should at least be wary of overstating the significance of recovering submerged DNA. For instance, the increased sensitivity of forensic DNA analysis to detect low levels of DNA has, in turn, permitted the recovery of DNA not deposited by direct contact . By assuming the recovery of DNA from an item, even if a full profile, was a result of the individual directly contacting the item, an analyst can overstate the significance of detecting that DNA.
Future studies could incorporate metal and plastic items of various smooth and textured surfaces to determine if any surface combinations provide the best conditions for DNA recovery. Additionally, future studies could evaluate how submersion in water in addition to other environmental factors influences DNA recovery which could provide a well-rounded view to submerged DNA behavior in natural environments. Introducing soap or detergents as variables may also be informative for submerged items recovered at indoor crime scenes. The results of an additional study could aid investigators in determining if a non-porous item recovered from water should be considered for DNA analysis, taking into account the item’s surface and environmental conditions.
In general, for controlled samples, DNA deposited onto glass and subsequently submerged in water experienced a decrease in DNA quantity over time, yet submersion did not have as strong of a negative effect on the amplification product. On average, over half the donor alleles were recovered at every submersion interval for both the stagnant and flowing conditions. The average percentage of donor alleles recovered was above 70% until the one-week submersion interval. In some instances, particularly at earlier submersion intervals, all donor alleles were recovered from the deposition slide samples. In most instances, flowing conditions either further negatively influenced DNA recovery or had little influence, in comparison to stagnant water alone. Of note is that in the mock casework samples, flow did not hinder DNA quantity, but the introduction of flow resulted in no alleles being recovered from the fingerprints despite alleles being recovered in the stagnant condition. Results of the controlled samples indicate that the evidentiary value of submerged non-porous items should not be discounted. Although DNA quantities and the number of alleles recovered were low throughout the mock casework samples, there were still instances where fingerprints yielded valuable DNA after submersion. For example, the average number of donor alleles recovered from the fingerprints after 72 h submersion was over 80% in the stagnant water condition. Even when trace DNA was not recovered from the mock casework samples, friction ridge detail was visible for all fingerprints without enhancement, even after one week submersion. This supports that friction ridge comparison may be more informative than or beneficial to perform in addition to trace DNA analysis for these sample types. Overall, trace DNA does appear to be recoverable from non-porous items after submersion; however, prolonged submersion can result in evidence loss. Blank slide samples experienced an increase in DNA quantity and the number of donor alleles over time. Because the blank slide samples were initially cleaned and a subset was swabbed, extracted, and quantified with no detectable DNA, the blank slides were assumed to be free of background human DNA. Due to this, in addition to all internal negative controls showing no contamination, the presence of these alleles was thought to be due to DNA transfer. Specifically, the possibility of transfer from deposition slide samples to the water to the blank slide samples which could be defined as an example of tertiary transfer. Investigating DNA transfer was not an initial objective for this study and was instead an observation when evaluating results. Due to this, statistical analyses could not be conducted on the limited number of blank slide samples. However, the observations of this study indicate the need to explore DNA transfer through water further. In multiple instances, deposition and blank slide samples yielded visually similar electropherograms, some to the extent that one would be unable to distinguish the deposition and blank slide samples without existing knowledge of which sample was which. In forensic DNA analysis, one is unable to know with certainty if trace DNA recovered from an evidence item was a result of direct contact or transfer, highlighting the importance of understanding the possibility of DNA transfer for interpretation purposes. The accumulation of results indicate that the behavior of low-level DNA and conditions for indirect DNA transfer may be unpredictable. Further, interpretations concerning the significance of DNA recovery from items submerged in water should not be overstated.
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Forensic Implications of the Discrepancies Caused between NGS and CE Results by New Microvariant Allele at Penta E Microsatellite
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1e79834d-e19e-4290-b9b8-f8bd929c9ea6
|
10218238
|
Forensic Medicine[mh]
|
The capillary electrophoresis based STR analysis (CE) was and still is the standard method used for personal identification in forensic genetics . However, the use of massively parallel sequencing (MPS) will provide a major boost to the field as the MPS STR analysis has many advantages over the traditional CE methods; for instance, it can define the complete nucleotide sequence of an allele genotyped encompassing the repeat units and the flanking regions as well . Although the discrimination power of the CE-based alleles is continuously growing with the introduction of new expanded multiplexes, their complexities caused by the ever-growing amplicon sizes and the used dyes may anticipate the limits of the traditional technology. Hence, there is no surprise that the willingness of the scientific community to use the new MPS technologies is increasing. Today, it is no question that by applying MPS STR typing in forensics, the highest discrimination power can be achieved in human identification. In addition, this modern technology is not limited to the repeat polymorphism, because single nucleotide polymorphisms (SNPs) can also be targeted together with the repeat numbers. Several studies demonstrated that MPS STR typing, compared to CE-based analysis, obtained an increase in allele variability . In 2016, because of the numerous newly discovered allele variants, considerations on minimal nomenclature requirements for forensic MPS STRs and the minimal requirement of data storage that laboratories should follow when adopting MPS of STRs were published by the DNA commission of the ISFG . In addition to the recommendations, the online STRidER database (STRs for Identity ENFSI Reference Database) has been created , which was an enlarged and improved version of the preceding ENFSI STRBase. Two years later, Phillips et al. released an expanded, enhanced and dynamically revised forensic STR Sequence Guide . In these early papers, principally, the sequence-specific allele variants detected in the repeat regions were described and published. However, variant sequences in the flanking regions cannot be neglected at all, which could be up to 20% of the newly described allele variants of the total SNP pool in Illumina chemistry and about 5% in Ion Torrent chemistry [ , , , , , ]. However, besides the advantages of this new method, it is necessary to discuss the challenges regarding sequence-based analyses, such as the backward compatibility of NGS data to CE results during a concordance validation. Some studies in which the Precision ID GlobalFiler NGS STR Panel v2 was used reported two types of discordances between CE and GlobalFiler panel data. Discordant genotypes were mostly due to observed base pair deletions in the flanking regions. In three cases, an allele 2.2 dropout was observed at Penta D locus during sequence evaluation of MPS STR using the Converge software (Thermo Fisher Scientific) where preceding CE genotypes were 2.2,11; 2.2,9; and 2.2,14, respectively . Further analyses of raw data and FASTQ files using STRaitRazor v3 proved the presence of allele 2.2 with a 13 bp deletion in the 5′ flanking region (rs119098807) in each case. Another study detected a 2 bp deletion at the D19S433 locus located in the 5′ flanking region (rs745607779) that was revealed via the IGV software (Integrative Genomic Viewer) and this resulted in a 13.2 allele in MPS STR typing compared to allele 14 determined using CE . Other cases demonstrated very low read count of some alleles during sequence evaluation and due to the extremely unbalanced amplification during library construction, allelic dropouts or 1–3 bp insertions had been observed which belonged to X1-3 type error, according to a previous study . In one case, Converge software did not detect a 1 bp insertion before the first complete repeat unit at the D2S441 locus but the analysis of the raw data and the FASTQ file showed that the result was in accordance with CE data and revealed the questioned 13.1 allele . During our internal laboratory validation, the Precision ID GlobalFiler NGS STR Panel v2 each genotype determined using the new MPS STR method was in accordance with the previous CE results, excepting two cases, in which allelic discrepancies were observed at Penta E locus between NGS and CE-based data. Instead of the prior 11.3,14 and 11.3,16 genotypes revealed via CE typing, NGS resulted in 12,14 and 12,16 genotypes, respectively.
For concordance testing during internal validation, our laboratory elimination database was used for 60 adult DNA samples with known family relations and with approved written consent. DNA was extracted from buccal swabs using EZ1 Advanced XL instrument with EZ1&2 DNA Investigator Kit according to the manufacturer’s recommendations (Qiagen, Hilden, Germany). Extracted genomic DNA was quantified using the Quantifiler Trio DNA Quantification kit (Thermo Fisher Scientific, Waltham, MA, USA) in the 7500 Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) and was analysed using the HID Real-Time PCR Analysis Software 1.2 (Thermo Fisher Scientific). For conventional STR typing, samples were amplified using the PowerPlex Fusion 6C and the PowerPlex 18D Systems according to the manufacturer’s recommendations (Promega, Madison, WI, USA). The GeneAmp PCR System 9700 thermal cycler was used for amplification and the electrophoresis was run on an ABI 3500xl Genetic Analyzer (Thermo Fisher Scientific). The electropherograms were analysed using GeneMapper ID-X software v1.6 (Thermo Fisher Scientific). For massive parallel sequencing, the library preparation was performed in 500 pg DNA template using the GlobalFiler NGS STR Panel v2 and the Precision ID Chef DL8 kits (Applied Biosystems, Waltham, MA, USA) with total reaction volumes of 15 µL. Each libraries contained a total mix of eight samples which were quantified using the Library TaqMan Quantification kit (Thermo Fisher Scientific) in the 7500 Real-Time PCR System. The E. coli DH10B control library was used as a quantification standard (Thermo Fisher Scientific). The libraries were diluted to 50 pM and then four libraries were mixed in equal proportions (6.25 µL per library) to a final volume of 25 µL for one sequencing run on Ion 530 chip. The Ion S5 Precision ID Chef Reagents were used for the automatic template preparation according to the manufacturer’s recommendations (Thermo Fisher Scientific). MPS was performed on the Ion S5 System using the Ion S5 Precision ID Sequencing Kit following the manufacturer’s instruction (Thermo Fisher Scientific). Sequence data were generated and evaluated using the Torrent Suite Software v5.10 (Thermo Fisher Scientific). For reference alignment, the Homo sapiens hg19 genome was applied (GCF_000001405.40). The HID STR Genotyper plugin v2.2 (Thermo Fisher Scientific) was used for STR typing at default thresholds excepting the analytical threshold (AT) and the stochastic threshold (ST) at some loci where the applied threshold values had been generated in course of the internal validation process. For PCR and Sanger sequencing of Penta E alleles, unlabelled oligonucleotide primer sequences were downloaded from STRBase ( https://strbase.nist.gov , accessed on 7 February 2023). PCR amplifications were performed on a GeneAmp 9700 PCR instrument with 0.5 ng template DNA using AmpliTaq Gold DNA Polymerase chemistry (Applied Biosystems) in 25 µL of total PCR volume. PCR conditions were denaturation for 10 min at 96 °C, then 94 °C at 20 s, 56 °C at 10 s, and 72 °C at 30 s for 32 cycles, and final extension at 72 °C for 10 min. PCR products were run on 3% agarose gel (SeaKem Agarose, Lonza, Basel, CH) to separate and visualise the heterozygous fragments. The two separate bands in each PCR product were excised and purified using Zymo Research Clean and Concentrator-25 Kit (Zymo Research, Seattle, WA, USA). In order to better sequence performance, the purified DNA fragments were reamplified and repurified as previously described. DNA sequencing were performed using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) in a 10 µL total volume with both forward and reverse primers. After cleaning up (Zymo Research DNA Sequencing Clean-up Kit), the reactions were analysed on an ABI Prism 3130xl Genetic Analyzer instrument and sequence data were evaluated using SeqScape v3.0 (Thermo Fisher Scientific) and Sequencing Analysis Software v6.0 (Thermo Fisher Scientific) and aligned to the Homo sapiens hg19 reference genome.
The CE-based PowerPlex Fusion 6C multiplex-PCR kit has already been validated earlier in the laboratory for routine casework. During the validation process, two samples in the elimination database carried a microvariant allele 11.3 at Penta E locus. The observed heterozygous genotypes with the microvariant allele were 11.3,16 and 11.3,14, respectively, and results were also confirmed using the PowerPlex 18D multiplex system. Although both CE-based multiplex-PCR kits were produced by the same manufacturer, the detected fragment sizes of the same alleles differed considerably between the two systems ( ). The approximately ten bases difference in size of the identical alleles can be due to the various primers applied, or due to adopted linker sequences. Unfortunately, there was no further information from the manufacturer regarding the oligos. The genetic relationship between the two samples harbouring the variant alleles were discovered and the mother/daughter descent was proven later via further DNA analyses. During the recent internal validation of the Precision ID GlobalFiler NGS STR Panel v2 kit on Ion S5 System platform, we received, however, the non-variant 12,16 and 12,14 genotypes at Penta E in the two samples, respectively, instead of the previously observed CE-based alleles. The Converge Software 2.2 was used primarily to evaluate NGS data in the laboratory. NGS genotypes in both samples were determined by normal read count for each allele ( ). All the other loci and genotypes in the two samples generated via the Converge were concordant with previous CE results. For secondary NGS analysis, FASTQ files were analysed with the freely available STRaitRazor v3 software which confirmed the same non-variant allele 12 in the samples ( ). Furthermore, the Binary Sequence Alignment/Map (BAM) files from these samples were analysed using the IGV v2.12.2 software, which determined the same non-variant allele 12 in both genotypes instead of the CE detected 11.3 microvariant ( ). IGV reads clearly revealed twelve repeat units in the samples, and neither SNP nor indel sites were detected in the approximately 160 bp long sequence read that covered the complete repeat structure and partly the flanking regions of the Penta E locus. To resolve the discrepant genotypes observed between the two methods, we decided to make further sequence analyses on the samples. For this purpose, traditional Sanger sequencing was the good choice to determine the accurate sequence of the PCR-amplified alleles. After separating the questioned alleles using electrophoresis, DNA fragments were isolated from the gel, purified and reamplified, and Sanger sequencing were conducted on each isolated samples, both on forward and reverse strands. The evaluated DNA sequences were aligned to the Penta E locus of the human hg19 reference genome (Penta E: chr15-hg19-97.374.245-97.374.269). The sequences of the variant alleles were in accordance with the complete repeat structure motifs, i.e., there were no base pair insertion/deletion variance observed in the repeat sequences at all. The sequenced repeat numbers of each allele were in accordance with the genotypes determined using MPS STR and CE. In case of the variant 11.3 alleles, the sequence data confirmed the complete twelve repeat unit structure in both samples, that corresponded with previous NGS sequence results. In parallel, the 14 and 16 repeat motifs have been identified in the homologous alleles. However, for PCR and Sanger sequencing, relatively long size DNA fragments were amplified (414 bp long fragment in case of allele 12), that covered the whole domain of the locus and made it possible to analyse the repeat structure of the nearby flanking regions in a more wide range. The extended sequencing of the flanking regions in discordant 11.3 allele revealed a two-bases GG deletion, 116–117 bases, downstream of the last TCTTT repeat motif in the forward strand. This dinucleotide deletion has been observed in both samples harbouring the microvariant 11.3 alleles, and the detected variant site was located at 203–204 bp distance from the 3′ end of the forward primer at the 97.374.386–97.374.387 chromosome positions on chromosome 15 aligned to the reference human genome hg19 ( ). Several searches have been conducted in the various important electronic available sequence databases to check the existence of this variant allele sequence (i.e., GenBank, STRBase, STRSeq BioProject, 1000 Genomes Browser, and UCSC Genome Browser). Some sequences in the databases revealed substitutions on this site, and there were deletions, insertions, or substitutions observed at the closest bases, but there was no similar dinucleotide deletion that we determined. Additionally, no references for this allele variant in the scientific literature were found. According to the manufacturer’s application guide for the Precision ID GlobalFiler NGS STR Panel v2 kit, the amplicon read sizes at Penta E locus were settled from 168 to 273 bases long-range depending on the sequenced allele. The amplicon read lengths also included the two primer oligos that were approximately 30 nt long. However, during the NGS data evaluation we were unable to determine fragments longer than 160 bp long in the raw data using IGV. This means that a considerable part of the allelic sequence reads fell out of the detected range. Indeed, inspecting the aligned IGV sequences, maximum 60 bp long fragments were readable at the forward strand downstream region of the last TCTTT repeat, and only 35 bp stretch upstream were readable from the starting repeat in the flanking region. Based on the Sanger sequencing results and considering the NGS amplicon sizes, the identified new mutation, i.e., GG deletion, must located in the downstream flanking region of the forward strand far away enough from the repeat structure that might have caused the kit amplicons did not cover the mutated site during library preparation. This phenomenon led to ignore the mutated Penta E alleles during NGS data evaluation and resulted in the misgenotyping of the two samples using the Precision ID GlobalFiler NGS STR Panel v2 kit that ultimately caused the discrepancy between NGS and CE-based results. Based on the literature, similar microvariant sites have been observed at almost all forensically relevant autosomal STRs, when usually base deletion in the flanking region far away from repeat structure was the reason for a discordant result. Nevertheless, according to the STRidER database ( https://strider.online/ , accessed on 7 February 2023), only three variant sites were found until now in the Penta E sequence downstream of the last TCTTT repeat unit. The reviewed rs188309642 and rs537369792 SNP positions were identified in African population samples, and the rs759423627 SNP in a European sample pool. However, the indel variation described in our study at 116–117 nucleotides downstream from the last repeat unit, just falls outside of the database sequence deposited in STRidER which ends at 115 nt position (GRCh38 coordinate: 96831154). The sequence of the new observed Penta E 11.3 allele variant has been annotated and deposited in the GenBank ( https://www.ncbi.nlm.nih.gov/genbank/ , accessed on 7 February 2023) with accession number OP700292.
In this study, we described an STR discrepancy case between NGS and traditional CE data that could not have been resolved using only different NGS software packages, and neither by re-analysis of the samples. Usually, microvariant alleles falling between two repeat units in size (such as 11.3 observed at Penta E locus) can be obtained in different ways using CE: as an insertion or deletion in the tandem repeat region, or as an indel variant in the flanking sequences that does not affect the repeat structure but still investigated using the muliplex-PCR systems. To determine the reason behind the genotype discrepancy in this case, an extended sequence analysis has been performed on the samples in question. Sanger sequencing of the variant alleles proved that they carry a GG deletion in the flanking region far away from the repeat stretch that resulted in the observed 11.3 intermediate alleles which were undetected via the applied MPS analysis software. Nevertheless, in forensic genetics the fragment length and repeat unit based allelic nomenclature is still accepted, but there is an increasing demand on comprehensive STR nomenclature systems to adopt MPS STR data in order to ensure compatibility with MPS and with traditional CE data . The intermediate alleles observed in this study were confirmed as having complete twelve repeat unit structure, therefore calling them as 12-2 or 12var corresponds to current nomenclature. It has to be also noted that relevant genetic variation outside the common repeat regions of STR sequences should be stored in databases as sequence strings that include flanking sequences according to the most recent recommendation . There were several reasons why we performed this deep analysis of the variant allele: (i) If an evidence sample carrying allele 11.3 at Penta E was genotyped using capillary electrophoresis but reference samples were genotyped using only NGS, an exclusion would be considered on this locus in a casework; (ii) The sequenced and annotated allele variant has been published and deposited in open databases that makes this observation available to the scientific community; and (iii) The results provide a clear explanation of the observed allele discrepancy to any another lab that would detect similar phenomenon. If a forensic lab using NGS for STR typing is aware of the potential pitfalls of the method used, similar issues can be avoided during interpretation of expert reports. However, the correct repeat numbers that were revealed using the NGS technology prove the usability of the technique in routine casework. In addition, our study highlights the necessity to consider applying several software packages or even traditional molecular methods for the evaluation of questionable NGS results or discordant genotypes.
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Evaluating the Usefulness of Human DNA Quantification to Predict DNA Profiling Success of Historical Bone Samples
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82c278ed-acea-494f-af41-b1f73f89cb0d
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10218391
|
Forensic Medicine[mh]
|
DNA profiling of historical remains can be notoriously difficult. As DNA degrades over time, only small fragments can be extracted from aged skeletal samples. Over the past 10 years, ancient DNA (aDNA) extraction methods have been developed and optimized to recover ultrashort (<40 bp) DNA molecules . These small DNA fragments are more conducive to shotgun library preparation and next-generation sequencing (NGS) than PCR-based DNA profiling methods that require intact amplicon targets. In addition to fragmentation, DNA damage such as cytosine deamination often affects ancient and historical remains (e.g., ). This can result in base substitutions in the replication process, which can impact the interpretation of sequence data. Poor environmental conditions, such as a warm and humid climate, can exacerbate DNA degradation and lead to microbial growth. Human DNA can be overwhelmed by co-extracted exogenous DNA, including DNA from the soil , which may be more abundant than the endogenous DNA in the specimen. The microenvironment and the skeletal element may also play a role in the human DNA content . Finally, human remains are sometimes stored in conditions that are not conducive to DNA preservation, such as in plastic bags , or they may be treated with chemical preservatives such as formalin that further diminish the ability to recover DNA. Due to these myriad factors, it can be difficult to gauge sample success prior to devoting resources to sample extraction, library preparation, and sequencing, as noted in Kapp et al. . In some situations, there may be an absence of contextual or historical information that could help determine the degree or types of damage in a sample prior to lab processing. Screening methods, such as whole genome sequencing (WGS), are often utilized in the aDNA community to predict sample success. However, this method is costly and minimally effective for samples with <1% endogenous DNA . Various aDNA laboratories have implemented hybridization capture for these compromised samples to target the endogenous portion. Some laboratories have implemented custom quantitative real-time PCR (qPCR) assays for a variety of species to quantify endogenous DNA in sample extracts and prepared libraries with the intent of predicting downstream success. Similarly, Enk et al. found that multi-copy qPCR assays can successfully estimate target DNA fragment length, which may improve the predictive power of qPCR as a screening tool. This conclusion was supported by a comparison of quantification methods conducted by Brzobohatá et al. using skeletal remains from a medieval burial site. Total double-stranded DNA (dsDNA) quantification methods resulted in the highest DNA concentrations due to the presence of exogenous DNA, while qPCR underestimated DNA quantity in degraded samples with small fragment sizes. A quantification system that utilized capillary electrophoresis to determine DNA concentration and fragment distribution was the most accurate and informative method of aDNA quantification for the data presented in . Within the forensic community, sample screening is traditionally performed with test PCR amplification events or qPCR. The most utilized human qPCR assays have been developed to work in tandem with short tandem repeat (STR) typing kits, such that qPCR results can identify the amount of target DNA of suitable size to amplify successfully with an STR multiplex. The forensic qPCR assays are furthermore designed with an internal positive control (IPC) that can alert the user to the presence of inhibitors common in forensic samples. Current qPCR kits include multi-copy autosomal and Y-chromosomal DNA targets, and often two autosomal targets of different fragment lengths are used for a degradation assessment (degradation index). The suitability of these traditional nuclear qPCR assays for highly degraded DNA has yet to be shown. As an alternative to nuclear DNA quantification, the use of qPCR to quantify mitochondrial DNA (mtDNA), which is more readily recovered from historical samples, has proven successful. MtDNA qPCR has been used to assess fragmentation and to direct the processing of degraded remains in both ancient and forensic settings. This proof-of-concept study evaluated DNA quantification as a screening method for historical remains when processed through a variety of downstream DNA profiling modalities including single nucleotide polymorphism (SNP), mtDNA, autosomal STR (auSTR), and Y-STR genotyping. A genomic DNA quantification method with an intercalating dye was used to determine the total dsDNA and well-characterized qPCR kits were used to determine human DNA quantity. The qPCR kits assessed included PowerQuant, Quantifiler Trio (Trio), and Investigator Quantiplex Pro (Pro). These assays are human-specific and unaffected by overwhelming quantities of non-human DNA. Together, this quantification data (total and human) can be used to calculate a human DNA (hDNA) ratio that also accounts for the proportion of exogenous DNA in a sample. This study tested whether a range of aged and highly degraded samples contained sufficient DNA to be detectable with three forensically oriented commercial qPCR kits. It then compared both the human DNA quantity and the human DNA ratio as potential screening tools for forensic DNA profiling to determine the most beneficial processing workflow. Such a predictive tool could increase efficiency and better direct resources, even in scenarios where little to no contextual sample information is available.
2.1. Skeletal Samples Thirty-one burials, from six historical contexts, were included in this study for DNA analysis ( ). These samples range in age from approximately 80 to 800 years postmortem and originated from multiple locations across the United States and Europe. The JB55 , Arch Street, and World War II (WWII) samples were previously processed at the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL), ensuring that a range in sample quality was assessed prior to inclusion in the study. The burial of JB55 also referred to as the New England vampire, was excavated in 1990 after being discovered during sand and gravel operations . In addition to JB55, a second bone known to be from a red deer through prior mtDNA testing, was included to test the specificity of the qPCR assay. Non-human bones may be submitted for forensic DNA analysis when they lack diagnostic features for definitive anthropological species identification. The Arch Street project consisted of remains from the First Baptist Church of Philadelphia that were unearthed during construction in 2017. WWII samples were comprised of disinterred remains from Germany and the National Memorial Cemetery of the Pacific, also known as the Punchbowl. The WWII Punchbowl samples were likely impacted by chemical treatments, such as formalin embalming and the application of hardening compounds, that inhibit DNA testing . However, the exact chemical treatment of each Punchbowl burial tested here was unknown, as these are true historical cases with only generalized contextual information . The Harewood Cemetery is located on the family estate of Samuel Washington, brother of George Washington who served as the first president of the United States. Of the three burials included in this study, a single skeletal element was selected per grave, aside from one burial which had two elements tested. The skeletal remains associated with the Basilica of St. Mary’s Assumption of the former monastery church in Fürstenfeldbruck, Germany, were collected from a walled-up, uninscribed coffin compartment in the crypt. The remains were collected in the presence of HRH Duke Franz of Bavaria and were radiocarbon dated to the mid-13th to early 14th centuries. Overall, samples exhibited varying levels of degradation due to environmental factors and chemical treatment. Additional information about each sample can be found in . 2.2. Sample Preparation and DNA Extraction All DNA extractions were performed at the AFMES-AFDIL, with the exception of the 12 Basilica samples that were extracted at the Institute of Legal Medicine in Innsbruck. DNA extractions were performed according to ancient and forensic DNA standards. This included the use of dedicated low-copy extraction laboratories with positive pressure for extraction and library preparation prior to amplification, appropriate personal protective equipment, sterilization of tools and laboratory consumables, and inclusion of extraction and library preparation controls processed alongside samples. For the extractions performed at the AFMES-AFDIL, the outer surface of the bone samples and excess spongy bone were sanded to remove exogenous DNA. Bone fragments were washed with sterile water and 100% ethanol, then ground to a powder in a Waring blender cup (Waring, Torrington, CT). Arch Street samples underwent a bleach wash with a 70 mM solution of household bleach (8.25% sodium hypochlorite) prior to the sterile water and ethanol washes. Bone powder aliquots and associated reagent blanks (RB) underwent the Dabney extraction protocol described in Rohland et al. to retain ultrashort DNA fragments. The volume of Dabney extraction buffer (0.46 M EDTA, 0.05% Tween-20) and proteinase K (20 mg/mL) differed across sample sets depending on whether the original AFDIL Dabney protocol or the modified AFDIL Dabney protocol was used ( ). The AFDIL Dabney protocol utilized 0.2 g of bone powder, 1 mL of Dabney extraction buffer, and 25 µL of proteinase K. The modified protocol utilized up to 0.4 g bone powder, 4 mL Dabney buffer, and 200 µL of proteinase K to enhance digestion. Both the AFDIL Dabney and the modified AFDIL Dabney methods maintained the same 10X binding buffer to lysate ratio as utilized in the Rohland et al. Dabney method for small fragment retention. All samples, across both protocols, were incubated at 56 °C overnight with constant agitation. Following overnight incubation, the supernatant was separated from the remaining bone powder and combined with 10X QIAGEN PB buffer (QIAGEN, Hilden, Germany) in place of binding buffer ‘D’ used in the Rohland et al. method . Samples were spun through a Roche High Pure Viral silica-based column (Roche, Pleasanton, CA, USA), and the bound DNA was washed twice with QIAGEN PE buffer. DNA was eluted in 50–60 µL Tris-EDTA (10 mM Tris, 0.1 mM EDTA, pH 7.5) following the Rohland et al. protocol . Extracts were stored at −20 °C immediately upon completion until downstream processing was initiated (approximately one to six months). Replicate extracts were prepared and homogenized to provide sufficient volume for quantification, STR typing and NGS analysis. The bone samples processed at the Institute of Legal Medicine in Innsbruck were sanded using a Dremel tool until 1–2 mm of the surface layer was removed. The bone powder was retrieved using a dental drill at low speed to avoid overheating the tissue. The resulting powder was lysed in three parallel 50 mg aliquots, and DNA was extracted by combining the aliquots through a filter, following the protocol described by Xavier et al. . Non-template controls and RBs were run in parallel throughout the experimental procedure to monitor possible contamination. Upon receipt at the AFMES-AFDIL, sample extracts ranged in volume from 13–34 µL and 10 mM Tris-HCl (pH 8.5) was added to reach a final volume of 45 µL prior to downstream processing. 2.3. DNA Quantification Human nuclear DNA-specific qPCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). For comparison purposes, DNA was quantified with three qPCR kits, unless indicated ( ). The Investigator Quantiplex Pro kit (QIAGEN), Quantifiler Trio kit (Thermo Fisher Scientific), and PowerQuant System (Promega Corporation, Madison, WI, USA) were used following their respective manufacturer’s recommendations. These three kits include both a small and large human autosomal target and a small Y-chromosomal target, all multi-copy. The PowerQuant System also includes a large Y-chromosomal target to assess Y chromosome-specific degradation. DNA was quantified using 2 µL extract. All kits include an IPC, a synthetic target that is used to detect the presence of some common inhibitors. A non-human bone sample was also tested in conjunction with standards and non-template controls. The size of each target can be found in . Where possible, the presentation of qPCR and sample processing information complies with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines . Results from the small autosomal target of each kit were compared in a pairwise fashion using a two-tailed, paired t -test. An α value of 0.05, consistent with a 95% confidence interval, was selected for assessing statistical significance. A p -value > 0.05 was considered ‘not significant’. The Basilica extracts were also quantified with SD quants, a tetraplex qPCR system, targeting an IPC, one nuclear (70 bp), and two mtDNA (69 bp and 143 bp) targets according to Xavier et al. . The mtDNA results were given in mitogenome equivalents (mtGE) while pg/µL were used for the autosomal target. A degradation index was calculated based on concentrations of the mtDNA targets. Total dsDNA was quantified using the Qubit 2.0 Fluorometer and the Qubit dsDNA HS or BR Assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific) using 2 µL of extract. To assess whether the proportion of human-to-exogenous DNA was predictive of downstream success, an hDNA ratio was determined using the formula below for each sample. The calculation required the quantification results from a target-specific qPCR assay (small autosomal target) and the total dsDNA quantity. The ratio compared the picograms of human autosomal DNA (auDNA) to nanograms of total dsDNA to make the resulting ratios manageable numbers. hDNA Ratio = pg input based on qPCR small autosomal target ng input of total dsDNA 2.4. Next Generation Sequencing 2.4.1. Library Preparation Sample and control libraries were prepared at the AFMES-AFDIL in a dedicated low copy, pre-PCR laboratory using the KAPA HyperPrep Kit (Roche Sequencing, Wilmington, MA, USA) following the procedure described in Tillmar et al. unless noted. No DNA repair was performed on the extracts. DNA input for end repair was based on the Qubit values. Adapter ligation was performed using 15 µM KAPA unique dual-indexed adapters (Roche Sequencing) for all samples and RBs. A 1:10 adapter dilution was used for all negative and positive controls based on a DNA input ≤ 1 ng. A 1.3x AMPure XP (Beckman Coulter, Brea, CA, USA) ratio was used for purification following adapter ligation to ensure the retention of small DNA fragments. Library PCR was performed using KAPA HiFi HotStart Uracil+ Ready Mix and 20 µM Illumina Primer mix (Roche Sequencing). The Uracil+ Ready Mix contains a uracil-tolerant polymerase that allows for the amplification of DNA with cytosine deamination, as characteristic of ancient and historical DNA . After PCR amplification, a 5X QIAGEN MinElute (QIAGEN) purification was performed with elution in 25 µL Tris-EDTA. Library quality was evaluated using the 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) before proceeding with hybridization capture. Additionally, a library input reduction test was performed to assess whether using the maximum 1 µg dsDNA input for library preparation resulted in inhibition and thus poor-quality libraries. Three samples from different historical contexts with initial library inputs of a maximum of 1 µg were diluted to 500 ng and 200 ng to evaluate reduced library input. Library preparation was performed on the 500 ng and 200 ng samples using the same procedures described previously. 2.4.2. Hybridization Capture Purified libraries were enriched via hybridization capture using myBaits v5 kits (Arbor Biosciences, Ann Arbor, MI, USA). Sample libraries were captured with two bait sets unless indicated ( ). The forensically oriented FORCE SNP panel β version contains ~20,000 unique baits targeting 5402 SNPs including identity, ancestry, phenotype, X- and Y-chromosomal SNPs. The β version excludes 44 tri-allelic, clinically relevant, uninformative and/or underperforming SNPs identified in . The second bait set was a custom panel that covers the human mitogenome with 1800 unique oligos . A maximum of 7 µL of the library was used for each capture reaction (FORCE and mitogenome). The capture product was amplified and purified according to , except the 20 µM Illumina Primer mix was used. Samples were eluted in 20 µL Tris-EDTA. All captured libraries were run on a 2100 Bioanalyzer using the 7500 assay. Any libraries with total DNA concentrations over 50 ng/µL were diluted and re-run using the 7500 assay for more accurate quantification. 2.4.3. Normalization and Pooling FORCE and mitogenome hybridization capture samples were primarily pooled by normalization, with a 50 nM target for the 170–1000 bp range. The mitogenome capture samples for the Basilica and Arch Street were pooled by volume instead of normalizing. Mitogenome sequencing pools contained 21–27 samples, while FORCE pools consisted of 11–16 samples. Positive controls were not sequenced and were used only to monitor library preparation and capture success. 2.4.4. Loading and Sequencing All pools were quantified on the 2100 Bioanalyzer using the DNA 7500 kit and diluted to 4 nM prior to loading. The 4 nM pools were denatured and diluted to 1.0 pM with 2.5% Illumina PhiX Control V3 (PhiX) (Illumina) or 1.2 pM with 5% PhiX. All runs were performed on a NextSeq 550 (Illumina) with Mid output v 2.5 kits. As general sample qualities were known, pools containing only highly degraded samples were sequenced with 75 × 75 paired-end cycles. For samples with moderately degraded DNA, 150 × 150 paired-end sequencing was used. 2.4.5. Sequence Data Analysis Following demultiplexing, FASTQ files were imported into the CLC Genomics Workbench v12.0.1 (QIAGEN) for analysis. FORCE analysis was completed with a custom workflow as described in with two modifications: (1) reads less than 30 bp were discarded to minimize the mapping of non-specific reads and (2) a custom de-duplication tool that mimics the functionality of markdup from SAMTools v1.61.1 ( http://www.htslib.org/doc/samtools-markdup.html ) (accessed on 14 February 2023) was used to more effectively remove PCR duplicates. Genotypes were called using the Known Mutations from Mapping tool, and a custom Microsoft Excel (Redmond, WA, USA) template as described in was used to apply additional analysis parameters to called SNPs and simplify the output. Calling of target SNPs required a minimum read count (coverage) of 10X. In addition to the minimum coverage requirement, an allele frequency of 90% or greater was necessary for homozygous genotypes. Heterozygous genotypes needed a minor allele frequency (MAF) of at least 30% to be called. Imbalanced genotypes (10–30% MAF) were excluded from analysis (not called), as heterozygosity could not be confidently determined. SNP coverage at 1X and 5X was also evaluated. Mitogenome analysis was also completed in the CLC Genomics Workbench v12.0.1 with the AFDIL-QIAGEN mtDNA Expert (AQME) plug-in and a custom workflow, as described in . Reads were mapped to the revised Cambridge Reference Sequence (rCRS) using the same stringent mapping parameters incorporated into the custom FORCE workflow. The custom de-duplication tool with local realignment was implemented to remove PCR duplicates and assist in indel alignment. A 10X coverage threshold, with a variant frequency ≥10% and a minimum variant count of four, was required for variant calling. 2.5. STR Typing To assess the suitability of DNA quantification to predict PCR enrichment success, short tandem repeat (STR) typing was performed. The PowerPlex Fusion kit (Promega) (PPF) amplifies 22 forensic auSTRs, one Y-STR, and one non-STR sex-determination locus (amelogenin). Samples, along with a 2800M positive control and negative control (sterile water), were amplified according to the manufacturer’s recommendations. The maximum sample volume of 15 µL was used when the extract volume allowed, unless indicated ( ). The Applied Biosystems’ AmpFLSTR Yfiler kit amplifies 17 forensic Y-chromosomal STRs (Y-STRs) for male identification. A modified Yfiler approach optimized for degraded, low copy number samples (LCNY) was developed at the AFMES-AFDIL . This LCNY protocol, which includes increased polymerase and cycles, was used to generate all Y-STR data. The maximum sample volume of 9.2 µL was utilized when extract the volume allowed, unless otherwise indicated ( ). If inhibition was suspected, a range of input volumes was amplified in a repeat amplification event to improve the number of loci obtained. The amplified product was prepared for capillary electrophoresis according to the manufacturer’s manual for each kit and loaded on a 3500xL Genetic Analyzer with POP-4 polymer (Applied Biosystems, Foster City, CA, USA). Samples were injected for 15 s (PPF) or 7 s (LCNY) using default injection parameters for each kit. STR data were analyzed using GeneMapper ID-X v1.4 (Applied Biosystems). Locus-specific stutter filters were determined by internal validation at the AFMES-AFDIL. For PPF, the reporting threshold was 70 relative fluorescent units (RFU) for heterozygous alleles and the Y-STR locus, and 550 RFU for homozygous alleles at the auSTR loci. For LCNY, peaks over 70 RFU were considered reportable.
Thirty-one burials, from six historical contexts, were included in this study for DNA analysis ( ). These samples range in age from approximately 80 to 800 years postmortem and originated from multiple locations across the United States and Europe. The JB55 , Arch Street, and World War II (WWII) samples were previously processed at the Armed Forces Medical Examiner System’s Armed Forces DNA Identification Laboratory (AFMES-AFDIL), ensuring that a range in sample quality was assessed prior to inclusion in the study. The burial of JB55 also referred to as the New England vampire, was excavated in 1990 after being discovered during sand and gravel operations . In addition to JB55, a second bone known to be from a red deer through prior mtDNA testing, was included to test the specificity of the qPCR assay. Non-human bones may be submitted for forensic DNA analysis when they lack diagnostic features for definitive anthropological species identification. The Arch Street project consisted of remains from the First Baptist Church of Philadelphia that were unearthed during construction in 2017. WWII samples were comprised of disinterred remains from Germany and the National Memorial Cemetery of the Pacific, also known as the Punchbowl. The WWII Punchbowl samples were likely impacted by chemical treatments, such as formalin embalming and the application of hardening compounds, that inhibit DNA testing . However, the exact chemical treatment of each Punchbowl burial tested here was unknown, as these are true historical cases with only generalized contextual information . The Harewood Cemetery is located on the family estate of Samuel Washington, brother of George Washington who served as the first president of the United States. Of the three burials included in this study, a single skeletal element was selected per grave, aside from one burial which had two elements tested. The skeletal remains associated with the Basilica of St. Mary’s Assumption of the former monastery church in Fürstenfeldbruck, Germany, were collected from a walled-up, uninscribed coffin compartment in the crypt. The remains were collected in the presence of HRH Duke Franz of Bavaria and were radiocarbon dated to the mid-13th to early 14th centuries. Overall, samples exhibited varying levels of degradation due to environmental factors and chemical treatment. Additional information about each sample can be found in .
All DNA extractions were performed at the AFMES-AFDIL, with the exception of the 12 Basilica samples that were extracted at the Institute of Legal Medicine in Innsbruck. DNA extractions were performed according to ancient and forensic DNA standards. This included the use of dedicated low-copy extraction laboratories with positive pressure for extraction and library preparation prior to amplification, appropriate personal protective equipment, sterilization of tools and laboratory consumables, and inclusion of extraction and library preparation controls processed alongside samples. For the extractions performed at the AFMES-AFDIL, the outer surface of the bone samples and excess spongy bone were sanded to remove exogenous DNA. Bone fragments were washed with sterile water and 100% ethanol, then ground to a powder in a Waring blender cup (Waring, Torrington, CT). Arch Street samples underwent a bleach wash with a 70 mM solution of household bleach (8.25% sodium hypochlorite) prior to the sterile water and ethanol washes. Bone powder aliquots and associated reagent blanks (RB) underwent the Dabney extraction protocol described in Rohland et al. to retain ultrashort DNA fragments. The volume of Dabney extraction buffer (0.46 M EDTA, 0.05% Tween-20) and proteinase K (20 mg/mL) differed across sample sets depending on whether the original AFDIL Dabney protocol or the modified AFDIL Dabney protocol was used ( ). The AFDIL Dabney protocol utilized 0.2 g of bone powder, 1 mL of Dabney extraction buffer, and 25 µL of proteinase K. The modified protocol utilized up to 0.4 g bone powder, 4 mL Dabney buffer, and 200 µL of proteinase K to enhance digestion. Both the AFDIL Dabney and the modified AFDIL Dabney methods maintained the same 10X binding buffer to lysate ratio as utilized in the Rohland et al. Dabney method for small fragment retention. All samples, across both protocols, were incubated at 56 °C overnight with constant agitation. Following overnight incubation, the supernatant was separated from the remaining bone powder and combined with 10X QIAGEN PB buffer (QIAGEN, Hilden, Germany) in place of binding buffer ‘D’ used in the Rohland et al. method . Samples were spun through a Roche High Pure Viral silica-based column (Roche, Pleasanton, CA, USA), and the bound DNA was washed twice with QIAGEN PE buffer. DNA was eluted in 50–60 µL Tris-EDTA (10 mM Tris, 0.1 mM EDTA, pH 7.5) following the Rohland et al. protocol . Extracts were stored at −20 °C immediately upon completion until downstream processing was initiated (approximately one to six months). Replicate extracts were prepared and homogenized to provide sufficient volume for quantification, STR typing and NGS analysis. The bone samples processed at the Institute of Legal Medicine in Innsbruck were sanded using a Dremel tool until 1–2 mm of the surface layer was removed. The bone powder was retrieved using a dental drill at low speed to avoid overheating the tissue. The resulting powder was lysed in three parallel 50 mg aliquots, and DNA was extracted by combining the aliquots through a filter, following the protocol described by Xavier et al. . Non-template controls and RBs were run in parallel throughout the experimental procedure to monitor possible contamination. Upon receipt at the AFMES-AFDIL, sample extracts ranged in volume from 13–34 µL and 10 mM Tris-HCl (pH 8.5) was added to reach a final volume of 45 µL prior to downstream processing.
Human nuclear DNA-specific qPCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). For comparison purposes, DNA was quantified with three qPCR kits, unless indicated ( ). The Investigator Quantiplex Pro kit (QIAGEN), Quantifiler Trio kit (Thermo Fisher Scientific), and PowerQuant System (Promega Corporation, Madison, WI, USA) were used following their respective manufacturer’s recommendations. These three kits include both a small and large human autosomal target and a small Y-chromosomal target, all multi-copy. The PowerQuant System also includes a large Y-chromosomal target to assess Y chromosome-specific degradation. DNA was quantified using 2 µL extract. All kits include an IPC, a synthetic target that is used to detect the presence of some common inhibitors. A non-human bone sample was also tested in conjunction with standards and non-template controls. The size of each target can be found in . Where possible, the presentation of qPCR and sample processing information complies with the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines . Results from the small autosomal target of each kit were compared in a pairwise fashion using a two-tailed, paired t -test. An α value of 0.05, consistent with a 95% confidence interval, was selected for assessing statistical significance. A p -value > 0.05 was considered ‘not significant’. The Basilica extracts were also quantified with SD quants, a tetraplex qPCR system, targeting an IPC, one nuclear (70 bp), and two mtDNA (69 bp and 143 bp) targets according to Xavier et al. . The mtDNA results were given in mitogenome equivalents (mtGE) while pg/µL were used for the autosomal target. A degradation index was calculated based on concentrations of the mtDNA targets. Total dsDNA was quantified using the Qubit 2.0 Fluorometer and the Qubit dsDNA HS or BR Assay kit according to the manufacturer’s instructions (Thermo Fisher Scientific) using 2 µL of extract. To assess whether the proportion of human-to-exogenous DNA was predictive of downstream success, an hDNA ratio was determined using the formula below for each sample. The calculation required the quantification results from a target-specific qPCR assay (small autosomal target) and the total dsDNA quantity. The ratio compared the picograms of human autosomal DNA (auDNA) to nanograms of total dsDNA to make the resulting ratios manageable numbers. hDNA Ratio = pg input based on qPCR small autosomal target ng input of total dsDNA
2.4.1. Library Preparation Sample and control libraries were prepared at the AFMES-AFDIL in a dedicated low copy, pre-PCR laboratory using the KAPA HyperPrep Kit (Roche Sequencing, Wilmington, MA, USA) following the procedure described in Tillmar et al. unless noted. No DNA repair was performed on the extracts. DNA input for end repair was based on the Qubit values. Adapter ligation was performed using 15 µM KAPA unique dual-indexed adapters (Roche Sequencing) for all samples and RBs. A 1:10 adapter dilution was used for all negative and positive controls based on a DNA input ≤ 1 ng. A 1.3x AMPure XP (Beckman Coulter, Brea, CA, USA) ratio was used for purification following adapter ligation to ensure the retention of small DNA fragments. Library PCR was performed using KAPA HiFi HotStart Uracil+ Ready Mix and 20 µM Illumina Primer mix (Roche Sequencing). The Uracil+ Ready Mix contains a uracil-tolerant polymerase that allows for the amplification of DNA with cytosine deamination, as characteristic of ancient and historical DNA . After PCR amplification, a 5X QIAGEN MinElute (QIAGEN) purification was performed with elution in 25 µL Tris-EDTA. Library quality was evaluated using the 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) before proceeding with hybridization capture. Additionally, a library input reduction test was performed to assess whether using the maximum 1 µg dsDNA input for library preparation resulted in inhibition and thus poor-quality libraries. Three samples from different historical contexts with initial library inputs of a maximum of 1 µg were diluted to 500 ng and 200 ng to evaluate reduced library input. Library preparation was performed on the 500 ng and 200 ng samples using the same procedures described previously. 2.4.2. Hybridization Capture Purified libraries were enriched via hybridization capture using myBaits v5 kits (Arbor Biosciences, Ann Arbor, MI, USA). Sample libraries were captured with two bait sets unless indicated ( ). The forensically oriented FORCE SNP panel β version contains ~20,000 unique baits targeting 5402 SNPs including identity, ancestry, phenotype, X- and Y-chromosomal SNPs. The β version excludes 44 tri-allelic, clinically relevant, uninformative and/or underperforming SNPs identified in . The second bait set was a custom panel that covers the human mitogenome with 1800 unique oligos . A maximum of 7 µL of the library was used for each capture reaction (FORCE and mitogenome). The capture product was amplified and purified according to , except the 20 µM Illumina Primer mix was used. Samples were eluted in 20 µL Tris-EDTA. All captured libraries were run on a 2100 Bioanalyzer using the 7500 assay. Any libraries with total DNA concentrations over 50 ng/µL were diluted and re-run using the 7500 assay for more accurate quantification. 2.4.3. Normalization and Pooling FORCE and mitogenome hybridization capture samples were primarily pooled by normalization, with a 50 nM target for the 170–1000 bp range. The mitogenome capture samples for the Basilica and Arch Street were pooled by volume instead of normalizing. Mitogenome sequencing pools contained 21–27 samples, while FORCE pools consisted of 11–16 samples. Positive controls were not sequenced and were used only to monitor library preparation and capture success. 2.4.4. Loading and Sequencing All pools were quantified on the 2100 Bioanalyzer using the DNA 7500 kit and diluted to 4 nM prior to loading. The 4 nM pools were denatured and diluted to 1.0 pM with 2.5% Illumina PhiX Control V3 (PhiX) (Illumina) or 1.2 pM with 5% PhiX. All runs were performed on a NextSeq 550 (Illumina) with Mid output v 2.5 kits. As general sample qualities were known, pools containing only highly degraded samples were sequenced with 75 × 75 paired-end cycles. For samples with moderately degraded DNA, 150 × 150 paired-end sequencing was used. 2.4.5. Sequence Data Analysis Following demultiplexing, FASTQ files were imported into the CLC Genomics Workbench v12.0.1 (QIAGEN) for analysis. FORCE analysis was completed with a custom workflow as described in with two modifications: (1) reads less than 30 bp were discarded to minimize the mapping of non-specific reads and (2) a custom de-duplication tool that mimics the functionality of markdup from SAMTools v1.61.1 ( http://www.htslib.org/doc/samtools-markdup.html ) (accessed on 14 February 2023) was used to more effectively remove PCR duplicates. Genotypes were called using the Known Mutations from Mapping tool, and a custom Microsoft Excel (Redmond, WA, USA) template as described in was used to apply additional analysis parameters to called SNPs and simplify the output. Calling of target SNPs required a minimum read count (coverage) of 10X. In addition to the minimum coverage requirement, an allele frequency of 90% or greater was necessary for homozygous genotypes. Heterozygous genotypes needed a minor allele frequency (MAF) of at least 30% to be called. Imbalanced genotypes (10–30% MAF) were excluded from analysis (not called), as heterozygosity could not be confidently determined. SNP coverage at 1X and 5X was also evaluated. Mitogenome analysis was also completed in the CLC Genomics Workbench v12.0.1 with the AFDIL-QIAGEN mtDNA Expert (AQME) plug-in and a custom workflow, as described in . Reads were mapped to the revised Cambridge Reference Sequence (rCRS) using the same stringent mapping parameters incorporated into the custom FORCE workflow. The custom de-duplication tool with local realignment was implemented to remove PCR duplicates and assist in indel alignment. A 10X coverage threshold, with a variant frequency ≥10% and a minimum variant count of four, was required for variant calling.
Sample and control libraries were prepared at the AFMES-AFDIL in a dedicated low copy, pre-PCR laboratory using the KAPA HyperPrep Kit (Roche Sequencing, Wilmington, MA, USA) following the procedure described in Tillmar et al. unless noted. No DNA repair was performed on the extracts. DNA input for end repair was based on the Qubit values. Adapter ligation was performed using 15 µM KAPA unique dual-indexed adapters (Roche Sequencing) for all samples and RBs. A 1:10 adapter dilution was used for all negative and positive controls based on a DNA input ≤ 1 ng. A 1.3x AMPure XP (Beckman Coulter, Brea, CA, USA) ratio was used for purification following adapter ligation to ensure the retention of small DNA fragments. Library PCR was performed using KAPA HiFi HotStart Uracil+ Ready Mix and 20 µM Illumina Primer mix (Roche Sequencing). The Uracil+ Ready Mix contains a uracil-tolerant polymerase that allows for the amplification of DNA with cytosine deamination, as characteristic of ancient and historical DNA . After PCR amplification, a 5X QIAGEN MinElute (QIAGEN) purification was performed with elution in 25 µL Tris-EDTA. Library quality was evaluated using the 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA) before proceeding with hybridization capture. Additionally, a library input reduction test was performed to assess whether using the maximum 1 µg dsDNA input for library preparation resulted in inhibition and thus poor-quality libraries. Three samples from different historical contexts with initial library inputs of a maximum of 1 µg were diluted to 500 ng and 200 ng to evaluate reduced library input. Library preparation was performed on the 500 ng and 200 ng samples using the same procedures described previously.
Purified libraries were enriched via hybridization capture using myBaits v5 kits (Arbor Biosciences, Ann Arbor, MI, USA). Sample libraries were captured with two bait sets unless indicated ( ). The forensically oriented FORCE SNP panel β version contains ~20,000 unique baits targeting 5402 SNPs including identity, ancestry, phenotype, X- and Y-chromosomal SNPs. The β version excludes 44 tri-allelic, clinically relevant, uninformative and/or underperforming SNPs identified in . The second bait set was a custom panel that covers the human mitogenome with 1800 unique oligos . A maximum of 7 µL of the library was used for each capture reaction (FORCE and mitogenome). The capture product was amplified and purified according to , except the 20 µM Illumina Primer mix was used. Samples were eluted in 20 µL Tris-EDTA. All captured libraries were run on a 2100 Bioanalyzer using the 7500 assay. Any libraries with total DNA concentrations over 50 ng/µL were diluted and re-run using the 7500 assay for more accurate quantification.
FORCE and mitogenome hybridization capture samples were primarily pooled by normalization, with a 50 nM target for the 170–1000 bp range. The mitogenome capture samples for the Basilica and Arch Street were pooled by volume instead of normalizing. Mitogenome sequencing pools contained 21–27 samples, while FORCE pools consisted of 11–16 samples. Positive controls were not sequenced and were used only to monitor library preparation and capture success.
All pools were quantified on the 2100 Bioanalyzer using the DNA 7500 kit and diluted to 4 nM prior to loading. The 4 nM pools were denatured and diluted to 1.0 pM with 2.5% Illumina PhiX Control V3 (PhiX) (Illumina) or 1.2 pM with 5% PhiX. All runs were performed on a NextSeq 550 (Illumina) with Mid output v 2.5 kits. As general sample qualities were known, pools containing only highly degraded samples were sequenced with 75 × 75 paired-end cycles. For samples with moderately degraded DNA, 150 × 150 paired-end sequencing was used.
Following demultiplexing, FASTQ files were imported into the CLC Genomics Workbench v12.0.1 (QIAGEN) for analysis. FORCE analysis was completed with a custom workflow as described in with two modifications: (1) reads less than 30 bp were discarded to minimize the mapping of non-specific reads and (2) a custom de-duplication tool that mimics the functionality of markdup from SAMTools v1.61.1 ( http://www.htslib.org/doc/samtools-markdup.html ) (accessed on 14 February 2023) was used to more effectively remove PCR duplicates. Genotypes were called using the Known Mutations from Mapping tool, and a custom Microsoft Excel (Redmond, WA, USA) template as described in was used to apply additional analysis parameters to called SNPs and simplify the output. Calling of target SNPs required a minimum read count (coverage) of 10X. In addition to the minimum coverage requirement, an allele frequency of 90% or greater was necessary for homozygous genotypes. Heterozygous genotypes needed a minor allele frequency (MAF) of at least 30% to be called. Imbalanced genotypes (10–30% MAF) were excluded from analysis (not called), as heterozygosity could not be confidently determined. SNP coverage at 1X and 5X was also evaluated. Mitogenome analysis was also completed in the CLC Genomics Workbench v12.0.1 with the AFDIL-QIAGEN mtDNA Expert (AQME) plug-in and a custom workflow, as described in . Reads were mapped to the revised Cambridge Reference Sequence (rCRS) using the same stringent mapping parameters incorporated into the custom FORCE workflow. The custom de-duplication tool with local realignment was implemented to remove PCR duplicates and assist in indel alignment. A 10X coverage threshold, with a variant frequency ≥10% and a minimum variant count of four, was required for variant calling.
To assess the suitability of DNA quantification to predict PCR enrichment success, short tandem repeat (STR) typing was performed. The PowerPlex Fusion kit (Promega) (PPF) amplifies 22 forensic auSTRs, one Y-STR, and one non-STR sex-determination locus (amelogenin). Samples, along with a 2800M positive control and negative control (sterile water), were amplified according to the manufacturer’s recommendations. The maximum sample volume of 15 µL was used when the extract volume allowed, unless indicated ( ). The Applied Biosystems’ AmpFLSTR Yfiler kit amplifies 17 forensic Y-chromosomal STRs (Y-STRs) for male identification. A modified Yfiler approach optimized for degraded, low copy number samples (LCNY) was developed at the AFMES-AFDIL . This LCNY protocol, which includes increased polymerase and cycles, was used to generate all Y-STR data. The maximum sample volume of 9.2 µL was utilized when extract the volume allowed, unless otherwise indicated ( ). If inhibition was suspected, a range of input volumes was amplified in a repeat amplification event to improve the number of loci obtained. The amplified product was prepared for capillary electrophoresis according to the manufacturer’s manual for each kit and loaded on a 3500xL Genetic Analyzer with POP-4 polymer (Applied Biosystems, Foster City, CA, USA). Samples were injected for 15 s (PPF) or 7 s (LCNY) using default injection parameters for each kit. STR data were analyzed using GeneMapper ID-X v1.4 (Applied Biosystems). Locus-specific stutter filters were determined by internal validation at the AFMES-AFDIL. For PPF, the reporting threshold was 70 relative fluorescent units (RFU) for heterozygous alleles and the Y-STR locus, and 550 RFU for homozygous alleles at the auSTR loci. For LCNY, peaks over 70 RFU were considered reportable.
3.1. DNA Quantification All 32 samples were quantified with Pro and PowerQuant; a total of 20 samples were quantified with Trio ( ). All human samples produced amplifiable DNA using the small autosomal target of each kit tested, except for one sample with PowerQuant and a different sample with Trio. These two samples were both from the WWII Punchbowl context and likely underwent chemical treatment known to cause inhibitory DNA-protein crosslinks . All human samples produced amplifiable DNA with the small target of Pro. At least one-third of samples did not produce amplifiable DNA (undetermined result) for the large autosomal target of each kit; therefore, the degradation index was incalculable. As a result, the small autosomal target was the focus of the comparisons and analyses presented in this study. A 1:1 relationship was observed between kits when DNA concentrations were compared in a pairwise fashion (Pro vs. PowerQuant, Pro vs. Trio, PowerQuant vs. Trio) ( and ), with R 2 values greater than 97%. Thus, the DNA concentrations obtained from the three qPCR kits were concordant. Nine of the 11 extracts with values less than 5 pg/µL with Pro resulted in significantly higher values with Trio ( p = 0.02; two-tailed, paired t -test). These nine samples were known to be highly degraded, and thus the smaller target size (80 bp for Trio compared to 91 bp for Pro) could explain this discrepancy. The PowerQuant system resulted in slightly higher quantification results for 71% of samples compared to Pro, perhaps due to the smaller target for PowerQuant relative to Pro; however, the difference was not significant ( p = 0.11; two-tailed, paired t -test). Quantification results from the small autosomal targets were compared to the approximate years postmortem to determine if age alone was predictive of DNA extract concentration. No direct correlation was observed ( ). As quantification results across the three kits were consistent, results from downstream processing will be presented primarily in reference to Pro since more bone sample data points were available for this kit. In addition to the three qPCR systems tested at the AFMES-AFDIL, one set of samples (Basilica) was quantified with the SD quants tetraplex at the Medical University of Innsbruck. Two extracts resulted in no quantification value with the SD quants system but did return a concentration with Pro ( ). Excluding these two outliers, the R 2 value improved from 0.435 to 0.974 ( and a). The DNA concentrations produced from the autosomal target of the SD quants tetraplex were approximately four times higher than Pro ( ). This may be due in part to the smaller target of the SD quants system (70 bp for SD quant and 91 bp for Pro), resulting in more amplifiable DNA from these degraded sample extracts. The mtDNA portion of the SD quants qPCR assay included both a small (69 bp) and a large (143 bp) target; however, only the small target was considered here due to the degraded nature of the ancient Basilica samples tested. While the R 2 between the small mtDNA target and Pro was only moderate (73%), the trend indicates approximately 1000 mtGE for each 5 pg/µL human autosomal DNA ( b). This ratio of mtDNA to nuclear DNA seems plausible and has been previously observed in a different context, (e.g., ). Quantification results for the small Y-DNA targets were compared across the three qPCR kits in a similar manner as the small autosomal targets. Trio and PowerQuant were both compared directly to Pro and to one another ( ), resulting in R 2 values of 0.95 and 0.92, respectively. Trio and PowerQuant both produced undetermined results for samples that generated a positive Y-DNA quantification result in Pro ( ). All RBs and the non-human bone sample failed to result in any amplifiable human DNA with the three qPCR kits tested. As the non-human sample was included to test the specificity of the qPCR assays, it will not be discussed further. include testing details and results. 3.2. NGS 3.2.1. FORCE Capture After library preparation, samples were run on the 2100 Bioanalyzer as a quality control check. The resulting library molarities were compared to the total dsDNA input ( ). One outlier, HC-1B, was identified with nearly maximum total DNA input (998 ng) but no visible library product. This sample and two others with maximum DNA input were subsequently processed with reduced DNA input (500 ng and 200 ng) to test for inhibition or DNA overload. These reduced-input libraries were then captured with the FORCE panel and sequenced; the data were then compared with the 1 µg (maximum input) results. For HC-1B, the 200 ng input library generated the highest library molarity (570 nM compared to 115 nM for 500 ng input and 32 nM for 1 µg input in the 170–1000 bp range). Therefore, in the HC-1B extract, an inhibitor was likely present that, when diluted, resulted in successful library preparation and 1X coverage of target SNP regions ( ). There was no indication of inhibition in HC-1B from any of the three qPCR kits, as the IPCs were within the expected ranges for all three. The typical forensically oriented qPCR and STR kits have buffers specifically designed to overcome inhibition common to forensic samples, such as humic acid, that can be co-extracted with DNA. However, other downstream assays with different chemistries, such as library preparation, may be negatively impacted by these common inhibitors. The other two samples included in the library input reduction test did not show indications of inhibition or overloaded library preparation ( ). Sample AS-9 behaved as expected, with reduced inputs resulting in lower SNP coverage. The average coverage for sample GE-1 was consistently low for all inputs and could possibly be explained by minimal human DNA input and off-target mapping of non-human DNA. Based on these findings, reduced volume input was used for HC-1B in all downstream tests. For the remaining 30 human sample extracts, the maximum input volume was utilized in all library preparation and PCR amplification events. Data quality metrics from all samples are presented in . The mean mappable fragment size was sample dependent and ranged between 55 and 155 bp. The proportion of reads mapped to the human genome also varied, from <1% to 85%. Thus, these 31 samples exhibit poor DNA quality which is expected from ancient and historical remains. As stated previously, it is important to note that each of the 31 samples produced amplifiable human autosomal DNA, albeit in very low quantities, despite the small mappable fragment size. SNP recovery was then assessed for all samples. To identify the best predictor of SNP recovery across both male and female samples, X- and Y-SNPs were ignored resulting in a maximum of 4342 autosomal SNPs (auSNPs) for analysis. The percentage of auSNPs called was plotted against the following: total dsDNA input from Qubit quantification, human DNA input based on the qPCR small autosomal target, and the hDNA ratio comparing the human DNA quantity (in pg) to total dsDNA quantity (in ng) as explained in the methods. Total dsDNA input was not a useful predictor, as these samples contain varying levels of exogenous DNA ( ). The human DNA (qPCR-based) input ( a) was the best predictor of SNP recovery. Samples with approximately 100 pg or higher human DNA input typically resulted in ≥80% called SNPs, while samples below 100 pg generally resulted in <40% called SNPs ( a). In terms of the hDNA ratio ( b), samples ≥ 0.7 resulted in >80% called SNPs, and those with hDNA ratios of <0.05 failed. However, ratios between these two values were less predictive of SNP recovery. Of note, two samples fell out of trend in terms of human DNA (qPCR-based) input. One sample with 114 pg input underperformed and a sample with 10 pg input overperformed. There is no clear explanation for the underperformance of the 114 pg input sample. Perhaps the root cause could be brought to light through replicate testing. The 10 pg sample had a mean paired distance of 80.8 bp, smaller than the small autosomal qPCR targets tested. Thus, the human DNA present in the 10 pg sample was likely too fragmented to be accurately quantified with the qPCR assays, yet sufficient fragmented human DNA was present to successfully sequence and cover the target SNPs. SNP recovery at 1X and 5X was used in an exploratory capacity to assess samples with less than 75% of the auSNPs called at a 10X level. Since a larger SNP panel was not tested here, this was conducted to mirror the lower thresholds that can be used with probabilistic approaches for SNP analysis, such as a genotype likelihood approach . For samples with at least 15 pg human DNA input (qPCR-based), >80% of SNPs were covered at a 1X threshold ( a). There were three samples that resulted in higher SNP recoveries than would have been expected at this threshold ( a, PB-1, HC-3, and FB-8). All three of these samples had total dsDNA inputs (Qubit-based) between 100 and 200 ng with mean paired distances between 60 and 80 bp. In these instances, the samples were fragmented beyond what could be detected with qPCR, but total dsDNA input was not as high as other samples that were found to contain high levels of exogenous DNA. For example, the other two samples with 2 ng input based on qPCR had total dsDNA inputs of 750–850 ng, indicating high levels of exogenous DNA. At human DNA inputs below 100 pg, samples may perform better than predicted by qPCR-based input alone if they have a relatively low exogenous DNA background and may be better characterized by considering both total dsDNA and human (qPCR-based) input. At the 5X threshold, aside from the 10 pg input outlier, samples with 57 pg and higher resulted in at least 40% of SNPs covered ( a). Conversely, samples with less than 57 pg input (all ≤ 15 pg) resulted in less than 20% of SNPs covered at the 5X threshold. There were no samples that had inputs between 57 pg and 15 pg in this dataset to evaluate 5X SNP recovery within this range. When assessing SNP recovery relative to the hDNA ratio ( b) there was a clearer trend than observed when considering only human DNA quantity, particularly for those samples discussed previously ( , PB-1, HC-3, and FB-8). Samples with an hDNA ratio ≥ 0.08 resulted in >40% SNP recovery at 5X and those < 0.08 resulted in <20% SNPs recovered. The exception was one sample with a ratio of 0.39 that produced only six SNPs covered at 5X. This sample had a very low DNA input (3 pg based on qPCR). Thus, the hDNA ratio may best be used as a measure of DNA quality for samples with 5–100 pg human DNA (qPCR-based) input. It is worth noting that the FORCE panel is relatively small with only 4342 auSNPs (5402 total nuclear SNPs), and >1000 called SNPs at a 10X threshold with sufficient intra-locus balance are needed for accurate and statistically supported kinship comparisons between case and reference samples . Those samples shown here with less than 20% of SNPs recovered (at a 10X calling threshold) would likely not result in sufficient SNPs for accurate predictions. As noted previously, samples with less than 100 pg of target input DNA may be better suited for whole genome enrichment and/or large SNP panels, such as the 95K panel , in order to obtain enough SNPs at 1X that they can be used for probabilistic kinship predictions. No controls resulted in usable data and were free of any detectable contamination. 3.2.2. Mitogenome Capture The results for the SD quants small mtDNA target were evaluated for a subset of Basilica samples with mitogenome hybridization capture data. The average coverage was plotted against mtDNA input based on the qPCR small mtDNA target ( a) and hDNA ratio ( b). For this analysis, the hDNA ratio was based on the qPCR small mtDNA target instead of the small autosomal target. The mtDNA input was positively correlated with the average coverage, but sample FB-5 fell slightly out of trend ( a). FB-5 had the highest mtDNA degradation index (69 bp mtDNA qPCR target/143 bp mtDNA qPCR target), indicating a greater degree of fragmentation than the other Basilica samples. FB-5 also had a greater amount of total dsDNA compared to FB-6, which suggests high exogenous DNA background. This was taken into account with the hDNA ratio ( b), and therefore, served as a slightly better predictor of average coverage of the mitogenome than mtDNA qPCR alone. Many laboratories, including the AFMES-AFDIL, do not have access to a mtDNA quantification system. Therefore, mitogenome capture results were also assessed using total dsDNA from Qubit and nuclear DNA qPCR kits that are more widely implemented. The average coverage was plotted against total dsDNA input from Qubit, human DNA input based on the qPCR small human autosomal target, and the hDNA ratio based on the qPCR small autosomal target. This was conducted for the full bone sample dataset since all samples were quantified with autosomal qPCR. Consistent with the SNP data shown in , total dsDNA from Qubit quantification ( ) was not a suitable predictor of average coverage of the mitogenome, due to varying levels of exogenous DNA background. DNA input by qPCR ( a) and the autosomal hDNA ratio ( b) were both strong predictors of average mitogenome coverage. In a, the majority of samples with ≥7 pg input resulted in average coverages above 500X; in b, this was achieved for the majority of samples with hDNA ratios ≥0.01. shows sample HC-1B having slightly lower average coverage than expected. This could be due to the sample inhibition identified in the library input reduction test. Sample PB-1 also fell out of trend in a with an average coverage of 1501X. This sample’s average coverage was better explained by the hDNA ratio ( b). PB-1 was one of the two samples originating from the WWII Punchbowl context in which burials were subjected to formalin preservation. The chemical treatment of PB-1 may explain this sample’s unique signature: higher than the expected 1X auSNP coverage and higher mitogenome average coverage when compared to historical samples with similar human DNA input. It is notable that the other Punchbowl sample, PB-2, was better explained by human DNA input ( a) than the hDNA ratio ( b). Across the entire dataset, the highest average coverage of the mitogenome observed was 11,515X, while the lowest was 95X—well above the 10X average coverage metric required by AFMES-AFDIL for data reporting. All samples except FB-1 generated full mitogenome coverage (16,569 bp) at a 10X threshold for the reported sequence range ( a). FB-1 had a 69 bp gap in the reported sequence range due to lower coverage (6–9X) observed in the polycytosine stretch in hypervariable region 1. Homopolymer regions often have lower coverage due to mapping difficulties , especially with very short fragments. All samples were single sourced with no indication of a mixture. No controls resulted in usable data and were free of any detectable contamination. 3.3. STR Typing Nineteen samples underwent auSTR typing, of which 14 resulted in at least one reportable locus. The percentage of auSTR loci recovered was plotted against human DNA input from the qPCR small autosomal target and the hDNA ratio based on the qPCR small autosomal target. DNA input by qPCR ( a) was the best predictor of STR recovery, as samples with ≥30 pg input resulted in >40% recovery of auSTR loci. Below this threshold, a maximum of one locus was recovered. The hDNA ratio ( b) was less predictive, which suggests that the non-human DNA background did not impact the percentage of STR loci recovered. It is worth noting that the 550 RFU homozygote stochastic reporting threshold validated at the AFMES-AFDIL for PowerPlex Fusion data may have a more noticeable impact on auSTR locus reportability for degraded samples, particularly those with many homozygous loci. Of the 14 samples that were tested using the LCNY protocol, including one suspected female, eight samples resulted in at least one reportable Y-STR locus. The small autosomal and Y-DNA targets were both strong predictors of the percentage of Y-STR loci recovered ( a,b), but a clearer trend was observed in the prediction from Y-qPCR. The Y-DNA target was also smaller than the autosomal target, benefiting more degraded samples. Greater than 58% of the Y-STR loci were recovered in samples with ≥19 pg autosomal DNA ( a) and ≥24 pg Y-DNA ( b). Below these input values, only one sample had a single locus recovered. As expected for the suspected female sample, zero Y-STR loci were recovered. The hDNA ratio by the small autosomal target ( c) and Y-DNA target ( d) were poorly correlated with Y-STR recovery. Additional LCNY amplifications were performed for sample HC-1B with reduced template volumes (1 µL and 5 µL) due to the inhibition observed during NGS processing. The 1 µL and 5 µL reduced template volumes resulted in Y-STR recoveries of 23.53% and 29.41%, respectively. As stated previously, the effects of inhibition were not observed in the IPCs for any of the qPCR assays. The LCNY protocol uses YFiler kit reagents, which is an older generation kit, and therefore, it may be less tolerant to inhibitors than current generation qPCR kits like those tested here. No controls resulted in usable data and were free of any detectable contamination
All 32 samples were quantified with Pro and PowerQuant; a total of 20 samples were quantified with Trio ( ). All human samples produced amplifiable DNA using the small autosomal target of each kit tested, except for one sample with PowerQuant and a different sample with Trio. These two samples were both from the WWII Punchbowl context and likely underwent chemical treatment known to cause inhibitory DNA-protein crosslinks . All human samples produced amplifiable DNA with the small target of Pro. At least one-third of samples did not produce amplifiable DNA (undetermined result) for the large autosomal target of each kit; therefore, the degradation index was incalculable. As a result, the small autosomal target was the focus of the comparisons and analyses presented in this study. A 1:1 relationship was observed between kits when DNA concentrations were compared in a pairwise fashion (Pro vs. PowerQuant, Pro vs. Trio, PowerQuant vs. Trio) ( and ), with R 2 values greater than 97%. Thus, the DNA concentrations obtained from the three qPCR kits were concordant. Nine of the 11 extracts with values less than 5 pg/µL with Pro resulted in significantly higher values with Trio ( p = 0.02; two-tailed, paired t -test). These nine samples were known to be highly degraded, and thus the smaller target size (80 bp for Trio compared to 91 bp for Pro) could explain this discrepancy. The PowerQuant system resulted in slightly higher quantification results for 71% of samples compared to Pro, perhaps due to the smaller target for PowerQuant relative to Pro; however, the difference was not significant ( p = 0.11; two-tailed, paired t -test). Quantification results from the small autosomal targets were compared to the approximate years postmortem to determine if age alone was predictive of DNA extract concentration. No direct correlation was observed ( ). As quantification results across the three kits were consistent, results from downstream processing will be presented primarily in reference to Pro since more bone sample data points were available for this kit. In addition to the three qPCR systems tested at the AFMES-AFDIL, one set of samples (Basilica) was quantified with the SD quants tetraplex at the Medical University of Innsbruck. Two extracts resulted in no quantification value with the SD quants system but did return a concentration with Pro ( ). Excluding these two outliers, the R 2 value improved from 0.435 to 0.974 ( and a). The DNA concentrations produced from the autosomal target of the SD quants tetraplex were approximately four times higher than Pro ( ). This may be due in part to the smaller target of the SD quants system (70 bp for SD quant and 91 bp for Pro), resulting in more amplifiable DNA from these degraded sample extracts. The mtDNA portion of the SD quants qPCR assay included both a small (69 bp) and a large (143 bp) target; however, only the small target was considered here due to the degraded nature of the ancient Basilica samples tested. While the R 2 between the small mtDNA target and Pro was only moderate (73%), the trend indicates approximately 1000 mtGE for each 5 pg/µL human autosomal DNA ( b). This ratio of mtDNA to nuclear DNA seems plausible and has been previously observed in a different context, (e.g., ). Quantification results for the small Y-DNA targets were compared across the three qPCR kits in a similar manner as the small autosomal targets. Trio and PowerQuant were both compared directly to Pro and to one another ( ), resulting in R 2 values of 0.95 and 0.92, respectively. Trio and PowerQuant both produced undetermined results for samples that generated a positive Y-DNA quantification result in Pro ( ). All RBs and the non-human bone sample failed to result in any amplifiable human DNA with the three qPCR kits tested. As the non-human sample was included to test the specificity of the qPCR assays, it will not be discussed further. include testing details and results.
3.2.1. FORCE Capture After library preparation, samples were run on the 2100 Bioanalyzer as a quality control check. The resulting library molarities were compared to the total dsDNA input ( ). One outlier, HC-1B, was identified with nearly maximum total DNA input (998 ng) but no visible library product. This sample and two others with maximum DNA input were subsequently processed with reduced DNA input (500 ng and 200 ng) to test for inhibition or DNA overload. These reduced-input libraries were then captured with the FORCE panel and sequenced; the data were then compared with the 1 µg (maximum input) results. For HC-1B, the 200 ng input library generated the highest library molarity (570 nM compared to 115 nM for 500 ng input and 32 nM for 1 µg input in the 170–1000 bp range). Therefore, in the HC-1B extract, an inhibitor was likely present that, when diluted, resulted in successful library preparation and 1X coverage of target SNP regions ( ). There was no indication of inhibition in HC-1B from any of the three qPCR kits, as the IPCs were within the expected ranges for all three. The typical forensically oriented qPCR and STR kits have buffers specifically designed to overcome inhibition common to forensic samples, such as humic acid, that can be co-extracted with DNA. However, other downstream assays with different chemistries, such as library preparation, may be negatively impacted by these common inhibitors. The other two samples included in the library input reduction test did not show indications of inhibition or overloaded library preparation ( ). Sample AS-9 behaved as expected, with reduced inputs resulting in lower SNP coverage. The average coverage for sample GE-1 was consistently low for all inputs and could possibly be explained by minimal human DNA input and off-target mapping of non-human DNA. Based on these findings, reduced volume input was used for HC-1B in all downstream tests. For the remaining 30 human sample extracts, the maximum input volume was utilized in all library preparation and PCR amplification events. Data quality metrics from all samples are presented in . The mean mappable fragment size was sample dependent and ranged between 55 and 155 bp. The proportion of reads mapped to the human genome also varied, from <1% to 85%. Thus, these 31 samples exhibit poor DNA quality which is expected from ancient and historical remains. As stated previously, it is important to note that each of the 31 samples produced amplifiable human autosomal DNA, albeit in very low quantities, despite the small mappable fragment size. SNP recovery was then assessed for all samples. To identify the best predictor of SNP recovery across both male and female samples, X- and Y-SNPs were ignored resulting in a maximum of 4342 autosomal SNPs (auSNPs) for analysis. The percentage of auSNPs called was plotted against the following: total dsDNA input from Qubit quantification, human DNA input based on the qPCR small autosomal target, and the hDNA ratio comparing the human DNA quantity (in pg) to total dsDNA quantity (in ng) as explained in the methods. Total dsDNA input was not a useful predictor, as these samples contain varying levels of exogenous DNA ( ). The human DNA (qPCR-based) input ( a) was the best predictor of SNP recovery. Samples with approximately 100 pg or higher human DNA input typically resulted in ≥80% called SNPs, while samples below 100 pg generally resulted in <40% called SNPs ( a). In terms of the hDNA ratio ( b), samples ≥ 0.7 resulted in >80% called SNPs, and those with hDNA ratios of <0.05 failed. However, ratios between these two values were less predictive of SNP recovery. Of note, two samples fell out of trend in terms of human DNA (qPCR-based) input. One sample with 114 pg input underperformed and a sample with 10 pg input overperformed. There is no clear explanation for the underperformance of the 114 pg input sample. Perhaps the root cause could be brought to light through replicate testing. The 10 pg sample had a mean paired distance of 80.8 bp, smaller than the small autosomal qPCR targets tested. Thus, the human DNA present in the 10 pg sample was likely too fragmented to be accurately quantified with the qPCR assays, yet sufficient fragmented human DNA was present to successfully sequence and cover the target SNPs. SNP recovery at 1X and 5X was used in an exploratory capacity to assess samples with less than 75% of the auSNPs called at a 10X level. Since a larger SNP panel was not tested here, this was conducted to mirror the lower thresholds that can be used with probabilistic approaches for SNP analysis, such as a genotype likelihood approach . For samples with at least 15 pg human DNA input (qPCR-based), >80% of SNPs were covered at a 1X threshold ( a). There were three samples that resulted in higher SNP recoveries than would have been expected at this threshold ( a, PB-1, HC-3, and FB-8). All three of these samples had total dsDNA inputs (Qubit-based) between 100 and 200 ng with mean paired distances between 60 and 80 bp. In these instances, the samples were fragmented beyond what could be detected with qPCR, but total dsDNA input was not as high as other samples that were found to contain high levels of exogenous DNA. For example, the other two samples with 2 ng input based on qPCR had total dsDNA inputs of 750–850 ng, indicating high levels of exogenous DNA. At human DNA inputs below 100 pg, samples may perform better than predicted by qPCR-based input alone if they have a relatively low exogenous DNA background and may be better characterized by considering both total dsDNA and human (qPCR-based) input. At the 5X threshold, aside from the 10 pg input outlier, samples with 57 pg and higher resulted in at least 40% of SNPs covered ( a). Conversely, samples with less than 57 pg input (all ≤ 15 pg) resulted in less than 20% of SNPs covered at the 5X threshold. There were no samples that had inputs between 57 pg and 15 pg in this dataset to evaluate 5X SNP recovery within this range. When assessing SNP recovery relative to the hDNA ratio ( b) there was a clearer trend than observed when considering only human DNA quantity, particularly for those samples discussed previously ( , PB-1, HC-3, and FB-8). Samples with an hDNA ratio ≥ 0.08 resulted in >40% SNP recovery at 5X and those < 0.08 resulted in <20% SNPs recovered. The exception was one sample with a ratio of 0.39 that produced only six SNPs covered at 5X. This sample had a very low DNA input (3 pg based on qPCR). Thus, the hDNA ratio may best be used as a measure of DNA quality for samples with 5–100 pg human DNA (qPCR-based) input. It is worth noting that the FORCE panel is relatively small with only 4342 auSNPs (5402 total nuclear SNPs), and >1000 called SNPs at a 10X threshold with sufficient intra-locus balance are needed for accurate and statistically supported kinship comparisons between case and reference samples . Those samples shown here with less than 20% of SNPs recovered (at a 10X calling threshold) would likely not result in sufficient SNPs for accurate predictions. As noted previously, samples with less than 100 pg of target input DNA may be better suited for whole genome enrichment and/or large SNP panels, such as the 95K panel , in order to obtain enough SNPs at 1X that they can be used for probabilistic kinship predictions. No controls resulted in usable data and were free of any detectable contamination. 3.2.2. Mitogenome Capture The results for the SD quants small mtDNA target were evaluated for a subset of Basilica samples with mitogenome hybridization capture data. The average coverage was plotted against mtDNA input based on the qPCR small mtDNA target ( a) and hDNA ratio ( b). For this analysis, the hDNA ratio was based on the qPCR small mtDNA target instead of the small autosomal target. The mtDNA input was positively correlated with the average coverage, but sample FB-5 fell slightly out of trend ( a). FB-5 had the highest mtDNA degradation index (69 bp mtDNA qPCR target/143 bp mtDNA qPCR target), indicating a greater degree of fragmentation than the other Basilica samples. FB-5 also had a greater amount of total dsDNA compared to FB-6, which suggests high exogenous DNA background. This was taken into account with the hDNA ratio ( b), and therefore, served as a slightly better predictor of average coverage of the mitogenome than mtDNA qPCR alone. Many laboratories, including the AFMES-AFDIL, do not have access to a mtDNA quantification system. Therefore, mitogenome capture results were also assessed using total dsDNA from Qubit and nuclear DNA qPCR kits that are more widely implemented. The average coverage was plotted against total dsDNA input from Qubit, human DNA input based on the qPCR small human autosomal target, and the hDNA ratio based on the qPCR small autosomal target. This was conducted for the full bone sample dataset since all samples were quantified with autosomal qPCR. Consistent with the SNP data shown in , total dsDNA from Qubit quantification ( ) was not a suitable predictor of average coverage of the mitogenome, due to varying levels of exogenous DNA background. DNA input by qPCR ( a) and the autosomal hDNA ratio ( b) were both strong predictors of average mitogenome coverage. In a, the majority of samples with ≥7 pg input resulted in average coverages above 500X; in b, this was achieved for the majority of samples with hDNA ratios ≥0.01. shows sample HC-1B having slightly lower average coverage than expected. This could be due to the sample inhibition identified in the library input reduction test. Sample PB-1 also fell out of trend in a with an average coverage of 1501X. This sample’s average coverage was better explained by the hDNA ratio ( b). PB-1 was one of the two samples originating from the WWII Punchbowl context in which burials were subjected to formalin preservation. The chemical treatment of PB-1 may explain this sample’s unique signature: higher than the expected 1X auSNP coverage and higher mitogenome average coverage when compared to historical samples with similar human DNA input. It is notable that the other Punchbowl sample, PB-2, was better explained by human DNA input ( a) than the hDNA ratio ( b). Across the entire dataset, the highest average coverage of the mitogenome observed was 11,515X, while the lowest was 95X—well above the 10X average coverage metric required by AFMES-AFDIL for data reporting. All samples except FB-1 generated full mitogenome coverage (16,569 bp) at a 10X threshold for the reported sequence range ( a). FB-1 had a 69 bp gap in the reported sequence range due to lower coverage (6–9X) observed in the polycytosine stretch in hypervariable region 1. Homopolymer regions often have lower coverage due to mapping difficulties , especially with very short fragments. All samples were single sourced with no indication of a mixture. No controls resulted in usable data and were free of any detectable contamination.
After library preparation, samples were run on the 2100 Bioanalyzer as a quality control check. The resulting library molarities were compared to the total dsDNA input ( ). One outlier, HC-1B, was identified with nearly maximum total DNA input (998 ng) but no visible library product. This sample and two others with maximum DNA input were subsequently processed with reduced DNA input (500 ng and 200 ng) to test for inhibition or DNA overload. These reduced-input libraries were then captured with the FORCE panel and sequenced; the data were then compared with the 1 µg (maximum input) results. For HC-1B, the 200 ng input library generated the highest library molarity (570 nM compared to 115 nM for 500 ng input and 32 nM for 1 µg input in the 170–1000 bp range). Therefore, in the HC-1B extract, an inhibitor was likely present that, when diluted, resulted in successful library preparation and 1X coverage of target SNP regions ( ). There was no indication of inhibition in HC-1B from any of the three qPCR kits, as the IPCs were within the expected ranges for all three. The typical forensically oriented qPCR and STR kits have buffers specifically designed to overcome inhibition common to forensic samples, such as humic acid, that can be co-extracted with DNA. However, other downstream assays with different chemistries, such as library preparation, may be negatively impacted by these common inhibitors. The other two samples included in the library input reduction test did not show indications of inhibition or overloaded library preparation ( ). Sample AS-9 behaved as expected, with reduced inputs resulting in lower SNP coverage. The average coverage for sample GE-1 was consistently low for all inputs and could possibly be explained by minimal human DNA input and off-target mapping of non-human DNA. Based on these findings, reduced volume input was used for HC-1B in all downstream tests. For the remaining 30 human sample extracts, the maximum input volume was utilized in all library preparation and PCR amplification events. Data quality metrics from all samples are presented in . The mean mappable fragment size was sample dependent and ranged between 55 and 155 bp. The proportion of reads mapped to the human genome also varied, from <1% to 85%. Thus, these 31 samples exhibit poor DNA quality which is expected from ancient and historical remains. As stated previously, it is important to note that each of the 31 samples produced amplifiable human autosomal DNA, albeit in very low quantities, despite the small mappable fragment size. SNP recovery was then assessed for all samples. To identify the best predictor of SNP recovery across both male and female samples, X- and Y-SNPs were ignored resulting in a maximum of 4342 autosomal SNPs (auSNPs) for analysis. The percentage of auSNPs called was plotted against the following: total dsDNA input from Qubit quantification, human DNA input based on the qPCR small autosomal target, and the hDNA ratio comparing the human DNA quantity (in pg) to total dsDNA quantity (in ng) as explained in the methods. Total dsDNA input was not a useful predictor, as these samples contain varying levels of exogenous DNA ( ). The human DNA (qPCR-based) input ( a) was the best predictor of SNP recovery. Samples with approximately 100 pg or higher human DNA input typically resulted in ≥80% called SNPs, while samples below 100 pg generally resulted in <40% called SNPs ( a). In terms of the hDNA ratio ( b), samples ≥ 0.7 resulted in >80% called SNPs, and those with hDNA ratios of <0.05 failed. However, ratios between these two values were less predictive of SNP recovery. Of note, two samples fell out of trend in terms of human DNA (qPCR-based) input. One sample with 114 pg input underperformed and a sample with 10 pg input overperformed. There is no clear explanation for the underperformance of the 114 pg input sample. Perhaps the root cause could be brought to light through replicate testing. The 10 pg sample had a mean paired distance of 80.8 bp, smaller than the small autosomal qPCR targets tested. Thus, the human DNA present in the 10 pg sample was likely too fragmented to be accurately quantified with the qPCR assays, yet sufficient fragmented human DNA was present to successfully sequence and cover the target SNPs. SNP recovery at 1X and 5X was used in an exploratory capacity to assess samples with less than 75% of the auSNPs called at a 10X level. Since a larger SNP panel was not tested here, this was conducted to mirror the lower thresholds that can be used with probabilistic approaches for SNP analysis, such as a genotype likelihood approach . For samples with at least 15 pg human DNA input (qPCR-based), >80% of SNPs were covered at a 1X threshold ( a). There were three samples that resulted in higher SNP recoveries than would have been expected at this threshold ( a, PB-1, HC-3, and FB-8). All three of these samples had total dsDNA inputs (Qubit-based) between 100 and 200 ng with mean paired distances between 60 and 80 bp. In these instances, the samples were fragmented beyond what could be detected with qPCR, but total dsDNA input was not as high as other samples that were found to contain high levels of exogenous DNA. For example, the other two samples with 2 ng input based on qPCR had total dsDNA inputs of 750–850 ng, indicating high levels of exogenous DNA. At human DNA inputs below 100 pg, samples may perform better than predicted by qPCR-based input alone if they have a relatively low exogenous DNA background and may be better characterized by considering both total dsDNA and human (qPCR-based) input. At the 5X threshold, aside from the 10 pg input outlier, samples with 57 pg and higher resulted in at least 40% of SNPs covered ( a). Conversely, samples with less than 57 pg input (all ≤ 15 pg) resulted in less than 20% of SNPs covered at the 5X threshold. There were no samples that had inputs between 57 pg and 15 pg in this dataset to evaluate 5X SNP recovery within this range. When assessing SNP recovery relative to the hDNA ratio ( b) there was a clearer trend than observed when considering only human DNA quantity, particularly for those samples discussed previously ( , PB-1, HC-3, and FB-8). Samples with an hDNA ratio ≥ 0.08 resulted in >40% SNP recovery at 5X and those < 0.08 resulted in <20% SNPs recovered. The exception was one sample with a ratio of 0.39 that produced only six SNPs covered at 5X. This sample had a very low DNA input (3 pg based on qPCR). Thus, the hDNA ratio may best be used as a measure of DNA quality for samples with 5–100 pg human DNA (qPCR-based) input. It is worth noting that the FORCE panel is relatively small with only 4342 auSNPs (5402 total nuclear SNPs), and >1000 called SNPs at a 10X threshold with sufficient intra-locus balance are needed for accurate and statistically supported kinship comparisons between case and reference samples . Those samples shown here with less than 20% of SNPs recovered (at a 10X calling threshold) would likely not result in sufficient SNPs for accurate predictions. As noted previously, samples with less than 100 pg of target input DNA may be better suited for whole genome enrichment and/or large SNP panels, such as the 95K panel , in order to obtain enough SNPs at 1X that they can be used for probabilistic kinship predictions. No controls resulted in usable data and were free of any detectable contamination.
The results for the SD quants small mtDNA target were evaluated for a subset of Basilica samples with mitogenome hybridization capture data. The average coverage was plotted against mtDNA input based on the qPCR small mtDNA target ( a) and hDNA ratio ( b). For this analysis, the hDNA ratio was based on the qPCR small mtDNA target instead of the small autosomal target. The mtDNA input was positively correlated with the average coverage, but sample FB-5 fell slightly out of trend ( a). FB-5 had the highest mtDNA degradation index (69 bp mtDNA qPCR target/143 bp mtDNA qPCR target), indicating a greater degree of fragmentation than the other Basilica samples. FB-5 also had a greater amount of total dsDNA compared to FB-6, which suggests high exogenous DNA background. This was taken into account with the hDNA ratio ( b), and therefore, served as a slightly better predictor of average coverage of the mitogenome than mtDNA qPCR alone. Many laboratories, including the AFMES-AFDIL, do not have access to a mtDNA quantification system. Therefore, mitogenome capture results were also assessed using total dsDNA from Qubit and nuclear DNA qPCR kits that are more widely implemented. The average coverage was plotted against total dsDNA input from Qubit, human DNA input based on the qPCR small human autosomal target, and the hDNA ratio based on the qPCR small autosomal target. This was conducted for the full bone sample dataset since all samples were quantified with autosomal qPCR. Consistent with the SNP data shown in , total dsDNA from Qubit quantification ( ) was not a suitable predictor of average coverage of the mitogenome, due to varying levels of exogenous DNA background. DNA input by qPCR ( a) and the autosomal hDNA ratio ( b) were both strong predictors of average mitogenome coverage. In a, the majority of samples with ≥7 pg input resulted in average coverages above 500X; in b, this was achieved for the majority of samples with hDNA ratios ≥0.01. shows sample HC-1B having slightly lower average coverage than expected. This could be due to the sample inhibition identified in the library input reduction test. Sample PB-1 also fell out of trend in a with an average coverage of 1501X. This sample’s average coverage was better explained by the hDNA ratio ( b). PB-1 was one of the two samples originating from the WWII Punchbowl context in which burials were subjected to formalin preservation. The chemical treatment of PB-1 may explain this sample’s unique signature: higher than the expected 1X auSNP coverage and higher mitogenome average coverage when compared to historical samples with similar human DNA input. It is notable that the other Punchbowl sample, PB-2, was better explained by human DNA input ( a) than the hDNA ratio ( b). Across the entire dataset, the highest average coverage of the mitogenome observed was 11,515X, while the lowest was 95X—well above the 10X average coverage metric required by AFMES-AFDIL for data reporting. All samples except FB-1 generated full mitogenome coverage (16,569 bp) at a 10X threshold for the reported sequence range ( a). FB-1 had a 69 bp gap in the reported sequence range due to lower coverage (6–9X) observed in the polycytosine stretch in hypervariable region 1. Homopolymer regions often have lower coverage due to mapping difficulties , especially with very short fragments. All samples were single sourced with no indication of a mixture. No controls resulted in usable data and were free of any detectable contamination.
Nineteen samples underwent auSTR typing, of which 14 resulted in at least one reportable locus. The percentage of auSTR loci recovered was plotted against human DNA input from the qPCR small autosomal target and the hDNA ratio based on the qPCR small autosomal target. DNA input by qPCR ( a) was the best predictor of STR recovery, as samples with ≥30 pg input resulted in >40% recovery of auSTR loci. Below this threshold, a maximum of one locus was recovered. The hDNA ratio ( b) was less predictive, which suggests that the non-human DNA background did not impact the percentage of STR loci recovered. It is worth noting that the 550 RFU homozygote stochastic reporting threshold validated at the AFMES-AFDIL for PowerPlex Fusion data may have a more noticeable impact on auSTR locus reportability for degraded samples, particularly those with many homozygous loci. Of the 14 samples that were tested using the LCNY protocol, including one suspected female, eight samples resulted in at least one reportable Y-STR locus. The small autosomal and Y-DNA targets were both strong predictors of the percentage of Y-STR loci recovered ( a,b), but a clearer trend was observed in the prediction from Y-qPCR. The Y-DNA target was also smaller than the autosomal target, benefiting more degraded samples. Greater than 58% of the Y-STR loci were recovered in samples with ≥19 pg autosomal DNA ( a) and ≥24 pg Y-DNA ( b). Below these input values, only one sample had a single locus recovered. As expected for the suspected female sample, zero Y-STR loci were recovered. The hDNA ratio by the small autosomal target ( c) and Y-DNA target ( d) were poorly correlated with Y-STR recovery. Additional LCNY amplifications were performed for sample HC-1B with reduced template volumes (1 µL and 5 µL) due to the inhibition observed during NGS processing. The 1 µL and 5 µL reduced template volumes resulted in Y-STR recoveries of 23.53% and 29.41%, respectively. As stated previously, the effects of inhibition were not observed in the IPCs for any of the qPCR assays. The LCNY protocol uses YFiler kit reagents, which is an older generation kit, and therefore, it may be less tolerant to inhibitors than current generation qPCR kits like those tested here. No controls resulted in usable data and were free of any detectable contamination
These results show that DNA quantification, both human-specific and total dsDNA content, can serve as a screening method for both high- and low-quality samples processed in ancient and forensic DNA laboratories. By testing a range of historical bone samples in terms of age and quality, the results from this study demonstrate that degraded and low template human DNA can be reliably quantified. Typical screening methods, such as WGS for aDNA laboratories, are costly and minimally effective for samples containing <1% endogenous DNA . Information obtained from WGS screening such as relative endogenous DNA content and fragment length can also be estimated with a refined qPCR multiplex and total dsDNA quantification . Maximizing the information that quantification systems offer allows the best downstream processing method to be selected. The quantification of extracts is a cost-effective and efficient approach since it precedes any downstream testing such as test amplification or library preparation and WGS. Additionally, many samples can be quantified simultaneously. This study consisted of samples with varying degrees of degradation. Samples were tested with several modalities of forensic DNA profiling (SNP genotyping, STR typing, and mtDNA sequencing) to comprehensively highlight the ways in which the qPCR system implemented can predict sample success. While this study highlights the potential benefits of qPCR screening, implementation in a forensic laboratory would require a number of studies to meet accreditation standards. These studies include an assessment of accuracy and precision with technical replicates, representative non-human samples for species specificity, mixtures, sensitivity, and case-type non-probative samples. Case samples vary between laboratories; therefore, ensuring the qPCR kit used is tailored to the samples and downstream processing can result in the most reliable prediction of success. Selecting a qPCR autosomal target of a similar size to the expected fragment length of sequenceable and informative fragments would best forecast success. For highly fragmented samples that are intended for NGS, a 60 bp qPCR target may better predict NGS success using a hybridization capture approach, while a 90 bp or larger target may be more appropriate for amplicon-based library preparation (or STR amplification). Where possible, having multiple targets of varying size would allow fragment size distributions to be inferred, which would help predict success as well as determine the number of cycles needed for sequencing (as longer DNA fragments require more cycles to sequence entire reads). Similarly, the qPCR kit will be more predictive if the qPCR buffer’s robustness to inhibition is consistent with the chemistry utilized in downstream processing methods to best alert the user when reduced input or dilution may be a benefit. For example, it is possible that current generation Y-STR kits, such as YFiler Plus (Thermo Fisher Scientific) and PowerPlex Y23 (Promega), have similar inhibitor tolerance to the qPCR assays tested in this study. Therefore, the newer Y-STR kits might also produce results in the presence of inhibitors that are more consistent with expectations based on current generation qPCR results. These considerations are true of traditional forensic STR typing and could also be applied to other processing streams such as NGS. Selecting a qPCR target specific to the data being generated is also beneficial in predicting sequencing results. For a subset of Basilica samples, the hDNA ratio determined by the small mtDNA qPCR target served as a better predictor of mtDNA capture results, versus the hDNA ratio or simply the quantification results of the small autosomal target. Likewise, the small Y-DNA target was a better predictor of LCNY success, as it was a more direct measurement of the target. Depending on the amount of exogenous DNA present in the samples being studied, the quantification results alone may serve as a sufficient predictor of on-target coverage. The DNA extraction method used can also affect fragment size distribution and thus the ideal qPCR target size. Extractions such as the Dabney method that prioritize small fragment retention may benefit from a smaller target compared to extracts created with other protocols. For example, a small subset of samples extracted with both the Dabney method and another common total demineralization extraction method , and processed through the FORCE hybridization capture method, showed that the average fragment length of DNA mapping to GRCh38 after FORCE SNP capture was approximately 40 bp smaller with the Dabney method (~110 bp vs. ~70 bp) ( ). While a direct comparison of DNA extraction methods was not an aim of this study, future work could include such a comparison of commonly utilized forensic and ancient DNA procedures . Future research could determine what impact any one DNA extraction method could have on the resulting qPCR and/or hDNA results, as well as downstream DNA profiling success. In conclusion, the three commercially available qPCR kits tested in this study were highly comparable. All three qPCR assays were appropriate for reliable quantification of historical DNA samples exhibiting varying levels of degradation. In scenarios where both nuclear DNA and mtDNA could be targets of interest, currently available qPCR kits with small autosomal targets were not only useful for predicting auDNA results but were also sufficient for predicting mtDNA success in most cases. Overall, qPCR quantification alone was the best predictor of sample success for both NGS and STR processing workflows. The hDNA ratio was used secondarily to predict SNP success where human DNA input was low (<100 pg) and in mitogenome capture. The qPCR-based DNA input requirements identified in this study can guide laboratories toward the downstream processing method(s) with the highest potential for success depending on the information required for a particular case. Toward this end of achieving a particular human DNA input target for predicted NGS or STR success, DNA extracts can be combined and/or concentrated to increase target DNA. Sample extracts with high exogenous DNA content that may exceed NGS library input recommendations can have libraries combined and/or concentrated for input into a single hybridization capture reaction for improved recovery. Alternatively, low quantity sample extracts can also benefit from concentrating sample library to increase input into SNP capture. If human DNA input is unavoidably low, mitogenome capture may be the best choice for obtaining forensically relevant genetic information from an ancient or historical case sample. Ultimately, these data may help to set a benchmark for bone sample success.
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Evaluation of Library Preparation Workflows and Applications to Different Sample Types Using the PowerSeq
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0f126023-3e7d-4570-899f-1ccf43acc4bd
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10218485
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Forensic Medicine[mh]
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Short tandem repeats (STRs) have been used for human identification in forensic science for over 20 years [ , , ] and are ideal for forensic casework because of the high power of discrimination they provide between individuals. Additionally, the Combined DNA Index System (CODIS) contains over 20 million STR reference profiles , and the system is nationally recognized. In 2011, a new technique referred to as next-generation sequencing (NGS), massively parallel sequencing (MPS), or second-generation sequencing (SGS) was first used for forensic applications to analyze multiple targeted STR loci simultaneously . Fordyce et al. reported the use of MPS to characterize ten individuals with five STR loci . They found that sequencing STRs, rather than using capillary electrophoresis (CE) or fragment analysis, provided better resolution of the STRs, e.g., sequence variation. In 2012, all 13 core CODIS STR loci and the amelogenin sex marker were successfully sequenced in individuals and mixtures . Since the discovery of MPS for use with forensic applications, adoption has been slow due to the rigorous testing and validation needed to incorporate new technology into forensic laboratories. In the past 10 years, substantial research and development has provided the forensic community with numerous panel options using markers for MPS analysis of STRs , single nucleotide polymorphisms (SNPs) , microhaplotypes , and mitochondrial DNA . Although STR analysis via CE is still the standard for forensic casework analysis, several studies have proposed MPS as an alternative because it offers a potential solution to many common challenges of STR analysis, including large multiplexes and sample mixtures , the increased genotyping success of degraded DNA and cost reduction compared to CE , and the increased success of challenging sample types . Additionally, challenges still exist for the implementation of MPS into forensic casework, which is one reason for the study presented here. To perform MPS analysis of a sample, a library of DNA fragments is constructed and sequenced. The forensic workflow of DNA library preparation prior to MPS includes several steps such as DNA extraction, PCR amplification of target regions, DNA purification, adaptor ligation, and library quantification. Numerous studies have focused on modifications to each of these steps for applications in human identification using various chemistries and across multiple sequencing platforms for autosomal analysis [ , , ]. Additionally, many laboratories have already implemented MPS technology for use with either criminal casework or human identification [ , , , , , , , , ]. In response to the increased interest in MPS, several manufacturers provide various kits. Some are amplification kits that require the purchase of a separate library preparation kit (PowerSeq ® 46GY System (Promega Corporation, Madison, WI, USA)), while others contain all the reagents necessary for DNA amplification and library preparation: Precision ID GlobalFiler™ NGS STR Panel v2 (Thermo Fisher Scientific, Waltham, MA, USA), and the ForenSeq DNA Signature Prep Kit (Verogen, Inc., San Diego, CA, USA). These products provide the user with a complete primer panel capable of amplifying makers commonly used in human identification. Several publications have reported on the use of the ForenSeq DNA Signature Prep Kit [ , , ] and Precision ID GlobalFiler NGS STR Panel [ , , ], among other panels. The PowerSeq ® 46GY System (previously the PowerSeq™ Auto/Y System) was evaluated for use with human population identification [ , , , , ], STR analysis of forensic-type samples , standard reference materials , and modifications of individual steps within the PowerSeq ® workflow . However, there are no published works on the optimization of the entire workflow of the PowerSeq ® 46GY System. Before any protocol is used for forensic casework, there is a need to assess the downstream effects of potential protocol-enhancing modifications to the manufacturers’ suggested protocols to ascertain if modifications will increase the quantity and quality of DNA libraries or affect the concordance of identification results. The goal of this study was to identify potential enhancements to the existing manufacturer protocol for the PowerSeq ® 46GY System and TruSeq workflow, then assess the effects of these modifications on casework-type samples through sequencing. To accomplish this, library preparation was performed using two kits, libraries were purified using three solid-phase reversible immobilization (SPRI) beads, and libraries were quantified with two kits. Each library was constructed using donor buccal samples, followed by sequencing on the Illumina MiSeq FGx™ (Illumina, San Diego, CA, USA). The most efficient protocol was selected based on sequencing data metrics obtained from buccal DNA-based libraries and applied to forensic-type bone and hair samples.
2.1. DNA Samples Two known buccal sources, one male and one female, were used as standards to test library preparation modifications. Single hairs were collected from four individuals, and bones from six individuals were provided by the Southeast Texas Applied Forensic Science Facility (STAFS) in Huntsville, TX, USA. Human hair and bone samples were used as casework-type samples to assess the downstream impact of library preparation modifications on sequencing results. Information regarding the bone and hair samples may be found in . Buccal and hair samples were collected with informed consent according to Internal Review Board (IRB) guidelines. 2.2. DNA Extraction and Quantification 2.2.1. Buccal Swab Extraction Buccal swabs were extracted using a semi-automated extraction protocol . The swab heads were placed in Investigator Lyse&Spin baskets (QIAGEN, Germantown, MD, USA) with 450 µL of lysis buffer (Buffer G2, Proteinase K (PK, 20 mg/mL) and dithiothreitol (DTT, 1 M)) and lysed for one hour at 56 °C with gentle agitation (~200 rpm). The purification of the lysates was performed with the EZ1 Advanced XL (QIAGEN, Hilden, Germany) using the Large Volume Protocol and eluting the DNA in 50 µL of nuclease-free water (hereafter termed ‘water’). 2.2.2. Hair Extraction Prior to use, hairs were stored at room temperature in individual, sealed and sterile plastic bags. Hair samples were prepared and extracted using a manual extraction and purification method . Hairs were cut approximately 2 cm from the root and individually placed in separate 1.5 mL tubes. Samples were purified using the PrepFiler ® DNA Extraction Kit (Life Technologies, Carlsbad, CA, USA) following and eluted in 65 µL of Elution Buffer. 2.2.3. Bone Extraction Bone powder (approximately 0.2 g) was weighed out (in triplicate) for each sample and placed in separate 15 mL conical tubes. To each tube, 3 mL demineralization (Demin) Buffer (0.5 M EDTA, 1% N-lauroylsarcosine sodium salt at pH 8) and 200 µL of PK were added. The tubes were gently vortexed, then incubated in a thermomixer with agitation at 900 rpm for 24 h at 56 °C. Extraction was completed following , using an Amicon Ultra-4 Centrifugal Filter Units with a 30 KDa membrane (MilliporeSigma, St. Louis, MO, USA) instead of a Vivacon ® 2 concentrator. Samples were eluted in 50 µL water according to . All bone samples were extracted in triplicate and combined for a larger volume. Reagent blanks were included for each extraction. Extracts were stored at −20 °C until use. 2.2.4. DNA Quantification All extracts were quantified using Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Thermo Fisher Scientific) as per the manufacturer’s protocol . Data were analyzed using HID Real-Time PCR Analysis Software v1.2. 2.3. PowerSeq ® Amplification and Library Preparation For the first three studies, male and female buccal extracts were amplified using 30 PCR cycles in duplicate at five DNA inputs (1 ng, 0.5 ng, 0.25 ng, 0.125 ng, 0.063 ng), and a negative and positive control were included with each experiment ( n = 22). For the final study, six bone and four hair extracts were amplified in duplicate in addition to a bone and hair reagent blank and a positive and negative control ( n = 24). Hair and bone extracts were amplified with 0.5 ng DNA or the maximum sample volume (15 µL) if DNA was less than 0.033 ng/µL, as per the manufacturer’s recommendations . For positive control samples, 0.5 ng of 2800 M Control DNA (Promega) was used. Note that the Promega Technical Manual has since been updated to target 1 ng of DNA input and 29 PCR cycles . 2.3.1. TruSeq DNA PCR-Free HT Library Preparation with SPBs Sample extracts (15 µL) were amplified using 5 µL PowerSeq ® 5X Master Mix and 5 µL PowerSeq ® 46GY 5X Primer Pair Mix with the following thermal cycling conditions: denaturation for 1 min at 96 °C, followed by 30 cycles of 96 °C for 5 s, 60 °C for 35 s, and 72 °C for 5 s, with a final extension for 2 min at 60 °C. Following amplification, libraries were prepared following the protocol described in the PowerSeq ® 46GY System Technical Manual using the TruSeq DNA PCR-Free HT Library Preparation Kit with Sample Purification Beads (SPBs, Illumina). Quantification of purified amplification products was performed using the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) on the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) or with the Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., Fairmont, WV, USA) using the Agilent High Sensitivity DNA Kit (Agilent). 2.3.2. KAPA HyperPrep Library Preparation with KPBs The KAPA HyperPrep Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) protocol recommends fragmentation of the DNA, followed by end repair and A-tailing, adapter ligation, post-ligation cleanup, library amplification, and post-amplification cleanup prior to sequencing. A modified version of the KAPA HyperPrep Kit protocol was adopted to better suit the library preparation workflow of the 46GY Panel and fragmentation was omitted. Buccal sample extracts (15 µL) were amplified using 25 µL KAPA HiFi HotStart ReadyMix (2×) and 10 µL PowerSeq ® 46GY 5× Primer Pair Mix with the following thermal cycling conditions: 1 min denaturation at 98 °C, followed by 30 cycles of 5 s at 98 °C, 35 s at 60 °C, and 5 s at 72 °C, and finally, a 1 min final extension at 72 °C. Post amplification required a 1× bead-based cleanup using 50 µL KAPA Pure Beads (KPBs, Roche Sequencing Solutions). The plate was incubated for 10 min at room temperature and then placed on a magnetic stand until the liquid was clear (~10 min). The supernatant was discarded and 200 µL 80% ethanol was added, followed by a 30 s incubation before removal, ensuring the beads remained attached to the tube wall. The ethanol cleanup was performed once more for a total of two cleanups. The beads were dried for 3 to 5 min at room temperature while residual ethanol evaporated. The plate was removed from the magnetic stand and the beads were resuspended in 55 µL water. The purified amplification product (50 µL) was transferred to a new plate. End repair and A-tailing were completed in a single step. Clean, amplified product (50 µL) was combined with 7 µL End Repair and A-Tailing Buffer and 3 µL End Repair and A-Tailing Enzyme Mix. The samples were incubated for 30 min at 20 °C followed by 30 min at 65 °C. Samples were quantified using the Qubit™ dsDNA High Sensitivity Assay Kit with the Qubit 3.0 Fluorometer to correctly dilute adapter stocks from the KAPA Dual Indexed Adapter Kit (Roche). Based on the quantities of the end repaired and A-tailed libraries, adapters were diluted 1:10, 1:20, or 1:40. For adapter ligation, 60 µL end repair and A-tailing reaction product was combined with 5 µL adapter stock, 5 µL water, 30 µL Ligation Buffer, and 10 µL DNA Ligase, which was incubated for 15 min at 20 °C. Next, a post-ligation cleanup was performed using 88 µL (0.8× ratio) KPBs with two ethanol cleanup steps, as described previously. The libraries were eluted in 55 µL water and 50 µL was transferred to a new plate for double-sided size selection. An aliquot (35 µL, 0.7× ratio) of KPBs was added to 50 µL library and incubated for 10 min at room temperature. The plate was placed on a magnetic stand and 80 µL supernatant was transferred to a new plate. A small aliquot (10 µL) KPBs was added to 80 µL supernatant from the first size selection. Two ethanol cleanup steps were performed as described previously. Samples were eluted in 25 µL Tris-HCl (pH 8.0–8.5) and 20 µL final product was transferred to a new plate. 2.3.3. TruSeq Library Preparation with AMPure XP Bead Purification This study was carried out exactly as the first TruSeq study except for the use of AMPure XP beads (Agencourt Bioscience Corporation, Beverly, MA, USA) in place of the SPBs included in the TruSeq DNA PCR-Free HT Library Preparation Kit. 2.3.4. Casework-Type Samples with Enhanced Library Preparation Method The library preparation protocol chosen for casework-type samples was based on quantification and sequencing results of buccal samples from the previous three studies. Based on these results, the TruSeq DNA PCR-Free HT Library Preparation Kit was chosen along with Illumina SPBs and the protocol described in 2.3.1. 2.4. Library Quantification, Normalization, and Illumina Sequencing 2.4.1. Library Quantification Libraries were quantified with two different MPS library quantification kits: the PowerSeq ® Quant MS System (Promega) and the KAPA Library Quantification Kit for Illumina ® Platforms (Roche) . Libraries were quantified in duplicate, using the respective MPS library quantification kits and protocols , on the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). Data were analyzed using QuantStudio™ Design & Analysis Software v1.4 and library concentrations were adjusted based on the dilution factor for each kit. Additionally, Roche Sequencing Solutions provided an Excel workbook (KAPA Library Quantification Data Analysis Template) that calculated the concentration of the undiluted library by multiplying the calculated average concentration (in pM) by the equation below for the size-adjusted concentration (in pM) and then by the dilution factor: (Size of DNA Standard in bp (452))/(Average fragment length of library in bp). 2.4.2. Library Normalization and Quality Check Libraries were diluted to 4 nM using Resuspension Buffer, and 5 µL of each dilution was pooled in a single tube. The diluted library pool and select samples were quality checked to determine the concentration of the sample pool after library dilution using the Agilent 2100 Bioanalyzer via the Agilent High Sensitivity DNA Kit. To denature the normalized libraries, an aliquot of the ~0.5 nM–4 nM pooled libraries (5 µL (4 nM)–40 µL (0.5 nM)) was combined with an equal volume of 0.2 N NaOH and incubated at room temperature for 5 min. Following denaturation, an equal volume (5 µL–40 µL) of 200 mM Tris-HCl, pH 7, was added to balance the pH of the solution for sequencing. Lastly, a Hybridization Buffer (880 µL–985 µL, depending on the other components added previously) was added to the tube containing the denatured library for a final library concentration of 20 pM. A PhiX Control (Illumina) was diluted and denatured by combining 2 µL PhiX Control (10 nM), 3 µL Resuspension Buffer, and 5 µL 0.2 M NaOH to incubate at room temperature for 5 min. Following incubation, 990 µL Hybridization Buffer was added for a final concentration of 20 pM. Finally, the sequencing dilution was created by combining 195 µL Hybridization Buffer, 365 µL of the 20 pM pooled and denatured libraries, and 40 µL of the 20 pM denatured PhiX Control. 2.4.3. Illumina Sequencing Sequencing was performed on an Illumina MiSeq FGx™ using a standard flow cell and 600-cycle v3 kit with 2 × 300 bp reads and 2 × 8 cycles for sample indices. For each of the first three studies ( , and ), 22 PowerSeq ® normalized sample libraries were pooled for sequencing. For the casework study ( ), 24 PowerSeq ® normalized sample libraries were pooled for sequencing. 2.5. Data Analysis Sequencing results and quality metrics were observed using Sequence Analysis Viewer software (Illumina) . FASTQ files were analyzed using STRait Razor Online (SRO) and sequences were aligned to human genome assembly GRCh38. An analytical threshold of 50 reads was applied to STR genotypes according to SWGDAM guidelines . An interpretation threshold of 500 reads for homozygotes and 100 reads for heterozygotes was applied to autosomal loci according to Hölzl-Müller et al. . Additionally, an interpretation threshold of 100 reads was also applied to Y-STRs according to Moon et al. . Reads above 50× coverage but below the interpretation threshold were considered “Below-Threshold” (BT). Loci below the analytical threshold or with no coverage were considered “Dropout” (DO). Heterozygote imbalance was called for ratios < 0.50, a threshold used by Zeng et al. . Furthermore, locus DYS389I is left out of the default STRait Razor analysis for the 46GY panel and therefore may not be optimally represented in the analyses of this study. This study assessed profile completeness (%), coverage range, average coverage, heterozygote balance (HB: the coverage of the lesser allele divided by the coverage of the larger sister allele), below-threshold alleles, drop-in alleles, and dropout. Data were compared to GlobalFiler CE reference profiles on file for buccal and hair samples, and STAFS provided GlobalFiler references for the bone samples. MPS references were produced by consensus from the replicates. Statistical significance was ascertained using an F-test Two-Sample for Variances followed by a Two-Sample, two tailed t -Test assuming equal (or unequal) variances or ANOVA using α 0.05. All statistical analyses were performed using IBM SPSS Statistics Version 29.0 (IBM Corporation, Armonk, NY, USA).
Two known buccal sources, one male and one female, were used as standards to test library preparation modifications. Single hairs were collected from four individuals, and bones from six individuals were provided by the Southeast Texas Applied Forensic Science Facility (STAFS) in Huntsville, TX, USA. Human hair and bone samples were used as casework-type samples to assess the downstream impact of library preparation modifications on sequencing results. Information regarding the bone and hair samples may be found in . Buccal and hair samples were collected with informed consent according to Internal Review Board (IRB) guidelines.
2.2.1. Buccal Swab Extraction Buccal swabs were extracted using a semi-automated extraction protocol . The swab heads were placed in Investigator Lyse&Spin baskets (QIAGEN, Germantown, MD, USA) with 450 µL of lysis buffer (Buffer G2, Proteinase K (PK, 20 mg/mL) and dithiothreitol (DTT, 1 M)) and lysed for one hour at 56 °C with gentle agitation (~200 rpm). The purification of the lysates was performed with the EZ1 Advanced XL (QIAGEN, Hilden, Germany) using the Large Volume Protocol and eluting the DNA in 50 µL of nuclease-free water (hereafter termed ‘water’). 2.2.2. Hair Extraction Prior to use, hairs were stored at room temperature in individual, sealed and sterile plastic bags. Hair samples were prepared and extracted using a manual extraction and purification method . Hairs were cut approximately 2 cm from the root and individually placed in separate 1.5 mL tubes. Samples were purified using the PrepFiler ® DNA Extraction Kit (Life Technologies, Carlsbad, CA, USA) following and eluted in 65 µL of Elution Buffer. 2.2.3. Bone Extraction Bone powder (approximately 0.2 g) was weighed out (in triplicate) for each sample and placed in separate 15 mL conical tubes. To each tube, 3 mL demineralization (Demin) Buffer (0.5 M EDTA, 1% N-lauroylsarcosine sodium salt at pH 8) and 200 µL of PK were added. The tubes were gently vortexed, then incubated in a thermomixer with agitation at 900 rpm for 24 h at 56 °C. Extraction was completed following , using an Amicon Ultra-4 Centrifugal Filter Units with a 30 KDa membrane (MilliporeSigma, St. Louis, MO, USA) instead of a Vivacon ® 2 concentrator. Samples were eluted in 50 µL water according to . All bone samples were extracted in triplicate and combined for a larger volume. Reagent blanks were included for each extraction. Extracts were stored at −20 °C until use. 2.2.4. DNA Quantification All extracts were quantified using Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Thermo Fisher Scientific) as per the manufacturer’s protocol . Data were analyzed using HID Real-Time PCR Analysis Software v1.2.
Buccal swabs were extracted using a semi-automated extraction protocol . The swab heads were placed in Investigator Lyse&Spin baskets (QIAGEN, Germantown, MD, USA) with 450 µL of lysis buffer (Buffer G2, Proteinase K (PK, 20 mg/mL) and dithiothreitol (DTT, 1 M)) and lysed for one hour at 56 °C with gentle agitation (~200 rpm). The purification of the lysates was performed with the EZ1 Advanced XL (QIAGEN, Hilden, Germany) using the Large Volume Protocol and eluting the DNA in 50 µL of nuclease-free water (hereafter termed ‘water’).
Prior to use, hairs were stored at room temperature in individual, sealed and sterile plastic bags. Hair samples were prepared and extracted using a manual extraction and purification method . Hairs were cut approximately 2 cm from the root and individually placed in separate 1.5 mL tubes. Samples were purified using the PrepFiler ® DNA Extraction Kit (Life Technologies, Carlsbad, CA, USA) following and eluted in 65 µL of Elution Buffer.
Bone powder (approximately 0.2 g) was weighed out (in triplicate) for each sample and placed in separate 15 mL conical tubes. To each tube, 3 mL demineralization (Demin) Buffer (0.5 M EDTA, 1% N-lauroylsarcosine sodium salt at pH 8) and 200 µL of PK were added. The tubes were gently vortexed, then incubated in a thermomixer with agitation at 900 rpm for 24 h at 56 °C. Extraction was completed following , using an Amicon Ultra-4 Centrifugal Filter Units with a 30 KDa membrane (MilliporeSigma, St. Louis, MO, USA) instead of a Vivacon ® 2 concentrator. Samples were eluted in 50 µL water according to . All bone samples were extracted in triplicate and combined for a larger volume. Reagent blanks were included for each extraction. Extracts were stored at −20 °C until use.
All extracts were quantified using Quantifiler™ Trio DNA Quantification Kit (Thermo Fisher Scientific) on a 7500 Real-Time PCR System (Thermo Fisher Scientific) as per the manufacturer’s protocol . Data were analyzed using HID Real-Time PCR Analysis Software v1.2.
® Amplification and Library Preparation For the first three studies, male and female buccal extracts were amplified using 30 PCR cycles in duplicate at five DNA inputs (1 ng, 0.5 ng, 0.25 ng, 0.125 ng, 0.063 ng), and a negative and positive control were included with each experiment ( n = 22). For the final study, six bone and four hair extracts were amplified in duplicate in addition to a bone and hair reagent blank and a positive and negative control ( n = 24). Hair and bone extracts were amplified with 0.5 ng DNA or the maximum sample volume (15 µL) if DNA was less than 0.033 ng/µL, as per the manufacturer’s recommendations . For positive control samples, 0.5 ng of 2800 M Control DNA (Promega) was used. Note that the Promega Technical Manual has since been updated to target 1 ng of DNA input and 29 PCR cycles . 2.3.1. TruSeq DNA PCR-Free HT Library Preparation with SPBs Sample extracts (15 µL) were amplified using 5 µL PowerSeq ® 5X Master Mix and 5 µL PowerSeq ® 46GY 5X Primer Pair Mix with the following thermal cycling conditions: denaturation for 1 min at 96 °C, followed by 30 cycles of 96 °C for 5 s, 60 °C for 35 s, and 72 °C for 5 s, with a final extension for 2 min at 60 °C. Following amplification, libraries were prepared following the protocol described in the PowerSeq ® 46GY System Technical Manual using the TruSeq DNA PCR-Free HT Library Preparation Kit with Sample Purification Beads (SPBs, Illumina). Quantification of purified amplification products was performed using the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) on the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) or with the Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., Fairmont, WV, USA) using the Agilent High Sensitivity DNA Kit (Agilent). 2.3.2. KAPA HyperPrep Library Preparation with KPBs The KAPA HyperPrep Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) protocol recommends fragmentation of the DNA, followed by end repair and A-tailing, adapter ligation, post-ligation cleanup, library amplification, and post-amplification cleanup prior to sequencing. A modified version of the KAPA HyperPrep Kit protocol was adopted to better suit the library preparation workflow of the 46GY Panel and fragmentation was omitted. Buccal sample extracts (15 µL) were amplified using 25 µL KAPA HiFi HotStart ReadyMix (2×) and 10 µL PowerSeq ® 46GY 5× Primer Pair Mix with the following thermal cycling conditions: 1 min denaturation at 98 °C, followed by 30 cycles of 5 s at 98 °C, 35 s at 60 °C, and 5 s at 72 °C, and finally, a 1 min final extension at 72 °C. Post amplification required a 1× bead-based cleanup using 50 µL KAPA Pure Beads (KPBs, Roche Sequencing Solutions). The plate was incubated for 10 min at room temperature and then placed on a magnetic stand until the liquid was clear (~10 min). The supernatant was discarded and 200 µL 80% ethanol was added, followed by a 30 s incubation before removal, ensuring the beads remained attached to the tube wall. The ethanol cleanup was performed once more for a total of two cleanups. The beads were dried for 3 to 5 min at room temperature while residual ethanol evaporated. The plate was removed from the magnetic stand and the beads were resuspended in 55 µL water. The purified amplification product (50 µL) was transferred to a new plate. End repair and A-tailing were completed in a single step. Clean, amplified product (50 µL) was combined with 7 µL End Repair and A-Tailing Buffer and 3 µL End Repair and A-Tailing Enzyme Mix. The samples were incubated for 30 min at 20 °C followed by 30 min at 65 °C. Samples were quantified using the Qubit™ dsDNA High Sensitivity Assay Kit with the Qubit 3.0 Fluorometer to correctly dilute adapter stocks from the KAPA Dual Indexed Adapter Kit (Roche). Based on the quantities of the end repaired and A-tailed libraries, adapters were diluted 1:10, 1:20, or 1:40. For adapter ligation, 60 µL end repair and A-tailing reaction product was combined with 5 µL adapter stock, 5 µL water, 30 µL Ligation Buffer, and 10 µL DNA Ligase, which was incubated for 15 min at 20 °C. Next, a post-ligation cleanup was performed using 88 µL (0.8× ratio) KPBs with two ethanol cleanup steps, as described previously. The libraries were eluted in 55 µL water and 50 µL was transferred to a new plate for double-sided size selection. An aliquot (35 µL, 0.7× ratio) of KPBs was added to 50 µL library and incubated for 10 min at room temperature. The plate was placed on a magnetic stand and 80 µL supernatant was transferred to a new plate. A small aliquot (10 µL) KPBs was added to 80 µL supernatant from the first size selection. Two ethanol cleanup steps were performed as described previously. Samples were eluted in 25 µL Tris-HCl (pH 8.0–8.5) and 20 µL final product was transferred to a new plate. 2.3.3. TruSeq Library Preparation with AMPure XP Bead Purification This study was carried out exactly as the first TruSeq study except for the use of AMPure XP beads (Agencourt Bioscience Corporation, Beverly, MA, USA) in place of the SPBs included in the TruSeq DNA PCR-Free HT Library Preparation Kit. 2.3.4. Casework-Type Samples with Enhanced Library Preparation Method The library preparation protocol chosen for casework-type samples was based on quantification and sequencing results of buccal samples from the previous three studies. Based on these results, the TruSeq DNA PCR-Free HT Library Preparation Kit was chosen along with Illumina SPBs and the protocol described in 2.3.1.
Sample extracts (15 µL) were amplified using 5 µL PowerSeq ® 5X Master Mix and 5 µL PowerSeq ® 46GY 5X Primer Pair Mix with the following thermal cycling conditions: denaturation for 1 min at 96 °C, followed by 30 cycles of 96 °C for 5 s, 60 °C for 35 s, and 72 °C for 5 s, with a final extension for 2 min at 60 °C. Following amplification, libraries were prepared following the protocol described in the PowerSeq ® 46GY System Technical Manual using the TruSeq DNA PCR-Free HT Library Preparation Kit with Sample Purification Beads (SPBs, Illumina). Quantification of purified amplification products was performed using the Qubit™ dsDNA High Sensitivity Assay Kit (Thermo Fisher Scientific) on the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) or with the Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., Fairmont, WV, USA) using the Agilent High Sensitivity DNA Kit (Agilent).
The KAPA HyperPrep Kit (Roche Sequencing Solutions, Pleasanton, CA, USA) protocol recommends fragmentation of the DNA, followed by end repair and A-tailing, adapter ligation, post-ligation cleanup, library amplification, and post-amplification cleanup prior to sequencing. A modified version of the KAPA HyperPrep Kit protocol was adopted to better suit the library preparation workflow of the 46GY Panel and fragmentation was omitted. Buccal sample extracts (15 µL) were amplified using 25 µL KAPA HiFi HotStart ReadyMix (2×) and 10 µL PowerSeq ® 46GY 5× Primer Pair Mix with the following thermal cycling conditions: 1 min denaturation at 98 °C, followed by 30 cycles of 5 s at 98 °C, 35 s at 60 °C, and 5 s at 72 °C, and finally, a 1 min final extension at 72 °C. Post amplification required a 1× bead-based cleanup using 50 µL KAPA Pure Beads (KPBs, Roche Sequencing Solutions). The plate was incubated for 10 min at room temperature and then placed on a magnetic stand until the liquid was clear (~10 min). The supernatant was discarded and 200 µL 80% ethanol was added, followed by a 30 s incubation before removal, ensuring the beads remained attached to the tube wall. The ethanol cleanup was performed once more for a total of two cleanups. The beads were dried for 3 to 5 min at room temperature while residual ethanol evaporated. The plate was removed from the magnetic stand and the beads were resuspended in 55 µL water. The purified amplification product (50 µL) was transferred to a new plate. End repair and A-tailing were completed in a single step. Clean, amplified product (50 µL) was combined with 7 µL End Repair and A-Tailing Buffer and 3 µL End Repair and A-Tailing Enzyme Mix. The samples were incubated for 30 min at 20 °C followed by 30 min at 65 °C. Samples were quantified using the Qubit™ dsDNA High Sensitivity Assay Kit with the Qubit 3.0 Fluorometer to correctly dilute adapter stocks from the KAPA Dual Indexed Adapter Kit (Roche). Based on the quantities of the end repaired and A-tailed libraries, adapters were diluted 1:10, 1:20, or 1:40. For adapter ligation, 60 µL end repair and A-tailing reaction product was combined with 5 µL adapter stock, 5 µL water, 30 µL Ligation Buffer, and 10 µL DNA Ligase, which was incubated for 15 min at 20 °C. Next, a post-ligation cleanup was performed using 88 µL (0.8× ratio) KPBs with two ethanol cleanup steps, as described previously. The libraries were eluted in 55 µL water and 50 µL was transferred to a new plate for double-sided size selection. An aliquot (35 µL, 0.7× ratio) of KPBs was added to 50 µL library and incubated for 10 min at room temperature. The plate was placed on a magnetic stand and 80 µL supernatant was transferred to a new plate. A small aliquot (10 µL) KPBs was added to 80 µL supernatant from the first size selection. Two ethanol cleanup steps were performed as described previously. Samples were eluted in 25 µL Tris-HCl (pH 8.0–8.5) and 20 µL final product was transferred to a new plate.
This study was carried out exactly as the first TruSeq study except for the use of AMPure XP beads (Agencourt Bioscience Corporation, Beverly, MA, USA) in place of the SPBs included in the TruSeq DNA PCR-Free HT Library Preparation Kit.
The library preparation protocol chosen for casework-type samples was based on quantification and sequencing results of buccal samples from the previous three studies. Based on these results, the TruSeq DNA PCR-Free HT Library Preparation Kit was chosen along with Illumina SPBs and the protocol described in 2.3.1.
2.4.1. Library Quantification Libraries were quantified with two different MPS library quantification kits: the PowerSeq ® Quant MS System (Promega) and the KAPA Library Quantification Kit for Illumina ® Platforms (Roche) . Libraries were quantified in duplicate, using the respective MPS library quantification kits and protocols , on the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). Data were analyzed using QuantStudio™ Design & Analysis Software v1.4 and library concentrations were adjusted based on the dilution factor for each kit. Additionally, Roche Sequencing Solutions provided an Excel workbook (KAPA Library Quantification Data Analysis Template) that calculated the concentration of the undiluted library by multiplying the calculated average concentration (in pM) by the equation below for the size-adjusted concentration (in pM) and then by the dilution factor: (Size of DNA Standard in bp (452))/(Average fragment length of library in bp). 2.4.2. Library Normalization and Quality Check Libraries were diluted to 4 nM using Resuspension Buffer, and 5 µL of each dilution was pooled in a single tube. The diluted library pool and select samples were quality checked to determine the concentration of the sample pool after library dilution using the Agilent 2100 Bioanalyzer via the Agilent High Sensitivity DNA Kit. To denature the normalized libraries, an aliquot of the ~0.5 nM–4 nM pooled libraries (5 µL (4 nM)–40 µL (0.5 nM)) was combined with an equal volume of 0.2 N NaOH and incubated at room temperature for 5 min. Following denaturation, an equal volume (5 µL–40 µL) of 200 mM Tris-HCl, pH 7, was added to balance the pH of the solution for sequencing. Lastly, a Hybridization Buffer (880 µL–985 µL, depending on the other components added previously) was added to the tube containing the denatured library for a final library concentration of 20 pM. A PhiX Control (Illumina) was diluted and denatured by combining 2 µL PhiX Control (10 nM), 3 µL Resuspension Buffer, and 5 µL 0.2 M NaOH to incubate at room temperature for 5 min. Following incubation, 990 µL Hybridization Buffer was added for a final concentration of 20 pM. Finally, the sequencing dilution was created by combining 195 µL Hybridization Buffer, 365 µL of the 20 pM pooled and denatured libraries, and 40 µL of the 20 pM denatured PhiX Control. 2.4.3. Illumina Sequencing Sequencing was performed on an Illumina MiSeq FGx™ using a standard flow cell and 600-cycle v3 kit with 2 × 300 bp reads and 2 × 8 cycles for sample indices. For each of the first three studies ( , and ), 22 PowerSeq ® normalized sample libraries were pooled for sequencing. For the casework study ( ), 24 PowerSeq ® normalized sample libraries were pooled for sequencing.
Libraries were quantified with two different MPS library quantification kits: the PowerSeq ® Quant MS System (Promega) and the KAPA Library Quantification Kit for Illumina ® Platforms (Roche) . Libraries were quantified in duplicate, using the respective MPS library quantification kits and protocols , on the QuantStudio™ 5 Real-Time PCR System (Applied Biosystems). Data were analyzed using QuantStudio™ Design & Analysis Software v1.4 and library concentrations were adjusted based on the dilution factor for each kit. Additionally, Roche Sequencing Solutions provided an Excel workbook (KAPA Library Quantification Data Analysis Template) that calculated the concentration of the undiluted library by multiplying the calculated average concentration (in pM) by the equation below for the size-adjusted concentration (in pM) and then by the dilution factor: (Size of DNA Standard in bp (452))/(Average fragment length of library in bp).
Libraries were diluted to 4 nM using Resuspension Buffer, and 5 µL of each dilution was pooled in a single tube. The diluted library pool and select samples were quality checked to determine the concentration of the sample pool after library dilution using the Agilent 2100 Bioanalyzer via the Agilent High Sensitivity DNA Kit. To denature the normalized libraries, an aliquot of the ~0.5 nM–4 nM pooled libraries (5 µL (4 nM)–40 µL (0.5 nM)) was combined with an equal volume of 0.2 N NaOH and incubated at room temperature for 5 min. Following denaturation, an equal volume (5 µL–40 µL) of 200 mM Tris-HCl, pH 7, was added to balance the pH of the solution for sequencing. Lastly, a Hybridization Buffer (880 µL–985 µL, depending on the other components added previously) was added to the tube containing the denatured library for a final library concentration of 20 pM. A PhiX Control (Illumina) was diluted and denatured by combining 2 µL PhiX Control (10 nM), 3 µL Resuspension Buffer, and 5 µL 0.2 M NaOH to incubate at room temperature for 5 min. Following incubation, 990 µL Hybridization Buffer was added for a final concentration of 20 pM. Finally, the sequencing dilution was created by combining 195 µL Hybridization Buffer, 365 µL of the 20 pM pooled and denatured libraries, and 40 µL of the 20 pM denatured PhiX Control.
Sequencing was performed on an Illumina MiSeq FGx™ using a standard flow cell and 600-cycle v3 kit with 2 × 300 bp reads and 2 × 8 cycles for sample indices. For each of the first three studies ( , and ), 22 PowerSeq ® normalized sample libraries were pooled for sequencing. For the casework study ( ), 24 PowerSeq ® normalized sample libraries were pooled for sequencing.
Sequencing results and quality metrics were observed using Sequence Analysis Viewer software (Illumina) . FASTQ files were analyzed using STRait Razor Online (SRO) and sequences were aligned to human genome assembly GRCh38. An analytical threshold of 50 reads was applied to STR genotypes according to SWGDAM guidelines . An interpretation threshold of 500 reads for homozygotes and 100 reads for heterozygotes was applied to autosomal loci according to Hölzl-Müller et al. . Additionally, an interpretation threshold of 100 reads was also applied to Y-STRs according to Moon et al. . Reads above 50× coverage but below the interpretation threshold were considered “Below-Threshold” (BT). Loci below the analytical threshold or with no coverage were considered “Dropout” (DO). Heterozygote imbalance was called for ratios < 0.50, a threshold used by Zeng et al. . Furthermore, locus DYS389I is left out of the default STRait Razor analysis for the 46GY panel and therefore may not be optimally represented in the analyses of this study. This study assessed profile completeness (%), coverage range, average coverage, heterozygote balance (HB: the coverage of the lesser allele divided by the coverage of the larger sister allele), below-threshold alleles, drop-in alleles, and dropout. Data were compared to GlobalFiler CE reference profiles on file for buccal and hair samples, and STAFS provided GlobalFiler references for the bone samples. MPS references were produced by consensus from the replicates. Statistical significance was ascertained using an F-test Two-Sample for Variances followed by a Two-Sample, two tailed t -Test assuming equal (or unequal) variances or ANOVA using α 0.05. All statistical analyses were performed using IBM SPSS Statistics Version 29.0 (IBM Corporation, Armonk, NY, USA).
3.1. TruSeq DNA PCR-Free HT Library Preparation Library concentrations ranged from 15 nM to 272 nM (averages 34 nM (63 pg)–188 nM (1 ng)) when using the PowerSeq ® Quant MS System (PowerSeq ® ), 19 nM to 1151 nM (averages 43 nM (63 pg)–506 nM (1 ng)) when using the KAPA quantification kit (KAPA) prior to size adjustment, and from 25 nM to 1530 nM (averages 58 nM (63 pg)–669 nM (1 ng)) when using KAPA’s data analysis template (fragment length adjustment) ( ). The KAPA size-adjustment method reported concentrations approximately 1.3× higher than the concentrations calculated prior to size adjustment and approximately 1.7×–5.6× higher than concentrations calculated using the PowerSeq ® kit. The KAPA size-adjusted concentrations were higher than most PowerSeq ® and all non-size-adjusted KAPA concentrations ( ). When samples were normalized to 4 nM and pooled, the final sample concentration was quantified via the DNA High Sensitivity Kit using the 2100 Bioanalyzer. The pooled libraries normalized by each library quantification method resulted in final library concentrations below the 4 nM target, meaning that libraries were overestimated by qPCR and thus overdiluted. However, PowerSeq-reported library concentrations were the most accurate, relative to final quantification with the bioanalyzer, and could be adjusted for sequencing during library denaturation and dilution. The reason the PowerSeq ® and KAPA kits resulted in largely different values, considering both chemistries use similar quantitative principles by measuring the number of adapter-ligated molecules available for sequencing, is uncertain. However, it may be due to the sensitivity of the KAPA kit to concatemers causing an artificially high quantification value per sample (personal communication—KAPA technical support specialist). Profile completeness using the TruSeq library preparation kit ranged from 98.5% to 100% ( ), with a single allele (DYS389I) below the interpretation threshold of 100 reads for six out of ten male samples ( , single blue bar). With future iterations of STRait Razor, utilizing each target of the 46GY panel, full profiles will likely be achievable at 63 pg DNA inputs and possibly lower. Additionally, all loci amplified ( , blue bars) and demonstrated high coverage at all five DNA inputs (0.063 ng–1 ng); however, the 0.5 ng input demonstrated significantly higher ( p < 0.001) coverage than all other inputs except for 1 ng ( ). This result was expected, as the PowerSeq ® 46GY Technical Manual had been optimized for an input of 0.5 ng of DNA. Coverage ranged from 101× (0.125 ng) to 28,079× (0.5 ng), with average coverage between 2690× (0.063 ng) and 4712× (0.5 ng) ( and , blue bars). Using the TruSeq library preparation, the locus with the highest average coverage was DYS437, whereas Moura-Neto et al. found that DYS439 demonstrated the highest coverage throughout all samples. However, they also found that DYS389I/II (combined data in their study) and DYS448 demonstrated the lowest read depth. We also observed this trend for DYS389I and DYS448, but not for DYS389II ( ). The heterozygote balance ranged from 0.41 (63 pg) to 0.99 (0.25, 0.5, and 1 ng). Additionally, the average heterozygote balance was >0.70 for all DNA input amounts and increased as the DNA concentration increased, ranging from 0.70 at 63 pg to 0.87 at 1 ng ( ). Moura-Neto et al. also observed heterozygosity of the autosomal loci, ranging from 0.70 to 0.85. In this study, all heterozygous loci had an average HB of ≥0.73, with the lowest being FGA and the highest being D5S818 with a 0.91 ratio, indicating no heterozygote imbalance (<0.50) ( ). Furthermore, there were no instances of dropout or drop-in using the TruSeq library preparation kit with SPBs. All data were concordant with the reference samples, and negative controls were free of contamination. Of note, at the time this work was completed, the suggested DNA input was 0.5 ng using 30 PCR cycles. However, the technical manual, revised 3/22, suggests a DNA input amount of 1 ng using 29 PCR cycles . 3.2. KAPA HyperPrep Library Preparation Library concentrations ranged from 0.1 nM to 13 nM (averages 0.6 nM (63 pg)–8 nM (0.5 ng)) using the PowerSeq ® quantification kit, 0.6 nM to 59 nM (averages 2 nM (63 pg)–29 nM (0.5 ng)) for the KAPA quantification kit, and from 1 nM to 78 nM (averages 3 nM (63 pg)–39 nM (0.5 ng)) using KAPA’s data analysis template ( ). The KAPA size-adjustment method reported concentrations approximately 1.5× higher than concentrations calculated prior to size adjustment, and approximately 6×–10× higher than concentrations calculated using the PowerSeq ® kit. Similar trends were observed when samples were prepared with the TruSeq library preparation kit. The profile completeness ranged from 0% to 92.7%, with an average profile completeness of 17% to 72% ( ). Unlike the TruSeq kit, coverage generally increased as the DNA concentration increased. Overall, for the KAPA HyperPrep kit, coverage was relatively low for all DNA inputs and the amplification of the loci was highly variable, resulting in inconsistent locus coverage across all samples ( ). Locus coverage ranged from 0× (dropout) to 5356× (1 ng), with the average coverage between 358× (0.125 ng) and 698× (1 ng) ( ), which is approximately 5×–8× lower than the average coverage obtained using the TruSeq kit and SPBs. Of the loci that amplified using the KAPA kit, Y-GATA-H4 yielded the highest average coverage and DYS448 yielded the lowest average coverage, the latter being similarly observed in Moura-Neto et al. and Silva et al. ( , orange bars). Excluding dropout (heterozygote balance of zero), heterozygote balance otherwise ranged from 0.40 (0.125 ng) to 1 (1 ng). Average HB was ≥0.14 for all DNA inputs, generally increasing as the DNA concentration increased, ranging from 0.14 (63 pg) to 0.74 (0.5 ng) ( ). DNA inputs of 63 pg and 0.125 ng resulted in <0.50 average heterozygote balance, i.e., imbalance, although Zeng et al. only observed more heterozygote imbalance at 62 pg and below. Additionally, two loci (D16S539 and D22S1045) exhibited complete dropout in all samples, as discussed below. Barring these two loci, all heterozygous loci had an average HB ≥ 0.11, with D18S51 being the lowest and D19S433 being the highest at 0.78 ( ). In addition, seven of the twenty-one remaining loci without dropout (33%) demonstrated heterozygote imbalance, including D10S1248, D12S391, D18S51, D1S1656, D2S441, FGA, and PentaE ( ). Zeng et al. noted that this imbalance is likely due to alleles having a greater size differential at certain loci, including D2S1338 and PentaE. However, this could depend on the genotype specific allele ranges, which vary based on the donor. We observed this trend in Penta E, but not in D2S1338. Coverage below 50× indicated dropout, and occurred in every sample and from many loci, totaling 317 instances with the HyperPrep kit, the most dropout of the three library preparation methods. For the female samples, there were 154 instances of dropout including two entire samples, which failed to amplify. For the male samples, there were 163 instances of dropout ( , orange bars); however, unlike the female samples, every sample amplified. A total of 43 (93.5%) of 46 loci resulted in dropout instances, including 29 at D16S539, 27 at D22S1045, 25 at D18S51, 24 at D10S1248, 20 at D12S391, and 14 or fewer for the remaining loci. Two Y-STRs (DYS392 and DYS389I) dropped out in every male sample ( ). No drop-in alleles were observed when the samples were prepared using the KAPA kit. Additionally, the KAPA kit produced the most instances of below-threshold alleles, with a total of 187 instances, occurring in every sample ( , orange bars). Below-threshold alleles were observed in 39 of the 46 loci (84.8%), with 13 instances in D5S818, 12 instances in PentaE and D2S441, 11 instances in D12S391, and 10 instances or fewer in the remaining loci ( ). All data were concordant with the reference samples, and negative controls were free of contamination. Riman et al. compared the TruSeq HT Library Preparation Kit with the KAPA HyperPrep Kit and found that the KAPA-prepared libraries exhibited higher yields of adapter-ligated libraries at all DNA input amounts, while our study revealed opposite findings ( ). However, in our study, the KAPA protocol was not optimized for the 46GY panel like the TruSeq protocol. Unlike TruSeq, modifications were required for the KAPA protocol to be compatible with the 46GY panel and a forensic workflow. These modifications included changes to the amplification temperatures and cycle number, adapter concentrations, and bead-to-template ratios. The optimization issues may have been the cause of KAPA’s noticeably lower library yield, profile completeness, and average coverage. In this study, the minimum modifications possible were made to the KAPA protocol to make it compatible with the 46GY workflow. It is possible that with further alterations and optimization, the KAPA kit may generate higher average coverage with the 46GY panel than that reported here. 3.3. TruSeq Library Preparation with AMPure XP Bead Purification Library concentrations ranged from 6 nM to 411 nM (averages 14 nM (63 pg)–313 nM (1 ng)) using the PowerSeq ® kit, 32 nM to 2004 nM (averages 67 nM (63 pg)–1432 nM (1 ng)) using the KAPA kit, and 43 nM to 2661 nM (averages 89 nM (63 pg)–1903 nM (1 ng)) using KAPA’s data analysis template ( ). KAPA size-adjusted concentrations were approximately 1.3× higher than concentrations calculated prior to size-adjustment, and approximately 6.3× higher than concentrations calculated using the PowerSeq ® kit, which was similar to what was observed in the two previous experiments when libraries were prepared via the TruSeq and KAPA library preparation kits. For the TruSeq library preparation with AMPure XP beads, profile completeness ranged from 36.4% to 100%, with an average of 84% to 100% ( ). Four male samples resulted in a profile completeness less than 100%. Three samples (one at 0.125 ng and two at 0.063 ng) were missing one allele (DYS389I). Additionally, a 1 ng sample was missing 42 out of 66 alleles (36.4% complete). This sample is likely a result of poor amplification efficiency, as the replicate resulted in 100% profile completeness. Overall, the TruSeq library preparation with AMPure XP purification resulted in the highest coverage across the three library preparation methods ( , gray bars). In contrast to the other two library preparation methods, 0.25 ng DNA input demonstrated the highest coverage, and was significantly higher ( p = 0.004) than all but the 0.5 ng input ( ). Coverage ranged from 103× (1 ng) to 22,320× (0.125 ng), with the 0.25 ng DNA input demonstrating the lowest and highest total coverage for a single concentration. The average coverage for these samples ranged from 4670× (1 ng) to 5673× (0.25 ng) ( ). The locus that yielded the highest average coverage was DYS392, which was similarly observed in Silva et al. . The locus that yielded the lowest average coverage, aside from DYS389I, was D3S1358 ( ). Additionally, the heterozygote balance ranged from 0.40 (63 pg) to 1 (1 ng). The average HB was ≥0.70 for all DNA inputs, generally increasing as the DNA concentration increased, ranging from 0.70 (63 pg) to 0.85 (0.5 ng) ( ). All heterozygous loci had an average HB ≥ 0.65, with Amelogenin being the lowest and CSF1PO being the highest at 0.90 ( ), showing no heterozygote Imbalance. In contrast, Silva et al. and Hölzl-Müller et al. found that D2S1338 was the most susceptible locus to heterozygote imbalance out of all the autosomal loci in the 46GY panel. The locus coverage ranged from 0× (dropout) to a maximum coverage of 22,320×, with a dropout in 3 of 20 samples, totaling 17 instances of dropout ( , gray bars). Dropout occurred in 13 of 46 loci (28.2%) and only in male samples, with 15 instances in a 1 ng sample, and one instance in replicate samples at 0.063 ng. Additionally, no drop-in alleles were observed using TruSeq library preparation with AMPure XP purification. However, there were 28 instances of below-threshold alleles in 2 out of 20 samples, both male, at 1 ng and 0.125 ng DNA inputs. A total of 21 of 46 loci (45.7%) resulted in two or fewer below-threshold alleles. The majority of below-threshold alleles (27 out of 28) were in the same 1 ng sample as the 15 instances of dropout, and it is believed that this was due to insufficient amplification ( , gray bars). All data were concordant with the reference samples, and negative controls were free of contamination. 3.4. Casework-Type Samples with the Enhanced Library Preparation Method One objective for this study was to determine the most accurate library quantification kit. Of the three quantification methods tested, the PowerSeq ® Quant MS System provided the most accurate library quantification results prior to library normalization and sequencing. Because of this, we quantified our bone and hair libraries with the PowerSeq ® kit only. Library concentrations ranged from 1 nM to 20 nM for bone samples and <1 nM to 24 nM for hair samples ( ). Of the six bone samples and four hair samples amplified in duplicate, one replicate set of hair samples (HQA and HQB) was contaminated and removed from analysis. Reagent blanks and negative controls were free of contamination. All bone and hair samples resulted in 100% profile completeness and high sample coverage for all samples. Coverage ranged from 388× to 128,906× for bone samples and 130× to 56,077× for hair samples. The average coverage for bone samples ranged from 7955× (B12A) to 34,332× (B9B), and from 8835× (HPB) to 16,102× (HKA) for hair samples ( ). In general, the coverage for hair samples was lower than that of bone samples. For all but two samples (B9 and B12), replicates resulted in similar coverage. For B9 replicate B the coverage was approximately 8000× higher than replicate A, and for B12 replicate B was approximately 26,000× higher than replicate A. This may be due to inefficient amplification, over dilution prior to end repair, stochastic variability, or inhibition from residual ethanol or cracked beads during cleanup. Additionally, the average HB was ≥0.74 for all samples, with bone and hair samples both yielding an overall average of ~0.80 ( ). All heterozygous loci had an average HB ≥ 0.67, with PentaE being the lowest and D1S1656 being the highest, at 0.89 ( ). Furthermore, PentaE was the only locus with an average HB below 0.70. All samples resulted in full profiles; therefore, no instances of dropout or below-threshold alleles were observed. Degradation Indices (DI) for these samples were between 0.78 and 2.57, with six samples indicating slight to moderate degradation (DI: 1–10) according to the Quantifiler Trio manual ( ) . However, the sequencing results displayed little to no indication of degradation. Additionally, when MPS profiles were compared to CE reference profiles, between one and three isoalleles were identified in all but one donor (B12). Isoalleles were identified in five autosomal STRs, including D2S441 (x1), D5S818 (x1), D7S820 (x1), D13S317 (x4), and D21S11 (x2), and one Y-STR, DYS393 (x3). The five autosomal STRs listed above were five of the thirteen STRs found to have isometric heterozygosity in Hölzl-Müller et al. .
Library concentrations ranged from 15 nM to 272 nM (averages 34 nM (63 pg)–188 nM (1 ng)) when using the PowerSeq ® Quant MS System (PowerSeq ® ), 19 nM to 1151 nM (averages 43 nM (63 pg)–506 nM (1 ng)) when using the KAPA quantification kit (KAPA) prior to size adjustment, and from 25 nM to 1530 nM (averages 58 nM (63 pg)–669 nM (1 ng)) when using KAPA’s data analysis template (fragment length adjustment) ( ). The KAPA size-adjustment method reported concentrations approximately 1.3× higher than the concentrations calculated prior to size adjustment and approximately 1.7×–5.6× higher than concentrations calculated using the PowerSeq ® kit. The KAPA size-adjusted concentrations were higher than most PowerSeq ® and all non-size-adjusted KAPA concentrations ( ). When samples were normalized to 4 nM and pooled, the final sample concentration was quantified via the DNA High Sensitivity Kit using the 2100 Bioanalyzer. The pooled libraries normalized by each library quantification method resulted in final library concentrations below the 4 nM target, meaning that libraries were overestimated by qPCR and thus overdiluted. However, PowerSeq-reported library concentrations were the most accurate, relative to final quantification with the bioanalyzer, and could be adjusted for sequencing during library denaturation and dilution. The reason the PowerSeq ® and KAPA kits resulted in largely different values, considering both chemistries use similar quantitative principles by measuring the number of adapter-ligated molecules available for sequencing, is uncertain. However, it may be due to the sensitivity of the KAPA kit to concatemers causing an artificially high quantification value per sample (personal communication—KAPA technical support specialist). Profile completeness using the TruSeq library preparation kit ranged from 98.5% to 100% ( ), with a single allele (DYS389I) below the interpretation threshold of 100 reads for six out of ten male samples ( , single blue bar). With future iterations of STRait Razor, utilizing each target of the 46GY panel, full profiles will likely be achievable at 63 pg DNA inputs and possibly lower. Additionally, all loci amplified ( , blue bars) and demonstrated high coverage at all five DNA inputs (0.063 ng–1 ng); however, the 0.5 ng input demonstrated significantly higher ( p < 0.001) coverage than all other inputs except for 1 ng ( ). This result was expected, as the PowerSeq ® 46GY Technical Manual had been optimized for an input of 0.5 ng of DNA. Coverage ranged from 101× (0.125 ng) to 28,079× (0.5 ng), with average coverage between 2690× (0.063 ng) and 4712× (0.5 ng) ( and , blue bars). Using the TruSeq library preparation, the locus with the highest average coverage was DYS437, whereas Moura-Neto et al. found that DYS439 demonstrated the highest coverage throughout all samples. However, they also found that DYS389I/II (combined data in their study) and DYS448 demonstrated the lowest read depth. We also observed this trend for DYS389I and DYS448, but not for DYS389II ( ). The heterozygote balance ranged from 0.41 (63 pg) to 0.99 (0.25, 0.5, and 1 ng). Additionally, the average heterozygote balance was >0.70 for all DNA input amounts and increased as the DNA concentration increased, ranging from 0.70 at 63 pg to 0.87 at 1 ng ( ). Moura-Neto et al. also observed heterozygosity of the autosomal loci, ranging from 0.70 to 0.85. In this study, all heterozygous loci had an average HB of ≥0.73, with the lowest being FGA and the highest being D5S818 with a 0.91 ratio, indicating no heterozygote imbalance (<0.50) ( ). Furthermore, there were no instances of dropout or drop-in using the TruSeq library preparation kit with SPBs. All data were concordant with the reference samples, and negative controls were free of contamination. Of note, at the time this work was completed, the suggested DNA input was 0.5 ng using 30 PCR cycles. However, the technical manual, revised 3/22, suggests a DNA input amount of 1 ng using 29 PCR cycles .
Library concentrations ranged from 0.1 nM to 13 nM (averages 0.6 nM (63 pg)–8 nM (0.5 ng)) using the PowerSeq ® quantification kit, 0.6 nM to 59 nM (averages 2 nM (63 pg)–29 nM (0.5 ng)) for the KAPA quantification kit, and from 1 nM to 78 nM (averages 3 nM (63 pg)–39 nM (0.5 ng)) using KAPA’s data analysis template ( ). The KAPA size-adjustment method reported concentrations approximately 1.5× higher than concentrations calculated prior to size adjustment, and approximately 6×–10× higher than concentrations calculated using the PowerSeq ® kit. Similar trends were observed when samples were prepared with the TruSeq library preparation kit. The profile completeness ranged from 0% to 92.7%, with an average profile completeness of 17% to 72% ( ). Unlike the TruSeq kit, coverage generally increased as the DNA concentration increased. Overall, for the KAPA HyperPrep kit, coverage was relatively low for all DNA inputs and the amplification of the loci was highly variable, resulting in inconsistent locus coverage across all samples ( ). Locus coverage ranged from 0× (dropout) to 5356× (1 ng), with the average coverage between 358× (0.125 ng) and 698× (1 ng) ( ), which is approximately 5×–8× lower than the average coverage obtained using the TruSeq kit and SPBs. Of the loci that amplified using the KAPA kit, Y-GATA-H4 yielded the highest average coverage and DYS448 yielded the lowest average coverage, the latter being similarly observed in Moura-Neto et al. and Silva et al. ( , orange bars). Excluding dropout (heterozygote balance of zero), heterozygote balance otherwise ranged from 0.40 (0.125 ng) to 1 (1 ng). Average HB was ≥0.14 for all DNA inputs, generally increasing as the DNA concentration increased, ranging from 0.14 (63 pg) to 0.74 (0.5 ng) ( ). DNA inputs of 63 pg and 0.125 ng resulted in <0.50 average heterozygote balance, i.e., imbalance, although Zeng et al. only observed more heterozygote imbalance at 62 pg and below. Additionally, two loci (D16S539 and D22S1045) exhibited complete dropout in all samples, as discussed below. Barring these two loci, all heterozygous loci had an average HB ≥ 0.11, with D18S51 being the lowest and D19S433 being the highest at 0.78 ( ). In addition, seven of the twenty-one remaining loci without dropout (33%) demonstrated heterozygote imbalance, including D10S1248, D12S391, D18S51, D1S1656, D2S441, FGA, and PentaE ( ). Zeng et al. noted that this imbalance is likely due to alleles having a greater size differential at certain loci, including D2S1338 and PentaE. However, this could depend on the genotype specific allele ranges, which vary based on the donor. We observed this trend in Penta E, but not in D2S1338. Coverage below 50× indicated dropout, and occurred in every sample and from many loci, totaling 317 instances with the HyperPrep kit, the most dropout of the three library preparation methods. For the female samples, there were 154 instances of dropout including two entire samples, which failed to amplify. For the male samples, there were 163 instances of dropout ( , orange bars); however, unlike the female samples, every sample amplified. A total of 43 (93.5%) of 46 loci resulted in dropout instances, including 29 at D16S539, 27 at D22S1045, 25 at D18S51, 24 at D10S1248, 20 at D12S391, and 14 or fewer for the remaining loci. Two Y-STRs (DYS392 and DYS389I) dropped out in every male sample ( ). No drop-in alleles were observed when the samples were prepared using the KAPA kit. Additionally, the KAPA kit produced the most instances of below-threshold alleles, with a total of 187 instances, occurring in every sample ( , orange bars). Below-threshold alleles were observed in 39 of the 46 loci (84.8%), with 13 instances in D5S818, 12 instances in PentaE and D2S441, 11 instances in D12S391, and 10 instances or fewer in the remaining loci ( ). All data were concordant with the reference samples, and negative controls were free of contamination. Riman et al. compared the TruSeq HT Library Preparation Kit with the KAPA HyperPrep Kit and found that the KAPA-prepared libraries exhibited higher yields of adapter-ligated libraries at all DNA input amounts, while our study revealed opposite findings ( ). However, in our study, the KAPA protocol was not optimized for the 46GY panel like the TruSeq protocol. Unlike TruSeq, modifications were required for the KAPA protocol to be compatible with the 46GY panel and a forensic workflow. These modifications included changes to the amplification temperatures and cycle number, adapter concentrations, and bead-to-template ratios. The optimization issues may have been the cause of KAPA’s noticeably lower library yield, profile completeness, and average coverage. In this study, the minimum modifications possible were made to the KAPA protocol to make it compatible with the 46GY workflow. It is possible that with further alterations and optimization, the KAPA kit may generate higher average coverage with the 46GY panel than that reported here.
Library concentrations ranged from 6 nM to 411 nM (averages 14 nM (63 pg)–313 nM (1 ng)) using the PowerSeq ® kit, 32 nM to 2004 nM (averages 67 nM (63 pg)–1432 nM (1 ng)) using the KAPA kit, and 43 nM to 2661 nM (averages 89 nM (63 pg)–1903 nM (1 ng)) using KAPA’s data analysis template ( ). KAPA size-adjusted concentrations were approximately 1.3× higher than concentrations calculated prior to size-adjustment, and approximately 6.3× higher than concentrations calculated using the PowerSeq ® kit, which was similar to what was observed in the two previous experiments when libraries were prepared via the TruSeq and KAPA library preparation kits. For the TruSeq library preparation with AMPure XP beads, profile completeness ranged from 36.4% to 100%, with an average of 84% to 100% ( ). Four male samples resulted in a profile completeness less than 100%. Three samples (one at 0.125 ng and two at 0.063 ng) were missing one allele (DYS389I). Additionally, a 1 ng sample was missing 42 out of 66 alleles (36.4% complete). This sample is likely a result of poor amplification efficiency, as the replicate resulted in 100% profile completeness. Overall, the TruSeq library preparation with AMPure XP purification resulted in the highest coverage across the three library preparation methods ( , gray bars). In contrast to the other two library preparation methods, 0.25 ng DNA input demonstrated the highest coverage, and was significantly higher ( p = 0.004) than all but the 0.5 ng input ( ). Coverage ranged from 103× (1 ng) to 22,320× (0.125 ng), with the 0.25 ng DNA input demonstrating the lowest and highest total coverage for a single concentration. The average coverage for these samples ranged from 4670× (1 ng) to 5673× (0.25 ng) ( ). The locus that yielded the highest average coverage was DYS392, which was similarly observed in Silva et al. . The locus that yielded the lowest average coverage, aside from DYS389I, was D3S1358 ( ). Additionally, the heterozygote balance ranged from 0.40 (63 pg) to 1 (1 ng). The average HB was ≥0.70 for all DNA inputs, generally increasing as the DNA concentration increased, ranging from 0.70 (63 pg) to 0.85 (0.5 ng) ( ). All heterozygous loci had an average HB ≥ 0.65, with Amelogenin being the lowest and CSF1PO being the highest at 0.90 ( ), showing no heterozygote Imbalance. In contrast, Silva et al. and Hölzl-Müller et al. found that D2S1338 was the most susceptible locus to heterozygote imbalance out of all the autosomal loci in the 46GY panel. The locus coverage ranged from 0× (dropout) to a maximum coverage of 22,320×, with a dropout in 3 of 20 samples, totaling 17 instances of dropout ( , gray bars). Dropout occurred in 13 of 46 loci (28.2%) and only in male samples, with 15 instances in a 1 ng sample, and one instance in replicate samples at 0.063 ng. Additionally, no drop-in alleles were observed using TruSeq library preparation with AMPure XP purification. However, there were 28 instances of below-threshold alleles in 2 out of 20 samples, both male, at 1 ng and 0.125 ng DNA inputs. A total of 21 of 46 loci (45.7%) resulted in two or fewer below-threshold alleles. The majority of below-threshold alleles (27 out of 28) were in the same 1 ng sample as the 15 instances of dropout, and it is believed that this was due to insufficient amplification ( , gray bars). All data were concordant with the reference samples, and negative controls were free of contamination.
One objective for this study was to determine the most accurate library quantification kit. Of the three quantification methods tested, the PowerSeq ® Quant MS System provided the most accurate library quantification results prior to library normalization and sequencing. Because of this, we quantified our bone and hair libraries with the PowerSeq ® kit only. Library concentrations ranged from 1 nM to 20 nM for bone samples and <1 nM to 24 nM for hair samples ( ). Of the six bone samples and four hair samples amplified in duplicate, one replicate set of hair samples (HQA and HQB) was contaminated and removed from analysis. Reagent blanks and negative controls were free of contamination. All bone and hair samples resulted in 100% profile completeness and high sample coverage for all samples. Coverage ranged from 388× to 128,906× for bone samples and 130× to 56,077× for hair samples. The average coverage for bone samples ranged from 7955× (B12A) to 34,332× (B9B), and from 8835× (HPB) to 16,102× (HKA) for hair samples ( ). In general, the coverage for hair samples was lower than that of bone samples. For all but two samples (B9 and B12), replicates resulted in similar coverage. For B9 replicate B the coverage was approximately 8000× higher than replicate A, and for B12 replicate B was approximately 26,000× higher than replicate A. This may be due to inefficient amplification, over dilution prior to end repair, stochastic variability, or inhibition from residual ethanol or cracked beads during cleanup. Additionally, the average HB was ≥0.74 for all samples, with bone and hair samples both yielding an overall average of ~0.80 ( ). All heterozygous loci had an average HB ≥ 0.67, with PentaE being the lowest and D1S1656 being the highest, at 0.89 ( ). Furthermore, PentaE was the only locus with an average HB below 0.70. All samples resulted in full profiles; therefore, no instances of dropout or below-threshold alleles were observed. Degradation Indices (DI) for these samples were between 0.78 and 2.57, with six samples indicating slight to moderate degradation (DI: 1–10) according to the Quantifiler Trio manual ( ) . However, the sequencing results displayed little to no indication of degradation. Additionally, when MPS profiles were compared to CE reference profiles, between one and three isoalleles were identified in all but one donor (B12). Isoalleles were identified in five autosomal STRs, including D2S441 (x1), D5S818 (x1), D7S820 (x1), D13S317 (x4), and D21S11 (x2), and one Y-STR, DYS393 (x3). The five autosomal STRs listed above were five of the thirteen STRs found to have isometric heterozygosity in Hölzl-Müller et al. .
The goal of this research was to determine whether modifications to the 46GY workflow could improve the overall result quality and sequencing coverage beyond what is achieved following the manufacturer’s protocol, especially for casework-type samples. However, there was at least one limitation of this study, and that was small sample size. Only two buccal donors were used to assess the three library preparation methods prior to evaluating casework-type samples. In the first study, the 46GY panel was found to be compatible with the TruSeq DNA PCR-Free HT Library Preparation Kit and TruSeq SPBs, with very few instances of below-threshold alleles and no instances of dropout. Coverage was high, and profiles were nearly complete for all DNA inputs from 1 ng down to 63 pg with a heterozygote balance ≥ 0.70. Additionally, all three quantification methods tested (PowerSeq ® Quant MS System, KAPA Library Quantification Kit for Illumina ® Platforms, and KAPA using a data analysis template) overquantified libraries resulting in excessive dilution; however, the PowerSeq ® quantification system was the most accurate and provided usable quantification values that produced quality sequencing results. While there was compatibility between the 46GY panel and the KAPA HyperPrep Kit, this combination was not optimized for the best results. Results demonstrated low coverage and over 500 instances of dropout and below-threshold alleles, with heterozygote balance as low as 0.14 at 63 pg input. This also confirmed that the PowerSeq ® quantification kit was the most accurate prior to sequencing. Following the establishment of the more optimal library preparation kit (TruSeq), the next step was to determine if the use of AMPure XP beads in lieu of SPBs would improve downstream results. This method resulted in the highest overall coverage and heterozygote balance ≥ 0.70 at all DNA input amounts; however, 45 instances of dropout and below-threshold alleles were observed due to inefficient amplification. Lastly, bone and hair samples were tested with the most optimal library and quantification methods, as determined by the previous three studies, i.e., the TruSeq library preparation with SPBs and PowerSeq ® quantification, respectively. All casework-type samples resulted in full profiles with high coverage, no instances of dropout or below-threshold alleles, and a heterozygote balance ≥ 0.75. Overall, the 46GY panel produced the highest quality results with the manufacturer’s protocol. However, while not stated in the protocol, the authors recommend additional QC steps post-normalization for the best quality sequencing results. Quantifying the normalized library pool ensures that the correct concentrations of sequencing pool components are used to dilute and denature the pool prior to sequencing.
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The Role of Pharmacogenetics in Personalizing the Antidepressant and Anxiolytic Therapy
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a7ee6812-524e-408b-a297-b4600d0e9f10
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10218654
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Pharmacology[mh]
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The pharmacotherapy of neuropsychiatric disorders, such as anxiety and depression, has been characterized by significant inter-individual variability in drug response and the development of severe adverse effects, which has been recognized as a major clinical problem . In addition to various environmental, physiological, and psychological factors, these individual differences might be largely due to genetic factors . Therefore, various pharmacogenetic studies have been conducted to identify genetic variants that can predict patients who may optimally benefit from specific, individually tailored treatments . Pharmacogenetic studies have focused primarily on candidate genes involved in drug metabolism and transport (pharmacokinetics) as well as drug action (pharmacodynamics), which can influence both treatment efficacy and the development of adverse drug effects . Pharmacokinetics addresses the variability in the drug’s absorption, distribution, metabolism, and elimination (ADME), which modulates the delivery of drugs and their active metabolites or their removal from action targets. The molecules involved in ADME processes include enzymes responsible for drug metabolism and drug transport molecules that mediate drug uptake and efflux . In this context, the cytochrome P450 (CYP) and multidrug resistance (MDR) gene families have been extensively studied . The CYP450 enzyme family in the liver is responsible for the metabolism of many psychotropic drugs . For certain CYPs, the genotype affects the serum/plasma drug levels, and consequently, its efficacy and development of adverse effects . There are four major CYP phenotypes produced by combinations of various alleles with different degrees of enzymatic activity: poor (PM), intermediate (IM), extensive (normal) (EM), and ultrarapid metabolizer (UM). PMs tend to accumulate higher drug levels in the blood and may require lower drug doses to achieve therapeutic effects, whereas UMs may require higher doses due to faster drug elimination . In addition, the therapeutic action of psychotropic drugs depends on their effective delivery to the brain. Although some substances may diffuse passively through the brain-blood barrier (BBB), the influx and efflux of most substances are actively regulated by a complex system of transporters, influencing both pharmacokinetics and pharmacodynamics. In the case of a genetically determined decrease in functional activity or expression of transport proteins in the BBB, drug efflux from the brain into the blood is disturbed. This could lead to increased drug exposure time in the brain, its accumulation during long-term therapy, and an increased risk of developing severe adverse effects . In contrast to pharmacokinetics, pharmacodynamics describes variability in drug action not dependent on variable drug concentrations but rather on the interaction of the active drug with its target molecules, including receptors, ion channels, and enzymes, and it can also influence both therapeutic responses and drug side effects. Single nucleotide polymorphisms (SNPs) are the most commonly investigated genetic variants in both pharmacokinetic and pharmacodynamic studies . Genetic polymorphism refers to the occurrence of two or more common variants (alleles) of a specific DNA sequence in a population with a frequency of more than 1% . Identifying SNPs associated with variability in drug response and toxicity has been the primary focus of a significant number of pharmacogenetic and pharmacogenomic studies . In these studies, two major research approaches have been used: the traditional candidate gene approach, which is hypothesis-driven and based on accumulated knowledge, and new methodologies such as genome-wide association studies (GWAS) or whole exome sequencing, which are data-driven and generate new hypotheses and knowledge . However, pharmacogenetics is not able to explain all observed heritable variations in drug responses, and there is growing evidence that responses to drugs could be influenced by individual epigenetic states . An emerging field of pharmacoepigenetics investigates how epigenetic mechanisms that modify gene expression without altering the genetic code might influence individual responses to drugs . Some of the most frequently studied epigenetic modifications include DNA methylation, histone modifications, and noncoding RNA actions . Various environmental factors, including drugs, nutrition, and stress, may induce epigenetic changes that can be transmitted over generations . Acute or chronic exposure to stressors can contribute to the development and progression of various neuropsychiatric disorders , including anxiety and depression, but it is also associated with alterations in the epigenome that may affect the expression of genes involved in drug metabolism, transport, and target molecules, and therefore impact the variability in antidepressant and anxiolytic drug responses . The most significant findings, obtained by both pharmacogenetic and pharmacoepigenetic research on antidepressants and anxiolytics, drugs commonly used for the therapy of various neuropsychiatric disorders, have been summarized and discussed in this review. Antidepressants are drugs commonly used to treat depression and anxiety disorders, although only half of patients respond to treatment, and only a third of patients experience symptom remission . Genetic factors seem to account for more than 60% of the variability in drug response and side effects for various types of antidepressant drugs, including selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), tricyclic (TCAs) and tetracyclic compounds, monoamine oxidase inhibitors (MAOIs), and noradrenergic and serotonergic modulators . Therefore, an individual’s genetic profile should be considered when choosing an antidepressant and determining the appropriate drug dosage. However, despite ongoing research, the effects of genetic variants on the pharmacokinetics and pharmacodynamics of antidepressant drugs remain unclear. Discovering the genetic factors that contribute to the variability of antidepressant responses can help clinicians select the most appropriate medication, dosage, and treatment duration for each individual patient. Therefore, the application of precision medicine may improve the treatment response rates to antidepressants and minimize the risk of adverse drug reactions. 2.1. Pharmacokinetic Variability 2.1.1. Cytochrome P450 Family The CYP450 family is a large group of enzymes responsible for the metabolism of various drugs and xenobiotics, including antidepressant drugs. Among the many identified CYP450 enzymes, the most important ones involved in the metabolism of a variety of psychotropic drugs are CYP2D6, CYP2C19, CYP3A4, CYP1A2, CYP2B6, CYP2C8, and CYP2C9 . Various CYP450 enzymes are capable of metabolizing more than one drug, and a single drug can be metabolized by multiple CYP enzymes . The activity of these enzymes could be influenced by genetic variations, which may result in individual differences in antidepressant drug metabolism and responses . Most genes encoding CYPs are highly polymorphic . The impact of CYP polymorphisms on drug metabolism is an important area of research in personalized medicine . While more than 2000 mutations have been found in CYP genes, only specific SNPs are known to affect CYP enzymatic activity . Depending on the genetic variants that influence enzyme activity, individuals can be classified into four main CYP phenotypes: from poor, via intermediate and extensive (normal), to ultrarapid metabolizers . Due to genetic variations, PMs have little to no CYP enzyme activity, whereas IMs have reduced enzyme activity. As a result of slower antidepressant drug metabolism, both PMs and IMs may be at an increased risk for adverse drug reactions and require lower dosages or less frequent dosing regimens for antidepressant medications. On the other hand, NMs possess typical enzyme activity and a normal drug metabolism rate. However, UMs exhibit increased enzymatic activity, resulting in faster drug metabolism. Therefore, they may require higher antidepressant doses or more frequent drug administration to achieve the desired therapeutic effect in patients with depression . The pharmacogenetics of the most important CYPs involved in the metabolism of antidepressant drugs are summarized below. CYP2D6 The CYP2D6 gene is highly polymorphic, meaning that it harbors genetic variations that can affect enzyme function . CYP2D6 enzyme is involved in the metabolism of SSRIs (paroxetine, fluvoxamine, and fluoxetine), amitriptyline (TCA), and venlafaxine (SNRI) . Different allele variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . CYP2D6*1 , *2 , *33 , and *35 allele variants are associated with normal function, whereas *9 , *10 , *14B , *17 , *29 , and *41 variants reduce CYP2D6 enzymatic capacity. Furthermore, CYP2D6*3 , *4 , *5 , *6 , *7 , *11 , *12 , *14A , *36 , and *68 variants are responsible for the loss of enzyme function . Apart from genetic variations, paroxetine and fluoxetine are potent inhibitors of CYP2D6, which may result in an “iatrogenic poor phenotype” or “phenocopy” when taken with drugs that are also metabolized by CYP2D6, such as venlafaxine. Patients with an “iatrogenic poor phenotype” may be at high risk of developing toxicity due to elevated plasma drug levels . Specifically, individuals with poor CYP2D6 function may have reduced metabolism of fluoxetine and paroxetine, resulting in higher plasma drug levels and an increased risk of side effects, such as the development of suicidal ideation or antidepressant-induced mania during the initiation of treatment. As a result, the U.S. Food and Drug Administration (FDA) has issued a black box warning for SSRIs cautioning that these side effects can occur, especially in the first few weeks of treatment. Therefore, close monitoring is required for all patients starting antidepressant therapy . Furthermore, studies have shown that poor metabolizers of fluoxetine may be at a risk of QT interval prolongation. . Similar to fluoxetine, poor metabolizers of venlafaxine (genotype CYP2D6 *6 / *4 , *5 / *4 , or *6 / *6 ) are also at risk for developing adverse effects, such as arrhythmias, gastrointestinal problems, and hyponatremia . Ahmed et al. showed that CYP2D6 UM status contributes to venlafaxine treatment remission in patients with major depressive disorder . Therefore, when prescribing SSRIs and other antidepressants, clinicians must consider a patient’s CYP2D6 genotype and adjust the dose or choose an alternative medication, if necessary, to minimize the risk of adverse effects while maximizing therapeutic benefit . Despite the growing literature on the clinical implications of the CYP2D6 genotype and phenoconversion on patient-related outcomes, implementation of pharmacogenetics to guide antidepressant prescribing is rare. CYP2C19 In addition to clinically significant variants of the CYP2D6 gene, a high number of polymorphisms in the CYP2C19 gene have also been discovered , resulting in different metabolizer phenotypes . Drugs that are mainly metabolized by CYP2C19 include SSRIs such as escitalopram, citalopram, and sertraline . Variations in the CYP2C19 gene can result in individuals having different levels of CYP2C19 enzyme activity, affecting how drugs such as escitalopram and sertraline are metabolized . According to previous research, the CYP2C19*1 allele variant is associated with normal function, whereas the CYP2C19*17 variant increases CYP2C19 enzymatic capacity. Furthermore, CYP2C19*2 and *3 variants are responsible for the loss of enzyme function . For example, PMs of escitalopram are more likely to have adverse effects and psychotherapy discontinuation. However, they respond better to escitalopram, provided the therapy is tolerated. A retrospective study involving more than 2000 escitalopram-treated patients demonstrated that those with CYP2C19*1 / *17 and CYP2C19*17 / *17 variants (IMs) were more likely to experience treatment failure , whereas UMs exhibited suicidal thoughts . For sertraline, recent findings demonstrated that CYP2C19 PMs had higher plasma levels in comparison to normal metabolizers . Ricardo-Silgado et al. suggested that CYP2C19 genotypes might be associated with an increased risk of weight gain in patients on citalopram therapy. These findings indicate the importance of CYP2C19 variants and different metabolic phenotypes in antidepressant treatment tolerability and outcome. The American Molecular Pathology published guidelines for testing CYP2C19*2 , *3 , and *17 variants and CYP2C19*4A-*4B , *5 , *6 , *7 , *8 , *9 , *10, and *35 variants . In addition, the relevance of CYP2C19 polymorphisms in inter-individual predisposition to mental diseases was also investigated. Sim et al. found that individuals who were poor CYP2C19 metabolizers displayed lower levels of depressive symptoms compared to individuals who were normal metabolizers. These results raise the possibility that CYP2C19 variations contribute to susceptibility to depression. Recent studies have also demonstrated a correlation between low CYP2C19 activity and the severity of depression symptoms . Moreover, a cross-sectional observational retrospective study, which included over 700 psychiatric patients with depression and anxiety, analyzed the frequencies of CYPC19*2 , *4 , and *17 , as well as CYP2D6*2 , *3 , *4 , *5 , *6 , *10 , and *41 variants . This study revealed that 77% of patients have at least one allele variant significantly affecting drug metabolism, with roughly half of the individuals with reduced CYP2D6 enzyme function and the majority of them being CYP2C19 rapid and ultrarapid metabolizers . Therefore, due to their clinical relevance, both CYP2D6 and CYP2C19 are included in pharmacogenetics guidelines and recommendations published by the Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), and regulatory agencies such as the FDA . CYP2C9 The CYP2C9 gene encodes an enzyme that is involved in the metabolism of many drugs, including antidepressants, such as MAOIs, TCAs, and SSRIs . The CYP2C9*1 variant is associated with normal enzyme function, whereas CYP2C9 * 2 , *5 , *8 , and *11 variants reduce CYP2C9 enzymatic capacity. Furthermore, CYP2D9*3 , *6 , and *13 variants are responsible for the loss of enzyme function . Different CYP2C9 variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . Moreover, variants of the CYP2C9 gene have been linked to mental disorders such as depression and psychosis . CYP1A2 The CYP1A2 enzyme, encoded by the CYP1A2 gene, is responsible for the metabolism of approximately 24% of antidepressant drugs, including agomelatine, escitalopram, venlafaxine, duloxetine, and mirtazapine . Several CYP1A2 variants, including * 1C , *1F , and *1B , have been identified as potential indicators of the CYP1A2 phenotype and agomelatine pharmacokinetics. Notably, the CYP1A2*1C variant is associated with reduced enzyme activity, resulting in higher plasma agomelatine concentrations and an increased risk of adverse effects. On the contrary, individuals with the CYP1A2 *1F and *1B variants, who are characterized by increased enzyme activity, may metabolize agomelatine more rapidly, leading to lower plasma concentrations and potentially reduced efficacy . Kuo et al. conducted a study to investigate the association between CYP1A2 polymorphisms and the metabolism of escitalopram in patients with major depressive disorder. The study found that several SNPs in the CYP1A2 gene, such as rs2069521, rs4646425, and rs4646427 polymorphisms, were associated with altered escitalopram metabolism and an increased risk of adverse effects, such as fatigue and nausea/vomiting, particularly during the initial stages of treatment. In addition, Lin et al. demonstrated that CYP1A2 SNPs, such as rs4646425, rs2472304, and rs2470890, are associated with a slower response to paroxetine treatment. Furthermore, the study of the linkage between CYP1A2 polymorphisms and the response to venlafaxine suggested that the rs2470890 polymorphism might be related to venlafaxine treatment remission . 2.1.2. P-glycoprotein P-glycoprotein, commonly referred to as P-gp, is a membrane transporter within the ATP-binding cassette (ABC) transporter family. It is encoded by the ABCB1 or MDR1 gene, located on chromosome 7. P-gp functions as an efflux pump, contributing to drug absorption, distribution, and elimination . P-gp has been found in various tissues throughout the body, including the liver, kidneys, intestines, and the blood-brain barrier. At the blood-brain barrier, P-gp can limit the entry of some drugs into the brain, which can affect their efficacy. The expression and activity of P-gp can vary widely between individuals, contributing to differences in drug responses and drug interactions . Genetic factors play a role in determining the level of P-gp expression in different tissues and individuals. Certain genetic variants of the ABCB1 gene have been associated with altered P-gp expression and activity, which can affect the pharmacokinetics and efficacy of drugs that are substrates for P-gp . P-gp has broad substrate specificity, including antidepressants such as escitalopram, fluvoxamine, paroxetine, amitriptyline, and imipramine. Therefore, when P-gp is absent or not functioning properly, these drugs can accumulate in the body, resulting in higher concentrations and potentially an increased risk of adverse effects. However, newer antidepressants (levomilnacipran, vortioxetine, and vilazodone) have been proven as poor P-gp substrates . There has been interest in studying the relationship between genetic variations in the MDR1 / ABCB1 gene and antidepressant treatment outcomes . Several genetic variants of the MDR1 / ABCB1 gene have been associated with altered P-gp activity. The three most common MDR1 / ABCB1 variants, C3435T (rs1045642), C1236T (rs1128503), and G2677T (rs2032582), have been the subject of extensive research . A recent study on experimental models suggested that the 2677G > T polymorphism in the ABCB1 gene has been associated with altered P-gp function and increased brain penetration of P-gp substrates without affecting P-gp protein expression in the blood-brain barrier . In addition, genetic variants of the ABCB1 gene, including the rs2235040 and rs4148739 polymorphisms, may be associated with the onset of response to antidepressant medications rather than the response rate . Furthermore, a significant link has been found between the ABCB1 rs2235015 GG genotype and better response to antidepressant treatment . Overall, while pharmacogenetic research on MDR1 / ABCB1 suggests the potential for improving outcomes of antidepressant treatment, further investigation is necessary to better comprehend the connection between genetic variations and treatment response, as well as to assess its clinical utility. 2.2. Pharmacodynamic Variability 2.2.1. Monoamine Metabolic Enzymes Tryptophan Hydroxylase Tryptophan hydroxylase (TPH) is an enzyme that catalyzes the conversion of the amino acid tryptophan to 5-hydroxytryptophan, which is the first step in the synthesis of serotonin, a neurotransmitter involved in the regulation of mood, appetite, sleep, and stress response . There are two isoforms of the TPH enzyme, TPH1 and TPH2, encoded by separate genes. TPH1 is primarily expressed in peripheral tissues, such as the pineal gland, skin, and gut, whereas TPH2 is mainly expressed in neurons in the central nervous system (CNS). The two isoforms have similar enzymatic activities but differ in regulation, tissue distribution, and developmental expression patterns . Overall, while TPH1 may be less expressed in the brain than TPH2, it appears to play an important role in the regulation of mood and stress responses and may contribute to antidepressant effects . Several variants of TPH genes associated with differences in serotonin production have been identified. Some of these variants have been linked to an increased risk of developing psychiatric disorders . The modulation of TPH genes has been studied as a potential approach for the treatment of various psychiatric disorders, including depression, anxiety, and addiction . However, several studies have demonstrated controversial results regarding TPH polymorphisms and response to SSRIs . Monoamine Oxidases Monoamine oxidases (MAOs) are a family of enzymes that play critical roles in the metabolism of monoamine neurotransmitters in the CNS . There are two types of MAOs, MAO-A and MAO-B, which are encoded by separate genes. MAO-A and MAO-B are found in the CNS, particularly in neurons and astroglia. MAO-A is primarily responsible for the metabolism of several important monoamine neurotransmitters, including serotonin, norepinephrine, and dopamine. These neurotransmitters play a crucial role in the regulation of mood, behavior, and cognition . Several genetic variants of the MAOA gene associated with differences in enzyme activity and monoamine metabolism have been identified. In addition, some of these variants have been linked to an increased risk of developing psychiatric disorders . However, the relationship between the MAOA gene variants and psychiatric disorders remains unclear. Polymorphisms in MAO genes have also been studied for their potential role in response to antidepressant medications. Some studies have suggested that certain genetic variants in MAO genes may affect the metabolism of antidepressants, potentially leading to differences in drug efficacy and side effects . For example, individuals with a specific variant of the MAOA gene had a better response to fluvoxamine compared to those without this variant . In addition, a recent study reported that the MAOA rs979605 polymorphism might modulate the response to antidepressant therapy in a sex-specific manner . Catechol-O-Methyltransferase Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the degradation of catecholamines such as dopamine, epinephrine, and norepinephrine. The COMT gene has several polymorphisms, and the most extensively studied is the rs4680 polymorphism, also known as Val158Met . This SNP causes the substitution of valine (Val) for methionine (Met) at position 158 in the COMT enzyme. The Val allele is associated with higher COMT activity, whereas the Met allele leads to lower activity. Therefore, individuals with the Val/Val genotype tend to have the highest level of COMT activity, followed by Val/Met individuals with intermediate activity, and Met/Met with the lowest activity . The COMT Val158Met polymorphism has been implicated in various psychiatric disorders, including depression, anxiety, and schizophrenia . Furthermore, studies have linked the COMT Val158Met polymorphism with response the to SSRIs such as fluoxetine and paroxetine . For instance, one study discovered that the Val/Met genotype significantly affected the response to fluvoxamine . Additionally, it has been suggested that the effects of the COMT genotype on antidepressant responses may depend on other factors, such as the type of medication, severity and duration of depression, and other genetic and environmental factors . However, on the contrary, Brunoni et al. did not observe a significant association between COMT variants and responses to escitalopram treatment. 2.2.2. Monoamine Transporters Serotonin Transporter The serotonin transporter (SERT) encoded by the SLC6A4 gene plays a critical role in regulating serotonin neurotransmission in the brain. SERT is responsible for serotonin reuptake from the synaptic cleft, thereby regulating the amount of serotonin available to bind to serotonin receptors in the brain . Several classes of antidepressant drugs, including SSRIs, SNRIs, and TCAs, target SERT and inhibit its activity . The SLC6A4 gene was the first gene genotyped as part of the Genome-Based Therapeutic Drugs for Depression (GENDEP), and several genetic variants have been identified . The most studied is the variant of the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR is located in the promoter region of the SLC6A4 gene and has two alleles: the short (S) and the long (L) allele. The S allele is associated with lower transcriptional efficiency and reduced expression of the SERT protein compared to the L allele. As a result, carriers of the S allele have been found to have lower serotonin reuptake efficiency, leading to higher serotonin levels. Therefore, individuals carrying the S/S genotype have been shown to have a poorer response to SSRI treatment and may experience more side effects compared to those with the L/L or L/S genotypes . Numerous studies have investigated the relationship between the 5-HTTLPR polymorphism and response to SSRIs . A recent case report suggested that the S/S genotype resulted in ineffective fluoxetine treatment and may have caused exacerbation of depression . Maron et al. investigated the association between 5-HTTLPR polymorphism and clinical response to escitalopram and found no significant association. However, they did observe a linkage between this polymorphism and a higher risk for adverse effects . This finding is consistent with other studies that have reported an association between SSRIs side effects and 5-HTTLPR polymorphism . Therefore, the 5-HTTLPR polymorphism may be a valuable marker for predicting antidepressant responses and tolerability in individuals with psychiatric disorders. In particular, genotyping for the 5-HTTLPR variant may help identify patients who are less likely to respond to SSRIs and may benefit from alternative treatment strategies, such as SNRIs or TCAs. Additionally, several studies have reported an association between the 5-HTTLPR S/S genotype and suicidal behavior , although other risk factors, such as life stressors, psychiatric disorders, and substance use, must be considered. Norepinephrine Transporter The norepinephrine transporter (NET) is a protein encoded by the SLC6A2 gene and plays an important role in the reuptake of norepinephrine (NE) . TCAs, such as desipramine and imipramine, work by inhibiting the reuptake of NE by the NET, which increases the levels of NE in the synapse and enhances its effects on target neurons. Although antidepressants, such as SSRIs, have largely replaced TCAs, they are still used in some cases where other treatments have been ineffective. Several genetic variants have been identified in the human SLC6A2 gene, which have functional consequences and can affect the efficacy of antidepressant drugs . A recent study found an association between certain SLC6A2 polymorphisms and antidepressant response variability . Although, the role of SLC6A2 variants in antidepressant responses is still being studied, current evidence suggests that these associations are complex and not fully understood. Dopamine Transporter The dopamine transporter (DAT), encoded by the SLC6A3 gene, is a protein responsible for dopamine reuptake from the synaptic cleft into the presynaptic neuron. To date, the SLC6A3 gene has been identified with at least 502 variations . However, the relationship between SLC6A3 polymorphisms and antidepressant responses is yet to be thoroughly researched. Nevertheless, some studies have suggested that specific SLC6A3 polymorphisms may influence the response to antidepressant therapy . For example, Kirchheiner et al. suggested that the SLC6A3 polymorphism increased the risk of a poorer and slower response to various antidepressants in individuals with the 9/10 and 9/9 genotypes compared to carriers of the 10/10 genotype. 2.2.3. Monoamine Receptors 5-HT 1A Receptor The serotonin 1A (5-HT 1A ) receptor is a G protein-coupled receptor found in the CNS and peripheral tissues. It is widely distributed in various brain regions, including the hippocampus, hypothalamus, cortex, and amygdala. It plays a crucial role in regulating the release of neurotransmitters, particularly serotonin, and is a target for many psychoactive drugs, including antidepressants and anxiolytics . The 5-HT 1A receptor is encoded by the HTR1A gene, and mutations in this gene have been linked to several neuropsychiatric disorders, including major depression, anxiety disorders, and autism spectrum disorder . The role of HTR1A polymorphisms in response to antidepressant therapy has also been investigated; however, a few recent studies found no significant associations . For instance, Scutt et al. reported no association between 5HT1A polymorphism and increased risk of SSRIs adverse effects. On the other hand, other studies have reported different findings . Villafuerte et al. suggested that the G allele of HTR1A rs1364043 polymorphism might predict citalopram response in depressed patients. This study showed that individuals homozygous for the G allele of the HTR1A rs1364043 polymorphism responded better to citalopram therapy. In patients with major depressive disorder, Kato et al. found an association between HTR1A rs1364043 polymorphism and a better response to antidepressant treatment. 5-HT 2A Receptor The HTR2A gene, coding for the 5-HT 2A receptor, contains several polymorphisms, some of which have been linked to altered receptor function or expression. However, the HTR2A rs6311 and rs6313 polymorphisms have been researched the most, especially their association with response to antidepressant therapy . Recent studies have shown that carriers of the HTR2A rs3803189 and rs7997012 polymorphisms responded better to SSRIs . On the other hand, a number of other studies reported no linkage between different HTR2A polymorphisms and a patient’s response to antidepressant treatment . Several studies have also investigated the relationship between different HTR2A SNPs and the tolerability of antidepressant therapy. According to their findings, specific HTR2A polymorphisms, such as rs7997012 and rs6314, may be associated with a reduced risk of adverse effects of antidepressant drugs . Dopamine Receptors Central dopamine receptors can be classified into two major families based on their structural and functional similarities: D1-like (D 1 and D 5 receptors) and D2-like receptors (D 2 , D 3 , and D 4 receptors). Dopamine D 2 receptors, encoded by the DRD2 gene, are among the most intensively researched receptors in depressive disorders. Several studies have pointed out the possibility that DRD2 polymorphisms play an important role in depressive disorder and response to antidepressant therapy ; however, other authors found no significant association . On the other hand, Wang et al. suggested that DRD2 rs1076562, rs2440390, and rs2734833 polymorphisms are associated with the onset time of the antidepressant response. In addition, according to Perlis et al. , DRD2 rs4245147 polymorphism has been linked to a better lamotrigine response in a group of patients with bipolar depression. 2.1.1. Cytochrome P450 Family The CYP450 family is a large group of enzymes responsible for the metabolism of various drugs and xenobiotics, including antidepressant drugs. Among the many identified CYP450 enzymes, the most important ones involved in the metabolism of a variety of psychotropic drugs are CYP2D6, CYP2C19, CYP3A4, CYP1A2, CYP2B6, CYP2C8, and CYP2C9 . Various CYP450 enzymes are capable of metabolizing more than one drug, and a single drug can be metabolized by multiple CYP enzymes . The activity of these enzymes could be influenced by genetic variations, which may result in individual differences in antidepressant drug metabolism and responses . Most genes encoding CYPs are highly polymorphic . The impact of CYP polymorphisms on drug metabolism is an important area of research in personalized medicine . While more than 2000 mutations have been found in CYP genes, only specific SNPs are known to affect CYP enzymatic activity . Depending on the genetic variants that influence enzyme activity, individuals can be classified into four main CYP phenotypes: from poor, via intermediate and extensive (normal), to ultrarapid metabolizers . Due to genetic variations, PMs have little to no CYP enzyme activity, whereas IMs have reduced enzyme activity. As a result of slower antidepressant drug metabolism, both PMs and IMs may be at an increased risk for adverse drug reactions and require lower dosages or less frequent dosing regimens for antidepressant medications. On the other hand, NMs possess typical enzyme activity and a normal drug metabolism rate. However, UMs exhibit increased enzymatic activity, resulting in faster drug metabolism. Therefore, they may require higher antidepressant doses or more frequent drug administration to achieve the desired therapeutic effect in patients with depression . The pharmacogenetics of the most important CYPs involved in the metabolism of antidepressant drugs are summarized below. CYP2D6 The CYP2D6 gene is highly polymorphic, meaning that it harbors genetic variations that can affect enzyme function . CYP2D6 enzyme is involved in the metabolism of SSRIs (paroxetine, fluvoxamine, and fluoxetine), amitriptyline (TCA), and venlafaxine (SNRI) . Different allele variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . CYP2D6*1 , *2 , *33 , and *35 allele variants are associated with normal function, whereas *9 , *10 , *14B , *17 , *29 , and *41 variants reduce CYP2D6 enzymatic capacity. Furthermore, CYP2D6*3 , *4 , *5 , *6 , *7 , *11 , *12 , *14A , *36 , and *68 variants are responsible for the loss of enzyme function . Apart from genetic variations, paroxetine and fluoxetine are potent inhibitors of CYP2D6, which may result in an “iatrogenic poor phenotype” or “phenocopy” when taken with drugs that are also metabolized by CYP2D6, such as venlafaxine. Patients with an “iatrogenic poor phenotype” may be at high risk of developing toxicity due to elevated plasma drug levels . Specifically, individuals with poor CYP2D6 function may have reduced metabolism of fluoxetine and paroxetine, resulting in higher plasma drug levels and an increased risk of side effects, such as the development of suicidal ideation or antidepressant-induced mania during the initiation of treatment. As a result, the U.S. Food and Drug Administration (FDA) has issued a black box warning for SSRIs cautioning that these side effects can occur, especially in the first few weeks of treatment. Therefore, close monitoring is required for all patients starting antidepressant therapy . Furthermore, studies have shown that poor metabolizers of fluoxetine may be at a risk of QT interval prolongation. . Similar to fluoxetine, poor metabolizers of venlafaxine (genotype CYP2D6 *6 / *4 , *5 / *4 , or *6 / *6 ) are also at risk for developing adverse effects, such as arrhythmias, gastrointestinal problems, and hyponatremia . Ahmed et al. showed that CYP2D6 UM status contributes to venlafaxine treatment remission in patients with major depressive disorder . Therefore, when prescribing SSRIs and other antidepressants, clinicians must consider a patient’s CYP2D6 genotype and adjust the dose or choose an alternative medication, if necessary, to minimize the risk of adverse effects while maximizing therapeutic benefit . Despite the growing literature on the clinical implications of the CYP2D6 genotype and phenoconversion on patient-related outcomes, implementation of pharmacogenetics to guide antidepressant prescribing is rare. CYP2C19 In addition to clinically significant variants of the CYP2D6 gene, a high number of polymorphisms in the CYP2C19 gene have also been discovered , resulting in different metabolizer phenotypes . Drugs that are mainly metabolized by CYP2C19 include SSRIs such as escitalopram, citalopram, and sertraline . Variations in the CYP2C19 gene can result in individuals having different levels of CYP2C19 enzyme activity, affecting how drugs such as escitalopram and sertraline are metabolized . According to previous research, the CYP2C19*1 allele variant is associated with normal function, whereas the CYP2C19*17 variant increases CYP2C19 enzymatic capacity. Furthermore, CYP2C19*2 and *3 variants are responsible for the loss of enzyme function . For example, PMs of escitalopram are more likely to have adverse effects and psychotherapy discontinuation. However, they respond better to escitalopram, provided the therapy is tolerated. A retrospective study involving more than 2000 escitalopram-treated patients demonstrated that those with CYP2C19*1 / *17 and CYP2C19*17 / *17 variants (IMs) were more likely to experience treatment failure , whereas UMs exhibited suicidal thoughts . For sertraline, recent findings demonstrated that CYP2C19 PMs had higher plasma levels in comparison to normal metabolizers . Ricardo-Silgado et al. suggested that CYP2C19 genotypes might be associated with an increased risk of weight gain in patients on citalopram therapy. These findings indicate the importance of CYP2C19 variants and different metabolic phenotypes in antidepressant treatment tolerability and outcome. The American Molecular Pathology published guidelines for testing CYP2C19*2 , *3 , and *17 variants and CYP2C19*4A-*4B , *5 , *6 , *7 , *8 , *9 , *10, and *35 variants . In addition, the relevance of CYP2C19 polymorphisms in inter-individual predisposition to mental diseases was also investigated. Sim et al. found that individuals who were poor CYP2C19 metabolizers displayed lower levels of depressive symptoms compared to individuals who were normal metabolizers. These results raise the possibility that CYP2C19 variations contribute to susceptibility to depression. Recent studies have also demonstrated a correlation between low CYP2C19 activity and the severity of depression symptoms . Moreover, a cross-sectional observational retrospective study, which included over 700 psychiatric patients with depression and anxiety, analyzed the frequencies of CYPC19*2 , *4 , and *17 , as well as CYP2D6*2 , *3 , *4 , *5 , *6 , *10 , and *41 variants . This study revealed that 77% of patients have at least one allele variant significantly affecting drug metabolism, with roughly half of the individuals with reduced CYP2D6 enzyme function and the majority of them being CYP2C19 rapid and ultrarapid metabolizers . Therefore, due to their clinical relevance, both CYP2D6 and CYP2C19 are included in pharmacogenetics guidelines and recommendations published by the Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), and regulatory agencies such as the FDA . CYP2C9 The CYP2C9 gene encodes an enzyme that is involved in the metabolism of many drugs, including antidepressants, such as MAOIs, TCAs, and SSRIs . The CYP2C9*1 variant is associated with normal enzyme function, whereas CYP2C9 * 2 , *5 , *8 , and *11 variants reduce CYP2C9 enzymatic capacity. Furthermore, CYP2D9*3 , *6 , and *13 variants are responsible for the loss of enzyme function . Different CYP2C9 variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . Moreover, variants of the CYP2C9 gene have been linked to mental disorders such as depression and psychosis . CYP1A2 The CYP1A2 enzyme, encoded by the CYP1A2 gene, is responsible for the metabolism of approximately 24% of antidepressant drugs, including agomelatine, escitalopram, venlafaxine, duloxetine, and mirtazapine . Several CYP1A2 variants, including * 1C , *1F , and *1B , have been identified as potential indicators of the CYP1A2 phenotype and agomelatine pharmacokinetics. Notably, the CYP1A2*1C variant is associated with reduced enzyme activity, resulting in higher plasma agomelatine concentrations and an increased risk of adverse effects. On the contrary, individuals with the CYP1A2 *1F and *1B variants, who are characterized by increased enzyme activity, may metabolize agomelatine more rapidly, leading to lower plasma concentrations and potentially reduced efficacy . Kuo et al. conducted a study to investigate the association between CYP1A2 polymorphisms and the metabolism of escitalopram in patients with major depressive disorder. The study found that several SNPs in the CYP1A2 gene, such as rs2069521, rs4646425, and rs4646427 polymorphisms, were associated with altered escitalopram metabolism and an increased risk of adverse effects, such as fatigue and nausea/vomiting, particularly during the initial stages of treatment. In addition, Lin et al. demonstrated that CYP1A2 SNPs, such as rs4646425, rs2472304, and rs2470890, are associated with a slower response to paroxetine treatment. Furthermore, the study of the linkage between CYP1A2 polymorphisms and the response to venlafaxine suggested that the rs2470890 polymorphism might be related to venlafaxine treatment remission . 2.1.2. P-glycoprotein P-glycoprotein, commonly referred to as P-gp, is a membrane transporter within the ATP-binding cassette (ABC) transporter family. It is encoded by the ABCB1 or MDR1 gene, located on chromosome 7. P-gp functions as an efflux pump, contributing to drug absorption, distribution, and elimination . P-gp has been found in various tissues throughout the body, including the liver, kidneys, intestines, and the blood-brain barrier. At the blood-brain barrier, P-gp can limit the entry of some drugs into the brain, which can affect their efficacy. The expression and activity of P-gp can vary widely between individuals, contributing to differences in drug responses and drug interactions . Genetic factors play a role in determining the level of P-gp expression in different tissues and individuals. Certain genetic variants of the ABCB1 gene have been associated with altered P-gp expression and activity, which can affect the pharmacokinetics and efficacy of drugs that are substrates for P-gp . P-gp has broad substrate specificity, including antidepressants such as escitalopram, fluvoxamine, paroxetine, amitriptyline, and imipramine. Therefore, when P-gp is absent or not functioning properly, these drugs can accumulate in the body, resulting in higher concentrations and potentially an increased risk of adverse effects. However, newer antidepressants (levomilnacipran, vortioxetine, and vilazodone) have been proven as poor P-gp substrates . There has been interest in studying the relationship between genetic variations in the MDR1 / ABCB1 gene and antidepressant treatment outcomes . Several genetic variants of the MDR1 / ABCB1 gene have been associated with altered P-gp activity. The three most common MDR1 / ABCB1 variants, C3435T (rs1045642), C1236T (rs1128503), and G2677T (rs2032582), have been the subject of extensive research . A recent study on experimental models suggested that the 2677G > T polymorphism in the ABCB1 gene has been associated with altered P-gp function and increased brain penetration of P-gp substrates without affecting P-gp protein expression in the blood-brain barrier . In addition, genetic variants of the ABCB1 gene, including the rs2235040 and rs4148739 polymorphisms, may be associated with the onset of response to antidepressant medications rather than the response rate . Furthermore, a significant link has been found between the ABCB1 rs2235015 GG genotype and better response to antidepressant treatment . Overall, while pharmacogenetic research on MDR1 / ABCB1 suggests the potential for improving outcomes of antidepressant treatment, further investigation is necessary to better comprehend the connection between genetic variations and treatment response, as well as to assess its clinical utility. The CYP450 family is a large group of enzymes responsible for the metabolism of various drugs and xenobiotics, including antidepressant drugs. Among the many identified CYP450 enzymes, the most important ones involved in the metabolism of a variety of psychotropic drugs are CYP2D6, CYP2C19, CYP3A4, CYP1A2, CYP2B6, CYP2C8, and CYP2C9 . Various CYP450 enzymes are capable of metabolizing more than one drug, and a single drug can be metabolized by multiple CYP enzymes . The activity of these enzymes could be influenced by genetic variations, which may result in individual differences in antidepressant drug metabolism and responses . Most genes encoding CYPs are highly polymorphic . The impact of CYP polymorphisms on drug metabolism is an important area of research in personalized medicine . While more than 2000 mutations have been found in CYP genes, only specific SNPs are known to affect CYP enzymatic activity . Depending on the genetic variants that influence enzyme activity, individuals can be classified into four main CYP phenotypes: from poor, via intermediate and extensive (normal), to ultrarapid metabolizers . Due to genetic variations, PMs have little to no CYP enzyme activity, whereas IMs have reduced enzyme activity. As a result of slower antidepressant drug metabolism, both PMs and IMs may be at an increased risk for adverse drug reactions and require lower dosages or less frequent dosing regimens for antidepressant medications. On the other hand, NMs possess typical enzyme activity and a normal drug metabolism rate. However, UMs exhibit increased enzymatic activity, resulting in faster drug metabolism. Therefore, they may require higher antidepressant doses or more frequent drug administration to achieve the desired therapeutic effect in patients with depression . The pharmacogenetics of the most important CYPs involved in the metabolism of antidepressant drugs are summarized below. CYP2D6 The CYP2D6 gene is highly polymorphic, meaning that it harbors genetic variations that can affect enzyme function . CYP2D6 enzyme is involved in the metabolism of SSRIs (paroxetine, fluvoxamine, and fluoxetine), amitriptyline (TCA), and venlafaxine (SNRI) . Different allele variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . CYP2D6*1 , *2 , *33 , and *35 allele variants are associated with normal function, whereas *9 , *10 , *14B , *17 , *29 , and *41 variants reduce CYP2D6 enzymatic capacity. Furthermore, CYP2D6*3 , *4 , *5 , *6 , *7 , *11 , *12 , *14A , *36 , and *68 variants are responsible for the loss of enzyme function . Apart from genetic variations, paroxetine and fluoxetine are potent inhibitors of CYP2D6, which may result in an “iatrogenic poor phenotype” or “phenocopy” when taken with drugs that are also metabolized by CYP2D6, such as venlafaxine. Patients with an “iatrogenic poor phenotype” may be at high risk of developing toxicity due to elevated plasma drug levels . Specifically, individuals with poor CYP2D6 function may have reduced metabolism of fluoxetine and paroxetine, resulting in higher plasma drug levels and an increased risk of side effects, such as the development of suicidal ideation or antidepressant-induced mania during the initiation of treatment. As a result, the U.S. Food and Drug Administration (FDA) has issued a black box warning for SSRIs cautioning that these side effects can occur, especially in the first few weeks of treatment. Therefore, close monitoring is required for all patients starting antidepressant therapy . Furthermore, studies have shown that poor metabolizers of fluoxetine may be at a risk of QT interval prolongation. . Similar to fluoxetine, poor metabolizers of venlafaxine (genotype CYP2D6 *6 / *4 , *5 / *4 , or *6 / *6 ) are also at risk for developing adverse effects, such as arrhythmias, gastrointestinal problems, and hyponatremia . Ahmed et al. showed that CYP2D6 UM status contributes to venlafaxine treatment remission in patients with major depressive disorder . Therefore, when prescribing SSRIs and other antidepressants, clinicians must consider a patient’s CYP2D6 genotype and adjust the dose or choose an alternative medication, if necessary, to minimize the risk of adverse effects while maximizing therapeutic benefit . Despite the growing literature on the clinical implications of the CYP2D6 genotype and phenoconversion on patient-related outcomes, implementation of pharmacogenetics to guide antidepressant prescribing is rare. CYP2C19 In addition to clinically significant variants of the CYP2D6 gene, a high number of polymorphisms in the CYP2C19 gene have also been discovered , resulting in different metabolizer phenotypes . Drugs that are mainly metabolized by CYP2C19 include SSRIs such as escitalopram, citalopram, and sertraline . Variations in the CYP2C19 gene can result in individuals having different levels of CYP2C19 enzyme activity, affecting how drugs such as escitalopram and sertraline are metabolized . According to previous research, the CYP2C19*1 allele variant is associated with normal function, whereas the CYP2C19*17 variant increases CYP2C19 enzymatic capacity. Furthermore, CYP2C19*2 and *3 variants are responsible for the loss of enzyme function . For example, PMs of escitalopram are more likely to have adverse effects and psychotherapy discontinuation. However, they respond better to escitalopram, provided the therapy is tolerated. A retrospective study involving more than 2000 escitalopram-treated patients demonstrated that those with CYP2C19*1 / *17 and CYP2C19*17 / *17 variants (IMs) were more likely to experience treatment failure , whereas UMs exhibited suicidal thoughts . For sertraline, recent findings demonstrated that CYP2C19 PMs had higher plasma levels in comparison to normal metabolizers . Ricardo-Silgado et al. suggested that CYP2C19 genotypes might be associated with an increased risk of weight gain in patients on citalopram therapy. These findings indicate the importance of CYP2C19 variants and different metabolic phenotypes in antidepressant treatment tolerability and outcome. The American Molecular Pathology published guidelines for testing CYP2C19*2 , *3 , and *17 variants and CYP2C19*4A-*4B , *5 , *6 , *7 , *8 , *9 , *10, and *35 variants . In addition, the relevance of CYP2C19 polymorphisms in inter-individual predisposition to mental diseases was also investigated. Sim et al. found that individuals who were poor CYP2C19 metabolizers displayed lower levels of depressive symptoms compared to individuals who were normal metabolizers. These results raise the possibility that CYP2C19 variations contribute to susceptibility to depression. Recent studies have also demonstrated a correlation between low CYP2C19 activity and the severity of depression symptoms . Moreover, a cross-sectional observational retrospective study, which included over 700 psychiatric patients with depression and anxiety, analyzed the frequencies of CYPC19*2 , *4 , and *17 , as well as CYP2D6*2 , *3 , *4 , *5 , *6 , *10 , and *41 variants . This study revealed that 77% of patients have at least one allele variant significantly affecting drug metabolism, with roughly half of the individuals with reduced CYP2D6 enzyme function and the majority of them being CYP2C19 rapid and ultrarapid metabolizers . Therefore, due to their clinical relevance, both CYP2D6 and CYP2C19 are included in pharmacogenetics guidelines and recommendations published by the Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), and regulatory agencies such as the FDA . CYP2C9 The CYP2C9 gene encodes an enzyme that is involved in the metabolism of many drugs, including antidepressants, such as MAOIs, TCAs, and SSRIs . The CYP2C9*1 variant is associated with normal enzyme function, whereas CYP2C9 * 2 , *5 , *8 , and *11 variants reduce CYP2C9 enzymatic capacity. Furthermore, CYP2D9*3 , *6 , and *13 variants are responsible for the loss of enzyme function . Different CYP2C9 variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . Moreover, variants of the CYP2C9 gene have been linked to mental disorders such as depression and psychosis . CYP1A2 The CYP1A2 enzyme, encoded by the CYP1A2 gene, is responsible for the metabolism of approximately 24% of antidepressant drugs, including agomelatine, escitalopram, venlafaxine, duloxetine, and mirtazapine . Several CYP1A2 variants, including * 1C , *1F , and *1B , have been identified as potential indicators of the CYP1A2 phenotype and agomelatine pharmacokinetics. Notably, the CYP1A2*1C variant is associated with reduced enzyme activity, resulting in higher plasma agomelatine concentrations and an increased risk of adverse effects. On the contrary, individuals with the CYP1A2 *1F and *1B variants, who are characterized by increased enzyme activity, may metabolize agomelatine more rapidly, leading to lower plasma concentrations and potentially reduced efficacy . Kuo et al. conducted a study to investigate the association between CYP1A2 polymorphisms and the metabolism of escitalopram in patients with major depressive disorder. The study found that several SNPs in the CYP1A2 gene, such as rs2069521, rs4646425, and rs4646427 polymorphisms, were associated with altered escitalopram metabolism and an increased risk of adverse effects, such as fatigue and nausea/vomiting, particularly during the initial stages of treatment. In addition, Lin et al. demonstrated that CYP1A2 SNPs, such as rs4646425, rs2472304, and rs2470890, are associated with a slower response to paroxetine treatment. Furthermore, the study of the linkage between CYP1A2 polymorphisms and the response to venlafaxine suggested that the rs2470890 polymorphism might be related to venlafaxine treatment remission . The CYP2D6 gene is highly polymorphic, meaning that it harbors genetic variations that can affect enzyme function . CYP2D6 enzyme is involved in the metabolism of SSRIs (paroxetine, fluvoxamine, and fluoxetine), amitriptyline (TCA), and venlafaxine (SNRI) . Different allele variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . CYP2D6*1 , *2 , *33 , and *35 allele variants are associated with normal function, whereas *9 , *10 , *14B , *17 , *29 , and *41 variants reduce CYP2D6 enzymatic capacity. Furthermore, CYP2D6*3 , *4 , *5 , *6 , *7 , *11 , *12 , *14A , *36 , and *68 variants are responsible for the loss of enzyme function . Apart from genetic variations, paroxetine and fluoxetine are potent inhibitors of CYP2D6, which may result in an “iatrogenic poor phenotype” or “phenocopy” when taken with drugs that are also metabolized by CYP2D6, such as venlafaxine. Patients with an “iatrogenic poor phenotype” may be at high risk of developing toxicity due to elevated plasma drug levels . Specifically, individuals with poor CYP2D6 function may have reduced metabolism of fluoxetine and paroxetine, resulting in higher plasma drug levels and an increased risk of side effects, such as the development of suicidal ideation or antidepressant-induced mania during the initiation of treatment. As a result, the U.S. Food and Drug Administration (FDA) has issued a black box warning for SSRIs cautioning that these side effects can occur, especially in the first few weeks of treatment. Therefore, close monitoring is required for all patients starting antidepressant therapy . Furthermore, studies have shown that poor metabolizers of fluoxetine may be at a risk of QT interval prolongation. . Similar to fluoxetine, poor metabolizers of venlafaxine (genotype CYP2D6 *6 / *4 , *5 / *4 , or *6 / *6 ) are also at risk for developing adverse effects, such as arrhythmias, gastrointestinal problems, and hyponatremia . Ahmed et al. showed that CYP2D6 UM status contributes to venlafaxine treatment remission in patients with major depressive disorder . Therefore, when prescribing SSRIs and other antidepressants, clinicians must consider a patient’s CYP2D6 genotype and adjust the dose or choose an alternative medication, if necessary, to minimize the risk of adverse effects while maximizing therapeutic benefit . Despite the growing literature on the clinical implications of the CYP2D6 genotype and phenoconversion on patient-related outcomes, implementation of pharmacogenetics to guide antidepressant prescribing is rare. In addition to clinically significant variants of the CYP2D6 gene, a high number of polymorphisms in the CYP2C19 gene have also been discovered , resulting in different metabolizer phenotypes . Drugs that are mainly metabolized by CYP2C19 include SSRIs such as escitalopram, citalopram, and sertraline . Variations in the CYP2C19 gene can result in individuals having different levels of CYP2C19 enzyme activity, affecting how drugs such as escitalopram and sertraline are metabolized . According to previous research, the CYP2C19*1 allele variant is associated with normal function, whereas the CYP2C19*17 variant increases CYP2C19 enzymatic capacity. Furthermore, CYP2C19*2 and *3 variants are responsible for the loss of enzyme function . For example, PMs of escitalopram are more likely to have adverse effects and psychotherapy discontinuation. However, they respond better to escitalopram, provided the therapy is tolerated. A retrospective study involving more than 2000 escitalopram-treated patients demonstrated that those with CYP2C19*1 / *17 and CYP2C19*17 / *17 variants (IMs) were more likely to experience treatment failure , whereas UMs exhibited suicidal thoughts . For sertraline, recent findings demonstrated that CYP2C19 PMs had higher plasma levels in comparison to normal metabolizers . Ricardo-Silgado et al. suggested that CYP2C19 genotypes might be associated with an increased risk of weight gain in patients on citalopram therapy. These findings indicate the importance of CYP2C19 variants and different metabolic phenotypes in antidepressant treatment tolerability and outcome. The American Molecular Pathology published guidelines for testing CYP2C19*2 , *3 , and *17 variants and CYP2C19*4A-*4B , *5 , *6 , *7 , *8 , *9 , *10, and *35 variants . In addition, the relevance of CYP2C19 polymorphisms in inter-individual predisposition to mental diseases was also investigated. Sim et al. found that individuals who were poor CYP2C19 metabolizers displayed lower levels of depressive symptoms compared to individuals who were normal metabolizers. These results raise the possibility that CYP2C19 variations contribute to susceptibility to depression. Recent studies have also demonstrated a correlation between low CYP2C19 activity and the severity of depression symptoms . Moreover, a cross-sectional observational retrospective study, which included over 700 psychiatric patients with depression and anxiety, analyzed the frequencies of CYPC19*2 , *4 , and *17 , as well as CYP2D6*2 , *3 , *4 , *5 , *6 , *10 , and *41 variants . This study revealed that 77% of patients have at least one allele variant significantly affecting drug metabolism, with roughly half of the individuals with reduced CYP2D6 enzyme function and the majority of them being CYP2C19 rapid and ultrarapid metabolizers . Therefore, due to their clinical relevance, both CYP2D6 and CYP2C19 are included in pharmacogenetics guidelines and recommendations published by the Clinical Pharmacogenetics Implementation Consortium (CPIC), Dutch Pharmacogenetics Working Group (DPWG), and regulatory agencies such as the FDA . The CYP2C9 gene encodes an enzyme that is involved in the metabolism of many drugs, including antidepressants, such as MAOIs, TCAs, and SSRIs . The CYP2C9*1 variant is associated with normal enzyme function, whereas CYP2C9 * 2 , *5 , *8 , and *11 variants reduce CYP2C9 enzymatic capacity. Furthermore, CYP2D9*3 , *6 , and *13 variants are responsible for the loss of enzyme function . Different CYP2C9 variants result in normal, decreased, and no enzyme function, characterized by extensive, intermediate, and poor metabolizer phenotypes, respectively . Moreover, variants of the CYP2C9 gene have been linked to mental disorders such as depression and psychosis . The CYP1A2 enzyme, encoded by the CYP1A2 gene, is responsible for the metabolism of approximately 24% of antidepressant drugs, including agomelatine, escitalopram, venlafaxine, duloxetine, and mirtazapine . Several CYP1A2 variants, including * 1C , *1F , and *1B , have been identified as potential indicators of the CYP1A2 phenotype and agomelatine pharmacokinetics. Notably, the CYP1A2*1C variant is associated with reduced enzyme activity, resulting in higher plasma agomelatine concentrations and an increased risk of adverse effects. On the contrary, individuals with the CYP1A2 *1F and *1B variants, who are characterized by increased enzyme activity, may metabolize agomelatine more rapidly, leading to lower plasma concentrations and potentially reduced efficacy . Kuo et al. conducted a study to investigate the association between CYP1A2 polymorphisms and the metabolism of escitalopram in patients with major depressive disorder. The study found that several SNPs in the CYP1A2 gene, such as rs2069521, rs4646425, and rs4646427 polymorphisms, were associated with altered escitalopram metabolism and an increased risk of adverse effects, such as fatigue and nausea/vomiting, particularly during the initial stages of treatment. In addition, Lin et al. demonstrated that CYP1A2 SNPs, such as rs4646425, rs2472304, and rs2470890, are associated with a slower response to paroxetine treatment. Furthermore, the study of the linkage between CYP1A2 polymorphisms and the response to venlafaxine suggested that the rs2470890 polymorphism might be related to venlafaxine treatment remission . P-glycoprotein, commonly referred to as P-gp, is a membrane transporter within the ATP-binding cassette (ABC) transporter family. It is encoded by the ABCB1 or MDR1 gene, located on chromosome 7. P-gp functions as an efflux pump, contributing to drug absorption, distribution, and elimination . P-gp has been found in various tissues throughout the body, including the liver, kidneys, intestines, and the blood-brain barrier. At the blood-brain barrier, P-gp can limit the entry of some drugs into the brain, which can affect their efficacy. The expression and activity of P-gp can vary widely between individuals, contributing to differences in drug responses and drug interactions . Genetic factors play a role in determining the level of P-gp expression in different tissues and individuals. Certain genetic variants of the ABCB1 gene have been associated with altered P-gp expression and activity, which can affect the pharmacokinetics and efficacy of drugs that are substrates for P-gp . P-gp has broad substrate specificity, including antidepressants such as escitalopram, fluvoxamine, paroxetine, amitriptyline, and imipramine. Therefore, when P-gp is absent or not functioning properly, these drugs can accumulate in the body, resulting in higher concentrations and potentially an increased risk of adverse effects. However, newer antidepressants (levomilnacipran, vortioxetine, and vilazodone) have been proven as poor P-gp substrates . There has been interest in studying the relationship between genetic variations in the MDR1 / ABCB1 gene and antidepressant treatment outcomes . Several genetic variants of the MDR1 / ABCB1 gene have been associated with altered P-gp activity. The three most common MDR1 / ABCB1 variants, C3435T (rs1045642), C1236T (rs1128503), and G2677T (rs2032582), have been the subject of extensive research . A recent study on experimental models suggested that the 2677G > T polymorphism in the ABCB1 gene has been associated with altered P-gp function and increased brain penetration of P-gp substrates without affecting P-gp protein expression in the blood-brain barrier . In addition, genetic variants of the ABCB1 gene, including the rs2235040 and rs4148739 polymorphisms, may be associated with the onset of response to antidepressant medications rather than the response rate . Furthermore, a significant link has been found between the ABCB1 rs2235015 GG genotype and better response to antidepressant treatment . Overall, while pharmacogenetic research on MDR1 / ABCB1 suggests the potential for improving outcomes of antidepressant treatment, further investigation is necessary to better comprehend the connection between genetic variations and treatment response, as well as to assess its clinical utility. 2.2.1. Monoamine Metabolic Enzymes Tryptophan Hydroxylase Tryptophan hydroxylase (TPH) is an enzyme that catalyzes the conversion of the amino acid tryptophan to 5-hydroxytryptophan, which is the first step in the synthesis of serotonin, a neurotransmitter involved in the regulation of mood, appetite, sleep, and stress response . There are two isoforms of the TPH enzyme, TPH1 and TPH2, encoded by separate genes. TPH1 is primarily expressed in peripheral tissues, such as the pineal gland, skin, and gut, whereas TPH2 is mainly expressed in neurons in the central nervous system (CNS). The two isoforms have similar enzymatic activities but differ in regulation, tissue distribution, and developmental expression patterns . Overall, while TPH1 may be less expressed in the brain than TPH2, it appears to play an important role in the regulation of mood and stress responses and may contribute to antidepressant effects . Several variants of TPH genes associated with differences in serotonin production have been identified. Some of these variants have been linked to an increased risk of developing psychiatric disorders . The modulation of TPH genes has been studied as a potential approach for the treatment of various psychiatric disorders, including depression, anxiety, and addiction . However, several studies have demonstrated controversial results regarding TPH polymorphisms and response to SSRIs . Monoamine Oxidases Monoamine oxidases (MAOs) are a family of enzymes that play critical roles in the metabolism of monoamine neurotransmitters in the CNS . There are two types of MAOs, MAO-A and MAO-B, which are encoded by separate genes. MAO-A and MAO-B are found in the CNS, particularly in neurons and astroglia. MAO-A is primarily responsible for the metabolism of several important monoamine neurotransmitters, including serotonin, norepinephrine, and dopamine. These neurotransmitters play a crucial role in the regulation of mood, behavior, and cognition . Several genetic variants of the MAOA gene associated with differences in enzyme activity and monoamine metabolism have been identified. In addition, some of these variants have been linked to an increased risk of developing psychiatric disorders . However, the relationship between the MAOA gene variants and psychiatric disorders remains unclear. Polymorphisms in MAO genes have also been studied for their potential role in response to antidepressant medications. Some studies have suggested that certain genetic variants in MAO genes may affect the metabolism of antidepressants, potentially leading to differences in drug efficacy and side effects . For example, individuals with a specific variant of the MAOA gene had a better response to fluvoxamine compared to those without this variant . In addition, a recent study reported that the MAOA rs979605 polymorphism might modulate the response to antidepressant therapy in a sex-specific manner . Catechol-O-Methyltransferase Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the degradation of catecholamines such as dopamine, epinephrine, and norepinephrine. The COMT gene has several polymorphisms, and the most extensively studied is the rs4680 polymorphism, also known as Val158Met . This SNP causes the substitution of valine (Val) for methionine (Met) at position 158 in the COMT enzyme. The Val allele is associated with higher COMT activity, whereas the Met allele leads to lower activity. Therefore, individuals with the Val/Val genotype tend to have the highest level of COMT activity, followed by Val/Met individuals with intermediate activity, and Met/Met with the lowest activity . The COMT Val158Met polymorphism has been implicated in various psychiatric disorders, including depression, anxiety, and schizophrenia . Furthermore, studies have linked the COMT Val158Met polymorphism with response the to SSRIs such as fluoxetine and paroxetine . For instance, one study discovered that the Val/Met genotype significantly affected the response to fluvoxamine . Additionally, it has been suggested that the effects of the COMT genotype on antidepressant responses may depend on other factors, such as the type of medication, severity and duration of depression, and other genetic and environmental factors . However, on the contrary, Brunoni et al. did not observe a significant association between COMT variants and responses to escitalopram treatment. 2.2.2. Monoamine Transporters Serotonin Transporter The serotonin transporter (SERT) encoded by the SLC6A4 gene plays a critical role in regulating serotonin neurotransmission in the brain. SERT is responsible for serotonin reuptake from the synaptic cleft, thereby regulating the amount of serotonin available to bind to serotonin receptors in the brain . Several classes of antidepressant drugs, including SSRIs, SNRIs, and TCAs, target SERT and inhibit its activity . The SLC6A4 gene was the first gene genotyped as part of the Genome-Based Therapeutic Drugs for Depression (GENDEP), and several genetic variants have been identified . The most studied is the variant of the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR is located in the promoter region of the SLC6A4 gene and has two alleles: the short (S) and the long (L) allele. The S allele is associated with lower transcriptional efficiency and reduced expression of the SERT protein compared to the L allele. As a result, carriers of the S allele have been found to have lower serotonin reuptake efficiency, leading to higher serotonin levels. Therefore, individuals carrying the S/S genotype have been shown to have a poorer response to SSRI treatment and may experience more side effects compared to those with the L/L or L/S genotypes . Numerous studies have investigated the relationship between the 5-HTTLPR polymorphism and response to SSRIs . A recent case report suggested that the S/S genotype resulted in ineffective fluoxetine treatment and may have caused exacerbation of depression . Maron et al. investigated the association between 5-HTTLPR polymorphism and clinical response to escitalopram and found no significant association. However, they did observe a linkage between this polymorphism and a higher risk for adverse effects . This finding is consistent with other studies that have reported an association between SSRIs side effects and 5-HTTLPR polymorphism . Therefore, the 5-HTTLPR polymorphism may be a valuable marker for predicting antidepressant responses and tolerability in individuals with psychiatric disorders. In particular, genotyping for the 5-HTTLPR variant may help identify patients who are less likely to respond to SSRIs and may benefit from alternative treatment strategies, such as SNRIs or TCAs. Additionally, several studies have reported an association between the 5-HTTLPR S/S genotype and suicidal behavior , although other risk factors, such as life stressors, psychiatric disorders, and substance use, must be considered. Norepinephrine Transporter The norepinephrine transporter (NET) is a protein encoded by the SLC6A2 gene and plays an important role in the reuptake of norepinephrine (NE) . TCAs, such as desipramine and imipramine, work by inhibiting the reuptake of NE by the NET, which increases the levels of NE in the synapse and enhances its effects on target neurons. Although antidepressants, such as SSRIs, have largely replaced TCAs, they are still used in some cases where other treatments have been ineffective. Several genetic variants have been identified in the human SLC6A2 gene, which have functional consequences and can affect the efficacy of antidepressant drugs . A recent study found an association between certain SLC6A2 polymorphisms and antidepressant response variability . Although, the role of SLC6A2 variants in antidepressant responses is still being studied, current evidence suggests that these associations are complex and not fully understood. Dopamine Transporter The dopamine transporter (DAT), encoded by the SLC6A3 gene, is a protein responsible for dopamine reuptake from the synaptic cleft into the presynaptic neuron. To date, the SLC6A3 gene has been identified with at least 502 variations . However, the relationship between SLC6A3 polymorphisms and antidepressant responses is yet to be thoroughly researched. Nevertheless, some studies have suggested that specific SLC6A3 polymorphisms may influence the response to antidepressant therapy . For example, Kirchheiner et al. suggested that the SLC6A3 polymorphism increased the risk of a poorer and slower response to various antidepressants in individuals with the 9/10 and 9/9 genotypes compared to carriers of the 10/10 genotype. 2.2.3. Monoamine Receptors 5-HT 1A Receptor The serotonin 1A (5-HT 1A ) receptor is a G protein-coupled receptor found in the CNS and peripheral tissues. It is widely distributed in various brain regions, including the hippocampus, hypothalamus, cortex, and amygdala. It plays a crucial role in regulating the release of neurotransmitters, particularly serotonin, and is a target for many psychoactive drugs, including antidepressants and anxiolytics . The 5-HT 1A receptor is encoded by the HTR1A gene, and mutations in this gene have been linked to several neuropsychiatric disorders, including major depression, anxiety disorders, and autism spectrum disorder . The role of HTR1A polymorphisms in response to antidepressant therapy has also been investigated; however, a few recent studies found no significant associations . For instance, Scutt et al. reported no association between 5HT1A polymorphism and increased risk of SSRIs adverse effects. On the other hand, other studies have reported different findings . Villafuerte et al. suggested that the G allele of HTR1A rs1364043 polymorphism might predict citalopram response in depressed patients. This study showed that individuals homozygous for the G allele of the HTR1A rs1364043 polymorphism responded better to citalopram therapy. In patients with major depressive disorder, Kato et al. found an association between HTR1A rs1364043 polymorphism and a better response to antidepressant treatment. 5-HT 2A Receptor The HTR2A gene, coding for the 5-HT 2A receptor, contains several polymorphisms, some of which have been linked to altered receptor function or expression. However, the HTR2A rs6311 and rs6313 polymorphisms have been researched the most, especially their association with response to antidepressant therapy . Recent studies have shown that carriers of the HTR2A rs3803189 and rs7997012 polymorphisms responded better to SSRIs . On the other hand, a number of other studies reported no linkage between different HTR2A polymorphisms and a patient’s response to antidepressant treatment . Several studies have also investigated the relationship between different HTR2A SNPs and the tolerability of antidepressant therapy. According to their findings, specific HTR2A polymorphisms, such as rs7997012 and rs6314, may be associated with a reduced risk of adverse effects of antidepressant drugs . Dopamine Receptors Central dopamine receptors can be classified into two major families based on their structural and functional similarities: D1-like (D 1 and D 5 receptors) and D2-like receptors (D 2 , D 3 , and D 4 receptors). Dopamine D 2 receptors, encoded by the DRD2 gene, are among the most intensively researched receptors in depressive disorders. Several studies have pointed out the possibility that DRD2 polymorphisms play an important role in depressive disorder and response to antidepressant therapy ; however, other authors found no significant association . On the other hand, Wang et al. suggested that DRD2 rs1076562, rs2440390, and rs2734833 polymorphisms are associated with the onset time of the antidepressant response. In addition, according to Perlis et al. , DRD2 rs4245147 polymorphism has been linked to a better lamotrigine response in a group of patients with bipolar depression. Tryptophan Hydroxylase Tryptophan hydroxylase (TPH) is an enzyme that catalyzes the conversion of the amino acid tryptophan to 5-hydroxytryptophan, which is the first step in the synthesis of serotonin, a neurotransmitter involved in the regulation of mood, appetite, sleep, and stress response . There are two isoforms of the TPH enzyme, TPH1 and TPH2, encoded by separate genes. TPH1 is primarily expressed in peripheral tissues, such as the pineal gland, skin, and gut, whereas TPH2 is mainly expressed in neurons in the central nervous system (CNS). The two isoforms have similar enzymatic activities but differ in regulation, tissue distribution, and developmental expression patterns . Overall, while TPH1 may be less expressed in the brain than TPH2, it appears to play an important role in the regulation of mood and stress responses and may contribute to antidepressant effects . Several variants of TPH genes associated with differences in serotonin production have been identified. Some of these variants have been linked to an increased risk of developing psychiatric disorders . The modulation of TPH genes has been studied as a potential approach for the treatment of various psychiatric disorders, including depression, anxiety, and addiction . However, several studies have demonstrated controversial results regarding TPH polymorphisms and response to SSRIs . Monoamine Oxidases Monoamine oxidases (MAOs) are a family of enzymes that play critical roles in the metabolism of monoamine neurotransmitters in the CNS . There are two types of MAOs, MAO-A and MAO-B, which are encoded by separate genes. MAO-A and MAO-B are found in the CNS, particularly in neurons and astroglia. MAO-A is primarily responsible for the metabolism of several important monoamine neurotransmitters, including serotonin, norepinephrine, and dopamine. These neurotransmitters play a crucial role in the regulation of mood, behavior, and cognition . Several genetic variants of the MAOA gene associated with differences in enzyme activity and monoamine metabolism have been identified. In addition, some of these variants have been linked to an increased risk of developing psychiatric disorders . However, the relationship between the MAOA gene variants and psychiatric disorders remains unclear. Polymorphisms in MAO genes have also been studied for their potential role in response to antidepressant medications. Some studies have suggested that certain genetic variants in MAO genes may affect the metabolism of antidepressants, potentially leading to differences in drug efficacy and side effects . For example, individuals with a specific variant of the MAOA gene had a better response to fluvoxamine compared to those without this variant . In addition, a recent study reported that the MAOA rs979605 polymorphism might modulate the response to antidepressant therapy in a sex-specific manner . Catechol-O-Methyltransferase Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the degradation of catecholamines such as dopamine, epinephrine, and norepinephrine. The COMT gene has several polymorphisms, and the most extensively studied is the rs4680 polymorphism, also known as Val158Met . This SNP causes the substitution of valine (Val) for methionine (Met) at position 158 in the COMT enzyme. The Val allele is associated with higher COMT activity, whereas the Met allele leads to lower activity. Therefore, individuals with the Val/Val genotype tend to have the highest level of COMT activity, followed by Val/Met individuals with intermediate activity, and Met/Met with the lowest activity . The COMT Val158Met polymorphism has been implicated in various psychiatric disorders, including depression, anxiety, and schizophrenia . Furthermore, studies have linked the COMT Val158Met polymorphism with response the to SSRIs such as fluoxetine and paroxetine . For instance, one study discovered that the Val/Met genotype significantly affected the response to fluvoxamine . Additionally, it has been suggested that the effects of the COMT genotype on antidepressant responses may depend on other factors, such as the type of medication, severity and duration of depression, and other genetic and environmental factors . However, on the contrary, Brunoni et al. did not observe a significant association between COMT variants and responses to escitalopram treatment. Tryptophan hydroxylase (TPH) is an enzyme that catalyzes the conversion of the amino acid tryptophan to 5-hydroxytryptophan, which is the first step in the synthesis of serotonin, a neurotransmitter involved in the regulation of mood, appetite, sleep, and stress response . There are two isoforms of the TPH enzyme, TPH1 and TPH2, encoded by separate genes. TPH1 is primarily expressed in peripheral tissues, such as the pineal gland, skin, and gut, whereas TPH2 is mainly expressed in neurons in the central nervous system (CNS). The two isoforms have similar enzymatic activities but differ in regulation, tissue distribution, and developmental expression patterns . Overall, while TPH1 may be less expressed in the brain than TPH2, it appears to play an important role in the regulation of mood and stress responses and may contribute to antidepressant effects . Several variants of TPH genes associated with differences in serotonin production have been identified. Some of these variants have been linked to an increased risk of developing psychiatric disorders . The modulation of TPH genes has been studied as a potential approach for the treatment of various psychiatric disorders, including depression, anxiety, and addiction . However, several studies have demonstrated controversial results regarding TPH polymorphisms and response to SSRIs . Monoamine oxidases (MAOs) are a family of enzymes that play critical roles in the metabolism of monoamine neurotransmitters in the CNS . There are two types of MAOs, MAO-A and MAO-B, which are encoded by separate genes. MAO-A and MAO-B are found in the CNS, particularly in neurons and astroglia. MAO-A is primarily responsible for the metabolism of several important monoamine neurotransmitters, including serotonin, norepinephrine, and dopamine. These neurotransmitters play a crucial role in the regulation of mood, behavior, and cognition . Several genetic variants of the MAOA gene associated with differences in enzyme activity and monoamine metabolism have been identified. In addition, some of these variants have been linked to an increased risk of developing psychiatric disorders . However, the relationship between the MAOA gene variants and psychiatric disorders remains unclear. Polymorphisms in MAO genes have also been studied for their potential role in response to antidepressant medications. Some studies have suggested that certain genetic variants in MAO genes may affect the metabolism of antidepressants, potentially leading to differences in drug efficacy and side effects . For example, individuals with a specific variant of the MAOA gene had a better response to fluvoxamine compared to those without this variant . In addition, a recent study reported that the MAOA rs979605 polymorphism might modulate the response to antidepressant therapy in a sex-specific manner . Catechol-O-methyltransferase (COMT) is an enzyme that plays a crucial role in the degradation of catecholamines such as dopamine, epinephrine, and norepinephrine. The COMT gene has several polymorphisms, and the most extensively studied is the rs4680 polymorphism, also known as Val158Met . This SNP causes the substitution of valine (Val) for methionine (Met) at position 158 in the COMT enzyme. The Val allele is associated with higher COMT activity, whereas the Met allele leads to lower activity. Therefore, individuals with the Val/Val genotype tend to have the highest level of COMT activity, followed by Val/Met individuals with intermediate activity, and Met/Met with the lowest activity . The COMT Val158Met polymorphism has been implicated in various psychiatric disorders, including depression, anxiety, and schizophrenia . Furthermore, studies have linked the COMT Val158Met polymorphism with response the to SSRIs such as fluoxetine and paroxetine . For instance, one study discovered that the Val/Met genotype significantly affected the response to fluvoxamine . Additionally, it has been suggested that the effects of the COMT genotype on antidepressant responses may depend on other factors, such as the type of medication, severity and duration of depression, and other genetic and environmental factors . However, on the contrary, Brunoni et al. did not observe a significant association between COMT variants and responses to escitalopram treatment. Serotonin Transporter The serotonin transporter (SERT) encoded by the SLC6A4 gene plays a critical role in regulating serotonin neurotransmission in the brain. SERT is responsible for serotonin reuptake from the synaptic cleft, thereby regulating the amount of serotonin available to bind to serotonin receptors in the brain . Several classes of antidepressant drugs, including SSRIs, SNRIs, and TCAs, target SERT and inhibit its activity . The SLC6A4 gene was the first gene genotyped as part of the Genome-Based Therapeutic Drugs for Depression (GENDEP), and several genetic variants have been identified . The most studied is the variant of the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR is located in the promoter region of the SLC6A4 gene and has two alleles: the short (S) and the long (L) allele. The S allele is associated with lower transcriptional efficiency and reduced expression of the SERT protein compared to the L allele. As a result, carriers of the S allele have been found to have lower serotonin reuptake efficiency, leading to higher serotonin levels. Therefore, individuals carrying the S/S genotype have been shown to have a poorer response to SSRI treatment and may experience more side effects compared to those with the L/L or L/S genotypes . Numerous studies have investigated the relationship between the 5-HTTLPR polymorphism and response to SSRIs . A recent case report suggested that the S/S genotype resulted in ineffective fluoxetine treatment and may have caused exacerbation of depression . Maron et al. investigated the association between 5-HTTLPR polymorphism and clinical response to escitalopram and found no significant association. However, they did observe a linkage between this polymorphism and a higher risk for adverse effects . This finding is consistent with other studies that have reported an association between SSRIs side effects and 5-HTTLPR polymorphism . Therefore, the 5-HTTLPR polymorphism may be a valuable marker for predicting antidepressant responses and tolerability in individuals with psychiatric disorders. In particular, genotyping for the 5-HTTLPR variant may help identify patients who are less likely to respond to SSRIs and may benefit from alternative treatment strategies, such as SNRIs or TCAs. Additionally, several studies have reported an association between the 5-HTTLPR S/S genotype and suicidal behavior , although other risk factors, such as life stressors, psychiatric disorders, and substance use, must be considered. Norepinephrine Transporter The norepinephrine transporter (NET) is a protein encoded by the SLC6A2 gene and plays an important role in the reuptake of norepinephrine (NE) . TCAs, such as desipramine and imipramine, work by inhibiting the reuptake of NE by the NET, which increases the levels of NE in the synapse and enhances its effects on target neurons. Although antidepressants, such as SSRIs, have largely replaced TCAs, they are still used in some cases where other treatments have been ineffective. Several genetic variants have been identified in the human SLC6A2 gene, which have functional consequences and can affect the efficacy of antidepressant drugs . A recent study found an association between certain SLC6A2 polymorphisms and antidepressant response variability . Although, the role of SLC6A2 variants in antidepressant responses is still being studied, current evidence suggests that these associations are complex and not fully understood. Dopamine Transporter The dopamine transporter (DAT), encoded by the SLC6A3 gene, is a protein responsible for dopamine reuptake from the synaptic cleft into the presynaptic neuron. To date, the SLC6A3 gene has been identified with at least 502 variations . However, the relationship between SLC6A3 polymorphisms and antidepressant responses is yet to be thoroughly researched. Nevertheless, some studies have suggested that specific SLC6A3 polymorphisms may influence the response to antidepressant therapy . For example, Kirchheiner et al. suggested that the SLC6A3 polymorphism increased the risk of a poorer and slower response to various antidepressants in individuals with the 9/10 and 9/9 genotypes compared to carriers of the 10/10 genotype. The serotonin transporter (SERT) encoded by the SLC6A4 gene plays a critical role in regulating serotonin neurotransmission in the brain. SERT is responsible for serotonin reuptake from the synaptic cleft, thereby regulating the amount of serotonin available to bind to serotonin receptors in the brain . Several classes of antidepressant drugs, including SSRIs, SNRIs, and TCAs, target SERT and inhibit its activity . The SLC6A4 gene was the first gene genotyped as part of the Genome-Based Therapeutic Drugs for Depression (GENDEP), and several genetic variants have been identified . The most studied is the variant of the serotonin transporter-linked polymorphic region (5-HTTLPR). The 5-HTTLPR is located in the promoter region of the SLC6A4 gene and has two alleles: the short (S) and the long (L) allele. The S allele is associated with lower transcriptional efficiency and reduced expression of the SERT protein compared to the L allele. As a result, carriers of the S allele have been found to have lower serotonin reuptake efficiency, leading to higher serotonin levels. Therefore, individuals carrying the S/S genotype have been shown to have a poorer response to SSRI treatment and may experience more side effects compared to those with the L/L or L/S genotypes . Numerous studies have investigated the relationship between the 5-HTTLPR polymorphism and response to SSRIs . A recent case report suggested that the S/S genotype resulted in ineffective fluoxetine treatment and may have caused exacerbation of depression . Maron et al. investigated the association between 5-HTTLPR polymorphism and clinical response to escitalopram and found no significant association. However, they did observe a linkage between this polymorphism and a higher risk for adverse effects . This finding is consistent with other studies that have reported an association between SSRIs side effects and 5-HTTLPR polymorphism . Therefore, the 5-HTTLPR polymorphism may be a valuable marker for predicting antidepressant responses and tolerability in individuals with psychiatric disorders. In particular, genotyping for the 5-HTTLPR variant may help identify patients who are less likely to respond to SSRIs and may benefit from alternative treatment strategies, such as SNRIs or TCAs. Additionally, several studies have reported an association between the 5-HTTLPR S/S genotype and suicidal behavior , although other risk factors, such as life stressors, psychiatric disorders, and substance use, must be considered. The norepinephrine transporter (NET) is a protein encoded by the SLC6A2 gene and plays an important role in the reuptake of norepinephrine (NE) . TCAs, such as desipramine and imipramine, work by inhibiting the reuptake of NE by the NET, which increases the levels of NE in the synapse and enhances its effects on target neurons. Although antidepressants, such as SSRIs, have largely replaced TCAs, they are still used in some cases where other treatments have been ineffective. Several genetic variants have been identified in the human SLC6A2 gene, which have functional consequences and can affect the efficacy of antidepressant drugs . A recent study found an association between certain SLC6A2 polymorphisms and antidepressant response variability . Although, the role of SLC6A2 variants in antidepressant responses is still being studied, current evidence suggests that these associations are complex and not fully understood. The dopamine transporter (DAT), encoded by the SLC6A3 gene, is a protein responsible for dopamine reuptake from the synaptic cleft into the presynaptic neuron. To date, the SLC6A3 gene has been identified with at least 502 variations . However, the relationship between SLC6A3 polymorphisms and antidepressant responses is yet to be thoroughly researched. Nevertheless, some studies have suggested that specific SLC6A3 polymorphisms may influence the response to antidepressant therapy . For example, Kirchheiner et al. suggested that the SLC6A3 polymorphism increased the risk of a poorer and slower response to various antidepressants in individuals with the 9/10 and 9/9 genotypes compared to carriers of the 10/10 genotype. 5-HT 1A Receptor The serotonin 1A (5-HT 1A ) receptor is a G protein-coupled receptor found in the CNS and peripheral tissues. It is widely distributed in various brain regions, including the hippocampus, hypothalamus, cortex, and amygdala. It plays a crucial role in regulating the release of neurotransmitters, particularly serotonin, and is a target for many psychoactive drugs, including antidepressants and anxiolytics . The 5-HT 1A receptor is encoded by the HTR1A gene, and mutations in this gene have been linked to several neuropsychiatric disorders, including major depression, anxiety disorders, and autism spectrum disorder . The role of HTR1A polymorphisms in response to antidepressant therapy has also been investigated; however, a few recent studies found no significant associations . For instance, Scutt et al. reported no association between 5HT1A polymorphism and increased risk of SSRIs adverse effects. On the other hand, other studies have reported different findings . Villafuerte et al. suggested that the G allele of HTR1A rs1364043 polymorphism might predict citalopram response in depressed patients. This study showed that individuals homozygous for the G allele of the HTR1A rs1364043 polymorphism responded better to citalopram therapy. In patients with major depressive disorder, Kato et al. found an association between HTR1A rs1364043 polymorphism and a better response to antidepressant treatment. 5-HT 2A Receptor The HTR2A gene, coding for the 5-HT 2A receptor, contains several polymorphisms, some of which have been linked to altered receptor function or expression. However, the HTR2A rs6311 and rs6313 polymorphisms have been researched the most, especially their association with response to antidepressant therapy . Recent studies have shown that carriers of the HTR2A rs3803189 and rs7997012 polymorphisms responded better to SSRIs . On the other hand, a number of other studies reported no linkage between different HTR2A polymorphisms and a patient’s response to antidepressant treatment . Several studies have also investigated the relationship between different HTR2A SNPs and the tolerability of antidepressant therapy. According to their findings, specific HTR2A polymorphisms, such as rs7997012 and rs6314, may be associated with a reduced risk of adverse effects of antidepressant drugs . Dopamine Receptors Central dopamine receptors can be classified into two major families based on their structural and functional similarities: D1-like (D 1 and D 5 receptors) and D2-like receptors (D 2 , D 3 , and D 4 receptors). Dopamine D 2 receptors, encoded by the DRD2 gene, are among the most intensively researched receptors in depressive disorders. Several studies have pointed out the possibility that DRD2 polymorphisms play an important role in depressive disorder and response to antidepressant therapy ; however, other authors found no significant association . On the other hand, Wang et al. suggested that DRD2 rs1076562, rs2440390, and rs2734833 polymorphisms are associated with the onset time of the antidepressant response. In addition, according to Perlis et al. , DRD2 rs4245147 polymorphism has been linked to a better lamotrigine response in a group of patients with bipolar depression. 1A Receptor The serotonin 1A (5-HT 1A ) receptor is a G protein-coupled receptor found in the CNS and peripheral tissues. It is widely distributed in various brain regions, including the hippocampus, hypothalamus, cortex, and amygdala. It plays a crucial role in regulating the release of neurotransmitters, particularly serotonin, and is a target for many psychoactive drugs, including antidepressants and anxiolytics . The 5-HT 1A receptor is encoded by the HTR1A gene, and mutations in this gene have been linked to several neuropsychiatric disorders, including major depression, anxiety disorders, and autism spectrum disorder . The role of HTR1A polymorphisms in response to antidepressant therapy has also been investigated; however, a few recent studies found no significant associations . For instance, Scutt et al. reported no association between 5HT1A polymorphism and increased risk of SSRIs adverse effects. On the other hand, other studies have reported different findings . Villafuerte et al. suggested that the G allele of HTR1A rs1364043 polymorphism might predict citalopram response in depressed patients. This study showed that individuals homozygous for the G allele of the HTR1A rs1364043 polymorphism responded better to citalopram therapy. In patients with major depressive disorder, Kato et al. found an association between HTR1A rs1364043 polymorphism and a better response to antidepressant treatment. 2A Receptor The HTR2A gene, coding for the 5-HT 2A receptor, contains several polymorphisms, some of which have been linked to altered receptor function or expression. However, the HTR2A rs6311 and rs6313 polymorphisms have been researched the most, especially their association with response to antidepressant therapy . Recent studies have shown that carriers of the HTR2A rs3803189 and rs7997012 polymorphisms responded better to SSRIs . On the other hand, a number of other studies reported no linkage between different HTR2A polymorphisms and a patient’s response to antidepressant treatment . Several studies have also investigated the relationship between different HTR2A SNPs and the tolerability of antidepressant therapy. According to their findings, specific HTR2A polymorphisms, such as rs7997012 and rs6314, may be associated with a reduced risk of adverse effects of antidepressant drugs . Central dopamine receptors can be classified into two major families based on their structural and functional similarities: D1-like (D 1 and D 5 receptors) and D2-like receptors (D 2 , D 3 , and D 4 receptors). Dopamine D 2 receptors, encoded by the DRD2 gene, are among the most intensively researched receptors in depressive disorders. Several studies have pointed out the possibility that DRD2 polymorphisms play an important role in depressive disorder and response to antidepressant therapy ; however, other authors found no significant association . On the other hand, Wang et al. suggested that DRD2 rs1076562, rs2440390, and rs2734833 polymorphisms are associated with the onset time of the antidepressant response. In addition, according to Perlis et al. , DRD2 rs4245147 polymorphism has been linked to a better lamotrigine response in a group of patients with bipolar depression. In comparison to antidepressants, pharmacogenetic research on anxiolytics is far less prevalent. This is due in part to the fact that benzodiazepines (BZDs), as a prototype of anxiolytic drugs, are administered for shorter periods. Some genes that have been studied regarding BZDs include those encoding drug-metabolizing enzymes, drug transporters, and drug targets . Certain gene polymorphisms that can alter BZDs pharmacokinetics and pharmacodynamics are reviewed in the following sections. 3.1. Pharmacokinetic Variability 3.1.1. UGT2B15 Enzyme UGT2B15 is an enzyme essential for the metabolism of many drugs, including anxiolytics. It belongs to the uridine diphosphate glucuronosyltransferase (UGT) family of enzymes, which catalyze the conjugation of lipophilic compounds with glucuronic acid, thereby facilitating their elimination from the body . UGT2B15 is primarily expressed in the liver and is encoded by the UGT2B15 gene. Genetic polymorphisms in the UGT2B15 gene can alter enzyme activity, leading to inter-individual variability in anxiolytic drug metabolism and response. For example, individuals with the UGT2B15*2 allele may have reduced UGT2B15 activity, resulting in slower drug metabolism and a potentially increased risk of adverse effects . The UGT2B15*2 variant is one of the most studied genetic polymorphisms in the UGT2B15 gene. This is due to a single nucleotide substitution (G > T) in the coding region of the gene, resulting in an amino acid change from aspartic acid (D) to tyrosine (Y) at position 85. This modification could alter the enzyme’s activity and therefore affect the metabolism of drugs that are substrates of UGT2B15 . Chung et al. were the first to demonstrate the effect of the UGT2B15 genotype on the pharmacokinetics of lorazepam in humans. Their study showed that homozygous carriers of the UGT2B15*2 variant had lower systemic clearance and metabolic activity of lorazepam in comparison to individuals without this variant. Since then, several other studies have also examined the association between the UGT2B15 genotype and the pharmacokinetics of lorazepam and other BZDs . Mijdervik et al. reported a linkage between the UGT2B15 genotype and the efficacy of lorazepam in reducing postoperative anxiety. Interestingly, the effects of the UGT2B15*2 variant on the response to lorazepam premedication appeared to differ between male and female patients. Specifically, whereas homozygous male carriers of the UGT2B15*2 variant had a lesser reduction in anxiety compared to placebo, female patients with the same genotype experienced greater anxiety reduction due to lorazepam premedication . However, Jackson et al. suggested that the greater clinical effect in females compared to males after a single dose of lorazepam is unlikely to be due to differences in pharmacokinetics. Instead, they proposed that differences in endogenous levels of neurosteroid hormones between males and females may play a role. Additionally, the UGT2B15*2 variant has also been shown to affect the metabolism of oxazepam. Several studies have shown that the UGT2B15*2 variant is associated with reduced oxazepam glucuronidation in human liver microsomes and decreased oxazepam clearance . A decrease in oxazepam metabolism could potentially affect the metabolism of other BZDs, where oxazepam is an active metabolite, including chlordiazepoxide, clorazepate, diazepam, and temazepam . This could lead to a potential increase in the adverse effects of these drugs, such as sedation, cognitive impairment, and impaired motor coordination . Therefore, patients carrying the UGT2B15*2 variant may require lower doses of these BZDs in order to achieve the same therapeutic effect and minimize adverse effects. 3.1.2. Cytochrome P450 Enzymes and P-glycoprotein As previously discussed, the metabolism of many drugs, including anxiolytics, involves numerous CYP enzymes. Anxiolytic diazepam, for instance, is primarily metabolized by the CYP2C19 enzyme. Other CYP enzymes involved in diazepam metabolism include CYP2C9, CYP2B6, CYP3A4, and CYP3A5 . The association between CYPC19 and CYP2B6 phenotypes and the safety of diazepam was the subject of a recent study by Zubair et al. . This was the first study that demonstrated a significant association between the CYP2B6 phenotype and the pharmacokinetics of diazepam. Their findings suggested that a dose reduction might be necessary for CYP2C19 and/or CYP2B6 PMs to avoid adverse drug reactions, such as dependence on and tolerance to BZDs. The CYP2C19 gene has various genetic variants, some of which have the potential to influence CYPC19 enzyme activity. For example, the CYP2C19*17 allele is associated with increased enzyme activity, whereas nonfunctional variants, such as CYP2C19*2 and *3 , have little or no enzyme function . As stated before, these CYP2C19 polymorphisms can result in altered rates of diazepam metabolism, leading to reduced drug effectiveness or potentially increased risk of adverse effects. Moreover, several recent studies have examined the effects of CYP2C19 polymorphisms, particularly CYP2C19*17 and CYP2C19*2 variants, on the efficacy and safety of diazepam in individuals with alcohol withdrawal syndrome . According to their findings, individuals with reduced CYP2C19 activity, such as carriers of the CYP2C19*2 allele, may be at an increased risk of adverse effects due to slower metabolism and potential drug accumulation. On the contrary, individuals with increased CYP2C19 activity, such as carriers of the CYP2C19*17 allele, may experience reduced efficacy with standard doses of diazepam due to faster metabolism and potentially lower plasma drug concentrations . These findings are consistent with previous studies that investigated the effects of CYP2C19 polymorphisms on diazepam metabolism and treatment outcomes in patients with alcohol withdrawal syndrome . Similarly, the CYP2C19*17 allele may also be responsible for the UM phenotype of clobazam . As a result, higher doses of clobazam may be required to achieve the same therapeutic effect. In contrast, CYP2C19*2 and CYP2C19*3 variants result in a nonfunctional or partially functional CYP2C19 enzyme. Individuals who are homozygous for CYP2C19*2 and CYP2C19*3 variants may have a PM phenotype with an increased risk of adverse effects and toxicity . Two case reports have shown an association between the CYP2C19*2 variant and an increased risk of adverse effects in clobazam-treated patients . As previously mentioned, the clearance of benzodiazepines, such as diazepam and clobazam, may be reduced in carriers of CYP2C19 nonfunctional variants. However, BZDs have a wide therapeutic window, indicating that many individuals with this variant can still tolerate standard doses without experiencing substantial adverse effects . Moreover, a recent study found an association between CYP3A5 polymorphism and the metabolism of midazolam . Specifically, carriers of the CYP3A5 rs776746 T allele had decreased plasma concentrations of midazolam in comparison to individuals with the C allele and may require higher doses of midazolam to achieve a sedative effect . The CYP3A5 polymorphism, particularly the CYP3A5*3 variant, has also been associated with the altered metabolism of alprazolam . Park et al. have shown that homozygous carriers of the CYP3A5*3 allele might have a slower metabolism of alprazolam, resulting in higher plasma concentrations. Regarding the CYP3A4 enzyme, the CYP3A4*22 gene variant has been frequently described in the literature . Individuals who carry the CYP3A4*22 allele may have reduced CYP3A4 enzyme activity and slower drug metabolism . Although, several studies have suggested that CYP3A4 polymorphisms may be associated with differences in response to BZDs, the evidence is not yet conclusive . In addition, there are currently no established pharmacogenetic biomarkers for BZDs such as bromazepam and lormetazepam . As in the case of antidepressants, BZDs, such as midazolam, are also substrates for P-gp . Several studies have investigated the potential role of P-gp polymorphisms in the response to midazolam . However, these findings are inconsistent and sometimes contradictory. Specifically, whereas Park et al. demonstrated that the polymorphism in the MDR1 / ABCB1 gene was linked to an increased midazolam concentration and higher sedation degree, Byon et al. found no significant association. 3.2. Pharmacodynamic Variability The pharmacodynamics of anxiolytic drugs are yet another target of pharmacogenetic studies investigating the treatment of various conditions . It is well known that BZDs enhance the inhibitory effects of γ-aminobutyric acid (GABA) through the allosteric modulation of GABA type A (GABA A ) receptors, resulting in anxiolytic, sedative, hypnotic, anticonvulsant, and myorelaxant effects . The majority of GABA A receptors are pentameric complexes composed of two α subunits, two β subunits, and one γ or δ subunit . Depending on the GABA A receptor subtype, the benzodiazepine effects can differ. The sedative effects of BZDs are primarily mediated by the α 1 subunit of GABA A receptors, whereas the α 2 and α 3 subunits are responsible for the anxiolytic effects of BZDs. Moreover, the α 5 subunit mediates the amnestic properties of BDZs . A study by Kelly et al. showed that certain polymorphisms in the GABA A receptor α 5 subunit might result in a conformational change of the receptor, altering the GABA binding site. Several animal models with genetic mutations affecting specific subunits of the GABA A receptor complex have been developed . For example, one study found that mice lacking the γ 2 subunit showed greatly reduced BZDs sensitivity, including the loss of sedative, anxiolytic, and anticonvulsant effects . On the other hand, Chandra et al. demonstrated that mice without the expression of γ 2 exhibited increased anxiety-like behaviors but did not show significant differences in the hypnotic response to BZDs. In addition, mice with a knock-in F77I mutation in the GABRG2 gene, coding for the GABA A receptor γ 2 subunit, were found to have reduced sensitivity to the hypnotic effects of zolpidem . Polymorphisms in the GABRA2 gene, encoding the α 2 subunit of the GABA A receptor, have been associated with substance abuse behaviors . Another study investigated the connection between polymorphisms in the GABRA1 gene and the response to zolpidem in patients with insomnia. The GABRA1 A15G variant has been identified as a polymorphism associated with complex sleep behaviors and amnesia . Choi et al. found that among five known SNPs in the GABRA1 gene, the rs4263535 polymorphism was associated with deeper sedation in response to intravenous midazolam. Moreover, Bowser et al. discovered that individuals with epilepsy who carried the R43Q mutation in the GABRG2 gene had reduced sensitivity to BZDs. Similarly, a recent study reported a possible association between two polymorphisms in GABRA2 and GABRA5 genes and drug-resistant epilepsy . Overall, research assessing the pharmacodynamic variability of anxiolytic drugs remains a relatively new field. Additional research is necessary to fully understand how genetic variations affect anxiolytic drug responses. 3.1.1. UGT2B15 Enzyme UGT2B15 is an enzyme essential for the metabolism of many drugs, including anxiolytics. It belongs to the uridine diphosphate glucuronosyltransferase (UGT) family of enzymes, which catalyze the conjugation of lipophilic compounds with glucuronic acid, thereby facilitating their elimination from the body . UGT2B15 is primarily expressed in the liver and is encoded by the UGT2B15 gene. Genetic polymorphisms in the UGT2B15 gene can alter enzyme activity, leading to inter-individual variability in anxiolytic drug metabolism and response. For example, individuals with the UGT2B15*2 allele may have reduced UGT2B15 activity, resulting in slower drug metabolism and a potentially increased risk of adverse effects . The UGT2B15*2 variant is one of the most studied genetic polymorphisms in the UGT2B15 gene. This is due to a single nucleotide substitution (G > T) in the coding region of the gene, resulting in an amino acid change from aspartic acid (D) to tyrosine (Y) at position 85. This modification could alter the enzyme’s activity and therefore affect the metabolism of drugs that are substrates of UGT2B15 . Chung et al. were the first to demonstrate the effect of the UGT2B15 genotype on the pharmacokinetics of lorazepam in humans. Their study showed that homozygous carriers of the UGT2B15*2 variant had lower systemic clearance and metabolic activity of lorazepam in comparison to individuals without this variant. Since then, several other studies have also examined the association between the UGT2B15 genotype and the pharmacokinetics of lorazepam and other BZDs . Mijdervik et al. reported a linkage between the UGT2B15 genotype and the efficacy of lorazepam in reducing postoperative anxiety. Interestingly, the effects of the UGT2B15*2 variant on the response to lorazepam premedication appeared to differ between male and female patients. Specifically, whereas homozygous male carriers of the UGT2B15*2 variant had a lesser reduction in anxiety compared to placebo, female patients with the same genotype experienced greater anxiety reduction due to lorazepam premedication . However, Jackson et al. suggested that the greater clinical effect in females compared to males after a single dose of lorazepam is unlikely to be due to differences in pharmacokinetics. Instead, they proposed that differences in endogenous levels of neurosteroid hormones between males and females may play a role. Additionally, the UGT2B15*2 variant has also been shown to affect the metabolism of oxazepam. Several studies have shown that the UGT2B15*2 variant is associated with reduced oxazepam glucuronidation in human liver microsomes and decreased oxazepam clearance . A decrease in oxazepam metabolism could potentially affect the metabolism of other BZDs, where oxazepam is an active metabolite, including chlordiazepoxide, clorazepate, diazepam, and temazepam . This could lead to a potential increase in the adverse effects of these drugs, such as sedation, cognitive impairment, and impaired motor coordination . Therefore, patients carrying the UGT2B15*2 variant may require lower doses of these BZDs in order to achieve the same therapeutic effect and minimize adverse effects. 3.1.2. Cytochrome P450 Enzymes and P-glycoprotein As previously discussed, the metabolism of many drugs, including anxiolytics, involves numerous CYP enzymes. Anxiolytic diazepam, for instance, is primarily metabolized by the CYP2C19 enzyme. Other CYP enzymes involved in diazepam metabolism include CYP2C9, CYP2B6, CYP3A4, and CYP3A5 . The association between CYPC19 and CYP2B6 phenotypes and the safety of diazepam was the subject of a recent study by Zubair et al. . This was the first study that demonstrated a significant association between the CYP2B6 phenotype and the pharmacokinetics of diazepam. Their findings suggested that a dose reduction might be necessary for CYP2C19 and/or CYP2B6 PMs to avoid adverse drug reactions, such as dependence on and tolerance to BZDs. The CYP2C19 gene has various genetic variants, some of which have the potential to influence CYPC19 enzyme activity. For example, the CYP2C19*17 allele is associated with increased enzyme activity, whereas nonfunctional variants, such as CYP2C19*2 and *3 , have little or no enzyme function . As stated before, these CYP2C19 polymorphisms can result in altered rates of diazepam metabolism, leading to reduced drug effectiveness or potentially increased risk of adverse effects. Moreover, several recent studies have examined the effects of CYP2C19 polymorphisms, particularly CYP2C19*17 and CYP2C19*2 variants, on the efficacy and safety of diazepam in individuals with alcohol withdrawal syndrome . According to their findings, individuals with reduced CYP2C19 activity, such as carriers of the CYP2C19*2 allele, may be at an increased risk of adverse effects due to slower metabolism and potential drug accumulation. On the contrary, individuals with increased CYP2C19 activity, such as carriers of the CYP2C19*17 allele, may experience reduced efficacy with standard doses of diazepam due to faster metabolism and potentially lower plasma drug concentrations . These findings are consistent with previous studies that investigated the effects of CYP2C19 polymorphisms on diazepam metabolism and treatment outcomes in patients with alcohol withdrawal syndrome . Similarly, the CYP2C19*17 allele may also be responsible for the UM phenotype of clobazam . As a result, higher doses of clobazam may be required to achieve the same therapeutic effect. In contrast, CYP2C19*2 and CYP2C19*3 variants result in a nonfunctional or partially functional CYP2C19 enzyme. Individuals who are homozygous for CYP2C19*2 and CYP2C19*3 variants may have a PM phenotype with an increased risk of adverse effects and toxicity . Two case reports have shown an association between the CYP2C19*2 variant and an increased risk of adverse effects in clobazam-treated patients . As previously mentioned, the clearance of benzodiazepines, such as diazepam and clobazam, may be reduced in carriers of CYP2C19 nonfunctional variants. However, BZDs have a wide therapeutic window, indicating that many individuals with this variant can still tolerate standard doses without experiencing substantial adverse effects . Moreover, a recent study found an association between CYP3A5 polymorphism and the metabolism of midazolam . Specifically, carriers of the CYP3A5 rs776746 T allele had decreased plasma concentrations of midazolam in comparison to individuals with the C allele and may require higher doses of midazolam to achieve a sedative effect . The CYP3A5 polymorphism, particularly the CYP3A5*3 variant, has also been associated with the altered metabolism of alprazolam . Park et al. have shown that homozygous carriers of the CYP3A5*3 allele might have a slower metabolism of alprazolam, resulting in higher plasma concentrations. Regarding the CYP3A4 enzyme, the CYP3A4*22 gene variant has been frequently described in the literature . Individuals who carry the CYP3A4*22 allele may have reduced CYP3A4 enzyme activity and slower drug metabolism . Although, several studies have suggested that CYP3A4 polymorphisms may be associated with differences in response to BZDs, the evidence is not yet conclusive . In addition, there are currently no established pharmacogenetic biomarkers for BZDs such as bromazepam and lormetazepam . As in the case of antidepressants, BZDs, such as midazolam, are also substrates for P-gp . Several studies have investigated the potential role of P-gp polymorphisms in the response to midazolam . However, these findings are inconsistent and sometimes contradictory. Specifically, whereas Park et al. demonstrated that the polymorphism in the MDR1 / ABCB1 gene was linked to an increased midazolam concentration and higher sedation degree, Byon et al. found no significant association. UGT2B15 is an enzyme essential for the metabolism of many drugs, including anxiolytics. It belongs to the uridine diphosphate glucuronosyltransferase (UGT) family of enzymes, which catalyze the conjugation of lipophilic compounds with glucuronic acid, thereby facilitating their elimination from the body . UGT2B15 is primarily expressed in the liver and is encoded by the UGT2B15 gene. Genetic polymorphisms in the UGT2B15 gene can alter enzyme activity, leading to inter-individual variability in anxiolytic drug metabolism and response. For example, individuals with the UGT2B15*2 allele may have reduced UGT2B15 activity, resulting in slower drug metabolism and a potentially increased risk of adverse effects . The UGT2B15*2 variant is one of the most studied genetic polymorphisms in the UGT2B15 gene. This is due to a single nucleotide substitution (G > T) in the coding region of the gene, resulting in an amino acid change from aspartic acid (D) to tyrosine (Y) at position 85. This modification could alter the enzyme’s activity and therefore affect the metabolism of drugs that are substrates of UGT2B15 . Chung et al. were the first to demonstrate the effect of the UGT2B15 genotype on the pharmacokinetics of lorazepam in humans. Their study showed that homozygous carriers of the UGT2B15*2 variant had lower systemic clearance and metabolic activity of lorazepam in comparison to individuals without this variant. Since then, several other studies have also examined the association between the UGT2B15 genotype and the pharmacokinetics of lorazepam and other BZDs . Mijdervik et al. reported a linkage between the UGT2B15 genotype and the efficacy of lorazepam in reducing postoperative anxiety. Interestingly, the effects of the UGT2B15*2 variant on the response to lorazepam premedication appeared to differ between male and female patients. Specifically, whereas homozygous male carriers of the UGT2B15*2 variant had a lesser reduction in anxiety compared to placebo, female patients with the same genotype experienced greater anxiety reduction due to lorazepam premedication . However, Jackson et al. suggested that the greater clinical effect in females compared to males after a single dose of lorazepam is unlikely to be due to differences in pharmacokinetics. Instead, they proposed that differences in endogenous levels of neurosteroid hormones between males and females may play a role. Additionally, the UGT2B15*2 variant has also been shown to affect the metabolism of oxazepam. Several studies have shown that the UGT2B15*2 variant is associated with reduced oxazepam glucuronidation in human liver microsomes and decreased oxazepam clearance . A decrease in oxazepam metabolism could potentially affect the metabolism of other BZDs, where oxazepam is an active metabolite, including chlordiazepoxide, clorazepate, diazepam, and temazepam . This could lead to a potential increase in the adverse effects of these drugs, such as sedation, cognitive impairment, and impaired motor coordination . Therefore, patients carrying the UGT2B15*2 variant may require lower doses of these BZDs in order to achieve the same therapeutic effect and minimize adverse effects. As previously discussed, the metabolism of many drugs, including anxiolytics, involves numerous CYP enzymes. Anxiolytic diazepam, for instance, is primarily metabolized by the CYP2C19 enzyme. Other CYP enzymes involved in diazepam metabolism include CYP2C9, CYP2B6, CYP3A4, and CYP3A5 . The association between CYPC19 and CYP2B6 phenotypes and the safety of diazepam was the subject of a recent study by Zubair et al. . This was the first study that demonstrated a significant association between the CYP2B6 phenotype and the pharmacokinetics of diazepam. Their findings suggested that a dose reduction might be necessary for CYP2C19 and/or CYP2B6 PMs to avoid adverse drug reactions, such as dependence on and tolerance to BZDs. The CYP2C19 gene has various genetic variants, some of which have the potential to influence CYPC19 enzyme activity. For example, the CYP2C19*17 allele is associated with increased enzyme activity, whereas nonfunctional variants, such as CYP2C19*2 and *3 , have little or no enzyme function . As stated before, these CYP2C19 polymorphisms can result in altered rates of diazepam metabolism, leading to reduced drug effectiveness or potentially increased risk of adverse effects. Moreover, several recent studies have examined the effects of CYP2C19 polymorphisms, particularly CYP2C19*17 and CYP2C19*2 variants, on the efficacy and safety of diazepam in individuals with alcohol withdrawal syndrome . According to their findings, individuals with reduced CYP2C19 activity, such as carriers of the CYP2C19*2 allele, may be at an increased risk of adverse effects due to slower metabolism and potential drug accumulation. On the contrary, individuals with increased CYP2C19 activity, such as carriers of the CYP2C19*17 allele, may experience reduced efficacy with standard doses of diazepam due to faster metabolism and potentially lower plasma drug concentrations . These findings are consistent with previous studies that investigated the effects of CYP2C19 polymorphisms on diazepam metabolism and treatment outcomes in patients with alcohol withdrawal syndrome . Similarly, the CYP2C19*17 allele may also be responsible for the UM phenotype of clobazam . As a result, higher doses of clobazam may be required to achieve the same therapeutic effect. In contrast, CYP2C19*2 and CYP2C19*3 variants result in a nonfunctional or partially functional CYP2C19 enzyme. Individuals who are homozygous for CYP2C19*2 and CYP2C19*3 variants may have a PM phenotype with an increased risk of adverse effects and toxicity . Two case reports have shown an association between the CYP2C19*2 variant and an increased risk of adverse effects in clobazam-treated patients . As previously mentioned, the clearance of benzodiazepines, such as diazepam and clobazam, may be reduced in carriers of CYP2C19 nonfunctional variants. However, BZDs have a wide therapeutic window, indicating that many individuals with this variant can still tolerate standard doses without experiencing substantial adverse effects . Moreover, a recent study found an association between CYP3A5 polymorphism and the metabolism of midazolam . Specifically, carriers of the CYP3A5 rs776746 T allele had decreased plasma concentrations of midazolam in comparison to individuals with the C allele and may require higher doses of midazolam to achieve a sedative effect . The CYP3A5 polymorphism, particularly the CYP3A5*3 variant, has also been associated with the altered metabolism of alprazolam . Park et al. have shown that homozygous carriers of the CYP3A5*3 allele might have a slower metabolism of alprazolam, resulting in higher plasma concentrations. Regarding the CYP3A4 enzyme, the CYP3A4*22 gene variant has been frequently described in the literature . Individuals who carry the CYP3A4*22 allele may have reduced CYP3A4 enzyme activity and slower drug metabolism . Although, several studies have suggested that CYP3A4 polymorphisms may be associated with differences in response to BZDs, the evidence is not yet conclusive . In addition, there are currently no established pharmacogenetic biomarkers for BZDs such as bromazepam and lormetazepam . As in the case of antidepressants, BZDs, such as midazolam, are also substrates for P-gp . Several studies have investigated the potential role of P-gp polymorphisms in the response to midazolam . However, these findings are inconsistent and sometimes contradictory. Specifically, whereas Park et al. demonstrated that the polymorphism in the MDR1 / ABCB1 gene was linked to an increased midazolam concentration and higher sedation degree, Byon et al. found no significant association. The pharmacodynamics of anxiolytic drugs are yet another target of pharmacogenetic studies investigating the treatment of various conditions . It is well known that BZDs enhance the inhibitory effects of γ-aminobutyric acid (GABA) through the allosteric modulation of GABA type A (GABA A ) receptors, resulting in anxiolytic, sedative, hypnotic, anticonvulsant, and myorelaxant effects . The majority of GABA A receptors are pentameric complexes composed of two α subunits, two β subunits, and one γ or δ subunit . Depending on the GABA A receptor subtype, the benzodiazepine effects can differ. The sedative effects of BZDs are primarily mediated by the α 1 subunit of GABA A receptors, whereas the α 2 and α 3 subunits are responsible for the anxiolytic effects of BZDs. Moreover, the α 5 subunit mediates the amnestic properties of BDZs . A study by Kelly et al. showed that certain polymorphisms in the GABA A receptor α 5 subunit might result in a conformational change of the receptor, altering the GABA binding site. Several animal models with genetic mutations affecting specific subunits of the GABA A receptor complex have been developed . For example, one study found that mice lacking the γ 2 subunit showed greatly reduced BZDs sensitivity, including the loss of sedative, anxiolytic, and anticonvulsant effects . On the other hand, Chandra et al. demonstrated that mice without the expression of γ 2 exhibited increased anxiety-like behaviors but did not show significant differences in the hypnotic response to BZDs. In addition, mice with a knock-in F77I mutation in the GABRG2 gene, coding for the GABA A receptor γ 2 subunit, were found to have reduced sensitivity to the hypnotic effects of zolpidem . Polymorphisms in the GABRA2 gene, encoding the α 2 subunit of the GABA A receptor, have been associated with substance abuse behaviors . Another study investigated the connection between polymorphisms in the GABRA1 gene and the response to zolpidem in patients with insomnia. The GABRA1 A15G variant has been identified as a polymorphism associated with complex sleep behaviors and amnesia . Choi et al. found that among five known SNPs in the GABRA1 gene, the rs4263535 polymorphism was associated with deeper sedation in response to intravenous midazolam. Moreover, Bowser et al. discovered that individuals with epilepsy who carried the R43Q mutation in the GABRG2 gene had reduced sensitivity to BZDs. Similarly, a recent study reported a possible association between two polymorphisms in GABRA2 and GABRA5 genes and drug-resistant epilepsy . Overall, research assessing the pharmacodynamic variability of anxiolytic drugs remains a relatively new field. Additional research is necessary to fully understand how genetic variations affect anxiolytic drug responses. An increasing number of both preclinical and clinical findings suggest that epigenetic changes might be useful for the prediction of treatment response. DNA methylation, one of the most investigated epigenetic modifications, has been shown to play a role in drug responses, including responses to antidepressants and anxiolytics . For instance, Takeuchi et al. found an association between DNA methylation at several CpG sites in specific genes and the therapeutic response to paroxetine in patients with major depressive disorder. Moreover, methylation at several CpG sites within the interleukin-11 ( IL11 ) promoter was found to be predictive of escitalopram or nortriptyline treatment response . Several studies have also investigated the potential effects of epigenetic regulation of the brain-derived neurotrophic factor ( BDNF ) gene on antidepressant treatment responses. For example, Wang et al. suggested that BDNF DNA hypomethylation leads to an impaired response to escitalopram. In addition, lower methylation levels at CpG sites within the BDNF promoter were associated with an antidepressant response and decreased BDNF plasma levels after one week of treatment . DNA methylation of BDNF seems to be epigenetically regulated by FK506 Binding Protein 5 (FKBP5), according to the antidepressant response . FKBP5 is a cochaperone protein of the glucocorticoid receptor complex involved in intracellular glucocorticoid signaling and the stress response . These findings are not surprising, since glucocorticoid secretion in response to stress, as well as genes involved in the glucocorticoid signaling pathway, are proposed to play an important role in shaping the epigenetic landscape . Moreover, there is strong evidence that antidepressants exert their effects by modulating the glucocorticoid receptor; therefore, epigenetic modifications of the glucocorticoid receptor could be of considerable importance for therapeutic efficacy . In the context of stress response, in addition to FKBP5 and BDNF , other extensively studied epigenetically altered genes include the NR3C1 gene coding for glucocorticoid receptor, CRH gene coding for corticotropin-releasing hormone, and MAOA and SLC6A4 genes. Specifically, the study of Elliott et al. (2010) demonstrated the demethylation of CRH gene, involved in the brain stress response, in mice exposed to chronic social stress and attenuation of CRH promoter methylation levels with the antidepressant imipramine . Moreover, hypomethylation in the promoter region of the SLC6A4 gene has been predictive of impaired response to SSRIs . On the other hand, investigations into the relationship between DNA methylation in the MAOA gene and the response to antidepressant drugs have produced conflicting results, with some studies reporting an association between MAOA DNA methylation and antidepressant response , while others observed no significant associations . Various studies have implicated histone modifications in stress and depressive phenotypes as well as in antidepressant treatment. As in the case of DNA methylation, the BDNF gene has also been investigated regarding histone modifications. For instance, in mice, chronic social defeat stress induced lasting downregulation of BDNF transcripts III and IV and increased repressive histone methylation at their corresponding promoters in the hippocampus . However, chronic imipramine treatment increased histone acetylation at the BDNF P3 and P4 promoters by selectively downregulating histone deacetylase (Hdac) . In addition, Chen et al. investigated the effects of antidepressant drugs on the human postmortem prefrontal cortex and reported increased expression of BDNF IV , which was associated with a decrease in histone H3 lysine 27 (H3K27) methylation at the promoter region . Moreover, a significant decrease in the peripheral blood H3K27 methylation levels at the promoter IV of the BDNF gene and a concomitant increase in BDNF mRNA expression were found in citalopram responders in comparison to nonresponders . These findings suggest that BDNF promoter H3K27 methylation levels could serve as a potential biomarker for citalopram response in depressive patients. Noncoding RNAs have also been investigated in relation to antidepressant responses . Several miRNAs, such as miR-1202, miR124, miR-135a, miR-145, and miR-20b, have been strongly associated with antidepressant responses . In addition, miR-146a5p, miR-146b-5p, miR-425-3p, and miR-24-3p expression levels decreased during antidepressant therapy and are therefore suggested as potential mediator biomarkers of antidepressant response . In contrast to antidepressants, pharmacoepigenetic studies on anxiolytic drugs are still relatively limited. However, some evidence suggests that epigenetic modifications may also play a role in the drug response and efficacy of anxiolytics . Pharmacogenetics can potentially optimize the treatment of neuropsychiatric disorders via the prediction of clinical outcomes of antidepressant and anxiolytic drug administration, including therapy response and development of drug side effects, without the need for conventional “trial-and-error” approaches. The PREPARE study, which covered a large number of drugs, demonstrated that genotype-guided treatment using a 12-gene pharmacogenetic panel significantly reduced the incidence of clinically relevant adverse drug reactions . These results show the large-scale feasibility and clinical usefulness of implementing of a panel-based pharmacogenetic testing strategy to make drug therapy increasingly safe . As shown in , for antidepressant and anxiolytic therapy, the most promising pharmacogenetic candidates are genetic polymorphisms affecting metabolizing cytochrome CYP450 and UGT enzymes, P-glycoprotein ABC transporter, as well as monoamine and GABA metabolic enzymes, transporters, and receptors. Some of these gene candidates, as well as many others, have been shown to be epigenetically altered in response to stress, suggesting the potential of epigenetic profiles for making clinically relevant decisions regarding antidepressant and anxiolytic therapy. Although pharmacogenetic research has revealed that more efficient and safer treatment with antidepressants and anxiolytics can be achieved through genotype-guided decisions, further well-designed trials are needed to overcome various methodological limitations and confirm the current pharmacogenetic findings. The other main constraints of pharmacogenetic studies targeting both pharmacokinetic and pharmacodynamic variability include the high cost of molecular testing, their relative inaccessibility, and the complexity of result interpretation. Nevertheless, with more evidence on the clinical and economic benefits, improved clinical guidelines, lower costs, and shorter delivery times, pharmacogenetics as a key part of personalized medicine may become a routine intervention in neuropsychiatric clinical practice. On the other hand, the pharmacoepigenetic field is still relatively new compared to pharmacogenetic research. Therefore, future research advances on the relationship between epigenetic modifications and drug responses are needed in order to personalize antidepressant and anxiolytic treatment.
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Patient-reported outcome measures and value-based medicine in paediatrics: a timely review
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a1be4fb3-31b8-4edf-b743-acfdd7606c8c
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10219120
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Pediatrics[mh]
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Timely, appropriate, evidence-based and cost-effective delivery of medical care for patients is the primary goal of healthcare providers. With advances in scientific research, medical knowledge has improved in leaps and bounds, further fuelled by technology, in a vastly interconnected medical milieu. In recent years, there is an increasing focus on integration of care — providing care closer to patients’ homes, as well as providing adequate support within communities. Healthcare providers need to go beyond the boundaries of traditional healthcare facilities and work with community partners. Many medical providers do not fully appreciate the impact that medical illnesses and treatment plans can have on their patients’ day-to-day functioning and lives. Often, quality in healthcare is measured by looking at easily quantifiable outcomes from the direct and immediate medical care that has been delivered. However, quality in healthcare should be measured not only by routine and well-established clinical outcomes such as survival rates, length of stay and complications such as healthcare-associated infections, but also take into account patients’ perspectives, experiences and other quality-of-life (QoL) parameters that are related to both healthcare and health. In other words, outcome measures in health and healthcare can be divided into three components: clinical outcome measures, patient-reported outcome measures (PROMs) and patient-reported experience measures (PREMs). The paediatric service has traditionally incorporated many patient-reported outcomes and experiences to improve the medical care rendered. However, there is a lack of systematic collection of patient-reported outcomes and experiences, as well as application of such data in quality improvement programmes. With increasing global awareness of the benefits of patient and public engagement in the design and delivery of healthcare services, this article discusses why PROMs matter, how PROMs can be applied, and what value-based medicine (VBM) means in paediatric clinical care today.
Health is “ a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity ”. Huber posits that “ health is the ability to adapt and to self-manage, in the face of social, physical and emotional challenges ”. Multiple factors affect health-related QoL . As clinicians, we often lack awareness about the consequences of a disease and treatment on our patients’ daily lives. We may take a comprehensive history, do a laudable physical examination and communicate effectively with our patients, but the extent to which we register and embed the effects of disease and care on the health of our patients is debatable. We should therefore not neglect the great significance of patient and public involvement in the remodelling of healthcare services. PROMs are used to objectively assess our patients’ perceptions of the status of their health and QoL, measured over a period of time. They are ‘ directly reported by the patient without interpretation of the patient’s response by a clinician or anyone else and pertain to the patient’s health, QoL, or functional status associated with healthcare or treatment ’. Validated measurement tools and questionnaires are used in PROMs to qualify and quantify these patient-reported outcomes. PROMs provide information that only patients themselves can provide to healthcare providers . They are relevant and crucial to allow healthcare providers to arrive at sound medical decisions with patients through better and effective conversations. PROMs help to bridge the gap by representing patients’ voices and views regarding their individual health. They help physicians to improve patient satisfaction with medical care. lists several pertinent reasons for why patients’ views matter. PROMs are not outcome measures per se but measurements of patients’ health journey at different time points in their life course. The choice of adopting PROMs at different time points is important to reduce subjectivity and recall bias in the patients involved. In general, PROMs can be any method in which patients’ input is obtained without interpretation by medical providers or anyone else — including from diaries and event logs using free-text input, to one-item or multi-item multi-domain scales. Data collection, analysis and interpretation are standardised and well documented to accurately represent the target patient population. Outcomes do not always need to be patient reported, but could also be observer or proxy reported. An observer-reported outcome is one where an observer (anyone other than the patient) is compiling the outcome from a patient, such as a doctor seeking a specific outcome from the patient. A proxy-reported outcome is one where someone other than the patient is reporting the outcome as if he/she is the patient, such as a parent reporting a specific outcome on behalf of a young child. This becomes especially relevant in the development of paediatric PROMs and is elaborated on later in this article. In comparison, PREMs assess the experiences patients go through and address ‘the humanity of care’. PREMs can be relational as well as functional. Relational PREMs measure patients’ perception of their healthcare journey from a relational perspective (e.g. did the patients feel that they were treated with respect?), while functional PREMs measure more operational issues, such as the facilities where the care was rendered. PROMs and PREMs can enhance the efficacy of the medical care given to patients because they enable more patient-centric care to be delivered, with treatment and care tailored to their specific and individual needs. Types of patient-reported outcome measures PROMs must be contextualised and determined by the specific patient needs and requirements of healthcare providers and their patients. They can be domain-specific, disease-specific, generic and preference-based . Commonly assessed areas in PROMs include: overall and health-related QoL; general perception and outlook on health; satisfaction with healthcare; physical functioning; mental well-being; psychosocial function and well-being; and others, including symptoms and impairments (e.g. pain, sleep), cognitive well-being and role activities (e.g. employment, financial well-being).
PROMs must be contextualised and determined by the specific patient needs and requirements of healthcare providers and their patients. They can be domain-specific, disease-specific, generic and preference-based . Commonly assessed areas in PROMs include: overall and health-related QoL; general perception and outlook on health; satisfaction with healthcare; physical functioning; mental well-being; psychosocial function and well-being; and others, including symptoms and impairments (e.g. pain, sleep), cognitive well-being and role activities (e.g. employment, financial well-being).
PROMs were originally developed for research and have been well established in clinical trials and pharmacological research for many years. Recently, regulatory bodies use PROMs to standardise interpretation in clinical trials. In health services research, PROMs facilitate swift and cost-effective evaluation of policies and services. The EuroQol 5D (five-dimension) instrument, for example, has been used for cost-effective evaluation in various programmes. The evolution of the applicability of PROMs in clinical practice demonstrates their critical role in the modern world. There is an unmet need to establish and maintain collaborative framework for validation of PROMs. Core outcome sets Core outcome sets (COS) are an accepted, consensus-based, standardised and minimum set of outcomes for a specific disease or trial population within a specified setting. Development of COS is a multi-step process to achieve consensus that includes a literature review of the evidence, followed by a series of Delphi questionnaires and rounds of discussion among a panel of experts. Delphi surveys aim to achieve consensus from among healthcare providers as well as the respective community on what outcomes to address. The expert panel deliberates and, once these core outcomes are agreed upon, determines the specific measurement domains and measurement instruments to use. The COS forms the basis upon which further domains and dimensions can be measured and subsequently developed. The Outcome Measures in Rheumatology (OMERACT) initiative, for instance, frames four key ‘impact of health’ aspects: survival, life impact, economic impact/resource use and pathophysiological manifestations. Applying to clinical practice Healthcare is strengthened by PROMs as they enable medical providers to deliver improved care that is patient-centric, and to evaluate and benchmark the quality of care by collecting information for assessing current practices and policies, providing the means to enhance healthcare productivity and quality of care. They can be translated into clinical practice in the monitoring and surveillance of an individual’s health and healthcare needs, and used in quality improvement and safety initiatives. Although some medical providers were initially skeptical about the usage of PROMs, most, if not all, will acknowledge the benefits of taking patients’ views in clinical practice. Selection of the relevant PROMs is crucial and will impact what the output is and how this eventually translates into care for the patients specifically and is scaled to the population at large. It is a multi-step process involving clear elucidation and comprehensive planning. The objectives and the population or patient group must first be clearly defined. A panel of experts must review the current evidence and literature, seeking feedback not only from healthcare practitioners but also patients, patient advocacy groups and the general population. The core outcomes to be measured and other important outcome parameters must be decided on, followed by choosing appropriate measurement instruments and tools. As there are many validated and well established outcome measurement tools, the challenge is to choose the most appropriate basket of measurement tools for the specific and required objectives. General considerations in choosing measurement tools There are some key principles when choosing patient-centred measurements. The choice of a PROM is dependent on various factors including ability to detect change, validity, reliability, acceptability and feasibility. The principles of patient-centred measurement state that measurements are: (a) patient-driven, with patients’ goals, preferences and priorities driving the metrics and assessment; (b) holistic, factoring aspects of the patient within and outside of the healthcare system; (c) transparent and accessible to all stakeholders; (d) timely and comprehensible; and (e) co-created with patients as equal partners in the enterprise. Firstly, the PROM tools must have been robustly validated and evaluated. They must also measure what they have been designed to assess. Other important considerations are compliance, preferences, implementation time and costs. Patients should be involved in its development. The PROM needs to be able to identify changes over time. The PROM must be clear and appropriately formatted so that there is patient participation and compliance. It must be consistent, reliable and reproducible. Finally, the PROM needs to be feasible to implement so that it is widely adopted and used by medical providers and administrators . Barriers, challenges and caveats in implementation There are many technical, logistical, social, cultural and legal barriers to translating PROMs into clinical practical in a sustainable manner. Varying definitions and terminologies currently exist in this field of practice. OMERACT, for instance, uses ‘core domain sets’, whereas the HOME (Harmonising Outcome Measures for Eczema) initiative describes ‘core outcome domains’. COSMIN (COnsensus-based Standards for the selection of health Measurement INstruments), COMET (Core Outcome Measures in Effectiveness Trials) and COMIS (Core Outcome Measurement Instrument Selection) are all initiatives that aim to set guidelines and consensus in a standardised way. In addition, there are also varied approaches in PROM methodologies. In order to strengthen and not compromise the validity of the PROMs collected, regulatory bodies ensure standardised and robust translation of the PROMs by qualified linguists. This should reflect the best practice to minimise inter-racial variability among various languages when collecting data. Other challenges and caveats in implementing PROMs include the following. (a) There must be high rates of patient participation. (b) The three dimensions of quality (safety, effectiveness and experience) must be incorporated. (c) The costs and time of data collection, analysis, interpretation and presentation must be manageable and sustainable. (d) Timely administration can avoid various confounding bias, including attribution bias and recall bias after specific events (for measurements such as emergency care) due to time lag with meaningful comparisons when attributing outcomes to quality of care. (e) PROMs should add value to patient care and not be used erroneously to simply ration medical care. To ensure the sustainability of PROMs in the clinical system, their incorporation should be embedded into existing clinical frameworks and workflows. They cannot exist as a standalone vicarious entity. Otherwise, the uptake by clinicians will be poor. PROMs should be seamlessly integrated into existing clinical notes and into patients’ electronic health records. PROMs take time out of clinical encounters, as they are usually administered during clinic visits and may pose delays and challenges for clinicians. Technology can help to reduce logistical barriers to PROM assessment with the use of Internet-based data collection platforms, thereby allowing completion of PROMs outside of clinical appointments and minimising administrative burdens. Experiences from centres that have integrated PROMs into clinical practice show that they can contribute to better care delivery by facilitating remote monitoring of progress by healthcare providers and the minimising of visits or rescheduling of earlier visits when indicated. PROMs enable better communication between healthcare provider and patient, enhance physicians’ awareness of patients’ problems, and allow for more consistent and real-time adjustments to healthcare. Patient-reported outcomes measurement information system Various centres have started using PROMs in clinical practice. The practice is growing in the United States and even more so in Europe, including efforts by the United Kingdom National Health Service. The Patient-Reported Outcomes Measurement Information System (PROMIS), from the National Institutes of Health in the United States, is a collection of person-centred measures that are accessible through the internet, assessing and monitoring “ physical, mental and social health in both adults and children ”. PROMIS aims to standardise and facilitate “ a common measurement system for patient-reported outcomes across clinical research ”. PROMIS uses cognitive and psychometric theories and tools, utilises ‘think-aloud’ and ‘respondent debriefing’ cognitive interviewing methods, and item response theory. Item response theory is also known as latent trait theory, strong true score theory or modern mental test theory, and is a paradigm for the design, analysis and scoring of various measurement variables. Computerised adaptive testing, also known as tailored testing in PROMIS, adapts to the respondent’s ability level . International consortium for health outcomes measurement The International Consortium for Health Outcomes Measurement (ICHOM) is a non-profit institution that plans to promote “ the potential of value-based healthcare by determining global standard sets of outcomes measures that are crucial to patients of various medical conditions ”, through facilitating endorsement and reporting of these measures worldwide to enable learning, choice and competition on value. To date, ICHOM has produced 28 standard sets covering various medical conditions and specific patient groups . Standard sets are derived through Delphi surveys and consensus in international expert panels. Case-mix factors and variables including demographic data (e.g. age and gender) as well as socioeconomic factors (e.g. employment and educational levels) are identified. Case-mix variables are important to provide clearer context and allow statistical adjustments for subsequent comparison and benchmarking between different cohorts to provide better and more accurate analysis.
Core outcome sets (COS) are an accepted, consensus-based, standardised and minimum set of outcomes for a specific disease or trial population within a specified setting. Development of COS is a multi-step process to achieve consensus that includes a literature review of the evidence, followed by a series of Delphi questionnaires and rounds of discussion among a panel of experts. Delphi surveys aim to achieve consensus from among healthcare providers as well as the respective community on what outcomes to address. The expert panel deliberates and, once these core outcomes are agreed upon, determines the specific measurement domains and measurement instruments to use. The COS forms the basis upon which further domains and dimensions can be measured and subsequently developed. The Outcome Measures in Rheumatology (OMERACT) initiative, for instance, frames four key ‘impact of health’ aspects: survival, life impact, economic impact/resource use and pathophysiological manifestations.
Healthcare is strengthened by PROMs as they enable medical providers to deliver improved care that is patient-centric, and to evaluate and benchmark the quality of care by collecting information for assessing current practices and policies, providing the means to enhance healthcare productivity and quality of care. They can be translated into clinical practice in the monitoring and surveillance of an individual’s health and healthcare needs, and used in quality improvement and safety initiatives. Although some medical providers were initially skeptical about the usage of PROMs, most, if not all, will acknowledge the benefits of taking patients’ views in clinical practice. Selection of the relevant PROMs is crucial and will impact what the output is and how this eventually translates into care for the patients specifically and is scaled to the population at large. It is a multi-step process involving clear elucidation and comprehensive planning. The objectives and the population or patient group must first be clearly defined. A panel of experts must review the current evidence and literature, seeking feedback not only from healthcare practitioners but also patients, patient advocacy groups and the general population. The core outcomes to be measured and other important outcome parameters must be decided on, followed by choosing appropriate measurement instruments and tools. As there are many validated and well established outcome measurement tools, the challenge is to choose the most appropriate basket of measurement tools for the specific and required objectives.
There are some key principles when choosing patient-centred measurements. The choice of a PROM is dependent on various factors including ability to detect change, validity, reliability, acceptability and feasibility. The principles of patient-centred measurement state that measurements are: (a) patient-driven, with patients’ goals, preferences and priorities driving the metrics and assessment; (b) holistic, factoring aspects of the patient within and outside of the healthcare system; (c) transparent and accessible to all stakeholders; (d) timely and comprehensible; and (e) co-created with patients as equal partners in the enterprise. Firstly, the PROM tools must have been robustly validated and evaluated. They must also measure what they have been designed to assess. Other important considerations are compliance, preferences, implementation time and costs. Patients should be involved in its development. The PROM needs to be able to identify changes over time. The PROM must be clear and appropriately formatted so that there is patient participation and compliance. It must be consistent, reliable and reproducible. Finally, the PROM needs to be feasible to implement so that it is widely adopted and used by medical providers and administrators .
There are many technical, logistical, social, cultural and legal barriers to translating PROMs into clinical practical in a sustainable manner. Varying definitions and terminologies currently exist in this field of practice. OMERACT, for instance, uses ‘core domain sets’, whereas the HOME (Harmonising Outcome Measures for Eczema) initiative describes ‘core outcome domains’. COSMIN (COnsensus-based Standards for the selection of health Measurement INstruments), COMET (Core Outcome Measures in Effectiveness Trials) and COMIS (Core Outcome Measurement Instrument Selection) are all initiatives that aim to set guidelines and consensus in a standardised way. In addition, there are also varied approaches in PROM methodologies. In order to strengthen and not compromise the validity of the PROMs collected, regulatory bodies ensure standardised and robust translation of the PROMs by qualified linguists. This should reflect the best practice to minimise inter-racial variability among various languages when collecting data. Other challenges and caveats in implementing PROMs include the following. (a) There must be high rates of patient participation. (b) The three dimensions of quality (safety, effectiveness and experience) must be incorporated. (c) The costs and time of data collection, analysis, interpretation and presentation must be manageable and sustainable. (d) Timely administration can avoid various confounding bias, including attribution bias and recall bias after specific events (for measurements such as emergency care) due to time lag with meaningful comparisons when attributing outcomes to quality of care. (e) PROMs should add value to patient care and not be used erroneously to simply ration medical care. To ensure the sustainability of PROMs in the clinical system, their incorporation should be embedded into existing clinical frameworks and workflows. They cannot exist as a standalone vicarious entity. Otherwise, the uptake by clinicians will be poor. PROMs should be seamlessly integrated into existing clinical notes and into patients’ electronic health records. PROMs take time out of clinical encounters, as they are usually administered during clinic visits and may pose delays and challenges for clinicians. Technology can help to reduce logistical barriers to PROM assessment with the use of Internet-based data collection platforms, thereby allowing completion of PROMs outside of clinical appointments and minimising administrative burdens. Experiences from centres that have integrated PROMs into clinical practice show that they can contribute to better care delivery by facilitating remote monitoring of progress by healthcare providers and the minimising of visits or rescheduling of earlier visits when indicated. PROMs enable better communication between healthcare provider and patient, enhance physicians’ awareness of patients’ problems, and allow for more consistent and real-time adjustments to healthcare.
Various centres have started using PROMs in clinical practice. The practice is growing in the United States and even more so in Europe, including efforts by the United Kingdom National Health Service. The Patient-Reported Outcomes Measurement Information System (PROMIS), from the National Institutes of Health in the United States, is a collection of person-centred measures that are accessible through the internet, assessing and monitoring “ physical, mental and social health in both adults and children ”. PROMIS aims to standardise and facilitate “ a common measurement system for patient-reported outcomes across clinical research ”. PROMIS uses cognitive and psychometric theories and tools, utilises ‘think-aloud’ and ‘respondent debriefing’ cognitive interviewing methods, and item response theory. Item response theory is also known as latent trait theory, strong true score theory or modern mental test theory, and is a paradigm for the design, analysis and scoring of various measurement variables. Computerised adaptive testing, also known as tailored testing in PROMIS, adapts to the respondent’s ability level .
The International Consortium for Health Outcomes Measurement (ICHOM) is a non-profit institution that plans to promote “ the potential of value-based healthcare by determining global standard sets of outcomes measures that are crucial to patients of various medical conditions ”, through facilitating endorsement and reporting of these measures worldwide to enable learning, choice and competition on value. To date, ICHOM has produced 28 standard sets covering various medical conditions and specific patient groups . Standard sets are derived through Delphi surveys and consensus in international expert panels. Case-mix factors and variables including demographic data (e.g. age and gender) as well as socioeconomic factors (e.g. employment and educational levels) are identified. Case-mix variables are important to provide clearer context and allow statistical adjustments for subsequent comparison and benchmarking between different cohorts to provide better and more accurate analysis.
Many PROMs exist in paediatrics and across its various subdisciplines, capturing a range of health outcomes including QoL and symptom severity in paediatrics and child health. They include generic as well as disease-specific instruments . Challenges in development and conduct Development of paediatric PROMs has peculiar considerations. Paediatric PROMs need to be tailored to varying developmental abilities of patients, even within the same age group. Not all children of varying ages are necessarily able to complete the PROMs reliably. As such, the age range of each PROM must be determined and evaluated within the intended target population prior to implementation. In the design of paediatric PROMs, due consideration should be given to making these paediatric PROMS relevant for the intended target group. There should be avoidance of medical jargon and use of words that are beyond the developmental reading ability of the child. Age-appropriate response options should be used with suitable recall periods, and PROM questionnaires should be of appropriate lengths. Use of illustrations and attention to the format of PROMs will affect the clarity and validity of the intended PROMs. The approach to PROM administration and data collection, including the use of electronic devices, should also be considered. The content of paediatric PROMs should be validated by involving children in the early qualitative research stages, so that the target group can understand the contents. This can be done via interviews with the children and their parents. Paediatric PROMs should be completed by the child as much as possible to avoid bias. Proxy reports should only be considered if the children cannot complete them due to young age or cognitive delays; when there are proxy reports, they should be analysed separately from self-reports. Patients and their caregivers (e.g. adolescents and their parents) may have different views about the content assessed in PROMs, and thus their reported outcomes should not be weighed equally. Lastly, paediatric PROMs may not be applicable if they are in a different culture or language and thus will need to be reviewed and calibrated accordingly. While many paediatric PROMs are available and in existence, there is limited data about their efficacy in clinical care. Studies have suggested that these PROMs need further validation even for research purposes. Inconsistencies in developing and validating PROMs have limited their use by the paediatric community, such as in the use of PROMs for genodermatosis. In addition, some PROMs that were developed for research may not have the ability to detect a difference in an individual’s QoL over time clinically, thus limiting their use in routine clinical settings. Gaps of knowledge on which PROMs to use and how to use them in clinical practice pose major obstacles to their systematic integration into routine clinical care. However, continued research is under way to determine how PROMs can affect health-related QoL, use of healthcare resources, patient outcomes and care quality in children with chronic diseases. Applying to clinical practice PROMIS is used by the Pediatric Patient Reported Outcomes in Chronic Diseases (PEPR) Consortium. The long-term goal of the PEPR Consortium is to conduct validation studies of PROMs in research and clinical care settings, and to determine “ the impact of environmental stressors (including socioeconomic aspects) on children’s symptoms and QoL in a variety of chronic diseases or conditions ”. Another example is KLIK, a web application used in Amsterdam. KLIK auto-generates an email to invite children and/or parents to complete electronic patient-reported outcomes that evaluate different aspects of health-related QoL. The responses generate information for medical providers to explore regarding QoL issues and allow monitoring of these outcomes over time. Use of KLIK in the juvenile idiopathic arthritis cohort has enabled physicians to better examine the emotional health and social functioning of their patients. The Web-based method not only improves patient outcomes but also gives physicians greater job satisfaction, particularly in their ability to provide emotional support. In addition to disease-specific standard sets, ICHOM recently established an Overall Paediatric Health Standard Set in February 2020. In summary, the role of PROMs in paediatrics and child health is to improve healthcare quality at the point of care and to serve as performance indicators for overall child health and paediatric care. Continued education of parents and children about the purpose of PROMs and how they contribute to their care, together with physician buy-in and training on PROMs, would help in their successful integration into clinical practice. VALUE-BASED MEDICINE AND PATIENT-REPORTED OUTCOME MEASURES Value is the translatable quality or practical quality physicians can provide to patients and populations, framed by costs to ensure it is sustainable in the long run. Value “ represents healthcare that has positive results (improved patient outcomes, safety and satisfaction) at a total cost that is reasonable and affordable ”. The value equation can be stated as: In recent years, there has been much talk about creating and developing value in healthcare, beyond just quality. Value in healthcare goes beyond quality or evidence-based practice, and incorporates both patients’ perceptions of the healthcare delivered and the resources utilised. VBM has five pillars and is defined as “ the practice of medicine incorporating the highest level of evidence-based data with the patient-perceived value conferred by healthcare interventions for the resources expended ”. Evidence-based data, patient perception, and resource utilisation are interconnected components of VBM. With this definition, VBM has three main stakeholders: the patients, the healthcare providers and the funding agency. Healthcare providers, by means of various diagnostic and therapeutic modalities based on the latest evidence, aim to produce a positive impact on patient outcomes. No one disputes the importance and relevance of evidence-based medicine (EBM) in delivering care to patients. However, in the pursuit of EBM, the importance of individualism and the needs of individual patients might arguably have been forgotten or misplaced. Quality in healthcare has been erroneously construed to mean compliance with EBM guidelines rather than a focus on outcomes and improvement in care for our patients. The consequent expanded volume of diagnostic and therapeutic interventions has generated an increase in healthcare expenditure, which does not support the perceived value in healthcare. With the final expenditure borne by the funding agency, there has been a paradigm shift in emphasis from the traditional volume-based funding to value-based funding in recent years. With rising costs in various healthcare systems, VBM can help to lower overall healthcare expenditure or optimise the appropriate use of limited resources in different countries. In our opinion, value should always be centred around the patient and “ since value depends on results, not inputs, value in healthcare is measured by the outcomes achieved, not the volume of services delivered, and shifting focus from volume to value is a central challenge ”. In other words, value to the patient is what matters, not the volume contributed to the system. Every patient can provide an input that can generate an improvement in the healthcare system. To practise and deliver patient-centric care, we need to focus on patients’ needs and what matters to them. The care we deliver must make sense to patients, and they must perceive the care rendered to be of value. This emerging concept of using PROMs as a tool to assess patient-reported outcomes in addition to clinical outcomes is appropriate and ultimately beneficial. Focusing on value in healthcare is therefore not just a method to contain costs in the system, regardless of the needs of the patient. Rather, it involves calibrating healthcare to the needs of the patient and ensuring the sustainability of the healthcare system as a whole in the long run, benefiting the whole community. Value-driven care, value-driven healthcare and value-driven outcomes are initiatives to focus health and healthcare back onto the healthcare and health needs of our patients. Value-driven care refers to a healthcare system that is driven by patients’ health and healthcare outcomes with the utility of PROMs. In paediatrics and child health, PROMs, if implemented well with appropriate measurement tools that are regularly updated and validated in a self-learning healthcare ecosystem, would help to enhance personalised healthcare delivery . Use of PROMs would complement practice of true VBM, thereby improving the health of the community at large.
Development of paediatric PROMs has peculiar considerations. Paediatric PROMs need to be tailored to varying developmental abilities of patients, even within the same age group. Not all children of varying ages are necessarily able to complete the PROMs reliably. As such, the age range of each PROM must be determined and evaluated within the intended target population prior to implementation. In the design of paediatric PROMs, due consideration should be given to making these paediatric PROMS relevant for the intended target group. There should be avoidance of medical jargon and use of words that are beyond the developmental reading ability of the child. Age-appropriate response options should be used with suitable recall periods, and PROM questionnaires should be of appropriate lengths. Use of illustrations and attention to the format of PROMs will affect the clarity and validity of the intended PROMs. The approach to PROM administration and data collection, including the use of electronic devices, should also be considered. The content of paediatric PROMs should be validated by involving children in the early qualitative research stages, so that the target group can understand the contents. This can be done via interviews with the children and their parents. Paediatric PROMs should be completed by the child as much as possible to avoid bias. Proxy reports should only be considered if the children cannot complete them due to young age or cognitive delays; when there are proxy reports, they should be analysed separately from self-reports. Patients and their caregivers (e.g. adolescents and their parents) may have different views about the content assessed in PROMs, and thus their reported outcomes should not be weighed equally. Lastly, paediatric PROMs may not be applicable if they are in a different culture or language and thus will need to be reviewed and calibrated accordingly. While many paediatric PROMs are available and in existence, there is limited data about their efficacy in clinical care. Studies have suggested that these PROMs need further validation even for research purposes. Inconsistencies in developing and validating PROMs have limited their use by the paediatric community, such as in the use of PROMs for genodermatosis. In addition, some PROMs that were developed for research may not have the ability to detect a difference in an individual’s QoL over time clinically, thus limiting their use in routine clinical settings. Gaps of knowledge on which PROMs to use and how to use them in clinical practice pose major obstacles to their systematic integration into routine clinical care. However, continued research is under way to determine how PROMs can affect health-related QoL, use of healthcare resources, patient outcomes and care quality in children with chronic diseases.
PROMIS is used by the Pediatric Patient Reported Outcomes in Chronic Diseases (PEPR) Consortium. The long-term goal of the PEPR Consortium is to conduct validation studies of PROMs in research and clinical care settings, and to determine “ the impact of environmental stressors (including socioeconomic aspects) on children’s symptoms and QoL in a variety of chronic diseases or conditions ”. Another example is KLIK, a web application used in Amsterdam. KLIK auto-generates an email to invite children and/or parents to complete electronic patient-reported outcomes that evaluate different aspects of health-related QoL. The responses generate information for medical providers to explore regarding QoL issues and allow monitoring of these outcomes over time. Use of KLIK in the juvenile idiopathic arthritis cohort has enabled physicians to better examine the emotional health and social functioning of their patients. The Web-based method not only improves patient outcomes but also gives physicians greater job satisfaction, particularly in their ability to provide emotional support. In addition to disease-specific standard sets, ICHOM recently established an Overall Paediatric Health Standard Set in February 2020. In summary, the role of PROMs in paediatrics and child health is to improve healthcare quality at the point of care and to serve as performance indicators for overall child health and paediatric care. Continued education of parents and children about the purpose of PROMs and how they contribute to their care, together with physician buy-in and training on PROMs, would help in their successful integration into clinical practice.
Value is the translatable quality or practical quality physicians can provide to patients and populations, framed by costs to ensure it is sustainable in the long run. Value “ represents healthcare that has positive results (improved patient outcomes, safety and satisfaction) at a total cost that is reasonable and affordable ”. The value equation can be stated as: In recent years, there has been much talk about creating and developing value in healthcare, beyond just quality. Value in healthcare goes beyond quality or evidence-based practice, and incorporates both patients’ perceptions of the healthcare delivered and the resources utilised. VBM has five pillars and is defined as “ the practice of medicine incorporating the highest level of evidence-based data with the patient-perceived value conferred by healthcare interventions for the resources expended ”. Evidence-based data, patient perception, and resource utilisation are interconnected components of VBM. With this definition, VBM has three main stakeholders: the patients, the healthcare providers and the funding agency. Healthcare providers, by means of various diagnostic and therapeutic modalities based on the latest evidence, aim to produce a positive impact on patient outcomes. No one disputes the importance and relevance of evidence-based medicine (EBM) in delivering care to patients. However, in the pursuit of EBM, the importance of individualism and the needs of individual patients might arguably have been forgotten or misplaced. Quality in healthcare has been erroneously construed to mean compliance with EBM guidelines rather than a focus on outcomes and improvement in care for our patients. The consequent expanded volume of diagnostic and therapeutic interventions has generated an increase in healthcare expenditure, which does not support the perceived value in healthcare. With the final expenditure borne by the funding agency, there has been a paradigm shift in emphasis from the traditional volume-based funding to value-based funding in recent years. With rising costs in various healthcare systems, VBM can help to lower overall healthcare expenditure or optimise the appropriate use of limited resources in different countries. In our opinion, value should always be centred around the patient and “ since value depends on results, not inputs, value in healthcare is measured by the outcomes achieved, not the volume of services delivered, and shifting focus from volume to value is a central challenge ”. In other words, value to the patient is what matters, not the volume contributed to the system. Every patient can provide an input that can generate an improvement in the healthcare system. To practise and deliver patient-centric care, we need to focus on patients’ needs and what matters to them. The care we deliver must make sense to patients, and they must perceive the care rendered to be of value. This emerging concept of using PROMs as a tool to assess patient-reported outcomes in addition to clinical outcomes is appropriate and ultimately beneficial. Focusing on value in healthcare is therefore not just a method to contain costs in the system, regardless of the needs of the patient. Rather, it involves calibrating healthcare to the needs of the patient and ensuring the sustainability of the healthcare system as a whole in the long run, benefiting the whole community. Value-driven care, value-driven healthcare and value-driven outcomes are initiatives to focus health and healthcare back onto the healthcare and health needs of our patients. Value-driven care refers to a healthcare system that is driven by patients’ health and healthcare outcomes with the utility of PROMs. In paediatrics and child health, PROMs, if implemented well with appropriate measurement tools that are regularly updated and validated in a self-learning healthcare ecosystem, would help to enhance personalised healthcare delivery . Use of PROMs would complement practice of true VBM, thereby improving the health of the community at large.
PROMs are key to delivering patient-centred and patient-centric care to meet the health and healthcare needs of our paediatric patients in the 21 st century. As healthcare providers, we need to understand the holistic needs of our patients and calibrate care accordingly. Development and implementation of PROMs pose major challenges, particularly in paediatrics. Although considerable progress has been made, much remains to be done to facilitate integration of appropriate PROMs into routine paediatric clinical practice. Effective PROMs enable us as healthcare providers to meaningfully meld ‘hard’ medical science with soft, ‘heart’ aspects to ensure optimal health for our patients. The practice of medicine in the final analysis is both a science as well as an art. As Paracelsus said, “ Medicine is not only a science; it is also an art. It does not consist of compounding pills and plasters; it deals with the very processes of life, which must be understood before they may be guided .” Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. About the first author Dr Tan Yi Hua is a Consultant at the Respiratory Medicine Service, Department of Paediatrics, KK Women’s and Children’s Hospital, Singapore. He treats a variety of paediatric respiratory disorders and sleep disorders. His academic appointment include Clinical Assistant Professor at Duke-NUS Medical School and Clinical Lecturer at NUS Yong Loo Lin School of Medicine and Lee Kong Chian School of Medicine. He is also a Clinical Core Faculty Member for the SingHealth Paediatric Medicine Residency Programme.
Nil.
There are no conflicts of interest.
Dr Tan Yi Hua is a Consultant at the Respiratory Medicine Service, Department of Paediatrics, KK Women’s and Children’s Hospital, Singapore. He treats a variety of paediatric respiratory disorders and sleep disorders. His academic appointment include Clinical Assistant Professor at Duke-NUS Medical School and Clinical Lecturer at NUS Yong Loo Lin School of Medicine and Lee Kong Chian School of Medicine. He is also a Clinical Core Faculty Member for the SingHealth Paediatric Medicine Residency Programme.
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State of the Art and New Trends from the 2022 Gism Annual Meeting
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da6dd609-fdad-4a58-82d2-3d3cd8f5bba8
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10219299
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Internal Medicine[mh]
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The 2022 Italian Mesenchymal Stem Cell Group (Gruppo Italiano Staminali Mesenchimali, GISM) annual meeting took place on 20–21 October 2022 in Turin, with the support of the University of Turin and the City of Health and Science of Turin. The novelty of this year’s meeting was its articulation reflecting the new structure of GISM, based on six sections, that have been implemented during the last few years to better cover the interests concerning the different aspects of mesenchymal stem/stromal cell (MSC) use. The meeting brought together GISM-Regenerative Medicine with the support of the Cell Factories and researchers close to the clinic, and the GISM-Next-Generation section reflects the willingness of young talented researchers on MSCs. The GISM-Veterinary section, a very active group that reached not only researchers but also clinical veterinarians, introduced research in animal models towards the GISM-Secretome , focused on paracrine mechanisms mediated by soluble molecules and extracellular vesicles of MSCs. The last section, GISM-Oncology , covered the discussed relations between MSCs and tumours as well as the use of MSCs as a tool for drug delivery in cancer. National and international speakers presented their scientific works with the aim of promoting an interactive discussion and training the youngest researchers.
The meeting was held in six different sessions in order to diversify the various study and research sectors (clinical, cell cultures and extra vesicles, veterinary, oncology, etc.) and to broaden the discussions and experiences of clinicians and researchers in the specific fields of study. 2.1. Session 1: “Bringing Advanced Therapies to the Clinic: Trends and Strategies” The first session started with Graziella Pellegrini (University of Modena and Reggio Emilia, Italy) who presented her work about ex vivo expanded autologous human corneal epithelial cells containing stem cells and their production process for marketing authorisation (Holoclar) . She and her group have been studying Limbal Stem Cell Deficiency (LSCD) since 1997 . Since the regulatory guidelines changed in 2008 , their focus was aimed to obtain EMA approval for Holoclar which happened in 2015 with a confirmatory trial of 80 patients. Their follow-up from 1997 to 2008 made it possible to have this court and, therefore, to already have in vivo studies, which allowed the facilitation of the approval. This allowed for real in vivo models as no animal eye can be compared to the human eye. She explained their procedure consisting of the isolation of epithelial cells from a very small biopsy from the healthy eye and their expansion and freezing. After thawing, the cells were transplanted with a special carrier on the damaged cornea . The patients had a fast recovery with re-epithelialization after one week and re-vascularization after a couple of months with restored vision and stability. The final costs showed that Holoclar is less expensive than surgery (O-01). Following, Stefano Cosma (University of Modena and Reggio Emilia, Italy) spoke about economic aspects and how researchers can bridge the funding gap. Scientific and operational factors, as well as economic factors (such as a lack or inadequacy of funding) represent risks of failure in medical research. Cosma and colleagues collected from the ClinicaTrial.gov database: 12,934 interventional studies were performed in Italy and 1137 in Europe, which were classified by keywords within advanced therapy medicinal product (ATMP) research. Through manual data reprocessing, they highlighted two important features: first, the information about failed and successful projects and their failure causes; second, the classification of the studies according to the funding source. An intersection of these data explained how financial resource allocations are correlated to the success or failure of clinical trials. This intervention was very important and elucidating, showing the point of view of an economist to understand and have indications of the economic aspects of the ATMPs (O-02). After a break, Nicholas Crippa Orlandi (University of Siena, Italy) presented three cases of different body segments obtained through orthopaedic surgery that benefit from the use of gelled preparations containing autologous bone marrow MSCs. Crippa Orlandi and colleagues optimised the isolation and expansion protocol of bone marrow MSCs using Platelet Rich Plasma (PRP) instead of Foetal Bovine Serum (FBS), characterising cells seeded on the bovine bone matrix (SmartBone) or on a lyophilized acellular matrix, and they increased cell differentiation and osteogenic properties by adding growth factors. On SmartBone, the cells differentiated into osteoblasts and produced collagen. These data suggest that MSCs can provide valid help in complicated orthopaedic surgeries (O-03). In order to have an overall view of the various sources of MSCs, Ilaria Roato (University of Turin, Italy) compared MSCs isolated from different districts of the oral cavity . Adult Dental Pulp Stem Cells (DPSCs) are less osteogenic than Buccal Fat Pad Stem Cells (BFPSCs) but are more indicated for the pulp–dentin complex regeneration. MSCs isolated from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed no differences in the MSC marker immunophenotype, but SHED and DPSCs had a higher percentage of endothelial precursors than PDLSCs. The capability of MSCs to differentiate into a specific tissue depends also on the different anatomical origins, therefore, MSCs isolated from the oral cavity might be more effective than MSCs isolated from other origins such as adipose-derived stem cells (ASCs). Furthermore, these cells can be harvested after tooth extraction without causing pain or additional manoeuvres to the patient (O-04). To conclude the first session of the congress, Marta Nardini (University of Genoa, Italy), selected as an oral communication, underlined the importance of cell behaviour after transplant because the distribution, engraftment, viability, and activity of transplanted cells are unclear. Nardini and colleagues developed a potency assay using Optoacoustic Imaging (OAI) in conjunction with rapid Gold-Nanostars (GNs). They characterised in vitro the complexes of GNs-labelled MSCs and extracellular vesicles (EVs), studied their distribution in sundry organs after a systemic injection using Multispectral Optoacoustic Tomography (MSOT), and tested in vivo toxicity. They isolated MSCs from bone marrow, cultured them with the addition of Platelet Lysate (PL) instead of FBS, and isolated the EVs from these cells. They injected labelled and unlabelled complexes into NuNu mice, and they tested in vivo toxicity at different time points with blood tests and histological analysis of the organs. GNs cannot overcome the Blood–Brain Barrier; after one day, they reached all organs and remained for all monitoring periods without any alteration, leading to a new perspective for tracking cell distribution (O-05). 2.2. Session 2: “Gism—Next Generation” This section was organised by young GISM members and was designed to improve general skills around publishing, writing patents, and fundraising. The session started with Filippo Piccinini (IRCCS IRST ‘‘Dino Amadori”, Meldola, Italy, and University of Bologna, Italy). His presentation had a very catchy title, “Scientific articles: how to choose the right journal taking advantage of some opportunities”. The main goal was to discuss how to speed up the publication process, defining the best journal for a specific article and increasing the chances of publication by exploiting the opportunities that technology and social networks today provide. In particular, he introduced the most popular freely available journal finders and with a case study, he showed the audience how to exploit them. Finally, he practically described the meaning of the journal IF (impact factor) and the journal quartiles for a better understanding of the non-written rules behind the publication process for increasing the chances of a successful submission (O-06). Next, Paola Bagnoli (IRCCS Ospedale Galeazzi Sant’Ambrogio, Milan, Italy) proceeded the section with a presentation related to the other side of research: “Technology transfer and intellectual property”. Besides summarising the main steps for patenting, she described the fundamental role of the Technology Transfer Offices (TTO) in scouting results, assisting researchers in protecting intellectual property, and activating stakeholders’ virtuous paths for the development of a project’s TRL (Technology Readiness Level) (O-07). Finally, the “GISM—Next Generation” section ended with a round table composed of senior researchers working at different institutions, in particular, Maddalena Mastrogiacomo (University of Genoa, Italy), Luca Battistelli (IRCCS IRST Meldola, Italy), Enrico Lucarelli (IRCCS IOR Bologna, Italy), and Roberta Visone (Politecnico of Milan, Italy). The discussion was about difficulties and opportunities for fundraising ideas for MSC-related research. Despite the general difficulties claimed by all the speakers, they gave the audience several hints for future submissions. In particular, they cited several MSC-specific calls for applications mainly dedicated to young researchers. 2.3. Session 3: “New Technologies for 3D Culture Systems” During this panel of the GISM congress, four innovative presentations illustrated new culture methods that could improve the expansion of mesenchymal cells for different purposes. The first presentation was by Matteo Moretti (I.R.C.C.S Istituto Ortopedico Galeazzi of Milan, Italy), a bioengineer who presented a joint-on-a-chip 3D model . This model was made by carefully layering chondrocytes, synovial fibroblasts, and synovial fluid in order to recreate on a small scale what is normally present in a patient and was used to test the effects of MSCs on osteoarthritis (OA) which is an inflammatory disease of the musculoskeletal system. In order to recreate the major hallmarks of this disease, injections of OA synovial fluid on the joint-on-chip were conducted and then the effect of MSCs was tested as a possible therapy option. These types of models could allow the personalization of the therapy for patients as the cells can be obtained directly from them and could allow the testing of innovative therapies (O-08). Afterwards, the word was given to Maria Harmati (Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary) who illustrated the crosstalk routes between tumour cells and stromal cells and vice versa. The crosstalk was conducted through extracellular vesicles tracked with specific dyes. She conducted her experiments not only on human ductal carcinoma cells but also on melanoma and osteosarcoma models. This provided more proof of the crosstalk that occurs and it provided proof on how the crosstalk changes in different tumour models (O-09). Subsequently, Lucia Ceresa (Charles River Microbial Solutions, Italy) illustrated the suitability of Rapid Microbial Methods as a method to test the quality and safety of new cell-based medicinal products, using an ATP-based luminescent platform such as the Celsis platform which depletes the presence of cellular ATP and also allows fast detection of microbial presence (O-10). Concluding this session, Silvia Scaglione (React4life startup, University of Genova, Italy) was chosen to discuss her poster as an oral communication. She presented her results on a novel Multi In Vivo Organ (MIVO) on a chip platform and how it could facilitate drug testing on cancer models . Indeed, the models proposed were a 3D ovarian model developed and treated with Cisplatin, and a 3D neuroblastoma cancer model which was used to coculture with immune cells. These studies obtained a relevant disease model that can be used to investigate crosstalk between healthy and pathogenic cells and can also be employed as a drug screening platform (O-11). 2.4. Session 4: “Therapeutic Applications of MSC-EVs in Veterinary and Human Medicine” Session 4 of the congress had a focus on the therapeutic applications of MSC-EVs in veterinary and human medicine. Stefania Bruno (University of Turin, Italy) presented the obstacles that can be found during the transfer of therapy with MSC-EVs from the laboratory to the clinic. In fact, MSC-EVs have been shown to have therapeutic effects in preclinical models of several diseases as they have pro-regenerative capacities and are considered therapeutic tools for various pathologies. To this end, EV characterisation is an area of intense investigation. To successfully translate EV research from the laboratory to patients, strategies must be designed to clinically test the safety and efficacy of MSC-EVs. Currently, manufacturing of cells and EVs, and quality controls, are being used for clinical testing of the feasibility of non-industrial processes. However, defining the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting (O-12). In this regard, Silvia Zia (StemSel srl, Bologna, Italy) presented the Celector ® instrument, useful for ATMPs quality control and standardisation. Since MSCs are a heterogeneous population, the identity/purity of the cell population and its yield are critical issues that may limit their clinical application. To improve the characterisation of MSCs and standardise protocols, it is necessary to develop functional assays that evaluate the biophysical properties, profile, and quality of the cells. The Celector ® instrument is able to analyse, discriminate, and tag free separate a wide range of cells based on their physical characteristics, with high resolution and without damage. She explained the separation method, imaging acquisition, post-processing, and data analysis underlining that Celector@ is able to highlight physical differences related to cell viability and regenerative potential (O-13). Afterwards, space was given to young researchers to orally communicate their posters. The first was the researcher Elena Ceccotti (University of Turin, Italy), who presented her work on chronic kidney diseases. In particular, she explained that human liver stem cells extracellular vesicles (HLSC-EVs) can be used as carriers for the transfer of active biological drugs both in vitro and in vivo models of renal ischemic reperfusion injury (IRI) associated with acute kidney injury (AKI) and chronic kidney disease (CKD). In this study, the results showed that in AKI mice, EV treatment attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation, and downregulated expression levels of fibrosis-related genes. In CKD mice, interstitial fibrosis and the expression levels of pro-fibrotic and pro-inflammatory genes were decreased . Thus, the administration of HLSC-EVs immediately after renal IRI protects the kidney from the development of AKI and interferes with the development of subsequent CKD (O-14). Proceeding with the session, Tarlan Eslami Arshaghi (University of Galway, Ireland) introduced the Aptamer approach: a fluorescence polarisation-based approach for EV quantification. This approach aimed to develop a novel high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific surface component, using fluorescence polarisation (FP) for detection. The method analysed the change in polarisation of the emitted light between unbound and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of bound probes. This property of FP allowed them to use it to quantify the amount of EVs in the solution. She explained the different strategies used to demonstrate the enhanced fluorescence polarisation in response to increasing EV concentration, quantified by NTA and the study of probe–target kinetics (O-15). Finally, the last speaker, Enrico Ragni (IRCCS-Galeazzi Hospital, Milan, Italy) talked about the MSC secretome for regenerative medicine related to orthopaedic conditions. He characterised adipose-derived MSCs (ASCs) and EV-miRNAs and their modulation after high levels of IFNy preconditioning and mimicking OA. The penetration of ASC-EVs was evaluated in cartilage explants. Bioinformatics tools were used to predict the modulatory effect of the identified molecules on pathological cartilage and to follow and quantify the incorporation of fluorescent EVs into cartilage explants. The ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes thanks to the detected presence of 50 cytokines/chemokines and more than 200 EV-miRNAs, and inflammatory preconditioning or OA-like conditions have been able to increase this ability. The ASC-secretome’s ability to stimulate healing and reduce inflammation allows it to be proposed as an ideal candidate for orthopaedic regenerative medicine (O-16). 2.5. Session 5: “Advancing MSC Therapies in Veterinary Medicine: Present Challenges and Future Perspectives” Laura Barrachina Porcar (University of Galway, Ireland) presented her work on MSCs and their immune properties. She explained that the use of allogeneic MSCs presented several advantages with respect to autologous cells as a possibility to have a ready-to-use product. Although cell-based products in the veterinary market are emerging, allogeneic therapy does not come without limitations. At first, MSCs, which were initially considered immune-privileged, can actually induce cellular and humoral immune responses. Based on these observations, she reported several human and animal studies with positive results after allogeneic MSC administration in the absence of adverse effects. A potential explanation for the mixed outcomes often seen was that MSCs can be recognised by the immune system (immunogenicity), but they can also regulate it (immunomodulation). She also explained the different strategies that could be designed to develop safer and more effective allogeneic therapies and how it could allow the creation of ‘haplo-banks’ of cells from donors. The understanding of the interactions between MSCs and the immune system could be a key to learn which factors we can manage, and how (O-17). Following, the first oral selected communication was from Barbara Merlo (University of Bologna, Italy) who presented the results of a pilot study concerning the effect of GM18, an α4β1 integrin agonist, on the adhesion properties of equine adipose tissue and Wharton’s jelly derived MSCs and on their ability to adhere to GM18- incorporated poly L-lactic acid (PLLA) scaffolds. The use of biomaterials with integrin agonists promoted cell adhesion in tissue repair processes confirming the presence of GM18-containing PLLA scaffolds. In conclusion, GM18 affects equine MSCs adhesion ability with donor-related variability. These preliminary results suggested that MSCs from Wharton Jelly might be more suitable than MSCs from adipose tissue (O-18). The second selected oral communication was presented by Gabriele Scattini (University of Perugia, Italy) which was about migrasomes (MG), a particular kind of EVs released by MSCs of different species and tissue sources possibly related to their migratory activity. He reported that MGs were isolated by differential ultracentrifugation of MSC supernatant and were observed by Transmission Electron Microscopy (TEM) as different from other microparticles in size and morphology. Their biogenesis and morphologic features were described in detail compared to the EV characteristics, although he underlined that their different functions are still to be clarified (O-19). 2.6. Session 6: “MSCs: A Double-Edged Sword: Friend or Foe in Oncology?” Roisin Dwyer (University of Galway, Ireland) opened the last session on the dual role of MSCs in oncology with a presentation of her research on the use of EVs isolated from MSCs as therapeutic delivery for cancer. MSC-derived EVs raised interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. The recent work presented by Dwyer’s group showed the development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy. Their objective was to develop an innovative approach based on MSC-EVs as a cancer treatment for patients with breast cancer who have limited treatment options. From her point of view, the use of more reflective pre-clinical models of the patient experience has to be developed to test new therapeutic approaches (O-20). Andrea Papait (Cattolica del Sacro Cuore University, Rome, Italy) demonstrated the MSC role in the tumour microenvironment and their complex interaction network in contact with anticancer agents. In his presentation, he discussed the use of MSCs considered as a double-edged sword: they can serve as a drug carrier while their immunomodulatory properties can participate in tumour initiation, development and progression, and metastasis formation . For him, in the era of immunotherapy, MSCs or their secretome could represent an adjuvant therapy in association with new drugs such as monoclonal antibodies aimed at re-educating the immune response (O-21). Finally, the meeting ended with the selected presentation by Valentina Coccè (University of Milan, Italy). She presented data on the inhibitory effect of adipose tissue-derived MSCs uploaded by paclitaxel (PTX) on malignant pleural mesothelioma in vitro and in vivo models. In particular, she saw how adipose tissue (FAT) after micro fragmentation (MFAT) was able to exert a dose-dependent inhibition on the growth of the human mesothelioma cell line (MSTO-211H). While, in xenografted Balb/c-Nude mice obtained after subcutaneous injection with MSTO- 211H, she observed a reduction of tumour mass volume measurements after treatment with MFAT loaded with PTX similar to that of free PTX. This could be a new therapeutic approach for a difficult to treat tumour (O-22).
The first session started with Graziella Pellegrini (University of Modena and Reggio Emilia, Italy) who presented her work about ex vivo expanded autologous human corneal epithelial cells containing stem cells and their production process for marketing authorisation (Holoclar) . She and her group have been studying Limbal Stem Cell Deficiency (LSCD) since 1997 . Since the regulatory guidelines changed in 2008 , their focus was aimed to obtain EMA approval for Holoclar which happened in 2015 with a confirmatory trial of 80 patients. Their follow-up from 1997 to 2008 made it possible to have this court and, therefore, to already have in vivo studies, which allowed the facilitation of the approval. This allowed for real in vivo models as no animal eye can be compared to the human eye. She explained their procedure consisting of the isolation of epithelial cells from a very small biopsy from the healthy eye and their expansion and freezing. After thawing, the cells were transplanted with a special carrier on the damaged cornea . The patients had a fast recovery with re-epithelialization after one week and re-vascularization after a couple of months with restored vision and stability. The final costs showed that Holoclar is less expensive than surgery (O-01). Following, Stefano Cosma (University of Modena and Reggio Emilia, Italy) spoke about economic aspects and how researchers can bridge the funding gap. Scientific and operational factors, as well as economic factors (such as a lack or inadequacy of funding) represent risks of failure in medical research. Cosma and colleagues collected from the ClinicaTrial.gov database: 12,934 interventional studies were performed in Italy and 1137 in Europe, which were classified by keywords within advanced therapy medicinal product (ATMP) research. Through manual data reprocessing, they highlighted two important features: first, the information about failed and successful projects and their failure causes; second, the classification of the studies according to the funding source. An intersection of these data explained how financial resource allocations are correlated to the success or failure of clinical trials. This intervention was very important and elucidating, showing the point of view of an economist to understand and have indications of the economic aspects of the ATMPs (O-02). After a break, Nicholas Crippa Orlandi (University of Siena, Italy) presented three cases of different body segments obtained through orthopaedic surgery that benefit from the use of gelled preparations containing autologous bone marrow MSCs. Crippa Orlandi and colleagues optimised the isolation and expansion protocol of bone marrow MSCs using Platelet Rich Plasma (PRP) instead of Foetal Bovine Serum (FBS), characterising cells seeded on the bovine bone matrix (SmartBone) or on a lyophilized acellular matrix, and they increased cell differentiation and osteogenic properties by adding growth factors. On SmartBone, the cells differentiated into osteoblasts and produced collagen. These data suggest that MSCs can provide valid help in complicated orthopaedic surgeries (O-03). In order to have an overall view of the various sources of MSCs, Ilaria Roato (University of Turin, Italy) compared MSCs isolated from different districts of the oral cavity . Adult Dental Pulp Stem Cells (DPSCs) are less osteogenic than Buccal Fat Pad Stem Cells (BFPSCs) but are more indicated for the pulp–dentin complex regeneration. MSCs isolated from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed no differences in the MSC marker immunophenotype, but SHED and DPSCs had a higher percentage of endothelial precursors than PDLSCs. The capability of MSCs to differentiate into a specific tissue depends also on the different anatomical origins, therefore, MSCs isolated from the oral cavity might be more effective than MSCs isolated from other origins such as adipose-derived stem cells (ASCs). Furthermore, these cells can be harvested after tooth extraction without causing pain or additional manoeuvres to the patient (O-04). To conclude the first session of the congress, Marta Nardini (University of Genoa, Italy), selected as an oral communication, underlined the importance of cell behaviour after transplant because the distribution, engraftment, viability, and activity of transplanted cells are unclear. Nardini and colleagues developed a potency assay using Optoacoustic Imaging (OAI) in conjunction with rapid Gold-Nanostars (GNs). They characterised in vitro the complexes of GNs-labelled MSCs and extracellular vesicles (EVs), studied their distribution in sundry organs after a systemic injection using Multispectral Optoacoustic Tomography (MSOT), and tested in vivo toxicity. They isolated MSCs from bone marrow, cultured them with the addition of Platelet Lysate (PL) instead of FBS, and isolated the EVs from these cells. They injected labelled and unlabelled complexes into NuNu mice, and they tested in vivo toxicity at different time points with blood tests and histological analysis of the organs. GNs cannot overcome the Blood–Brain Barrier; after one day, they reached all organs and remained for all monitoring periods without any alteration, leading to a new perspective for tracking cell distribution (O-05).
This section was organised by young GISM members and was designed to improve general skills around publishing, writing patents, and fundraising. The session started with Filippo Piccinini (IRCCS IRST ‘‘Dino Amadori”, Meldola, Italy, and University of Bologna, Italy). His presentation had a very catchy title, “Scientific articles: how to choose the right journal taking advantage of some opportunities”. The main goal was to discuss how to speed up the publication process, defining the best journal for a specific article and increasing the chances of publication by exploiting the opportunities that technology and social networks today provide. In particular, he introduced the most popular freely available journal finders and with a case study, he showed the audience how to exploit them. Finally, he practically described the meaning of the journal IF (impact factor) and the journal quartiles for a better understanding of the non-written rules behind the publication process for increasing the chances of a successful submission (O-06). Next, Paola Bagnoli (IRCCS Ospedale Galeazzi Sant’Ambrogio, Milan, Italy) proceeded the section with a presentation related to the other side of research: “Technology transfer and intellectual property”. Besides summarising the main steps for patenting, she described the fundamental role of the Technology Transfer Offices (TTO) in scouting results, assisting researchers in protecting intellectual property, and activating stakeholders’ virtuous paths for the development of a project’s TRL (Technology Readiness Level) (O-07). Finally, the “GISM—Next Generation” section ended with a round table composed of senior researchers working at different institutions, in particular, Maddalena Mastrogiacomo (University of Genoa, Italy), Luca Battistelli (IRCCS IRST Meldola, Italy), Enrico Lucarelli (IRCCS IOR Bologna, Italy), and Roberta Visone (Politecnico of Milan, Italy). The discussion was about difficulties and opportunities for fundraising ideas for MSC-related research. Despite the general difficulties claimed by all the speakers, they gave the audience several hints for future submissions. In particular, they cited several MSC-specific calls for applications mainly dedicated to young researchers.
During this panel of the GISM congress, four innovative presentations illustrated new culture methods that could improve the expansion of mesenchymal cells for different purposes. The first presentation was by Matteo Moretti (I.R.C.C.S Istituto Ortopedico Galeazzi of Milan, Italy), a bioengineer who presented a joint-on-a-chip 3D model . This model was made by carefully layering chondrocytes, synovial fibroblasts, and synovial fluid in order to recreate on a small scale what is normally present in a patient and was used to test the effects of MSCs on osteoarthritis (OA) which is an inflammatory disease of the musculoskeletal system. In order to recreate the major hallmarks of this disease, injections of OA synovial fluid on the joint-on-chip were conducted and then the effect of MSCs was tested as a possible therapy option. These types of models could allow the personalization of the therapy for patients as the cells can be obtained directly from them and could allow the testing of innovative therapies (O-08). Afterwards, the word was given to Maria Harmati (Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary) who illustrated the crosstalk routes between tumour cells and stromal cells and vice versa. The crosstalk was conducted through extracellular vesicles tracked with specific dyes. She conducted her experiments not only on human ductal carcinoma cells but also on melanoma and osteosarcoma models. This provided more proof of the crosstalk that occurs and it provided proof on how the crosstalk changes in different tumour models (O-09). Subsequently, Lucia Ceresa (Charles River Microbial Solutions, Italy) illustrated the suitability of Rapid Microbial Methods as a method to test the quality and safety of new cell-based medicinal products, using an ATP-based luminescent platform such as the Celsis platform which depletes the presence of cellular ATP and also allows fast detection of microbial presence (O-10). Concluding this session, Silvia Scaglione (React4life startup, University of Genova, Italy) was chosen to discuss her poster as an oral communication. She presented her results on a novel Multi In Vivo Organ (MIVO) on a chip platform and how it could facilitate drug testing on cancer models . Indeed, the models proposed were a 3D ovarian model developed and treated with Cisplatin, and a 3D neuroblastoma cancer model which was used to coculture with immune cells. These studies obtained a relevant disease model that can be used to investigate crosstalk between healthy and pathogenic cells and can also be employed as a drug screening platform (O-11).
Session 4 of the congress had a focus on the therapeutic applications of MSC-EVs in veterinary and human medicine. Stefania Bruno (University of Turin, Italy) presented the obstacles that can be found during the transfer of therapy with MSC-EVs from the laboratory to the clinic. In fact, MSC-EVs have been shown to have therapeutic effects in preclinical models of several diseases as they have pro-regenerative capacities and are considered therapeutic tools for various pathologies. To this end, EV characterisation is an area of intense investigation. To successfully translate EV research from the laboratory to patients, strategies must be designed to clinically test the safety and efficacy of MSC-EVs. Currently, manufacturing of cells and EVs, and quality controls, are being used for clinical testing of the feasibility of non-industrial processes. However, defining the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting (O-12). In this regard, Silvia Zia (StemSel srl, Bologna, Italy) presented the Celector ® instrument, useful for ATMPs quality control and standardisation. Since MSCs are a heterogeneous population, the identity/purity of the cell population and its yield are critical issues that may limit their clinical application. To improve the characterisation of MSCs and standardise protocols, it is necessary to develop functional assays that evaluate the biophysical properties, profile, and quality of the cells. The Celector ® instrument is able to analyse, discriminate, and tag free separate a wide range of cells based on their physical characteristics, with high resolution and without damage. She explained the separation method, imaging acquisition, post-processing, and data analysis underlining that Celector@ is able to highlight physical differences related to cell viability and regenerative potential (O-13). Afterwards, space was given to young researchers to orally communicate their posters. The first was the researcher Elena Ceccotti (University of Turin, Italy), who presented her work on chronic kidney diseases. In particular, she explained that human liver stem cells extracellular vesicles (HLSC-EVs) can be used as carriers for the transfer of active biological drugs both in vitro and in vivo models of renal ischemic reperfusion injury (IRI) associated with acute kidney injury (AKI) and chronic kidney disease (CKD). In this study, the results showed that in AKI mice, EV treatment attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation, and downregulated expression levels of fibrosis-related genes. In CKD mice, interstitial fibrosis and the expression levels of pro-fibrotic and pro-inflammatory genes were decreased . Thus, the administration of HLSC-EVs immediately after renal IRI protects the kidney from the development of AKI and interferes with the development of subsequent CKD (O-14). Proceeding with the session, Tarlan Eslami Arshaghi (University of Galway, Ireland) introduced the Aptamer approach: a fluorescence polarisation-based approach for EV quantification. This approach aimed to develop a novel high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific surface component, using fluorescence polarisation (FP) for detection. The method analysed the change in polarisation of the emitted light between unbound and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of bound probes. This property of FP allowed them to use it to quantify the amount of EVs in the solution. She explained the different strategies used to demonstrate the enhanced fluorescence polarisation in response to increasing EV concentration, quantified by NTA and the study of probe–target kinetics (O-15). Finally, the last speaker, Enrico Ragni (IRCCS-Galeazzi Hospital, Milan, Italy) talked about the MSC secretome for regenerative medicine related to orthopaedic conditions. He characterised adipose-derived MSCs (ASCs) and EV-miRNAs and their modulation after high levels of IFNy preconditioning and mimicking OA. The penetration of ASC-EVs was evaluated in cartilage explants. Bioinformatics tools were used to predict the modulatory effect of the identified molecules on pathological cartilage and to follow and quantify the incorporation of fluorescent EVs into cartilage explants. The ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes thanks to the detected presence of 50 cytokines/chemokines and more than 200 EV-miRNAs, and inflammatory preconditioning or OA-like conditions have been able to increase this ability. The ASC-secretome’s ability to stimulate healing and reduce inflammation allows it to be proposed as an ideal candidate for orthopaedic regenerative medicine (O-16).
Laura Barrachina Porcar (University of Galway, Ireland) presented her work on MSCs and their immune properties. She explained that the use of allogeneic MSCs presented several advantages with respect to autologous cells as a possibility to have a ready-to-use product. Although cell-based products in the veterinary market are emerging, allogeneic therapy does not come without limitations. At first, MSCs, which were initially considered immune-privileged, can actually induce cellular and humoral immune responses. Based on these observations, she reported several human and animal studies with positive results after allogeneic MSC administration in the absence of adverse effects. A potential explanation for the mixed outcomes often seen was that MSCs can be recognised by the immune system (immunogenicity), but they can also regulate it (immunomodulation). She also explained the different strategies that could be designed to develop safer and more effective allogeneic therapies and how it could allow the creation of ‘haplo-banks’ of cells from donors. The understanding of the interactions between MSCs and the immune system could be a key to learn which factors we can manage, and how (O-17). Following, the first oral selected communication was from Barbara Merlo (University of Bologna, Italy) who presented the results of a pilot study concerning the effect of GM18, an α4β1 integrin agonist, on the adhesion properties of equine adipose tissue and Wharton’s jelly derived MSCs and on their ability to adhere to GM18- incorporated poly L-lactic acid (PLLA) scaffolds. The use of biomaterials with integrin agonists promoted cell adhesion in tissue repair processes confirming the presence of GM18-containing PLLA scaffolds. In conclusion, GM18 affects equine MSCs adhesion ability with donor-related variability. These preliminary results suggested that MSCs from Wharton Jelly might be more suitable than MSCs from adipose tissue (O-18). The second selected oral communication was presented by Gabriele Scattini (University of Perugia, Italy) which was about migrasomes (MG), a particular kind of EVs released by MSCs of different species and tissue sources possibly related to their migratory activity. He reported that MGs were isolated by differential ultracentrifugation of MSC supernatant and were observed by Transmission Electron Microscopy (TEM) as different from other microparticles in size and morphology. Their biogenesis and morphologic features were described in detail compared to the EV characteristics, although he underlined that their different functions are still to be clarified (O-19).
Roisin Dwyer (University of Galway, Ireland) opened the last session on the dual role of MSCs in oncology with a presentation of her research on the use of EVs isolated from MSCs as therapeutic delivery for cancer. MSC-derived EVs raised interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. The recent work presented by Dwyer’s group showed the development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy. Their objective was to develop an innovative approach based on MSC-EVs as a cancer treatment for patients with breast cancer who have limited treatment options. From her point of view, the use of more reflective pre-clinical models of the patient experience has to be developed to test new therapeutic approaches (O-20). Andrea Papait (Cattolica del Sacro Cuore University, Rome, Italy) demonstrated the MSC role in the tumour microenvironment and their complex interaction network in contact with anticancer agents. In his presentation, he discussed the use of MSCs considered as a double-edged sword: they can serve as a drug carrier while their immunomodulatory properties can participate in tumour initiation, development and progression, and metastasis formation . For him, in the era of immunotherapy, MSCs or their secretome could represent an adjuvant therapy in association with new drugs such as monoclonal antibodies aimed at re-educating the immune response (O-21). Finally, the meeting ended with the selected presentation by Valentina Coccè (University of Milan, Italy). She presented data on the inhibitory effect of adipose tissue-derived MSCs uploaded by paclitaxel (PTX) on malignant pleural mesothelioma in vitro and in vivo models. In particular, she saw how adipose tissue (FAT) after micro fragmentation (MFAT) was able to exert a dose-dependent inhibition on the growth of the human mesothelioma cell line (MSTO-211H). While, in xenografted Balb/c-Nude mice obtained after subcutaneous injection with MSTO- 211H, she observed a reduction of tumour mass volume measurements after treatment with MFAT loaded with PTX similar to that of free PTX. This could be a new therapeutic approach for a difficult to treat tumour (O-22).
After each session, at least 30 min were dedicated to questions from the audience and replies from the speakers, making the discussion very open and engaging for all the presentations and also helping to draw out take-home messages. In particular: The first session about the source of funding for the research projects was very animated and of great interest for all the attendees. The “GISM—Next Generation” section, which primarily concentrates on improving general skills related to publishing, patent writing, fundraising, and knowledge transferring, provided valuable insights based on the latest technological advancements and opportunities presented by social networks. The third session, dedicated to new 3D culture systems, gave insight on the current 3D strategies that could aid in the creation of personalised therapy options with systems such as joints-on-chip or the MIVO system. The session on therapeutic applications of MSC-EVs in veterinary and human medicine provided that the mode of action in different diseases is essential for the MSC-EVs translation from the laboratory to the clinical setting The fifth session showed the advancing MSC therapies in veterinary medicine present challenges and future perspectives not only in the veterinary field, but also for translation research. In fact, the MSC therapy in animals represents an excellent approach to have results describe an experimental investigation brochure dossier for an experimental clinical trial with secretome or MSCs The last session was dedicated to how EVs can be used as drug carrier systems and how they interact with the tumour microenvironment, showing interesting new discoveries especially in breast and mesothelioma. summarises the positive and negative aspects in mesenchymal stem cells utilisation and MSC secretome (microvesicles and exosomes), as raised from the six sections discussed here. In general, the congress was a great success thanks to lively participation of researchers of all ages, demonstrating that the topics of the congress were very attractive in the scientific community. In fact, more than 100 researchers participated in the meeting and 51 posters were exposed and presented in a dedicated section of the congress. Overall, the atmosphere was interactive and full of young researchers who felt at ease sharing their ideas and questions with senior mentors in all moments of the congress, including pleasant moments of relationship. All the abstracts of oral and poster presentations, with written consent for the publication here.
During the 2022 GISM annual meeting, three “Young Investigator Awards” of 500 euros each were assigned. In order to be eligible, researchers had to ( a ) submit a spontaneous candidature; ( b ) be the first author of an accepted abstract; ( c ) be younger than 35 years on 20 October 2022; ( d ) be present at the Award Ceremony held during the “GISM—Next Generation” section. The three winners of the 2022 GISM annual meeting “Young Investigator Awards” were: Priscilla Berni (University of Parma, Italy), Elena Ceccotti (University of Turin, Italy), and Gianluca Cidonio (CLN2S, Fondazione Istituto Italiano di Tecnologia, Rome, Italy).
5.1. Oral Presentation O-01. BRIDGING THE GAP IN MEDICAL RESEARCH ON ADVANCED THERAPY MEDICINAL PRODUCTS: A BIOLOGICAL, REGULATORY AND MEDICAL EXPERIENCE Graziella Pellegrini Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena, Italy Abstract Gene therapy, cell therapy, and tissue engineering have the potential to revolutionise the treatment of disease and injury. Attaining marketing authorisation for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agency—authorisation is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means such as “hospital exemption” schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorisation and is the first to contain stem cells. This review highlights the differences in standards between an authorised and unauthorised medicinal product and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorisation. The result is that patients will have access to a therapy that is manufactured to high commercial standards and is supported by robust clinical safety and efficacy data. O-02. BRIDGING THE FUNDING GAP IN MEDICAL RESEARCH IN ADVANCED THERAPY MEDICINAL PRODUCTS: TOWARDS A FAIR MEASUREMENT OF VALUE CREATION Stefano Cosma, Daniela Pennetta, Francesca Guida University of Modena and Reggio Emilia, Modena, Italy Abstract The right to healthcare for every individual is crucial for social inclusion and sustainable development. Despite this, innovation and medical research face several difficulties in their path. The risks of failure in medical research are due to scientific and operational factors, but also to economic factors, particularly the lack or inadequacy of funding. In Finance, the prerequisite for a project to be funded is the possibility of correctly determining its overall value. This paper is the first step of a wider project that aims to measure the operational and financial needs of the research into ATMPs with a Capital budgeting approach in order to correctly determine the ability to generate (economic and social) value. The paper aims to explore the causes of failure of the various phases of medical research and if and how the current funding schemes or financial partners may affect them. The purpose is achieved with the support of the ClinicaTrial.gov database, which includes privately and publicly funded clinical studies conducted worldwide. As a preliminary study, we focused our attention on the Italian context, collecting information on interventional studies carried out by Italian research groups during the entire period of database coverage (since 1998 to 2021). We collected a total of 12,934 interventional studies, 344 of which we classified by keywords within ATMPs research. The analysis was also extended to a sample of 1137 European studies classified by keywords within ATMPs research. The study builds a complete, general picture of the current funding of clinical research, the current role of Finance and the types of involvement of the public sector and other non-profit partners. Through manual data reprocessing, we were able to highlight two important features of the studies in the sample. First, the information on their current status allowed us to pinpoint failed and succeeded projects, while also understanding failure causes. Second, information on their sponsor was re-elaborated in order to classify the studies according to the source of funding (e.g., industry, university, research centres, hospitals, and so on). Thanks to an intersection of this data, our work provides insights into how the current financial resource allocation may be correlated to the success or failure of clinical trials, with a focus on advanced therapies, revealing not only potential funding gaps but also implications for involved researchers, policymakers, and stakeholders. O-03. MY EXPERIENCE WITH MSCS IN ORTHOPAEDICS: PAST, PRESENT AND FUTURE Nicholas Crippa Orlandi, Nicola Mondanelli, Stefano Giannotti Department of Orthopaedics and Traumatology, University of Siena, Italy Abstract Orthopaedic surgery can benefit a lot from the use of stem cells. Three case reports drawn from the experience of the authors’ Orthopaedic Clinic are illustrated to highlight the benefits of applying this technology. Drawing on the extensive experience gained within the authors’ Operating Unit, three cases regarding different body segments have been selected to prove the benefits deriving from the use of gelled preparations containing autologous MSC from bone marrow. A case of humeral shaft non-union, the management of an atypical proximal femur fracture in congenital hip dysplasia, and a case of rupture of the patellar tendon and consequent reconstruction of the extensor apparatus with a cadaveric transplant. The experimental study, whose three cornerstones are mesenchymal stem cells (hMSCs), scaffolds, and growth factors, aims to develop new tissue engineering strategies to be applied in orthopaedic surgery. In the first part of the work, the focus was on the optimisation of the isolation and expansion protocol of the hMSCs, taken from the bone marrow and deposited in the Biobank present inside the laboratory which is part of the Network Telethon (TNGB), evaluating the replacement of the FBS (fetal bovine serum) with autologous PRP (platelet rich plasma). Then, the focus shifted to the characterisation of cells seeded on two types of scaffolds: bovine bone matrix (SmartBone) and lyophilized acellular dermis. The final goal of the work is to develop strategies to increase the osteogenic properties of the support by adding growth factors that enhance cell differentiation on the scaffold. Our technique of applying stem cells to cases of complex orthopaedic surgery has shown excellent results both in a clinical functional objective and subjective evaluations and in radiographic evaluations. The experimental study, currently underway, is providing excellent results. The data suggest PRP as a valid alternative to FBS, it supports the expansion of mesenchymal stem cells without compromising their capacity since cells loaded on SmartBone differentiate into osteoblasts and produce collagen. In our opinion, MCSs are and will become more and more a valid tool to provide the surgeon with important help in cases of great complexity and, therefore, to obtain the tailored care that every patient needs and deserves. O-04. ORAL CAVITY MSC PHENOTYPING FOR REGENERATIVE MEDICINE APPLICATIONS Ilaria Roato, Tullio Genova, Beatrice Masante, Giacomo Baima, Alessandro Mosca Balma, Federico Mussano University of Turin, Italy Abstract The oral cavity contains multiple sites of MSCs, which have been studied as promising candidates for tissue regeneration in the dental/maxillofacial and neuroregenerative field due to the neural crest derivation of most of them. Adult dental pulp stem cells (DPSCs) are more indicated for the pulp–dentin complex regeneration while proved to be less osteogenic compared to buccal fat pad stem cells (BFPSCs). The analysis of the immunophenotype of in vitro expanded stem cells from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed a comparable expression of MSC markers among them and a higher percentage of endothelial precursor subset in SHED and DPSCs than in PDLSCs. Owing to their peculiar origin, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, even though MSCs retrieved from different tissues show comparable immunophenotype and multilineage differentiation ability, their capability to differentiate into a specific tissue depends also on the different anatomical origins. Moreover, often, these cells may be harvested without any burden or additional discomfort for the patient who undergoes a tooth extraction for orthodontic indications. O-05. A NEW STRATEGY TO MONITOR MESENCHYMAL STEM CELLS AND EXTRACELLULAR VESICLES IN ADVANCED THERAPIES APPLICATION Marta Nardini 1 , Maria Elisabetta Federica Palamà 2 , Vipul Gujrati 3 , Vasilis Ntziachristos 3 , Mary Murphy 4 , Niamh Duffy 4 , Ignacio de Miguel 5 , Martin Leahy 6 , Chiara Gentili 2 and Maddalena Mastrogiacomo 1 1 Department of Internal Medicine (DIMI), University of Genoa, Italy 2 Department of Experimental Medicine (DIMES) University of Genova, Italy 3 Chair of Biological Imaging at the Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Germany 4 Regenerative Medicine Institute, School of Medicine, University of Galway, Ireland 5 Knowledge & Technology Transfer department. Institut de Ciències Fotòniques (ICFO), The Barcelona Institute of Science and Technology, Barcelona, Spain 6 National University of Ireland, National Biophotonics and Imaging Platform, School of Physics, Tissue Optics and Microcirculation Imaging Group, Galway, Ireland Objective An important challenge in regenerative medicine to the regulatory approval and widespread clinical acceptance of cell therapies is surrounding the behaviour of cells after transplant. The distribution, engraftment, viability, and activity of transplanted cells are unclear. This reduces confidence in the safety of cell therapies and makes it difficult to understand the mechanism of action for developing potency assays that will be required for advanced clinical testing and the approval of cell therapy. When Extracellular Vesicles (EVs) are used as therapeutic agents, similar issues arise. Optoacoustic imaging (OAI) is a rapidly developing technology with world-leading capabilities in functional imaging that is uniquely informative and lower cost, convenient, and rapid. Gold-Nanostars (GNs) are suited for use as a contrast medium for optoacoustic imaging that generates a very strong photo-thermal signal in response to light of longer wavelengths. The combination of OAI and GNs offers a class-leading imaging solution for cell therapies in regenerative medicine. In this work after a characterisation in vitro of the complexes, GNs-labelled Mesenchymal Stem Cells (MSC) and EVs were studied for their distribution in different organs after a systemic injection by visualisation with Multispectral Optoacoustic Tomography (MSOT) and tested for the in vivo toxicity. Materials and Methods MSCs were isolated from human bone marrow, expanded in Platelet Lysates (PL), and derived from the same cell EVs. The MSC and EVs were labelled with GNs and by in vitro tests with the right concentration of particles, in terms of capability to be visualised by MSOT and no cytotoxicity, was defined. Labelled and unlabelled complexes were injected into the caudal vein of male and female Nu/Nu mice. Blood tests and histological analysis of the organs were performed at different time points to test the in vivo toxicity. Results No alterations were observed in terms of biochemical and blood markers. MSOT analysis showed that after one day, GNs reached all organs and remained for all monitoring periods without any alteration as revealed by histological analyses. In particular, MSOT analysis showed that GNs cannot overcome Blood–Brain Barrier (BBB). Conclusions The results showed that the GNs can reach the various organs and that they persist there for some time, but although they persist, they cannot overcome BBB and does not lead to any type of alteration. This approach holds promise for tracking cell distribution in the tissue repair process. O-06. SCIENTIFIC ARTICLES: HOW TO CHOOSE THE RIGHT JOURNAL TAKING ADVANTAGE OF SOME OPPORTUNITIES Filippo Piccinini 1,2 1 IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) ‘‘Dino Amadori”, 47014 Meldola, Forlì-Cesena, Italy 2 Department of Medical and Surgical Sciences (DIMEC), University of Bologna, 40136 Bologna, Italy Abstract “Scientific Articles” are not the final goal but a necessary intermediate step for worldwide progress. “Scientific journals” are the right place to communicate discoveries to society. “Editors and Reviewers” are important people for evaluating the results. “Impact Factor” is one of the most common words in the life of a researcher. These terms are just a few of many behind the real important things in research including “Hypotheses and Results”. However, a large part of the working life of a researcher is spent writing scientific articles and finding the right journals for them. In this presentation, we will discuss how to speed up the publication process, define the best journal, and increase the chances of publication by exploiting opportunities that technology and social networks today provide us. O-07. TECHNOLOGY TRANSFER AND INTELLECTUAL PROPERTY: LET’S TALK TO AN EXPERT Paola Bagnoli IRCCS Ospedale Galeazzi Sant’Ambrogio of Milan, Italy Abstract Technology transfer in the Life Science field is a demanding but very stimulating challenge for both researchers and technology transfer experts. The sector has peculiarities that differentiate it from most other technological fields and make the exploitation of research results even more challenging. The results obtained by universities and research hospitals, while promising, are often premature and require a long development path to be commercialised and brought to the clinic. It is well known that very long research and development times and high costs are required to bring a medical device or drug to the market. This gap between proposals from the research world and needs from industry is well known by the technology transfer experts and often discourages the researchers who do not see their research reaching the patient, although a strong clinical need is well identified. Many factors are crucial to fill this gap, not just the need for dedicated funding. The design according to the correct principles is fundamental to allow the industrialisation of the prototype, the scale up of the processes, and the subsequent certification phases. Last but not least, the protection of industrial property (IP) is crucial. Indeed, a company would be unlikely to invest millions of euros in a new life science project without the certainty of exclusivity on the results that would guarantee a competitive advantage over its competitors. The patent is the main tool to obtain this exclusivity, together with the copyright, the design and the trade secret. The proper protection of the research results is an essential step in the technology transfer process. Researchers can have support from the Technology Transfer Offices (TTO) of their institutions, which have the fundamental role of scouting the results, assisting researchers in protecting IP and then activating virtuous paths for the development of the Technology Readiness Level (TRL) of the projects. These paths have to be walked together with several stakeholders such as corporates, start-up incubators, business angels, venture capitalists, etc. The activation of co-development processes with companies and grants dedicated to the development of the Proof of Concept of the patented technologies are key steps in making the project attractive for a company that can licence it or in creating a startup to develop and commercialise the new technology. O-08. RECAPITULATING MUSCULOSKELETAL TISSUE COMPLEXITY THROUGH MICROFLUIDIC AND BIO-FABRICATED 3D MODELS Matteo Moretti IRCCS Istituto Ortopedico Galeazzi of Milan, Italy Abstract The musculoskeletal system is composed of different tissues and organs, i.e., bones, muscles, tendons, and joints. Several pathologies can affect its homeostasis including traumatic injuries and inflammatory diseases such as osteoarthritis (OA). In this context, mesenchymal stem cells have been considered either as building blocks to fabricate biological substitutes for damaged musculoskeletal tissues due to their differentiation potential towards bone and cartilage or as a possible therapy due to their anti-inflammatory properties. On the other hand, 3D in vitro models are increasingly being considered a powerful tool to investigate pathological mechanisms and therapeutic efficacy, allowing to overcome the excessive simplification of standard in vitro models and species-specific differences of animal models. Thus, we developed 3D in vitro models of joints to test the potential of mesenchymal cells as anti-OA therapy and of the muscle–tendon–bone junction, based on differentiated mesenchymal cells. We generated a microfluidic joint-on-a-chip model including cartilage, synovial membrane and synovial fluid, based on patient-matched synovial fluid and cells embedded in gels mimicking the composition of cartilage ECM. The injection of OA synovial fluid allowed for the reproducing of the main hallmarks of early OA as the production of degradative enzymes and an increase in inflammatory cytokines. Furthermore, we were able to detect the effects of mesenchymal cell injection, measuring MMPs and inflammatory cytokine release, on a patient basis, opening new possibilities for the establishment of personalised treatments. We also fabricated a 3D bioprinted muscle–tendon–bone model embedded in a microfluidic chip, allowing the fabrication and culture of three different connective tissues in their specific culture media. Computational simulations were performed to design the chip, keeping the culture media separated during perfusion. The chip also allowed to apply compression to the bone and stretch to muscle and tendon in physiological and pathological ranges, simulating traumatic conditions. The 3D bioprinting procedure resulted in three separated cell compartments, maintaining a good shape fidelity and high cell viability. In conclusion, biofabricated 3D in vitro models could help foster the application of mesenchymal cells as anti-inflammatory therapy and could benefit from their potential as starting material for the reconstruction of musculoskeletal tissue units. O-09. EXTRACELLULAR VESICLE-MEDIATED COMMUNICATION ROUTES IN 3D TUMOUR MODELS Maria Harmati 1 , Akos Diosdi 1,2,3 , Ede Migh 1 , Gabriella Dobra 1,4 , Timea Böröczky 1,4 , Edina Gyukity-Sebestyen 1 , Matyas Bukva 1,4 , Sandor Körmöndi 5 , Peter Horvath 1,3,6 , Krisztina Buzas 1,7 1 Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary 2 Doctoral School of Biology, University of Szeged, Hungary 3 Single-Cell Technologies Ltd., Szeged, Hungary 4 Doctoral School of Interdisciplinary Medicine, University of Szeged, Hungary 5 Department of Traumatology, University of Szeged, Hungary 6 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland 7 Department of Immunology, University of Szeged, Hungary Objective The evolutionary process of solid tumours highly relies on the extracellular vesicle (EV)-mediated crosstalk between malignant cells and stromal cells in the tumour microenvironment (TME). In this study, we aimed to establish a multicellular three-dimensional (3D) tumour model system for tracking the EV communication network of different tumour tissues under physiological conditions and cytostatic treatments. Materials and Methods Human ductal carcinoma, melanoma, and osteosarcoma models were established via co-culturing the respective tumour cell line (T-47D/A375/MG-63) with MRC-5 fibroblasts and EA.hy926 endothelial cells on flat- or U-bottom plates after staining with CellTracker dyes (Orange CMTMR, Deep Red, Green CMFDA). To mimic chemotherapeutic stress, low dose doxorubicin was used and the 2D and 3D cultures were imaged daily by a PerkinElmer Operetta High Content Screening System and a Leica SP8 Digital LightSheet microscope, respectively. Results Preliminary experiments showed that CellTracker dyes can be used for in-cell labelling of EVs, allowing the quantitative monitoring of EV crosstalk, i.e., EV routes between each cell type and in both directions. The three types of tumour models showed differences in their 3D structure, EV crosstalk activity, and drug-induced effects as well. We could observe distinct temporal kinetics in the development of the EV communication network in 2D and 3D, also priorities of the investigated EV routes varied between the two co-culture systems. Conclusions The developed 3D model system is suitable for live tracking of EV crosstalk in the TME, which enables the comparison of the primary EV communication routes in different tumour types and drug treatments. Further data will help (i) to identify potential targets of EV-blocking therapies, which may increase the efficacy of chemotherapies, and (ii) to predict the drug-induced changes of the communication activity in different tumour tissues. O-10. DEMONSTRATING METHOD SUITABILITY FOR A RAPID MICROBIAL DETECTION METHOD APPLICABLE TO BIOLOGIC PRODUCTS Lucia Ceresa, Senior Technology and Market Development Manager Microbial Solutions, Charles River, Italy Abstract New and emerging cell therapies and medicinal products present new challenges in the assurance of quality and safety in terms of end-product testing. While traditional pharmaceutical drug products have long-established standards for sterility assurance, these established processes are not optimised for cell therapies. For example, the Compendial Sterility Test requires more than fourteen days of incubation for a reliable result, making it a rate limiter in the distribution of therapies to patients. Rapid Microbial Methods (RMM’s) offer reliable alternatives to ageing microbiological methods to solve the problem of reducing cycle time in cell therapy manufacture. ATP Bioluminescence is a matured technology that is used in the quality testing of various product samples in different industries. Detection of microbial ATP using the luciferin-luciferase reaction allows for the detection of microbes before they can be cultured to visual detection levels on microbial media. Until recently, ATP bioluminescence was not a viable contamination detection option for cell-based products because these samples also contain cellular ATP. The established ATP Bioluminescence platform, Celsis ® , was further developed to address this limitation. A sample cell lysing procedure allows for the extraction and, more important, the depletion of “non-microbial-ATP”, while leaving microbial ATP intact. A case study on tests performed on different cell lines demonstrate the detection of the “slow growing” C. acnes as well as a wide panel of other typical and critical microbial species. Studies performed using the Celsis ® platform and the Celsis Adapt™ complimentary technology demonstrated the successful depletion of cellular ATP from samples, while also allowing fast detection of microbial presence contamination for superior microbiological contamination control. O-11. A NOVEL ORGAN ON CHIP PLATFORM FOR CULTURING 3D HUMAN TISSUES UNDER PHYSIOLOGICAL FLOW-CONDITIONS FOR MORE PREDICTIVE DRUG TESTING AND HUMAN DISEASE MODELLING Silvia Scaglione 1,2 , Monica Marzagalli 1 1 React4life srl, Genoa, Italy 2 CNR-IEIIT Institute, National Research Council of Italy, Genoa, Italy Objectives The human disease modelling for basic research and drug testing purposes is currently carried out through 2D cell culture in static conditions and in vivo xenografts or genetically engineered animal models, but predictability, reliability, and complete immune compatibility remain important challenges. For this aim, novel 3D, fully humanised in vitro tissue models have been recently investigated by adopting emerging technologies such as bioprinting and microphysiological systems, also named organ on chips. A novel Multi-In Vitro Organ (MIVO) organ on a chip platform has been recently developed to culture 3D clinically relevant size tissues under proper physiological culture conditions. Material and Methods Biologically relevant cancer samples (up to 5 mm), and patient biopsies of scaffold-based tissues have been cultured within the MIVO chamber, while either testing molecules or human immune cells (e.g., Natural Killer cells, NK) can circulate in the OOC mimicking the blood capillary flow. The cell proliferation, viability, and migration within 3D matrixes were investigated in such dynamic cell culture conditions. When systemic drug administration was simulated within the OOC, the anticancer drug efficacy was tested and compared to the animal model. When NK cells were placed in circulation, their extravasation through a permeable barrier resembling the vascular barrier and infiltration within the cancer tissue were analysed. Results A human 3D ovarian model was developed and treated with Cisplatin in static conditions within MIVO and in the xenograft model. Similar tumour regression was observed in MIVO and in mice, while the static culture displayed an unpredicted chemoresistance, due to unreliable drug diffusion. A human 3D neuroblastoma cancer model with proper immunophenotype was optimised to develop a complex tumour/immune cell co-culture as a paradigm of an immune-oncology screening platform. Importantly, a tumour-specific NK cell extravasation was observed with a tumour-specific NK cell infiltration within 3D tumour tissue and cancer cells apoptosis induction. Conclusions We generated a relevant human disease mode, through the adoption of the MIVO device, that can be efficiently employed as a drug screening platform, but also for better investigating crosstalk among immune cells and other healthy/pathological tissues. O-12. TRANSLATIONAL LAB-TO-CLINIC HURDLES IN THERAPY WITH MSC-EVS Stefania Bruno Department of Medical Sciences, University of Turin, Italy Abstract Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to have therapeutic effects in pre-clinical models of different diseases. EVs derived from various types of MSCs have pro-regenerative capabilities comparable to cells of origin and are considered promising tools for the treatment of a variety of acute and chronic pathologies. To this end, the characterisation (size, phenotype, molecular content, etc.) of EVs and the evaluation of their biological effects are areas of intense investigation. To successfully translate EV research along the path from the laboratory to the patients, strategies should be designed to reach the aim of clinical testing of MSC-EVs safety and efficacy. Currently, there are emerging strategies for manufacturing cells and EVs and quality controls for practicability of clinical testing of the EVs, mainly related to non-industrial processes. Moreover, the identification of the mode of action in the therapeutic approach in the different diseases remains a major challenge to the translation path of EVs from the laboratory toward the clinical setting. O-13. CELECTOR ® , AN INSTRUMENT FOR QUALITY CONTROL AND STANDARDISATION OF ATMPS Silvia Zia, Pasquale Marrazzo, Laura Bonsi, Francesco Alviano, Barbara Roda, Andrea Zattoni, Pierluigi Reschiglian Stem Sel srl, Bologna, Italy Abstract Cell therapy represents innovative medical approaches in many clinical fields such as osteo-articular reconstructive surgery, tissue engineering, and cancer. Advanced therapy medicinal products (ATMPs) are cell and tissue products that are considered new types of drugs. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials. Besides their regenerative properties, hMSCs have also shown high immunomodulatory potential. The ATMP products should follow requirements including sterility, identity, purity, viability, potency, and reproducibility. Because stem cells are a heterogenous population that can differ depending on the origin and manipulation of the starting material, the identity/purity of the final cell population and its yield is still a critical issue which can limit clinical application of MSC-based products. New approaches for the standardisation of cell-based protocols may allow for the development of new high-efficiency drug systems. To improve MSC characterisation, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. In this work, we present a new technology, Celector ® , for the quality control of the cell population. Celector ® has been shown to be able to tag-less analyse, discriminate, and separate a wide size range of cells based on their physical characteristics, with high resolution and throughput and with total maintenance of native properties. The separation is obtained in a short time (around 15 min) in a rectangular shape capillary device, through to the combined action of gravity, acting perpendicularly to the flow and opposing lift forces that depend on the morphological features of the sample. A micro-camera is connected as a detector and specifically designed software was developed for image acquisition, post-processing and data analysis, and the fingerprint of the biological sample. Celector ® is able to highlight physical differences that can be correlated to cell viability and regenerative potential. In addition, cells with stable and reproducible doubling time analysis can be collected and used as standardised systems for the development of high-quality clinical protocols. O-14. HUMAN LIVER STEM CELL-DERIVED EXTRACELLULAR VESICLES INTERFERE WITH THE DEVELOPMENT OF CHRONIC KIDNEY DISEASE IN AN IN VIVO EXPERIMENTAL MODEL OF RENAL ISCHEMIA AND REPERFUSION INJURY Elena Ceccotti 1 , Massimo Cedrino 2 , Giulia Chiabotto 1 , Cristina Grange 1,3 , Samuela De Rosa 1 , Giovanni Camussi 1,3 , Stefania Bruno 1,3 1 Department of Medical Sciences, University of Turin, Italy 2 Unicyte AG, Obendorf, Switzerland 3 Molecular Biotechnology Center, University of Turin, Italy Objective Renal ischemia reperfusion injury (IRI) is the major cause of acute kidney injury (AKI), and it increases the risk of progression to chronic kidney disease (CKD). Human liver stem cells (HLSCs) are a mesenchymal stromal cell (MSC)-like population isolated from adult liver biopsy. HLSCs share with MSCs the same phenotype, gene expression profile, and differentiation capabilities. As shown in previous studies, HLSCs improved the recovery in different experimental models of liver and kidney injury. HLSC-derived extracellular vesicles (HLSC-EVs) have been studied as vehicles for transferring active biological materials both in vitro and in vivo. In this study, we set up an in vivo murine model of IRI-AKI, which subsequently developed into IRI-CKD and we investigated the potential therapeutic effect of HLSC-EVs. Materials and Methods EVs were purified from HLSC supernatant by ultracentrifugation. They were analysed through flow cytometry and Western Blotting to detect the expression of the main mesenchymal and EV surface markers. Transmission electron microscopy was performed to analyse EV size and morphology. Male BALB-c mice were subjected to 30 min ischemia followed by reperfusion. HLSC-EVs were intravenously administered immediately after the surgery and three days after. To evaluate AKI, mice were sacrificed two and three days after the surgery; while to assess the development of CKD, mice were sacrificed two months after. The histological analyses were performed on tissue sections stained with hematoxylin and eosin and Masson’s trichrome for collagen detection. Expression of specific markers of fibrosis development (alpha-Smooth Muscle Actin (alpha-SMA), collagen I and transforming growth factor-beta) and inflammation (interleukin-1 beta, interleukin-6, and tumour necrosis factor-alpha) were evaluated at mRNA and protein levels. Results In AKI mice, the EV treatment-attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation. We also noticed the downregulation of the expression levels of fibrosis-related genes. In CKD mice, EVs effectively reduced the development of interstitial fibrosis at the histological level and reduced the expression levels of pro-fibrotic and pro-inflammatory genes. Conclusions The administration of HLSC-EVs immediately after renal IRI protects the kidney from AKI development and interferes with the development of subsequent CKD. O-15. FLUORESCENCE POLARISATION-BASED EXTRACELLULAR VESICLES QUANTIFICATION: APTAMER APPROACH Tarlan Eslami Arshaghi 1 , Jerry Clifford 2 , Stephanie J Davies 2 , Frank Barry 1 1 Regenerative Medicine Institute, University of Galway, Ireland 2 Valitacell Ltd., Dublin, Ireland Objective Extracellular vesicles (EVs) have attracted wide interest in recent years due to their potential applications in regenerative medicine, as biomarkers for disease diagnosis, and their role in cell–cell interactions. However, EV isolation, quantification, and characterisation remain challenging in terms of purity and specificity as well as time- and cost-effectiveness. This work aims to develop a novel and high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific EV surface component, using fluorescence polarisation (FP) for detection. The method analyses the change in the polarisation of emitted light, between unbound, and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of the bound probe. This property of FP allows us to use it to quantify the amount of EVs in the solution. Materials and Methods Two distinct strategies have been investigated, with probes targeting (i) specific EV surface markers (Tetraspanins, e.g., CD63) for EV sub-population quantification or (ii) the EV phospholipid bilayer membrane for total EV quantification. Commercially available fluorescently labelled CD63 binding aptamers and proteins and lipophilic dyes have been evaluated. EVs derived from HEK and MSC cultures have been purchased or isolated through PEG-precipitation and ultracentrifugation, and particles quantified through Nano Tracking Analysis (NTA) or Imaging Flow Cytometry. Each probe candidate was incubated with EVs, and FP was measured over time using a Spark Cyto plate reader. Results Tetraspanin specific (Anti-CD63) aptamer probes and lipophilic dyes have demonstrated increased fluorescence polarisation in response to increasing EV concentration quantified by NTA. Conclusions This initial proof of concept supports the use of FP as a high throughput EV detection and quantification method, with the ability to provide both total particle and CD63 +ve particle numbers. Further investigation is required to demonstrate the specific binding of each probe to its target to benchmark the FP assay against currently available methods. For this purpose, Bio-Layer Interferometry (BLI) assay is under examination to study the probe–target kinetic. O-16. MESENCHYMAL STROMAL CELL SECRETOME FOR REGENERATIVE MEDICINE: MODULATION OF SOLUBLE FACTORS AND EXTRACELLULAR VESICLES EMBEDDED-miRNAs BY DIFFERENT CULTURING CONDITIONS FOR JOINT DISEASES Enrico Ragni 1 , Paola De Luca 1 , Carlotta Perucca Orfei 1 , Alessandra Colombini 1 , Marco Viganò 1 , Francesca Libonati 1 , Stefania Cicolari 1 , Leonardo Mortati 2 , Laura de Girolamo 1 1 IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, Milan, Italy 2 INRIM, Istituto Nazionale di Ricerca Metrologica, Turin, Italy Objective In regenerative medicine approaches related to orthopaedic conditions, mesenchymal stromal cells (MSCs) showed positive outcomes due to the secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. MSC-derived factors may be modulated by both culturing and in vivo conditions. Nevertheless, a homogenous and comprehensive fingerprint in the frame of orthopaedic applications is missing. The aim of this work was to characterise adipose-derived MSC, (ASC)-secreted factors, and EV-miRNAs and their modulation after high levels of IFNγ preconditioning, as proposed for clinical-grade production of secretome with improved potential or low levels inflammatory conditions, mimicking osteoarthritis (OA) and synovial fluid (SF). In addition, ASC-EVs penetration in cartilage explants was scored. Material and Methods ASCs were cultured with and without IFNγ (1 ng/mL) or TNFα (5 pg/mL) + IL1β (10 pg/mL) + IFNγ (40 pg/mL) mimicking OA-SF. First, 200 secreted factors were assayed by ELISA. Second, 754 miRNAs were searched by qRT-PCR in ultracentrifuge-purified EVs. Bioinformatics tools were used to predict the modulatory effect of identified molecules on pathologic cartilage and synovial macrophages. Time-lapse coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence were used to follow and quantify fluorescent EVs incorporation into cartilage explants. Results Data showed that more than 50 cytokines/chemokines and more than 200 EV-miRNAs could be identified. The vast majority of molecules are involved in extracellular matrix remodelling and homeostasis of inflammatory cells. Inflammatory priming and synovial fluid-like conditions were able to further increase the capacity of the secretome to stimulate healing and inflammation reduction. Eventually, EV penetration was monitored as a fast process, starting in a few minutes, and reaching 30–40 μm depth after 5 h and plateau at 16 h in both cells and matrix of the cartilage explants. Conclusions Due to the portfolio of soluble factors and EV-miRNAs, the ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes. Inflammatory preconditioning or OA-like conditions were able to increase this ability. Eventually, microscopy data supported the capacity of MSC-EVs to influence the chondrocytes embedded in their native ECM by active interaction and eventual therapeutic cargo release. O-17. MESENCHYMAL STEM CELLS AND IMMUNE PROPERTIES: WHAT HAVE WE LEARNED FOR THEIR ALLOGENEIC APPLICATION Laura Barrachina Porcar Regenerative Medicine Institute (Remedi), University of Galway, Ireland Abstract Allogeneic MSCs present several advantages and the first allogeneic cell-based products in the veterinary market are emerging. Quality cells can be banked to be ready to use, avoiding the delay inherent to expanding autologous cells, which in addition may be unsuitable due to the patient’s age, genetic, or metabolic disorders. However, allogeneic therapy does not come without limitations. At first, MSCs were considered immune-privileged, but currently, we know that allogeneic MSCs can induce cellular and humoral immune responses. The immune targeting of MSCs can impact their properties and lead to adverse events, thus affecting their therapeutic efficacy and safety. Nevertheless, several human and animal studies report positive results after allogeneic MSC administration. A potential explanation for the mixed outcomes often seen is that MSCs can be immunogenic (i.e., able to raise an immune response) but they also are immunomodulatory. The less immunogenic and the more regulatory MSCs are, the better chances they have to elicit their therapeutic actions. However, several factors can affect the balance between these two immune properties. MSC immune recognition is mainly mediated by the major histocompatibility complex (MHC), which is variably expressed depending on the donor, source, or in vitro/in vivo conditions. In addition, the MHC matching between donor and recipient determines the immune recognition as in organ transplants. Although MSCs can be recognised by the immune system, they can also regulate it. Factors such as inflammation or differentiation can affect both MSC immunogenicity and immunomodulation, so it is key to learning how different conditions affect this balance. Based on this knowledge, different strategies can be designed to develop safer and more effective allogeneic therapies. The selection of the donor based on its MHC haplotype and expression pattern would allow the creation of ‘haplo-banks’ of cells from donors homozygous for the most common MHC haplotypes, as it has been proposed for human iPSCs. Other strategies aim at either decreasing the immunogenicity or increasing the immunomodulation of MSCs. For example, some growth factors can decrease the MHC expression and pro-inflammatory cytokines can increase MSC immune suppression. Understanding the interactions between MSCs and the immune system is key to learning which factors we can manage and how in order to enhance the clinical safety and effectiveness of allogeneic cell therapies. O-18. PEPTIDE MEDIATED ADHESION TO BETA-LACTAM RING OF EQUINE MESENCHYMAL STROMAL CELLS: A PILOT STUDY Barbara Merlo 1,2 , Vito Antonio Baldassarro 1,2,3 , Alessandra Flagelli 2 , Romina Marcoccia 2 , Valentina Giraldi 2,4 , Maria Letizia Focarete 2,4 , Daria Giacomini 2,4 , Eleonora Iacono 1,2 1 Department of Veterinary Medical Sciences, University of Bologna, Italy 2 Interdepartmental Center for Industrial Research in Health Sciences and Technologies, University of Bologna, Italy 3 IRET Foundation, Ozzano Emilia, Italy 4 Department of Chemistry “Giacomo Ciamician” and INSTM UdR of Bologna, University of Bologna, Italy Objective The use of biomaterials with integrin agonists could promote cell adhesion in tissue repair processes. Studies on the use of scaffolds with integrin agonists based on β-lactams in equines have never been reported. The aim of this study was to analyse the effect of GM18 (α4β1 integrin agonist) on cell adhesion of equine adipose tissue (AT) and Wharton’s jelly (WJ) mesenchymal stromal cells (MSCs) and to investigate the cell adhesion to GM18-incorporated poly L-lactic acid (PLLA) scaffolds. Materials and Methods Scaffold was fabricated using a homemade electrospinning apparatus consisting of a high-voltage power supply, a syringe pump, a glass syringe containing the PLLA solution, and connected to a stainless steel blunt-ended needle through a PTFE tube. Adhesion assays were performed after culturing AT and WJMSCs with coating or soluble GM18. Gene expression of the target integrins was evaluated. Cell adhesion on GM18 containing PLLA scaffolds after 20 min and 24 h coincubation was assessed using the two samples of AT and WJMCSs more sensitive to GM18. A cell-based high content screening was used for analyses. For statistical analyses, 2- or 1- Way ANOVA was used. Results were considered significant for p < 0.05. Results Soluble GM18 affects the adhesion of equine AT and WJMSCs, even if its effect is variable between donors. For ATMSCs, the presence of GM18 did not affect the adhesion to the PLLA scaffold, mainly due to a high variability detected at a concentration of 10%. Moreover, cultures seeded on the PLLA control scaffold cannot increase cell number after 24 h, reflecting an impairment in the early proliferation. However, the presence of GM18 in all the analysed concentrations induces an increase in cell number after 24 h (5%, p = 0.0170; 10%, p = 0.0144; 15%, p = 0.0395). For WJMSCs, the cell adhesion was affected after 24 h ( p = 0.0427), drastically increasing due to the highest concentration of GM18 ( p = 0.0368). The same concentration is also the only condition producing an increase in cell number after 24 h ( p = 0.0021). Conclusions In conclusion, the α4β1 integrin agonist GM18 affects equine AT and WJMSCs adhesion ability with a donor-related variability. These preliminary results represent a first step in the study of equine MSCs adhesion to PLLA scaffolds containing GM18, suggesting that WJMSCs might be more suitable than ATMSCs. However, the results need to be confirmed by increasing the number of samples before drawing definite conclusions. O-19. MESENCHYMAL STROMAL CELL-DERIVED MIGRASOMES: A MORPHOLOGICAL STUDY Gabriele Scattini, Luisa Pascucci University of Perugia, Department of Veterinary Medicine, Perugia, Italy Objective Cell migration is fundamental in numerous physiological and pathological processes, including tissue regeneration. Migrasomes (MG) are large structures growing on the cell surface and on the tip of nanotubes emerging from cell body during migration. MG represents a distinct type of extracellular vesicles (EVs) and are significantly different from other microparticles in size, morphology, and function. In this study, a morphological analysis of MG generated by Mesenchymal Stromal Cells (MSC) of different species was performed. Material and Methods EVs were isolated by differential ultracentrifugation. Briefly, the conditioned medium was centrifuged at 300× g to pellet cells. The supernatant was centrifuged at 2000× g (2 K EVs fraction), at 10,000× g (10 K EVs fraction), and finally at 100,000× g (100 K EVs fraction). EVs suspensions were placed on formvar-coated copper grids, contrasted with 2% uranyl acetate, and observed under a Philips EM 208 electron microscope (TEM) equipped with a digital camera. MSC monolayers were also fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated, and resin embedded. 80 nm thick sections were contrasted with 2% uranyl acetate and observed at TEM. Results Ultrastructural analysis revealed that MSC produces a previously unrecognised kind of vesicles, referred to as “migrasomes” that originate from the cell surface. The biogenesis and morphologic features of these vesicles are completely different from typical EVs. In fact, they are very large in diameter (500–2000 nm) and contain a variable number of luminal vesicles. MG were also detected in the 2 k fraction obtained by supernatant centrifugation. Conclusions Migrasomes are a special kind of EVs that are released by migrating cells. Determining their molecular content and signalling potential clearly need extensive research. The most crucial things to comprehend are how MG works, which signals are triggered by coming into contact with or ingesting MG, what messages they transport between cells, and so on. The discovery of these EVs raises many new questions for future research. In fact, although the heterogeneity of EVs has become obvious, as highlighted by the International Society for Extracellular Vesicles, specific tools to distinguish EVs of different origins are still lacking, and thus different functions are probably not correctly evaluated at the moment. O-20. MSC-EVS AS DELIVERY VEHICLES FOR TUMOUR TARGETED THERAPY Róisín Dwyer University of Galway, Ireland Abstract Despite improvements in treatments for breast cancer, when patients are diagnosed with metastatic disease that has spread to distant sites there are limited treatment options and no cure available. Extracellular Vesicles (EVs) hold immense potential as cancer therapeutics due to their small size, biocompatibility, and potential for manipulation of content and surface characteristics. Tumours actively recruit stromal cells including Mesenchymal Stromal Cells (MSCs) into the tumour microenvironment. The tumour-targeted tropism of MSCs is thought to be due to high local concentrations of inflammatory chemokines and growth factors. This tumour tropism combined with the apparent immunosuppressive characteristics of the cells raised remarkable interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. MSC-derived EVs (MSC-EVs) will potentially retain the tumour targeting and immune privilege associated with MSCs while overcoming challenges associated with the use of cells. Our recent work in development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy will be discussed. The impact of human- and murine-derived MSC-EVs on the immune system and the potential for scale up of EV production will also be highlighted. An approach to support sustained delivery of EVs over time would be very beneficial to prevent cancer resurgence. The use of pre-clinical models that are more reflective of the patient experience will be critical for testing novel approaches to breast cancer treatment. Progress and challenges in the development of MSC-EVs as cancer therapeutics will also be addressed. While we require further understanding of factors mediating EV content, persistence, and uptake, this exciting approach holds tremendous potential for patients with limited existing treatment options. O-21. MESENCHYMAL STROMAL CELLS AND THEIR ROLE IN THE TUMOUR MICROENVIRONMENT: CHALLENGES AND OPPORTUNITIES IN THE IMMUNOTHERAPY ERA Andrea Papait Cattolica del Sacro Cuore University, Rome, Italy Abstract Mesenchymal stromal cells (MSCs) have long been studied for their applications in regenerative medicine, exploiting their unique ability to modulate the immune response in a large number of diseases in which the immune response is dysregulated. In addition, MSCs have been studied for their potential applications as an anticancer therapeutic strategy. Indeed, MSCs have been shown to exhibit a natural tropism toward sites of inflammation, and this property has been exploited for their potential use as a vehicle for the selective delivery of anticancer drugs. In addition, MSCs can be genetically modified in an attempt to reactivate the antitumour immune response. Unfortunately, however, the application of MSCs as anticancer therapy has often achieved mixed results. In addition, the fight against cancer must take into account the complex network of interactions that exist between the different actors that are part of the tumour microenvironment (TME) and that are represented by tumour cells, stromal cells, endothelial cells, and immune cells. In fact, the use of MSCs in this regard can be considered a double-edged sword: on the one hand, they can serve as a carrier of drugs or chemotherapeutic compounds; while on the other hand, their immunomodulatory properties can participate in tumour initiation, development, and progression, even contributing to metastasis formation. In this presentation, we will discuss all these merits and demerits of MSCs by contextualising them in the era of immunotherapy, which sees the use of new drugs, mostly monoclonal antibodies, aimed at re-educating the immune response, thus evaluating the possibility of using MSCs or their secretome as adjuvant therapy. O-22. INHIBITION OF HUMAN MESOTHELIOMA PROGRESSION IN A MOUSE XENOGRAFT MODEL BY MICRO-FRAGMENTED FAT (MFAT) Valentina Coccè 1 , Silvia La Monica 2 , Mara Bonelli 2 , Giulio Alessandri 1,3 , Roberta Alfieri 2 , Costanza Annamaria Lagrasta 2 , Caterina Frati 2 , Lisa Flammini 4 , Aldo Giannì 1,5 , Luisa Doneda 1 , Francesco Petrella 1,6,7 , Francesca Paino 1 , Augusto Pessina 1 1 CRC StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Italy 2 Department of Medicine and Surgery, University of Parma, Italy 3 Image Regenerative Clinic, Milan, Italy 4 Food and Drug Department, University of Parma, Italy 5 Maxillo-Facial and Dental Unit, Fondazione Ca’ Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy 6 Department of Thoracic Surgery, IRCCS European Institute of Oncology, Milan, Italy 7 Department of Oncology and Hemato-Oncology, University of Milan, Italy Objective Malignant Pleural Mesothelioma (MPM) is a tumour related to asbestos exposure with no effective therapy and a poor prognosis. Our previous studies demonstrated an in vitro and in vivo inhibitory effect of adipose tissue-derived Mesenchymal Stromal Cells (MSCs) or their derivatives (conditioned media, cellular lysates) on MPM. The purpose of this study was to verify whether fat tissue (FAT), a natural container of MSCs after micro-fragmentation (MFAT) was able to exert a similar inhibitory action on the growth of the human MPM cell line (MSTO-211H) xeno-transplanted in immunodeficient mice. Materials and Methods MFAT was prepared according to standardised methods using Lipogems device. MSCs were obtained by enzymatic digestion of MFAT. The in vitro effect of MFAT on MSTO-211H cell proliferation was analysed using transwell inserts and measuring the absorbance by a crystal violet assay. PBS were used as negative control. For in vivo experiments, Balb/c-Nude female mice were subcutaneously injected with 10 6 MSTO-211H cells suspended in Matrigel/PBS. Mice were randomised into 4 groups: control, paclitaxel (PTX), MSCs and MFAT. After a week from injection (time 0), vehicle alone (control group) or PTX (20 mg/kg) were administered intraperitoneally (IP) and MSCs (5 × 10 5 ) or MFAT (200 µL) were subcutaneously injected close to the tumour. On days 0, 7, and 14, the size of tumour nodules was measured and on day 20, nodules were collected. Morphometric evaluation of xenograft composition was performed on Masson’s trichrome-stained sections. Results The in vitro exposure of MSTO-211H cells to MFAT produced a dose-dependent inhibition of cell proliferation. In the in vivo study, the measures of volume of growing tumour mass indicated that the in situ treatment with MFAT produced an important inhibition similar to those obtained in mice treated with the anticancer drug PTX. A trend of inhibition, but not significant, was also observed in mice treated with free MFAT derived MSCs. The morphometric analysis of the tumour xenograft did not show significant differences among groups. Conclusions Our results show that MFAT, injected in situ, produced a significant ( p < 0.05) inhibition of the MSTO-211H growth both in vitro and in vivo, and was even comparable to IP PTX treatment. Interestingly, the treatment with free MSCs (5 × 10 5 ), at a similar amount contained in around 1 mL of MFAT, exerted only a little anticancer activity. 5.2. Posters Communications P-01. ANTIMICROBIAL ACTIVITY OF CANINE ADIPOSE TISSUE-DERIVED LYOSECRETOME Valentina Andreoli 1 , Costanza Spadini 1 , Mattia Iannarelli 1 , Priscilla Berni 1 , Virna Conti 1 , Roberto Ramoni 1 , Elia Bari 2 , Silvia Dotti 3 , Clotilde Cabassi 1 , Maria Luisa Torre 2 and Stefano Grolli 1 1 Department of Veterinary Science, University of Parma, Italy 2 Department of Pharmaceuticals Sciences, University of Piemonte Orientale, Novara, Italy 3 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy Objective Mesenchymal Stem/Stromal Cells (MSCs) have been studied and applied as therapeutics in regenerative medicine based on their peculiar properties. Although several papers demonstrate the efficacy of MSC-based therapy in preclinical models, clinical applications are still limited due to doubts about the safety of treatment with viable cells. MSCs secretome is a cell-free product that maintains a large part of cells’ therapeutic properties providing soluble and insoluble bioactive molecules involved in cellular crosstalk. Recent data support an antibacterial activity for both MSCs and their secretome. In this work, canine Lyosecretome (c-Lyo), a freeze-dried secretome prepared from adipose tissue-derived MSCs, has been tested to evaluate its antimicrobial activity against some of the most common canine pathogens involved in infections of the gastrointestinal tract, skin, and ears. Pathogens were subjected to a Minimal Inhibitory Concentration (MIC) assay to evaluate the amount of c-Lyo necessary to inhibit bacterial/fungal growth. Materials and Methods c-Lyo was resuspended in 500 µL of sterile saline (0.9%) and tested at a concentration range of 20–0.04 mg/mL. Nine replicates for each assay were performed. Tested reference bacteria and yeasts were E. coli , S. Typhimurium , S. aureus , Methicillin-Resistant S. aureus (MRSA), S. pseudintermedius , P. aeruginosa, and M. pachydermatis . Strains were amplified for 18–24 h at 37 °C to bring them into logarithmic growth phase and added to the plate at 5 × 10 5 CFU/mL and incubated at 37 °C overnight. MIC plate reading was performed with a reverse mirror. Mannitol was used as an internal control. Results A fair inhibitory activity of c-Lyo against both Gram-positive and Gram-negative bacteria was observed. Growth inhibition was higher for Gram-positive (2.2 < MIC < 9.8 mg/mL) but positive results were obtained even on Gram-negative bacteria (10.5 < MIC < 26.1 mg/mL). c-Lyo demonstrated an inhibitory activity also against the yeast M. pachydermatis (MIC = 13.3 mg/mL) supporting a possible efficacy in skin infections. Conclusions MIC data suggest that canine c-Lyo exerts inhibitory activity against various bacterial and yeast pathogens. The availability of an off-the-shelf, ready-to-use MSCs secretome acting as an antibacterial agent could help in replacing/supporting traditional antibiotics therapy, decreasing their use in veterinary medicine, as requested to control the spread of antibiotic resistance. P-02. SECRETOME ISOLATED FROM MESENCHYMAL STROMAL CELLS LOADED WITH PACLITAXEL HAVE CYTOTOXIC EFFECT ON OSTEOSARCOMA CELL LINES Alessia Giovanna Santa Banche Niclot 1 , Elena Marini 1 , Ivana Ferrero 2 , Camilla Francesca Proto 1 , Francesco Barbero 3 , Ivana Fenoglio 3 , Alessandro Barge 4 , Valentina Coccè 5 , Francesca Paino 5 , Katia Mareschi 1,2 and Franca Fagioli 1,2 1 Department of Public Health and Paediatrics, The University of Turin, Italy 2 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 3 Department of Chemistry, University of Turin, Italy 4 Department of Drug Science and Technology, University of Turin, Italy 5 CRC StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Italy Objective Bone sarcomas are rare tumours that constitute a very aggressive disease for children and adolescents and represent still an important challenge for clinicians. Our aim is to develop a new method of drug delivery (DDS) based on the Extracellular Vesicles (EVs) loaded with Paclitaxel (PTX) to use for osteosarcoma (OS) treatment. We performed pre-clinical studies to test the cytotoxic effect of Secretome isolated from Mesenchymal Stromal Cells (MSCs) loaded with PTX (PTX-MSCSecr) on OS cell lines (MG63 and SJSA). Materials and Methods We first calculated the PTX-IC50 in 3 MSC batches, then, we isolated PTX-MSC-Secr by loading MSCs with PTX at the concentration of 15 µg and we analysed its cytotoxic effect on MG63 and SJSA after treatment for 5 days using MTT test. We also analysed the size distribution, particle concentration, and Zeta potential of EVs present in PTX-MSC-Secr by Nanoparticle Tracking Analysis (NTA) instrument. The secretome ability to have EVs with encapsulated PTX was analysed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results PTX-IC50 for MSCs was a mean of 25.1 ± 3.6 µg and for the OS cell lines was, respectively a mean of 17.5 ± 1.0 µg for MG63 and 30 ± 3.1 µg for SJSA. PTX-MSC-Secr induced a decrease of the viability of cells 43 ± 11% and 36 ± 22%, respectively in SJSA and MG63 after five days of treatment. This effect was dose-dependent because scalar dilutions of PTX-MSC-Secr reduced the cytotoxic effect. Cell viability did not decrease after treatment with Secr isolated from CTRL-MSCs (viability of 87 ± 5% in SJSA and of 91 ± 12% in MG63). The dimensional analyses, the result of three independent experiments, indicated EVs mean sizes of 175.8 ± 13 nm (CTRL) and 165.7 ± 11 nm (PTX loaded). EVs Zeta potentials of both CTRL and PTX loaded, as expected, were found to be negative with mean values of −40 ± 2 mV and −38 ± 5 mV. Conclusions We demonstrated the cytotoxic effect of PTX-MSC-Secr in OS cell lines. The NTA analyses showed a typical mean size of EVs and that no significant differences between the secretome batches and between experimental conditions (CTRL and PTX loaded) were observed. The experiments we performed have provided promising preliminary data that need further investigation but the EVs ability to encapsulate PTX allows us to propose the EVs-PTX isolated from the MSCs as ideal candidates for drug delivery as innovative paediatric sarcomas treatment. P-03. 3D BIOPRINTED CONTROLLED RELEASE SCAFFOLD CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME FOR BONE REGENERATION: STERILE MANUFACTURING AND IN VITRO BIOLOGICAL EFFICACY Elia Bari 1 , Franca Scocozza 2,3 , Sara Perteghella 4,5 , Marzio Sorlini 5,6 , Lorena Segale 1 , Lorella Giovannelli 1 , Ferdinando Auricchio 2,3 , Michele Conti 2,3 and Maria Luisa Torre 4,5 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Pavia, Department of Civil Engineering and Architecture, Pavia, Italy 3 P4P S.r.l., Pavia, Italy 4 University of Pavia, Department of Drug Sciences, Pavia, Italy 5 PharmaExceed S.r.l., Pavia, Italy 6 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland Objective The present study proposes the design and sterile manufacturing of 3D-printed polycaprolactone (PCL) scaffolds enriched with mesenchymal stem cell (MSC)-secretome for bone tissue regeneration and evaluates their in vitro biological efficacy. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Porous parallelepiped-shaped PCL scaffolds (10 × 10 × 3 mm) were prepared by co-printing PCL with an alginate hydrogel (10% w / v ) containing lyosecretome (0.25 mg). Scaffolds were tested for sterility and microbiological tests, as indicated in the EuPh 2.6.27 and 2.6.1 chapters. In addition, the scaffold colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy, and dosage of osteocalcin by ELISA). Scaffolds without lyosecretome were used as a control. Results Sterile scaffolds have been obtained and lyosecretome enhanced their colonisation by MSCs: MSCs showed a spread morphology with the initial formation of filopodia, with more frequent and complex cellular processes, overall indicating the cytocompatibility of the scaffold. Lyosecretome also sustained MSC differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralised matrix detected by alizarin red was significantly higher for lyosecretome scaffolds. Likely, the amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with lyosecretome scaffolds after 14 and 28 days. Conclusions Overall, these results prove the role of MSC-secretome, co-printed in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC-secretome are now available for in vivo preclinical tests of bone regeneration. P-04. OSTEOINDUCTIVE AND OSTEOCONDUCTIVE PROPERTIES OF TITANIUM CAGES CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME Elia Bari 1 , Sara Perteghella 2,3 , Marzio Sorlini 3,4 , Delia Mandracchia 5 , Lorella Giovannelli 1 , Lorena Segale 1 and Maria Luisa Torre 2,3 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Pavia, Department of Drug Sciences, Pavia, Italy 3 PharmaExceed S.r.l., Pavia, Italy 4 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland 5 University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy Objective The present study investigates the capacity of the mesenchymal stem cell (MSC)-secretome, formulated as a ready-to-use and freeze-dried medicinal product (the lyosecretome), to promote the osteoinductive and osteoconductive properties of titanium cages. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Lyosecretome was added to titanium cages (1 × 1 × 0.3 cm in size) kindly provided by MT Ortho and manufactured by an additive manufacturing technology called electron beam melting. The cages colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy and dosage of osteocalcin by ELISA). Cages without lyosecretome were used as a control. Results After 14 days, in the presence of lyosecretome, significant cell proliferation improvement was observed. Scanning electron microscopy revealed the cytocompatibility of titanium cages: the MSCs seeded showed a spread morphology and the initial formation of filopodia. After seven days, in the presence of lyosecretome, more frequent and complex cellular processes forming bridges across the porous surface of the scaffold were revealed. Moreover, after 14 and 28 days of a culture in an osteogenic medium, the amount of mineralised matrix detected by alizarin red was significantly higher when lyosecretome was used. Finally, improved osteogenesis with lyosecretome was confirmed by confocal analysis after 28 and 56 days of treatment and demonstrating the production by osteoblast-differentiated MSCs of osteocalcin, a specific bone matrix protein. Conclusions Overall, these results confirm the role of MSC-secretome in combination with titanium cages in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing. P-05. OSTEOINDUCTIVE AND OSTEOCONDUCTIVE PROPERTIES OF BIOHYBRID BOVINE MATRIX CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME Elia Bari 1 , Ilaria Roato 2 , Giuseppe Perale 3,4,5 , Filippo Rossi 6 , Tullio Genova 7 , Federico Mussano 2 , Riccardo Ferracini 8 , Marzio Sorlini 9,10 , Sara Perteghella 1,10 and Maria Luisa Torre 1,10 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Torino, Department of Surgical Sciences, CIR-Dental School, Torino, Italy 3 Industrie Biomediche Insubri SA, Mezzovico-Vira, Switzerland 4 University of Southern Switzerland (USI), Faculty of Biomedical Sciences, Lugano, Italy 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria 6 Politecnico di Milano, Department of Chemistry, Materials and Chemical Engineering Milan, Italy 7 University of Torino, Department of Life Sciences and Systems Biology, Turin, Italy 8 University of Genova, Department of Surgical Sciences and Integrated Diagnostics, Genoa, Italy 9 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland 10 PharmaExceed Srl, Pavia, Italy Objective The present study combines biohybrid bone substitute scaffolds (SB) with lyosecretome, a freeze-dried MSC-secretome formulation containing proteins and extracellular vesicles and evaluates the osteoinductive and osteoconductive in vitro. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining Lyosecretome (0.1 × 10 6 cell equivalents/mg). Each SB scaffold (1 × 1 × 0.3 cm in size) was loaded with 16 × 10 3 cell equivalents of Lyosecretome by an absorption method, obtaining SBlyo. 1 × 10 6 MSCs were seeded onto the upper surface of SB in an osteogenic medium, and after 14 and 60 days of cultures, gene expression for osteocalcin (OCN), alkaline phosphatase (ALP), and collagen 1 (COLL-1) was evaluated. After 60 days, a high-resolution X-ray microtomography was used to identify the newly formed mineralised tissue after cell colonisation on SB and SBlyo. Moreover, SB and SBlyo were fixed, decalcified, dehydrated, cut into thin sections, and stained with hematoxylin and eosin (H&E) for morphological analyses. Immunohistochemical analysis was also performed using antibodies for osteocalcin and TGF-β. Results After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. For SB, on the other hand, the process was still present, but tissue formation was less organised at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralise after 60 days. SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralised tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β. Conclusions Overall, these results confirm the role of MSC-secretome loaded on biohybrid bovine matrix in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing. P-06. LOCAL AND SYSTEMIC APPLICATION OF AUTOLOGOUS MESENCHYMAL STROMAL CELLS IN CATS SUFFERING FROM CHRONIC GINGIVOSTOMATITIS: A PILOT STUDY Priscilla Berni 1 , Tommaso Magni 2 , Maurizio Del Bue 3 , Virna Conti 1 , Valentina Andreoli 1 , Rosanna Di Lecce 1 , Anna Maria Cantoni 1 , Roberto Ramoni 1 , Stefano Grolli 1 1 Department of Veterinary Medical Science, University of Parma, Italy 2 Veterinary Practitioner, Clinica Veterinaria Pet Care, Bologna, Italy 3 Freelance Veterinary Medical Doctor, Parma, Italy Objective Feline Chronic Gingivostomatitis (FCGS) is a severe inflammatory oral disease characterised by painful mucosal lesions, oral discomfort, inappetence, reduced grooming, weight loss, and hypersalivation, seriously affecting the patient’s quality of life. The current standard of care is invasive full/near-full mouth tooth extraction and long-term pharmacological treatments, with a high rate of relapse. Since FCGS is probably immune-mediated, Mesenchymal Stromal Cells (MSCs) represent a promising tool for this disorder. Different studies have reported the efficacy of systemic administration of adipose-derived MSCs (Ad-MSCs) in cats with FCGS, while a pilot study reported a lack of efficacy when the treatment is performed prior to full-mouth tooth extraction. This study aims to determine the efficacy of local and systemic administration of Ad-MSCs in cats with FCGS, with or without teeth. Materials and Methods Eleven client-owned cats with FCGS and with long-term pharmacological clinical history, with or without teeth, were treated with a double application of autologous Ad-MSCs at 30-day intervals. The cats were enrolled in two groups: one was treated with local injections of 5 × 10 6 autologous Ad-MSCs and the other was treated with local injections associated with systemic infusions of 2 × 10 6 /Kg autologous Ad-MSCs. An oral examination with photographs and oral biopsies was performed at the enrolment and 30 days after each treatment. A SDAI (Stomatitis index) scoring was calculated at the same intervals, in addition to a brief owner questionnaire and a veterinarian scoring. Furthermore, a complete blood count, blood immune cell phenotyping, and biochemical profile were planned on day zero and three months after the first treatment. Results At the time of writing, eight cats have been treated with double MSCs application. Seven cats have completely suspended any pharmacological treatment after the first application. The clinical assessment at day 60 showed a marked clinical improvement reported by the owners, except for one patient that showed the maximum SDAI score at the enrolling who improved only in the body weight parameter. A statistically significant difference was observed in the SDAI between day 0 and 60 for seven cats, two with a complete resolution of the oral inflammation ( p < 0.05). Conclusions Immunohistochemical analysis and blood immune cell phenotyping are needed to confirm the observed clinical improvement. P-07. A NEW PROTOCOL FOR VALIDATION OF CHONDRO, ADIPO AND OSTEO DIFFERENTIATION KIT OF CULTURED ADIPOSE-DERIVED STEM CELLS (ADSC) BY REAL-TIME RT-QPCR Carlotta Castagnoli 1 , Valentina Daprà 2,3 , Daniela Alotto 1 , Stefania Casarin 1 , Stefano Gambarino 2,3 , Mara Fumagalli 1 , Sara Castiglia 4 , Deborah Rustichelli 4 , Maddalena Dini 3 , Ilaria Galliano 2 , Massimiliano Bergallo 2,3 1 Skin Bank, Department of General and Specialized Surgery, University Hospital City of Health and Science of Turin, Italy 2 Department of Public Health and Pediatric Sciences, University of Turin, Italy 3 BioMole srl, Spin-off University of Turin, Italy 4 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy Objective Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose-derived Stem Cells) show great potential for degenerative disease treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validate a new commercially available kit able to explore changes in gene expression in human ADSC subjected to osteogenic, adipogenic, and chondrogenic differentiation. Materials and Methods As the primary outcome of the study, we selected better indicators of trilineage differentiation using the third passage of cultured ADSC isolated from the stromal vascular fraction (SVF) by enzymatic digestion. ACAN, FABP4A, and Col11a1 were selected as indicators of chondrogenic, adipogenic, and osteogenic differentiation, respectively based on statistically significant results. As a secondary outcome of the study, an in vitro ageing test was performed from passage 2 to 6 to evaluate the highest differentiation potential. Total RNA extraction from differentiated and control ADSC were performed. Relative quantifications of mRNA expression of selected genes was completed according to rt-PCR kit protocol. Results ACAN detection, a test for chondrogenic differentiation, revealed equivalent ∆∆Ct values between P3 and P6. These were considered equivalent passages for induction differentiation tests. FABP4 detection, an assay for adipogenic differentiation, showed similar results for all cell passages tested so they can all be considered suitable in differentiation assay induction; on the contrary, only passage P6 for Col11a1 was suitable for osteogenic differentiation. Conclusions In conclusion, we validated a new real-time rt-QPCR protocol able to evaluate osteogenic, chondrogenic, and adipogenic ADSC differentiation in vitro. P-08. EVALUATION OF THE EFFECTS OF HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS AND AMNIOTIC MEMBRANE CONDITIONED MEDIUM ON OVARIAN CANCER CELLS USING 2D AND 3D CELL MODELS Paola Chiodelli 1 , Patrizia Bonassi Signoroni 1 , Silvia De Munari 1 , Andrea Papait 2 , Sara Ficai 2 , Antonietta Silini 1 and Ornella Parolini 2,3 1 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy 2 Department of Life science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 3 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy Objective Ovarian cancer is the seventh most common cancer and sixth most common cause of cancer death for women globally. Nowadays, surgical resection followed by chemotherapy is the standard of care. However, a number of patients are faced with recurrence due to tumour dissemination and acquired chemoresistance. Therefore, the novel alternative approaches targeting ovarian cancer cells are urgently needed to improve the standard of care for patients. With this regard, mesenchymal stromal cells (MSC) constitute a compelling therapeutic option. Of particular interest, MSC isolated from the amniotic membrane of the human term placenta (hAMSC) are of therapeutic interest and present noteworthy advantages when compared to MSC from other sources, such as their ease of recovery from biological waste. In addition, we previously reported that the hAMSC secretome has antiproliferative effects in vitro when co-cultured with different tumour cells. Material and Methods We decided to evaluate the possible anti-proliferative effects of the secretome (conditioned medium, CM) from hAMSC in 2D and 3D models of ovarian cancer. In parallel, we evaluated the CM from the intact amniotic membrane (hAM) to see if antiproliferative effects could be maintained without the need to perform MSC isolation. Results Both CM-hAMSC and CM-hAM inhibit the proliferation and migration of ovarian cancer cells (HEY and SKOV-3) in 2D scratch assays. Moreover, both CM-hAMSC and CM-hAM affect the apoptotic process in HEY and SKOV-3. In the SKOV-3 spheroid 3D model, CM-hAMSC and CM-hAM significantly decrease the spheroid area inducing also a change in spheroid morphology. Conclusions The data so far collected indicated that CM-hAMSC and CM-hAM impact the growth and activity of ovarian cancer cells. Further experiments are needed to better understand their inhibitory capacity on ovarian cancer cells in 2D and in 3D models and clarify their use as a potential adjuvant therapeutic strategy able to target tumour cells. P-09. EXTRACELLULAR VESICLES FROM CORD BLOOD MESENCHYMAL STROMAL CELLS STIMULATE REGENERATION PROCESS IN ORGANOTYPIC MODEL OF NEURONAL INJURY Stefania D’Agostino 1,2 , Francesca Cecchinato 2,4 , Beatrice Auletta 2,4 , Marcin Jurga 3 , Maurizio Muraca 1,2 , Anna Urciuolo 2,4 , Michela Pozzobon 1,2 1 Department of Women’s and Children’s Health, University of Padova, Italy 2 Institute of Pediatric Research (IRP) Città della Speranza, Padova, Italy 3 EXO Biologics (SA), Belgium 4 Department of Molecular Medicine, University of Padova, Italy Objective The central nervous system (CNS) has only a limited capacity to regenerate, hence, after injury, a progressive loss of neurons, due to homeostatic imbalance, leads to neurodegenerative pathology. The homeostasis process critically depends on the interaction between neurons and glial cells. Novel treatment suggestions for neurodegenerative disorders consider the use of cell-derived products, relying on the beneficial paracrine effects of the applied products. Extracellular vesicles (EVs) recently emerged as versatile messengers in CNS cell communication. These nanoparticles, defined by a phospholipid bilayer, can convey signals by triggering surface receptors, activating second messenger signalling cascades or delivering their cargo, such as proteins, nucleic acids, and small molecules. In this work, we considered an organotypic in vitro model of spinal injury treated with EVs derived by Wharton Jelly Mesenchymal stromal cells. Materials and Methods Isolated spinal cords from rat foetuses were cut into small pieces and cultured on a bed of Matrigel. Organotypic spinal cord (oSpC) were treated with EVs soon after the beginning and after 24 h of culture. To monitor the early effect of the EVs on neural axon sprouting, samples were analysed 48 h after the seeding. Results Our results showed that neural axon sprouting was significantly increased in EV-treated samples when compared to untreated oSpCs. Moreover, the neural protein TujI/TUBB3 expressed in the developing neurons and the glia marker GFAP, that identified new astrocytes, were differently detected in the presence of EVs. Fluo-4 imaging revealed a more controlled calcium flux in oSpC treated with EVs at the axonal projection compared to the untreated SpC. Real-time PCR on neural miRNA highlighted how the EVs can be responsible for miRNA mediator of neural regeneration. Conclusions Our results suggest a possible role of perinatal EVs in promoting neural axon sprouting, opening new perspectives for their application in neural regeneration, and as new exciting signalling modalities that add a new dimension to the interaction between neurons and glial cells. P-10. 3D ORGANOIDS: A NEW TRANSLATIONAL APPROACH FOR THE ASSESSMENT OF THE IMMUNOMODULATORY ACTIVITY OF MESENCHYMAL STROMAL CELLS DERIVED EXTRACELLULAR VESICLES IN INFLAMMATORY BOWEL DISEASE Giada de lazzari 1,2,3,5,6 , David Sagnat 5,6 , Ricardo Malvicini 1,2,3 , Michela Pozzobon 1,2 , Gustavo Yannarelli 4 , Anna Maria Tolomeo 1,2,3 , Nathalie Vergnolle 5,6,7 , Maurizio Muraca 1,2,3 1 Foundation Institute of Pediatric Research Città della Speranza, Padova, Italy 2 Dept of women’s and children’s health, University of Padova, Italy 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (coris), Veneto Region, Padova, Italy 4 Instituto de Medicina Traslacional, Trasplante y Bioingeniería (imettyb-conicet), Buenos Aires, Argentina 5 Toulouse Organoid Platform, France 6 Inserm, Toulouse, France 7 University of Calgary, Faculty of Medicine, Department of Physiology and Pharmacology, Calgary, AB, Canada Objective Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and Ulcerative Colitis (UC), are chronic relapsing–remitting disorders characterised by inflammation of the gut. Different factors contribute to IBD development, such as deficiencies in epithelial integrity, immune response mechanisms, and mucosal barrier function, whose complexity is difficult to reproduce in experimental conditions. The need to overcome the typical limits of cell lines, animal models, and organ culture, led to the development of a 3D culture system capable of maintaining the characteristics of the target organ in the long term. We thus explored the feasibility of this tool to evaluate the therapeutic potential of extracellular vesicles derived from mesenchymal stromal cells (MSC-EVs) whose immunomodulatory activity is the object of increasing interest. Materials and Methods MSC-EVs were isolated from human umbilical cord MSCs by ultrafiltration (100 kD cutoff), quantified by TRPS and characterised for established markers by MACSPlex. Colon organoids were derived from human colon samples’ extracted crypts and seeded in Matrigel beads. Inflamed (IBD) organoids, in particular, were obtained from the stimulation of control organoids with a pro-inflammatory cocktail (IL-1β, TNF-α, and IL-6, at 100 ng/mL) on days 3, 5, and 7 to reproduce a chronic inflammatory state. To evaluate the effect of MSC-EVs, IBD organoids were then treated on day 7 with a dose of 1 × 10 9 MSC-EVs/mL at different time points (3 h, 6 h, 24 h, and 48 h). As readouts, we evaluated the effect on proliferation, differentiation, inflammation, barrier, and growth by immunofluorescence and molecular biology. Results We found that the stimulation with the pro-inflammatory cocktail increases inflammation (IL8, MCP1, and TNF- α), stress (Chop), carcinogenesis (Wnt5a), growth (OCT4, SOX9), proliferation (EGF), and decreases differentiation (MUC2) and epithelial barrier marker (Epcam) compared to control organoids. Treatment with MSC-EVs at 24 h decreased inflammation, stress, growth, proliferation, carcinogenesis, and increased differentiation and epithelial barrier marker. Conclusions In conclusion, we have provided preliminary evidence of the therapeutic effect of MSC-EVs in inflamed intestinal organoids. These results suggest that the present 3D model could represent a useful experimental tool closely reproducing some critical features of human IBD. P-11. CHARACTERIZATION OF HUMAN DENTAL PULP STEM CELLS MULTICELLULAR SPHEROIDS AS ORGANOTYPIC THREE-DIMENSIONAL IN VITRO MODEL Ilaria De Santis 1,2 , Alessandro Bevilacqua 3,4 , Thimios A. Mitsiadis 2 , Deborah Stanco 2 1 University of Bologna, Interdepartmental Centre Alma Mater Research Institute on Global Challenges and Climate Change (Alma Climate), Bologna, Italy 2 University of Zürich, Orofacial Development and Regeneration, Institute of Oral Biology, Zürich, Switzerland 3 University of Bologna, Advanced Research Center on Electronic Systems (ARCES) for Information and Communication Technologies “E. De Castro”, Bologna, Italy 4 University of Bologna, Department of Computer Science and Engineering (DISI), Bologna, Italy Objective Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) of neural crest origin. High availability, multipotency, and plasticity make them a promising source of patient-specific MSCs for personalised regenerative medicine strategies. However, their clinical translation still faces many challenges due to a lack of deep understanding of their niche microenvironment, biology, and functionality in vivo. By recapitulating the complex in vivo-like microenvironment, three-dimensional (3D) multicellular spheroids open to new opportunities for MSCs translation in clinical and preclinical settings. In this context, the development of human DPSC multicellular spheroids as organotypic 3D in vitro models was assessed. Material and Methods 2 × 10 4 DPSCs at passage IV were used for spheroid creation by hanging drop technique and transferred to 96 ULA plates after 24 h. Spheroid morphology, viability (FDA-PI staining), and metabolic activity (Alamar Blue assay) were evaluated at day 1, 2, 3, 4, and 7 of culture. Tissue-specific (nestin, vimentin, collagen I and IV, laminin, and fibronectin) and stem-related (BMI1, CD90, SOX2, NANOG, and OCT4) markers were evaluated at gene (qRT-PCR) and protein level (IF). After bright field imaging and by on-purpose method for automated image analysis, spheroid dimension, morphology, and compactness were quantified by their equivalent diameter (ED), sphericity (S), and border indentation (BI), respectively. After normality check by Shapiro–Wilk test, statistical significance was assessed by t -test or Wilcoxon test ( p < 0.05). Results The hanging drop technique has a mean efficiency of spherical (S ≥ 80%) spheroid creation at 82% in 24 h. ED progressively shrinks, while S rises up to day four. At day three, spheroids show highest viability (I FDA /I PI = 12.8, p < 10 −6 ) and the most stable morphology (ED = 488 ± 43 µm, S = 0.87 ± 0.04, BI = 0.79 ± 0.03). Spheroid metabolic activity was stable in time and significantly lower than standard 2D culture (−35% avg, p < 0.03). DPSC spheroids expressed high levels of nestin, vimentin, collagen I and IV, laminin, and fibronectin, beside significantly higher mRNA levels of BMI1, CD90, SOX2, NANOG, and OCT4. Conclusions The human DPSC spheroids can be easily and quickly created by a low-cost procedure in 24 h and they may efficiently enhance the therapeutic action of DPSCs. Moreover, automated image analysis here proves as a valuable tool for the finest analysis of DPSC spheroid morphology in future preclinical setting applications. P-12. CELL-FREE ORTHOBIOLOGICS FROM ADIPOSE MESENCHYMAL STEM/STROMAL CELLS: THE ROAD SO FAR AND FUTURE PERSPECTIVES IN OSTEOARTHRITIS TREATMENT Elena Della Morte 1 , Chiara Giannasi 1,2 , Stefania Niada 1 and Anna Teresa Brini 1,2 1 IRCCS Istituto Ortopedico Galeazzi, Milan, Italy 2 University of Milan, Department of Biomedical Surgical and Dental Sciences, Milan, Italy Objective Mesenchymal Stem/stromal Cells, and, in particular, adipose-derived ones (ASCs), show great therapeutic potential in counteracting orthopaedic conditions. Since a large part of ASC action is exerted through paracrine signalling, in the last years, we focused on the study of their conditioned medium (CM) in terms of molecular composition and biological action on experimental models of osteoarthritis (OA). Materials and Methods The CM was obtained from confluent ASCs cultured for 72 h in the absence of FBS, then concentrated through filter devices with a 3 kDa cut off. Its components were investigated through Raman Spectroscopy, NTA, -omic approaches, and ELISA. Articular chondrocytes (CHs) and osteochondral explants (OEs) were harvested from patients undergoing arthroplasty at IRCCS Istituto Ortopedico Galeazzi prior to approval of the ethics committee. OA phenotype was induced by stimulation with 10 ng/mL TNFα, and specimens were treated with the CM derived from 0.5–1 × 10 6 ASCs. The levels of inflammatory, hypertrophic, and catabolic factors were monitored through time by immunological or enzymatic assays. Results ASC-CM peculiar Raman fingerprint and its characteristic vesicular profile were depicted. The analysis of ASC-CM composition showed the presence of stable levels of bioactive factors that can be putative players in counteracting the OA process. Among other potential effectors, the abundance of the chondroprotective factors Dkk-1 and TIMP-1/2 was particularly intriguing. Accordingly, in both experimental OA models, i.e., articular chondrocytes (CHs) and osteochondral explants (OEs), ASC-CM efficiently buffered the TNFα-induced aberrant activity of matrix metalloproteinases. Furthermore, ASC-CM reverted TNFα-induced expression of PGE2 and COL10A1 in CHs, while it lowered the catabolic release of glycosaminoglycans on OEs. Conclusions These in vitro and ex vivo experiments confirm ASC-CM beneficial potential in dampening OA-related hallmarks, encouraging deeper investigations of this product in the perspective of its future clinical translation as a cell-free orthobiologic. P-13. LATTICE-BASED SCAFFOLDS FOR THE BULK REINFORCEMENT OF SOFT MATERIALS FOR CARTILAGE REGENERATION Stephanie E. Doyle 1,2 , Finn Snow 1 , Rhyys Turner 1 , Carmine Onofrillo 2,3,4 , Cathal D. O’Connell 1,2 , Claudia Di Bella 2,3,5 , Elena Pirogova 1 , Serena Duchi 2,3,4 1 Electrical and Biomedical Engineering, School of Engineering, RMIT University, Melbourne, Victoria, Australia 2 ACMD, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia 3 Department of Surgery, The University of Melbourne, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia 4 ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Wollongong, NSW, Australia 5 Department of Orthopaedics, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia Objective Hydrogels are a fundamental element of cartilage engineering by providing a suitable environment for cells to proliferate, migrate, and differentiate. However, this typically soft environment is often not suitable under high mechanical loads. This can present an issue for implantable scaffolds where a large stiffness mismatch between the implant and native tissue can contribute to its failure. With this work we prioritise the bulk mechanical properties of the implant to reduce the stiffness mismatch with the native tissue. We aim to do this without negatively impacting the environment, previously established by our team, for mesenchymal stem/stromal cells (MSCs) to create new cartilaginous tissue. Materials and Methods Lattice scaffolds, designed to minimise the total volume fraction of the reinforcement scaffold, are made via the Negative Embodied Sacrificial Template (NEST) 3D printing process from polycaprolactone. The NEST scaffolds are combined with MSCs of adipose origin and embedded in a soft, 6% gelatin methacryloyl (GelMA) hydrogel and UV photo crosslinked. After six weeks of culture, samples are analysed by a combination of imaging (immunohistochemistry and histology staining), metabolic activity, glycosaminoglycan (GAG) content, compression testing, and quantitative polymerase chain reaction (qPCR). Results Using highly porous (up to 90%) lattice designs we have demonstrated the biocompatibility of the NEST scaffolds where we recorded no significant difference in metabolic activity between reinforced and control samples after six weeks in in vitro culture. Secondly, limited to no interference of the NEST scaffold on bulk cell differentiation as measured by GAG production, qPCR, and imaging. Third, the ability to match the native tissue stiffness from day 0, for example, native articular cartilage of the knee has a compressive modulus of ≈400–800 kPa and our reinforced NEST scaffold of a clinically relevant size embedded in 6% GelMA has a compressive modulus of ≈480 kPa. Conclusions Our reinforced hydrogels retain the ideal soft environment for cells to differentiate down the chondrogenic lineage while reducing the stiffness mismatch between the implant and native tissue. Therefore, we address one area, stiffness mismatch, where implants can fail when moving from an in vitro model to in vivo. P-14. MESENCHYMAL STEM CELLS AS A SYSTEM FOR DELIVERING NAB-PACLITAXEL IN A METASTATIC MODEL OF PDAC Benedetta Ferrara, Smeralda Rapisarda, Antonio Citro, Martina Policardi, Fabio Manenti, Chiara Gnasso, Annapaola Andolfo, Denise Drago, Lorenzo Piemonti Diabetes Research Institute, IRCCS San Raffaele, Milan, Italy Objective Current therapy for metastatic pancreatic ductal adenocarcinoma (PDAC) is limited by drug toxicity and resistance. A delivery system might improve drug bioavailability. The aim of this study was to investigate mesenchymal stem cells (MSCs) for delivering nab-paclitaxel (nPTX) in metastatic PDAC. The feasible manipulation of MSCs, their ability to deliver drugs and to do homing in the damaged site make them a suitable candidate for an autologous cell-therapy approach. Materials and Methods An in vivo model of liver metastases was generated by injection of K8484 murine PDAC cells derived from KPC mice into the portal vein of C57BL/6N mice. Firstly, tumour-bearing mice were treated with nPTX to assess their sensitivity to this drug by monitoring the metastatic volume by magnetic resonance. Secondly, the MSCs viability after treatment with nPTX, as well as drug uptake and release, were investigated in vitro. The biodistribution of luciferase (LUC)-transduced MSCs intraportally injected in tumour-bearing mice was investigated by in vivo imaging systems (IVIS). Finally, the effect on the metastatic reduction of nPTX-loaded MSCs was evaluated on tumour-bearing mice after intraportal injection. Results NPTX significantly reduced the metastatic volume of tumour-bearing mice, demonstrating the responsiveness of our model to this drug. In vitro, after 24 h, we could appreciate a high uptake of nPTX by MSCs without a reduction of cell viability. NPTX release by MSCs was higher after 24 h but sustained until 72 h, as reported by mass-spectrometry analysis. Biodistribution studies revealed a high and prolonged accumulation of MSCs in the liver after intraportal injection. Results obtained in vivo on the antitumour efficacy of this system reported a significantly higher metastatic reduction in mice treated with nPTXloaded MSCs with respect to control mice treated with not loaded MSCs or free nPTX. Conclusions The ability of MSCs to incorporate nPTX to a high extent and without reporting toxicity is promising. This system allowed to lower the drug dose, thus reducing nPTX toxicity and specifically targeting the tumour site reporting an effective reduction of the metastatic burden. Obtained results open new insights into the use of MSCs for delivering nPTX as a suitable and promising therapeutic option for PDAC. P-15. DISSECTING THE EFFECT OF MICROPLASTICS ON HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS Sara Ficai 1 , Andrea Papait 1,2 , Alice Masserdotti 1 , Patrizia Bonassi 3 , Antonietta Silini 3 , Ornella Parolini 1,2 1 Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy 3 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy Objective Environmental microplastic (MPs, 1 µm–5 mm) degradation has become a problem for human health due to the possible production of toxic metabolites, contaminating water, air, food, and several daily used products. Bisphenol A (BPA) is the most representative chemical component of MPs debris, and it is reported to alter cellular functions by acting as an endocrine disruptor. Endocrine disrupting chemicals (EDC) are able to interfere with endogenous hormone biosynthesis, metabolism, and functions through the binding with typical and peculiar oestrogen receptors, triggering a dysregulation in cellular physiological processes such as oxidative stress. Placental tissues are supposed to be susceptible to EDC for the abundance of hormone receptor expression. Thus, it has been hypothesised that exposure of the mother to MPs-derived chemicals, such as BPA, can lead to an imbalance of the physiological processes that contribute to a successful pregnancy, increasing the risk of gestation-related complications. Based on this evidence, our purpose is to evaluate the effects of BPA on mesenchymal stromal cells isolated from the amniotic membrane of the human term placenta (hAMSC). Materials and Methods Our preliminary experiments aimed to evaluate the impact of BPA on hAMSC properties and functions in vitro. Cellular viability, metabolism, and apoptotic rate were analysed after a 24 h exposure to increasing concentrations of BPA (0.1; 0.2; 0.3; 0.4 µM) by MTT assay, ATP lite assay, and PI/Annexin kit, respectively. Concomitantly, the loss of cellular oxidative balance has been assessed by flow cytometry 3 h and 24 h after BPA exposure. In addition, dysregulation in the expression of cell cycle mediators was evaluated by RT-PCR. Results We first observed a dose-dependent reduction in hAMSC viability and metabolic capacity as well as an enhancement in cellular apoptotic rate after the treatment with increasing concentrations of BPA. At the highest concentration of BPA used in our study, hAMSC-intracellular oxidative stress and the gene expression of typical cell cycle regulators both increased. Conclusions Our preliminary data suggest that BPA may affect hAMSC functions. In light of these observations, additional studies will be performed to evaluate intracellular pathways through which BPA can act, supposing that adverse consequences on placenta resident MSC may be related to a negative outcome of gestation and riskiness for the baby. P-16. HINTS ON THE METABOLIC STATUS OF SERUM-STARVED MESENCHYMAL STEM/STROMAL CELLS Chiara Giannasi 1,2 , Stefania Niada 2 , Elena Della Morte 2 and Anna Teresa Brini 1,2 1 University of Milan, Department of Biomedical Surgical and Dental Sciences, Milan, Italy 2 IRCCS Istituto Ortopedico Galeazzi, Milan, Italy Objective In the last decade, the scientific interest in the secretome of Mesenchymal Stem/stromal Cells (MSCs) has increased tremendously due to its promising potential as an alternative to cell therapy. Mid-term serum starvation represents a convenient strategy for secretome production since it stimulates cell secretion while reducing the drawbacks associated with the use of animal-derived supplements. Nevertheless, the impact of this procedure on the metabolic status of donor cells still needs to be defined. Here, we investigate this aspect through metabolomics by comparing MSCs cultured with 10% foetal bovine serum (FBS) and serum-starved ones. Materials and Methods Primary human adipose-derived MSCs were grown in complete culture medium until confluence. Then, cells were either collected and analysed or rinsed and cultured for three days in the absence of FBS. Samples were screened for polar and apolar molecules by untargeted metabolomics at the Proteomics and Metabolomics Facility of IRCCS Ospedale San Raffaele. The differences revealed by this analysis were further validated by ad hoc biochemical and functional assays. Results Differential metabolomics shows a clear clustering between samples grown in standard conditions and under serum deprivation. Metabolite set enrichment analysis reveals several processes affected by serum withdrawal, most of them occurring at the mitochondrial level such as Mitochondrial Electron Transport Chain, Oxidation of Branched Chain Fatty Acids, and Citric Acid Cycle. The impairment of mitochondrial metabolism is further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. At last, cells exposed to serum starvation show higher expression levels of mitochondrial superoxide dismutase. Conclusions Mid-term serum deprivation affects cell metabolomes by impairing mitochondrial activity and inducing oxidative stress. We hypothesise that the metabolic stress occurring during serum starvation may trigger the release by donor cells of multiple bioactive factors mediating the pro-angiogenic, trophic, and antioxidant effects of their secretome. P-17. MESENCHYMAL STEM CELL CONDITIONED MEDIUM PROMOTES VASCULARIZATION OF BIO-COMPATIBLE SCAFFOLDS TRANSPLANTED INTO NUDE MICE Ludovica Barone, Federica Rossi, Marina Borgese, Luca Buonarrivo, Mario Raspanti, Piero Antonio Zecca, Giovanni Bernardini, Rosalba Gornati Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy Objective Human adult mesenchymal stem cells (MSCs) have been largely studied over the past decades, for regenerative medicine applications, due to their multilineage differentiation and their potential use in several cell-based therapies. However, in the last few years, the increasing evidence showing the potential of MSC secretome has led to the acknowledgement that the use of MSC conditioned medium may represent a valid alternative to the use of stem cells, overcoming the main obstacles related to cell samples handling, survival, and rejection. Materials and Methods Accordingly, this study focuses on the characterization and in vivo application of MSC conditioned medium (CM). To this aim, MSCs have been isolated from two different sources, adipose tissue (ASCs) and dental pulp (DPSCs). Although ASCs have been largely studied, very little is known about DPSCs, therefore, DPSCs have been characterised by FACS, qPCR, and immunofluorescence up to their 30th passage to confirm their stemness maintenance over long culture. Afterward, to compare the pro-angiogenic potential of the ASC-CM and DPSC-CM vs. the cells, conditioned media, obtained after 48 h of starvation, have been mixed with a collagen scaffold, INTEGRA ® Flowable Wound Matrix (FWM), grafted in BALB-C nude athymic mice for 28 days and then removed, observed, and processed for gene expression and microscopy analysis. Furthermore, ASC and DPSC CMs, obtained after 72 h of starvation in both normoxic and hypoxic conditions, have been characterised by ELISA to evaluate the effect of oxygen concentration on the release of pro-angiogenic factors; then, their pro-angiogenic potential has been evaluated in vivo using the Ultimatrix sponge assay. Results and Conclusions Even though an exhaustive characterisation of the CM, which also includes the microvesicle fraction, is still in progress, the data obtained demonstrated that the scaffolds associated with CM showed the same efficiency of the ones associated with cells in promoting cellular invasion and capillary growth. Furthermore, the CMs produced under hypoxic conditions seem to promote more efficiently the angiogenesis in vivo. P-18. MELANOMA EXOSOMES INDUCE PD-1 OVEREXPRESSION AND TUMOUR PROGRESSION VIA MESENCHYMAL STEM CELL ONCOGENIC REPROGRAMMING Edina Gyukity-Sebestyén 1 , Mária Harmati 1 , Gabriella Dobra 1,2 , István B Németh 3 , Ágnes Zvara 1 , Éva Hunyadi-Gulyás 1 , Péter Horváth 1,3 , Tibor Pankotai 4 , Barbara Borsos 4 , Miklós Erdélyi 5 , Edit I Buzás 6 , Lajos Kemény 3 , Krisztina Buzás 1,7 1 Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary 2 Doctoral School of Interdisciplinary Medicine, University of Szeged, Hungary 3 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland 4 Institute of Pathology, University of Szeged, Hungary 5 Faculty of Science and Informatics, University of Szeged, Hungary 6 Faculty of Medicine, Semmelweis University, Budapest, Hungary 7 Department of Immunology, University of Szeged, Hungary Objective Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive. However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations. Methods Murine primary mesenchymal stem cells (MSCs) from adipose tissue were pretreated with B16F1 melanoma cell-derived exosomes (mcde). Exosomes were stained by lipophilic dyes, and their uptake into recipient cells was visualised. The rate of apoptosis and expression of multipotent stromal cell markers were analysed by flow cytofluorometry. Tumour-bearing mice were injected with mcde-conditioned MSCs i.v.; the control mice received untreated MSCs. The whole miRNA spectra and the proteome of mcde were analysed by SOLiD5500xl technology and LC-MS/MS, respectively. Results Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSC PD-1+ ) from naïve MSCs. Exosomes and mMSC PD-1+ cells induce tumour progression and expression of oncogenic factors in vivo. Finally, we revealed a characteristic, tumorigenic signalling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, and MTOR) and their upstream exosomal regulating proteins and miRNAs. Conclusions Our study highlights the complexity of exosomal communication during tumour progression and contributes to the detailed understanding of metastatic processes. P-19. CELLULAR AND STRUCTURAL CHANGES OF THE TENDON IN A RAT MODEL OF ACHILLES TENDINOPATHY: PRESENCE OF TENDON STEM/PROGENITOR CELLS AND MACROPHAGES Francesca Libonati 1 , Carlotta Perucca Orfei 1 , Dimitrios Kouroupis 2,3 , Enrico Ragni 1 , Paola De Luca 1 , Laura de Girolamo 1 1 Laboratorio di Biotecnologie Applicate all’Ortopedia, IRCCS Ospedale Galeazzi Sant’Ambrogio, Milan, Italy 2 Department of Orthopedics, UHealth Sports Medicine Institute, University of Miami, Miller School of Medicine, Miami, FL, USA 3 Diabetes Research Institute & Cell Transplantation Center, University of Miami, Miller School of Medicine, Miami, FL, USA Objective Achilles tendinopathy is one of the most common tendon disorders of the lower limb in both the athletic and general population, requiring new and effective therapies. In this context, the recent evidence on the presence of tendon stem/progenitor cells (TSPCs) in tendons and the involvement of immune cells in the early stages of tendinopathy paves the way for future therapies. For this purpose, this study is intended to highlight the structural and cellular alteration occurring in a model of Achilles tendinopathy in rats in order to implement the knowledge about tendinopathy onset. Materials and Methods A total of 12 Sprague Dawley rats were treated to induce the Achilles tendinopathy by injecting type I Collagenase (3 mg/mL) in the right tendon, while the contralateral limb was either untreated (healthy control group) or saline injected (sham group). On day 7, tendon explants were histologically evaluated by 4 blinded observers using a semi-quantitative score, to highlight the structural and cellular alterations of the tissue. Immunohistochemical analysis was performed to localise TSPCs (CD90, CD146) and M1 (CD86) and M2 (CD206) macrophages within the tissue. Results The injection of type I Collagenase induced substantial structural damage with cellular alterations, disorganisation of collagen fibers, and a thickening of the paratenon region. An increase in the number of rounded cells was observed in both tendon and paratenon regions together with a higher cell density. TSPCs were more visible in paratenon than in the tendon proper, suggesting a possible aggregation of these cells in the vascularised area typically seen in a paratenon. A slight difference was observed in the presence of TSPCs between pathological and healthy tendons even if not significant. The presence of immune cells was increased in pathological tissues with visible infiltrative (M1) and resident macrophages (M2); however, there is no significant difference in the M1/M2 ratio in this model at 7 days. Conclusions Type I collagenase injection induced Achilles tendinopathy since structural changes and cellular alterations were clearly visible. As regards the inflammation and the recruitment of immune cells, the data obtained suggest their partial involvement, which should be further investigated at other timing of the tendinopathy to understand possible interaction between TSPCs and macrophages. P-20. COMPARATIVE ANALYSIS OF HUMAN MESENCHYMAL STROMAL CELLS DERIVED FROM ADIPOSE TISSUE AND DENTAL PULP: PHENOTYPIC CHARACTERIZATION AND SECRETOME EVALUATION FOR CELL-FREE THERAPY Serena Marcozzi 1 , Maria Assunta Ucci 1 , Francesca Gioia Klinger 2 , Vincenzo Campanella 3 , Antonio Libonati 3 , Cosimo Tudisco 2 , Manuel Scimeca 4 , Giulia Salvatore 1 , Simone Vumbaca 5 , Rosita Russo 6 , Mario Picozza 7 , Angela Chambery 6 , Giovanna Borsellino 7 , Massimo De Felici 1 , Antonella Camaioni 1 1 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 2 Saint Camillus International, University of Health Sciences, Rome, Italy 3 Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy 4 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy 5 Department of Biology, University of Rome Tor Vergata, Rome, Italy 6 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania Luigi Vanvitelli, Caserta, Italy 7 Neuroimmunology Unit, Santa Lucia Foundation I.R.C.C.S., Rome, Italy Objective The purpose of the present study was a comparative analysis of the biological characteristics of human Mesenchymal Stromal Cells (MSCs) derived from Adipose Tissue (ADSCs) and Dental Pulp (DPSCs) in order to evaluate their use in regenerative medicine. Materials and Methods ADSCs and DPSCs were obtained from healthy patients. ADSCs were isolated by two methods known as Stromal Vascular Fraction (SVF) and Mechanical Fragmentation (MF), while DPSCs were obtained from open apex third molars by MF after separation of the radicular (RPSCs) and the coronal region (CPSCs). These populations were compared based on their morphological features by TEM and IF, and their capacity to proliferate by WST-1 assay. Expression of surface markers was measured by flow cytometry, and the ability to undergo trilineage differentiation was also studied. Conditioned medium (CM) was collected after 3 days of culture and the Bio-Plex Pro Human Cytokine 27-plex Assay (Bio-Rad) was performed. Extracellular vesicles were separated by ExoQuick-TC and suspensions were examined by flow cytometry. Results Both ADSC cell types were able to differentiate towards adipogenic, chondrogenic, and osteogenic lineages as expected, whereas DPSC populations were unable to differentiate into adipocytes. Interestingly, the doubling time of DPSCs calculated during 96 hrs of culture was significantly lower (RPSCs = 27.72 ± 1.34 h vs. CPSCs = 35.26 ± 2.00 h) in comparison to both ADSCs (ADSCs-SVF = 69.71 ± 4.16 h; ADSCs-MF = 65.97 ± 3.75 h). In DPSCs, more than 90% were Nestin + and only 10% αSMA + compared with <1% and ~30% in ADSCs, respectively. Multivariate analysis by PCA for the expression of 16 surface markers measured by flow cytometry showed that organ source (dental pulp or adipose tissue) was the most critical factor that discriminates cell phenotypes. The Bio-Plex assay showed that, whereas the analyte composition of CMs from the two DPSCs appeared very similar, the isolation method was responsible for large differences among CMs from ADSCs in terms of cytokines, chemokines, and growth factors secretion. Finally, ADSCs released a significantly higher number of the smaller EVs named exosomes, ≤100 nm in diameter, than DPSCs. Conclusions These results indicate that the four MSC populations show different phenotypic and functional signatures depending on both the tissue source and the extraction method. These differences may be crucial for cell-free therapies using CMs from such cells. P-21. (YIA) HUMAN PLATELET LYSATE DEPLETED FROM FIBRINOGEN IS A GOOD ADDITIVE FOR LARGE-SCALE PRODUCTION OF MESENCHYMAL STEM CELLS UNDER GOOD MANUFACTURING PRACTICE CONDITIONS AND FOR LYO-SECRETOME ISOLATION, PRESERVING ITS IMMUNOMODULANT PROPERTIES Elena Marini 1 , Alessia Giovanna Santa Banche Niclot 1 , Ivana Ferrero 2 , Camilla Francesca Proto 1 , Marta Barone 1 , Giuseppe Pinnetta 1 , Luciana Labanca 3 , Elia Bari 4 , Maria Luisa Torre 4 , Katia Mareschi 1,2 and Franca Fagioli 1,2 1 Department of Public Health and Paediatrics, University of Turin, Italy 2 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 3 Blood Component Production and Validation Centre, City of Health and Science of Turin, S. Anna Hospital, Turin, Italy 4 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy Objective Human Platelet Lysate (HPL) is an additive, rich in growth factors, cytokines, and proteins used to expand Mesenchymal Stem Cells (MSCs) under Good Manufacturing Practice (GMP) conditions. Preparation techniques may influence HPL composition and, therefore, the biological properties of cultured cells. Standard HPL (HPL-E) production consists of repeated freezing/thawing cycles of platelets and heparin in addition to avoiding culture medium gelling. The new method (HPL-S) consists of making platelets coagulate through the addition of Ca-Gluconate and mechanical squeezing. MSC properties are associated with their secretome and we defined a GMPprocess for freeze-dried MSC-secretome (lyo-secretome). In this work, we verified if the new HPL production method is effective and preserves the chemical and biological properties of MSCs and of their secretome. Materials and Methods From the same platelet pool, we obtained the standard HPL-E and the new HPL-S. We investigated if the treatment with Ca-Gluconate could interfere with the chemical characteristics and the growth factor amounts. Moreover, we compared the cellular growth, immunophenotype, and multipotent capacity of MSCs isolated and expanded in HPL-E and -S. We also isolated lyo-secretome from cell supernatant after serum starvation, ultrafiltration, and freeze-drying from MSCs. Lyo-secretome was analysed for lipid, protein, and growth factor contents, extra-vesicle size and concentrations, immunophenotype, anti-elastase activity, and immunomodulant properties. Results HPL-S did not contain PLTs and fibrinogen; total protein and growth factor amounts were comparable with HPL-E. The number of colony-forming unit fibroblasts showed no significant differences between the two groups. MSCs with HPL-E showed a cumulative Population Doubling higher in the earlier passages with an inversion of the growth trend in passage 4. Stem cell markers were maintained during expansion. Immunophenotypic analysis showed a significantly different expression of HLA-DR (1.30% with HPL-S, 14.10% with HPL-E) and of CD146 (>50% with HPL-S, <30% with HPL-E). Lyo-secretome obtained from MSCs with HPL-E or HPL-S did not show significant chemical/biological differences. Conclusions The use of HPL-S is an effective alternative for MSC production in GMP conditions and the obtained lyo-secretome could replace MSC-cellular therapy as a cell-free surrogate. P-22. BIOCOMPATIBILITY ASSESSMENT OF BIOMATERIALS OBTAINED FROM DISCARDED NATURAL SOURCES FOR DENTAL PULP STEM CELL CULTURE Pasquale Marrazzo 1 , Francesca Paris 1 , Valeria Pizzuti 1 , Silvia Zia 2 , Barbara Roda 1,2 , Francesco Alviano 1 and Laura Bonsi 1 1 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 2 Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, Italy 3 Stem Sel s.r.l., Bologna, Italy Objective The growth in vitro of medicinal stem cells depends on the culture method, including the attachment substrate required for adhering to cell culture. Strategies that involve the testing of new biocompatible substrates for stem cell modulation or expansion, for example, nature-inspired biomaterials, may improve the phenotype and the functionality of these cells. The chicken eggshells and honey bee pollen were usually considered waste-like materials. We aim to evaluate these two organic sources obtainable by the food industry for supporting mesenchymal stem culture. Materials and Methods Human Dental Pulp Stem Cells (DP) were selected as a mesenchymal stem cell culture model. Resazurin reduction assay was used to assess the adhesion to the eggshell membrane by living cells and, in parallel, to evaluate cytotoxicity in presence of bee pollen solution. Immunofluorescence staining was performed to display morphology of cells attached to the eggshell membrane. Monochlorobimane probe was used to evaluate glutathione levels after exposure to bee pollen. Results The viability signal of DP stem cells plated on the eggshell membrane increased according to seeded cell density. The cell attachment to the membrane was also confirmed by immunofluorescence performed on fixed cultured membranes. Bee pollen solution was able to increase metabolism-based signal of viability, as well as to increase antioxidant cellular glutathione content. Conclusions The biocompatibility of both eggshell membranes and bee pollen treatment in DP stem cells was confirmed. The experiments with both the organic materials showed positive results in terms of cell metabolism stimulation and no significant alteration of classical morphology of mesenchymal stem cells in vitro. We conclude that these data can be promising for continuing this study and starting new analyses concerning specific stem cell properties changes, in comparison to standard tissue culture plates. P-23. (YIA) INVESTIGATION OF THE EFFECTS OF CONTROLLED UNIAXIAL STRETCH STIMULI ON HUMAN PERIODONTAL LIGAMENT AND ADIPOSE-DERIVED STAMINAL CELLS Beatrice Masante 1,2 , Ilaria Roato 2 , Giovanni Putame 1 , Andrea Tancredi Lugas 1 , Marta Tosini 1 , Mara Terzini 1 , Alberto Audenino 1 , Diana Massai 1 , Federico Mussano 2 1 PolitoBIOMed Lab, Department of Mechanical and Aerospace Engineering, Politecnico of Turin, Italy 2 Bone and Dental Bioengineering Lab, CIR-Dental School, Department of Surgical Sciences, University of Turin, Italy Objective The periodontal ligament (PDL) plays a key role in providing mechanical stability and absorbing the high forces associated with mastication. These capacities deteriorate if PDL is affected by periodontitis, a degenerating disease that leads to loss of the PDL and the supporting alveolar bone. In view of PDL engineering and PDL regeneration, we investigated the hPDLSCs and adipose-derived stem cells (ASCs) behaviour under controlled uniaxial stretch stimuli, exploiting a previously developed bioreactor and customized flexible substrates. Materials and Methods ASCs (ASC52 -telo hTERT, ATCC) and primary hPDLSCs were characterised for the expression of mesenchymal markers by means of a FACS analysis. The same cell types were seeded on customised flexible substrates and exposed to a controlled uniaxial stretch stimulus (15% of strain, 1 Hz, for 90 s every 6 h for 3 days, n = 3) using a previously in-house developed bioreactor. Cells cultured in static conditions were used as control (n = 3). For both cell types and both conditions, DMEM was used as a culture medium. At the end of the culture period, cells were collected and the expression of stemness markers (NANOG, SOX2, and OCT3/4) and osteogenic markers (ALP, OCN, and RUNX2) were assessed through Real-Time PCR. Results FACS analysis highlighted that the phenotypes of ASCs and hPDLSCs are comparable, both expressed CD73, CD90, CD105, and CD44, while they were negative for CD45 and HLADR. The basal stemness marker expression seems to be higher for hPDLSCs than ASCs, nonetheless, both cell types showed an increasing trend in their expression after stretch stimulation. After the stretch stimulus, the expression of the osteogenic genes was increased, especially the OCN ( p < 0.05), in the hPDLSCs, but not in the ASCs. Conclusions The results obtained by FACS analysis demonstrated that both cell types expressed the typical mesenchymal markers, and the expression of the stemness genes showed an increasing trend after stretch stimulation. Osteogenic genes were upregulated in hPDLSCs when cultured under controlled uniaxial stretch conditions. According to these results, hPDLSCs showed more osteo differentiating ability than ASCs. Further studies are ongoing to define the response of hPDLSCs and ASCs to combined stimuli, such as mechanical (stretch) and chemical (tenogenic medium) ones, to obtain an effective system for PDL regeneration. P-24. 3D BRAIN ORGANOID MODELS AS A TOOL TO SCREEN FOR NUTRACEUTICALS Arianna Minoia 1 , Luca Dalle Carbonare 1 , Jens C. Schwamborn 2 , Silvia Bolognin 2 and Maria Teresa Valenti 3 1 Department of Medicine, University of Verona, University of Verona, Italy 2 Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 4365 Belvaux, Luxembourg 3 Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy Objective Degenerative conditions of the skeleton and the brain are significant issues with significant socioeconomic effects. This study would underline the significance of the interaction between nerve and bone cells in 3D brain organoid models as a tool to screen for nutraceuticals which have an impact on skeletal metabolism. Knowing that one of the main and important pathways that link bone metabolism and the brain is the Wnt/β-catenin pathway and plays a crucial role in the development of many aspects of midbrain DA development, we would try to understand the effect of some molecules chronically treating 3D organoids derived from two different cell lines for up to 40 days, focusing on the study of genes involved in the Wnt/b-catenin pathway and in neuronal degeneration. Material and Methods Generation organoids: NESCs derived from iPSCs. We cultured organoids for 40 days. Sample preparation: we extract total RNA using the mRNA Qiagen-Kit. Reverse transcription was done using High-Capacity cDNA Transcription Kit. Real-time data: TaqMan probes and TaqMan Universal Master Mix to analyse the gene expression. Flow cytometry: Flow cytometry was performed using BD LSR-Fortessa. Sectioning organoids. Immunofluorescence staining. Confocal imaging Results We investigated the gene expression of PARK2, NR2F1, CTNNB1, and LRP5 for untreated 3D organoids and treated 3D organoids with DMSO, lipoic acid, and JH-II in the two cell lines. Comparing the expression of PARK2 between the organoids deriving from the Bil-WT line towards the treated organoids with mutation LRRK2-G2019S, we note a higher expression of PARK2 in the samples treated with the molecules of interest. We also found this trend of a significant increase in expression for the NR2F1 gene. As regards the LRP5 gene, we note an identification in the samples treated with lipoic acid and an increase in expression trend for the organoids treated with the JH-II inhibitor. As regards the last gene analysed, CTNNB1, there is a higher expression for the samples treated with the inhibitor of the LRRK2-G2019S mutation. Conclusions The molecules impact on the expression of the genes associated with the Wnt/β-catenin pathway upstream and downstream appear to have a beneficial impact on the rise in the expression of the target genes. Considering an improvement in the conditions of PD patients, the treatment with the molecules we examined would result in an increase in the expression of the gene of interest. P-25. BIOLOGICAL EVALUATION OF 3D-BIOPRINTED SCAFFOLD FOR PERIODONTAL REGENERATION Alessandro Mosca Balma 1 , Ilaria Roato 1 , Beatrice Masante 1,2,3 , Federico Mussano 1 1 Department of Surgical Science, C.I.R. Dental School, University of Turin, Italy 2 PolitoBIOMed Lab, Department of Mechanical and Aerospace Engineering, Polytechnic of Turin, Italy 3 Interuniversity Centre for the Promotion of the 3Rs Principles in Teaching and Research, Pisa, Italy Objective Periodontitis is one of the most common diseases worldwide, causing a progressive destruction of the tooth supporting tissues. Thus, different regenerative approaches are under investigation, and this study aims to evaluate the biological properties of polycaprolactone (PCL) 3D-Bioprinted scaffolds w/or w/o alumina toughened zirconia (ATZ) filler, as suitable materials for alveolar bone and periodontal ligament (PDL) regeneration. Materials and Methods Three different types of blends were compared to each other, respectively: pure PCL, 80/20 w / w PCL/ATZ, and 60/40 w / w PCL/ATZ. The ATZ was incorporated in the PCL matrix through dissolution in chloroform by solvent casting method, then the bioink was 3D bioprinted to generate scaffolds with a standardised porous and circular geometry. ASC52 hTert cells (ASCs) were seeded on these scaffolds for 24 h to test their adhesion on the material surface. To evaluate the biocompatibility of the scaffolds, ASCs were cultured for 3, 7, and 14 days to allow scaffold colonisation, and registered their growth through the quantification of the ATP release in culture by viable cells (CellTiter-Glo kit). To investigate whether the scaffold had osteoinductive properties, ASCs were cultured in an osteogenic medium for 2 months, then RNA was extracted and the expression of osteogenic genes (ALP, COLL1, OCN, and RUNX2) were quantified with Real-Time PCR. SEM and EDX analysis were performed to evaluate the newly formed bone and to quantify the presence of calcium deposition, respectively. Results All the scaffolds allowed ASC adhesion and growth, particularly the ones with 80/20 w / w PCL/ATZ showed better biocompatibility in the long term. The addition of ATZ to the PCL matrix reduced the osteoinductive property of the materials. After two months in the osteogenic medium, the expression of ALP and RUNX2 was not different among the three polymeric scaffolds, while both COLL1 and OCN expressions were reduced in the presence of ATZ filler. These results were confirmed by SEM images and EDX analysis. Conclusions The presence of ATZ seems to reduce the osteoinductivity of the scaffolds, pointing out the importance of the material chosen as a support of new PDL regeneration, because this could limit the possibility of tooth ankylosis. Future tests will be focused on the quantification of scaffolds’ mechanical properties improvement in the presence of a toughening filler as ATZ and on tenogenic differentiation capability with PDL mesenchymal stem cells. P-26. HA AND PRP COMBINATIONS AS “OFF THE SHELF” DEVICE FOR CLINICAL APPLICATIONS Marta Nardini 1 , Anita Muraglia 1,2 , Antonella D’Agostino 4 , D’Agostino Maria 4 , Gilberto Filaci 1,2 , Ranieri Cancedda 3 , Chiara Schiraldi 4 and Maddalena Mastrogiacomo 1 1 Department of Internal Medicine (DIMI), University of Genoa, Italy 2 Biotherapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy 3 Emeritus professor at the University of Genoa, Italy 4 Department of Experimental Medicine, University of Campania “L. Vanvitelli”, Naples, Italy Objective Platelet Rich Plasma (PRP) is a well-known natural product, optimised, standardised, and used for regenerative medicine. The current goal is to identify substrates as vehicles for a gradual platelet content release. Hyaluronic acid (HA) is proposed thanks to its viscoelastic and biological properties and biocompatibility. This study aimed to set up and characterise an “off the shelf” freeze-dried and injectable device based on HA and PRP for tissue regeneration. Materials and Methods Different Molecular Weights of HA (Low and High– HA-HMW and HA-LMW) were used in combination with a standardised PRP in the ratio 1:1. Rheological analysis of compounds was performed, and the products were lyophilised. After reconstitution by water, the HA/PRP mixtures were tested in terms of cell proliferation capability and wound scratch recovery on human primary fibroblasts. With the aim of validating the methods of storage of the products, the biological activity of freeze-dried HA/PRP formulations was tested at different temperatures of conservation (25 °C, 4 °C and −20 °C) for up to 6 months. Results HA-HMW/PRP compound prompted human dermal fibroblast proliferation such as PRP alone, but the formulations needed almost half an hour for full reconstitution and strong pressure to be extruded by a 21-gauge needle. To overcome these limitations, different HA-LMW/PRP formulations were characterised and tested in terms of biological activity. These formulations are capable of sustaining cell proliferation and are stable at different temperatures and lengths of storage compared to the loss of PRP activity. Interestingly, only the lowest HA-LMW tested (56 KDa) in combination with PRP showed from one up to six months of significant preservation of the proliferation activity compared to PRP alone. This was also confirmed by in vitro scratch assay using a time-lapse video microscopy station. Conclusions In conclusion, we developed a lyophilised HA-based/PRP device that improves and preserves the PRP activity over time. HA/PRP allows the development of promising products for topical, intradermic, and intra-articular applications. P-27. THERAPEUTIC EFFECTS OF HUMAN PLACENTAL-DERIVED MESENCHYMAL STROMAL CELLS (PDMSCS) ON A LIPOPOLYSACCHARIDE INDUCED MOUSE MODEL OF PREECLAMPSIA (PE) Anna Maria Nuzzo, Laura Moretti, Ilaria Faletti, Alberto Revelli, Alessandro Rolfo Dept. Surgical Sciences, University of Turin, Italy Objective Preeclampsia (PE), the most severe human pregnancy-related syndrome, is a leading cause of foetal-maternal mortality and morbidity and lack of an effective therapy. The main hallmarks of PE are severe maternal hypertension and proteinuria, expression of generalised endothelial damage, and inflammation that could lead to Foetal Growth Restriction (FGR). Human Placenta-Derived Mesenchymal Stromal Cells (hPDMSCs) are well renowned for their pro-angiogenic and anti-inflammatory effects exerted via paracrine interactions. Herein, we tested the effects of hPDMSCs-CM (Conditioned-Media) on a mouse model of preeclampsia. Materials and Methods PDMSCs were isolated from control placentae and plated (1 × 10 5 cells/mL) in DMEM without FBS at passage 5. After 48 h, CM was collected. Preeclampsia was induced in pregnant C57BL/6NCrl mice by intravenous bacterial Lipopolysaccharide (LPS) injection. Starting from d9, maternal blood pressure and proteinuria were monitored until d19. At d11 of pregnancy, dams were injected with E.Coli LPS (1 µg/Kg). At d12, mice were randomly divided into two groups (n = 7 each) and treated intravenously as follows: plain vehicle (300 µL, placebo) and hPDMSCs-CM (300 µL, treated). At d19, mice were sacrificed. A number of foetuses, FGR, foetal reabsorption, and placental weight were evaluated. Next, placentae were processed for mRNA and protein isolation. sFlt-1, IL-6, and TNF-α gene and protein expression were evaluated by Real-Time PCR and Enzyme-Linked Immunosorbent Assay (ELISA). Results Injection of hPDMSCs-CM on d12 significantly decreased maternal systolic blood pressure and proteinuria by day 13 until term relative to placebo group. No FGR and/or reabsorbed foetuses were delivered by hPDMSCs-CM treated PE mice, while 5 FGR foetuses were found in the placebo group. No differences were found in placental weight between groups. hPDMSCs-CM injection significantly decreased sFlt-1, IL-6, and TNF-α levels in PE mice. Conclusions Our data indicate that hPDMSCs-derived trophic mediators can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia. Supported by Corion Biotech s.r.l. P-28. OXIDATIVE STRESS AND PLACENTAL-DERIVED MESENCHYMAL STROMAL CELLS (PDMSCS): NEW PERSPECTIVE FOR PREECLAMPSIA (PE) ETIOPATHOGENESIS Anna Maria Nuzzo, Laura Moretti, Ilaria Faletti, Alberto Revelli, Alessandro Rolfo Dept. Surgical Sciences, University of Turin, Italy Objective Mesenchymal Stromal Cells have been highlighted as an effective antioxidant therapy. Indeed, anomalies in PDMSCs antioxidant defences might cause/contribute to the increased oxidative stress (OxS) typical of PE placentae. Herein, we compared the release of antioxidant proteins and investigated the expression of the antioxidant enzymes Catalase (CAT) and Superoxide Dismutase1 (SOD1) and of OxS-triggered cell death modulators PARP1, Caspase3, and LDOC1 in normal and PE-PDMSCs. Finally, we tested the hypothesis that PDMSCs-CM (Conditioned-Media) could restore CAT and SOD1 in H2O2-treated villous explants. Materials and Methods PDMSCs were isolated from control (n = 10) and PE (n = 10) placentae. At passage 5, cells were plated (1 × 10 5 cells/mL) in DMEM without FBS. After 48 h, CM was collected, and total antioxidant capacity was tested by Cayman’s Antioxidant Assay. Control (n = 24) villous explants were treated for 24 h by H2O2 and next by control PDMSCs-CM for 48 h. CAT, SOD1, PARP1, Caspase3, and LDOC1 gene expression were evaluated by Real Time PCR. Results We reported a lower total antioxidant capacity (1.42 Fold Decrease) in PE-PDMSCs relative to control. We reported decreased CAT and SOD1 and increased PARP1 ( p = 0.03), Caspase3 ( p = 0.04), and LDOC1 ( p = 0.05) gene levels in PE relative to control PDMSCs. After 24 h with H2O2, normal PDMSCs-CM treatment increased CAT and SOD1 and significantly decreased PARP1 ( p = 0.02), Caspase3, and LDOC1 mRNA levels relative to controls. Conclusions Herein, we demonstrated that pathological PE-PDMSCs are characterised by aberrant antioxidant properties followed by increased production of OxS-related cell death effectors. Moreover, PDMSCs-CM promotes the expression of antioxidant enzymes, thus inhibiting OxS-mediated cell death. Indeed, our data suggest that PDMSCs-CM could be used to neutralise the exacerbated OxS typical of PE placentae, thus opening to novel PDMSCs-based therapeutic options. P-29. MESENCHYMAL STEM CELLS IN WOUND HEALING: FOCUS ON MSC-MEDIATED SKIN REGENERATION AFTER DERMATO-ONCOLOGICAL SURGICAL PROCEDURES Alessia Paganelli, Cristina Magnoni UO di Chirurgia Dermatologico a Indirizzo Oncologico e Rigenerativo, AOU Policlinico di Modena, Università degli Studi di Modena e Reggio Emilia, Modena, Italy Objective To assess the role of mesenchymal stem cells (MSCs) on skin wound healing both in vitro and in vivo after dermatological surgical procedures performed for oncological purposes. Materials and Methods MSCs were obtained from discarded adipose tissue during dermato-surgical procedures. After isolation, MSCs were cultured in ascorbic-acid enriched medium in order to obtain MSC-based dermal scaffolds. Organotypic cultures were also performed to assess the pro-epithelizing properties of MSCs. MSCs were also seeded on commercially available acellular dermal matrices (ADMs) to assess whether they could exert a synergistic action. Finally, we also looked at whether MSCs were spontaneously recruited in vivo at wound sites in the presence of ADMs. Classical histology, immunofluorescence, and ELISA tests have been employed in the aforementioned experimental settings. Results MSCs can efficiently be used to produce dermal equivalents. MSCs secrete all the main components (collagen and fibronectin) of the extracellular matrix upon stimulation. MSCs guide wound re-epithelialization in vitro in the presence of keratinocytes, mainly through a paracrine action on epidermal basal stem cells. When seeded on acellular dermal collagenic substitutes, MSCs significantly increase extracellular-matrix production, therefore, confirming the potential effectiveness of combination treatments. CD90+ STRO-1+ cells were detected in the neodermis after ADM positioning, therefore suggesting efficient ADM-mediated MSC recruitment. Conclusions MSCs represent a promising tool in the regenerative setting, especially for the treatment of large surgical wounds after dermatological surgical procedures. However, further studies are needed to confirm their safety in oncological patients since a potential tumour-promoting role has recently been postulated for MSCs. P-30. PRODUCTION OF CLINICAL GRADE EXTRACELLULAR VESICLES (EVS) SECRETED BY MESENCHYMAL STROMAL CELLS AND INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF OSTEOARTHRITIS Maria Elisabetta Federica Palamà 1 , Cansu Gorgun 1 , Georgina Shaw 2 , Mary Murphy 2 , Chiara Gentili 1 1 Department of Experimental Medicine (DIMES), University of Genova, Italy 2 Regenerative Medicine Institute (REMEDI), University of Galway, Ireland Objective Mesenchymal stromal cells (MSCs) derived extracellular vesicles (EVs) have been studied for the treatment of Osteoarthritis (OA), the most common chronic disease of the joint cartilage. A large-scale expansion of MSCs is required to meet clinical demand and this could affect the effectiveness of cells and cell products. Therefore, MSC generated from induced pluripotent stem cells (iMSC) represent a promising cellular source for the manufacture of EV therapeutics. In this study, we isolated and tested the efficacy of EV secreted by MSCs and iMSC in the treatment of OA in vitro. Methods MSCs and iMSCs were cultured in vitro in serum-free clinical grade conditions. Cells were characterised during long-term in vitro expansion for surface expression pattern, proliferation ability, senescence rate, and differentiation capacity. EVs were isolated using an FPLC-anion exchange chromatography (AEX) approach and their biological effect on IL-1α treated human chondrocytes was examined. Results The use of a serum-free, chemically defined medium for isolation and culture of hMSCs allowed us to expand a population with a stable phenotype from early to late passages. It is already well known that MSC proliferation, differentiation, and function decline with passaging. After three passages, we indeed observed a drastic impact on cell growth and differentiation. The paracrine activity of hMSCs during long-term expansion was also evaluated. The number and size of vesicles released by hMSCs increased proportionally with their age. The anti-inflammatory activity of MSC-EVs was investigated in an in vitro model on osteoarthritic chondrocytes and the expression of inflammatory cytokines such as IL-6 and IL-8 were measured. Administration of hMSC-EVs showed relevant anti-inflammatory effects only for early passages-derived vesicles (until passage three). iMSCs were also expanded for the long term to define the best culture conditions and the best time window for the isolation of EVs with maximum biological activity. Conclusions Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. The focus of our study was directed on the determination of the optimal range of time in which MSCs and iMSC are biologically functionally in a serum-free culture system. This paracrine application may represent a novel therapeutic approach for the treatment of OA. P-31. MESENCHYMAL STEM CELLS COMBINED WITH ENDOTHELIAL CELLS SUPPORT SPHEROID FORMATION OF OSTEOSARCOMA CELLS Micaela Pannella, Chiara Bellotti, Ania Naila Guerrieri, Toni Ibrahim, Enrico Lucarelli Terapie Rigenerative in Oncologia, Struttura Complessa di Osteoncologia, Sarcomi dell’osso e dei tessuti molli, e Terapie Innovative, IRCCS, Istituto Ortopedico Rizzoli, Bologna, Italy Background and Objectives The tumour microenvironment (TME) is a complex milieu that contains cancer cells and non-malignant cellular and non-cellular components which together orchestrate a complex dialog. In the last decades, several papers have proposed that mesenchymal stem cells (MSCs) play a critical role in TME formation and function. In our work, we evaluated the influence of MSCs in combination with endothelial cells on Osteosarcoma (OS) cells using multicellular spheroids. Our hypothesis is that MSCs may have a pro-tumorigenic action. Materials and Methods The OS cell lines MG-63, U-2 OS, SaOS-2, and 143B were engineered to express GFP, Endothelial Cells (HUVEC) were purchased, while MSCs were isolated at the Rizzoli. Spheroids were made by self assembly in ultra-low attachment 96 well plates. Based on previous experience in generating multicellular spheroids, we combined OS/HUVEC/MSCs with a ratio of 5:3:2. Results Commercially available OS cell lines show a different ability to grow as stable spheroids; only some of them grow rapidly as spheroids but develop necrotic cores over time. Our results revealed that the metabolic activity of cells in OS cell-only spheroids decreased with time, however, increased in hybrid spheroids with MSCs/HUVEC. OS spheroids composed of U-2 OS and SaOS-2 cells formed irregular spheroids while in the hybrid configuration, with MSCs and HUVEC, they assembled forming regular spheroids. OS spheroids composed by 143B and MG-63 cells only had a rounded and compact morphology. The hybrid spheroids of 143B cells and MSCs/HUVEC had an extremely regular and smooth surface, and over a long time, they began to form buds. The hybrid spheroids composed of MG-63 and MSCs/HUVEC had a smooth and regular surface; interestingly, after 96 h MG-63 cells started to leave the spheroid’s core and after 7 and 12 days, they formed buds. Bud formation and OS cell migration suggest an increase in OS cell motility and migration when they are cultured with MSCs/HUVEC. Several studies have shown that spheroids’ roughness is indicative of cellular invasiveness. Thus, we compared the roughness between simple and hybrid spheroids demonstrating that all hybrid spheroids exhibit greater roughness than spheroids composed of OS cells only. Conclusions Data obtained shows that combined MSCs and HUVEC support OS growth and may influence the spheroid morphology and invasiveness, thus sustaining the hypothesis of the pro-tumorigenic effect of these cells. P-32. DEVELOPMENT OF A PRECLINICAL MODEL OF PDAC TO INVESTIGATE MSC-BASED DELIVERY SYSTEMS Smeralda Rapisarda 1 , Benedetta Ferrara 1 , Antonio Citro 1 , Fabio Manenti 1 , Chiara Gnasso 2 , Lorenzo Piemonti 1 1 Diabetes Research Institute, IRCCS San Raffaele, Milan, Italy 2 Experimental Imaging Centre, IRCCS San Raffaele, Milan, Italy Objective Conventional therapies for pancreatic ductal adenocarcinoma (PDAC) present limits due to drug toxicity and the chemoresistance of PDAC cells. That can be due to the PDAC stroma, which constitutes a barrier for the transport of drugs to tumour cells and a drug-delivery system might improve the treatment. The aims of this study were firstly to develop a preclinical metastatic model of PDAC and secondly to set up a model of treatment with mesenchymal stem cells (MSCs) for future applications as a drug-delivery tool. Materials and Methods To generate the liver metastatic model, three different cell lines derived from KPC mice were used: K8484 (from PdxCre/LSL-KrasG12D-Trp53R172H), DT6606 (from PdxCre/LSL-KrasG12D), and DT6606lm (from liver metastases after intraportal injection of DT6606). Cells were injected into the portal vein of immunocompetent eight weeks C57BL/6N male mice in a dose range of 1 × 10 3 –5 × 10 5 . On day 20, after tumour induction, the metastatic growth was assessed by seven-tesla magnetic resonance (MR). On day 21, the first group of tumour-bearing mice underwent intravenous (i.v.) injection of luciferase-transduced MSCs (LUC + MSCs) to evaluate the MSCs biodistribution using in vivo imaging system (IVIS). A second group of tumour-bearing mice underwent an intraportal injection of LUC + MSCs and the LUC signal intensity of the two groups was compared. Results Among the three investigated cell lines, only the K8484 cell line was chosen to generate the metastatic model. K8484 cell-derived liver metastases, indeed, exhibited a largely glandular architecture, more similar to human metastases. Moreover, the use of the intraportal model allowed a homogeneous and synchronous metastatic growth in mice 20 days after the injection. Studies on biodistribution of i.v. injected MSCs revealed a cell accumulation in the lung after injection with a low-intensity signal and several animals died after cell administration. The mice subjected to intraportal injection of MSCs reported a high cell accumulation in the liver with a prolonged signal up to six days after injection, making the intraportal injection preferable to allow MSCs to reach the tumour site. Conclusions The development of this metastatic model of PDAC in the liver allowed us to evaluate MSCs as a promising therapeutic option for PDAC, especially using intraportal injection to deliver them directly to the tumour site. Obtained results may open up new insights into the use of MSCs as tools to treat PDAC. P-33. HUMAN AMNIOTIC MESENCHYMAL STROMAL CELL CONDITIONED MEDIUM MODIFIES CANCER ASSOCIATED FIBROBLAST GENE EXPRESSION AND CYTOSKELETAL ORGANIZATION Jacopo Romoli 1 , Andrea Papait 1,2 , Paola Chiodelli 3 , Patrizia Bonassi 3 , Silvia De Munari 3 , Elsa Vertua 3 , Serafina Farigu 3 , Antonietta Silini 3 , Ornella Parolini 1,2 1 Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy 3 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy Objective Tumour-associated stromal cells, also known as carcinoma-associated fibroblasts (CAFs), play a pivotal role in favouring tumour growth by their interactions with both tumour cells and cells present in the tumour microenvironment (TME). Recent studies demonstrate that CAFs can favour metastasis and are responsible for chemo-resistance mechanisms. Human amniotic mesenchymal stromal cells (hAMSC) are known to exert immune-modulatory and anti-fibrotic effects, targeting not only immune cells but also stromal cells in damaged tissues. The aim of our study was to determine if hAMSC could also exert effects on stromal cells within the tumour microenvironment, namely CAFs. To this end, we in vitro stimulated the differentiation of normal adult fibroblasts towards CAFs with exogenous TGF-β. The acquisition of CAF features was assessed by immunofluorescence for αSMA expression and for cytoskeleton organisation. In addition, we used Real-Time PCR and flow cytometry to assess the expression of the most relevant CAF markers. Finally, we evaluated the ability of hAMSC conditioned medium (CM-hAMSC) to counteract the acquisition of CAF-like gene expression and functionality. Materials and Methods Conditioned medium from hAMSC was obtained by culturing hAMSC at passage 2 in DMEM-F12 without serum for 5 days. Human dermal fibroblasts were treated with TGF-β ± CM-hAMSC for 3, 7, and 11 days. At each timepoint, flow cytometry, immunofluorescence, and RT-PCR were performed to assess CAF-like phenotype. Results Flow cytometry showed a high percentage of αSMA + cells after TGF-β treatment, which decreased after CM-hAMSC treatment. Concurrently TGF-β-treated fibroblasts displayed well-organised cytoskeletal αSMA and presented extracellular MFAP5 deposition, whereas CM-hAMSC treatment interfered with these processes. These data are supported by RT-PCR analysis which showed αSMA and MFAP5 downregulation when dermal fibroblasts were treated with CM-hAMSC. On the other hand, we observed an increase of PDPN gene expression and positive cells after CM-hAMSC treatment. Conclusions Our preliminary results suggest that hAMSC-secreted factors are able to inhibit CAF activation. Further studies will be aimed at confirming these results and understanding if hAMSC-secreted factors inhibit CAF migration and ultimately clarify the use of hAMSC as a potential therapeutic strategy able to not only target tumour cells, but also stromal cells within the tumour microenvironment. P-34. MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF CRANIAL CRUCIATE LIGAMENT RUPTURE OF DOGS Gabriele Scattini 1 , Piero Boni 2 , Alessandro Fruganti 3 , Fabrizio Dini 3 , Luisa Pascucci 1 1 University of Perugia, Department of Veterinary Medicine, Perugia, Italy 2 Veterinary practitioner, Cannara (PG), Italy 3 University of Camerino, School of Biosciences and Veterinary Medicine, Matelica, Italy Objective In recent years, mesenchymal stromal cells (MSC) have received a strong boost in veterinary medicine due to their pro-regenerative properties. MSCs are virtually present in all the organs possessing a vascular stroma. The aim of this study was to evaluate the potential therapeutic use of MSC from adipose tissue in dogs with complete cranial cruciate ligament (CCL) ruptures that had not undergone surgical treatment. CCL rupture is the most common cause of lameness in dogs and can be treated conservatively, but surgical therapy is the treatment of choice to restore stability and functionality of the joint. However, surgery does not prevent the development of osteoarthritis, which over time leads to relapse of lameness and pain. It is worth mentioning that an increasing number of public and private veterinary hospitals all over the world treat partial CCL rupture with MSC as a second-line or even first-line therapy. There is no evidence of MSC use in complete ruptures. Material and Methods Tyson, an American Staffordshire Terrier, male, 4 years old, received a diagnosis of complete CCL rupture followed by 4 intra-articular infiltrations of autologous MSC. Post-infiltration monitoring was performed by orthopaedic, ultrasound, radiography examination, and nuclear magnetic resonance (NMR). A 20-month follow up was performed. Results MSC did not trigger adverse effects in the short to medium term, nor they cause regeneration of the CCL. However, they displayed a strong anti-inflammatory activity responsible for symptom remission. The therapeutic effectiveness of MSC seems to be attributable to their chondroprotective effect which slows down the development and progression of osteoarthritis. Conclusions The results described in this study suggest that the use of MSC for the treatment of complete rupture of the CCL could represent a valid option: (i) As an alternative to surgery when the patient or owners are unable to deal with it; (ii) As an adjuvant to surgical therapy both in the peri-operative and in the post-operative in order to obtain a stabilisation and reduce the development of osteoarthritis; (iii) as a substitute of non-steroidal anti-inflammatory drugs with the advantage of reducing side effects of pharmacological therapy. P-35. MIR-214 MEDIATED STROMA-TUMOUR CELL CROSSTALK DURING TUMOUR PROGRESSION Francesca Orso 1,2 , Federico Virga 1,2,10 , Daniela Dettori 1,2 , Alessandra Dalmasso 1,2 , Mladen Paradzik 1,2 , Aurora Savino 1,2 , Stefania Cucinelli 1,2 , Maura Coco 1,2 , Iris Chiara Salaroglio 3 , Joanna Kopecka 3 , Margherita Aalba Carlotta Pomatto 4 , Giovanni Camussi 4 , Katia Mareschi 5,6 , Leonardo Salmena 7 , Paolo Provero 8,9 , Valeria Poli 1,2 , Chiara Riganti 3 , Massimiliano Mazzone 1,2,10 Pier Paolo Pandolfi 1,2,11 , Daniela Taverna 1,2 1 Molecular Biotechnology Center (MBC), Turin, Italy 2 Dept. Molecular Biotechnology and Health Sciences, University of Turin, Italy 3 Department of Oncology, University of Turin, Italy 4 Department of Medical Sciences, University of Turin, Italy 5 Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 6 Department of Public Health and Paediatrics, University of Turin, Italy 7 Princess Margaret Cancer Centre, University Health Network, ON, Canada 8 Centre for Omics Sciences, IRCCS San Raffaele Scientific Institute, Milan, Italy 9 Department of Neurosciences “Rita Levi Montalcini”, University of Turin, Italy 10 Center for Cancer Biology (CCB), VIB, Leuven, Belgium 11 Renown Institute for Cancer, Nevada System of Higher Education, Reno, NV, USA Objective Cancer and stroma cells continuously interact during tumour progression and influence each other. Secreted microRNAs (miRNAs) have recently been implicated in the tumour–stroma crosstalk. Here, we show that miR-214 is highly expressed in stromal cells such as Mesenchymal Stem Cells (MSCs) or Cancer-Associated Fibroblasts (CAFs), often derived from CAFs, and that it correlates with stromal signatures in human breast cancers and melanomas. Upon tumour cell signals, stroma miR-214 is released via Extracellular Vesicles (EVs) and is instrumental for cancer cells to promote metastasis formation through the activation of a pro-metastatic pathway which involves the protein-coding genes TFAP2C, ITGA5, and ALCAM and the anti-metastatic small non-coding RNA, miR-148b. Metabolic rewiring and, particularly, reprogrammed glucose metabolism is a hallmark of cancer, crucial for tumour progression. Material and Methods We analysed the impact of stroma-derived miR-214 on the metabolic status of tumour cells and observed glycolysis enhancement and an Oxidative Phosphorylation (OXPHOS) impairment linked to metastatic traits. Results and Conclusions Our results underline the relevance of “stroma miR-214” for tumour dissemination and metastasis formation and suggest the possibility of a double-edge therapeutic approach based on the targeting of miR-214 and of major metabolic players in tumour and/or stroma cells. P-36. CELLULAR SENESCENCE IN SYNOVIAL FLUID MESENCHYMAL STROMAL CELLS AND POTENTIAL IMPLICATIONS IN THE PROGRESSION OF OSTEOARTHRITIS Gabriella Teti 1 , Valentina Gatta 1 , Francesca Chiarini 2 , Mirella Falconi 3 1 Department of Biomedical and Neuromotor Sciences, University di Bologna, Italy 2 Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, University of Bologna, Italy 3 Department of Experimental, Diagnostic and Specialty Medicine—DIMES, Bologna, Italy Objective Osteoarthritis (OA) is characterised by cartilage degradation, joint inflammation, subchondral bone remodelling, and fibrosis. Current therapies for OA are mainly focused on treating symptoms of pain rather than to counteract the progression of the disease. Recently, OA has been associated with the accumulation of senescent cells in joint tissues but how senescence can affect each resident joint cell and its link with the progression of OA are still poorly described. With the advance of tissue engineering and regenerative medicine, mesenchymal stem/stromal cells (MSCs) have represented a promising candidate for cartilage repair and regeneration. However, most of the current research has been focused on the application of MSCs derived from bone marrow, and just a few studies have investigated the potential therapeutic properties of MSCs isolated from synovial fluid (sf-MSCs). Although sf-MSCs have demonstrated higher chondrogenic capabilities their accumulation as senescent cells in synovial fluid and their correlation with OA progression has never been investigated. Thus, the aim of the study was to verify the presence of senescent sf-MSCs isolated from OA joints and compare their chondrogenic capabilities with sf- MSCs isolated from healthy joints. Material and Methods Sf-MSCs were isolated from tibia-tarsal joints of healthy and diseased horses with an established diagnosis of OA. Cells were cultured in vitro and characterised for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and evaluation of senescent markers. To evaluate the influence of OA on chondrogenic differentiation, sf-MSCs isolated from diseased joints were in vitro stimulated to chondrogenic differentiation and the expression of chondrogenic markers was checked and compared to healthy sf-MSCs. Results Results clearly showed an arrest of the cell cycle in combination with upregulation of the senescent markers p21 WAF1/Cip1 and p16 INK4 , reduced autophagy and increased ROS production in OA sf-MSCs, demonstrating a senescent state. Furthermore, OA sf-MSCs showed a reduced ability in synthesising proteoglycans while the ability to express collagen II is upregulated, suggesting the involvement of OA sf-MSCs in developing a fibrotic joint environment. Conclusions In conclusion, our results demonstrate the presence of senescent sf-MSCs in OA joints which could have a key role in the progression of the disorder. P-37. LASER DISSECTION-COUPLED QUANTITATIVE MICROLIPIDOMIC METHOD TO RESOLVE TUMOUR HETEROGENEITY Vanda Varga-Zsíros 1,2 , Mária Péter 1 , Ede Migh 1 , Annamária Marton 1 , Aladár Pettkó-Szandtner 3 , Imre Gombos 1 , Zoltán Kóta 4 , Péter Horváth 1 , László Tiszlavicz 5 , Zsuzsanna Darula 3,4 , Csaba Vizler 1 , Zsolt Török 1 , László Vígh 1 , Gábor Balogh 1 1 Biological Research Centre, Institute of Biochemistry, ELKH, Szeged, Hungary 2 University of Szeged, Ph.D. School in Biology, Szeged, Hungary 3 Biological Research Centre, Laboratory of Proteomics Research, Szeged, Hungary 4 Single Cell Omics Advanced Core Facility, HCEMM, Szeged, Hungary 5 University of Szeged, Department of Pathology, Szeged, Hungary Objective Lipid metabolic reprogramming is a newly recognized hallmark of malignancy. Most normal cells build up their membranes from dietary lipids. In contrast, cancer cells reactivate the de novo lipogenesis. To understand the aggressiveness and metastatic potential of tumours, the exploration of tumour heterogeneity is of great interest but also a great challenge. Materials and Methods We developed and validated a novel laser microdissection-coupled multi omics platform which combines quantitative and comprehensive lipidome, proteome, and/or transcriptome analysis with spatial resolution. The multistep approach involves the preparation of parallel cryosections from spheroids or different tissue samples, cross-referencing of hematoxylin–eosin-stained and native images, laser microdissection of marked regions (down to ~15 cells), in situ lipid microextraction/protein digestion or RNA isolation, and high-performance mass spectrometry (direct injection-based shotgun lipidomics and UPLC-coupled proteomics on an Orbitrap Lumos instrument) or transcriptomics. Results We identified a radial gradient in the lipidomic profile of 4T1 mouse breast cancer spheroids which correlated well with nutrient availability. By using mouse allografts injected with 4T1 cells, we observed substantially different lipidomic patterns not only between tumorous and non-tumorous areas of the liver or spleen but also between tumorous regions grown in different microenvironments. Close-distant parallels of cryosections were subjected to other omics and/or staining procedures. Conclusions The integration of lipidomic results with transcriptomic, proteomic, and immunohistological data can improve our understanding in tumour heterogeneity as well as in the pathology of various lipid-related disorders.
O-01. BRIDGING THE GAP IN MEDICAL RESEARCH ON ADVANCED THERAPY MEDICINAL PRODUCTS: A BIOLOGICAL, REGULATORY AND MEDICAL EXPERIENCE Graziella Pellegrini Centre for Regenerative Medicine “Stefano Ferrari”, University of Modena and Reggio Emilia, Modena, Italy Abstract Gene therapy, cell therapy, and tissue engineering have the potential to revolutionise the treatment of disease and injury. Attaining marketing authorisation for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agency—authorisation is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means such as “hospital exemption” schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorisation and is the first to contain stem cells. This review highlights the differences in standards between an authorised and unauthorised medicinal product and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorisation. The result is that patients will have access to a therapy that is manufactured to high commercial standards and is supported by robust clinical safety and efficacy data. O-02. BRIDGING THE FUNDING GAP IN MEDICAL RESEARCH IN ADVANCED THERAPY MEDICINAL PRODUCTS: TOWARDS A FAIR MEASUREMENT OF VALUE CREATION Stefano Cosma, Daniela Pennetta, Francesca Guida University of Modena and Reggio Emilia, Modena, Italy Abstract The right to healthcare for every individual is crucial for social inclusion and sustainable development. Despite this, innovation and medical research face several difficulties in their path. The risks of failure in medical research are due to scientific and operational factors, but also to economic factors, particularly the lack or inadequacy of funding. In Finance, the prerequisite for a project to be funded is the possibility of correctly determining its overall value. This paper is the first step of a wider project that aims to measure the operational and financial needs of the research into ATMPs with a Capital budgeting approach in order to correctly determine the ability to generate (economic and social) value. The paper aims to explore the causes of failure of the various phases of medical research and if and how the current funding schemes or financial partners may affect them. The purpose is achieved with the support of the ClinicaTrial.gov database, which includes privately and publicly funded clinical studies conducted worldwide. As a preliminary study, we focused our attention on the Italian context, collecting information on interventional studies carried out by Italian research groups during the entire period of database coverage (since 1998 to 2021). We collected a total of 12,934 interventional studies, 344 of which we classified by keywords within ATMPs research. The analysis was also extended to a sample of 1137 European studies classified by keywords within ATMPs research. The study builds a complete, general picture of the current funding of clinical research, the current role of Finance and the types of involvement of the public sector and other non-profit partners. Through manual data reprocessing, we were able to highlight two important features of the studies in the sample. First, the information on their current status allowed us to pinpoint failed and succeeded projects, while also understanding failure causes. Second, information on their sponsor was re-elaborated in order to classify the studies according to the source of funding (e.g., industry, university, research centres, hospitals, and so on). Thanks to an intersection of this data, our work provides insights into how the current financial resource allocation may be correlated to the success or failure of clinical trials, with a focus on advanced therapies, revealing not only potential funding gaps but also implications for involved researchers, policymakers, and stakeholders. O-03. MY EXPERIENCE WITH MSCS IN ORTHOPAEDICS: PAST, PRESENT AND FUTURE Nicholas Crippa Orlandi, Nicola Mondanelli, Stefano Giannotti Department of Orthopaedics and Traumatology, University of Siena, Italy Abstract Orthopaedic surgery can benefit a lot from the use of stem cells. Three case reports drawn from the experience of the authors’ Orthopaedic Clinic are illustrated to highlight the benefits of applying this technology. Drawing on the extensive experience gained within the authors’ Operating Unit, three cases regarding different body segments have been selected to prove the benefits deriving from the use of gelled preparations containing autologous MSC from bone marrow. A case of humeral shaft non-union, the management of an atypical proximal femur fracture in congenital hip dysplasia, and a case of rupture of the patellar tendon and consequent reconstruction of the extensor apparatus with a cadaveric transplant. The experimental study, whose three cornerstones are mesenchymal stem cells (hMSCs), scaffolds, and growth factors, aims to develop new tissue engineering strategies to be applied in orthopaedic surgery. In the first part of the work, the focus was on the optimisation of the isolation and expansion protocol of the hMSCs, taken from the bone marrow and deposited in the Biobank present inside the laboratory which is part of the Network Telethon (TNGB), evaluating the replacement of the FBS (fetal bovine serum) with autologous PRP (platelet rich plasma). Then, the focus shifted to the characterisation of cells seeded on two types of scaffolds: bovine bone matrix (SmartBone) and lyophilized acellular dermis. The final goal of the work is to develop strategies to increase the osteogenic properties of the support by adding growth factors that enhance cell differentiation on the scaffold. Our technique of applying stem cells to cases of complex orthopaedic surgery has shown excellent results both in a clinical functional objective and subjective evaluations and in radiographic evaluations. The experimental study, currently underway, is providing excellent results. The data suggest PRP as a valid alternative to FBS, it supports the expansion of mesenchymal stem cells without compromising their capacity since cells loaded on SmartBone differentiate into osteoblasts and produce collagen. In our opinion, MCSs are and will become more and more a valid tool to provide the surgeon with important help in cases of great complexity and, therefore, to obtain the tailored care that every patient needs and deserves. O-04. ORAL CAVITY MSC PHENOTYPING FOR REGENERATIVE MEDICINE APPLICATIONS Ilaria Roato, Tullio Genova, Beatrice Masante, Giacomo Baima, Alessandro Mosca Balma, Federico Mussano University of Turin, Italy Abstract The oral cavity contains multiple sites of MSCs, which have been studied as promising candidates for tissue regeneration in the dental/maxillofacial and neuroregenerative field due to the neural crest derivation of most of them. Adult dental pulp stem cells (DPSCs) are more indicated for the pulp–dentin complex regeneration while proved to be less osteogenic compared to buccal fat pad stem cells (BFPSCs). The analysis of the immunophenotype of in vitro expanded stem cells from human exfoliated teeth (SHED), DPSCs, and periodontal ligament stem cells (PDLSCs) showed a comparable expression of MSC markers among them and a higher percentage of endothelial precursor subset in SHED and DPSCs than in PDLSCs. Owing to their peculiar origin, MSCs isolated from the oral cavity might be more effective than adipose-derived stem cells (ASCs) for the treatment of dental defects. Indeed, even though MSCs retrieved from different tissues show comparable immunophenotype and multilineage differentiation ability, their capability to differentiate into a specific tissue depends also on the different anatomical origins. Moreover, often, these cells may be harvested without any burden or additional discomfort for the patient who undergoes a tooth extraction for orthodontic indications. O-05. A NEW STRATEGY TO MONITOR MESENCHYMAL STEM CELLS AND EXTRACELLULAR VESICLES IN ADVANCED THERAPIES APPLICATION Marta Nardini 1 , Maria Elisabetta Federica Palamà 2 , Vipul Gujrati 3 , Vasilis Ntziachristos 3 , Mary Murphy 4 , Niamh Duffy 4 , Ignacio de Miguel 5 , Martin Leahy 6 , Chiara Gentili 2 and Maddalena Mastrogiacomo 1 1 Department of Internal Medicine (DIMI), University of Genoa, Italy 2 Department of Experimental Medicine (DIMES) University of Genova, Italy 3 Chair of Biological Imaging at the Central Institute for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, Germany 4 Regenerative Medicine Institute, School of Medicine, University of Galway, Ireland 5 Knowledge & Technology Transfer department. Institut de Ciències Fotòniques (ICFO), The Barcelona Institute of Science and Technology, Barcelona, Spain 6 National University of Ireland, National Biophotonics and Imaging Platform, School of Physics, Tissue Optics and Microcirculation Imaging Group, Galway, Ireland Objective An important challenge in regenerative medicine to the regulatory approval and widespread clinical acceptance of cell therapies is surrounding the behaviour of cells after transplant. The distribution, engraftment, viability, and activity of transplanted cells are unclear. This reduces confidence in the safety of cell therapies and makes it difficult to understand the mechanism of action for developing potency assays that will be required for advanced clinical testing and the approval of cell therapy. When Extracellular Vesicles (EVs) are used as therapeutic agents, similar issues arise. Optoacoustic imaging (OAI) is a rapidly developing technology with world-leading capabilities in functional imaging that is uniquely informative and lower cost, convenient, and rapid. Gold-Nanostars (GNs) are suited for use as a contrast medium for optoacoustic imaging that generates a very strong photo-thermal signal in response to light of longer wavelengths. The combination of OAI and GNs offers a class-leading imaging solution for cell therapies in regenerative medicine. In this work after a characterisation in vitro of the complexes, GNs-labelled Mesenchymal Stem Cells (MSC) and EVs were studied for their distribution in different organs after a systemic injection by visualisation with Multispectral Optoacoustic Tomography (MSOT) and tested for the in vivo toxicity. Materials and Methods MSCs were isolated from human bone marrow, expanded in Platelet Lysates (PL), and derived from the same cell EVs. The MSC and EVs were labelled with GNs and by in vitro tests with the right concentration of particles, in terms of capability to be visualised by MSOT and no cytotoxicity, was defined. Labelled and unlabelled complexes were injected into the caudal vein of male and female Nu/Nu mice. Blood tests and histological analysis of the organs were performed at different time points to test the in vivo toxicity. Results No alterations were observed in terms of biochemical and blood markers. MSOT analysis showed that after one day, GNs reached all organs and remained for all monitoring periods without any alteration as revealed by histological analyses. In particular, MSOT analysis showed that GNs cannot overcome Blood–Brain Barrier (BBB). Conclusions The results showed that the GNs can reach the various organs and that they persist there for some time, but although they persist, they cannot overcome BBB and does not lead to any type of alteration. This approach holds promise for tracking cell distribution in the tissue repair process. O-06. SCIENTIFIC ARTICLES: HOW TO CHOOSE THE RIGHT JOURNAL TAKING ADVANTAGE OF SOME OPPORTUNITIES Filippo Piccinini 1,2 1 IRCCS Istituto Romagnolo per lo Studio dei Tumori (IRST) ‘‘Dino Amadori”, 47014 Meldola, Forlì-Cesena, Italy 2 Department of Medical and Surgical Sciences (DIMEC), University of Bologna, 40136 Bologna, Italy Abstract “Scientific Articles” are not the final goal but a necessary intermediate step for worldwide progress. “Scientific journals” are the right place to communicate discoveries to society. “Editors and Reviewers” are important people for evaluating the results. “Impact Factor” is one of the most common words in the life of a researcher. These terms are just a few of many behind the real important things in research including “Hypotheses and Results”. However, a large part of the working life of a researcher is spent writing scientific articles and finding the right journals for them. In this presentation, we will discuss how to speed up the publication process, define the best journal, and increase the chances of publication by exploiting opportunities that technology and social networks today provide us. O-07. TECHNOLOGY TRANSFER AND INTELLECTUAL PROPERTY: LET’S TALK TO AN EXPERT Paola Bagnoli IRCCS Ospedale Galeazzi Sant’Ambrogio of Milan, Italy Abstract Technology transfer in the Life Science field is a demanding but very stimulating challenge for both researchers and technology transfer experts. The sector has peculiarities that differentiate it from most other technological fields and make the exploitation of research results even more challenging. The results obtained by universities and research hospitals, while promising, are often premature and require a long development path to be commercialised and brought to the clinic. It is well known that very long research and development times and high costs are required to bring a medical device or drug to the market. This gap between proposals from the research world and needs from industry is well known by the technology transfer experts and often discourages the researchers who do not see their research reaching the patient, although a strong clinical need is well identified. Many factors are crucial to fill this gap, not just the need for dedicated funding. The design according to the correct principles is fundamental to allow the industrialisation of the prototype, the scale up of the processes, and the subsequent certification phases. Last but not least, the protection of industrial property (IP) is crucial. Indeed, a company would be unlikely to invest millions of euros in a new life science project without the certainty of exclusivity on the results that would guarantee a competitive advantage over its competitors. The patent is the main tool to obtain this exclusivity, together with the copyright, the design and the trade secret. The proper protection of the research results is an essential step in the technology transfer process. Researchers can have support from the Technology Transfer Offices (TTO) of their institutions, which have the fundamental role of scouting the results, assisting researchers in protecting IP and then activating virtuous paths for the development of the Technology Readiness Level (TRL) of the projects. These paths have to be walked together with several stakeholders such as corporates, start-up incubators, business angels, venture capitalists, etc. The activation of co-development processes with companies and grants dedicated to the development of the Proof of Concept of the patented technologies are key steps in making the project attractive for a company that can licence it or in creating a startup to develop and commercialise the new technology. O-08. RECAPITULATING MUSCULOSKELETAL TISSUE COMPLEXITY THROUGH MICROFLUIDIC AND BIO-FABRICATED 3D MODELS Matteo Moretti IRCCS Istituto Ortopedico Galeazzi of Milan, Italy Abstract The musculoskeletal system is composed of different tissues and organs, i.e., bones, muscles, tendons, and joints. Several pathologies can affect its homeostasis including traumatic injuries and inflammatory diseases such as osteoarthritis (OA). In this context, mesenchymal stem cells have been considered either as building blocks to fabricate biological substitutes for damaged musculoskeletal tissues due to their differentiation potential towards bone and cartilage or as a possible therapy due to their anti-inflammatory properties. On the other hand, 3D in vitro models are increasingly being considered a powerful tool to investigate pathological mechanisms and therapeutic efficacy, allowing to overcome the excessive simplification of standard in vitro models and species-specific differences of animal models. Thus, we developed 3D in vitro models of joints to test the potential of mesenchymal cells as anti-OA therapy and of the muscle–tendon–bone junction, based on differentiated mesenchymal cells. We generated a microfluidic joint-on-a-chip model including cartilage, synovial membrane and synovial fluid, based on patient-matched synovial fluid and cells embedded in gels mimicking the composition of cartilage ECM. The injection of OA synovial fluid allowed for the reproducing of the main hallmarks of early OA as the production of degradative enzymes and an increase in inflammatory cytokines. Furthermore, we were able to detect the effects of mesenchymal cell injection, measuring MMPs and inflammatory cytokine release, on a patient basis, opening new possibilities for the establishment of personalised treatments. We also fabricated a 3D bioprinted muscle–tendon–bone model embedded in a microfluidic chip, allowing the fabrication and culture of three different connective tissues in their specific culture media. Computational simulations were performed to design the chip, keeping the culture media separated during perfusion. The chip also allowed to apply compression to the bone and stretch to muscle and tendon in physiological and pathological ranges, simulating traumatic conditions. The 3D bioprinting procedure resulted in three separated cell compartments, maintaining a good shape fidelity and high cell viability. In conclusion, biofabricated 3D in vitro models could help foster the application of mesenchymal cells as anti-inflammatory therapy and could benefit from their potential as starting material for the reconstruction of musculoskeletal tissue units. O-09. EXTRACELLULAR VESICLE-MEDIATED COMMUNICATION ROUTES IN 3D TUMOUR MODELS Maria Harmati 1 , Akos Diosdi 1,2,3 , Ede Migh 1 , Gabriella Dobra 1,4 , Timea Böröczky 1,4 , Edina Gyukity-Sebestyen 1 , Matyas Bukva 1,4 , Sandor Körmöndi 5 , Peter Horvath 1,3,6 , Krisztina Buzas 1,7 1 Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary 2 Doctoral School of Biology, University of Szeged, Hungary 3 Single-Cell Technologies Ltd., Szeged, Hungary 4 Doctoral School of Interdisciplinary Medicine, University of Szeged, Hungary 5 Department of Traumatology, University of Szeged, Hungary 6 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland 7 Department of Immunology, University of Szeged, Hungary Objective The evolutionary process of solid tumours highly relies on the extracellular vesicle (EV)-mediated crosstalk between malignant cells and stromal cells in the tumour microenvironment (TME). In this study, we aimed to establish a multicellular three-dimensional (3D) tumour model system for tracking the EV communication network of different tumour tissues under physiological conditions and cytostatic treatments. Materials and Methods Human ductal carcinoma, melanoma, and osteosarcoma models were established via co-culturing the respective tumour cell line (T-47D/A375/MG-63) with MRC-5 fibroblasts and EA.hy926 endothelial cells on flat- or U-bottom plates after staining with CellTracker dyes (Orange CMTMR, Deep Red, Green CMFDA). To mimic chemotherapeutic stress, low dose doxorubicin was used and the 2D and 3D cultures were imaged daily by a PerkinElmer Operetta High Content Screening System and a Leica SP8 Digital LightSheet microscope, respectively. Results Preliminary experiments showed that CellTracker dyes can be used for in-cell labelling of EVs, allowing the quantitative monitoring of EV crosstalk, i.e., EV routes between each cell type and in both directions. The three types of tumour models showed differences in their 3D structure, EV crosstalk activity, and drug-induced effects as well. We could observe distinct temporal kinetics in the development of the EV communication network in 2D and 3D, also priorities of the investigated EV routes varied between the two co-culture systems. Conclusions The developed 3D model system is suitable for live tracking of EV crosstalk in the TME, which enables the comparison of the primary EV communication routes in different tumour types and drug treatments. Further data will help (i) to identify potential targets of EV-blocking therapies, which may increase the efficacy of chemotherapies, and (ii) to predict the drug-induced changes of the communication activity in different tumour tissues. O-10. DEMONSTRATING METHOD SUITABILITY FOR A RAPID MICROBIAL DETECTION METHOD APPLICABLE TO BIOLOGIC PRODUCTS Lucia Ceresa, Senior Technology and Market Development Manager Microbial Solutions, Charles River, Italy Abstract New and emerging cell therapies and medicinal products present new challenges in the assurance of quality and safety in terms of end-product testing. While traditional pharmaceutical drug products have long-established standards for sterility assurance, these established processes are not optimised for cell therapies. For example, the Compendial Sterility Test requires more than fourteen days of incubation for a reliable result, making it a rate limiter in the distribution of therapies to patients. Rapid Microbial Methods (RMM’s) offer reliable alternatives to ageing microbiological methods to solve the problem of reducing cycle time in cell therapy manufacture. ATP Bioluminescence is a matured technology that is used in the quality testing of various product samples in different industries. Detection of microbial ATP using the luciferin-luciferase reaction allows for the detection of microbes before they can be cultured to visual detection levels on microbial media. Until recently, ATP bioluminescence was not a viable contamination detection option for cell-based products because these samples also contain cellular ATP. The established ATP Bioluminescence platform, Celsis ® , was further developed to address this limitation. A sample cell lysing procedure allows for the extraction and, more important, the depletion of “non-microbial-ATP”, while leaving microbial ATP intact. A case study on tests performed on different cell lines demonstrate the detection of the “slow growing” C. acnes as well as a wide panel of other typical and critical microbial species. Studies performed using the Celsis ® platform and the Celsis Adapt™ complimentary technology demonstrated the successful depletion of cellular ATP from samples, while also allowing fast detection of microbial presence contamination for superior microbiological contamination control. O-11. A NOVEL ORGAN ON CHIP PLATFORM FOR CULTURING 3D HUMAN TISSUES UNDER PHYSIOLOGICAL FLOW-CONDITIONS FOR MORE PREDICTIVE DRUG TESTING AND HUMAN DISEASE MODELLING Silvia Scaglione 1,2 , Monica Marzagalli 1 1 React4life srl, Genoa, Italy 2 CNR-IEIIT Institute, National Research Council of Italy, Genoa, Italy Objectives The human disease modelling for basic research and drug testing purposes is currently carried out through 2D cell culture in static conditions and in vivo xenografts or genetically engineered animal models, but predictability, reliability, and complete immune compatibility remain important challenges. For this aim, novel 3D, fully humanised in vitro tissue models have been recently investigated by adopting emerging technologies such as bioprinting and microphysiological systems, also named organ on chips. A novel Multi-In Vitro Organ (MIVO) organ on a chip platform has been recently developed to culture 3D clinically relevant size tissues under proper physiological culture conditions. Material and Methods Biologically relevant cancer samples (up to 5 mm), and patient biopsies of scaffold-based tissues have been cultured within the MIVO chamber, while either testing molecules or human immune cells (e.g., Natural Killer cells, NK) can circulate in the OOC mimicking the blood capillary flow. The cell proliferation, viability, and migration within 3D matrixes were investigated in such dynamic cell culture conditions. When systemic drug administration was simulated within the OOC, the anticancer drug efficacy was tested and compared to the animal model. When NK cells were placed in circulation, their extravasation through a permeable barrier resembling the vascular barrier and infiltration within the cancer tissue were analysed. Results A human 3D ovarian model was developed and treated with Cisplatin in static conditions within MIVO and in the xenograft model. Similar tumour regression was observed in MIVO and in mice, while the static culture displayed an unpredicted chemoresistance, due to unreliable drug diffusion. A human 3D neuroblastoma cancer model with proper immunophenotype was optimised to develop a complex tumour/immune cell co-culture as a paradigm of an immune-oncology screening platform. Importantly, a tumour-specific NK cell extravasation was observed with a tumour-specific NK cell infiltration within 3D tumour tissue and cancer cells apoptosis induction. Conclusions We generated a relevant human disease mode, through the adoption of the MIVO device, that can be efficiently employed as a drug screening platform, but also for better investigating crosstalk among immune cells and other healthy/pathological tissues. O-12. TRANSLATIONAL LAB-TO-CLINIC HURDLES IN THERAPY WITH MSC-EVS Stefania Bruno Department of Medical Sciences, University of Turin, Italy Abstract Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have been demonstrated to have therapeutic effects in pre-clinical models of different diseases. EVs derived from various types of MSCs have pro-regenerative capabilities comparable to cells of origin and are considered promising tools for the treatment of a variety of acute and chronic pathologies. To this end, the characterisation (size, phenotype, molecular content, etc.) of EVs and the evaluation of their biological effects are areas of intense investigation. To successfully translate EV research along the path from the laboratory to the patients, strategies should be designed to reach the aim of clinical testing of MSC-EVs safety and efficacy. Currently, there are emerging strategies for manufacturing cells and EVs and quality controls for practicability of clinical testing of the EVs, mainly related to non-industrial processes. Moreover, the identification of the mode of action in the therapeutic approach in the different diseases remains a major challenge to the translation path of EVs from the laboratory toward the clinical setting. O-13. CELECTOR ® , AN INSTRUMENT FOR QUALITY CONTROL AND STANDARDISATION OF ATMPS Silvia Zia, Pasquale Marrazzo, Laura Bonsi, Francesco Alviano, Barbara Roda, Andrea Zattoni, Pierluigi Reschiglian Stem Sel srl, Bologna, Italy Abstract Cell therapy represents innovative medical approaches in many clinical fields such as osteo-articular reconstructive surgery, tissue engineering, and cancer. Advanced therapy medicinal products (ATMPs) are cell and tissue products that are considered new types of drugs. Mesenchymal stromal stem cells (MSCs) are the most promising candidates in current clinical trials. Besides their regenerative properties, hMSCs have also shown high immunomodulatory potential. The ATMP products should follow requirements including sterility, identity, purity, viability, potency, and reproducibility. Because stem cells are a heterogenous population that can differ depending on the origin and manipulation of the starting material, the identity/purity of the final cell population and its yield is still a critical issue which can limit clinical application of MSC-based products. New approaches for the standardisation of cell-based protocols may allow for the development of new high-efficiency drug systems. To improve MSC characterisation, novel label-free functional tests, evaluating the biophysical properties of the cells, will be advantageous for their cell profiling, population sorting, and quality control. In this work, we present a new technology, Celector ® , for the quality control of the cell population. Celector ® has been shown to be able to tag-less analyse, discriminate, and separate a wide size range of cells based on their physical characteristics, with high resolution and throughput and with total maintenance of native properties. The separation is obtained in a short time (around 15 min) in a rectangular shape capillary device, through to the combined action of gravity, acting perpendicularly to the flow and opposing lift forces that depend on the morphological features of the sample. A micro-camera is connected as a detector and specifically designed software was developed for image acquisition, post-processing and data analysis, and the fingerprint of the biological sample. Celector ® is able to highlight physical differences that can be correlated to cell viability and regenerative potential. In addition, cells with stable and reproducible doubling time analysis can be collected and used as standardised systems for the development of high-quality clinical protocols. O-14. HUMAN LIVER STEM CELL-DERIVED EXTRACELLULAR VESICLES INTERFERE WITH THE DEVELOPMENT OF CHRONIC KIDNEY DISEASE IN AN IN VIVO EXPERIMENTAL MODEL OF RENAL ISCHEMIA AND REPERFUSION INJURY Elena Ceccotti 1 , Massimo Cedrino 2 , Giulia Chiabotto 1 , Cristina Grange 1,3 , Samuela De Rosa 1 , Giovanni Camussi 1,3 , Stefania Bruno 1,3 1 Department of Medical Sciences, University of Turin, Italy 2 Unicyte AG, Obendorf, Switzerland 3 Molecular Biotechnology Center, University of Turin, Italy Objective Renal ischemia reperfusion injury (IRI) is the major cause of acute kidney injury (AKI), and it increases the risk of progression to chronic kidney disease (CKD). Human liver stem cells (HLSCs) are a mesenchymal stromal cell (MSC)-like population isolated from adult liver biopsy. HLSCs share with MSCs the same phenotype, gene expression profile, and differentiation capabilities. As shown in previous studies, HLSCs improved the recovery in different experimental models of liver and kidney injury. HLSC-derived extracellular vesicles (HLSC-EVs) have been studied as vehicles for transferring active biological materials both in vitro and in vivo. In this study, we set up an in vivo murine model of IRI-AKI, which subsequently developed into IRI-CKD and we investigated the potential therapeutic effect of HLSC-EVs. Materials and Methods EVs were purified from HLSC supernatant by ultracentrifugation. They were analysed through flow cytometry and Western Blotting to detect the expression of the main mesenchymal and EV surface markers. Transmission electron microscopy was performed to analyse EV size and morphology. Male BALB-c mice were subjected to 30 min ischemia followed by reperfusion. HLSC-EVs were intravenously administered immediately after the surgery and three days after. To evaluate AKI, mice were sacrificed two and three days after the surgery; while to assess the development of CKD, mice were sacrificed two months after. The histological analyses were performed on tissue sections stained with hematoxylin and eosin and Masson’s trichrome for collagen detection. Expression of specific markers of fibrosis development (alpha-Smooth Muscle Actin (alpha-SMA), collagen I and transforming growth factor-beta) and inflammation (interleukin-1 beta, interleukin-6, and tumour necrosis factor-alpha) were evaluated at mRNA and protein levels. Results In AKI mice, the EV treatment-attenuated kidney damage by reducing tubular necrosis and increasing tubular cell proliferation. We also noticed the downregulation of the expression levels of fibrosis-related genes. In CKD mice, EVs effectively reduced the development of interstitial fibrosis at the histological level and reduced the expression levels of pro-fibrotic and pro-inflammatory genes. Conclusions The administration of HLSC-EVs immediately after renal IRI protects the kidney from AKI development and interferes with the development of subsequent CKD. O-15. FLUORESCENCE POLARISATION-BASED EXTRACELLULAR VESICLES QUANTIFICATION: APTAMER APPROACH Tarlan Eslami Arshaghi 1 , Jerry Clifford 2 , Stephanie J Davies 2 , Frank Barry 1 1 Regenerative Medicine Institute, University of Galway, Ireland 2 Valitacell Ltd., Dublin, Ireland Objective Extracellular vesicles (EVs) have attracted wide interest in recent years due to their potential applications in regenerative medicine, as biomarkers for disease diagnosis, and their role in cell–cell interactions. However, EV isolation, quantification, and characterisation remain challenging in terms of purity and specificity as well as time- and cost-effectiveness. This work aims to develop a novel and high-throughput EV quantification tool based on the interaction between a fluorescently labelled probe and a specific EV surface component, using fluorescence polarisation (FP) for detection. The method analyses the change in the polarisation of emitted light, between unbound, and bound probes, with the observed polarisation in a mixture of the labelled probe and target being proportional to the fraction of the bound probe. This property of FP allows us to use it to quantify the amount of EVs in the solution. Materials and Methods Two distinct strategies have been investigated, with probes targeting (i) specific EV surface markers (Tetraspanins, e.g., CD63) for EV sub-population quantification or (ii) the EV phospholipid bilayer membrane for total EV quantification. Commercially available fluorescently labelled CD63 binding aptamers and proteins and lipophilic dyes have been evaluated. EVs derived from HEK and MSC cultures have been purchased or isolated through PEG-precipitation and ultracentrifugation, and particles quantified through Nano Tracking Analysis (NTA) or Imaging Flow Cytometry. Each probe candidate was incubated with EVs, and FP was measured over time using a Spark Cyto plate reader. Results Tetraspanin specific (Anti-CD63) aptamer probes and lipophilic dyes have demonstrated increased fluorescence polarisation in response to increasing EV concentration quantified by NTA. Conclusions This initial proof of concept supports the use of FP as a high throughput EV detection and quantification method, with the ability to provide both total particle and CD63 +ve particle numbers. Further investigation is required to demonstrate the specific binding of each probe to its target to benchmark the FP assay against currently available methods. For this purpose, Bio-Layer Interferometry (BLI) assay is under examination to study the probe–target kinetic. O-16. MESENCHYMAL STROMAL CELL SECRETOME FOR REGENERATIVE MEDICINE: MODULATION OF SOLUBLE FACTORS AND EXTRACELLULAR VESICLES EMBEDDED-miRNAs BY DIFFERENT CULTURING CONDITIONS FOR JOINT DISEASES Enrico Ragni 1 , Paola De Luca 1 , Carlotta Perucca Orfei 1 , Alessandra Colombini 1 , Marco Viganò 1 , Francesca Libonati 1 , Stefania Cicolari 1 , Leonardo Mortati 2 , Laura de Girolamo 1 1 IRCCS Istituto Ortopedico Galeazzi, Laboratorio di Biotecnologie Applicate all’Ortopedia, Milan, Italy 2 INRIM, Istituto Nazionale di Ricerca Metrologica, Turin, Italy Objective In regenerative medicine approaches related to orthopaedic conditions, mesenchymal stromal cells (MSCs) showed positive outcomes due to the secretion of therapeutic factors, both free and conveyed within extracellular vesicles (EVs), collectively termed secretome. MSC-derived factors may be modulated by both culturing and in vivo conditions. Nevertheless, a homogenous and comprehensive fingerprint in the frame of orthopaedic applications is missing. The aim of this work was to characterise adipose-derived MSC, (ASC)-secreted factors, and EV-miRNAs and their modulation after high levels of IFNγ preconditioning, as proposed for clinical-grade production of secretome with improved potential or low levels inflammatory conditions, mimicking osteoarthritis (OA) and synovial fluid (SF). In addition, ASC-EVs penetration in cartilage explants was scored. Material and Methods ASCs were cultured with and without IFNγ (1 ng/mL) or TNFα (5 pg/mL) + IL1β (10 pg/mL) + IFNγ (40 pg/mL) mimicking OA-SF. First, 200 secreted factors were assayed by ELISA. Second, 754 miRNAs were searched by qRT-PCR in ultracentrifuge-purified EVs. Bioinformatics tools were used to predict the modulatory effect of identified molecules on pathologic cartilage and synovial macrophages. Time-lapse coherent anti-Stokes Raman scattering, second harmonic generation, and two-photon excited fluorescence were used to follow and quantify fluorescent EVs incorporation into cartilage explants. Results Data showed that more than 50 cytokines/chemokines and more than 200 EV-miRNAs could be identified. The vast majority of molecules are involved in extracellular matrix remodelling and homeostasis of inflammatory cells. Inflammatory priming and synovial fluid-like conditions were able to further increase the capacity of the secretome to stimulate healing and inflammation reduction. Eventually, EV penetration was monitored as a fast process, starting in a few minutes, and reaching 30–40 μm depth after 5 h and plateau at 16 h in both cells and matrix of the cartilage explants. Conclusions Due to the portfolio of soluble factors and EV-miRNAs, the ASC secretome showed a strong propensity to modulate inflammatory and degenerative processes. Inflammatory preconditioning or OA-like conditions were able to increase this ability. Eventually, microscopy data supported the capacity of MSC-EVs to influence the chondrocytes embedded in their native ECM by active interaction and eventual therapeutic cargo release. O-17. MESENCHYMAL STEM CELLS AND IMMUNE PROPERTIES: WHAT HAVE WE LEARNED FOR THEIR ALLOGENEIC APPLICATION Laura Barrachina Porcar Regenerative Medicine Institute (Remedi), University of Galway, Ireland Abstract Allogeneic MSCs present several advantages and the first allogeneic cell-based products in the veterinary market are emerging. Quality cells can be banked to be ready to use, avoiding the delay inherent to expanding autologous cells, which in addition may be unsuitable due to the patient’s age, genetic, or metabolic disorders. However, allogeneic therapy does not come without limitations. At first, MSCs were considered immune-privileged, but currently, we know that allogeneic MSCs can induce cellular and humoral immune responses. The immune targeting of MSCs can impact their properties and lead to adverse events, thus affecting their therapeutic efficacy and safety. Nevertheless, several human and animal studies report positive results after allogeneic MSC administration. A potential explanation for the mixed outcomes often seen is that MSCs can be immunogenic (i.e., able to raise an immune response) but they also are immunomodulatory. The less immunogenic and the more regulatory MSCs are, the better chances they have to elicit their therapeutic actions. However, several factors can affect the balance between these two immune properties. MSC immune recognition is mainly mediated by the major histocompatibility complex (MHC), which is variably expressed depending on the donor, source, or in vitro/in vivo conditions. In addition, the MHC matching between donor and recipient determines the immune recognition as in organ transplants. Although MSCs can be recognised by the immune system, they can also regulate it. Factors such as inflammation or differentiation can affect both MSC immunogenicity and immunomodulation, so it is key to learning how different conditions affect this balance. Based on this knowledge, different strategies can be designed to develop safer and more effective allogeneic therapies. The selection of the donor based on its MHC haplotype and expression pattern would allow the creation of ‘haplo-banks’ of cells from donors homozygous for the most common MHC haplotypes, as it has been proposed for human iPSCs. Other strategies aim at either decreasing the immunogenicity or increasing the immunomodulation of MSCs. For example, some growth factors can decrease the MHC expression and pro-inflammatory cytokines can increase MSC immune suppression. Understanding the interactions between MSCs and the immune system is key to learning which factors we can manage and how in order to enhance the clinical safety and effectiveness of allogeneic cell therapies. O-18. PEPTIDE MEDIATED ADHESION TO BETA-LACTAM RING OF EQUINE MESENCHYMAL STROMAL CELLS: A PILOT STUDY Barbara Merlo 1,2 , Vito Antonio Baldassarro 1,2,3 , Alessandra Flagelli 2 , Romina Marcoccia 2 , Valentina Giraldi 2,4 , Maria Letizia Focarete 2,4 , Daria Giacomini 2,4 , Eleonora Iacono 1,2 1 Department of Veterinary Medical Sciences, University of Bologna, Italy 2 Interdepartmental Center for Industrial Research in Health Sciences and Technologies, University of Bologna, Italy 3 IRET Foundation, Ozzano Emilia, Italy 4 Department of Chemistry “Giacomo Ciamician” and INSTM UdR of Bologna, University of Bologna, Italy Objective The use of biomaterials with integrin agonists could promote cell adhesion in tissue repair processes. Studies on the use of scaffolds with integrin agonists based on β-lactams in equines have never been reported. The aim of this study was to analyse the effect of GM18 (α4β1 integrin agonist) on cell adhesion of equine adipose tissue (AT) and Wharton’s jelly (WJ) mesenchymal stromal cells (MSCs) and to investigate the cell adhesion to GM18-incorporated poly L-lactic acid (PLLA) scaffolds. Materials and Methods Scaffold was fabricated using a homemade electrospinning apparatus consisting of a high-voltage power supply, a syringe pump, a glass syringe containing the PLLA solution, and connected to a stainless steel blunt-ended needle through a PTFE tube. Adhesion assays were performed after culturing AT and WJMSCs with coating or soluble GM18. Gene expression of the target integrins was evaluated. Cell adhesion on GM18 containing PLLA scaffolds after 20 min and 24 h coincubation was assessed using the two samples of AT and WJMCSs more sensitive to GM18. A cell-based high content screening was used for analyses. For statistical analyses, 2- or 1- Way ANOVA was used. Results were considered significant for p < 0.05. Results Soluble GM18 affects the adhesion of equine AT and WJMSCs, even if its effect is variable between donors. For ATMSCs, the presence of GM18 did not affect the adhesion to the PLLA scaffold, mainly due to a high variability detected at a concentration of 10%. Moreover, cultures seeded on the PLLA control scaffold cannot increase cell number after 24 h, reflecting an impairment in the early proliferation. However, the presence of GM18 in all the analysed concentrations induces an increase in cell number after 24 h (5%, p = 0.0170; 10%, p = 0.0144; 15%, p = 0.0395). For WJMSCs, the cell adhesion was affected after 24 h ( p = 0.0427), drastically increasing due to the highest concentration of GM18 ( p = 0.0368). The same concentration is also the only condition producing an increase in cell number after 24 h ( p = 0.0021). Conclusions In conclusion, the α4β1 integrin agonist GM18 affects equine AT and WJMSCs adhesion ability with a donor-related variability. These preliminary results represent a first step in the study of equine MSCs adhesion to PLLA scaffolds containing GM18, suggesting that WJMSCs might be more suitable than ATMSCs. However, the results need to be confirmed by increasing the number of samples before drawing definite conclusions. O-19. MESENCHYMAL STROMAL CELL-DERIVED MIGRASOMES: A MORPHOLOGICAL STUDY Gabriele Scattini, Luisa Pascucci University of Perugia, Department of Veterinary Medicine, Perugia, Italy Objective Cell migration is fundamental in numerous physiological and pathological processes, including tissue regeneration. Migrasomes (MG) are large structures growing on the cell surface and on the tip of nanotubes emerging from cell body during migration. MG represents a distinct type of extracellular vesicles (EVs) and are significantly different from other microparticles in size, morphology, and function. In this study, a morphological analysis of MG generated by Mesenchymal Stromal Cells (MSC) of different species was performed. Material and Methods EVs were isolated by differential ultracentrifugation. Briefly, the conditioned medium was centrifuged at 300× g to pellet cells. The supernatant was centrifuged at 2000× g (2 K EVs fraction), at 10,000× g (10 K EVs fraction), and finally at 100,000× g (100 K EVs fraction). EVs suspensions were placed on formvar-coated copper grids, contrasted with 2% uranyl acetate, and observed under a Philips EM 208 electron microscope (TEM) equipped with a digital camera. MSC monolayers were also fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated, and resin embedded. 80 nm thick sections were contrasted with 2% uranyl acetate and observed at TEM. Results Ultrastructural analysis revealed that MSC produces a previously unrecognised kind of vesicles, referred to as “migrasomes” that originate from the cell surface. The biogenesis and morphologic features of these vesicles are completely different from typical EVs. In fact, they are very large in diameter (500–2000 nm) and contain a variable number of luminal vesicles. MG were also detected in the 2 k fraction obtained by supernatant centrifugation. Conclusions Migrasomes are a special kind of EVs that are released by migrating cells. Determining their molecular content and signalling potential clearly need extensive research. The most crucial things to comprehend are how MG works, which signals are triggered by coming into contact with or ingesting MG, what messages they transport between cells, and so on. The discovery of these EVs raises many new questions for future research. In fact, although the heterogeneity of EVs has become obvious, as highlighted by the International Society for Extracellular Vesicles, specific tools to distinguish EVs of different origins are still lacking, and thus different functions are probably not correctly evaluated at the moment. O-20. MSC-EVS AS DELIVERY VEHICLES FOR TUMOUR TARGETED THERAPY Róisín Dwyer University of Galway, Ireland Abstract Despite improvements in treatments for breast cancer, when patients are diagnosed with metastatic disease that has spread to distant sites there are limited treatment options and no cure available. Extracellular Vesicles (EVs) hold immense potential as cancer therapeutics due to their small size, biocompatibility, and potential for manipulation of content and surface characteristics. Tumours actively recruit stromal cells including Mesenchymal Stromal Cells (MSCs) into the tumour microenvironment. The tumour-targeted tropism of MSCs is thought to be due to high local concentrations of inflammatory chemokines and growth factors. This tumour tropism combined with the apparent immunosuppressive characteristics of the cells raised remarkable interest in their potential as tumour-targeted delivery vehicles for therapeutic agents. MSC-derived EVs (MSC-EVs) will potentially retain the tumour targeting and immune privilege associated with MSCs while overcoming challenges associated with the use of cells. Our recent work in development of MSC-EVs enriched with a tumour suppressor microRNA for breast cancer therapy will be discussed. The impact of human- and murine-derived MSC-EVs on the immune system and the potential for scale up of EV production will also be highlighted. An approach to support sustained delivery of EVs over time would be very beneficial to prevent cancer resurgence. The use of pre-clinical models that are more reflective of the patient experience will be critical for testing novel approaches to breast cancer treatment. Progress and challenges in the development of MSC-EVs as cancer therapeutics will also be addressed. While we require further understanding of factors mediating EV content, persistence, and uptake, this exciting approach holds tremendous potential for patients with limited existing treatment options. O-21. MESENCHYMAL STROMAL CELLS AND THEIR ROLE IN THE TUMOUR MICROENVIRONMENT: CHALLENGES AND OPPORTUNITIES IN THE IMMUNOTHERAPY ERA Andrea Papait Cattolica del Sacro Cuore University, Rome, Italy Abstract Mesenchymal stromal cells (MSCs) have long been studied for their applications in regenerative medicine, exploiting their unique ability to modulate the immune response in a large number of diseases in which the immune response is dysregulated. In addition, MSCs have been studied for their potential applications as an anticancer therapeutic strategy. Indeed, MSCs have been shown to exhibit a natural tropism toward sites of inflammation, and this property has been exploited for their potential use as a vehicle for the selective delivery of anticancer drugs. In addition, MSCs can be genetically modified in an attempt to reactivate the antitumour immune response. Unfortunately, however, the application of MSCs as anticancer therapy has often achieved mixed results. In addition, the fight against cancer must take into account the complex network of interactions that exist between the different actors that are part of the tumour microenvironment (TME) and that are represented by tumour cells, stromal cells, endothelial cells, and immune cells. In fact, the use of MSCs in this regard can be considered a double-edged sword: on the one hand, they can serve as a carrier of drugs or chemotherapeutic compounds; while on the other hand, their immunomodulatory properties can participate in tumour initiation, development, and progression, even contributing to metastasis formation. In this presentation, we will discuss all these merits and demerits of MSCs by contextualising them in the era of immunotherapy, which sees the use of new drugs, mostly monoclonal antibodies, aimed at re-educating the immune response, thus evaluating the possibility of using MSCs or their secretome as adjuvant therapy. O-22. INHIBITION OF HUMAN MESOTHELIOMA PROGRESSION IN A MOUSE XENOGRAFT MODEL BY MICRO-FRAGMENTED FAT (MFAT) Valentina Coccè 1 , Silvia La Monica 2 , Mara Bonelli 2 , Giulio Alessandri 1,3 , Roberta Alfieri 2 , Costanza Annamaria Lagrasta 2 , Caterina Frati 2 , Lisa Flammini 4 , Aldo Giannì 1,5 , Luisa Doneda 1 , Francesco Petrella 1,6,7 , Francesca Paino 1 , Augusto Pessina 1 1 CRC StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Italy 2 Department of Medicine and Surgery, University of Parma, Italy 3 Image Regenerative Clinic, Milan, Italy 4 Food and Drug Department, University of Parma, Italy 5 Maxillo-Facial and Dental Unit, Fondazione Ca’ Granda IRCCS Ospedale Maggiore Policlinico, Milan, Italy 6 Department of Thoracic Surgery, IRCCS European Institute of Oncology, Milan, Italy 7 Department of Oncology and Hemato-Oncology, University of Milan, Italy Objective Malignant Pleural Mesothelioma (MPM) is a tumour related to asbestos exposure with no effective therapy and a poor prognosis. Our previous studies demonstrated an in vitro and in vivo inhibitory effect of adipose tissue-derived Mesenchymal Stromal Cells (MSCs) or their derivatives (conditioned media, cellular lysates) on MPM. The purpose of this study was to verify whether fat tissue (FAT), a natural container of MSCs after micro-fragmentation (MFAT) was able to exert a similar inhibitory action on the growth of the human MPM cell line (MSTO-211H) xeno-transplanted in immunodeficient mice. Materials and Methods MFAT was prepared according to standardised methods using Lipogems device. MSCs were obtained by enzymatic digestion of MFAT. The in vitro effect of MFAT on MSTO-211H cell proliferation was analysed using transwell inserts and measuring the absorbance by a crystal violet assay. PBS were used as negative control. For in vivo experiments, Balb/c-Nude female mice were subcutaneously injected with 10 6 MSTO-211H cells suspended in Matrigel/PBS. Mice were randomised into 4 groups: control, paclitaxel (PTX), MSCs and MFAT. After a week from injection (time 0), vehicle alone (control group) or PTX (20 mg/kg) were administered intraperitoneally (IP) and MSCs (5 × 10 5 ) or MFAT (200 µL) were subcutaneously injected close to the tumour. On days 0, 7, and 14, the size of tumour nodules was measured and on day 20, nodules were collected. Morphometric evaluation of xenograft composition was performed on Masson’s trichrome-stained sections. Results The in vitro exposure of MSTO-211H cells to MFAT produced a dose-dependent inhibition of cell proliferation. In the in vivo study, the measures of volume of growing tumour mass indicated that the in situ treatment with MFAT produced an important inhibition similar to those obtained in mice treated with the anticancer drug PTX. A trend of inhibition, but not significant, was also observed in mice treated with free MFAT derived MSCs. The morphometric analysis of the tumour xenograft did not show significant differences among groups. Conclusions Our results show that MFAT, injected in situ, produced a significant ( p < 0.05) inhibition of the MSTO-211H growth both in vitro and in vivo, and was even comparable to IP PTX treatment. Interestingly, the treatment with free MSCs (5 × 10 5 ), at a similar amount contained in around 1 mL of MFAT, exerted only a little anticancer activity.
P-01. ANTIMICROBIAL ACTIVITY OF CANINE ADIPOSE TISSUE-DERIVED LYOSECRETOME Valentina Andreoli 1 , Costanza Spadini 1 , Mattia Iannarelli 1 , Priscilla Berni 1 , Virna Conti 1 , Roberto Ramoni 1 , Elia Bari 2 , Silvia Dotti 3 , Clotilde Cabassi 1 , Maria Luisa Torre 2 and Stefano Grolli 1 1 Department of Veterinary Science, University of Parma, Italy 2 Department of Pharmaceuticals Sciences, University of Piemonte Orientale, Novara, Italy 3 Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Brescia, Italy Objective Mesenchymal Stem/Stromal Cells (MSCs) have been studied and applied as therapeutics in regenerative medicine based on their peculiar properties. Although several papers demonstrate the efficacy of MSC-based therapy in preclinical models, clinical applications are still limited due to doubts about the safety of treatment with viable cells. MSCs secretome is a cell-free product that maintains a large part of cells’ therapeutic properties providing soluble and insoluble bioactive molecules involved in cellular crosstalk. Recent data support an antibacterial activity for both MSCs and their secretome. In this work, canine Lyosecretome (c-Lyo), a freeze-dried secretome prepared from adipose tissue-derived MSCs, has been tested to evaluate its antimicrobial activity against some of the most common canine pathogens involved in infections of the gastrointestinal tract, skin, and ears. Pathogens were subjected to a Minimal Inhibitory Concentration (MIC) assay to evaluate the amount of c-Lyo necessary to inhibit bacterial/fungal growth. Materials and Methods c-Lyo was resuspended in 500 µL of sterile saline (0.9%) and tested at a concentration range of 20–0.04 mg/mL. Nine replicates for each assay were performed. Tested reference bacteria and yeasts were E. coli , S. Typhimurium , S. aureus , Methicillin-Resistant S. aureus (MRSA), S. pseudintermedius , P. aeruginosa, and M. pachydermatis . Strains were amplified for 18–24 h at 37 °C to bring them into logarithmic growth phase and added to the plate at 5 × 10 5 CFU/mL and incubated at 37 °C overnight. MIC plate reading was performed with a reverse mirror. Mannitol was used as an internal control. Results A fair inhibitory activity of c-Lyo against both Gram-positive and Gram-negative bacteria was observed. Growth inhibition was higher for Gram-positive (2.2 < MIC < 9.8 mg/mL) but positive results were obtained even on Gram-negative bacteria (10.5 < MIC < 26.1 mg/mL). c-Lyo demonstrated an inhibitory activity also against the yeast M. pachydermatis (MIC = 13.3 mg/mL) supporting a possible efficacy in skin infections. Conclusions MIC data suggest that canine c-Lyo exerts inhibitory activity against various bacterial and yeast pathogens. The availability of an off-the-shelf, ready-to-use MSCs secretome acting as an antibacterial agent could help in replacing/supporting traditional antibiotics therapy, decreasing their use in veterinary medicine, as requested to control the spread of antibiotic resistance. P-02. SECRETOME ISOLATED FROM MESENCHYMAL STROMAL CELLS LOADED WITH PACLITAXEL HAVE CYTOTOXIC EFFECT ON OSTEOSARCOMA CELL LINES Alessia Giovanna Santa Banche Niclot 1 , Elena Marini 1 , Ivana Ferrero 2 , Camilla Francesca Proto 1 , Francesco Barbero 3 , Ivana Fenoglio 3 , Alessandro Barge 4 , Valentina Coccè 5 , Francesca Paino 5 , Katia Mareschi 1,2 and Franca Fagioli 1,2 1 Department of Public Health and Paediatrics, The University of Turin, Italy 2 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 3 Department of Chemistry, University of Turin, Italy 4 Department of Drug Science and Technology, University of Turin, Italy 5 CRC StaMeTec, Department of Biomedical, Surgical and Dental Sciences, University of Milan, Italy Objective Bone sarcomas are rare tumours that constitute a very aggressive disease for children and adolescents and represent still an important challenge for clinicians. Our aim is to develop a new method of drug delivery (DDS) based on the Extracellular Vesicles (EVs) loaded with Paclitaxel (PTX) to use for osteosarcoma (OS) treatment. We performed pre-clinical studies to test the cytotoxic effect of Secretome isolated from Mesenchymal Stromal Cells (MSCs) loaded with PTX (PTX-MSCSecr) on OS cell lines (MG63 and SJSA). Materials and Methods We first calculated the PTX-IC50 in 3 MSC batches, then, we isolated PTX-MSC-Secr by loading MSCs with PTX at the concentration of 15 µg and we analysed its cytotoxic effect on MG63 and SJSA after treatment for 5 days using MTT test. We also analysed the size distribution, particle concentration, and Zeta potential of EVs present in PTX-MSC-Secr by Nanoparticle Tracking Analysis (NTA) instrument. The secretome ability to have EVs with encapsulated PTX was analysed by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Results PTX-IC50 for MSCs was a mean of 25.1 ± 3.6 µg and for the OS cell lines was, respectively a mean of 17.5 ± 1.0 µg for MG63 and 30 ± 3.1 µg for SJSA. PTX-MSC-Secr induced a decrease of the viability of cells 43 ± 11% and 36 ± 22%, respectively in SJSA and MG63 after five days of treatment. This effect was dose-dependent because scalar dilutions of PTX-MSC-Secr reduced the cytotoxic effect. Cell viability did not decrease after treatment with Secr isolated from CTRL-MSCs (viability of 87 ± 5% in SJSA and of 91 ± 12% in MG63). The dimensional analyses, the result of three independent experiments, indicated EVs mean sizes of 175.8 ± 13 nm (CTRL) and 165.7 ± 11 nm (PTX loaded). EVs Zeta potentials of both CTRL and PTX loaded, as expected, were found to be negative with mean values of −40 ± 2 mV and −38 ± 5 mV. Conclusions We demonstrated the cytotoxic effect of PTX-MSC-Secr in OS cell lines. The NTA analyses showed a typical mean size of EVs and that no significant differences between the secretome batches and between experimental conditions (CTRL and PTX loaded) were observed. The experiments we performed have provided promising preliminary data that need further investigation but the EVs ability to encapsulate PTX allows us to propose the EVs-PTX isolated from the MSCs as ideal candidates for drug delivery as innovative paediatric sarcomas treatment. P-03. 3D BIOPRINTED CONTROLLED RELEASE SCAFFOLD CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME FOR BONE REGENERATION: STERILE MANUFACTURING AND IN VITRO BIOLOGICAL EFFICACY Elia Bari 1 , Franca Scocozza 2,3 , Sara Perteghella 4,5 , Marzio Sorlini 5,6 , Lorena Segale 1 , Lorella Giovannelli 1 , Ferdinando Auricchio 2,3 , Michele Conti 2,3 and Maria Luisa Torre 4,5 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Pavia, Department of Civil Engineering and Architecture, Pavia, Italy 3 P4P S.r.l., Pavia, Italy 4 University of Pavia, Department of Drug Sciences, Pavia, Italy 5 PharmaExceed S.r.l., Pavia, Italy 6 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland Objective The present study proposes the design and sterile manufacturing of 3D-printed polycaprolactone (PCL) scaffolds enriched with mesenchymal stem cell (MSC)-secretome for bone tissue regeneration and evaluates their in vitro biological efficacy. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Porous parallelepiped-shaped PCL scaffolds (10 × 10 × 3 mm) were prepared by co-printing PCL with an alginate hydrogel (10% w / v ) containing lyosecretome (0.25 mg). Scaffolds were tested for sterility and microbiological tests, as indicated in the EuPh 2.6.27 and 2.6.1 chapters. In addition, the scaffold colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy, and dosage of osteocalcin by ELISA). Scaffolds without lyosecretome were used as a control. Results Sterile scaffolds have been obtained and lyosecretome enhanced their colonisation by MSCs: MSCs showed a spread morphology with the initial formation of filopodia, with more frequent and complex cellular processes, overall indicating the cytocompatibility of the scaffold. Lyosecretome also sustained MSC differentiation towards the bone line in an osteogenic medium. Indeed, after 14 days, the amount of mineralised matrix detected by alizarin red was significantly higher for lyosecretome scaffolds. Likely, the amount of osteocalcin, a specific bone matrix protein, was significantly higher at all the times considered (14 and 28 days) for the lyosecretome scaffolds. Confocal microscopy further confirmed such results, demonstrating improved osteogenesis with lyosecretome scaffolds after 14 and 28 days. Conclusions Overall, these results prove the role of MSC-secretome, co-printed in PCL/alginate scaffolds, in inducing bone regeneration; sterile scaffolds containing MSC-secretome are now available for in vivo preclinical tests of bone regeneration. P-04. OSTEOINDUCTIVE AND OSTEOCONDUCTIVE PROPERTIES OF TITANIUM CAGES CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME Elia Bari 1 , Sara Perteghella 2,3 , Marzio Sorlini 3,4 , Delia Mandracchia 5 , Lorella Giovannelli 1 , Lorena Segale 1 and Maria Luisa Torre 2,3 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Pavia, Department of Drug Sciences, Pavia, Italy 3 PharmaExceed S.r.l., Pavia, Italy 4 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland 5 University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy Objective The present study investigates the capacity of the mesenchymal stem cell (MSC)-secretome, formulated as a ready-to-use and freeze-dried medicinal product (the lyosecretome), to promote the osteoinductive and osteoconductive properties of titanium cages. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining lyosecretome (0.1 × 10 6 cell equivalents/mg). Lyosecretome was added to titanium cages (1 × 1 × 0.3 cm in size) kindly provided by MT Ortho and manufactured by an additive manufacturing technology called electron beam melting. The cages colonisation by MSCs was investigated by SEM, and in vitro biological efficacy was investigated by MSC osteogenic differentiation and matrix production tests (alizarin red, confocal microscopy and dosage of osteocalcin by ELISA). Cages without lyosecretome were used as a control. Results After 14 days, in the presence of lyosecretome, significant cell proliferation improvement was observed. Scanning electron microscopy revealed the cytocompatibility of titanium cages: the MSCs seeded showed a spread morphology and the initial formation of filopodia. After seven days, in the presence of lyosecretome, more frequent and complex cellular processes forming bridges across the porous surface of the scaffold were revealed. Moreover, after 14 and 28 days of a culture in an osteogenic medium, the amount of mineralised matrix detected by alizarin red was significantly higher when lyosecretome was used. Finally, improved osteogenesis with lyosecretome was confirmed by confocal analysis after 28 and 56 days of treatment and demonstrating the production by osteoblast-differentiated MSCs of osteocalcin, a specific bone matrix protein. Conclusions Overall, these results confirm the role of MSC-secretome in combination with titanium cages in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing. P-05. OSTEOINDUCTIVE AND OSTEOCONDUCTIVE PROPERTIES OF BIOHYBRID BOVINE MATRIX CONTAINING MESENCHYMAL STEM/STROMAL LYOSECRETOME Elia Bari 1 , Ilaria Roato 2 , Giuseppe Perale 3,4,5 , Filippo Rossi 6 , Tullio Genova 7 , Federico Mussano 2 , Riccardo Ferracini 8 , Marzio Sorlini 9,10 , Sara Perteghella 1,10 and Maria Luisa Torre 1,10 1 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy 2 University of Torino, Department of Surgical Sciences, CIR-Dental School, Torino, Italy 3 Industrie Biomediche Insubri SA, Mezzovico-Vira, Switzerland 4 University of Southern Switzerland (USI), Faculty of Biomedical Sciences, Lugano, Italy 5 Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria 6 Politecnico di Milano, Department of Chemistry, Materials and Chemical Engineering Milan, Italy 7 University of Torino, Department of Life Sciences and Systems Biology, Turin, Italy 8 University of Genova, Department of Surgical Sciences and Integrated Diagnostics, Genoa, Italy 9 University of Applied Sciences and Arts of Southern Switzerland, SUPSI, Department of Innovative Technologies, Lugano, Switzerland 10 PharmaExceed Srl, Pavia, Italy Objective The present study combines biohybrid bone substitute scaffolds (SB) with lyosecretome, a freeze-dried MSC-secretome formulation containing proteins and extracellular vesicles and evaluates the osteoinductive and osteoconductive in vitro. Materials and Methods Adipose MSCs were cultured in DMEM/F12 with 5% platelet lysate (PL); secretome release was obtained by 48 h PL starvation. Supernatants were ultrafiltered, cryoprotectant was added, and freeze-dried, obtaining Lyosecretome (0.1 × 10 6 cell equivalents/mg). Each SB scaffold (1 × 1 × 0.3 cm in size) was loaded with 16 × 10 3 cell equivalents of Lyosecretome by an absorption method, obtaining SBlyo. 1 × 10 6 MSCs were seeded onto the upper surface of SB in an osteogenic medium, and after 14 and 60 days of cultures, gene expression for osteocalcin (OCN), alkaline phosphatase (ALP), and collagen 1 (COLL-1) was evaluated. After 60 days, a high-resolution X-ray microtomography was used to identify the newly formed mineralised tissue after cell colonisation on SB and SBlyo. Moreover, SB and SBlyo were fixed, decalcified, dehydrated, cut into thin sections, and stained with hematoxylin and eosin (H&E) for morphological analyses. Immunohistochemical analysis was also performed using antibodies for osteocalcin and TGF-β. Results After 14 days, significant cell proliferation improvement was observed on SBlyo with respect to SB, where cells filled the cavities between the native trabeculae. For SB, on the other hand, the process was still present, but tissue formation was less organised at 60 days. On both scaffolds, cells differentiated into osteoblasts and were able to mineralise after 60 days. SBlyo showed a higher expression of osteoblast markers and a higher quantity of newly formed trabeculae than SB alone. The quantification analysis of the newly formed mineralised tissue and the immunohistochemical studies demonstrated that SBlyo induces bone formation more effectively. This osteoinductive effect is likely due to the osteogenic factors in the lyosecretome, such as fibronectin, alpha-2-macroglobulin, apolipoprotein A, and TGF-β. Conclusions Overall, these results confirm the role of MSC-secretome loaded on biohybrid bovine matrix in inducing bone regeneration. Such scaffold prototypes for bone regenerative medicine are now available for further in vivo safety and efficacy testing. P-06. LOCAL AND SYSTEMIC APPLICATION OF AUTOLOGOUS MESENCHYMAL STROMAL CELLS IN CATS SUFFERING FROM CHRONIC GINGIVOSTOMATITIS: A PILOT STUDY Priscilla Berni 1 , Tommaso Magni 2 , Maurizio Del Bue 3 , Virna Conti 1 , Valentina Andreoli 1 , Rosanna Di Lecce 1 , Anna Maria Cantoni 1 , Roberto Ramoni 1 , Stefano Grolli 1 1 Department of Veterinary Medical Science, University of Parma, Italy 2 Veterinary Practitioner, Clinica Veterinaria Pet Care, Bologna, Italy 3 Freelance Veterinary Medical Doctor, Parma, Italy Objective Feline Chronic Gingivostomatitis (FCGS) is a severe inflammatory oral disease characterised by painful mucosal lesions, oral discomfort, inappetence, reduced grooming, weight loss, and hypersalivation, seriously affecting the patient’s quality of life. The current standard of care is invasive full/near-full mouth tooth extraction and long-term pharmacological treatments, with a high rate of relapse. Since FCGS is probably immune-mediated, Mesenchymal Stromal Cells (MSCs) represent a promising tool for this disorder. Different studies have reported the efficacy of systemic administration of adipose-derived MSCs (Ad-MSCs) in cats with FCGS, while a pilot study reported a lack of efficacy when the treatment is performed prior to full-mouth tooth extraction. This study aims to determine the efficacy of local and systemic administration of Ad-MSCs in cats with FCGS, with or without teeth. Materials and Methods Eleven client-owned cats with FCGS and with long-term pharmacological clinical history, with or without teeth, were treated with a double application of autologous Ad-MSCs at 30-day intervals. The cats were enrolled in two groups: one was treated with local injections of 5 × 10 6 autologous Ad-MSCs and the other was treated with local injections associated with systemic infusions of 2 × 10 6 /Kg autologous Ad-MSCs. An oral examination with photographs and oral biopsies was performed at the enrolment and 30 days after each treatment. A SDAI (Stomatitis index) scoring was calculated at the same intervals, in addition to a brief owner questionnaire and a veterinarian scoring. Furthermore, a complete blood count, blood immune cell phenotyping, and biochemical profile were planned on day zero and three months after the first treatment. Results At the time of writing, eight cats have been treated with double MSCs application. Seven cats have completely suspended any pharmacological treatment after the first application. The clinical assessment at day 60 showed a marked clinical improvement reported by the owners, except for one patient that showed the maximum SDAI score at the enrolling who improved only in the body weight parameter. A statistically significant difference was observed in the SDAI between day 0 and 60 for seven cats, two with a complete resolution of the oral inflammation ( p < 0.05). Conclusions Immunohistochemical analysis and blood immune cell phenotyping are needed to confirm the observed clinical improvement. P-07. A NEW PROTOCOL FOR VALIDATION OF CHONDRO, ADIPO AND OSTEO DIFFERENTIATION KIT OF CULTURED ADIPOSE-DERIVED STEM CELLS (ADSC) BY REAL-TIME RT-QPCR Carlotta Castagnoli 1 , Valentina Daprà 2,3 , Daniela Alotto 1 , Stefania Casarin 1 , Stefano Gambarino 2,3 , Mara Fumagalli 1 , Sara Castiglia 4 , Deborah Rustichelli 4 , Maddalena Dini 3 , Ilaria Galliano 2 , Massimiliano Bergallo 2,3 1 Skin Bank, Department of General and Specialized Surgery, University Hospital City of Health and Science of Turin, Italy 2 Department of Public Health and Pediatric Sciences, University of Turin, Italy 3 BioMole srl, Spin-off University of Turin, Italy 4 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy Objective Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose-derived Stem Cells) show great potential for degenerative disease treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validate a new commercially available kit able to explore changes in gene expression in human ADSC subjected to osteogenic, adipogenic, and chondrogenic differentiation. Materials and Methods As the primary outcome of the study, we selected better indicators of trilineage differentiation using the third passage of cultured ADSC isolated from the stromal vascular fraction (SVF) by enzymatic digestion. ACAN, FABP4A, and Col11a1 were selected as indicators of chondrogenic, adipogenic, and osteogenic differentiation, respectively based on statistically significant results. As a secondary outcome of the study, an in vitro ageing test was performed from passage 2 to 6 to evaluate the highest differentiation potential. Total RNA extraction from differentiated and control ADSC were performed. Relative quantifications of mRNA expression of selected genes was completed according to rt-PCR kit protocol. Results ACAN detection, a test for chondrogenic differentiation, revealed equivalent ∆∆Ct values between P3 and P6. These were considered equivalent passages for induction differentiation tests. FABP4 detection, an assay for adipogenic differentiation, showed similar results for all cell passages tested so they can all be considered suitable in differentiation assay induction; on the contrary, only passage P6 for Col11a1 was suitable for osteogenic differentiation. Conclusions In conclusion, we validated a new real-time rt-QPCR protocol able to evaluate osteogenic, chondrogenic, and adipogenic ADSC differentiation in vitro. P-08. EVALUATION OF THE EFFECTS OF HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS AND AMNIOTIC MEMBRANE CONDITIONED MEDIUM ON OVARIAN CANCER CELLS USING 2D AND 3D CELL MODELS Paola Chiodelli 1 , Patrizia Bonassi Signoroni 1 , Silvia De Munari 1 , Andrea Papait 2 , Sara Ficai 2 , Antonietta Silini 1 and Ornella Parolini 2,3 1 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy 2 Department of Life science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 3 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy Objective Ovarian cancer is the seventh most common cancer and sixth most common cause of cancer death for women globally. Nowadays, surgical resection followed by chemotherapy is the standard of care. However, a number of patients are faced with recurrence due to tumour dissemination and acquired chemoresistance. Therefore, the novel alternative approaches targeting ovarian cancer cells are urgently needed to improve the standard of care for patients. With this regard, mesenchymal stromal cells (MSC) constitute a compelling therapeutic option. Of particular interest, MSC isolated from the amniotic membrane of the human term placenta (hAMSC) are of therapeutic interest and present noteworthy advantages when compared to MSC from other sources, such as their ease of recovery from biological waste. In addition, we previously reported that the hAMSC secretome has antiproliferative effects in vitro when co-cultured with different tumour cells. Material and Methods We decided to evaluate the possible anti-proliferative effects of the secretome (conditioned medium, CM) from hAMSC in 2D and 3D models of ovarian cancer. In parallel, we evaluated the CM from the intact amniotic membrane (hAM) to see if antiproliferative effects could be maintained without the need to perform MSC isolation. Results Both CM-hAMSC and CM-hAM inhibit the proliferation and migration of ovarian cancer cells (HEY and SKOV-3) in 2D scratch assays. Moreover, both CM-hAMSC and CM-hAM affect the apoptotic process in HEY and SKOV-3. In the SKOV-3 spheroid 3D model, CM-hAMSC and CM-hAM significantly decrease the spheroid area inducing also a change in spheroid morphology. Conclusions The data so far collected indicated that CM-hAMSC and CM-hAM impact the growth and activity of ovarian cancer cells. Further experiments are needed to better understand their inhibitory capacity on ovarian cancer cells in 2D and in 3D models and clarify their use as a potential adjuvant therapeutic strategy able to target tumour cells. P-09. EXTRACELLULAR VESICLES FROM CORD BLOOD MESENCHYMAL STROMAL CELLS STIMULATE REGENERATION PROCESS IN ORGANOTYPIC MODEL OF NEURONAL INJURY Stefania D’Agostino 1,2 , Francesca Cecchinato 2,4 , Beatrice Auletta 2,4 , Marcin Jurga 3 , Maurizio Muraca 1,2 , Anna Urciuolo 2,4 , Michela Pozzobon 1,2 1 Department of Women’s and Children’s Health, University of Padova, Italy 2 Institute of Pediatric Research (IRP) Città della Speranza, Padova, Italy 3 EXO Biologics (SA), Belgium 4 Department of Molecular Medicine, University of Padova, Italy Objective The central nervous system (CNS) has only a limited capacity to regenerate, hence, after injury, a progressive loss of neurons, due to homeostatic imbalance, leads to neurodegenerative pathology. The homeostasis process critically depends on the interaction between neurons and glial cells. Novel treatment suggestions for neurodegenerative disorders consider the use of cell-derived products, relying on the beneficial paracrine effects of the applied products. Extracellular vesicles (EVs) recently emerged as versatile messengers in CNS cell communication. These nanoparticles, defined by a phospholipid bilayer, can convey signals by triggering surface receptors, activating second messenger signalling cascades or delivering their cargo, such as proteins, nucleic acids, and small molecules. In this work, we considered an organotypic in vitro model of spinal injury treated with EVs derived by Wharton Jelly Mesenchymal stromal cells. Materials and Methods Isolated spinal cords from rat foetuses were cut into small pieces and cultured on a bed of Matrigel. Organotypic spinal cord (oSpC) were treated with EVs soon after the beginning and after 24 h of culture. To monitor the early effect of the EVs on neural axon sprouting, samples were analysed 48 h after the seeding. Results Our results showed that neural axon sprouting was significantly increased in EV-treated samples when compared to untreated oSpCs. Moreover, the neural protein TujI/TUBB3 expressed in the developing neurons and the glia marker GFAP, that identified new astrocytes, were differently detected in the presence of EVs. Fluo-4 imaging revealed a more controlled calcium flux in oSpC treated with EVs at the axonal projection compared to the untreated SpC. Real-time PCR on neural miRNA highlighted how the EVs can be responsible for miRNA mediator of neural regeneration. Conclusions Our results suggest a possible role of perinatal EVs in promoting neural axon sprouting, opening new perspectives for their application in neural regeneration, and as new exciting signalling modalities that add a new dimension to the interaction between neurons and glial cells. P-10. 3D ORGANOIDS: A NEW TRANSLATIONAL APPROACH FOR THE ASSESSMENT OF THE IMMUNOMODULATORY ACTIVITY OF MESENCHYMAL STROMAL CELLS DERIVED EXTRACELLULAR VESICLES IN INFLAMMATORY BOWEL DISEASE Giada de lazzari 1,2,3,5,6 , David Sagnat 5,6 , Ricardo Malvicini 1,2,3 , Michela Pozzobon 1,2 , Gustavo Yannarelli 4 , Anna Maria Tolomeo 1,2,3 , Nathalie Vergnolle 5,6,7 , Maurizio Muraca 1,2,3 1 Foundation Institute of Pediatric Research Città della Speranza, Padova, Italy 2 Dept of women’s and children’s health, University of Padova, Italy 3 L.I.F.E.L.A.B. Program, Consorzio per la Ricerca Sanitaria (coris), Veneto Region, Padova, Italy 4 Instituto de Medicina Traslacional, Trasplante y Bioingeniería (imettyb-conicet), Buenos Aires, Argentina 5 Toulouse Organoid Platform, France 6 Inserm, Toulouse, France 7 University of Calgary, Faculty of Medicine, Department of Physiology and Pharmacology, Calgary, AB, Canada Objective Inflammatory bowel diseases (IBD), including Crohn’s disease (CD) and Ulcerative Colitis (UC), are chronic relapsing–remitting disorders characterised by inflammation of the gut. Different factors contribute to IBD development, such as deficiencies in epithelial integrity, immune response mechanisms, and mucosal barrier function, whose complexity is difficult to reproduce in experimental conditions. The need to overcome the typical limits of cell lines, animal models, and organ culture, led to the development of a 3D culture system capable of maintaining the characteristics of the target organ in the long term. We thus explored the feasibility of this tool to evaluate the therapeutic potential of extracellular vesicles derived from mesenchymal stromal cells (MSC-EVs) whose immunomodulatory activity is the object of increasing interest. Materials and Methods MSC-EVs were isolated from human umbilical cord MSCs by ultrafiltration (100 kD cutoff), quantified by TRPS and characterised for established markers by MACSPlex. Colon organoids were derived from human colon samples’ extracted crypts and seeded in Matrigel beads. Inflamed (IBD) organoids, in particular, were obtained from the stimulation of control organoids with a pro-inflammatory cocktail (IL-1β, TNF-α, and IL-6, at 100 ng/mL) on days 3, 5, and 7 to reproduce a chronic inflammatory state. To evaluate the effect of MSC-EVs, IBD organoids were then treated on day 7 with a dose of 1 × 10 9 MSC-EVs/mL at different time points (3 h, 6 h, 24 h, and 48 h). As readouts, we evaluated the effect on proliferation, differentiation, inflammation, barrier, and growth by immunofluorescence and molecular biology. Results We found that the stimulation with the pro-inflammatory cocktail increases inflammation (IL8, MCP1, and TNF- α), stress (Chop), carcinogenesis (Wnt5a), growth (OCT4, SOX9), proliferation (EGF), and decreases differentiation (MUC2) and epithelial barrier marker (Epcam) compared to control organoids. Treatment with MSC-EVs at 24 h decreased inflammation, stress, growth, proliferation, carcinogenesis, and increased differentiation and epithelial barrier marker. Conclusions In conclusion, we have provided preliminary evidence of the therapeutic effect of MSC-EVs in inflamed intestinal organoids. These results suggest that the present 3D model could represent a useful experimental tool closely reproducing some critical features of human IBD. P-11. CHARACTERIZATION OF HUMAN DENTAL PULP STEM CELLS MULTICELLULAR SPHEROIDS AS ORGANOTYPIC THREE-DIMENSIONAL IN VITRO MODEL Ilaria De Santis 1,2 , Alessandro Bevilacqua 3,4 , Thimios A. Mitsiadis 2 , Deborah Stanco 2 1 University of Bologna, Interdepartmental Centre Alma Mater Research Institute on Global Challenges and Climate Change (Alma Climate), Bologna, Italy 2 University of Zürich, Orofacial Development and Regeneration, Institute of Oral Biology, Zürich, Switzerland 3 University of Bologna, Advanced Research Center on Electronic Systems (ARCES) for Information and Communication Technologies “E. De Castro”, Bologna, Italy 4 University of Bologna, Department of Computer Science and Engineering (DISI), Bologna, Italy Objective Dental pulp stem cells (DPSCs) are mesenchymal stem cells (MSCs) of neural crest origin. High availability, multipotency, and plasticity make them a promising source of patient-specific MSCs for personalised regenerative medicine strategies. However, their clinical translation still faces many challenges due to a lack of deep understanding of their niche microenvironment, biology, and functionality in vivo. By recapitulating the complex in vivo-like microenvironment, three-dimensional (3D) multicellular spheroids open to new opportunities for MSCs translation in clinical and preclinical settings. In this context, the development of human DPSC multicellular spheroids as organotypic 3D in vitro models was assessed. Material and Methods 2 × 10 4 DPSCs at passage IV were used for spheroid creation by hanging drop technique and transferred to 96 ULA plates after 24 h. Spheroid morphology, viability (FDA-PI staining), and metabolic activity (Alamar Blue assay) were evaluated at day 1, 2, 3, 4, and 7 of culture. Tissue-specific (nestin, vimentin, collagen I and IV, laminin, and fibronectin) and stem-related (BMI1, CD90, SOX2, NANOG, and OCT4) markers were evaluated at gene (qRT-PCR) and protein level (IF). After bright field imaging and by on-purpose method for automated image analysis, spheroid dimension, morphology, and compactness were quantified by their equivalent diameter (ED), sphericity (S), and border indentation (BI), respectively. After normality check by Shapiro–Wilk test, statistical significance was assessed by t -test or Wilcoxon test ( p < 0.05). Results The hanging drop technique has a mean efficiency of spherical (S ≥ 80%) spheroid creation at 82% in 24 h. ED progressively shrinks, while S rises up to day four. At day three, spheroids show highest viability (I FDA /I PI = 12.8, p < 10 −6 ) and the most stable morphology (ED = 488 ± 43 µm, S = 0.87 ± 0.04, BI = 0.79 ± 0.03). Spheroid metabolic activity was stable in time and significantly lower than standard 2D culture (−35% avg, p < 0.03). DPSC spheroids expressed high levels of nestin, vimentin, collagen I and IV, laminin, and fibronectin, beside significantly higher mRNA levels of BMI1, CD90, SOX2, NANOG, and OCT4. Conclusions The human DPSC spheroids can be easily and quickly created by a low-cost procedure in 24 h and they may efficiently enhance the therapeutic action of DPSCs. Moreover, automated image analysis here proves as a valuable tool for the finest analysis of DPSC spheroid morphology in future preclinical setting applications. P-12. CELL-FREE ORTHOBIOLOGICS FROM ADIPOSE MESENCHYMAL STEM/STROMAL CELLS: THE ROAD SO FAR AND FUTURE PERSPECTIVES IN OSTEOARTHRITIS TREATMENT Elena Della Morte 1 , Chiara Giannasi 1,2 , Stefania Niada 1 and Anna Teresa Brini 1,2 1 IRCCS Istituto Ortopedico Galeazzi, Milan, Italy 2 University of Milan, Department of Biomedical Surgical and Dental Sciences, Milan, Italy Objective Mesenchymal Stem/stromal Cells, and, in particular, adipose-derived ones (ASCs), show great therapeutic potential in counteracting orthopaedic conditions. Since a large part of ASC action is exerted through paracrine signalling, in the last years, we focused on the study of their conditioned medium (CM) in terms of molecular composition and biological action on experimental models of osteoarthritis (OA). Materials and Methods The CM was obtained from confluent ASCs cultured for 72 h in the absence of FBS, then concentrated through filter devices with a 3 kDa cut off. Its components were investigated through Raman Spectroscopy, NTA, -omic approaches, and ELISA. Articular chondrocytes (CHs) and osteochondral explants (OEs) were harvested from patients undergoing arthroplasty at IRCCS Istituto Ortopedico Galeazzi prior to approval of the ethics committee. OA phenotype was induced by stimulation with 10 ng/mL TNFα, and specimens were treated with the CM derived from 0.5–1 × 10 6 ASCs. The levels of inflammatory, hypertrophic, and catabolic factors were monitored through time by immunological or enzymatic assays. Results ASC-CM peculiar Raman fingerprint and its characteristic vesicular profile were depicted. The analysis of ASC-CM composition showed the presence of stable levels of bioactive factors that can be putative players in counteracting the OA process. Among other potential effectors, the abundance of the chondroprotective factors Dkk-1 and TIMP-1/2 was particularly intriguing. Accordingly, in both experimental OA models, i.e., articular chondrocytes (CHs) and osteochondral explants (OEs), ASC-CM efficiently buffered the TNFα-induced aberrant activity of matrix metalloproteinases. Furthermore, ASC-CM reverted TNFα-induced expression of PGE2 and COL10A1 in CHs, while it lowered the catabolic release of glycosaminoglycans on OEs. Conclusions These in vitro and ex vivo experiments confirm ASC-CM beneficial potential in dampening OA-related hallmarks, encouraging deeper investigations of this product in the perspective of its future clinical translation as a cell-free orthobiologic. P-13. LATTICE-BASED SCAFFOLDS FOR THE BULK REINFORCEMENT OF SOFT MATERIALS FOR CARTILAGE REGENERATION Stephanie E. Doyle 1,2 , Finn Snow 1 , Rhyys Turner 1 , Carmine Onofrillo 2,3,4 , Cathal D. O’Connell 1,2 , Claudia Di Bella 2,3,5 , Elena Pirogova 1 , Serena Duchi 2,3,4 1 Electrical and Biomedical Engineering, School of Engineering, RMIT University, Melbourne, Victoria, Australia 2 ACMD, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia 3 Department of Surgery, The University of Melbourne, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia 4 ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, University of Wollongong, Wollongong, NSW, Australia 5 Department of Orthopaedics, St Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia Objective Hydrogels are a fundamental element of cartilage engineering by providing a suitable environment for cells to proliferate, migrate, and differentiate. However, this typically soft environment is often not suitable under high mechanical loads. This can present an issue for implantable scaffolds where a large stiffness mismatch between the implant and native tissue can contribute to its failure. With this work we prioritise the bulk mechanical properties of the implant to reduce the stiffness mismatch with the native tissue. We aim to do this without negatively impacting the environment, previously established by our team, for mesenchymal stem/stromal cells (MSCs) to create new cartilaginous tissue. Materials and Methods Lattice scaffolds, designed to minimise the total volume fraction of the reinforcement scaffold, are made via the Negative Embodied Sacrificial Template (NEST) 3D printing process from polycaprolactone. The NEST scaffolds are combined with MSCs of adipose origin and embedded in a soft, 6% gelatin methacryloyl (GelMA) hydrogel and UV photo crosslinked. After six weeks of culture, samples are analysed by a combination of imaging (immunohistochemistry and histology staining), metabolic activity, glycosaminoglycan (GAG) content, compression testing, and quantitative polymerase chain reaction (qPCR). Results Using highly porous (up to 90%) lattice designs we have demonstrated the biocompatibility of the NEST scaffolds where we recorded no significant difference in metabolic activity between reinforced and control samples after six weeks in in vitro culture. Secondly, limited to no interference of the NEST scaffold on bulk cell differentiation as measured by GAG production, qPCR, and imaging. Third, the ability to match the native tissue stiffness from day 0, for example, native articular cartilage of the knee has a compressive modulus of ≈400–800 kPa and our reinforced NEST scaffold of a clinically relevant size embedded in 6% GelMA has a compressive modulus of ≈480 kPa. Conclusions Our reinforced hydrogels retain the ideal soft environment for cells to differentiate down the chondrogenic lineage while reducing the stiffness mismatch between the implant and native tissue. Therefore, we address one area, stiffness mismatch, where implants can fail when moving from an in vitro model to in vivo. P-14. MESENCHYMAL STEM CELLS AS A SYSTEM FOR DELIVERING NAB-PACLITAXEL IN A METASTATIC MODEL OF PDAC Benedetta Ferrara, Smeralda Rapisarda, Antonio Citro, Martina Policardi, Fabio Manenti, Chiara Gnasso, Annapaola Andolfo, Denise Drago, Lorenzo Piemonti Diabetes Research Institute, IRCCS San Raffaele, Milan, Italy Objective Current therapy for metastatic pancreatic ductal adenocarcinoma (PDAC) is limited by drug toxicity and resistance. A delivery system might improve drug bioavailability. The aim of this study was to investigate mesenchymal stem cells (MSCs) for delivering nab-paclitaxel (nPTX) in metastatic PDAC. The feasible manipulation of MSCs, their ability to deliver drugs and to do homing in the damaged site make them a suitable candidate for an autologous cell-therapy approach. Materials and Methods An in vivo model of liver metastases was generated by injection of K8484 murine PDAC cells derived from KPC mice into the portal vein of C57BL/6N mice. Firstly, tumour-bearing mice were treated with nPTX to assess their sensitivity to this drug by monitoring the metastatic volume by magnetic resonance. Secondly, the MSCs viability after treatment with nPTX, as well as drug uptake and release, were investigated in vitro. The biodistribution of luciferase (LUC)-transduced MSCs intraportally injected in tumour-bearing mice was investigated by in vivo imaging systems (IVIS). Finally, the effect on the metastatic reduction of nPTX-loaded MSCs was evaluated on tumour-bearing mice after intraportal injection. Results NPTX significantly reduced the metastatic volume of tumour-bearing mice, demonstrating the responsiveness of our model to this drug. In vitro, after 24 h, we could appreciate a high uptake of nPTX by MSCs without a reduction of cell viability. NPTX release by MSCs was higher after 24 h but sustained until 72 h, as reported by mass-spectrometry analysis. Biodistribution studies revealed a high and prolonged accumulation of MSCs in the liver after intraportal injection. Results obtained in vivo on the antitumour efficacy of this system reported a significantly higher metastatic reduction in mice treated with nPTXloaded MSCs with respect to control mice treated with not loaded MSCs or free nPTX. Conclusions The ability of MSCs to incorporate nPTX to a high extent and without reporting toxicity is promising. This system allowed to lower the drug dose, thus reducing nPTX toxicity and specifically targeting the tumour site reporting an effective reduction of the metastatic burden. Obtained results open new insights into the use of MSCs for delivering nPTX as a suitable and promising therapeutic option for PDAC. P-15. DISSECTING THE EFFECT OF MICROPLASTICS ON HUMAN AMNIOTIC MESENCHYMAL STROMAL CELLS Sara Ficai 1 , Andrea Papait 1,2 , Alice Masserdotti 1 , Patrizia Bonassi 3 , Antonietta Silini 3 , Ornella Parolini 1,2 1 Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy 3 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy Objective Environmental microplastic (MPs, 1 µm–5 mm) degradation has become a problem for human health due to the possible production of toxic metabolites, contaminating water, air, food, and several daily used products. Bisphenol A (BPA) is the most representative chemical component of MPs debris, and it is reported to alter cellular functions by acting as an endocrine disruptor. Endocrine disrupting chemicals (EDC) are able to interfere with endogenous hormone biosynthesis, metabolism, and functions through the binding with typical and peculiar oestrogen receptors, triggering a dysregulation in cellular physiological processes such as oxidative stress. Placental tissues are supposed to be susceptible to EDC for the abundance of hormone receptor expression. Thus, it has been hypothesised that exposure of the mother to MPs-derived chemicals, such as BPA, can lead to an imbalance of the physiological processes that contribute to a successful pregnancy, increasing the risk of gestation-related complications. Based on this evidence, our purpose is to evaluate the effects of BPA on mesenchymal stromal cells isolated from the amniotic membrane of the human term placenta (hAMSC). Materials and Methods Our preliminary experiments aimed to evaluate the impact of BPA on hAMSC properties and functions in vitro. Cellular viability, metabolism, and apoptotic rate were analysed after a 24 h exposure to increasing concentrations of BPA (0.1; 0.2; 0.3; 0.4 µM) by MTT assay, ATP lite assay, and PI/Annexin kit, respectively. Concomitantly, the loss of cellular oxidative balance has been assessed by flow cytometry 3 h and 24 h after BPA exposure. In addition, dysregulation in the expression of cell cycle mediators was evaluated by RT-PCR. Results We first observed a dose-dependent reduction in hAMSC viability and metabolic capacity as well as an enhancement in cellular apoptotic rate after the treatment with increasing concentrations of BPA. At the highest concentration of BPA used in our study, hAMSC-intracellular oxidative stress and the gene expression of typical cell cycle regulators both increased. Conclusions Our preliminary data suggest that BPA may affect hAMSC functions. In light of these observations, additional studies will be performed to evaluate intracellular pathways through which BPA can act, supposing that adverse consequences on placenta resident MSC may be related to a negative outcome of gestation and riskiness for the baby. P-16. HINTS ON THE METABOLIC STATUS OF SERUM-STARVED MESENCHYMAL STEM/STROMAL CELLS Chiara Giannasi 1,2 , Stefania Niada 2 , Elena Della Morte 2 and Anna Teresa Brini 1,2 1 University of Milan, Department of Biomedical Surgical and Dental Sciences, Milan, Italy 2 IRCCS Istituto Ortopedico Galeazzi, Milan, Italy Objective In the last decade, the scientific interest in the secretome of Mesenchymal Stem/stromal Cells (MSCs) has increased tremendously due to its promising potential as an alternative to cell therapy. Mid-term serum starvation represents a convenient strategy for secretome production since it stimulates cell secretion while reducing the drawbacks associated with the use of animal-derived supplements. Nevertheless, the impact of this procedure on the metabolic status of donor cells still needs to be defined. Here, we investigate this aspect through metabolomics by comparing MSCs cultured with 10% foetal bovine serum (FBS) and serum-starved ones. Materials and Methods Primary human adipose-derived MSCs were grown in complete culture medium until confluence. Then, cells were either collected and analysed or rinsed and cultured for three days in the absence of FBS. Samples were screened for polar and apolar molecules by untargeted metabolomics at the Proteomics and Metabolomics Facility of IRCCS Ospedale San Raffaele. The differences revealed by this analysis were further validated by ad hoc biochemical and functional assays. Results Differential metabolomics shows a clear clustering between samples grown in standard conditions and under serum deprivation. Metabolite set enrichment analysis reveals several processes affected by serum withdrawal, most of them occurring at the mitochondrial level such as Mitochondrial Electron Transport Chain, Oxidation of Branched Chain Fatty Acids, and Citric Acid Cycle. The impairment of mitochondrial metabolism is further confirmed by the significant accumulation of reactive oxygen species and the reduction of succinate dehydrogenase activity. At last, cells exposed to serum starvation show higher expression levels of mitochondrial superoxide dismutase. Conclusions Mid-term serum deprivation affects cell metabolomes by impairing mitochondrial activity and inducing oxidative stress. We hypothesise that the metabolic stress occurring during serum starvation may trigger the release by donor cells of multiple bioactive factors mediating the pro-angiogenic, trophic, and antioxidant effects of their secretome. P-17. MESENCHYMAL STEM CELL CONDITIONED MEDIUM PROMOTES VASCULARIZATION OF BIO-COMPATIBLE SCAFFOLDS TRANSPLANTED INTO NUDE MICE Ludovica Barone, Federica Rossi, Marina Borgese, Luca Buonarrivo, Mario Raspanti, Piero Antonio Zecca, Giovanni Bernardini, Rosalba Gornati Department of Biotechnology and Life Sciences, University of Insubria, Varese, Italy Objective Human adult mesenchymal stem cells (MSCs) have been largely studied over the past decades, for regenerative medicine applications, due to their multilineage differentiation and their potential use in several cell-based therapies. However, in the last few years, the increasing evidence showing the potential of MSC secretome has led to the acknowledgement that the use of MSC conditioned medium may represent a valid alternative to the use of stem cells, overcoming the main obstacles related to cell samples handling, survival, and rejection. Materials and Methods Accordingly, this study focuses on the characterization and in vivo application of MSC conditioned medium (CM). To this aim, MSCs have been isolated from two different sources, adipose tissue (ASCs) and dental pulp (DPSCs). Although ASCs have been largely studied, very little is known about DPSCs, therefore, DPSCs have been characterised by FACS, qPCR, and immunofluorescence up to their 30th passage to confirm their stemness maintenance over long culture. Afterward, to compare the pro-angiogenic potential of the ASC-CM and DPSC-CM vs. the cells, conditioned media, obtained after 48 h of starvation, have been mixed with a collagen scaffold, INTEGRA ® Flowable Wound Matrix (FWM), grafted in BALB-C nude athymic mice for 28 days and then removed, observed, and processed for gene expression and microscopy analysis. Furthermore, ASC and DPSC CMs, obtained after 72 h of starvation in both normoxic and hypoxic conditions, have been characterised by ELISA to evaluate the effect of oxygen concentration on the release of pro-angiogenic factors; then, their pro-angiogenic potential has been evaluated in vivo using the Ultimatrix sponge assay. Results and Conclusions Even though an exhaustive characterisation of the CM, which also includes the microvesicle fraction, is still in progress, the data obtained demonstrated that the scaffolds associated with CM showed the same efficiency of the ones associated with cells in promoting cellular invasion and capillary growth. Furthermore, the CMs produced under hypoxic conditions seem to promote more efficiently the angiogenesis in vivo. P-18. MELANOMA EXOSOMES INDUCE PD-1 OVEREXPRESSION AND TUMOUR PROGRESSION VIA MESENCHYMAL STEM CELL ONCOGENIC REPROGRAMMING Edina Gyukity-Sebestyén 1 , Mária Harmati 1 , Gabriella Dobra 1,2 , István B Németh 3 , Ágnes Zvara 1 , Éva Hunyadi-Gulyás 1 , Péter Horváth 1,3 , Tibor Pankotai 4 , Barbara Borsos 4 , Miklós Erdélyi 5 , Edit I Buzás 6 , Lajos Kemény 3 , Krisztina Buzás 1,7 1 Biological Research Centre, Eötvös Lorand Research Network, Szeged, Hungary 2 Doctoral School of Interdisciplinary Medicine, University of Szeged, Hungary 3 Institute for Molecular Medicine Finland (FIMM), University of Helsinki, Finland 4 Institute of Pathology, University of Szeged, Hungary 5 Faculty of Science and Informatics, University of Szeged, Hungary 6 Faculty of Medicine, Semmelweis University, Budapest, Hungary 7 Department of Immunology, University of Szeged, Hungary Objective Recently, it has been described that programmed cell death protein 1 (PD-1) overexpressing melanoma cells are highly aggressive. However, until now it has not been defined which factors lead to the generation of PD-1 overexpressing subpopulations. Methods Murine primary mesenchymal stem cells (MSCs) from adipose tissue were pretreated with B16F1 melanoma cell-derived exosomes (mcde). Exosomes were stained by lipophilic dyes, and their uptake into recipient cells was visualised. The rate of apoptosis and expression of multipotent stromal cell markers were analysed by flow cytofluorometry. Tumour-bearing mice were injected with mcde-conditioned MSCs i.v.; the control mice received untreated MSCs. The whole miRNA spectra and the proteome of mcde were analysed by SOLiD5500xl technology and LC-MS/MS, respectively. Results Here, we present that melanoma-derived exosomes, conveying oncogenic molecular reprogramming, induce the formation of a melanoma-like, PD-1 overexpressing cell population (mMSC PD-1+ ) from naïve MSCs. Exosomes and mMSC PD-1+ cells induce tumour progression and expression of oncogenic factors in vivo. Finally, we revealed a characteristic, tumorigenic signalling network combining the upregulated molecules (e.g., PD-1, MET, RAF1, BCL2, and MTOR) and their upstream exosomal regulating proteins and miRNAs. Conclusions Our study highlights the complexity of exosomal communication during tumour progression and contributes to the detailed understanding of metastatic processes. P-19. CELLULAR AND STRUCTURAL CHANGES OF THE TENDON IN A RAT MODEL OF ACHILLES TENDINOPATHY: PRESENCE OF TENDON STEM/PROGENITOR CELLS AND MACROPHAGES Francesca Libonati 1 , Carlotta Perucca Orfei 1 , Dimitrios Kouroupis 2,3 , Enrico Ragni 1 , Paola De Luca 1 , Laura de Girolamo 1 1 Laboratorio di Biotecnologie Applicate all’Ortopedia, IRCCS Ospedale Galeazzi Sant’Ambrogio, Milan, Italy 2 Department of Orthopedics, UHealth Sports Medicine Institute, University of Miami, Miller School of Medicine, Miami, FL, USA 3 Diabetes Research Institute & Cell Transplantation Center, University of Miami, Miller School of Medicine, Miami, FL, USA Objective Achilles tendinopathy is one of the most common tendon disorders of the lower limb in both the athletic and general population, requiring new and effective therapies. In this context, the recent evidence on the presence of tendon stem/progenitor cells (TSPCs) in tendons and the involvement of immune cells in the early stages of tendinopathy paves the way for future therapies. For this purpose, this study is intended to highlight the structural and cellular alteration occurring in a model of Achilles tendinopathy in rats in order to implement the knowledge about tendinopathy onset. Materials and Methods A total of 12 Sprague Dawley rats were treated to induce the Achilles tendinopathy by injecting type I Collagenase (3 mg/mL) in the right tendon, while the contralateral limb was either untreated (healthy control group) or saline injected (sham group). On day 7, tendon explants were histologically evaluated by 4 blinded observers using a semi-quantitative score, to highlight the structural and cellular alterations of the tissue. Immunohistochemical analysis was performed to localise TSPCs (CD90, CD146) and M1 (CD86) and M2 (CD206) macrophages within the tissue. Results The injection of type I Collagenase induced substantial structural damage with cellular alterations, disorganisation of collagen fibers, and a thickening of the paratenon region. An increase in the number of rounded cells was observed in both tendon and paratenon regions together with a higher cell density. TSPCs were more visible in paratenon than in the tendon proper, suggesting a possible aggregation of these cells in the vascularised area typically seen in a paratenon. A slight difference was observed in the presence of TSPCs between pathological and healthy tendons even if not significant. The presence of immune cells was increased in pathological tissues with visible infiltrative (M1) and resident macrophages (M2); however, there is no significant difference in the M1/M2 ratio in this model at 7 days. Conclusions Type I collagenase injection induced Achilles tendinopathy since structural changes and cellular alterations were clearly visible. As regards the inflammation and the recruitment of immune cells, the data obtained suggest their partial involvement, which should be further investigated at other timing of the tendinopathy to understand possible interaction between TSPCs and macrophages. P-20. COMPARATIVE ANALYSIS OF HUMAN MESENCHYMAL STROMAL CELLS DERIVED FROM ADIPOSE TISSUE AND DENTAL PULP: PHENOTYPIC CHARACTERIZATION AND SECRETOME EVALUATION FOR CELL-FREE THERAPY Serena Marcozzi 1 , Maria Assunta Ucci 1 , Francesca Gioia Klinger 2 , Vincenzo Campanella 3 , Antonio Libonati 3 , Cosimo Tudisco 2 , Manuel Scimeca 4 , Giulia Salvatore 1 , Simone Vumbaca 5 , Rosita Russo 6 , Mario Picozza 7 , Angela Chambery 6 , Giovanna Borsellino 7 , Massimo De Felici 1 , Antonella Camaioni 1 1 Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy 2 Saint Camillus International, University of Health Sciences, Rome, Italy 3 Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, Rome, Italy 4 Department of Experimental Medicine, University of Rome Tor Vergata, Rome, Italy 5 Department of Biology, University of Rome Tor Vergata, Rome, Italy 6 Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania Luigi Vanvitelli, Caserta, Italy 7 Neuroimmunology Unit, Santa Lucia Foundation I.R.C.C.S., Rome, Italy Objective The purpose of the present study was a comparative analysis of the biological characteristics of human Mesenchymal Stromal Cells (MSCs) derived from Adipose Tissue (ADSCs) and Dental Pulp (DPSCs) in order to evaluate their use in regenerative medicine. Materials and Methods ADSCs and DPSCs were obtained from healthy patients. ADSCs were isolated by two methods known as Stromal Vascular Fraction (SVF) and Mechanical Fragmentation (MF), while DPSCs were obtained from open apex third molars by MF after separation of the radicular (RPSCs) and the coronal region (CPSCs). These populations were compared based on their morphological features by TEM and IF, and their capacity to proliferate by WST-1 assay. Expression of surface markers was measured by flow cytometry, and the ability to undergo trilineage differentiation was also studied. Conditioned medium (CM) was collected after 3 days of culture and the Bio-Plex Pro Human Cytokine 27-plex Assay (Bio-Rad) was performed. Extracellular vesicles were separated by ExoQuick-TC and suspensions were examined by flow cytometry. Results Both ADSC cell types were able to differentiate towards adipogenic, chondrogenic, and osteogenic lineages as expected, whereas DPSC populations were unable to differentiate into adipocytes. Interestingly, the doubling time of DPSCs calculated during 96 hrs of culture was significantly lower (RPSCs = 27.72 ± 1.34 h vs. CPSCs = 35.26 ± 2.00 h) in comparison to both ADSCs (ADSCs-SVF = 69.71 ± 4.16 h; ADSCs-MF = 65.97 ± 3.75 h). In DPSCs, more than 90% were Nestin + and only 10% αSMA + compared with <1% and ~30% in ADSCs, respectively. Multivariate analysis by PCA for the expression of 16 surface markers measured by flow cytometry showed that organ source (dental pulp or adipose tissue) was the most critical factor that discriminates cell phenotypes. The Bio-Plex assay showed that, whereas the analyte composition of CMs from the two DPSCs appeared very similar, the isolation method was responsible for large differences among CMs from ADSCs in terms of cytokines, chemokines, and growth factors secretion. Finally, ADSCs released a significantly higher number of the smaller EVs named exosomes, ≤100 nm in diameter, than DPSCs. Conclusions These results indicate that the four MSC populations show different phenotypic and functional signatures depending on both the tissue source and the extraction method. These differences may be crucial for cell-free therapies using CMs from such cells. P-21. (YIA) HUMAN PLATELET LYSATE DEPLETED FROM FIBRINOGEN IS A GOOD ADDITIVE FOR LARGE-SCALE PRODUCTION OF MESENCHYMAL STEM CELLS UNDER GOOD MANUFACTURING PRACTICE CONDITIONS AND FOR LYO-SECRETOME ISOLATION, PRESERVING ITS IMMUNOMODULANT PROPERTIES Elena Marini 1 , Alessia Giovanna Santa Banche Niclot 1 , Ivana Ferrero 2 , Camilla Francesca Proto 1 , Marta Barone 1 , Giuseppe Pinnetta 1 , Luciana Labanca 3 , Elia Bari 4 , Maria Luisa Torre 4 , Katia Mareschi 1,2 and Franca Fagioli 1,2 1 Department of Public Health and Paediatrics, University of Turin, Italy 2 Stem Cell Transplantation and Cellular Therapy Laboratory, Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 3 Blood Component Production and Validation Centre, City of Health and Science of Turin, S. Anna Hospital, Turin, Italy 4 University of Piemonte Orientale, Department of Pharmaceutical Sciences, Novara, Italy Objective Human Platelet Lysate (HPL) is an additive, rich in growth factors, cytokines, and proteins used to expand Mesenchymal Stem Cells (MSCs) under Good Manufacturing Practice (GMP) conditions. Preparation techniques may influence HPL composition and, therefore, the biological properties of cultured cells. Standard HPL (HPL-E) production consists of repeated freezing/thawing cycles of platelets and heparin in addition to avoiding culture medium gelling. The new method (HPL-S) consists of making platelets coagulate through the addition of Ca-Gluconate and mechanical squeezing. MSC properties are associated with their secretome and we defined a GMPprocess for freeze-dried MSC-secretome (lyo-secretome). In this work, we verified if the new HPL production method is effective and preserves the chemical and biological properties of MSCs and of their secretome. Materials and Methods From the same platelet pool, we obtained the standard HPL-E and the new HPL-S. We investigated if the treatment with Ca-Gluconate could interfere with the chemical characteristics and the growth factor amounts. Moreover, we compared the cellular growth, immunophenotype, and multipotent capacity of MSCs isolated and expanded in HPL-E and -S. We also isolated lyo-secretome from cell supernatant after serum starvation, ultrafiltration, and freeze-drying from MSCs. Lyo-secretome was analysed for lipid, protein, and growth factor contents, extra-vesicle size and concentrations, immunophenotype, anti-elastase activity, and immunomodulant properties. Results HPL-S did not contain PLTs and fibrinogen; total protein and growth factor amounts were comparable with HPL-E. The number of colony-forming unit fibroblasts showed no significant differences between the two groups. MSCs with HPL-E showed a cumulative Population Doubling higher in the earlier passages with an inversion of the growth trend in passage 4. Stem cell markers were maintained during expansion. Immunophenotypic analysis showed a significantly different expression of HLA-DR (1.30% with HPL-S, 14.10% with HPL-E) and of CD146 (>50% with HPL-S, <30% with HPL-E). Lyo-secretome obtained from MSCs with HPL-E or HPL-S did not show significant chemical/biological differences. Conclusions The use of HPL-S is an effective alternative for MSC production in GMP conditions and the obtained lyo-secretome could replace MSC-cellular therapy as a cell-free surrogate. P-22. BIOCOMPATIBILITY ASSESSMENT OF BIOMATERIALS OBTAINED FROM DISCARDED NATURAL SOURCES FOR DENTAL PULP STEM CELL CULTURE Pasquale Marrazzo 1 , Francesca Paris 1 , Valeria Pizzuti 1 , Silvia Zia 2 , Barbara Roda 1,2 , Francesco Alviano 1 and Laura Bonsi 1 1 Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy 2 Department of Chemistry “G. Ciamician”, University of Bologna, Bologna, Italy 3 Stem Sel s.r.l., Bologna, Italy Objective The growth in vitro of medicinal stem cells depends on the culture method, including the attachment substrate required for adhering to cell culture. Strategies that involve the testing of new biocompatible substrates for stem cell modulation or expansion, for example, nature-inspired biomaterials, may improve the phenotype and the functionality of these cells. The chicken eggshells and honey bee pollen were usually considered waste-like materials. We aim to evaluate these two organic sources obtainable by the food industry for supporting mesenchymal stem culture. Materials and Methods Human Dental Pulp Stem Cells (DP) were selected as a mesenchymal stem cell culture model. Resazurin reduction assay was used to assess the adhesion to the eggshell membrane by living cells and, in parallel, to evaluate cytotoxicity in presence of bee pollen solution. Immunofluorescence staining was performed to display morphology of cells attached to the eggshell membrane. Monochlorobimane probe was used to evaluate glutathione levels after exposure to bee pollen. Results The viability signal of DP stem cells plated on the eggshell membrane increased according to seeded cell density. The cell attachment to the membrane was also confirmed by immunofluorescence performed on fixed cultured membranes. Bee pollen solution was able to increase metabolism-based signal of viability, as well as to increase antioxidant cellular glutathione content. Conclusions The biocompatibility of both eggshell membranes and bee pollen treatment in DP stem cells was confirmed. The experiments with both the organic materials showed positive results in terms of cell metabolism stimulation and no significant alteration of classical morphology of mesenchymal stem cells in vitro. We conclude that these data can be promising for continuing this study and starting new analyses concerning specific stem cell properties changes, in comparison to standard tissue culture plates. P-23. (YIA) INVESTIGATION OF THE EFFECTS OF CONTROLLED UNIAXIAL STRETCH STIMULI ON HUMAN PERIODONTAL LIGAMENT AND ADIPOSE-DERIVED STAMINAL CELLS Beatrice Masante 1,2 , Ilaria Roato 2 , Giovanni Putame 1 , Andrea Tancredi Lugas 1 , Marta Tosini 1 , Mara Terzini 1 , Alberto Audenino 1 , Diana Massai 1 , Federico Mussano 2 1 PolitoBIOMed Lab, Department of Mechanical and Aerospace Engineering, Politecnico of Turin, Italy 2 Bone and Dental Bioengineering Lab, CIR-Dental School, Department of Surgical Sciences, University of Turin, Italy Objective The periodontal ligament (PDL) plays a key role in providing mechanical stability and absorbing the high forces associated with mastication. These capacities deteriorate if PDL is affected by periodontitis, a degenerating disease that leads to loss of the PDL and the supporting alveolar bone. In view of PDL engineering and PDL regeneration, we investigated the hPDLSCs and adipose-derived stem cells (ASCs) behaviour under controlled uniaxial stretch stimuli, exploiting a previously developed bioreactor and customized flexible substrates. Materials and Methods ASCs (ASC52 -telo hTERT, ATCC) and primary hPDLSCs were characterised for the expression of mesenchymal markers by means of a FACS analysis. The same cell types were seeded on customised flexible substrates and exposed to a controlled uniaxial stretch stimulus (15% of strain, 1 Hz, for 90 s every 6 h for 3 days, n = 3) using a previously in-house developed bioreactor. Cells cultured in static conditions were used as control (n = 3). For both cell types and both conditions, DMEM was used as a culture medium. At the end of the culture period, cells were collected and the expression of stemness markers (NANOG, SOX2, and OCT3/4) and osteogenic markers (ALP, OCN, and RUNX2) were assessed through Real-Time PCR. Results FACS analysis highlighted that the phenotypes of ASCs and hPDLSCs are comparable, both expressed CD73, CD90, CD105, and CD44, while they were negative for CD45 and HLADR. The basal stemness marker expression seems to be higher for hPDLSCs than ASCs, nonetheless, both cell types showed an increasing trend in their expression after stretch stimulation. After the stretch stimulus, the expression of the osteogenic genes was increased, especially the OCN ( p < 0.05), in the hPDLSCs, but not in the ASCs. Conclusions The results obtained by FACS analysis demonstrated that both cell types expressed the typical mesenchymal markers, and the expression of the stemness genes showed an increasing trend after stretch stimulation. Osteogenic genes were upregulated in hPDLSCs when cultured under controlled uniaxial stretch conditions. According to these results, hPDLSCs showed more osteo differentiating ability than ASCs. Further studies are ongoing to define the response of hPDLSCs and ASCs to combined stimuli, such as mechanical (stretch) and chemical (tenogenic medium) ones, to obtain an effective system for PDL regeneration. P-24. 3D BRAIN ORGANOID MODELS AS A TOOL TO SCREEN FOR NUTRACEUTICALS Arianna Minoia 1 , Luca Dalle Carbonare 1 , Jens C. Schwamborn 2 , Silvia Bolognin 2 and Maria Teresa Valenti 3 1 Department of Medicine, University of Verona, University of Verona, Italy 2 Luxembourg Centre for Systems Biomedicine (LCSB), Developmental and Cellular Biology, University of Luxembourg, 4365 Belvaux, Luxembourg 3 Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Italy Objective Degenerative conditions of the skeleton and the brain are significant issues with significant socioeconomic effects. This study would underline the significance of the interaction between nerve and bone cells in 3D brain organoid models as a tool to screen for nutraceuticals which have an impact on skeletal metabolism. Knowing that one of the main and important pathways that link bone metabolism and the brain is the Wnt/β-catenin pathway and plays a crucial role in the development of many aspects of midbrain DA development, we would try to understand the effect of some molecules chronically treating 3D organoids derived from two different cell lines for up to 40 days, focusing on the study of genes involved in the Wnt/b-catenin pathway and in neuronal degeneration. Material and Methods Generation organoids: NESCs derived from iPSCs. We cultured organoids for 40 days. Sample preparation: we extract total RNA using the mRNA Qiagen-Kit. Reverse transcription was done using High-Capacity cDNA Transcription Kit. Real-time data: TaqMan probes and TaqMan Universal Master Mix to analyse the gene expression. Flow cytometry: Flow cytometry was performed using BD LSR-Fortessa. Sectioning organoids. Immunofluorescence staining. Confocal imaging Results We investigated the gene expression of PARK2, NR2F1, CTNNB1, and LRP5 for untreated 3D organoids and treated 3D organoids with DMSO, lipoic acid, and JH-II in the two cell lines. Comparing the expression of PARK2 between the organoids deriving from the Bil-WT line towards the treated organoids with mutation LRRK2-G2019S, we note a higher expression of PARK2 in the samples treated with the molecules of interest. We also found this trend of a significant increase in expression for the NR2F1 gene. As regards the LRP5 gene, we note an identification in the samples treated with lipoic acid and an increase in expression trend for the organoids treated with the JH-II inhibitor. As regards the last gene analysed, CTNNB1, there is a higher expression for the samples treated with the inhibitor of the LRRK2-G2019S mutation. Conclusions The molecules impact on the expression of the genes associated with the Wnt/β-catenin pathway upstream and downstream appear to have a beneficial impact on the rise in the expression of the target genes. Considering an improvement in the conditions of PD patients, the treatment with the molecules we examined would result in an increase in the expression of the gene of interest. P-25. BIOLOGICAL EVALUATION OF 3D-BIOPRINTED SCAFFOLD FOR PERIODONTAL REGENERATION Alessandro Mosca Balma 1 , Ilaria Roato 1 , Beatrice Masante 1,2,3 , Federico Mussano 1 1 Department of Surgical Science, C.I.R. Dental School, University of Turin, Italy 2 PolitoBIOMed Lab, Department of Mechanical and Aerospace Engineering, Polytechnic of Turin, Italy 3 Interuniversity Centre for the Promotion of the 3Rs Principles in Teaching and Research, Pisa, Italy Objective Periodontitis is one of the most common diseases worldwide, causing a progressive destruction of the tooth supporting tissues. Thus, different regenerative approaches are under investigation, and this study aims to evaluate the biological properties of polycaprolactone (PCL) 3D-Bioprinted scaffolds w/or w/o alumina toughened zirconia (ATZ) filler, as suitable materials for alveolar bone and periodontal ligament (PDL) regeneration. Materials and Methods Three different types of blends were compared to each other, respectively: pure PCL, 80/20 w / w PCL/ATZ, and 60/40 w / w PCL/ATZ. The ATZ was incorporated in the PCL matrix through dissolution in chloroform by solvent casting method, then the bioink was 3D bioprinted to generate scaffolds with a standardised porous and circular geometry. ASC52 hTert cells (ASCs) were seeded on these scaffolds for 24 h to test their adhesion on the material surface. To evaluate the biocompatibility of the scaffolds, ASCs were cultured for 3, 7, and 14 days to allow scaffold colonisation, and registered their growth through the quantification of the ATP release in culture by viable cells (CellTiter-Glo kit). To investigate whether the scaffold had osteoinductive properties, ASCs were cultured in an osteogenic medium for 2 months, then RNA was extracted and the expression of osteogenic genes (ALP, COLL1, OCN, and RUNX2) were quantified with Real-Time PCR. SEM and EDX analysis were performed to evaluate the newly formed bone and to quantify the presence of calcium deposition, respectively. Results All the scaffolds allowed ASC adhesion and growth, particularly the ones with 80/20 w / w PCL/ATZ showed better biocompatibility in the long term. The addition of ATZ to the PCL matrix reduced the osteoinductive property of the materials. After two months in the osteogenic medium, the expression of ALP and RUNX2 was not different among the three polymeric scaffolds, while both COLL1 and OCN expressions were reduced in the presence of ATZ filler. These results were confirmed by SEM images and EDX analysis. Conclusions The presence of ATZ seems to reduce the osteoinductivity of the scaffolds, pointing out the importance of the material chosen as a support of new PDL regeneration, because this could limit the possibility of tooth ankylosis. Future tests will be focused on the quantification of scaffolds’ mechanical properties improvement in the presence of a toughening filler as ATZ and on tenogenic differentiation capability with PDL mesenchymal stem cells. P-26. HA AND PRP COMBINATIONS AS “OFF THE SHELF” DEVICE FOR CLINICAL APPLICATIONS Marta Nardini 1 , Anita Muraglia 1,2 , Antonella D’Agostino 4 , D’Agostino Maria 4 , Gilberto Filaci 1,2 , Ranieri Cancedda 3 , Chiara Schiraldi 4 and Maddalena Mastrogiacomo 1 1 Department of Internal Medicine (DIMI), University of Genoa, Italy 2 Biotherapy Unit, IRCCS Ospedale Policlinico San Martino, Genoa, Italy 3 Emeritus professor at the University of Genoa, Italy 4 Department of Experimental Medicine, University of Campania “L. Vanvitelli”, Naples, Italy Objective Platelet Rich Plasma (PRP) is a well-known natural product, optimised, standardised, and used for regenerative medicine. The current goal is to identify substrates as vehicles for a gradual platelet content release. Hyaluronic acid (HA) is proposed thanks to its viscoelastic and biological properties and biocompatibility. This study aimed to set up and characterise an “off the shelf” freeze-dried and injectable device based on HA and PRP for tissue regeneration. Materials and Methods Different Molecular Weights of HA (Low and High– HA-HMW and HA-LMW) were used in combination with a standardised PRP in the ratio 1:1. Rheological analysis of compounds was performed, and the products were lyophilised. After reconstitution by water, the HA/PRP mixtures were tested in terms of cell proliferation capability and wound scratch recovery on human primary fibroblasts. With the aim of validating the methods of storage of the products, the biological activity of freeze-dried HA/PRP formulations was tested at different temperatures of conservation (25 °C, 4 °C and −20 °C) for up to 6 months. Results HA-HMW/PRP compound prompted human dermal fibroblast proliferation such as PRP alone, but the formulations needed almost half an hour for full reconstitution and strong pressure to be extruded by a 21-gauge needle. To overcome these limitations, different HA-LMW/PRP formulations were characterised and tested in terms of biological activity. These formulations are capable of sustaining cell proliferation and are stable at different temperatures and lengths of storage compared to the loss of PRP activity. Interestingly, only the lowest HA-LMW tested (56 KDa) in combination with PRP showed from one up to six months of significant preservation of the proliferation activity compared to PRP alone. This was also confirmed by in vitro scratch assay using a time-lapse video microscopy station. Conclusions In conclusion, we developed a lyophilised HA-based/PRP device that improves and preserves the PRP activity over time. HA/PRP allows the development of promising products for topical, intradermic, and intra-articular applications. P-27. THERAPEUTIC EFFECTS OF HUMAN PLACENTAL-DERIVED MESENCHYMAL STROMAL CELLS (PDMSCS) ON A LIPOPOLYSACCHARIDE INDUCED MOUSE MODEL OF PREECLAMPSIA (PE) Anna Maria Nuzzo, Laura Moretti, Ilaria Faletti, Alberto Revelli, Alessandro Rolfo Dept. Surgical Sciences, University of Turin, Italy Objective Preeclampsia (PE), the most severe human pregnancy-related syndrome, is a leading cause of foetal-maternal mortality and morbidity and lack of an effective therapy. The main hallmarks of PE are severe maternal hypertension and proteinuria, expression of generalised endothelial damage, and inflammation that could lead to Foetal Growth Restriction (FGR). Human Placenta-Derived Mesenchymal Stromal Cells (hPDMSCs) are well renowned for their pro-angiogenic and anti-inflammatory effects exerted via paracrine interactions. Herein, we tested the effects of hPDMSCs-CM (Conditioned-Media) on a mouse model of preeclampsia. Materials and Methods PDMSCs were isolated from control placentae and plated (1 × 10 5 cells/mL) in DMEM without FBS at passage 5. After 48 h, CM was collected. Preeclampsia was induced in pregnant C57BL/6NCrl mice by intravenous bacterial Lipopolysaccharide (LPS) injection. Starting from d9, maternal blood pressure and proteinuria were monitored until d19. At d11 of pregnancy, dams were injected with E.Coli LPS (1 µg/Kg). At d12, mice were randomly divided into two groups (n = 7 each) and treated intravenously as follows: plain vehicle (300 µL, placebo) and hPDMSCs-CM (300 µL, treated). At d19, mice were sacrificed. A number of foetuses, FGR, foetal reabsorption, and placental weight were evaluated. Next, placentae were processed for mRNA and protein isolation. sFlt-1, IL-6, and TNF-α gene and protein expression were evaluated by Real-Time PCR and Enzyme-Linked Immunosorbent Assay (ELISA). Results Injection of hPDMSCs-CM on d12 significantly decreased maternal systolic blood pressure and proteinuria by day 13 until term relative to placebo group. No FGR and/or reabsorbed foetuses were delivered by hPDMSCs-CM treated PE mice, while 5 FGR foetuses were found in the placebo group. No differences were found in placental weight between groups. hPDMSCs-CM injection significantly decreased sFlt-1, IL-6, and TNF-α levels in PE mice. Conclusions Our data indicate that hPDMSCs-derived trophic mediators can reverse PE-like features during pregnancy, suggesting a therapeutic role for hPDMSCs for the treatment of preeclampsia. Supported by Corion Biotech s.r.l. P-28. OXIDATIVE STRESS AND PLACENTAL-DERIVED MESENCHYMAL STROMAL CELLS (PDMSCS): NEW PERSPECTIVE FOR PREECLAMPSIA (PE) ETIOPATHOGENESIS Anna Maria Nuzzo, Laura Moretti, Ilaria Faletti, Alberto Revelli, Alessandro Rolfo Dept. Surgical Sciences, University of Turin, Italy Objective Mesenchymal Stromal Cells have been highlighted as an effective antioxidant therapy. Indeed, anomalies in PDMSCs antioxidant defences might cause/contribute to the increased oxidative stress (OxS) typical of PE placentae. Herein, we compared the release of antioxidant proteins and investigated the expression of the antioxidant enzymes Catalase (CAT) and Superoxide Dismutase1 (SOD1) and of OxS-triggered cell death modulators PARP1, Caspase3, and LDOC1 in normal and PE-PDMSCs. Finally, we tested the hypothesis that PDMSCs-CM (Conditioned-Media) could restore CAT and SOD1 in H2O2-treated villous explants. Materials and Methods PDMSCs were isolated from control (n = 10) and PE (n = 10) placentae. At passage 5, cells were plated (1 × 10 5 cells/mL) in DMEM without FBS. After 48 h, CM was collected, and total antioxidant capacity was tested by Cayman’s Antioxidant Assay. Control (n = 24) villous explants were treated for 24 h by H2O2 and next by control PDMSCs-CM for 48 h. CAT, SOD1, PARP1, Caspase3, and LDOC1 gene expression were evaluated by Real Time PCR. Results We reported a lower total antioxidant capacity (1.42 Fold Decrease) in PE-PDMSCs relative to control. We reported decreased CAT and SOD1 and increased PARP1 ( p = 0.03), Caspase3 ( p = 0.04), and LDOC1 ( p = 0.05) gene levels in PE relative to control PDMSCs. After 24 h with H2O2, normal PDMSCs-CM treatment increased CAT and SOD1 and significantly decreased PARP1 ( p = 0.02), Caspase3, and LDOC1 mRNA levels relative to controls. Conclusions Herein, we demonstrated that pathological PE-PDMSCs are characterised by aberrant antioxidant properties followed by increased production of OxS-related cell death effectors. Moreover, PDMSCs-CM promotes the expression of antioxidant enzymes, thus inhibiting OxS-mediated cell death. Indeed, our data suggest that PDMSCs-CM could be used to neutralise the exacerbated OxS typical of PE placentae, thus opening to novel PDMSCs-based therapeutic options. P-29. MESENCHYMAL STEM CELLS IN WOUND HEALING: FOCUS ON MSC-MEDIATED SKIN REGENERATION AFTER DERMATO-ONCOLOGICAL SURGICAL PROCEDURES Alessia Paganelli, Cristina Magnoni UO di Chirurgia Dermatologico a Indirizzo Oncologico e Rigenerativo, AOU Policlinico di Modena, Università degli Studi di Modena e Reggio Emilia, Modena, Italy Objective To assess the role of mesenchymal stem cells (MSCs) on skin wound healing both in vitro and in vivo after dermatological surgical procedures performed for oncological purposes. Materials and Methods MSCs were obtained from discarded adipose tissue during dermato-surgical procedures. After isolation, MSCs were cultured in ascorbic-acid enriched medium in order to obtain MSC-based dermal scaffolds. Organotypic cultures were also performed to assess the pro-epithelizing properties of MSCs. MSCs were also seeded on commercially available acellular dermal matrices (ADMs) to assess whether they could exert a synergistic action. Finally, we also looked at whether MSCs were spontaneously recruited in vivo at wound sites in the presence of ADMs. Classical histology, immunofluorescence, and ELISA tests have been employed in the aforementioned experimental settings. Results MSCs can efficiently be used to produce dermal equivalents. MSCs secrete all the main components (collagen and fibronectin) of the extracellular matrix upon stimulation. MSCs guide wound re-epithelialization in vitro in the presence of keratinocytes, mainly through a paracrine action on epidermal basal stem cells. When seeded on acellular dermal collagenic substitutes, MSCs significantly increase extracellular-matrix production, therefore, confirming the potential effectiveness of combination treatments. CD90+ STRO-1+ cells were detected in the neodermis after ADM positioning, therefore suggesting efficient ADM-mediated MSC recruitment. Conclusions MSCs represent a promising tool in the regenerative setting, especially for the treatment of large surgical wounds after dermatological surgical procedures. However, further studies are needed to confirm their safety in oncological patients since a potential tumour-promoting role has recently been postulated for MSCs. P-30. PRODUCTION OF CLINICAL GRADE EXTRACELLULAR VESICLES (EVS) SECRETED BY MESENCHYMAL STROMAL CELLS AND INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF OSTEOARTHRITIS Maria Elisabetta Federica Palamà 1 , Cansu Gorgun 1 , Georgina Shaw 2 , Mary Murphy 2 , Chiara Gentili 1 1 Department of Experimental Medicine (DIMES), University of Genova, Italy 2 Regenerative Medicine Institute (REMEDI), University of Galway, Ireland Objective Mesenchymal stromal cells (MSCs) derived extracellular vesicles (EVs) have been studied for the treatment of Osteoarthritis (OA), the most common chronic disease of the joint cartilage. A large-scale expansion of MSCs is required to meet clinical demand and this could affect the effectiveness of cells and cell products. Therefore, MSC generated from induced pluripotent stem cells (iMSC) represent a promising cellular source for the manufacture of EV therapeutics. In this study, we isolated and tested the efficacy of EV secreted by MSCs and iMSC in the treatment of OA in vitro. Methods MSCs and iMSCs were cultured in vitro in serum-free clinical grade conditions. Cells were characterised during long-term in vitro expansion for surface expression pattern, proliferation ability, senescence rate, and differentiation capacity. EVs were isolated using an FPLC-anion exchange chromatography (AEX) approach and their biological effect on IL-1α treated human chondrocytes was examined. Results The use of a serum-free, chemically defined medium for isolation and culture of hMSCs allowed us to expand a population with a stable phenotype from early to late passages. It is already well known that MSC proliferation, differentiation, and function decline with passaging. After three passages, we indeed observed a drastic impact on cell growth and differentiation. The paracrine activity of hMSCs during long-term expansion was also evaluated. The number and size of vesicles released by hMSCs increased proportionally with their age. The anti-inflammatory activity of MSC-EVs was investigated in an in vitro model on osteoarthritic chondrocytes and the expression of inflammatory cytokines such as IL-6 and IL-8 were measured. Administration of hMSC-EVs showed relevant anti-inflammatory effects only for early passages-derived vesicles (until passage three). iMSCs were also expanded for the long term to define the best culture conditions and the best time window for the isolation of EVs with maximum biological activity. Conclusions Despite the promising potential of EVs for therapeutic applications, robust manufacturing processes that would increase the consistency and scalability of EV production are still lacking. The focus of our study was directed on the determination of the optimal range of time in which MSCs and iMSC are biologically functionally in a serum-free culture system. This paracrine application may represent a novel therapeutic approach for the treatment of OA. P-31. MESENCHYMAL STEM CELLS COMBINED WITH ENDOTHELIAL CELLS SUPPORT SPHEROID FORMATION OF OSTEOSARCOMA CELLS Micaela Pannella, Chiara Bellotti, Ania Naila Guerrieri, Toni Ibrahim, Enrico Lucarelli Terapie Rigenerative in Oncologia, Struttura Complessa di Osteoncologia, Sarcomi dell’osso e dei tessuti molli, e Terapie Innovative, IRCCS, Istituto Ortopedico Rizzoli, Bologna, Italy Background and Objectives The tumour microenvironment (TME) is a complex milieu that contains cancer cells and non-malignant cellular and non-cellular components which together orchestrate a complex dialog. In the last decades, several papers have proposed that mesenchymal stem cells (MSCs) play a critical role in TME formation and function. In our work, we evaluated the influence of MSCs in combination with endothelial cells on Osteosarcoma (OS) cells using multicellular spheroids. Our hypothesis is that MSCs may have a pro-tumorigenic action. Materials and Methods The OS cell lines MG-63, U-2 OS, SaOS-2, and 143B were engineered to express GFP, Endothelial Cells (HUVEC) were purchased, while MSCs were isolated at the Rizzoli. Spheroids were made by self assembly in ultra-low attachment 96 well plates. Based on previous experience in generating multicellular spheroids, we combined OS/HUVEC/MSCs with a ratio of 5:3:2. Results Commercially available OS cell lines show a different ability to grow as stable spheroids; only some of them grow rapidly as spheroids but develop necrotic cores over time. Our results revealed that the metabolic activity of cells in OS cell-only spheroids decreased with time, however, increased in hybrid spheroids with MSCs/HUVEC. OS spheroids composed of U-2 OS and SaOS-2 cells formed irregular spheroids while in the hybrid configuration, with MSCs and HUVEC, they assembled forming regular spheroids. OS spheroids composed by 143B and MG-63 cells only had a rounded and compact morphology. The hybrid spheroids of 143B cells and MSCs/HUVEC had an extremely regular and smooth surface, and over a long time, they began to form buds. The hybrid spheroids composed of MG-63 and MSCs/HUVEC had a smooth and regular surface; interestingly, after 96 h MG-63 cells started to leave the spheroid’s core and after 7 and 12 days, they formed buds. Bud formation and OS cell migration suggest an increase in OS cell motility and migration when they are cultured with MSCs/HUVEC. Several studies have shown that spheroids’ roughness is indicative of cellular invasiveness. Thus, we compared the roughness between simple and hybrid spheroids demonstrating that all hybrid spheroids exhibit greater roughness than spheroids composed of OS cells only. Conclusions Data obtained shows that combined MSCs and HUVEC support OS growth and may influence the spheroid morphology and invasiveness, thus sustaining the hypothesis of the pro-tumorigenic effect of these cells. P-32. DEVELOPMENT OF A PRECLINICAL MODEL OF PDAC TO INVESTIGATE MSC-BASED DELIVERY SYSTEMS Smeralda Rapisarda 1 , Benedetta Ferrara 1 , Antonio Citro 1 , Fabio Manenti 1 , Chiara Gnasso 2 , Lorenzo Piemonti 1 1 Diabetes Research Institute, IRCCS San Raffaele, Milan, Italy 2 Experimental Imaging Centre, IRCCS San Raffaele, Milan, Italy Objective Conventional therapies for pancreatic ductal adenocarcinoma (PDAC) present limits due to drug toxicity and the chemoresistance of PDAC cells. That can be due to the PDAC stroma, which constitutes a barrier for the transport of drugs to tumour cells and a drug-delivery system might improve the treatment. The aims of this study were firstly to develop a preclinical metastatic model of PDAC and secondly to set up a model of treatment with mesenchymal stem cells (MSCs) for future applications as a drug-delivery tool. Materials and Methods To generate the liver metastatic model, three different cell lines derived from KPC mice were used: K8484 (from PdxCre/LSL-KrasG12D-Trp53R172H), DT6606 (from PdxCre/LSL-KrasG12D), and DT6606lm (from liver metastases after intraportal injection of DT6606). Cells were injected into the portal vein of immunocompetent eight weeks C57BL/6N male mice in a dose range of 1 × 10 3 –5 × 10 5 . On day 20, after tumour induction, the metastatic growth was assessed by seven-tesla magnetic resonance (MR). On day 21, the first group of tumour-bearing mice underwent intravenous (i.v.) injection of luciferase-transduced MSCs (LUC + MSCs) to evaluate the MSCs biodistribution using in vivo imaging system (IVIS). A second group of tumour-bearing mice underwent an intraportal injection of LUC + MSCs and the LUC signal intensity of the two groups was compared. Results Among the three investigated cell lines, only the K8484 cell line was chosen to generate the metastatic model. K8484 cell-derived liver metastases, indeed, exhibited a largely glandular architecture, more similar to human metastases. Moreover, the use of the intraportal model allowed a homogeneous and synchronous metastatic growth in mice 20 days after the injection. Studies on biodistribution of i.v. injected MSCs revealed a cell accumulation in the lung after injection with a low-intensity signal and several animals died after cell administration. The mice subjected to intraportal injection of MSCs reported a high cell accumulation in the liver with a prolonged signal up to six days after injection, making the intraportal injection preferable to allow MSCs to reach the tumour site. Conclusions The development of this metastatic model of PDAC in the liver allowed us to evaluate MSCs as a promising therapeutic option for PDAC, especially using intraportal injection to deliver them directly to the tumour site. Obtained results may open up new insights into the use of MSCs as tools to treat PDAC. P-33. HUMAN AMNIOTIC MESENCHYMAL STROMAL CELL CONDITIONED MEDIUM MODIFIES CANCER ASSOCIATED FIBROBLAST GENE EXPRESSION AND CYTOSKELETAL ORGANIZATION Jacopo Romoli 1 , Andrea Papait 1,2 , Paola Chiodelli 3 , Patrizia Bonassi 3 , Silvia De Munari 3 , Elsa Vertua 3 , Serafina Farigu 3 , Antonietta Silini 3 , Ornella Parolini 1,2 1 Department of Life Science and Public Health, Università Cattolica del Sacro Cuore, Rome, Italy 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, Rome, Italy 3 Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy Objective Tumour-associated stromal cells, also known as carcinoma-associated fibroblasts (CAFs), play a pivotal role in favouring tumour growth by their interactions with both tumour cells and cells present in the tumour microenvironment (TME). Recent studies demonstrate that CAFs can favour metastasis and are responsible for chemo-resistance mechanisms. Human amniotic mesenchymal stromal cells (hAMSC) are known to exert immune-modulatory and anti-fibrotic effects, targeting not only immune cells but also stromal cells in damaged tissues. The aim of our study was to determine if hAMSC could also exert effects on stromal cells within the tumour microenvironment, namely CAFs. To this end, we in vitro stimulated the differentiation of normal adult fibroblasts towards CAFs with exogenous TGF-β. The acquisition of CAF features was assessed by immunofluorescence for αSMA expression and for cytoskeleton organisation. In addition, we used Real-Time PCR and flow cytometry to assess the expression of the most relevant CAF markers. Finally, we evaluated the ability of hAMSC conditioned medium (CM-hAMSC) to counteract the acquisition of CAF-like gene expression and functionality. Materials and Methods Conditioned medium from hAMSC was obtained by culturing hAMSC at passage 2 in DMEM-F12 without serum for 5 days. Human dermal fibroblasts were treated with TGF-β ± CM-hAMSC for 3, 7, and 11 days. At each timepoint, flow cytometry, immunofluorescence, and RT-PCR were performed to assess CAF-like phenotype. Results Flow cytometry showed a high percentage of αSMA + cells after TGF-β treatment, which decreased after CM-hAMSC treatment. Concurrently TGF-β-treated fibroblasts displayed well-organised cytoskeletal αSMA and presented extracellular MFAP5 deposition, whereas CM-hAMSC treatment interfered with these processes. These data are supported by RT-PCR analysis which showed αSMA and MFAP5 downregulation when dermal fibroblasts were treated with CM-hAMSC. On the other hand, we observed an increase of PDPN gene expression and positive cells after CM-hAMSC treatment. Conclusions Our preliminary results suggest that hAMSC-secreted factors are able to inhibit CAF activation. Further studies will be aimed at confirming these results and understanding if hAMSC-secreted factors inhibit CAF migration and ultimately clarify the use of hAMSC as a potential therapeutic strategy able to not only target tumour cells, but also stromal cells within the tumour microenvironment. P-34. MESENCHYMAL STROMAL CELLS FOR THE TREATMENT OF CRANIAL CRUCIATE LIGAMENT RUPTURE OF DOGS Gabriele Scattini 1 , Piero Boni 2 , Alessandro Fruganti 3 , Fabrizio Dini 3 , Luisa Pascucci 1 1 University of Perugia, Department of Veterinary Medicine, Perugia, Italy 2 Veterinary practitioner, Cannara (PG), Italy 3 University of Camerino, School of Biosciences and Veterinary Medicine, Matelica, Italy Objective In recent years, mesenchymal stromal cells (MSC) have received a strong boost in veterinary medicine due to their pro-regenerative properties. MSCs are virtually present in all the organs possessing a vascular stroma. The aim of this study was to evaluate the potential therapeutic use of MSC from adipose tissue in dogs with complete cranial cruciate ligament (CCL) ruptures that had not undergone surgical treatment. CCL rupture is the most common cause of lameness in dogs and can be treated conservatively, but surgical therapy is the treatment of choice to restore stability and functionality of the joint. However, surgery does not prevent the development of osteoarthritis, which over time leads to relapse of lameness and pain. It is worth mentioning that an increasing number of public and private veterinary hospitals all over the world treat partial CCL rupture with MSC as a second-line or even first-line therapy. There is no evidence of MSC use in complete ruptures. Material and Methods Tyson, an American Staffordshire Terrier, male, 4 years old, received a diagnosis of complete CCL rupture followed by 4 intra-articular infiltrations of autologous MSC. Post-infiltration monitoring was performed by orthopaedic, ultrasound, radiography examination, and nuclear magnetic resonance (NMR). A 20-month follow up was performed. Results MSC did not trigger adverse effects in the short to medium term, nor they cause regeneration of the CCL. However, they displayed a strong anti-inflammatory activity responsible for symptom remission. The therapeutic effectiveness of MSC seems to be attributable to their chondroprotective effect which slows down the development and progression of osteoarthritis. Conclusions The results described in this study suggest that the use of MSC for the treatment of complete rupture of the CCL could represent a valid option: (i) As an alternative to surgery when the patient or owners are unable to deal with it; (ii) As an adjuvant to surgical therapy both in the peri-operative and in the post-operative in order to obtain a stabilisation and reduce the development of osteoarthritis; (iii) as a substitute of non-steroidal anti-inflammatory drugs with the advantage of reducing side effects of pharmacological therapy. P-35. MIR-214 MEDIATED STROMA-TUMOUR CELL CROSSTALK DURING TUMOUR PROGRESSION Francesca Orso 1,2 , Federico Virga 1,2,10 , Daniela Dettori 1,2 , Alessandra Dalmasso 1,2 , Mladen Paradzik 1,2 , Aurora Savino 1,2 , Stefania Cucinelli 1,2 , Maura Coco 1,2 , Iris Chiara Salaroglio 3 , Joanna Kopecka 3 , Margherita Aalba Carlotta Pomatto 4 , Giovanni Camussi 4 , Katia Mareschi 5,6 , Leonardo Salmena 7 , Paolo Provero 8,9 , Valeria Poli 1,2 , Chiara Riganti 3 , Massimiliano Mazzone 1,2,10 Pier Paolo Pandolfi 1,2,11 , Daniela Taverna 1,2 1 Molecular Biotechnology Center (MBC), Turin, Italy 2 Dept. Molecular Biotechnology and Health Sciences, University of Turin, Italy 3 Department of Oncology, University of Turin, Italy 4 Department of Medical Sciences, University of Turin, Italy 5 Paediatric Onco-Haematology Division, Regina Margherita Children’s Hospital, City of Health and Science of Turin, Italy 6 Department of Public Health and Paediatrics, University of Turin, Italy 7 Princess Margaret Cancer Centre, University Health Network, ON, Canada 8 Centre for Omics Sciences, IRCCS San Raffaele Scientific Institute, Milan, Italy 9 Department of Neurosciences “Rita Levi Montalcini”, University of Turin, Italy 10 Center for Cancer Biology (CCB), VIB, Leuven, Belgium 11 Renown Institute for Cancer, Nevada System of Higher Education, Reno, NV, USA Objective Cancer and stroma cells continuously interact during tumour progression and influence each other. Secreted microRNAs (miRNAs) have recently been implicated in the tumour–stroma crosstalk. Here, we show that miR-214 is highly expressed in stromal cells such as Mesenchymal Stem Cells (MSCs) or Cancer-Associated Fibroblasts (CAFs), often derived from CAFs, and that it correlates with stromal signatures in human breast cancers and melanomas. Upon tumour cell signals, stroma miR-214 is released via Extracellular Vesicles (EVs) and is instrumental for cancer cells to promote metastasis formation through the activation of a pro-metastatic pathway which involves the protein-coding genes TFAP2C, ITGA5, and ALCAM and the anti-metastatic small non-coding RNA, miR-148b. Metabolic rewiring and, particularly, reprogrammed glucose metabolism is a hallmark of cancer, crucial for tumour progression. Material and Methods We analysed the impact of stroma-derived miR-214 on the metabolic status of tumour cells and observed glycolysis enhancement and an Oxidative Phosphorylation (OXPHOS) impairment linked to metastatic traits. Results and Conclusions Our results underline the relevance of “stroma miR-214” for tumour dissemination and metastasis formation and suggest the possibility of a double-edge therapeutic approach based on the targeting of miR-214 and of major metabolic players in tumour and/or stroma cells. P-36. CELLULAR SENESCENCE IN SYNOVIAL FLUID MESENCHYMAL STROMAL CELLS AND POTENTIAL IMPLICATIONS IN THE PROGRESSION OF OSTEOARTHRITIS Gabriella Teti 1 , Valentina Gatta 1 , Francesca Chiarini 2 , Mirella Falconi 3 1 Department of Biomedical and Neuromotor Sciences, University di Bologna, Italy 2 Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze, University of Bologna, Italy 3 Department of Experimental, Diagnostic and Specialty Medicine—DIMES, Bologna, Italy Objective Osteoarthritis (OA) is characterised by cartilage degradation, joint inflammation, subchondral bone remodelling, and fibrosis. Current therapies for OA are mainly focused on treating symptoms of pain rather than to counteract the progression of the disease. Recently, OA has been associated with the accumulation of senescent cells in joint tissues but how senescence can affect each resident joint cell and its link with the progression of OA are still poorly described. With the advance of tissue engineering and regenerative medicine, mesenchymal stem/stromal cells (MSCs) have represented a promising candidate for cartilage repair and regeneration. However, most of the current research has been focused on the application of MSCs derived from bone marrow, and just a few studies have investigated the potential therapeutic properties of MSCs isolated from synovial fluid (sf-MSCs). Although sf-MSCs have demonstrated higher chondrogenic capabilities their accumulation as senescent cells in synovial fluid and their correlation with OA progression has never been investigated. Thus, the aim of the study was to verify the presence of senescent sf-MSCs isolated from OA joints and compare their chondrogenic capabilities with sf- MSCs isolated from healthy joints. Material and Methods Sf-MSCs were isolated from tibia-tarsal joints of healthy and diseased horses with an established diagnosis of OA. Cells were cultured in vitro and characterised for cell proliferation assay, cell cycle analysis, ROS detection assay, ultrastructure analysis, and evaluation of senescent markers. To evaluate the influence of OA on chondrogenic differentiation, sf-MSCs isolated from diseased joints were in vitro stimulated to chondrogenic differentiation and the expression of chondrogenic markers was checked and compared to healthy sf-MSCs. Results Results clearly showed an arrest of the cell cycle in combination with upregulation of the senescent markers p21 WAF1/Cip1 and p16 INK4 , reduced autophagy and increased ROS production in OA sf-MSCs, demonstrating a senescent state. Furthermore, OA sf-MSCs showed a reduced ability in synthesising proteoglycans while the ability to express collagen II is upregulated, suggesting the involvement of OA sf-MSCs in developing a fibrotic joint environment. Conclusions In conclusion, our results demonstrate the presence of senescent sf-MSCs in OA joints which could have a key role in the progression of the disorder. P-37. LASER DISSECTION-COUPLED QUANTITATIVE MICROLIPIDOMIC METHOD TO RESOLVE TUMOUR HETEROGENEITY Vanda Varga-Zsíros 1,2 , Mária Péter 1 , Ede Migh 1 , Annamária Marton 1 , Aladár Pettkó-Szandtner 3 , Imre Gombos 1 , Zoltán Kóta 4 , Péter Horváth 1 , László Tiszlavicz 5 , Zsuzsanna Darula 3,4 , Csaba Vizler 1 , Zsolt Török 1 , László Vígh 1 , Gábor Balogh 1 1 Biological Research Centre, Institute of Biochemistry, ELKH, Szeged, Hungary 2 University of Szeged, Ph.D. School in Biology, Szeged, Hungary 3 Biological Research Centre, Laboratory of Proteomics Research, Szeged, Hungary 4 Single Cell Omics Advanced Core Facility, HCEMM, Szeged, Hungary 5 University of Szeged, Department of Pathology, Szeged, Hungary Objective Lipid metabolic reprogramming is a newly recognized hallmark of malignancy. Most normal cells build up their membranes from dietary lipids. In contrast, cancer cells reactivate the de novo lipogenesis. To understand the aggressiveness and metastatic potential of tumours, the exploration of tumour heterogeneity is of great interest but also a great challenge. Materials and Methods We developed and validated a novel laser microdissection-coupled multi omics platform which combines quantitative and comprehensive lipidome, proteome, and/or transcriptome analysis with spatial resolution. The multistep approach involves the preparation of parallel cryosections from spheroids or different tissue samples, cross-referencing of hematoxylin–eosin-stained and native images, laser microdissection of marked regions (down to ~15 cells), in situ lipid microextraction/protein digestion or RNA isolation, and high-performance mass spectrometry (direct injection-based shotgun lipidomics and UPLC-coupled proteomics on an Orbitrap Lumos instrument) or transcriptomics. Results We identified a radial gradient in the lipidomic profile of 4T1 mouse breast cancer spheroids which correlated well with nutrient availability. By using mouse allografts injected with 4T1 cells, we observed substantially different lipidomic patterns not only between tumorous and non-tumorous areas of the liver or spleen but also between tumorous regions grown in different microenvironments. Close-distant parallels of cryosections were subjected to other omics and/or staining procedures. Conclusions The integration of lipidomic results with transcriptomic, proteomic, and immunohistological data can improve our understanding in tumour heterogeneity as well as in the pathology of various lipid-related disorders.
This conference report reflects the consensus viewpoint of the authors and scientists participating in the 2022 GISM annual meeting: Tarlan Eslami Arshaghi, Paola Bagnoli, Laura Barrachina Porcar, Luca Battistelli, Anna Brini, Stefania Bruno, Elena Ceccotti, Lucia Ceresa, Valentina Coccè, Stefano Cosma, Nicholas Crippa Orlandi, Maurizio Del Bue, Laura De Girolamo, Silvia Dotti, Roisin Dwyer, Franca Fagioli, Andrea Grosso, Maria Harmati, Ana Ivanoska, Enrico Lucarelli, Maddalena Mastrogiacomo, Barbara Merlo, Matteo Moretti, Marta Nardini, Andrea Papait, Graziella Pellegrini, Augusto Pessina, Michela Pozzobon, Enrico Ragni, Ilaria Roato, Silvia Scaglione, Gabriele Scattini, Maria Luisa Torre, Roberta Visone, and Silvia Zia.
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Management of children with poor prognosis first permanent molars: an interdisciplinary approach is the key
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cef72e45-482b-4e64-b8ef-60b09c760df8
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10219812
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Dental[mh]
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Dental care for any child, especially those with high caries risk, should be founded on personalised and evidence-based prevention, aimed at averting disease and a host of potential negative impacts for the child, their family and service providers. A sizeable body of evidence supports the effectiveness of various professionally applied and home-care preventive regimens to help reduce caries and improve oral health outcomes for children. , Given this preventive-based ethos, one may ask why dental health professionals are still seeing so many children with compromised first permanent molars (FPMs) in their daily practice. Moreover, how should these children be managed, given the complexity of decision-making in relation to long-term prognosis, orthodontic status, and relevant child/parental factors? Here, we provide a pragmatic commentary on the broad principles of dental care for children with FPMs of poor prognosis.
General dental practitioners (GDPs) in the UK have recently reported that around 10% of the children that they see will have compromised FPMs. Data from the Office of National Statistics (2015) corroborate this clinical impression, with the finding that 5% of eight-year-olds, and an alarming 25% of 15-year-olds, have some form of caries in their FPMs. It is also important to recognise that carious FPMs may have an underlying enamel defect, which can predispose them to a greater risk of caries. Molar incisor hypomineralisation (MIH) is an increasingly common systemic condition, characterised by qualitative enamel defects predominating in the FPMs and incisor teeth. Not only are affected molars more likely to develop caries (reportedly up to six times), but they are also prone to rapid and extensive post-eruptive enamel breakdown, which can cause extreme dentine hypersensitivity. , Epidemiological data suggest that MIH affects around 13% of children worldwide, so even in communities with decreasing caries rates, clinicians will continue to face the challenge of managing children with poor prognosis FPMs.
Treatment planning for children with carious and/or hypomineralised FPMs relies on the assimilation of social, behavioural, medical and dental factors, alongside child and family preferences. The European Academy of Paediatric Dentistry has recently published Best clinical practice guidance , specific to children with MIH, which provides a consensus for treatment alongside the quality of the supporting evidence for each option. It is interesting to reflect on reported differences in management approaches between various clinician groups and between different countries. Notwithstanding these acknowledged disparities, an early diagnosis of enamel hypomineralisation and/or caries is paramount to inform pre-emptive (simple) treatment and maximising best clinical and patient-reported outcomes over the longer-term. The initial assessment It is important to carry out a comprehensive and timely history and examination for any child with FPMs of concern. As will be discussed later, the stage of dental development is an important factor when planning the timing of any extractions. Furthermore, clinicians should be aware of the potential for congenitally missing second premolars in children with MIH which would represent a contraindication to FPM extraction. highlights some of the factors that should be elicited and taken into consideration for all children with compromised FPMs. Caries prevention and management of dentine hypersensitivity Having addressed any acute presenting complaint, the first phase of any treatment plan is to establish a preventive programme. Children with carious and/or hypomineralised FPMs require optimal topical fluoride regimens, including professionally applied fluoride varnish at least twice a year, 2800 ppm fluoride toothpaste (if older than ten years old), and, ideally, a daily fluoridated mouthwash, in conjunction with dietary advice and toothbrushing instruction. Fissure sealants should be applied on any permanent molars not requiring restoration or extraction, although bonding to hypomineralised enamel can be unpredictable. This, together with poor moisture control (stemming from an underlying dentine hypersensitivity and/or child anxiety) can lead to higher failure rates of conventional resin-based fissure sealants. An alternative and less technique-sensitive approach for both child and clinician is the interim use of a resin-modified glass ionomer sealant restoration ( ). Although some clinicians advocate the use of remineralising products (casein phosphopeptide-amorphous calcium phosphate products), desensitising toothpastes, or silver fluoride preparations for the management of MIH hypersensitivity, the evidence base has not been established. Having carried out an initial clinical and radiographic assessment (ideally soon after eruption of the FPMs) the initial phase of treatment aims to manage any symptoms or anxiety, establish a personalised preventive strategy, and protect the teeth from any further post-eruptive breakdown, caries or erosion. The next consideration is to evaluate the likely long-term prognosis and treatment need for each FPM, alongside the variables outlined in . However, a definitive decision may not be appropriate at the first assessment, so the child should be kept under regular review and the family made aware that there are several future treatment options. Taking a restorative approach Current thinking regarding dentine or cavitated caries management is orientated towards minimally invasive approaches, which favour selective or stepwise caries removal, rather than complete. However, in the case of deep caries in asymptomatic vital FPMs, partial or coronal pulpotomies (using materials such as mineral trioxide aggregate or biodentine) have been reported to have variable success rates of around 60-80% at five years. , Crucial to the success of these techniques is optimal moisture control with rubber dam and the use of restorative materials that provide an hermetic seal. The use of amalgam is no longer supported for children under the age of 15 in the UK. It is not common practice to embark on a pulpectomy for FPMs for this young age group in the UK and while endodontic treatment is possible (and sometimes indicated), extraction of these teeth (with or without orthodontic space closure) is likely to achieve better patient and cost outcomes in the longer-term. More challenging than simple caries management is the restoration of hypomineralised FPMs. A recent systematic review provides a comprehensive critique of the various restorative options for children with MIH. For mildly affected FPMs (minimal post-eruptive breakdown), a composite resin restoration, extending beyond the visibly affected enamel opacity, would seem to be the best option. In cases where the opacities involve multiple surfaces, together with rapid post-eruptive breakdown and hypersensitivity, direct or indirect composite resin restorations may be considered. Expert opinion seems to support the removal of any soft hypomineralised enamel before the placement of an indirect restoration with optimal rubber dam moisture control. For some children with severe MIH, full coronal coverage using a preformed metal crown (PMC) may offer a simple medium-term restoration. In such cases, the non-invasive Hall technique for PMC placement usually obviates the need for local anaesthetic and tooth tissue removal; beneficial for young and/or anxious children. A PMC is not considered a definitive restoration (due to potential wear and periodontal damage), but it can be advantageous in situations where the FPM needs to be retained for several years until the optimal time for its planned removal ( ). Any restorative intervention for a young child with compromised FPMs will confer a long-term treatment burden for that patient. Indeed, between the age of 9-18-years old, children with MIH can undergo four times as many treatment episodes (usually retreatment of failed restorations) for these teeth compared to a control group. By the age of 18 years, the MIH group continued to face an ongoing cycle of restorative interventions. Indications for extraction The removal of one or more compromised FPMs is not common practice outside the UK, and a restorative approach is generally favoured in Europe. This may be partly explained by the higher caries prevalence in the UK and more widespread societal acceptance of extractions under sedation or general anaesthetic. Nonetheless, it is argued that extractions may offer the most appropriate treatment for some children with extensive caries and/or hypomineralisation, particularly those experiencing symptoms. The rationale for FPM 'interceptive' extraction is that it obviates the need for ongoing restorative and endodontic care, and encourages second permanent molar eruption with space closure between this tooth and the second premolar, particularly if undertaken at the 'ideal' stage of dental development (that is, around the age of 8-10 years, with the second permanent molar still developing within alveolar bone). Variable success rates have been reported in the maxillary and mandibular arches, with most researchers citing an 80-90% chance of contact in the maxillary arch and around 50-60% in the mandible. , , However, the evidence base for clinical and patient-based outcomes associated with FPMs remains surprisingly sparse, with a lack of randomised clinical trials (RCTs) ( ). In general, there are some acknowledged clinical and patient-related factors which tend to favour the extraction of one or more compromised FPMs ( ). Box 1 Why are there no randomised clinical trials investigating the consequences of FPM extraction? Given the number of children seen every year in both the UK and internationally with compromised FPMs, it would seem surprising that there is a lack of high-quality data investigating the outcomes of treatment. Currently, much of what we know is based upon retrospective cohort data collected from busy hospital departments - particularly in relation to occlusal outcomes following enforced loss of these teeth. Not only do we need more robust data on the long-term occlusal and oral health-related sequelae of interceptive extractions or restorative care, but also more data relating to quality of life outcomes in relation to management decisions for children affected by this common condition over both the immediate and longer-term. It is generally acknowledged that a RCT represents the most robust method of investigating the effects of a treatment intervention, but there are significant challenges associated with applying this methodology to the management of compromised FPMs. Indeed, while attempts have been made to apply this methodology to FPM extraction, no results have yet been published. A significant issue is the ethics of randomising children to extractions or restoration, which is exacerbated by fundamentally different approaches to treatment in different parts of the world. Perhaps the solution might be an international prospective multicentre trial with good control of the variables involved, rather than the ethically more challenging imposition of a RCT. Orthodontic considerations The role of the orthodontist in managing poor prognosis FPMs is to liaise with the paediatric dentist or GDP, and give advice within the context of any potential interceptive extractions and overall management of any underlying malocclusion. It is important to state that the key to orthodontic decision-making is clear direction on the long-term prognosis of each affected tooth and this should come from the paediatric dentist or GDP, particularly in relation to teeth affected by MIH ( ). In addition, the presence of any acute symptoms, the ability of a child to accept restorative care, and of course, any requirement for a general anaesthetic as part of their management, will have a significant influence on fundamental treatment planning decisions. Guidelines from the Faculty of Dental Surgery, Royal College of Surgeons of England, describe best practice on the timing, compensation and balancing of FPM extractions; however, the evidence base is generally low quality, with a preponderance of retrospective investigations currently populating the literature. In terms of interceptive extractions, predictors for successful eruption of the second permanent molar have always been more important in the mandibular arch. Classically, the child should have a Class I malocclusion and be between the ages of 8-10 years old to ensure minimal disruption to occlusal development. In addition, radiographic evidence of the second permanent molar unerupted in alveolar bone and early mineralisation of the bifurcation represent an optimal time for FPM extraction to ensure a good eruptive position of the second molar. , More recent evidence would suggest that the window of opportunity in relation to radiographic development of the second permanent molar is wider in terms of bifurcation mineralisation, and that the mesiodistal angulation of these teeth and presence of a third permanent molar can offer further useful prediction of favourable second permanent molar eruption. , , All these predictive factors are more relevant in the mandibular arch, as the maxillary second permanent molar will generally achieve a good eruptive position over a wider range of extraction timings. , , In terms of interceptive treatment, routine balancing extraction of a sound FPM to preserve a dental centreline is not recommended. Compensating extraction of a sound upper FPM has been suggested to prevent over-eruption of this tooth when extraction of the lower FPM is required. For an upper FPM that will remain unopposed for some time, significant over-eruption can cause interferences with the erupting lower second permanent molar, impeding space closure and potentially contributing to other occlusal interferences. Current evidence would suggest that the risk of upper FPM over-eruption, as a consequence of lower FPM extraction, is small, and decisions should be made on a case-by-case basis. The widespread use of modern fixed appliances and fixed anchorage in orthodontics has meant that the incorporation of FPM extractions has become more routine in the management of malocclusion. , Indeed, with radiographic evidence of third permanent molar development and a requirement for extraction-based fixed appliance treatment, the presence of caries, MIH, or a restoration in any FPM should elicit serious consideration of its elective extraction as part of an orthodontic treatment plan incorporating fixed appliances. When considering orthodontic treatment, there is no doubt that occlusal outcomes are generally easier to control in Class I cases, and those cases associated with any degree of sagittal discrepancy that are at the milder end of the spectrum. In general, the higher the anchorage requirements, the more difficult FPM extraction cases become to manage with fixed appliances, particularly those associated with the presence of a significant overjet and/or crowding. The reliance upon anchorage reinforcement with headgear, transpalatal arches and mini implants becomes more important for achieving a successful outcome, particularly in the older child ( ). However, depending upon severity of the malocclusion, even poorly positioned second permanent molars can be relatively easily managed with fixed appliances ( ). Space closure can be prolonged, particularly in the mandibular arch, but careful anchorage management and patient mechanics can produce good occlusal results, even in the adult dentition ( ). It should also be remembered that for some children presenting in the established permanent dentition with high caries risk and/or poor oral hygiene, fixed appliances might not be appropriate and sometimes compromises will need to be made when FPMs cannot be restored.
It is important to carry out a comprehensive and timely history and examination for any child with FPMs of concern. As will be discussed later, the stage of dental development is an important factor when planning the timing of any extractions. Furthermore, clinicians should be aware of the potential for congenitally missing second premolars in children with MIH which would represent a contraindication to FPM extraction. highlights some of the factors that should be elicited and taken into consideration for all children with compromised FPMs.
Having addressed any acute presenting complaint, the first phase of any treatment plan is to establish a preventive programme. Children with carious and/or hypomineralised FPMs require optimal topical fluoride regimens, including professionally applied fluoride varnish at least twice a year, 2800 ppm fluoride toothpaste (if older than ten years old), and, ideally, a daily fluoridated mouthwash, in conjunction with dietary advice and toothbrushing instruction. Fissure sealants should be applied on any permanent molars not requiring restoration or extraction, although bonding to hypomineralised enamel can be unpredictable. This, together with poor moisture control (stemming from an underlying dentine hypersensitivity and/or child anxiety) can lead to higher failure rates of conventional resin-based fissure sealants. An alternative and less technique-sensitive approach for both child and clinician is the interim use of a resin-modified glass ionomer sealant restoration ( ). Although some clinicians advocate the use of remineralising products (casein phosphopeptide-amorphous calcium phosphate products), desensitising toothpastes, or silver fluoride preparations for the management of MIH hypersensitivity, the evidence base has not been established. Having carried out an initial clinical and radiographic assessment (ideally soon after eruption of the FPMs) the initial phase of treatment aims to manage any symptoms or anxiety, establish a personalised preventive strategy, and protect the teeth from any further post-eruptive breakdown, caries or erosion. The next consideration is to evaluate the likely long-term prognosis and treatment need for each FPM, alongside the variables outlined in . However, a definitive decision may not be appropriate at the first assessment, so the child should be kept under regular review and the family made aware that there are several future treatment options.
Current thinking regarding dentine or cavitated caries management is orientated towards minimally invasive approaches, which favour selective or stepwise caries removal, rather than complete. However, in the case of deep caries in asymptomatic vital FPMs, partial or coronal pulpotomies (using materials such as mineral trioxide aggregate or biodentine) have been reported to have variable success rates of around 60-80% at five years. , Crucial to the success of these techniques is optimal moisture control with rubber dam and the use of restorative materials that provide an hermetic seal. The use of amalgam is no longer supported for children under the age of 15 in the UK. It is not common practice to embark on a pulpectomy for FPMs for this young age group in the UK and while endodontic treatment is possible (and sometimes indicated), extraction of these teeth (with or without orthodontic space closure) is likely to achieve better patient and cost outcomes in the longer-term. More challenging than simple caries management is the restoration of hypomineralised FPMs. A recent systematic review provides a comprehensive critique of the various restorative options for children with MIH. For mildly affected FPMs (minimal post-eruptive breakdown), a composite resin restoration, extending beyond the visibly affected enamel opacity, would seem to be the best option. In cases where the opacities involve multiple surfaces, together with rapid post-eruptive breakdown and hypersensitivity, direct or indirect composite resin restorations may be considered. Expert opinion seems to support the removal of any soft hypomineralised enamel before the placement of an indirect restoration with optimal rubber dam moisture control. For some children with severe MIH, full coronal coverage using a preformed metal crown (PMC) may offer a simple medium-term restoration. In such cases, the non-invasive Hall technique for PMC placement usually obviates the need for local anaesthetic and tooth tissue removal; beneficial for young and/or anxious children. A PMC is not considered a definitive restoration (due to potential wear and periodontal damage), but it can be advantageous in situations where the FPM needs to be retained for several years until the optimal time for its planned removal ( ). Any restorative intervention for a young child with compromised FPMs will confer a long-term treatment burden for that patient. Indeed, between the age of 9-18-years old, children with MIH can undergo four times as many treatment episodes (usually retreatment of failed restorations) for these teeth compared to a control group. By the age of 18 years, the MIH group continued to face an ongoing cycle of restorative interventions.
The removal of one or more compromised FPMs is not common practice outside the UK, and a restorative approach is generally favoured in Europe. This may be partly explained by the higher caries prevalence in the UK and more widespread societal acceptance of extractions under sedation or general anaesthetic. Nonetheless, it is argued that extractions may offer the most appropriate treatment for some children with extensive caries and/or hypomineralisation, particularly those experiencing symptoms. The rationale for FPM 'interceptive' extraction is that it obviates the need for ongoing restorative and endodontic care, and encourages second permanent molar eruption with space closure between this tooth and the second premolar, particularly if undertaken at the 'ideal' stage of dental development (that is, around the age of 8-10 years, with the second permanent molar still developing within alveolar bone). Variable success rates have been reported in the maxillary and mandibular arches, with most researchers citing an 80-90% chance of contact in the maxillary arch and around 50-60% in the mandible. , , However, the evidence base for clinical and patient-based outcomes associated with FPMs remains surprisingly sparse, with a lack of randomised clinical trials (RCTs) ( ). In general, there are some acknowledged clinical and patient-related factors which tend to favour the extraction of one or more compromised FPMs ( ). Box 1 Why are there no randomised clinical trials investigating the consequences of FPM extraction? Given the number of children seen every year in both the UK and internationally with compromised FPMs, it would seem surprising that there is a lack of high-quality data investigating the outcomes of treatment. Currently, much of what we know is based upon retrospective cohort data collected from busy hospital departments - particularly in relation to occlusal outcomes following enforced loss of these teeth. Not only do we need more robust data on the long-term occlusal and oral health-related sequelae of interceptive extractions or restorative care, but also more data relating to quality of life outcomes in relation to management decisions for children affected by this common condition over both the immediate and longer-term. It is generally acknowledged that a RCT represents the most robust method of investigating the effects of a treatment intervention, but there are significant challenges associated with applying this methodology to the management of compromised FPMs. Indeed, while attempts have been made to apply this methodology to FPM extraction, no results have yet been published. A significant issue is the ethics of randomising children to extractions or restoration, which is exacerbated by fundamentally different approaches to treatment in different parts of the world. Perhaps the solution might be an international prospective multicentre trial with good control of the variables involved, rather than the ethically more challenging imposition of a RCT.
The role of the orthodontist in managing poor prognosis FPMs is to liaise with the paediatric dentist or GDP, and give advice within the context of any potential interceptive extractions and overall management of any underlying malocclusion. It is important to state that the key to orthodontic decision-making is clear direction on the long-term prognosis of each affected tooth and this should come from the paediatric dentist or GDP, particularly in relation to teeth affected by MIH ( ). In addition, the presence of any acute symptoms, the ability of a child to accept restorative care, and of course, any requirement for a general anaesthetic as part of their management, will have a significant influence on fundamental treatment planning decisions. Guidelines from the Faculty of Dental Surgery, Royal College of Surgeons of England, describe best practice on the timing, compensation and balancing of FPM extractions; however, the evidence base is generally low quality, with a preponderance of retrospective investigations currently populating the literature. In terms of interceptive extractions, predictors for successful eruption of the second permanent molar have always been more important in the mandibular arch. Classically, the child should have a Class I malocclusion and be between the ages of 8-10 years old to ensure minimal disruption to occlusal development. In addition, radiographic evidence of the second permanent molar unerupted in alveolar bone and early mineralisation of the bifurcation represent an optimal time for FPM extraction to ensure a good eruptive position of the second molar. , More recent evidence would suggest that the window of opportunity in relation to radiographic development of the second permanent molar is wider in terms of bifurcation mineralisation, and that the mesiodistal angulation of these teeth and presence of a third permanent molar can offer further useful prediction of favourable second permanent molar eruption. , , All these predictive factors are more relevant in the mandibular arch, as the maxillary second permanent molar will generally achieve a good eruptive position over a wider range of extraction timings. , , In terms of interceptive treatment, routine balancing extraction of a sound FPM to preserve a dental centreline is not recommended. Compensating extraction of a sound upper FPM has been suggested to prevent over-eruption of this tooth when extraction of the lower FPM is required. For an upper FPM that will remain unopposed for some time, significant over-eruption can cause interferences with the erupting lower second permanent molar, impeding space closure and potentially contributing to other occlusal interferences. Current evidence would suggest that the risk of upper FPM over-eruption, as a consequence of lower FPM extraction, is small, and decisions should be made on a case-by-case basis. The widespread use of modern fixed appliances and fixed anchorage in orthodontics has meant that the incorporation of FPM extractions has become more routine in the management of malocclusion. , Indeed, with radiographic evidence of third permanent molar development and a requirement for extraction-based fixed appliance treatment, the presence of caries, MIH, or a restoration in any FPM should elicit serious consideration of its elective extraction as part of an orthodontic treatment plan incorporating fixed appliances. When considering orthodontic treatment, there is no doubt that occlusal outcomes are generally easier to control in Class I cases, and those cases associated with any degree of sagittal discrepancy that are at the milder end of the spectrum. In general, the higher the anchorage requirements, the more difficult FPM extraction cases become to manage with fixed appliances, particularly those associated with the presence of a significant overjet and/or crowding. The reliance upon anchorage reinforcement with headgear, transpalatal arches and mini implants becomes more important for achieving a successful outcome, particularly in the older child ( ). However, depending upon severity of the malocclusion, even poorly positioned second permanent molars can be relatively easily managed with fixed appliances ( ). Space closure can be prolonged, particularly in the mandibular arch, but careful anchorage management and patient mechanics can produce good occlusal results, even in the adult dentition ( ). It should also be remembered that for some children presenting in the established permanent dentition with high caries risk and/or poor oral hygiene, fixed appliances might not be appropriate and sometimes compromises will need to be made when FPMs cannot be restored.
Within the dental literature, there is growing emphasis on how dental conditions may impact on children's oral and general health-related quality of life. It is now well-recognised that both untreated caries and MIH can have profoundly negative impacts on a child's social, emotional and functional wellbeing. , More research is needed to better understand how interventions can improve patient-reported outcomes and experiences for children with compromised FPMs, both in the short- and long-term.
Compromised FPMs can have a negative impact on a child's quality of life and present significant management challenges for the dental team. Although a high-quality evidence base is still lacking to support all the different treatment options, early diagnosis and multidisciplinary treatment planning are key to achieving the best possible outcomes.
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Comparative pathology and immunohistochemistry of Newcastle disease in domestic chicken (
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2908f7c7-4e0b-4a8a-860f-629dd8945327
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10219819
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Anatomy[mh]
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Newcastle disease (ND) is highly pathogenic in reared chicken but less pathogenic in other birds that are important in spreading the Newcastle disease virus (NDV). It has high morbidity and mortality and can spread rapidly . NDV has widely infected bird species, such as chickens, ducks, geese, pigeons, parrots, and several other birds . Ducks and geese act as a natural reservoir that does not show clinical symptoms but can transmit the deadly disease to chickens . compared six breeds of ducks and found mallard ducks were the most susceptible and Pekin ducks were the more resistant; they found that the susceptibility of ducks to NDV decreased with age, and most deaths occurred between 15 and 30 days of age. reported that 10 domestic chicken samples collected from field cases in 2008–2009 in Bali, Indonesia, were positive to be infected by the acute NDV. NDV infection in domestic chicken, broiler chicken, and waterfowl in Aceh, Indonesia, is dominated by virulent strains . In addition, several pathogenic isolates have been isolated from reared ducks ( ; Zhang et al. , 2011; ). ND can cause damage to lymphoid tissue and macrophages. Previous studies reported that histopathologically ND causes lymphoid follicular depletion, necrosis, and apoptosis in the caecal tonsil, thymus, and bursa of Fabricius and spleen chicken . Skeletal muscle congestion, mild intestinal erosion, and mild hemorrhagic cecal tonsils in ducks . Apoptosis is a significant factor in viral pathogenesis, especially in the mechanism of clearance virus by the immune system . NDV causes apoptosis in the spleen of domestic chickens , chicken embryo fibroblast cells , and chicken macrophages . There are many things yet unknown on the cause of why ducks are more resistant to ND disease compared to chickens. Therefore, this study was designed to compare the clinical symptoms features, pathological lesions, viral distribution, and apoptosis response due to NDV in domestic chickens and Alabio ducks.
Research procedure One-day-old domestic chickens ( Gallus gallus domesticus) and 1-day-old Alabio duck ( Anas platyrhynchos Borneo) were reared until 6 weeks in semi-isolated cages in groups. Feed and drinking water were provided ad libitum. The treatment groups are: AC-A (Domestic chicken group, n = 20) and AC-I (Alabio duck group, n = 20), were infected by NDV velogenic isolate Ducks/Aceh Besar_IND/2013/eoAC080721 under 10 6 ELD 50 dosage. The K-A (Domestic chicken control group, n = 20) and K-I (Alabio duck control group, n = 20) were inoculated by PBS. All inoculations were performed via intraorbital as much as 0.1 ml. Before being infected by NDV, the domestic chickens and Alabio ducks tested negative for NDV antibody by hemaglutinin inhibition test. Clinical symptoms and gross anatomy observation Clinical symptoms were observed from day 1 until day 7 post-infection (PI). Three individuals from each group were necropsied on days 1, 2, 3, 5, and 7 PI. Gross pathological changes in the proventriculus, duodenum, ceca tonsil, trachea, lung, heart, thymus, Fabricius bursa, spleen, kidney, and brain were observed. All organ samples were cut into 1 × 1 × 0.5 cm sizes and fixed in neutral buffered formalin 10% for a minimum of 24 hours to be made into histopathology preparations in paraffin blocks. Histopathology examination Each organ was trimmed into 5 mm size and put inside a tissue cassette, then put into an automatic tissue processor for dehydration, clearing, paraffin infiltration, embedding, and paraffin blocking. Finally, the blocks were cut into 5 µm with a rotary microtome to be stained with hematoxylin staining or immunohistochemistry. Hematoxylin and eosin staining Hematoxylin and eosin staining starter by deparaffinization by xylol and ethanol rehydration. Staining was performed by submerging preparations inside Mayer's hematoxylin stain, followed by eosin. The tissues were then dehydrated with ethanol 96% and absolute ethanol 2. The clearing was performed by submerging the tissue in xylol. The last process was mounting using gum and cover glass. Histopathology observation was performed by examining the lesion severity. The following criteria had determined: if the lesion was spread locally, multifocally, or diffuse, the seriousness in that order was light, moderate, or severe. The examination was conducted under 100 times magnification with five fields of view repetition. Immunohistochemistry staining The immunohistochemistry staining referred to the procedures recommended in the catalog from Dako, North America Inc. (Dako) with several modifications. Tissue slices attached to poly-L-Lysine 1% spread object glass was deparaffinized by xylol and then rehydrated by ethanol. The antigen retrieval process was performed by boiling the preparations in citrate buffer at 100°C for 15 minutes. Blocking of endogenous activity was performed by submerging the preparations in H 2 O 2 3% for 35 minutes at room temperature and washing them with PBS in three repetitions for 5 minutes each. Blocking of non-specific protein bonds was conducted using normal fetal bovine serum 10% for 35 minutes at room temperature and then washed once more with PBS in three repetitions for 5 minutes each. Each tissue was given drops of primary antibody rabbit anti-NDV polyclonal antibody (1:250 in PBS), and for caspase-3, was given drops of primary antibody Polyclonal Anti-Casp3 (HPA002643, Sigma-Aldrich; 1:250 in PBS). The preparations were then incubated overnight at −5°C. The preparations were washed with PBS in three repetitions for 5 minutes each at room temperature. The preparations were then dropped with secondary antibody Dako REAL™ envision™/HRP, Rabbit/Mouse (K5007) for 40 minutes at room temperature, then washed with PBS in three repetitions 5 minutes each at room temperature. The preparations were then given Dako REAL™ DAB+chromogen in Dako REAL™ substrate buffer (K5007) for 40 seconds at room temperature and then washed with flowing water for 10 minutes and washed by PBS in three repetitions for 5 minutes each at room temperature. Mayer’s Hematoxylin was used as counterstaining. The examination was regarded as positive if, during preparation, reading brown stained antigen was found and regarded as negative if all preparation appeared blue. The immunopositivity against NDV on each organ was scored with light severity (1–10 immunopositive cells), moderate (11–20 immunopositive cells), and severe (more than 20 immunopositive cells) . The immunohistochemistry results against NDV and caspase-3 were examined under 400 times magnification with five field view repetitions. Data analysis The data from clinical symptoms and pathological lesions examinations were analyzed descriptively. The immunohistochemistry results against the NDV were scored based on the immunopositive cells’ level, while for caspase-3, the immunopositive reaction was analyzed based on the positive area percentage against caspase-3. Ethical approval The use and treatment of experimental animals in this study have approval from the animal ethics committee of the Institute for Research and Community Service, Bogor Agricultural University.
One-day-old domestic chickens ( Gallus gallus domesticus) and 1-day-old Alabio duck ( Anas platyrhynchos Borneo) were reared until 6 weeks in semi-isolated cages in groups. Feed and drinking water were provided ad libitum. The treatment groups are: AC-A (Domestic chicken group, n = 20) and AC-I (Alabio duck group, n = 20), were infected by NDV velogenic isolate Ducks/Aceh Besar_IND/2013/eoAC080721 under 10 6 ELD 50 dosage. The K-A (Domestic chicken control group, n = 20) and K-I (Alabio duck control group, n = 20) were inoculated by PBS. All inoculations were performed via intraorbital as much as 0.1 ml. Before being infected by NDV, the domestic chickens and Alabio ducks tested negative for NDV antibody by hemaglutinin inhibition test.
Clinical symptoms were observed from day 1 until day 7 post-infection (PI). Three individuals from each group were necropsied on days 1, 2, 3, 5, and 7 PI. Gross pathological changes in the proventriculus, duodenum, ceca tonsil, trachea, lung, heart, thymus, Fabricius bursa, spleen, kidney, and brain were observed. All organ samples were cut into 1 × 1 × 0.5 cm sizes and fixed in neutral buffered formalin 10% for a minimum of 24 hours to be made into histopathology preparations in paraffin blocks.
Each organ was trimmed into 5 mm size and put inside a tissue cassette, then put into an automatic tissue processor for dehydration, clearing, paraffin infiltration, embedding, and paraffin blocking. Finally, the blocks were cut into 5 µm with a rotary microtome to be stained with hematoxylin staining or immunohistochemistry.
Hematoxylin and eosin staining starter by deparaffinization by xylol and ethanol rehydration. Staining was performed by submerging preparations inside Mayer's hematoxylin stain, followed by eosin. The tissues were then dehydrated with ethanol 96% and absolute ethanol 2. The clearing was performed by submerging the tissue in xylol. The last process was mounting using gum and cover glass. Histopathology observation was performed by examining the lesion severity. The following criteria had determined: if the lesion was spread locally, multifocally, or diffuse, the seriousness in that order was light, moderate, or severe. The examination was conducted under 100 times magnification with five fields of view repetition.
The immunohistochemistry staining referred to the procedures recommended in the catalog from Dako, North America Inc. (Dako) with several modifications. Tissue slices attached to poly-L-Lysine 1% spread object glass was deparaffinized by xylol and then rehydrated by ethanol. The antigen retrieval process was performed by boiling the preparations in citrate buffer at 100°C for 15 minutes. Blocking of endogenous activity was performed by submerging the preparations in H 2 O 2 3% for 35 minutes at room temperature and washing them with PBS in three repetitions for 5 minutes each. Blocking of non-specific protein bonds was conducted using normal fetal bovine serum 10% for 35 minutes at room temperature and then washed once more with PBS in three repetitions for 5 minutes each. Each tissue was given drops of primary antibody rabbit anti-NDV polyclonal antibody (1:250 in PBS), and for caspase-3, was given drops of primary antibody Polyclonal Anti-Casp3 (HPA002643, Sigma-Aldrich; 1:250 in PBS). The preparations were then incubated overnight at −5°C. The preparations were washed with PBS in three repetitions for 5 minutes each at room temperature. The preparations were then dropped with secondary antibody Dako REAL™ envision™/HRP, Rabbit/Mouse (K5007) for 40 minutes at room temperature, then washed with PBS in three repetitions 5 minutes each at room temperature. The preparations were then given Dako REAL™ DAB+chromogen in Dako REAL™ substrate buffer (K5007) for 40 seconds at room temperature and then washed with flowing water for 10 minutes and washed by PBS in three repetitions for 5 minutes each at room temperature. Mayer’s Hematoxylin was used as counterstaining. The examination was regarded as positive if, during preparation, reading brown stained antigen was found and regarded as negative if all preparation appeared blue. The immunopositivity against NDV on each organ was scored with light severity (1–10 immunopositive cells), moderate (11–20 immunopositive cells), and severe (more than 20 immunopositive cells) . The immunohistochemistry results against NDV and caspase-3 were examined under 400 times magnification with five field view repetitions.
The data from clinical symptoms and pathological lesions examinations were analyzed descriptively. The immunohistochemistry results against the NDV were scored based on the immunopositive cells’ level, while for caspase-3, the immunopositive reaction was analyzed based on the positive area percentage against caspase-3.
The use and treatment of experimental animals in this study have approval from the animal ethics committee of the Institute for Research and Community Service, Bogor Agricultural University.
Clinical symptoms observation On day 1, domestic chicken suffered from depression and lethargy; on day 2, PI conjunctivitis appeared; on day 3, PI had difficulty breathing, greenish-white diarrhea, and anorexia appeared; on days 5 and 7, PI nervous symptoms such as muscle tremor, difficulty in standing, and wings dropping along with light edema on the head appeared. Death in domestic chickens started to appear on day 4 PI . Alabio duck groups only seemed to be depressed and lightly lethargic starting from day 5 PI. Control domestic chicken and Alabio duck group did not show any clinical symptoms. Gross anatomy observation The control domestic chicken and Alabio duck group showed no gross anatomy lesions. Proventriculus, duodenum, and cecal tonsil did not show lesions on day 1 PI in domestic chickens, but in Alabio ducks, proventriculus appeared to have diffuse catarrhal exudation on the mucosal layer. Proventriculus on day 3 PI showed multifocal petechiae. On days 5 and 7, PI showed diffuse hemorrhage on domestic chickens . Alabio duck proventriculus on days 3 and 5 PI showed diffuse catarrhal exudation, and no lesion appeared on day 7 PI . Duodenum on day 3 until day 7 PI appeared hemorrhagic with multifocal necrosis on domestic chickens. In contrast, in Alabio ducks, no lesion appeared on all observation days. Cecal tonsil on day 3 PI until the last observation day seemed to have a multifocal hemorrhage in domestic chicken, but in Alabio ducks, no lesion appeared on all observation days . On day 5 PI, the trachea suffered multifocal diffuse congestion on domestic chickens and focal congestion on Alabio ducks . Lungs on days 1 and 3 PI appeared abnormal from multifocal congestion. On every following observation day, the lesions spread diffusely on domestic chickens, whereas, on Alabio ducks, multifocal congestion only appeared on day 1 PI. On all observation days, the Thymus of domestic chicken appeared to have half of its lobes atrophied and had petechiae/hemorrhage. In contrast, in Alabio ducks, part of the lobus showed petechiae on all observation days. The spleen of domestic chickens on days 1, 3, and 5 PI showed congestion, swelling, and multifocal necrosis, while the days after were followed by atrophy. In Alabio ducks, the spleen showed swelling and multifocal necrosis only during day 3 PI. The Fabricius bursa of domestic chicken on day 1 PI appeared to be swelling with focal hemorrhage. On the rest of the observation days, it was followed by atrophy and diffuse hemorrhage, whereas in Alabio ducks, no lesion was found on all observation days. Starting from day 3 PI, the domestic chicken heart showed swelling, while the Alabio duck heart showed no gross pathology changes on every observation day. Domestic chicken kidneys on all observation days appeared swelled with multifocal paleness, while in Alabio ducks, no lesion was seen on any observation day. Domestic chicken brain from day 5 PI started to show edema and multifocal congestion, while in Alabio duck, the lesion was focal Histopathology examination The control domestic chicken and Alabio duck group showed no histopathology lesions. Domestic chicken proventriculus on day 1 PI showed congestion, focal mononuclear proliferation on the mucosal layer, necrosis, and focal epithelial cell desquamation on proventriculus glands. On day 3 PI, the lesions spread in a multifocal pattern; on days 5 and 7 PI epithelial cells showed necrosis and diffuse hemorrhage by the muscle layer and multifocal epithelial cell desquamation as well as necrosis, congestion, and multifocal mononuclear cell infiltration by the proventiculus gland. Alabio duck proventriculus on day 1 PI showed focal epithelial cell desquamation; on days 3 and 5 PI showed congestion, epithelial cell desquamation, and multifocal mononuclear cell proliferation, while proventriculus glands showed multifocal necrosis, epithelial cell desquamation, and focal congestion. On day 7 PI, no lesion was seen by the proventriculus. On days 1 and 3 PI, domestic chicken duodenum showed hemorrhage, congestion, and focal crypt epithelial cell necrosis. On all following observation days, multifocal hemorrhage with intestine villus desquamation, diffuse crypt epithelial cell necrosis, and multifocal proliferation of crypt epithelial cells in lamina propia was observed. Alabio duck duodenum from days 3 to 5 PI showed multifocal crypt epithelial necrosis, congestion, hemorrhage, and focal goblet cell proliferation. On day 7 PI, there were multifocal crypt epithelial cell necrosis and focal mononuclear cell proliferation in lamina propria. The cecal tonsil of domestic chicken on all observation days histopathologically showed congestion, hemorrhage, necrosis (karyopicnosis) of crypt epithelial cells, mononuclear cell proliferation on the lamina propria, and lymphocyte cell depletion inside the lymphoid follicle, which spread in the multifocal pattern. The Alabio duck cecal tonsil on days 1 and 3 PI showed congestion, necrosis (Karyopicnosis) of crypt cells, and multifocal mononuclear cell proliferation by the lamina propria. On days 5 and 7, multifocal mononuclear cell proliferation in lamina propria and depletion of lymphocyte cells in lymphoid follicles were seen . Domestic chicken trachea on day 1 PI showed congestion, epithelial cell desquamation, focal inflammatory cell infiltration, and diffuse edema. On the following observation days, the lesions spread in multifocal patterns. Alabio duck trachea on all observation days showed congestion, epithelial desquamation, focal inflammatory cell infiltration, goblet cell proliferation, and diffuse edema . Domestic chicken lung on day 1 PI showed hemorrhage, congestion, edema, and multifocal mononuclear cell proliferation. On all following observation days, the lesion spread in a diffuse pattern. Alabio duck lungs on all observation days showed congestion, edema, hemorrhage, and multifocal mononuclear cell proliferation. The domestic chicken's thymus, Fabricius bursa, and spleen on days 1 and 3 PI showed lymphoid depletion, congestion, and multifocal vasculitis. On days 5 and 7, the lesion spread in a diffuse pattern accompanied by cyst formation, lysing cells, and part of the tissue being replaced by connective tissue. The ducks' Thymus, Fabricius bursae, and spleen on day 1 appeared to suffer lymphoid depletion, congestion, and focal vasculitis. On days 3 and 5 PI, the lesions were spread in a multifocal pattern, and on day 7, the lesions were spread in a focal pattern. The heart of domestic chicken on day 3 PI started to show edema and focal degeneration; on day 5 PI, it was accompanied by hemorrhage, congestion, and focal mononuclear cell infiltration. On day 7, the lesions spread in a multifocal pattern accompanied by pericarditis and endothelial hypertrophy. However, in Alabio duck, the lesions were only spread in a focal pattern without pericarditis. The kidney of domestic chicken on day 1 PI appeared to suffer from edema, congestion, and focal hemorrhage. On day 3 PI, the lesions were spread in a multifocal pattern. Every following observation day, the lesions spread diffusely, followed by multifocal mononuclear cell infiltration by the interstitial area of the kidney. Alabio duck kidney appeared to suffer from edema and focal congestion on all observation days, and focal mononuclear infiltration by kidney interstitial started to appear from day 3 PI. Domestic chicken brain on days 1 and 3 PI showed neuron degeneration, congestion, edema, and multifocal endothelial hypertrophy. On days 5 and 7 PI, they were followed by neuron cells necrosis, multifocal gliosis, and focal perivascular cuffing . The Alabio duck brain on every observation day appeared to suffer from congestion, edema, endothelial cell hypertrophy, and multifocal gliosis. NDV distribution Immunohistochemistry staining showed that immunopositive reaction against NDV was found in all treatment groups from day 1 until the last observation day, with severity ranging from light to severe, while on all control groups, immunonegative. The immunopositive location against NDV was not different between domestic chicken and Alabio duck. A positive reaction was found in epithelial cells and mononuclear inflammation in the intestinal organs . Cilia epithelial cells, mucosal layer mononuclear cells, and in the spaces of tracheal goblet calls . Parabronchi epithelial cells, pneumocystis, and inflammatory cells in lung alveoli. Heart blood vessel endothelial cells and the urinary. Reticular epithelial cells at the medulla layer and mononuclear cells by the thymus cortex. Lymphoid cells of white pulp and lymphoid cells inside the Fabricius bursa lymphoid follicle. The plica epithelial cells and mononuclear cells are suffering from depletion within the lymphoid follicle of the Fabricius bursa: the Virchow-Robin endothelial cell, necrotic glial cells, and neurons in the brain . The caspase-3 expression in lymphoreticular organs The percentage of caspase-3 in lymphoreticular organs peaked earlier in the Alabio duck group, which was on day 2 PI, compared to the domestic chicken group, which on average, occurred after day 3 PI. The caspase-3 expression in domestic chicken and Alabio duck thymus was more dominant in the medullar area and rarely by the cortex. The caspase-3 expression in domestic chicken and Alabio duck Fabricius bursa by the plica epithelial and lymphoid follicle, while in the cecal tonsil by the mucosa epithelial cell, lamina propria inflammatory cells, and crypt epithelial cells. The caspase-3 expression of domestic chicken spleen was more dominant around the germinal center, whereas caspase-3 expression in Alabio ducks was more dominant in the germinal center.
On day 1, domestic chicken suffered from depression and lethargy; on day 2, PI conjunctivitis appeared; on day 3, PI had difficulty breathing, greenish-white diarrhea, and anorexia appeared; on days 5 and 7, PI nervous symptoms such as muscle tremor, difficulty in standing, and wings dropping along with light edema on the head appeared. Death in domestic chickens started to appear on day 4 PI . Alabio duck groups only seemed to be depressed and lightly lethargic starting from day 5 PI. Control domestic chicken and Alabio duck group did not show any clinical symptoms.
The control domestic chicken and Alabio duck group showed no gross anatomy lesions. Proventriculus, duodenum, and cecal tonsil did not show lesions on day 1 PI in domestic chickens, but in Alabio ducks, proventriculus appeared to have diffuse catarrhal exudation on the mucosal layer. Proventriculus on day 3 PI showed multifocal petechiae. On days 5 and 7, PI showed diffuse hemorrhage on domestic chickens . Alabio duck proventriculus on days 3 and 5 PI showed diffuse catarrhal exudation, and no lesion appeared on day 7 PI . Duodenum on day 3 until day 7 PI appeared hemorrhagic with multifocal necrosis on domestic chickens. In contrast, in Alabio ducks, no lesion appeared on all observation days. Cecal tonsil on day 3 PI until the last observation day seemed to have a multifocal hemorrhage in domestic chicken, but in Alabio ducks, no lesion appeared on all observation days . On day 5 PI, the trachea suffered multifocal diffuse congestion on domestic chickens and focal congestion on Alabio ducks . Lungs on days 1 and 3 PI appeared abnormal from multifocal congestion. On every following observation day, the lesions spread diffusely on domestic chickens, whereas, on Alabio ducks, multifocal congestion only appeared on day 1 PI. On all observation days, the Thymus of domestic chicken appeared to have half of its lobes atrophied and had petechiae/hemorrhage. In contrast, in Alabio ducks, part of the lobus showed petechiae on all observation days. The spleen of domestic chickens on days 1, 3, and 5 PI showed congestion, swelling, and multifocal necrosis, while the days after were followed by atrophy. In Alabio ducks, the spleen showed swelling and multifocal necrosis only during day 3 PI. The Fabricius bursa of domestic chicken on day 1 PI appeared to be swelling with focal hemorrhage. On the rest of the observation days, it was followed by atrophy and diffuse hemorrhage, whereas in Alabio ducks, no lesion was found on all observation days. Starting from day 3 PI, the domestic chicken heart showed swelling, while the Alabio duck heart showed no gross pathology changes on every observation day. Domestic chicken kidneys on all observation days appeared swelled with multifocal paleness, while in Alabio ducks, no lesion was seen on any observation day. Domestic chicken brain from day 5 PI started to show edema and multifocal congestion, while in Alabio duck, the lesion was focal
The control domestic chicken and Alabio duck group showed no histopathology lesions. Domestic chicken proventriculus on day 1 PI showed congestion, focal mononuclear proliferation on the mucosal layer, necrosis, and focal epithelial cell desquamation on proventriculus glands. On day 3 PI, the lesions spread in a multifocal pattern; on days 5 and 7 PI epithelial cells showed necrosis and diffuse hemorrhage by the muscle layer and multifocal epithelial cell desquamation as well as necrosis, congestion, and multifocal mononuclear cell infiltration by the proventiculus gland. Alabio duck proventriculus on day 1 PI showed focal epithelial cell desquamation; on days 3 and 5 PI showed congestion, epithelial cell desquamation, and multifocal mononuclear cell proliferation, while proventriculus glands showed multifocal necrosis, epithelial cell desquamation, and focal congestion. On day 7 PI, no lesion was seen by the proventriculus. On days 1 and 3 PI, domestic chicken duodenum showed hemorrhage, congestion, and focal crypt epithelial cell necrosis. On all following observation days, multifocal hemorrhage with intestine villus desquamation, diffuse crypt epithelial cell necrosis, and multifocal proliferation of crypt epithelial cells in lamina propia was observed. Alabio duck duodenum from days 3 to 5 PI showed multifocal crypt epithelial necrosis, congestion, hemorrhage, and focal goblet cell proliferation. On day 7 PI, there were multifocal crypt epithelial cell necrosis and focal mononuclear cell proliferation in lamina propria. The cecal tonsil of domestic chicken on all observation days histopathologically showed congestion, hemorrhage, necrosis (karyopicnosis) of crypt epithelial cells, mononuclear cell proliferation on the lamina propria, and lymphocyte cell depletion inside the lymphoid follicle, which spread in the multifocal pattern. The Alabio duck cecal tonsil on days 1 and 3 PI showed congestion, necrosis (Karyopicnosis) of crypt cells, and multifocal mononuclear cell proliferation by the lamina propria. On days 5 and 7, multifocal mononuclear cell proliferation in lamina propria and depletion of lymphocyte cells in lymphoid follicles were seen . Domestic chicken trachea on day 1 PI showed congestion, epithelial cell desquamation, focal inflammatory cell infiltration, and diffuse edema. On the following observation days, the lesions spread in multifocal patterns. Alabio duck trachea on all observation days showed congestion, epithelial desquamation, focal inflammatory cell infiltration, goblet cell proliferation, and diffuse edema . Domestic chicken lung on day 1 PI showed hemorrhage, congestion, edema, and multifocal mononuclear cell proliferation. On all following observation days, the lesion spread in a diffuse pattern. Alabio duck lungs on all observation days showed congestion, edema, hemorrhage, and multifocal mononuclear cell proliferation. The domestic chicken's thymus, Fabricius bursa, and spleen on days 1 and 3 PI showed lymphoid depletion, congestion, and multifocal vasculitis. On days 5 and 7, the lesion spread in a diffuse pattern accompanied by cyst formation, lysing cells, and part of the tissue being replaced by connective tissue. The ducks' Thymus, Fabricius bursae, and spleen on day 1 appeared to suffer lymphoid depletion, congestion, and focal vasculitis. On days 3 and 5 PI, the lesions were spread in a multifocal pattern, and on day 7, the lesions were spread in a focal pattern. The heart of domestic chicken on day 3 PI started to show edema and focal degeneration; on day 5 PI, it was accompanied by hemorrhage, congestion, and focal mononuclear cell infiltration. On day 7, the lesions spread in a multifocal pattern accompanied by pericarditis and endothelial hypertrophy. However, in Alabio duck, the lesions were only spread in a focal pattern without pericarditis. The kidney of domestic chicken on day 1 PI appeared to suffer from edema, congestion, and focal hemorrhage. On day 3 PI, the lesions were spread in a multifocal pattern. Every following observation day, the lesions spread diffusely, followed by multifocal mononuclear cell infiltration by the interstitial area of the kidney. Alabio duck kidney appeared to suffer from edema and focal congestion on all observation days, and focal mononuclear infiltration by kidney interstitial started to appear from day 3 PI. Domestic chicken brain on days 1 and 3 PI showed neuron degeneration, congestion, edema, and multifocal endothelial hypertrophy. On days 5 and 7 PI, they were followed by neuron cells necrosis, multifocal gliosis, and focal perivascular cuffing . The Alabio duck brain on every observation day appeared to suffer from congestion, edema, endothelial cell hypertrophy, and multifocal gliosis.
Immunohistochemistry staining showed that immunopositive reaction against NDV was found in all treatment groups from day 1 until the last observation day, with severity ranging from light to severe, while on all control groups, immunonegative. The immunopositive location against NDV was not different between domestic chicken and Alabio duck. A positive reaction was found in epithelial cells and mononuclear inflammation in the intestinal organs . Cilia epithelial cells, mucosal layer mononuclear cells, and in the spaces of tracheal goblet calls . Parabronchi epithelial cells, pneumocystis, and inflammatory cells in lung alveoli. Heart blood vessel endothelial cells and the urinary. Reticular epithelial cells at the medulla layer and mononuclear cells by the thymus cortex. Lymphoid cells of white pulp and lymphoid cells inside the Fabricius bursa lymphoid follicle. The plica epithelial cells and mononuclear cells are suffering from depletion within the lymphoid follicle of the Fabricius bursa: the Virchow-Robin endothelial cell, necrotic glial cells, and neurons in the brain .
The percentage of caspase-3 in lymphoreticular organs peaked earlier in the Alabio duck group, which was on day 2 PI, compared to the domestic chicken group, which on average, occurred after day 3 PI. The caspase-3 expression in domestic chicken and Alabio duck thymus was more dominant in the medullar area and rarely by the cortex. The caspase-3 expression in domestic chicken and Alabio duck Fabricius bursa by the plica epithelial and lymphoid follicle, while in the cecal tonsil by the mucosa epithelial cell, lamina propria inflammatory cells, and crypt epithelial cells. The caspase-3 expression of domestic chicken spleen was more dominant around the germinal center, whereas caspase-3 expression in Alabio ducks was more dominant in the germinal center.
The chains of pathological changes occurring in the respiratory, circulation, gastrointestinal, urinary, and nervous systems are closely related to the clinical symptoms. For example, Conjunctivitis appearing on day 2 PI occurred because the infection route was intraorbital, causing a local immune response by the eye region. Depression, lethargy, difficulty breathing, and catarrhal exudation in the nose on AC-A align with the gross pathological changes, which were congestion and catarrhal exudation by the trachea. This is related to the increase of goblet cells initiated by viruses attaching to the epithelial cells via the use of sialic acid on the host cells as receptors. Clinical symptoms such as greenish-white diarrhea, nervous disorder, death, and head edema only appeared in domestic chickens but were not found in Alabio ducks. Diarrhea and anorexia on day 3 PI in domestic chicken were in line with gross pathological changes in skeletal muscle, which looked pale and emaciated. Anorexia was indicated by depression in the chicken period. The low feed and drinking water consumption was due to the chicken feeling sick from septicemia or viremia. The sickness in domestic chicken continued after day 3 PI and caused death starting from day 4 PI, followed by nervous symptoms on day 5 PI. Death with the nervous disorder is a clinical manifestation of neuron degeneration, edema, congestion, thickening blood vessel walls, perivascular cuffing, the proliferation of glial cells (gliosis), and necrosis in the brain. The presence of NDV in the brain can cause vascular and neuron damage, further causing an inflammatory response. The replication of NDV in internal organs initiated the pathogenesis of ND in the gastrointestinal tract. The NDV was distributed from the respiratory to the gastrointestinal system, possibly through the circulatory system or directly into the chicken’s internal organs. NDV replication in the gastrointestinal system is indicated by catarrhal exudation to widespread hemorrhage by the viscera due to blood vessel damage. The gross anatomy lesion that appeared by the gastrointestinal tract is in line with a histopathological lesion found: congestion, edema, epithelial cell necrosis and desquamation, the proliferation of mononuclear cells, and goblet cell hyperplasia from light to severe level. The findings matched a previous report by about goblet cell hyperplasia and severe desquamation of intestinal epithelial cells. Necrosis was in the form of karyorrhexis debris and ulceration of intestine epithelial cells . The NDV immunopositive reaction in gastrointestinal organs was also distributed in severe severity. The immunopositive response in the gastrointestinal tract matched the previous report by on inflammatory and gastrointestinal system epithelial cells. reported in the duodenum, proventriculus, and heart. said that in the esophagus, crop, pancreas, and proventriculus. reported in the cecal tonsil. Kidney lesions also occur due to viremia, which allows NDV to be spread from the respiratory or gastrointestinal tract via the blood circulation system to the kidney. We assume that one of the reasons Alabio ducks are more resistant than local chickens is the lack of severe structural and histological abnormalities in the Alabio duck kidney. However, we are still unsure of the mechanism that causes the absence of these lesions. The immunopositive reaction in the kidney is similar to a previous report by about chicken kidney tubules epithelial cells and in the cytoplasm and nucleolus of duck kidney tubules cells . The pathogenesis of a disease is also closely related the lymphoreticular organ damage as they produce immunity compounds to eliminate infectious agents. This research showed that all lymphoreticular organs generally suffered from changes in gross pathology and histopathology. The gross pathology and histopathology lesions generally spread lightly in Alabio duck groups, different from immunohistochemistry results whose distribution was light to severe. This data showed that although no severe lesion was found according to gross pathology and histopathology, the viral concentration within the lymphoreticular organ was high. The different bird species used in this research showed that the severe immunopositive reaction found in the gastrointestinal and lymphoreticular organs in Alabio duck proved that although Alabio ducks do not show ND clinical symptoms, inside their body, NDV was present in high concentration. The differences in clinical symptoms and lesion features that appeared on domestic chickens and Alabio ducks may be caused by different genes or protein expressions in domestic chickens and Alabio ducks that fight viral infection. According to , the interferon-ß expression is earlier, stronger, and more intensive in duck tissue (Japanese commercial duck) compared to chicken (white leghorn SPF) upon infection by virulent NDV. In chicken, retinoic acid-induced gene-I (RIG-I) was reported to be absent. However, it was highly expressed in the duck spleen and heart . The absence of RIG-I is hypothesized to make chicken less resistant to the influenza virus than duck as its natural reservoir . explained that RIG-I is identified as a cytoplasmic censor against RNA virus, which is important in initiating the nonspecific immune response. The caspase-3 expression as an apoptosis indicator in this research added information that the clinical expression and lesion differences in domestic chicken and Alabio duck infected by a local isolate from duck might be caused by the time difference of the highest progressive increase of apoptosis response, which was on day 2 PI in Alabio duck and day 3 PI in domestic chicken by the thymus, Fabricius bursa, and spleen; all of them crucial as primary and secondary systems in fighting infections. Ideally, when an infectious agent contacts host cells, an apoptosis process for clearance rapidly occurs to prevent viral aggression from continuing. The apoptosis percentage in the lymphoreticular organ continued to show a regressive decrease after reaching the peak, probably caused by two conditions. In domestic chickens, histopathologically, on days 1 and 3, PI lymphoid depletion, congestion, and multifocal vasculitis were found; on days 5 and 7, PI lesions were spread in diffuse accompanied by cells undergoing lysis and partly replaced by connective tissue. In the Fabricius bursa, the process was accompanied by cyst formation. It was different in Alabio ducks. In Alabio ducks, the apoptosis response decrease at the end of observation was probably caused by the rapid NDV clearance, which prompts earlier cell regeneration. stated that several duck organs showed increased mitosis compared to ducks, allowing rapid tissue regeneration. The lowering apoptosis responses in Alabio duck after reaching its peak align with the lowering lesion severity into focal in the thymus, Fabricius bursa, and spleen. This allows the lymphoreticular organ ability as the defensive organ to return to normal, so Alabio ducks turn healthy again until the last observation day. In closing, it can be said that generally, the difference in lesion pattern between domestic chickens and ducks infected by the velogenic NDV Ducks/Aceh Besar_IND/2013/eoAC080721 is that in domestic chickens until the last observation day, the lesion progressively turned severe, while in Alabio ducks the lesions showed improvements. In summary, the clinical symptoms, pathological lesions, and histopathology of ND are more severe in domestic chickens compared to Alabio ducks in the treatment group. The immunopositive reaction against the NDV in domestic chicken continued to increase, while in Alabio ducks, it decreased until the last observation day, especially in gastrointestinal tracts. The apoptosis response showed earlier in Alabio ducks compared to domestic chickens.
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Under-representation of classical hematology training on hematology-oncology fellowship program websites in the United States
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4152286e-c843-4152-bfd7-c4f2d2f1f664
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10220257
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Internal Medicine[mh]
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Pan-Asian adapted ESMO Clinical Practice Guidelines for the diagnosis, treatment and follow-up of patients with metastatic colorectal cancer
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9f0bfa65-cd40-4ec5-91fd-8c2a47b98d0b
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10220270
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Internal Medicine[mh]
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There were an estimated 19.3 million new cases and 10 million cancer deaths worldwide in 2020. Overall, for both sexes combined, colorectal cancer (CRC) globally ranks third as the most commonly diagnosed cancer (10.0% of new cases) and second as a leading cause of cancer death (9.4% of global cancer deaths). For both sexes combined the highest incidence of CRC cases (51.8%) and of CRC deaths (52.4%) are estimated to occur in Asia. The estimated epidemiology for CRC in Asia for 2018 showed China to have the highest 5-year prevalence, number of new cases and deaths from CRC, followed by, in terms of new cases, Japan, India, Korea, Indonesia, Thailand and the Philippines. The increase in the crude incidence of CRC across Asia can be attributed in large part to an increasingly westernised dietary lifestyle, smoking, high alcohol consumption, physical inactivity, obesity and diabetes and also to increasingly aged populations. On an individual level, China showed an increasing incidence and mortality, Singapore a rising incidence but reduced mortality and Japan a decreasing incidence and mortality. Japan, South Korea, Singapore, Taiwan and certain provinces in China , , , , have population-based screening programmes which together with the Asia-Pacific consensus recommendations for CRC screening, which involved 13 countries in the Asia-Pacific region, should facilitate an improvement in early tumour detection. Also, recently a Chinese study reported a higher incidence of adenomas in the right- versus left-sided colon (44% versus 39%). Whilst, a Korean study reported a higher proportion of right-sided adenomas in older subjects when compared with younger subjects. Significantly, a multinational study involving Hong Kong, Taiwan, Korea and Japan identified an increasing trend in early-onset (<50 years of age) colon and rectal cancers. The most recent European Society for Medical Oncology (ESMO) guidelines for the diagnosis treatment and follow up of patients with mCRC were published in 2022, and a decision was taken by ESMO and the Japanese Society of Medical Oncology (JSMO) that these latest ESMO guidelines should be adapted for the management and treatment of patients of Asian ethnicity. This manuscript summarises the Pan-Asian adapted guidelines developed and agreed at a hybrid virtual/face-to-face working meeting that took place in Singapore on 01 December 2022, hosted by JSMO. Each recommendation is accompanied by the level of evidence (LoE), grade of recommendation (GoR) ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) and the percentage consensus reached.
This Pan-Asian adaptation of the current ESMO Clinical Practice Guidelines, and associated updates, was prepared in accordance with the principles of ESMO standard operating procedures ( https://www.esmo.org/Guidelines/ESMO-Guidelines-Methodology ) and was a JSMO-ESMO initiative endorsed by the Chinese Society of Clinical Oncology (CSCO), the Indonesian Society of Hematology and Medical Oncology (ISHMO), the Indian Society of Medical and Paediatric Oncology (ISMPO), the Korean Society of Medical Oncology (KSMO), the Malaysian Oncological Society (MOS), the Philippine Society of Medical Oncology (PSMO), the Singapore Society of Oncology (SSO), the Taiwan Oncology Society (TOS) and the Thailand Society of Clinical Oncology (TSCO). An international panel of experts was selected from the JSMO ( n = 10), the ESMO ( n = 5), and two experts from each of the nine other oncological societies. Only two of the six expert members from the JSMO (HB and EO) were allowed to vote on the recommendations together with the experts from each of the nine other Asian oncology societies ( n = 20). None of the additional JSMO members present and none of the ESMO experts were allowed to vote and were present in an advisory role only (see Methodology, available at https://doi.org/10.1016/j.esmoop.2023.101558 ).
A Scientific adaptations of the ESMO recommendations In the initial pre-meeting survey, the 20 voting Asian experts reported on the ‘acceptability’ of the 68 recommendations for the diagnosis, treatment and follow-up of patients with mCRC from the most recent ESMO Clinical Practice Guidelines ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), in the five categories outlined in the text below and in . A lack of agreement in the pre-meeting survey was established for 25 recommendations, 21 of which were discussed at the hybrid virtual/face-to-face face working meeting in Singapore to adapt the recently published ESMO Clinical Practice Guidelines and four after the meeting. Two new recommendations were also added (see Results, available at https://doi.org/10.1016/j.esmoop.2023.101558 ) 1 Diagnosis, pathology and molecular biology—recommendations 1a-k The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 1a-g and 1i, and 1j’ , without change. In relation to ‘recommendation 1h’, however, concern was raised over restricting the recommendation to patients with RAS wild-type (wt) disease. Although the trials have mainly been conducted in patients with RAS wt disease, the MyPathway study reported a HER2 amplification in 23% of patients with RAS -mutant disease and an overall response rate for these patients of 8% compared with 40% for those with HER2 -amplified, RAS wt disease. Thus, although it was accepted that ‘recommendation 1h’ should remain unchanged ( 100% consensus ) , it was agreed that testing for HER2 amplification should not be excluded for patients with RAS -mutant disease but should be conducted subject to resource availability and the reimbursement and diagnostic testing policies of the individual Asian countries. Also, where HER2 testing is not reimbursed consideration should be given to referring patients to centres conducting clinical trials. In some Asian countries HER2 testing is carried out at the same time as RAS , BRAF and mismatch repair deficiency (dMMR)/microsatellite instability (MSI) testing to minimise the loss of tumour specimens. Although, it should be noted that in some Asian countries HER2 testing is only carried out in patients with RAS wt disease for the same reason. Furthermore, a recent Asian phase II study has shown that circulating tumour cell DNA (ctDNA) genotyping can identify patients who can benefit from dual human epidermal growth factor receptor 2 (HER2) blockade as well as monitor treatment response. These results warrant the further investigation of ctDNA genotyping for patients with HER2 - amplified mCRC in clinical trials. The representatives of 9 of the 10 Asian countries, however, did not consider ‘recommendation 1k’ acceptable in the pre-meeting survey ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) on the basis that the incidence of dihydropyrimidine dehydrogenase (DPD) deficiency is estimated to be very low in Asian populations compared with non-Asian populations. A Japanese study investigated the incidence of DPD deficiency in 1362 Asian colon cancer patients who were enrolled in the JOIN and ACHIEVE adjuvant chemotherapy trials and suggested that the incidence of DPD deficiency for these patients was in the region of 0.6%, with no clear association observed between DPD deficiency and safety. In addition, DPD deficiency was not detected by analysing DPD full-length RNA polymorphisms in peripheral blood mononuclear cells in 67 Taiwanese patients using multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) and non-isotopic RNase cleavage assays (NIRCA). DPD deficiency is also reported not to be common in Korea . Thus, due to the low incidence of DPD deficiency in Asian patients, DPD genotyping and phenotyping is not carried out in routine daily practice in Asia, but is recommended for patients, who experience severe 5-fluorouracil (5-FU) toxicity during and after their first cycle of chemotherapy. ‘Recommendation 1k’ was therefore revised from the original ESMO recommendation ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), to be consistent with the Pan-Asian adaptation of the ESMO Clinical Practice Guidelines for the diagnosis treatment and follow-up of patients with localised colon cancer, as per the bold text below, and the GoR revised to C and a new statement added. 1k. Depending on the anticipated genetic profile of a specific Asian patient population , DPD genotyping or phenotyping may be considered before initiating fluoropyrimidine-based therapy [III, C ]. DPD genotyping or phenotyping should be implemented in patients who experience severe fluoropyrimidine toxicity [V; consensus = 100%] . The experts from JSMO also proposed the addition of a new recommendation, ‘recommendation 1l’ below and , based on data from several Asian studies regarding irinotecan toxicity. , , , , , , Genetic variations within the UDP glucuronosyltransferase 1 family, polypeptide A1 ( UGT1A1 ) gene have been associated with the development of certain drug toxicities, with the UGT1A1 ∗ 6 variant, common in Asian populations. UGT1A1 gene polymorphisms are predictive of irinotecan-related side-effects, including diarrhoea, neutropaenia and vomiting. A systematic review and meta-analysis has shown the increased risk of severe neutropaenia in cancer patients with UGT1A1∗6 polymorphisms. 1l. UGT1A1 genotyping remains an option and it is recommended that it is carried out in patients with a suspicion of UGT1A1 deficiency as reflected by low conjugated bilirubin or in patients where an irinotecan dose of >180 mg/m 2 per administration is planned [III, C; consensus = 100%] . It was also recommended that depending on the prevalence of UGT1A1 polymorphisms per country, a lower irinotecan threshold dose for UGT1A1 genotyping may be used. , available at https://doi.org/10.1016/j.esmoop.2023.101558 describes the biomarkers and molecular targets for precision medicine and the corresponding ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) scores. 2 Staging and risk assessment—recommendations 2a-d The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 2a-d’ , available at https://doi.org/10.1016/j.esmoop.2023.101558 and . 3 Management of resectable/potentially resectable disease—recommendations 3a–f Treatment of patients with potentially resectable mCRC The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3a-d and 3f’ . There was, however, much discussion about the precise meaning of the ESMO ‘recommendation 3e’ below (and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) which states that: 3e. Patients unresponsive to first-line chemotherapy should not be denied resection or ablation of their metastases since the outcome of resected patients after second-line treatment could be also favourable. Intra-arterial chemotherapy could be an option in such patients, not only to recover a response but also to achieve liver resection [III, C]. It was agreed by the experts that patients unresponsive to first-line therapy should be assessed for resection after a second-line treatment benefit is observed in the absence of contraindications. Thus, ‘recommendation 3e’ was reworded as per the bold text, below and . 3e. Patients with metastases progressing on first-line chemotherapy should be assessed for resection after second-line treatment benefit . Intra-arterial chemotherapy may be used as a second-line option not only to achieve a response but also liver resection [III, C; consensus = 100%] . Intent and choice of local treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3g and 3i’ , but discussions at the hybrid virtual/face-to-face meeting led to the rewording of the original ‘recommendation 3h’. The original ‘recommendation 3h’ below was revised and shortened (as per the bold text) for better understanding from: 3h. Local treatment can be used as a primary or metastasis-specific treatment to halt further dissemination, and/or following systemic therapy as a consolidation treatment, to delay or pause further treatment [III, C]. to read as follows: 3h. Local treatment may be used as a primary or metastasis-specific treatment or following systemic therapy as a consolidation strategy [III, C; consensus = 100 %] . Local ablation treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3j – m’ . An algorithm summarising the options for local ablation treatment is presented in . Intra-arterial therapies The Pan-Asian panel of experts revised ‘recommendation 3n’ below to more accurately reflect the situation in their countries. In Korea and Taiwan intra-arterial therapies are only used within the remit of a clinical trial, whilst in Japan they are not commonly used at all. The feeling amongst the experts was that hepatic arterial infusion chemotherapy (HAIC) in particular was limited by a lack of expertise. The experts from TSCO considered that the evidence to support the use of transarterial chemoembolisation (TACE) was inadequate. Thus, the consensus was that such procedures should be limited to expert centres or clinical trials. The wording of the original ‘recommendation 3n’ below: 3n. TACE, TARE/SIRT and HAIC may be also considered as treatment options with non-curative intent [III, B], was revised to read: 3n. TACE, transarterial radioembolisation (TARE)/selective internal radiotherapy (SIRT) and HAIC may be considered as treatment options with non-curative intent, if available in expert centres [ III, C; consensus = 100%]. Patient participation in related clinical trials should be encouraged. The GoR was revised from B to C . The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the ESMO ‘recommendation 3o’ in the pre-meeting survey without change. The options for intra-arterial therapy are summarised in . 4 Management of advanced and metastatic disease without potential for conversion—recommendations 4a–jj First-line therapy The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 4a-d’ and 4f-j, . The Asian experts also accepted the original ‘recommendation 4e’ without change , after discussion of whether there was evidence to support the benefit of the addition of anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy (either cetuximab or panitumumab) to doublet chemotherapy in patients with right-sided RAS wt primary tumours. Data from the Japanese PARADIGM trial (NCT02394795), the first prospective trial to test the superiority of panitumumab versus bevacizumab in combination with standard doublet (mFOLFOX6) first-line chemotherapy for patients with RAS wt mCRC and left-sided primary tumours, showed panitumumab to statistically significantly improve overall survival and improve response rate and R0 resection rate compared with bevacizumab when used in combination with mFOLFOX6. The statistically significant overall survival benefit and improved response rate and R0 resection rates was retained in the full analysis population, consistent with the data that support the use of anti-EGFR mAb therapy in combination with chemotherapy first line in patients with RAS wt/ BRAF wt left-sided primary tumours. Although, additional data showed that patients with right-sided tumours did not derive an overall survival benefit when treated with panitumumab in combination with chemotherapy, the R0 resection rate for patients with right-sided tumours was similar for patients who received either panitumumab or bevacizumab. More recently, a biomarker study of PARADIGM trial patients has shown that overall survival tended to be longer for selected patients with no gene alterations treated with panitumumab than for those treated with bevacizumab, irrespective of tumour sidedness. This suggests that selection of patients with RAS wt mCRC using ctDNA analysis may further refine the selection of patients for treatment with panitumumab rather than bevacizumab in the first-line treatment setting. There was considerable discussion around ‘recommendation 4k’, however, with the available clinical data presenting conflicting results regarding the addition of anti-EGFR mAbs to triplet FOLFOXIRI (FOLFOX plus irinotecan) therapy first line. FOLFOXIRI, combined with bevacizumab or panitumumab, has been shown to be superior when compared with doublet combinations in patients with RAS wt mCRC. The Japanese randomised phase II DEEPER trial (NCT02515734) of FOLFOXIRI plus cetuximab versus FOLFOXIRI plus bevacizumab first line in patients with RAS wt mCRC showed FOLFOXIRI plus cetuximab to be significantly superior to FOLFOXIRI plus bevacizumab in terms of depth of response (DpR), the primary endpoint. There is no phase III evidence, however, to support the use of anti-EGFR mAbs in combination with triplet chemotherapy as demonstrated by the European TRIPLETE trial which failed to show a benefit for FOLFOXIRI plus panitumumab versus FOLFOX plus panitumumab in terms of early tumour shrinkage and DpR. Thus, the word ‘currently’ was added to the original recommendation 4k as per the bold text below, and also . 4k. Triplets with FOLFOXIRI and anti-EGFR mAbs are not currently recommended [I, D; consensus 100% ]. The Asian experts also accepted ‘recommendation 4l’ unchanged ( 100% consensus ), supported by data from the Japanese phase II OGSG 1602 study (UMIN000024528) of panitumumab monotherapy in chemotherapy-naive frail/elderly patients with unresectable RAS wt metastatic/advanced CRC. The LoE however, was revised to III , C . The Asian experts were did not agree with the wording of the original ‘recommendation 4m’ and thought that the wording should reflect attempts at dose modification. The oral fluoropyrimidine S-1, developed as a prodrug of 5-FU, is frequently used in Asia and can be used when i.v. 5-FU or capecitabine-based chemotherapy cannot be tolerated, due to cardiotoxicity and/or hand-foot syndrome. Thus, the original wording of ‘recommendation 4m’ below: 4m. In patients presenting with cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B], was revised to read: 4m. In patients unable to tolerate cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B; consensus = 100% ]. The Asian experts also accepted ‘recommendations 4n and 4o’ unchanged ( 100% consensus ) with the observation with regard to ‘recommendation 4o’, in patients with dMMR/MSI disease, that although the overall survival of the patients receiving the immune checkpoint inhibitor (ICI) pembrolizumab was not significantly longer in the KEYNOTE-177 study (probably because of the 60% cross-over), it was superior, in those patients receiving pembrolizumab. The primary endpoint prolongation of progression-free survival was met. , As a consequence the consensus was that the ICI pembrolizumab can be recommended as a standard of care, for the treatment of patients with dMMR/MSI mCRC in the first-line setting. A summary of the first-line treatment options for the management of patients with stage IV unresectable mCRC is presented in . Maintenance therapy Consideration of maintenance therapy is generally applicable to those patients who are not amenable to surgery or local therapy and involves a de-escalation in the intensity of their systemic treatment with a concomitant improvement in treatment-related side-effects. The Asian experts accepted completely ( 100% consensus ) ‘recommendations 4p and 4q’ relating to maintenance therapy, without change , in the pre-meeting survey. The Asian experts thought the wording of ‘recommendations 4r and 4s’ was unclear ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), however, and they were revised accordingly as indicated by the bold text below and . 4r. When FOLFIRI is used in first-line treatment, it can be continued until disease progression if well tolerated [V, B; consensus = 100% ]. 4s. Reintroduction of an initial successful induction therapy should be done after progressive disease while on durable maintenance therapy [III, B; consensus = 100% ]. A summary of the maintenance treatment options according to prior therapy are summarised in . Second-line therapy The Asian experts accepted ‘recommendations 4t-y’ relating to second-line therapy, unchanged ( 100% consensus ), and after discussion, ‘recommendations 4u, 4v and 4w’. A modification was made to ‘recommendation 4z’ below and , however, as per the bold text, based on data from the BEACON trial (NCT02928224), at the request of the JSMO experts, as encorafenib–cetuximab plus or minus binimetinib, is reimbursed in Japan for patients with ECOG performance status of 1, >3 metastatic sites, high serum creatinine protein levels (>1 mg/dL), or no history of primary tumour resection. 4z. For pre-treated mCRC patients with BRAF V600E-mutated tumours, encorafenib–cetuximab is recommended as the best option in second line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available (consensus = 100%) . Recommendation 4aa was discussed and an additional statement added to the recommendation, as per the bold text below and , to include the option to use nivolumab or pembrolizumab monotherapy second line for patients with dMMR/MSI-H mCRC. 4aa. For dMMR/MSI-H tumours progressing after first-line chemotherapy, ipilimumab–nivolumab is recommended [III, B; ESMO-MCBS v1.1 score 3]. Nivolumab [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] or pembrolizumab monotherapy is an option [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] . An algorithm of proposed second-line treatment options according to prior therapy and primary tumour mutation profile is presented in . Third-line and further therapy Recently, the addition of bevacizumab to trifluridine–tipiracil has been shown to improve overall and progression-free survival, as well as objective response rate, when compared with trifluridine–tipiracil alone in the randomised phase III SUNLIGHT trial. Also, cetuximab and panitumumab have both shown efficacy in this treatment setting in patients with RAS wt mCRC, as single agents. , , In addition, treatment involving dual HER2 blockade using a combination of trastuzumab an anti-HER2 mAb and the tyrosine kinase inhibitor lapatinib has been shown to be effective in patients with RAS wt, HER2-positve, treatment-refractory mCRC. An algorithm of proposed third-line and later-line treatment options according to the molecular profile of the primary tumour is presented in . The Asian experts accepted the original ESMO recommendations, ‘recommendations 4bb, 4cc, 4gg and 4hh’ without change ( 100% consensus ) with the observation from the JSMO experts that the combination cetuximab–encorafenib–binimetinib , is also available in Japan in relation to ‘recommendation 4ee’. ‘Recommendation 4dd’ was revised retrospectively as per the bold text below and , based on the data published from the SUNLIGHT trial. 4dd. Trifluridine–tipiracil plus or minus bevacizumab is recommended in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics, if available, or in earlier lines of therapy following oxaliplatin and irinotecan regimen failure, depending on local approvals [I, A; consensus = 100%] . ‘Recommendation 4ee’ was revised as per the bold text below and , as per ‘recommendation 4z’ above, based on data from the Beacon trial. 4ee. For BRAF V600E-mutated, pre-treated mCRC patients, encorafenib–cetuximab is recommended as the best option in third line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available . The Asian experts requested the minor modification to ‘recommendation 4ff’ with ‘and’ replaced by ‘or’ as per the bold text below and . 4ff. In RAS wt and BRAF wt patients not previously treated with the EGFR antibodies, cetuximab or panitumumab are recommended as single agents [I, A; panitumumab ESMO-MCBS v1.1 score: 3; consensus = 100% ]. At the request of the experts from JSMO ‘recommendation 4kk’ was added below, and , for patients who had received prior fluoropyrimidine-, oxaliplatin- and irinotecan-based and anti-vascular endothelial growth factor (anti-VEGF) mAb therapy (and anti-EGFR-mAb therapy if RAS wt) based on data from the randomised Chinese FRESCO-1 and global FRESCO-2 trials of fruquintinib versus placebo plus or minus best supportive care, both of which reported a statistically significant improvement in overall survival. 4kk. Fruquintinib is an additional option in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics , [I, B; consensus = 100%] . Also, it was noted that the phase II TRIUMPH study (UMIN000027887) of dual HER2 antibodies (pertuzumab plus trastuzumab) demonstrated promising activity in patients with HER2-positive mCRC, which led to the regulatory approval of this combination therapy in Japan. Whilst, the durable response reported for the recent multicentre, open-label, phase II MOUNTAINEER trial (NCT03043313) in patients with HER2-positive RAS wt mCRC who had progressed on, or were intolerant to, fluoropyrimidine, oxaliplatin, irinotecan and anti-VEGF mAb therapy, treated with tucatinib and trastuzumab, means that this combination is likely to become a new treatment option for patients with chemotherapy-refractory HER2-positive RAS wt mCRC. 5 Follow-up, long-term implications and survivorship—recommendations 5a–b The Asian experts had concerns over the clarity of ‘recommendation 5a’ and the frequency of the proposed radiological evaluations. Several of the experts considered 8-12 weeks to be too frequent for patients receiving active treatment Thus, the original ‘recommendation 5a’ ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) was revised, as per the bold text, to read as follows and . 5a. For patients receiving active treatment, radiological evaluation should be carried out at least every 12 weeks , including (in most cases) a CT scan or MRI scan , as well as the measurement of tumour marker levels [IV, B; consensus = 100% ]. The Asian experts accepted completely ( 100% consensus ) the ESMO ‘recommendation 5b’ . A clinical assessment of the toxicities resulting from both systemic treatment and surgery should be conducted whenever possible together with an assessment of long-term survivors. B Applicability of the recommendations Following the hybrid virtual/face-to-face working meeting, hosted by JSMO, the Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the adapted ESMO guidelines listed in above. The applicability of each of the guideline recommendations, however, is highly dependent on the drug and testing approvals for each Asian country and their reimbursement policies both of which differ markedly across the 10 countries represented. The drug and treatment availability for the 10 Asian countries is summarised in , available at https://doi.org/10.1016/j.esmoop.2023.101558 and outlined in the text below for each country individually and summarised individually for each country in , available at https://doi.org/10.1016/j.esmoop.2023.101558 . Most notable are the striking differences between the individual countries in terms of their availability and reimbursement policies with patients in Indonesia, Malaysia, The Philippines and Thailand having fewer approvals for testing and treatments, whilst also receiving very little reimbursement ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In contrast, patients in India, Japan and Singapore have access to nearly all the cancer therapies and testing services at modest or no personal cost ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). For example, in Singapore, testing is approved but not reimbursed and most cancer treatments are approved. Patients can pay out of their national savings scheme and basic health insurance plan for those agents that are approved (i.e. nearly all the drugs discussed in the main text above). There is also a complex insurance system for people who can afford it, and a means tested ‘safety net’ for those that cannot ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In Korea, nearly all tests and treatments are approved, but although the testing is reimbursed, the reimbursement of treatment is limited beyond first line ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In the case of Taiwan, there are limited approvals for both testing and therapy, with EGFR inhibitors and bevacizumab approved in first and third line, and first line only, respectively ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In China, most drugs and a number of targeted agents are available for the treatment of patients with mCRC, although several targeted agents as well as several genetic tests are not reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). The differences between the countries reflect the way the different countries manage their health care systems and the money allocated from the individual governments to cancer care. Significantly, the health care systems of the more affluent countries offer a higher proportion of their populations access to all levels of cancer care due not only to them having the highest rates of drug approvals and/or better public reimbursement policies, but also due to a higher percentage of their populations being able to purchase or obtain through their employment, private medical cover. The individual statements, from the experts of each country, describing the availability and access to optimal diagnostic and molecular testing and the latest drug therapies for their individual countries, are described in the paragraphs below together with some of the details of their reimbursement policies. China In China, the drugs available for the treatment of patients with mCRC include the following, namely the chemotherapy drugs 5-FU, oxaliplatin, irinotecan, capecitabine and trifluridine–tipiracil, and the targeted agents cetuximab (for patients with RAS wt mCRC), bevacizumab, regorafenib and fruquintinib, and are reimbursed by medical insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Pembrolizumab is also available for those patients with MSI-H mCRC but is not reimbursed. To date, the agents aflibercept, panitumumab, ramucirumab, encorafenib, adagrasib, sotorasib, lapatinib, tucatinib, trastuzumab deruxtecan and larotrectinib are still not available to Chinese patients. Patients with mCRC can undergo recommended genetic testing, including for their RAS , BRAF , HER2 , MSI status and for NTRK fusions. Currently, however, medical insurance in China only covers PCR testing for RAS ( KRAS/NRAS ) and BRAF alterations, and MSI testing. Medical insurance also covers immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) detection of HER2 alterations, and IHC detection of MMR. Next generation sequencing (NGS) testing is not reimbursed by medical insurance. There are different policies in terms of reimbursement across the different provinces depending on province-based approvals. Indonesia In Indonesia the universal health care system (UHC) covers most of the health services. Although almost 80% of Indonesians are covered by the UHC, however, amongst them are individuals who also have their own private medical insurance or whose health care is covered by their employer. The use of 5-FU-based chemotherapy (including oxaliplatin or irinotecan regimens) as first-line or second-line therapy is reimbursed, whilst targeted therapies and checkpoint inhibitors are not. Biomarker testing is available but not all tests are reimbursed. New technology-based tests (i.e. NGS) are not reimbursed. Also, after a Health Technology Assessment (HTA), bevacizumab and cetuximab for the treatment of mCRC have been excluded from the UHC scheme. In terms of availability, in Indonesia new drugs/agents firstly have to receive approval from the Indonesian Food and Drug Authority (FDA). Then after 2 years an application can be made for the drug to be included in the national formulary which is a list of medications that are eligible to be given to patients under the UHC scheme. Due to the high burden of health care costs, however, especially for cancer treatment, it can sometimes take years, multiple scientific evaluations and cost effectiveness analyses/HTA, for a new drug to be listed under the national formulary for UHC. Thus, most of the drugs are not available or reimbursed for patients with CRC. Drugs are approved for very specific indications. India Most of the drugs (chemotherapy and biologicals) for the treatment of mCRC discussed in the ESMO guidelines and Section A above are available in India ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Chemotherapy drugs and some of the biologicals are available in the government hospitals with cancer services and hence accessible to most patients at modest or no cost. India has a graded payment/insurance scheme based on patient income, decided by the government. Also, the wide availability of cheaper and good quality generics and biosimilars have improved patient access to better treatment. Drugs not covered by the government centres are covered by private insurance or by the patient as an out-of-pocket payment. The government health scheme covers about 30%-35% of the population, with the remainder taken care of by the private sector. Most tests including NGS are available but are expensive. Limited PCR panels are widely available. Some of the tests are reimbursed by insurance providers, whereas some drug companies offer coupons to subsidise certain tests. Many centres conduct investigator-initiated studies to generate Indian patient data including on biomarkers, facilitating increased patient access to newer therapeutic strategies. Japan In Japan, all the drugs recommended in the ESMO Clinical Practice Guideline, except for trastuzumab plus lapatinib and trastuzumab deruxtecan are approved and reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In addition, the triplet combination of cetuximab plus encorafenib plus binimetinib in second or later line, , and trastuzumab plus pertuzumab in third or later line, are approved and reimbursed. All the recommended assays for biomarker analysis (except primary NGS), are approved and reimbursed. Also, repeated plasma-based digital PCR RAS testing for rechallenge of anti-EGFR therapies and NGS for comprehensive genome profiling after standard therapies are also both approved and reimbursed. Patients pay 0%-30% of the total cost for diagnostic/molecular testing and treatment, depending on their age and income. Korea All drugs and assays discussed above in Section A of this manuscript are available in Korea, except for ramucirumab and trastuzumab deruxtecan, as indicated in , available at https://doi.org/10.1016/j.esmoop.2023.101558 . Some biological agents or ICIs are also available beyond second line for patients with mCRC, but their cost is not reimbursed yet. Malaysia In Malaysia there are two types of health care system. The government provides universal health care under the Ministry of Health (MOH) for all Malaysians for a very low fee. Alternatively there is private health care, which is not affiliated with MOH, for those who wish to and are able to pay for it. Targeted drugs are very limited and are often not available widely under the MOH due to cost issues and are therefore generally not reimbursable. Malaysia does not practice a partly funded government health insurance policy for individual citizens. Common systemic chemotherapy regimens are covered but not the newer targeted therapies. Patients therefore often pay privately, often out of their own pocket or using some form of insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Thus, in the private sector in Malaysia, targeted drugs are more readily available as the patients pay using their own money or private insurance. The MOH tries to negotiate with the pharmaceutical companies, to lower their prices, while at the same time considering generic options whenever possible. Health Intervention Technology, a division in the MOH, often discusses cost–benefit ratios when coming to a decision to include certain drugs for use by the MOH. The Philippines Most of the drugs enumerated ( and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) are available in the Philippines, although not reimbursed, resulting in 100% out-of-pocket patient payments. Most laboratory tests and diagnostics are available in the big cities like Manila and in big treatment centres, thus, ‘availability for all patients’ is an issue. Some agents can be obtained for compassionate use from Hong Kong or Singapore. The Philippines comprises >7000 islands with a range of different procurement policies. Singapore In Singapore, ∼90% of Health Sciences Authority-approved cancer treatments are on the Cancer Drug List (CDL), a list of clinically proven and cost-effective cancer treatments. Only indication-specific treatments in the CDL can be claimed under MediSave (MSV), MediShield Life (MSHL) and Integrated Shield Plans (IPs). MSV is a national medical savings scheme that mandates individuals to set aside part of their income to pay for their hospitalisation and outpatient expenses including cancer drugs listed on the CDL. MediShield Life is a basic health insurance plan which helps to pay for large hospital bills and outpatient treatments including selected cancer drugs. IPs include MSHL and private insurance plans that provide additional cover for cancer diagnostics and drugs. Patients receiving government-subsidised care in public health care institutions utilising drugs which are listed under the Standard Drug List and/or covered under the Medication Assistance Fund may receive an up to 75% subsidy in drug costs (based on their monthly per capita household income). Those in need of financial assistance who fulfil the means testing requirements are eligible for government financial subsidies such as Medifund. In Singapore, molecular assays (e.g. IHC, PCR and NGS) are not subsidised or reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Indication-specific drugs that are not on the CDL such as lapatinib, pertuzumab and trastuzumab deruxtecan for CRC treatment are not subsidised or reimbursed. Taiwan All drugs and tests are available in Taiwan but not always covered by public insurance. The coverage of National Health Insurance in Taiwan is basically ‘ALL or NONE’ and the financial burden is huge and expected to increase further in the era of precision medicine and immuno-oncology. The availability of other medications for the same indication and future budget burden are the most important considerations, as well as the scientific results of pivotal trials, for decisions on reimbursement. This explains the reasons for the relatively limited coverage of expensive biologics (a maximum of 36 weeks reimbursement for bevacizumab), some of the new technology-based tests (i.e. NGS) and new treatments (e.g. ICIs in MSI-H mCRC) on the reimbursement list ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Also, in the case of patients receiving triplet therapy, only two agents are reimbursed with the cost of the third agent having to be paid for. Thailand In Thailand’s health care system, there are three main reimbursement schemes including the Universal Coverage (UC) scheme, the Social Security Scheme (SSS) and the Civil Servant Medical Benefit Scheme (CSMBS). The majority of Thai people were covered by the UC and the SSS. All government employees and their dependents are covered by the CSMBS. For the UC and SSS, the use of oxaliplatin- and irinotecan-based regimens as first-line or second-line therapy are reimbursable. Targeted therapies, ICIs and biomarker testing are not reimbursable. For those under the CSMBS, in addition to chemotherapy, they are able to access targeted therapy and companion biomarker testing using a pre-authorised system. Six months of bevacizumab therapy is eligible for reimbursement for second-line therapy. Panitumumab is the only anti-EGFR agent that is reimbursed for patients with RAS wt advanced CRC, with two indications including first-line therapy with doublet chemotherapy only for patients with potentially resectable advanced CRC, and third-line therapy in combination with single-agent irinotecan. Regorafenib is also reimbursable for patients with chemotherapy-refractory disease ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Otherwise the medical expenses are not reimbursed. The ESMO-MCBSs for the different systemic therapy options and new therapy combinations for the treatment of patients with mCRC are to be found at https://www.esmo.org/guidelines/esmo-mcbs/esmo-mcbs-scorecards?mcbs_score_cards_form%5BsearchText%5D=&mcbs_score_cards_form%5Btumour-sub-type%5D=Colon+and+R .
Scientific adaptations of the ESMO recommendations In the initial pre-meeting survey, the 20 voting Asian experts reported on the ‘acceptability’ of the 68 recommendations for the diagnosis, treatment and follow-up of patients with mCRC from the most recent ESMO Clinical Practice Guidelines ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), in the five categories outlined in the text below and in . A lack of agreement in the pre-meeting survey was established for 25 recommendations, 21 of which were discussed at the hybrid virtual/face-to-face face working meeting in Singapore to adapt the recently published ESMO Clinical Practice Guidelines and four after the meeting. Two new recommendations were also added (see Results, available at https://doi.org/10.1016/j.esmoop.2023.101558 ) 1 Diagnosis, pathology and molecular biology—recommendations 1a-k The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 1a-g and 1i, and 1j’ , without change. In relation to ‘recommendation 1h’, however, concern was raised over restricting the recommendation to patients with RAS wild-type (wt) disease. Although the trials have mainly been conducted in patients with RAS wt disease, the MyPathway study reported a HER2 amplification in 23% of patients with RAS -mutant disease and an overall response rate for these patients of 8% compared with 40% for those with HER2 -amplified, RAS wt disease. Thus, although it was accepted that ‘recommendation 1h’ should remain unchanged ( 100% consensus ) , it was agreed that testing for HER2 amplification should not be excluded for patients with RAS -mutant disease but should be conducted subject to resource availability and the reimbursement and diagnostic testing policies of the individual Asian countries. Also, where HER2 testing is not reimbursed consideration should be given to referring patients to centres conducting clinical trials. In some Asian countries HER2 testing is carried out at the same time as RAS , BRAF and mismatch repair deficiency (dMMR)/microsatellite instability (MSI) testing to minimise the loss of tumour specimens. Although, it should be noted that in some Asian countries HER2 testing is only carried out in patients with RAS wt disease for the same reason. Furthermore, a recent Asian phase II study has shown that circulating tumour cell DNA (ctDNA) genotyping can identify patients who can benefit from dual human epidermal growth factor receptor 2 (HER2) blockade as well as monitor treatment response. These results warrant the further investigation of ctDNA genotyping for patients with HER2 - amplified mCRC in clinical trials. The representatives of 9 of the 10 Asian countries, however, did not consider ‘recommendation 1k’ acceptable in the pre-meeting survey ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) on the basis that the incidence of dihydropyrimidine dehydrogenase (DPD) deficiency is estimated to be very low in Asian populations compared with non-Asian populations. A Japanese study investigated the incidence of DPD deficiency in 1362 Asian colon cancer patients who were enrolled in the JOIN and ACHIEVE adjuvant chemotherapy trials and suggested that the incidence of DPD deficiency for these patients was in the region of 0.6%, with no clear association observed between DPD deficiency and safety. In addition, DPD deficiency was not detected by analysing DPD full-length RNA polymorphisms in peripheral blood mononuclear cells in 67 Taiwanese patients using multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) and non-isotopic RNase cleavage assays (NIRCA). DPD deficiency is also reported not to be common in Korea . Thus, due to the low incidence of DPD deficiency in Asian patients, DPD genotyping and phenotyping is not carried out in routine daily practice in Asia, but is recommended for patients, who experience severe 5-fluorouracil (5-FU) toxicity during and after their first cycle of chemotherapy. ‘Recommendation 1k’ was therefore revised from the original ESMO recommendation ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), to be consistent with the Pan-Asian adaptation of the ESMO Clinical Practice Guidelines for the diagnosis treatment and follow-up of patients with localised colon cancer, as per the bold text below, and the GoR revised to C and a new statement added. 1k. Depending on the anticipated genetic profile of a specific Asian patient population , DPD genotyping or phenotyping may be considered before initiating fluoropyrimidine-based therapy [III, C ]. DPD genotyping or phenotyping should be implemented in patients who experience severe fluoropyrimidine toxicity [V; consensus = 100%] . The experts from JSMO also proposed the addition of a new recommendation, ‘recommendation 1l’ below and , based on data from several Asian studies regarding irinotecan toxicity. , , , , , , Genetic variations within the UDP glucuronosyltransferase 1 family, polypeptide A1 ( UGT1A1 ) gene have been associated with the development of certain drug toxicities, with the UGT1A1 ∗ 6 variant, common in Asian populations. UGT1A1 gene polymorphisms are predictive of irinotecan-related side-effects, including diarrhoea, neutropaenia and vomiting. A systematic review and meta-analysis has shown the increased risk of severe neutropaenia in cancer patients with UGT1A1∗6 polymorphisms. 1l. UGT1A1 genotyping remains an option and it is recommended that it is carried out in patients with a suspicion of UGT1A1 deficiency as reflected by low conjugated bilirubin or in patients where an irinotecan dose of >180 mg/m 2 per administration is planned [III, C; consensus = 100%] . It was also recommended that depending on the prevalence of UGT1A1 polymorphisms per country, a lower irinotecan threshold dose for UGT1A1 genotyping may be used. , available at https://doi.org/10.1016/j.esmoop.2023.101558 describes the biomarkers and molecular targets for precision medicine and the corresponding ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) scores. 2 Staging and risk assessment—recommendations 2a-d The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 2a-d’ , available at https://doi.org/10.1016/j.esmoop.2023.101558 and . 3 Management of resectable/potentially resectable disease—recommendations 3a–f Treatment of patients with potentially resectable mCRC The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3a-d and 3f’ . There was, however, much discussion about the precise meaning of the ESMO ‘recommendation 3e’ below (and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) which states that: 3e. Patients unresponsive to first-line chemotherapy should not be denied resection or ablation of their metastases since the outcome of resected patients after second-line treatment could be also favourable. Intra-arterial chemotherapy could be an option in such patients, not only to recover a response but also to achieve liver resection [III, C]. It was agreed by the experts that patients unresponsive to first-line therapy should be assessed for resection after a second-line treatment benefit is observed in the absence of contraindications. Thus, ‘recommendation 3e’ was reworded as per the bold text, below and . 3e. Patients with metastases progressing on first-line chemotherapy should be assessed for resection after second-line treatment benefit . Intra-arterial chemotherapy may be used as a second-line option not only to achieve a response but also liver resection [III, C; consensus = 100%] . Intent and choice of local treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3g and 3i’ , but discussions at the hybrid virtual/face-to-face meeting led to the rewording of the original ‘recommendation 3h’. The original ‘recommendation 3h’ below was revised and shortened (as per the bold text) for better understanding from: 3h. Local treatment can be used as a primary or metastasis-specific treatment to halt further dissemination, and/or following systemic therapy as a consolidation treatment, to delay or pause further treatment [III, C]. to read as follows: 3h. Local treatment may be used as a primary or metastasis-specific treatment or following systemic therapy as a consolidation strategy [III, C; consensus = 100 %] . Local ablation treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3j – m’ . An algorithm summarising the options for local ablation treatment is presented in . Intra-arterial therapies The Pan-Asian panel of experts revised ‘recommendation 3n’ below to more accurately reflect the situation in their countries. In Korea and Taiwan intra-arterial therapies are only used within the remit of a clinical trial, whilst in Japan they are not commonly used at all. The feeling amongst the experts was that hepatic arterial infusion chemotherapy (HAIC) in particular was limited by a lack of expertise. The experts from TSCO considered that the evidence to support the use of transarterial chemoembolisation (TACE) was inadequate. Thus, the consensus was that such procedures should be limited to expert centres or clinical trials. The wording of the original ‘recommendation 3n’ below: 3n. TACE, TARE/SIRT and HAIC may be also considered as treatment options with non-curative intent [III, B], was revised to read: 3n. TACE, transarterial radioembolisation (TARE)/selective internal radiotherapy (SIRT) and HAIC may be considered as treatment options with non-curative intent, if available in expert centres [ III, C; consensus = 100%]. Patient participation in related clinical trials should be encouraged. The GoR was revised from B to C . The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the ESMO ‘recommendation 3o’ in the pre-meeting survey without change. The options for intra-arterial therapy are summarised in . 4 Management of advanced and metastatic disease without potential for conversion—recommendations 4a–jj First-line therapy The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 4a-d’ and 4f-j, . The Asian experts also accepted the original ‘recommendation 4e’ without change , after discussion of whether there was evidence to support the benefit of the addition of anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy (either cetuximab or panitumumab) to doublet chemotherapy in patients with right-sided RAS wt primary tumours. Data from the Japanese PARADIGM trial (NCT02394795), the first prospective trial to test the superiority of panitumumab versus bevacizumab in combination with standard doublet (mFOLFOX6) first-line chemotherapy for patients with RAS wt mCRC and left-sided primary tumours, showed panitumumab to statistically significantly improve overall survival and improve response rate and R0 resection rate compared with bevacizumab when used in combination with mFOLFOX6. The statistically significant overall survival benefit and improved response rate and R0 resection rates was retained in the full analysis population, consistent with the data that support the use of anti-EGFR mAb therapy in combination with chemotherapy first line in patients with RAS wt/ BRAF wt left-sided primary tumours. Although, additional data showed that patients with right-sided tumours did not derive an overall survival benefit when treated with panitumumab in combination with chemotherapy, the R0 resection rate for patients with right-sided tumours was similar for patients who received either panitumumab or bevacizumab. More recently, a biomarker study of PARADIGM trial patients has shown that overall survival tended to be longer for selected patients with no gene alterations treated with panitumumab than for those treated with bevacizumab, irrespective of tumour sidedness. This suggests that selection of patients with RAS wt mCRC using ctDNA analysis may further refine the selection of patients for treatment with panitumumab rather than bevacizumab in the first-line treatment setting. There was considerable discussion around ‘recommendation 4k’, however, with the available clinical data presenting conflicting results regarding the addition of anti-EGFR mAbs to triplet FOLFOXIRI (FOLFOX plus irinotecan) therapy first line. FOLFOXIRI, combined with bevacizumab or panitumumab, has been shown to be superior when compared with doublet combinations in patients with RAS wt mCRC. The Japanese randomised phase II DEEPER trial (NCT02515734) of FOLFOXIRI plus cetuximab versus FOLFOXIRI plus bevacizumab first line in patients with RAS wt mCRC showed FOLFOXIRI plus cetuximab to be significantly superior to FOLFOXIRI plus bevacizumab in terms of depth of response (DpR), the primary endpoint. There is no phase III evidence, however, to support the use of anti-EGFR mAbs in combination with triplet chemotherapy as demonstrated by the European TRIPLETE trial which failed to show a benefit for FOLFOXIRI plus panitumumab versus FOLFOX plus panitumumab in terms of early tumour shrinkage and DpR. Thus, the word ‘currently’ was added to the original recommendation 4k as per the bold text below, and also . 4k. Triplets with FOLFOXIRI and anti-EGFR mAbs are not currently recommended [I, D; consensus 100% ]. The Asian experts also accepted ‘recommendation 4l’ unchanged ( 100% consensus ), supported by data from the Japanese phase II OGSG 1602 study (UMIN000024528) of panitumumab monotherapy in chemotherapy-naive frail/elderly patients with unresectable RAS wt metastatic/advanced CRC. The LoE however, was revised to III , C . The Asian experts were did not agree with the wording of the original ‘recommendation 4m’ and thought that the wording should reflect attempts at dose modification. The oral fluoropyrimidine S-1, developed as a prodrug of 5-FU, is frequently used in Asia and can be used when i.v. 5-FU or capecitabine-based chemotherapy cannot be tolerated, due to cardiotoxicity and/or hand-foot syndrome. Thus, the original wording of ‘recommendation 4m’ below: 4m. In patients presenting with cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B], was revised to read: 4m. In patients unable to tolerate cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B; consensus = 100% ]. The Asian experts also accepted ‘recommendations 4n and 4o’ unchanged ( 100% consensus ) with the observation with regard to ‘recommendation 4o’, in patients with dMMR/MSI disease, that although the overall survival of the patients receiving the immune checkpoint inhibitor (ICI) pembrolizumab was not significantly longer in the KEYNOTE-177 study (probably because of the 60% cross-over), it was superior, in those patients receiving pembrolizumab. The primary endpoint prolongation of progression-free survival was met. , As a consequence the consensus was that the ICI pembrolizumab can be recommended as a standard of care, for the treatment of patients with dMMR/MSI mCRC in the first-line setting. A summary of the first-line treatment options for the management of patients with stage IV unresectable mCRC is presented in . Maintenance therapy Consideration of maintenance therapy is generally applicable to those patients who are not amenable to surgery or local therapy and involves a de-escalation in the intensity of their systemic treatment with a concomitant improvement in treatment-related side-effects. The Asian experts accepted completely ( 100% consensus ) ‘recommendations 4p and 4q’ relating to maintenance therapy, without change , in the pre-meeting survey. The Asian experts thought the wording of ‘recommendations 4r and 4s’ was unclear ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), however, and they were revised accordingly as indicated by the bold text below and . 4r. When FOLFIRI is used in first-line treatment, it can be continued until disease progression if well tolerated [V, B; consensus = 100% ]. 4s. Reintroduction of an initial successful induction therapy should be done after progressive disease while on durable maintenance therapy [III, B; consensus = 100% ]. A summary of the maintenance treatment options according to prior therapy are summarised in . Second-line therapy The Asian experts accepted ‘recommendations 4t-y’ relating to second-line therapy, unchanged ( 100% consensus ), and after discussion, ‘recommendations 4u, 4v and 4w’. A modification was made to ‘recommendation 4z’ below and , however, as per the bold text, based on data from the BEACON trial (NCT02928224), at the request of the JSMO experts, as encorafenib–cetuximab plus or minus binimetinib, is reimbursed in Japan for patients with ECOG performance status of 1, >3 metastatic sites, high serum creatinine protein levels (>1 mg/dL), or no history of primary tumour resection. 4z. For pre-treated mCRC patients with BRAF V600E-mutated tumours, encorafenib–cetuximab is recommended as the best option in second line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available (consensus = 100%) . Recommendation 4aa was discussed and an additional statement added to the recommendation, as per the bold text below and , to include the option to use nivolumab or pembrolizumab monotherapy second line for patients with dMMR/MSI-H mCRC. 4aa. For dMMR/MSI-H tumours progressing after first-line chemotherapy, ipilimumab–nivolumab is recommended [III, B; ESMO-MCBS v1.1 score 3]. Nivolumab [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] or pembrolizumab monotherapy is an option [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] . An algorithm of proposed second-line treatment options according to prior therapy and primary tumour mutation profile is presented in . Third-line and further therapy Recently, the addition of bevacizumab to trifluridine–tipiracil has been shown to improve overall and progression-free survival, as well as objective response rate, when compared with trifluridine–tipiracil alone in the randomised phase III SUNLIGHT trial. Also, cetuximab and panitumumab have both shown efficacy in this treatment setting in patients with RAS wt mCRC, as single agents. , , In addition, treatment involving dual HER2 blockade using a combination of trastuzumab an anti-HER2 mAb and the tyrosine kinase inhibitor lapatinib has been shown to be effective in patients with RAS wt, HER2-positve, treatment-refractory mCRC. An algorithm of proposed third-line and later-line treatment options according to the molecular profile of the primary tumour is presented in . The Asian experts accepted the original ESMO recommendations, ‘recommendations 4bb, 4cc, 4gg and 4hh’ without change ( 100% consensus ) with the observation from the JSMO experts that the combination cetuximab–encorafenib–binimetinib , is also available in Japan in relation to ‘recommendation 4ee’. ‘Recommendation 4dd’ was revised retrospectively as per the bold text below and , based on the data published from the SUNLIGHT trial. 4dd. Trifluridine–tipiracil plus or minus bevacizumab is recommended in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics, if available, or in earlier lines of therapy following oxaliplatin and irinotecan regimen failure, depending on local approvals [I, A; consensus = 100%] . ‘Recommendation 4ee’ was revised as per the bold text below and , as per ‘recommendation 4z’ above, based on data from the Beacon trial. 4ee. For BRAF V600E-mutated, pre-treated mCRC patients, encorafenib–cetuximab is recommended as the best option in third line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available . The Asian experts requested the minor modification to ‘recommendation 4ff’ with ‘and’ replaced by ‘or’ as per the bold text below and . 4ff. In RAS wt and BRAF wt patients not previously treated with the EGFR antibodies, cetuximab or panitumumab are recommended as single agents [I, A; panitumumab ESMO-MCBS v1.1 score: 3; consensus = 100% ]. At the request of the experts from JSMO ‘recommendation 4kk’ was added below, and , for patients who had received prior fluoropyrimidine-, oxaliplatin- and irinotecan-based and anti-vascular endothelial growth factor (anti-VEGF) mAb therapy (and anti-EGFR-mAb therapy if RAS wt) based on data from the randomised Chinese FRESCO-1 and global FRESCO-2 trials of fruquintinib versus placebo plus or minus best supportive care, both of which reported a statistically significant improvement in overall survival. 4kk. Fruquintinib is an additional option in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics , [I, B; consensus = 100%] . Also, it was noted that the phase II TRIUMPH study (UMIN000027887) of dual HER2 antibodies (pertuzumab plus trastuzumab) demonstrated promising activity in patients with HER2-positive mCRC, which led to the regulatory approval of this combination therapy in Japan. Whilst, the durable response reported for the recent multicentre, open-label, phase II MOUNTAINEER trial (NCT03043313) in patients with HER2-positive RAS wt mCRC who had progressed on, or were intolerant to, fluoropyrimidine, oxaliplatin, irinotecan and anti-VEGF mAb therapy, treated with tucatinib and trastuzumab, means that this combination is likely to become a new treatment option for patients with chemotherapy-refractory HER2-positive RAS wt mCRC. 5 Follow-up, long-term implications and survivorship—recommendations 5a–b The Asian experts had concerns over the clarity of ‘recommendation 5a’ and the frequency of the proposed radiological evaluations. Several of the experts considered 8-12 weeks to be too frequent for patients receiving active treatment Thus, the original ‘recommendation 5a’ ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) was revised, as per the bold text, to read as follows and . 5a. For patients receiving active treatment, radiological evaluation should be carried out at least every 12 weeks , including (in most cases) a CT scan or MRI scan , as well as the measurement of tumour marker levels [IV, B; consensus = 100% ]. The Asian experts accepted completely ( 100% consensus ) the ESMO ‘recommendation 5b’ . A clinical assessment of the toxicities resulting from both systemic treatment and surgery should be conducted whenever possible together with an assessment of long-term survivors.
Diagnosis, pathology and molecular biology—recommendations 1a-k The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 1a-g and 1i, and 1j’ , without change. In relation to ‘recommendation 1h’, however, concern was raised over restricting the recommendation to patients with RAS wild-type (wt) disease. Although the trials have mainly been conducted in patients with RAS wt disease, the MyPathway study reported a HER2 amplification in 23% of patients with RAS -mutant disease and an overall response rate for these patients of 8% compared with 40% for those with HER2 -amplified, RAS wt disease. Thus, although it was accepted that ‘recommendation 1h’ should remain unchanged ( 100% consensus ) , it was agreed that testing for HER2 amplification should not be excluded for patients with RAS -mutant disease but should be conducted subject to resource availability and the reimbursement and diagnostic testing policies of the individual Asian countries. Also, where HER2 testing is not reimbursed consideration should be given to referring patients to centres conducting clinical trials. In some Asian countries HER2 testing is carried out at the same time as RAS , BRAF and mismatch repair deficiency (dMMR)/microsatellite instability (MSI) testing to minimise the loss of tumour specimens. Although, it should be noted that in some Asian countries HER2 testing is only carried out in patients with RAS wt disease for the same reason. Furthermore, a recent Asian phase II study has shown that circulating tumour cell DNA (ctDNA) genotyping can identify patients who can benefit from dual human epidermal growth factor receptor 2 (HER2) blockade as well as monitor treatment response. These results warrant the further investigation of ctDNA genotyping for patients with HER2 - amplified mCRC in clinical trials. The representatives of 9 of the 10 Asian countries, however, did not consider ‘recommendation 1k’ acceptable in the pre-meeting survey ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) on the basis that the incidence of dihydropyrimidine dehydrogenase (DPD) deficiency is estimated to be very low in Asian populations compared with non-Asian populations. A Japanese study investigated the incidence of DPD deficiency in 1362 Asian colon cancer patients who were enrolled in the JOIN and ACHIEVE adjuvant chemotherapy trials and suggested that the incidence of DPD deficiency for these patients was in the region of 0.6%, with no clear association observed between DPD deficiency and safety. In addition, DPD deficiency was not detected by analysing DPD full-length RNA polymorphisms in peripheral blood mononuclear cells in 67 Taiwanese patients using multiplex nested reverse transcription-polymerase chain reaction (RT-PCR) and non-isotopic RNase cleavage assays (NIRCA). DPD deficiency is also reported not to be common in Korea . Thus, due to the low incidence of DPD deficiency in Asian patients, DPD genotyping and phenotyping is not carried out in routine daily practice in Asia, but is recommended for patients, who experience severe 5-fluorouracil (5-FU) toxicity during and after their first cycle of chemotherapy. ‘Recommendation 1k’ was therefore revised from the original ESMO recommendation ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), to be consistent with the Pan-Asian adaptation of the ESMO Clinical Practice Guidelines for the diagnosis treatment and follow-up of patients with localised colon cancer, as per the bold text below, and the GoR revised to C and a new statement added. 1k. Depending on the anticipated genetic profile of a specific Asian patient population , DPD genotyping or phenotyping may be considered before initiating fluoropyrimidine-based therapy [III, C ]. DPD genotyping or phenotyping should be implemented in patients who experience severe fluoropyrimidine toxicity [V; consensus = 100%] . The experts from JSMO also proposed the addition of a new recommendation, ‘recommendation 1l’ below and , based on data from several Asian studies regarding irinotecan toxicity. , , , , , , Genetic variations within the UDP glucuronosyltransferase 1 family, polypeptide A1 ( UGT1A1 ) gene have been associated with the development of certain drug toxicities, with the UGT1A1 ∗ 6 variant, common in Asian populations. UGT1A1 gene polymorphisms are predictive of irinotecan-related side-effects, including diarrhoea, neutropaenia and vomiting. A systematic review and meta-analysis has shown the increased risk of severe neutropaenia in cancer patients with UGT1A1∗6 polymorphisms. 1l. UGT1A1 genotyping remains an option and it is recommended that it is carried out in patients with a suspicion of UGT1A1 deficiency as reflected by low conjugated bilirubin or in patients where an irinotecan dose of >180 mg/m 2 per administration is planned [III, C; consensus = 100%] . It was also recommended that depending on the prevalence of UGT1A1 polymorphisms per country, a lower irinotecan threshold dose for UGT1A1 genotyping may be used. , available at https://doi.org/10.1016/j.esmoop.2023.101558 describes the biomarkers and molecular targets for precision medicine and the corresponding ESMO Scale for Clinical Actionability of molecular Targets (ESCAT) scores.
Staging and risk assessment—recommendations 2a-d The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 2a-d’ , available at https://doi.org/10.1016/j.esmoop.2023.101558 and .
Management of resectable/potentially resectable disease—recommendations 3a–f Treatment of patients with potentially resectable mCRC The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3a-d and 3f’ . There was, however, much discussion about the precise meaning of the ESMO ‘recommendation 3e’ below (and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) which states that: 3e. Patients unresponsive to first-line chemotherapy should not be denied resection or ablation of their metastases since the outcome of resected patients after second-line treatment could be also favourable. Intra-arterial chemotherapy could be an option in such patients, not only to recover a response but also to achieve liver resection [III, C]. It was agreed by the experts that patients unresponsive to first-line therapy should be assessed for resection after a second-line treatment benefit is observed in the absence of contraindications. Thus, ‘recommendation 3e’ was reworded as per the bold text, below and . 3e. Patients with metastases progressing on first-line chemotherapy should be assessed for resection after second-line treatment benefit . Intra-arterial chemotherapy may be used as a second-line option not only to achieve a response but also liver resection [III, C; consensus = 100%] . Intent and choice of local treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3g and 3i’ , but discussions at the hybrid virtual/face-to-face meeting led to the rewording of the original ‘recommendation 3h’. The original ‘recommendation 3h’ below was revised and shortened (as per the bold text) for better understanding from: 3h. Local treatment can be used as a primary or metastasis-specific treatment to halt further dissemination, and/or following systemic therapy as a consolidation treatment, to delay or pause further treatment [III, C]. to read as follows: 3h. Local treatment may be used as a primary or metastasis-specific treatment or following systemic therapy as a consolidation strategy [III, C; consensus = 100 %] . Local ablation treatment The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3j – m’ . An algorithm summarising the options for local ablation treatment is presented in . Intra-arterial therapies The Pan-Asian panel of experts revised ‘recommendation 3n’ below to more accurately reflect the situation in their countries. In Korea and Taiwan intra-arterial therapies are only used within the remit of a clinical trial, whilst in Japan they are not commonly used at all. The feeling amongst the experts was that hepatic arterial infusion chemotherapy (HAIC) in particular was limited by a lack of expertise. The experts from TSCO considered that the evidence to support the use of transarterial chemoembolisation (TACE) was inadequate. Thus, the consensus was that such procedures should be limited to expert centres or clinical trials. The wording of the original ‘recommendation 3n’ below: 3n. TACE, TARE/SIRT and HAIC may be also considered as treatment options with non-curative intent [III, B], was revised to read: 3n. TACE, transarterial radioembolisation (TARE)/selective internal radiotherapy (SIRT) and HAIC may be considered as treatment options with non-curative intent, if available in expert centres [ III, C; consensus = 100%]. Patient participation in related clinical trials should be encouraged. The GoR was revised from B to C . The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the ESMO ‘recommendation 3o’ in the pre-meeting survey without change. The options for intra-arterial therapy are summarised in .
The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3a-d and 3f’ . There was, however, much discussion about the precise meaning of the ESMO ‘recommendation 3e’ below (and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) which states that: 3e. Patients unresponsive to first-line chemotherapy should not be denied resection or ablation of their metastases since the outcome of resected patients after second-line treatment could be also favourable. Intra-arterial chemotherapy could be an option in such patients, not only to recover a response but also to achieve liver resection [III, C]. It was agreed by the experts that patients unresponsive to first-line therapy should be assessed for resection after a second-line treatment benefit is observed in the absence of contraindications. Thus, ‘recommendation 3e’ was reworded as per the bold text, below and . 3e. Patients with metastases progressing on first-line chemotherapy should be assessed for resection after second-line treatment benefit . Intra-arterial chemotherapy may be used as a second-line option not only to achieve a response but also liver resection [III, C; consensus = 100%] .
The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3g and 3i’ , but discussions at the hybrid virtual/face-to-face meeting led to the rewording of the original ‘recommendation 3h’. The original ‘recommendation 3h’ below was revised and shortened (as per the bold text) for better understanding from: 3h. Local treatment can be used as a primary or metastasis-specific treatment to halt further dissemination, and/or following systemic therapy as a consolidation treatment, to delay or pause further treatment [III, C]. to read as follows: 3h. Local treatment may be used as a primary or metastasis-specific treatment or following systemic therapy as a consolidation strategy [III, C; consensus = 100 %] .
The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 3j – m’ . An algorithm summarising the options for local ablation treatment is presented in .
The Pan-Asian panel of experts revised ‘recommendation 3n’ below to more accurately reflect the situation in their countries. In Korea and Taiwan intra-arterial therapies are only used within the remit of a clinical trial, whilst in Japan they are not commonly used at all. The feeling amongst the experts was that hepatic arterial infusion chemotherapy (HAIC) in particular was limited by a lack of expertise. The experts from TSCO considered that the evidence to support the use of transarterial chemoembolisation (TACE) was inadequate. Thus, the consensus was that such procedures should be limited to expert centres or clinical trials. The wording of the original ‘recommendation 3n’ below: 3n. TACE, TARE/SIRT and HAIC may be also considered as treatment options with non-curative intent [III, B], was revised to read: 3n. TACE, transarterial radioembolisation (TARE)/selective internal radiotherapy (SIRT) and HAIC may be considered as treatment options with non-curative intent, if available in expert centres [ III, C; consensus = 100%]. Patient participation in related clinical trials should be encouraged. The GoR was revised from B to C . The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the ESMO ‘recommendation 3o’ in the pre-meeting survey without change. The options for intra-arterial therapy are summarised in .
Management of advanced and metastatic disease without potential for conversion—recommendations 4a–jj First-line therapy The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 4a-d’ and 4f-j, . The Asian experts also accepted the original ‘recommendation 4e’ without change , after discussion of whether there was evidence to support the benefit of the addition of anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy (either cetuximab or panitumumab) to doublet chemotherapy in patients with right-sided RAS wt primary tumours. Data from the Japanese PARADIGM trial (NCT02394795), the first prospective trial to test the superiority of panitumumab versus bevacizumab in combination with standard doublet (mFOLFOX6) first-line chemotherapy for patients with RAS wt mCRC and left-sided primary tumours, showed panitumumab to statistically significantly improve overall survival and improve response rate and R0 resection rate compared with bevacizumab when used in combination with mFOLFOX6. The statistically significant overall survival benefit and improved response rate and R0 resection rates was retained in the full analysis population, consistent with the data that support the use of anti-EGFR mAb therapy in combination with chemotherapy first line in patients with RAS wt/ BRAF wt left-sided primary tumours. Although, additional data showed that patients with right-sided tumours did not derive an overall survival benefit when treated with panitumumab in combination with chemotherapy, the R0 resection rate for patients with right-sided tumours was similar for patients who received either panitumumab or bevacizumab. More recently, a biomarker study of PARADIGM trial patients has shown that overall survival tended to be longer for selected patients with no gene alterations treated with panitumumab than for those treated with bevacizumab, irrespective of tumour sidedness. This suggests that selection of patients with RAS wt mCRC using ctDNA analysis may further refine the selection of patients for treatment with panitumumab rather than bevacizumab in the first-line treatment setting. There was considerable discussion around ‘recommendation 4k’, however, with the available clinical data presenting conflicting results regarding the addition of anti-EGFR mAbs to triplet FOLFOXIRI (FOLFOX plus irinotecan) therapy first line. FOLFOXIRI, combined with bevacizumab or panitumumab, has been shown to be superior when compared with doublet combinations in patients with RAS wt mCRC. The Japanese randomised phase II DEEPER trial (NCT02515734) of FOLFOXIRI plus cetuximab versus FOLFOXIRI plus bevacizumab first line in patients with RAS wt mCRC showed FOLFOXIRI plus cetuximab to be significantly superior to FOLFOXIRI plus bevacizumab in terms of depth of response (DpR), the primary endpoint. There is no phase III evidence, however, to support the use of anti-EGFR mAbs in combination with triplet chemotherapy as demonstrated by the European TRIPLETE trial which failed to show a benefit for FOLFOXIRI plus panitumumab versus FOLFOX plus panitumumab in terms of early tumour shrinkage and DpR. Thus, the word ‘currently’ was added to the original recommendation 4k as per the bold text below, and also . 4k. Triplets with FOLFOXIRI and anti-EGFR mAbs are not currently recommended [I, D; consensus 100% ]. The Asian experts also accepted ‘recommendation 4l’ unchanged ( 100% consensus ), supported by data from the Japanese phase II OGSG 1602 study (UMIN000024528) of panitumumab monotherapy in chemotherapy-naive frail/elderly patients with unresectable RAS wt metastatic/advanced CRC. The LoE however, was revised to III , C . The Asian experts were did not agree with the wording of the original ‘recommendation 4m’ and thought that the wording should reflect attempts at dose modification. The oral fluoropyrimidine S-1, developed as a prodrug of 5-FU, is frequently used in Asia and can be used when i.v. 5-FU or capecitabine-based chemotherapy cannot be tolerated, due to cardiotoxicity and/or hand-foot syndrome. Thus, the original wording of ‘recommendation 4m’ below: 4m. In patients presenting with cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B], was revised to read: 4m. In patients unable to tolerate cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B; consensus = 100% ]. The Asian experts also accepted ‘recommendations 4n and 4o’ unchanged ( 100% consensus ) with the observation with regard to ‘recommendation 4o’, in patients with dMMR/MSI disease, that although the overall survival of the patients receiving the immune checkpoint inhibitor (ICI) pembrolizumab was not significantly longer in the KEYNOTE-177 study (probably because of the 60% cross-over), it was superior, in those patients receiving pembrolizumab. The primary endpoint prolongation of progression-free survival was met. , As a consequence the consensus was that the ICI pembrolizumab can be recommended as a standard of care, for the treatment of patients with dMMR/MSI mCRC in the first-line setting. A summary of the first-line treatment options for the management of patients with stage IV unresectable mCRC is presented in . Maintenance therapy Consideration of maintenance therapy is generally applicable to those patients who are not amenable to surgery or local therapy and involves a de-escalation in the intensity of their systemic treatment with a concomitant improvement in treatment-related side-effects. The Asian experts accepted completely ( 100% consensus ) ‘recommendations 4p and 4q’ relating to maintenance therapy, without change , in the pre-meeting survey. The Asian experts thought the wording of ‘recommendations 4r and 4s’ was unclear ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), however, and they were revised accordingly as indicated by the bold text below and . 4r. When FOLFIRI is used in first-line treatment, it can be continued until disease progression if well tolerated [V, B; consensus = 100% ]. 4s. Reintroduction of an initial successful induction therapy should be done after progressive disease while on durable maintenance therapy [III, B; consensus = 100% ]. A summary of the maintenance treatment options according to prior therapy are summarised in . Second-line therapy The Asian experts accepted ‘recommendations 4t-y’ relating to second-line therapy, unchanged ( 100% consensus ), and after discussion, ‘recommendations 4u, 4v and 4w’. A modification was made to ‘recommendation 4z’ below and , however, as per the bold text, based on data from the BEACON trial (NCT02928224), at the request of the JSMO experts, as encorafenib–cetuximab plus or minus binimetinib, is reimbursed in Japan for patients with ECOG performance status of 1, >3 metastatic sites, high serum creatinine protein levels (>1 mg/dL), or no history of primary tumour resection. 4z. For pre-treated mCRC patients with BRAF V600E-mutated tumours, encorafenib–cetuximab is recommended as the best option in second line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available (consensus = 100%) . Recommendation 4aa was discussed and an additional statement added to the recommendation, as per the bold text below and , to include the option to use nivolumab or pembrolizumab monotherapy second line for patients with dMMR/MSI-H mCRC. 4aa. For dMMR/MSI-H tumours progressing after first-line chemotherapy, ipilimumab–nivolumab is recommended [III, B; ESMO-MCBS v1.1 score 3]. Nivolumab [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] or pembrolizumab monotherapy is an option [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] . An algorithm of proposed second-line treatment options according to prior therapy and primary tumour mutation profile is presented in . Third-line and further therapy Recently, the addition of bevacizumab to trifluridine–tipiracil has been shown to improve overall and progression-free survival, as well as objective response rate, when compared with trifluridine–tipiracil alone in the randomised phase III SUNLIGHT trial. Also, cetuximab and panitumumab have both shown efficacy in this treatment setting in patients with RAS wt mCRC, as single agents. , , In addition, treatment involving dual HER2 blockade using a combination of trastuzumab an anti-HER2 mAb and the tyrosine kinase inhibitor lapatinib has been shown to be effective in patients with RAS wt, HER2-positve, treatment-refractory mCRC. An algorithm of proposed third-line and later-line treatment options according to the molecular profile of the primary tumour is presented in . The Asian experts accepted the original ESMO recommendations, ‘recommendations 4bb, 4cc, 4gg and 4hh’ without change ( 100% consensus ) with the observation from the JSMO experts that the combination cetuximab–encorafenib–binimetinib , is also available in Japan in relation to ‘recommendation 4ee’. ‘Recommendation 4dd’ was revised retrospectively as per the bold text below and , based on the data published from the SUNLIGHT trial. 4dd. Trifluridine–tipiracil plus or minus bevacizumab is recommended in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics, if available, or in earlier lines of therapy following oxaliplatin and irinotecan regimen failure, depending on local approvals [I, A; consensus = 100%] . ‘Recommendation 4ee’ was revised as per the bold text below and , as per ‘recommendation 4z’ above, based on data from the Beacon trial. 4ee. For BRAF V600E-mutated, pre-treated mCRC patients, encorafenib–cetuximab is recommended as the best option in third line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available . The Asian experts requested the minor modification to ‘recommendation 4ff’ with ‘and’ replaced by ‘or’ as per the bold text below and . 4ff. In RAS wt and BRAF wt patients not previously treated with the EGFR antibodies, cetuximab or panitumumab are recommended as single agents [I, A; panitumumab ESMO-MCBS v1.1 score: 3; consensus = 100% ]. At the request of the experts from JSMO ‘recommendation 4kk’ was added below, and , for patients who had received prior fluoropyrimidine-, oxaliplatin- and irinotecan-based and anti-vascular endothelial growth factor (anti-VEGF) mAb therapy (and anti-EGFR-mAb therapy if RAS wt) based on data from the randomised Chinese FRESCO-1 and global FRESCO-2 trials of fruquintinib versus placebo plus or minus best supportive care, both of which reported a statistically significant improvement in overall survival. 4kk. Fruquintinib is an additional option in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics , [I, B; consensus = 100%] . Also, it was noted that the phase II TRIUMPH study (UMIN000027887) of dual HER2 antibodies (pertuzumab plus trastuzumab) demonstrated promising activity in patients with HER2-positive mCRC, which led to the regulatory approval of this combination therapy in Japan. Whilst, the durable response reported for the recent multicentre, open-label, phase II MOUNTAINEER trial (NCT03043313) in patients with HER2-positive RAS wt mCRC who had progressed on, or were intolerant to, fluoropyrimidine, oxaliplatin, irinotecan and anti-VEGF mAb therapy, treated with tucatinib and trastuzumab, means that this combination is likely to become a new treatment option for patients with chemotherapy-refractory HER2-positive RAS wt mCRC.
The Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the original ESMO recommendations, ‘recommendations 4a-d’ and 4f-j, . The Asian experts also accepted the original ‘recommendation 4e’ without change , after discussion of whether there was evidence to support the benefit of the addition of anti-epidermal growth factor receptor monoclonal antibody (anti-EGFR mAb) therapy (either cetuximab or panitumumab) to doublet chemotherapy in patients with right-sided RAS wt primary tumours. Data from the Japanese PARADIGM trial (NCT02394795), the first prospective trial to test the superiority of panitumumab versus bevacizumab in combination with standard doublet (mFOLFOX6) first-line chemotherapy for patients with RAS wt mCRC and left-sided primary tumours, showed panitumumab to statistically significantly improve overall survival and improve response rate and R0 resection rate compared with bevacizumab when used in combination with mFOLFOX6. The statistically significant overall survival benefit and improved response rate and R0 resection rates was retained in the full analysis population, consistent with the data that support the use of anti-EGFR mAb therapy in combination with chemotherapy first line in patients with RAS wt/ BRAF wt left-sided primary tumours. Although, additional data showed that patients with right-sided tumours did not derive an overall survival benefit when treated with panitumumab in combination with chemotherapy, the R0 resection rate for patients with right-sided tumours was similar for patients who received either panitumumab or bevacizumab. More recently, a biomarker study of PARADIGM trial patients has shown that overall survival tended to be longer for selected patients with no gene alterations treated with panitumumab than for those treated with bevacizumab, irrespective of tumour sidedness. This suggests that selection of patients with RAS wt mCRC using ctDNA analysis may further refine the selection of patients for treatment with panitumumab rather than bevacizumab in the first-line treatment setting. There was considerable discussion around ‘recommendation 4k’, however, with the available clinical data presenting conflicting results regarding the addition of anti-EGFR mAbs to triplet FOLFOXIRI (FOLFOX plus irinotecan) therapy first line. FOLFOXIRI, combined with bevacizumab or panitumumab, has been shown to be superior when compared with doublet combinations in patients with RAS wt mCRC. The Japanese randomised phase II DEEPER trial (NCT02515734) of FOLFOXIRI plus cetuximab versus FOLFOXIRI plus bevacizumab first line in patients with RAS wt mCRC showed FOLFOXIRI plus cetuximab to be significantly superior to FOLFOXIRI plus bevacizumab in terms of depth of response (DpR), the primary endpoint. There is no phase III evidence, however, to support the use of anti-EGFR mAbs in combination with triplet chemotherapy as demonstrated by the European TRIPLETE trial which failed to show a benefit for FOLFOXIRI plus panitumumab versus FOLFOX plus panitumumab in terms of early tumour shrinkage and DpR. Thus, the word ‘currently’ was added to the original recommendation 4k as per the bold text below, and also . 4k. Triplets with FOLFOXIRI and anti-EGFR mAbs are not currently recommended [I, D; consensus 100% ]. The Asian experts also accepted ‘recommendation 4l’ unchanged ( 100% consensus ), supported by data from the Japanese phase II OGSG 1602 study (UMIN000024528) of panitumumab monotherapy in chemotherapy-naive frail/elderly patients with unresectable RAS wt metastatic/advanced CRC. The LoE however, was revised to III , C . The Asian experts were did not agree with the wording of the original ‘recommendation 4m’ and thought that the wording should reflect attempts at dose modification. The oral fluoropyrimidine S-1, developed as a prodrug of 5-FU, is frequently used in Asia and can be used when i.v. 5-FU or capecitabine-based chemotherapy cannot be tolerated, due to cardiotoxicity and/or hand-foot syndrome. Thus, the original wording of ‘recommendation 4m’ below: 4m. In patients presenting with cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B], was revised to read: 4m. In patients unable to tolerate cardiotoxicity and/or hand-foot syndrome on 5-FU or capecitabine-based chemotherapy, S-1 may be used as an alternative [III, B; consensus = 100% ]. The Asian experts also accepted ‘recommendations 4n and 4o’ unchanged ( 100% consensus ) with the observation with regard to ‘recommendation 4o’, in patients with dMMR/MSI disease, that although the overall survival of the patients receiving the immune checkpoint inhibitor (ICI) pembrolizumab was not significantly longer in the KEYNOTE-177 study (probably because of the 60% cross-over), it was superior, in those patients receiving pembrolizumab. The primary endpoint prolongation of progression-free survival was met. , As a consequence the consensus was that the ICI pembrolizumab can be recommended as a standard of care, for the treatment of patients with dMMR/MSI mCRC in the first-line setting. A summary of the first-line treatment options for the management of patients with stage IV unresectable mCRC is presented in .
Consideration of maintenance therapy is generally applicable to those patients who are not amenable to surgery or local therapy and involves a de-escalation in the intensity of their systemic treatment with a concomitant improvement in treatment-related side-effects. The Asian experts accepted completely ( 100% consensus ) ‘recommendations 4p and 4q’ relating to maintenance therapy, without change , in the pre-meeting survey. The Asian experts thought the wording of ‘recommendations 4r and 4s’ was unclear ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ), however, and they were revised accordingly as indicated by the bold text below and . 4r. When FOLFIRI is used in first-line treatment, it can be continued until disease progression if well tolerated [V, B; consensus = 100% ]. 4s. Reintroduction of an initial successful induction therapy should be done after progressive disease while on durable maintenance therapy [III, B; consensus = 100% ]. A summary of the maintenance treatment options according to prior therapy are summarised in .
The Asian experts accepted ‘recommendations 4t-y’ relating to second-line therapy, unchanged ( 100% consensus ), and after discussion, ‘recommendations 4u, 4v and 4w’. A modification was made to ‘recommendation 4z’ below and , however, as per the bold text, based on data from the BEACON trial (NCT02928224), at the request of the JSMO experts, as encorafenib–cetuximab plus or minus binimetinib, is reimbursed in Japan for patients with ECOG performance status of 1, >3 metastatic sites, high serum creatinine protein levels (>1 mg/dL), or no history of primary tumour resection. 4z. For pre-treated mCRC patients with BRAF V600E-mutated tumours, encorafenib–cetuximab is recommended as the best option in second line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available (consensus = 100%) . Recommendation 4aa was discussed and an additional statement added to the recommendation, as per the bold text below and , to include the option to use nivolumab or pembrolizumab monotherapy second line for patients with dMMR/MSI-H mCRC. 4aa. For dMMR/MSI-H tumours progressing after first-line chemotherapy, ipilimumab–nivolumab is recommended [III, B; ESMO-MCBS v1.1 score 3]. Nivolumab [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] or pembrolizumab monotherapy is an option [III, C; ESMO-MCBS v1.1 score: 3; consensus = 100%] . An algorithm of proposed second-line treatment options according to prior therapy and primary tumour mutation profile is presented in .
Recently, the addition of bevacizumab to trifluridine–tipiracil has been shown to improve overall and progression-free survival, as well as objective response rate, when compared with trifluridine–tipiracil alone in the randomised phase III SUNLIGHT trial. Also, cetuximab and panitumumab have both shown efficacy in this treatment setting in patients with RAS wt mCRC, as single agents. , , In addition, treatment involving dual HER2 blockade using a combination of trastuzumab an anti-HER2 mAb and the tyrosine kinase inhibitor lapatinib has been shown to be effective in patients with RAS wt, HER2-positve, treatment-refractory mCRC. An algorithm of proposed third-line and later-line treatment options according to the molecular profile of the primary tumour is presented in . The Asian experts accepted the original ESMO recommendations, ‘recommendations 4bb, 4cc, 4gg and 4hh’ without change ( 100% consensus ) with the observation from the JSMO experts that the combination cetuximab–encorafenib–binimetinib , is also available in Japan in relation to ‘recommendation 4ee’. ‘Recommendation 4dd’ was revised retrospectively as per the bold text below and , based on the data published from the SUNLIGHT trial. 4dd. Trifluridine–tipiracil plus or minus bevacizumab is recommended in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics, if available, or in earlier lines of therapy following oxaliplatin and irinotecan regimen failure, depending on local approvals [I, A; consensus = 100%] . ‘Recommendation 4ee’ was revised as per the bold text below and , as per ‘recommendation 4z’ above, based on data from the Beacon trial. 4ee. For BRAF V600E-mutated, pre-treated mCRC patients, encorafenib–cetuximab is recommended as the best option in third line [I, A; ESMO-MCBS v1.1 score: 4; ESCAT: I-A]. The addition of binimetinib may be an option, if available . The Asian experts requested the minor modification to ‘recommendation 4ff’ with ‘and’ replaced by ‘or’ as per the bold text below and . 4ff. In RAS wt and BRAF wt patients not previously treated with the EGFR antibodies, cetuximab or panitumumab are recommended as single agents [I, A; panitumumab ESMO-MCBS v1.1 score: 3; consensus = 100% ]. At the request of the experts from JSMO ‘recommendation 4kk’ was added below, and , for patients who had received prior fluoropyrimidine-, oxaliplatin- and irinotecan-based and anti-vascular endothelial growth factor (anti-VEGF) mAb therapy (and anti-EGFR-mAb therapy if RAS wt) based on data from the randomised Chinese FRESCO-1 and global FRESCO-2 trials of fruquintinib versus placebo plus or minus best supportive care, both of which reported a statistically significant improvement in overall survival. 4kk. Fruquintinib is an additional option in patients pre-treated with fluoropyrimidines, oxaliplatin, irinotecan and biologics , [I, B; consensus = 100%] . Also, it was noted that the phase II TRIUMPH study (UMIN000027887) of dual HER2 antibodies (pertuzumab plus trastuzumab) demonstrated promising activity in patients with HER2-positive mCRC, which led to the regulatory approval of this combination therapy in Japan. Whilst, the durable response reported for the recent multicentre, open-label, phase II MOUNTAINEER trial (NCT03043313) in patients with HER2-positive RAS wt mCRC who had progressed on, or were intolerant to, fluoropyrimidine, oxaliplatin, irinotecan and anti-VEGF mAb therapy, treated with tucatinib and trastuzumab, means that this combination is likely to become a new treatment option for patients with chemotherapy-refractory HER2-positive RAS wt mCRC.
Follow-up, long-term implications and survivorship—recommendations 5a–b The Asian experts had concerns over the clarity of ‘recommendation 5a’ and the frequency of the proposed radiological evaluations. Several of the experts considered 8-12 weeks to be too frequent for patients receiving active treatment Thus, the original ‘recommendation 5a’ ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) was revised, as per the bold text, to read as follows and . 5a. For patients receiving active treatment, radiological evaluation should be carried out at least every 12 weeks , including (in most cases) a CT scan or MRI scan , as well as the measurement of tumour marker levels [IV, B; consensus = 100% ]. The Asian experts accepted completely ( 100% consensus ) the ESMO ‘recommendation 5b’ . A clinical assessment of the toxicities resulting from both systemic treatment and surgery should be conducted whenever possible together with an assessment of long-term survivors.
Applicability of the recommendations Following the hybrid virtual/face-to-face working meeting, hosted by JSMO, the Pan-Asian panel of experts agreed with and accepted completely ( 100% consensus ) the adapted ESMO guidelines listed in above. The applicability of each of the guideline recommendations, however, is highly dependent on the drug and testing approvals for each Asian country and their reimbursement policies both of which differ markedly across the 10 countries represented. The drug and treatment availability for the 10 Asian countries is summarised in , available at https://doi.org/10.1016/j.esmoop.2023.101558 and outlined in the text below for each country individually and summarised individually for each country in , available at https://doi.org/10.1016/j.esmoop.2023.101558 . Most notable are the striking differences between the individual countries in terms of their availability and reimbursement policies with patients in Indonesia, Malaysia, The Philippines and Thailand having fewer approvals for testing and treatments, whilst also receiving very little reimbursement ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In contrast, patients in India, Japan and Singapore have access to nearly all the cancer therapies and testing services at modest or no personal cost ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). For example, in Singapore, testing is approved but not reimbursed and most cancer treatments are approved. Patients can pay out of their national savings scheme and basic health insurance plan for those agents that are approved (i.e. nearly all the drugs discussed in the main text above). There is also a complex insurance system for people who can afford it, and a means tested ‘safety net’ for those that cannot ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In Korea, nearly all tests and treatments are approved, but although the testing is reimbursed, the reimbursement of treatment is limited beyond first line ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In the case of Taiwan, there are limited approvals for both testing and therapy, with EGFR inhibitors and bevacizumab approved in first and third line, and first line only, respectively ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In China, most drugs and a number of targeted agents are available for the treatment of patients with mCRC, although several targeted agents as well as several genetic tests are not reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). The differences between the countries reflect the way the different countries manage their health care systems and the money allocated from the individual governments to cancer care. Significantly, the health care systems of the more affluent countries offer a higher proportion of their populations access to all levels of cancer care due not only to them having the highest rates of drug approvals and/or better public reimbursement policies, but also due to a higher percentage of their populations being able to purchase or obtain through their employment, private medical cover. The individual statements, from the experts of each country, describing the availability and access to optimal diagnostic and molecular testing and the latest drug therapies for their individual countries, are described in the paragraphs below together with some of the details of their reimbursement policies. China In China, the drugs available for the treatment of patients with mCRC include the following, namely the chemotherapy drugs 5-FU, oxaliplatin, irinotecan, capecitabine and trifluridine–tipiracil, and the targeted agents cetuximab (for patients with RAS wt mCRC), bevacizumab, regorafenib and fruquintinib, and are reimbursed by medical insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Pembrolizumab is also available for those patients with MSI-H mCRC but is not reimbursed. To date, the agents aflibercept, panitumumab, ramucirumab, encorafenib, adagrasib, sotorasib, lapatinib, tucatinib, trastuzumab deruxtecan and larotrectinib are still not available to Chinese patients. Patients with mCRC can undergo recommended genetic testing, including for their RAS , BRAF , HER2 , MSI status and for NTRK fusions. Currently, however, medical insurance in China only covers PCR testing for RAS ( KRAS/NRAS ) and BRAF alterations, and MSI testing. Medical insurance also covers immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) detection of HER2 alterations, and IHC detection of MMR. Next generation sequencing (NGS) testing is not reimbursed by medical insurance. There are different policies in terms of reimbursement across the different provinces depending on province-based approvals. Indonesia In Indonesia the universal health care system (UHC) covers most of the health services. Although almost 80% of Indonesians are covered by the UHC, however, amongst them are individuals who also have their own private medical insurance or whose health care is covered by their employer. The use of 5-FU-based chemotherapy (including oxaliplatin or irinotecan regimens) as first-line or second-line therapy is reimbursed, whilst targeted therapies and checkpoint inhibitors are not. Biomarker testing is available but not all tests are reimbursed. New technology-based tests (i.e. NGS) are not reimbursed. Also, after a Health Technology Assessment (HTA), bevacizumab and cetuximab for the treatment of mCRC have been excluded from the UHC scheme. In terms of availability, in Indonesia new drugs/agents firstly have to receive approval from the Indonesian Food and Drug Authority (FDA). Then after 2 years an application can be made for the drug to be included in the national formulary which is a list of medications that are eligible to be given to patients under the UHC scheme. Due to the high burden of health care costs, however, especially for cancer treatment, it can sometimes take years, multiple scientific evaluations and cost effectiveness analyses/HTA, for a new drug to be listed under the national formulary for UHC. Thus, most of the drugs are not available or reimbursed for patients with CRC. Drugs are approved for very specific indications. India Most of the drugs (chemotherapy and biologicals) for the treatment of mCRC discussed in the ESMO guidelines and Section A above are available in India ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Chemotherapy drugs and some of the biologicals are available in the government hospitals with cancer services and hence accessible to most patients at modest or no cost. India has a graded payment/insurance scheme based on patient income, decided by the government. Also, the wide availability of cheaper and good quality generics and biosimilars have improved patient access to better treatment. Drugs not covered by the government centres are covered by private insurance or by the patient as an out-of-pocket payment. The government health scheme covers about 30%-35% of the population, with the remainder taken care of by the private sector. Most tests including NGS are available but are expensive. Limited PCR panels are widely available. Some of the tests are reimbursed by insurance providers, whereas some drug companies offer coupons to subsidise certain tests. Many centres conduct investigator-initiated studies to generate Indian patient data including on biomarkers, facilitating increased patient access to newer therapeutic strategies. Japan In Japan, all the drugs recommended in the ESMO Clinical Practice Guideline, except for trastuzumab plus lapatinib and trastuzumab deruxtecan are approved and reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In addition, the triplet combination of cetuximab plus encorafenib plus binimetinib in second or later line, , and trastuzumab plus pertuzumab in third or later line, are approved and reimbursed. All the recommended assays for biomarker analysis (except primary NGS), are approved and reimbursed. Also, repeated plasma-based digital PCR RAS testing for rechallenge of anti-EGFR therapies and NGS for comprehensive genome profiling after standard therapies are also both approved and reimbursed. Patients pay 0%-30% of the total cost for diagnostic/molecular testing and treatment, depending on their age and income. Korea All drugs and assays discussed above in Section A of this manuscript are available in Korea, except for ramucirumab and trastuzumab deruxtecan, as indicated in , available at https://doi.org/10.1016/j.esmoop.2023.101558 . Some biological agents or ICIs are also available beyond second line for patients with mCRC, but their cost is not reimbursed yet. Malaysia In Malaysia there are two types of health care system. The government provides universal health care under the Ministry of Health (MOH) for all Malaysians for a very low fee. Alternatively there is private health care, which is not affiliated with MOH, for those who wish to and are able to pay for it. Targeted drugs are very limited and are often not available widely under the MOH due to cost issues and are therefore generally not reimbursable. Malaysia does not practice a partly funded government health insurance policy for individual citizens. Common systemic chemotherapy regimens are covered but not the newer targeted therapies. Patients therefore often pay privately, often out of their own pocket or using some form of insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Thus, in the private sector in Malaysia, targeted drugs are more readily available as the patients pay using their own money or private insurance. The MOH tries to negotiate with the pharmaceutical companies, to lower their prices, while at the same time considering generic options whenever possible. Health Intervention Technology, a division in the MOH, often discusses cost–benefit ratios when coming to a decision to include certain drugs for use by the MOH. The Philippines Most of the drugs enumerated ( and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) are available in the Philippines, although not reimbursed, resulting in 100% out-of-pocket patient payments. Most laboratory tests and diagnostics are available in the big cities like Manila and in big treatment centres, thus, ‘availability for all patients’ is an issue. Some agents can be obtained for compassionate use from Hong Kong or Singapore. The Philippines comprises >7000 islands with a range of different procurement policies. Singapore In Singapore, ∼90% of Health Sciences Authority-approved cancer treatments are on the Cancer Drug List (CDL), a list of clinically proven and cost-effective cancer treatments. Only indication-specific treatments in the CDL can be claimed under MediSave (MSV), MediShield Life (MSHL) and Integrated Shield Plans (IPs). MSV is a national medical savings scheme that mandates individuals to set aside part of their income to pay for their hospitalisation and outpatient expenses including cancer drugs listed on the CDL. MediShield Life is a basic health insurance plan which helps to pay for large hospital bills and outpatient treatments including selected cancer drugs. IPs include MSHL and private insurance plans that provide additional cover for cancer diagnostics and drugs. Patients receiving government-subsidised care in public health care institutions utilising drugs which are listed under the Standard Drug List and/or covered under the Medication Assistance Fund may receive an up to 75% subsidy in drug costs (based on their monthly per capita household income). Those in need of financial assistance who fulfil the means testing requirements are eligible for government financial subsidies such as Medifund. In Singapore, molecular assays (e.g. IHC, PCR and NGS) are not subsidised or reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Indication-specific drugs that are not on the CDL such as lapatinib, pertuzumab and trastuzumab deruxtecan for CRC treatment are not subsidised or reimbursed. Taiwan All drugs and tests are available in Taiwan but not always covered by public insurance. The coverage of National Health Insurance in Taiwan is basically ‘ALL or NONE’ and the financial burden is huge and expected to increase further in the era of precision medicine and immuno-oncology. The availability of other medications for the same indication and future budget burden are the most important considerations, as well as the scientific results of pivotal trials, for decisions on reimbursement. This explains the reasons for the relatively limited coverage of expensive biologics (a maximum of 36 weeks reimbursement for bevacizumab), some of the new technology-based tests (i.e. NGS) and new treatments (e.g. ICIs in MSI-H mCRC) on the reimbursement list ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Also, in the case of patients receiving triplet therapy, only two agents are reimbursed with the cost of the third agent having to be paid for. Thailand In Thailand’s health care system, there are three main reimbursement schemes including the Universal Coverage (UC) scheme, the Social Security Scheme (SSS) and the Civil Servant Medical Benefit Scheme (CSMBS). The majority of Thai people were covered by the UC and the SSS. All government employees and their dependents are covered by the CSMBS. For the UC and SSS, the use of oxaliplatin- and irinotecan-based regimens as first-line or second-line therapy are reimbursable. Targeted therapies, ICIs and biomarker testing are not reimbursable. For those under the CSMBS, in addition to chemotherapy, they are able to access targeted therapy and companion biomarker testing using a pre-authorised system. Six months of bevacizumab therapy is eligible for reimbursement for second-line therapy. Panitumumab is the only anti-EGFR agent that is reimbursed for patients with RAS wt advanced CRC, with two indications including first-line therapy with doublet chemotherapy only for patients with potentially resectable advanced CRC, and third-line therapy in combination with single-agent irinotecan. Regorafenib is also reimbursable for patients with chemotherapy-refractory disease ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Otherwise the medical expenses are not reimbursed. The ESMO-MCBSs for the different systemic therapy options and new therapy combinations for the treatment of patients with mCRC are to be found at https://www.esmo.org/guidelines/esmo-mcbs/esmo-mcbs-scorecards?mcbs_score_cards_form%5BsearchText%5D=&mcbs_score_cards_form%5Btumour-sub-type%5D=Colon+and+R .
In China, the drugs available for the treatment of patients with mCRC include the following, namely the chemotherapy drugs 5-FU, oxaliplatin, irinotecan, capecitabine and trifluridine–tipiracil, and the targeted agents cetuximab (for patients with RAS wt mCRC), bevacizumab, regorafenib and fruquintinib, and are reimbursed by medical insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Pembrolizumab is also available for those patients with MSI-H mCRC but is not reimbursed. To date, the agents aflibercept, panitumumab, ramucirumab, encorafenib, adagrasib, sotorasib, lapatinib, tucatinib, trastuzumab deruxtecan and larotrectinib are still not available to Chinese patients. Patients with mCRC can undergo recommended genetic testing, including for their RAS , BRAF , HER2 , MSI status and for NTRK fusions. Currently, however, medical insurance in China only covers PCR testing for RAS ( KRAS/NRAS ) and BRAF alterations, and MSI testing. Medical insurance also covers immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH) detection of HER2 alterations, and IHC detection of MMR. Next generation sequencing (NGS) testing is not reimbursed by medical insurance. There are different policies in terms of reimbursement across the different provinces depending on province-based approvals.
In Indonesia the universal health care system (UHC) covers most of the health services. Although almost 80% of Indonesians are covered by the UHC, however, amongst them are individuals who also have their own private medical insurance or whose health care is covered by their employer. The use of 5-FU-based chemotherapy (including oxaliplatin or irinotecan regimens) as first-line or second-line therapy is reimbursed, whilst targeted therapies and checkpoint inhibitors are not. Biomarker testing is available but not all tests are reimbursed. New technology-based tests (i.e. NGS) are not reimbursed. Also, after a Health Technology Assessment (HTA), bevacizumab and cetuximab for the treatment of mCRC have been excluded from the UHC scheme. In terms of availability, in Indonesia new drugs/agents firstly have to receive approval from the Indonesian Food and Drug Authority (FDA). Then after 2 years an application can be made for the drug to be included in the national formulary which is a list of medications that are eligible to be given to patients under the UHC scheme. Due to the high burden of health care costs, however, especially for cancer treatment, it can sometimes take years, multiple scientific evaluations and cost effectiveness analyses/HTA, for a new drug to be listed under the national formulary for UHC. Thus, most of the drugs are not available or reimbursed for patients with CRC. Drugs are approved for very specific indications.
Most of the drugs (chemotherapy and biologicals) for the treatment of mCRC discussed in the ESMO guidelines and Section A above are available in India ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Chemotherapy drugs and some of the biologicals are available in the government hospitals with cancer services and hence accessible to most patients at modest or no cost. India has a graded payment/insurance scheme based on patient income, decided by the government. Also, the wide availability of cheaper and good quality generics and biosimilars have improved patient access to better treatment. Drugs not covered by the government centres are covered by private insurance or by the patient as an out-of-pocket payment. The government health scheme covers about 30%-35% of the population, with the remainder taken care of by the private sector. Most tests including NGS are available but are expensive. Limited PCR panels are widely available. Some of the tests are reimbursed by insurance providers, whereas some drug companies offer coupons to subsidise certain tests. Many centres conduct investigator-initiated studies to generate Indian patient data including on biomarkers, facilitating increased patient access to newer therapeutic strategies.
In Japan, all the drugs recommended in the ESMO Clinical Practice Guideline, except for trastuzumab plus lapatinib and trastuzumab deruxtecan are approved and reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). In addition, the triplet combination of cetuximab plus encorafenib plus binimetinib in second or later line, , and trastuzumab plus pertuzumab in third or later line, are approved and reimbursed. All the recommended assays for biomarker analysis (except primary NGS), are approved and reimbursed. Also, repeated plasma-based digital PCR RAS testing for rechallenge of anti-EGFR therapies and NGS for comprehensive genome profiling after standard therapies are also both approved and reimbursed. Patients pay 0%-30% of the total cost for diagnostic/molecular testing and treatment, depending on their age and income.
All drugs and assays discussed above in Section A of this manuscript are available in Korea, except for ramucirumab and trastuzumab deruxtecan, as indicated in , available at https://doi.org/10.1016/j.esmoop.2023.101558 . Some biological agents or ICIs are also available beyond second line for patients with mCRC, but their cost is not reimbursed yet.
In Malaysia there are two types of health care system. The government provides universal health care under the Ministry of Health (MOH) for all Malaysians for a very low fee. Alternatively there is private health care, which is not affiliated with MOH, for those who wish to and are able to pay for it. Targeted drugs are very limited and are often not available widely under the MOH due to cost issues and are therefore generally not reimbursable. Malaysia does not practice a partly funded government health insurance policy for individual citizens. Common systemic chemotherapy regimens are covered but not the newer targeted therapies. Patients therefore often pay privately, often out of their own pocket or using some form of insurance ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Thus, in the private sector in Malaysia, targeted drugs are more readily available as the patients pay using their own money or private insurance. The MOH tries to negotiate with the pharmaceutical companies, to lower their prices, while at the same time considering generic options whenever possible. Health Intervention Technology, a division in the MOH, often discusses cost–benefit ratios when coming to a decision to include certain drugs for use by the MOH.
Most of the drugs enumerated ( and , available at https://doi.org/10.1016/j.esmoop.2023.101558 ) are available in the Philippines, although not reimbursed, resulting in 100% out-of-pocket patient payments. Most laboratory tests and diagnostics are available in the big cities like Manila and in big treatment centres, thus, ‘availability for all patients’ is an issue. Some agents can be obtained for compassionate use from Hong Kong or Singapore. The Philippines comprises >7000 islands with a range of different procurement policies.
In Singapore, ∼90% of Health Sciences Authority-approved cancer treatments are on the Cancer Drug List (CDL), a list of clinically proven and cost-effective cancer treatments. Only indication-specific treatments in the CDL can be claimed under MediSave (MSV), MediShield Life (MSHL) and Integrated Shield Plans (IPs). MSV is a national medical savings scheme that mandates individuals to set aside part of their income to pay for their hospitalisation and outpatient expenses including cancer drugs listed on the CDL. MediShield Life is a basic health insurance plan which helps to pay for large hospital bills and outpatient treatments including selected cancer drugs. IPs include MSHL and private insurance plans that provide additional cover for cancer diagnostics and drugs. Patients receiving government-subsidised care in public health care institutions utilising drugs which are listed under the Standard Drug List and/or covered under the Medication Assistance Fund may receive an up to 75% subsidy in drug costs (based on their monthly per capita household income). Those in need of financial assistance who fulfil the means testing requirements are eligible for government financial subsidies such as Medifund. In Singapore, molecular assays (e.g. IHC, PCR and NGS) are not subsidised or reimbursed ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Indication-specific drugs that are not on the CDL such as lapatinib, pertuzumab and trastuzumab deruxtecan for CRC treatment are not subsidised or reimbursed.
All drugs and tests are available in Taiwan but not always covered by public insurance. The coverage of National Health Insurance in Taiwan is basically ‘ALL or NONE’ and the financial burden is huge and expected to increase further in the era of precision medicine and immuno-oncology. The availability of other medications for the same indication and future budget burden are the most important considerations, as well as the scientific results of pivotal trials, for decisions on reimbursement. This explains the reasons for the relatively limited coverage of expensive biologics (a maximum of 36 weeks reimbursement for bevacizumab), some of the new technology-based tests (i.e. NGS) and new treatments (e.g. ICIs in MSI-H mCRC) on the reimbursement list ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Also, in the case of patients receiving triplet therapy, only two agents are reimbursed with the cost of the third agent having to be paid for.
In Thailand’s health care system, there are three main reimbursement schemes including the Universal Coverage (UC) scheme, the Social Security Scheme (SSS) and the Civil Servant Medical Benefit Scheme (CSMBS). The majority of Thai people were covered by the UC and the SSS. All government employees and their dependents are covered by the CSMBS. For the UC and SSS, the use of oxaliplatin- and irinotecan-based regimens as first-line or second-line therapy are reimbursable. Targeted therapies, ICIs and biomarker testing are not reimbursable. For those under the CSMBS, in addition to chemotherapy, they are able to access targeted therapy and companion biomarker testing using a pre-authorised system. Six months of bevacizumab therapy is eligible for reimbursement for second-line therapy. Panitumumab is the only anti-EGFR agent that is reimbursed for patients with RAS wt advanced CRC, with two indications including first-line therapy with doublet chemotherapy only for patients with potentially resectable advanced CRC, and third-line therapy in combination with single-agent irinotecan. Regorafenib is also reimbursable for patients with chemotherapy-refractory disease ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Otherwise the medical expenses are not reimbursed. The ESMO-MCBSs for the different systemic therapy options and new therapy combinations for the treatment of patients with mCRC are to be found at https://www.esmo.org/guidelines/esmo-mcbs/esmo-mcbs-scorecards?mcbs_score_cards_form%5BsearchText%5D=&mcbs_score_cards_form%5Btumour-sub-type%5D=Colon+and+R .
The results of the voting by the Asian experts both before and after the hybrid virtual/face-to-face working meeting showed >80% concordance with the ESMO recommendations for the treatment of patients with mCRC ( , available at https://doi.org/10.1016/j.esmoop.2023.101558 ). Following the virtual ‘face-to-face’ discussions, revisions were made to the wording of ‘recommendations 1k, 1l (new recommendation), 3e, 3h, 3n, 4k, 4m, 4r, 4s, 4z, 4aa, 4k (new recommendation) and 5a and resulted in a 100% consensus being achieved in terms of acceptability for all the recommendations listed in , which constitute the consensus clinical practice guidelines for the treatment of patients with mCRC in Asia. The variations in the availability for the patients of the testing, drugs and therefore treatment possibilities, between the different countries, reflect the differences in the organisation of their health care systems and their reimbursement strategies and will have a significant impact on the implementation of the scientific recommendations. Thus, policy initiatives are advised in order to improve patient access to state-of-the-art cancer care, adapted for the heterogeneous socioeconomic situations in the different Asian countries.
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Ethnobotany, Biological Activities and Phytochemical Compounds of Some Species of the Genus
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5702afdb-2bb4-4e59-b406-648d64b9cd95
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10221068
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Pharmacology[mh]
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Plants constitute a valuable resource in the health systems of developing countries, and in this sense, the World Health Organization (WHO) has estimated that more than 80% of the world population uses traditional medicine for primary health care, mainly through treatments with plant extracts or their active ingredients . According to the WHO (1979) a medicinal plant is defined as any plant species that contains substances which can be used for therapeutic purposes or whose active ingredients can serve as precursors for the synthesis of new drugs. Therefore, drugs derived from plants have gained relevance both in traditional medicine and in modern medicine . However, Ref. the massive use of medicinal plants is limited by procedures such as the classification, identification and characterization of these and their active principles; therefore, the generation of knowledge about medicinal plants in different latitudes is highly relevant. In Mexico, medicinal plants have been historically important even before colonization of the New World; thus, knowledge of plants and their usefulness as medicine, have passed from generation to generation as part of inherited traditions. There are people who master the empirical knowledge of medicinal plants, while others only sell them unaware of their properties and health effects . The diversity of plants is very high in Mexico, and between 3000 and 5000 species are plants with therapeutic potential and only 1% have been studied in depth to discover their medicinal properties . Among the plants with medicinal properties are those belonging to the genus Eryngium , the largest and most complex of the Apiaceae family. There are 250 species of which 28 are distributed in the central-western region of the country and 21–22 in Michoacán . Therapeutic uses have been reported for some of these Eryngium species mostly in rural communities, and some of them have been studied due to their beneficial properties, mainly to treat diabetes, dyslipidemia, and kidney conditions . Extracts from some Eryngium spp. have been shown to have biological activities and various bioactive compounds . However, only a few species have been investigated to ensure the efficacy and safety of the treatments for which they are prescribed. Consistent with the above information, the main objectives of the present work were to review (1) the traditional uses associated with the Eryngium spp. distributed in the central-western region of the country; (2) the advances in the detection and identification of their bioactive compounds; and (3) the biological activities that reveal their healing potential. Having this information available will allow us to propose future research to contribute to the development of safe products and therapies aimed at treating chronic degenerative diseases, among others. 2.1. The Genus Eryngium Worldwide and in Mexico There are approximately 230–250 species of Eryngium L. distributed in tropical and temperate regions in Eurasia, North Africa, North and South America, and Australia . Except in the tropics and Southern Africa , Eryngium L. is the largest genus and possibly the most taxonomically complex of the Apiaceae family . In his treatise, Wolff (1913) considered Eryngium one of the most complex, and he recognized two groups within the genus: “Species Gerontogeae” representing 12 sections of the Old World (Africa, Europe, and Asia) and “Species Americanae and Australienses” including 22 sections from the New World (America and Australia). According to Wörz (1999) , and in agreement with updated information, there are about 100 South American and 85 North American species. In Mexico, there are around 50–60 species: between 27 and 28 in the central-western region of the country, and 21–22 in the state of Michoacán . 2.2. The Genus Eryngium Worldwide and in Mexico Eryngium L. Creeping to erect, herbaceous, caulescent or acaulescent, these plants are usually glabrous biennials or perennials, which grow from stout taproots or rootstocks bearing fibrous roots. The leaves are coriaceous or membranaceous, entire, or pinnately or palmately lobed to divided, often ciliate to spinose, and the venation parallel or reticulate. The petiole’s sheathing is sometimes septate. It has an inflorescence capitate, with the heads either solitary or in cymes or racemes. It has an involucre of one or more series of entire or lobed bracts subtending the head and an involucel of entire or lobed bractlets subtending the flowers. The flowers are white to purple and sessile; the petals ovate to oblong with variously inflexed and lobed to fimbriate tips; the sepals ovate to lanceolate, acute to obtuse, entire, or rarely spinescent; the styles are shorter and do not exceed the sepals, as they lack a stylopodium; and the carpophore is absent. The fruit are globose to obovoid, scarcely flattened laterally, and variously covered with scales or tubercles; the ribs are obsolete; the commissure is broad; and the five oil-tubes are mostly inconspicuous. The seeds subterete in cross-section, and the face is plane or slightly concave . Worldwide, several species of the genus Eryngium have been used in traditional medicine, especially to treat cholesterol and diabetes problems. In Mexico there are about 50 species, of which about 27 taxa are considered for the central-west region, which includes the states of Michoacán and Jalisco, as well as areas adjacent to the neighboring states: State of Mexico, Guanajuato, and the southern zone of Nayarit . Various properties have been reported for only eight taxa since they are traditionally used as an auxiliary or complement to family health. The species reported in this review fall into the section and subsection at the infrageneric level, according to Wolff (1913) and Calviño et al. (2008) , as “American and Australian species (new world)” as follows . Some characteristics on the biology and distribution of the Eryngium species reported in the central-western region of Mexico are shown in . 2.3. Traditional Uses Many Eryngium species have traditionally been used as ornamental, edible, or medicinal plants . Various Eryngium spp. are used for the treatment of different inflammatory diseases around the world . In Mexico, the use of different Eryngium species in traditional medicine is very important, some of them are distributed in the central-western region . In general, they are known as “hierba del sapo” (toad grass) regardless of the species. Such is the case of Eryngium beecheyanum Hook. F. and Arn., a species mostly preferred by the population of the Purépecha Plateau in Michoacán, Mexico, to treat skin inflammation; consumed orally as an infusion of the aerial part of the plant, it is also applied in fomentation form to the affected area . It has been reported as an antipyretic , and the farmers of the Sierra de Huautla, Morelos, use it to treat renal inflammation . Eryngium carlinae F. Delaroche, also known as “hierba del sapo” or “mosquitas” , whose medicinal properties have been widely studied, is also one of the most used in indigenous communities in central Mexico as well as in Michoacán, Hidalgo, State of Mexico and Querétaro . It is mainly used in the treatment of kidney problems such as cystitis known as “mal de orín” in Puebla and Tlaxcala, kidney pain in Hidalgo, and as a diuretic in the State of Mexico. In general, the whole plant is used with or without the roots and taken orally as an infusion. It is also used to reduce inflammation of the stomach in the State of Mexico, and to treat biliary disease, taken on an empty stomach. In the case of inflammation caused by blows, it is topically applied to the affected area, using hot fomentations, and adding salt to the infusion. E. carlinae is also used to treat inflammatory conditions of the bowel, back pain, pain of the bones, chest, ear, and hernia, and against snake bites and fever . One of the most popular uses is in the treatment of diabetes and dyslipidemias as an infusion taken throughout the day called “agua de uso” . Additional uses have been reported, including as an aphrodisiac, an anticrotalic antidote, antispasmodic, antipodagric, antitumor, carminative, diuretic, expectorant, increased heat in the stomach, cold-caused illnesses, asystole, and gastroenteritis, among others . Eryngium comosum F. Delaroche, is used as an aphrodisiac, antigonorrheal, antipyretic, antipodagric, diuretic and oxytocic , for lipid-lowering, and to treat “mal de orín” (cysititis) related to urinary tract infections and kidney pain . Eryngium longifolium is another of the species used to treat diabetes in Hidalgo, where it is given the name “piñuela”, where the form of use is as an infusion of the dried plant (aerial part) taken throughout the day (“agua de uso”) . In addition, its use as a diuretic, emmenagogue, and alexiteric has been reported , similar to Eryngium fluitans . Another empirically well-known species is Eryngium heterophyllum Engel, also called “toad grass”, which is considered useful for the treatment of diabetes, arthritis, and hypercholesterolemia, among other diseases. The commonly recommended form of use is to prepare the aerial part of the plant in its natural state, pulverized and boiled in water, and the infusion is supplied according to the condition . It has also been reported to be useful for the control of gallstones . Eryngium nasturtiifolium is known to be used locally as a traditional medicine against type 2 diabetes mellitus and “mal de orín” , similar to other Eryngium species. The traditional medicinal uses of Eryngium species reviewed are summarized in . There are approximately 230–250 species of Eryngium L. distributed in tropical and temperate regions in Eurasia, North Africa, North and South America, and Australia . Except in the tropics and Southern Africa , Eryngium L. is the largest genus and possibly the most taxonomically complex of the Apiaceae family . In his treatise, Wolff (1913) considered Eryngium one of the most complex, and he recognized two groups within the genus: “Species Gerontogeae” representing 12 sections of the Old World (Africa, Europe, and Asia) and “Species Americanae and Australienses” including 22 sections from the New World (America and Australia). According to Wörz (1999) , and in agreement with updated information, there are about 100 South American and 85 North American species. In Mexico, there are around 50–60 species: between 27 and 28 in the central-western region of the country, and 21–22 in the state of Michoacán . Eryngium L. Creeping to erect, herbaceous, caulescent or acaulescent, these plants are usually glabrous biennials or perennials, which grow from stout taproots or rootstocks bearing fibrous roots. The leaves are coriaceous or membranaceous, entire, or pinnately or palmately lobed to divided, often ciliate to spinose, and the venation parallel or reticulate. The petiole’s sheathing is sometimes septate. It has an inflorescence capitate, with the heads either solitary or in cymes or racemes. It has an involucre of one or more series of entire or lobed bracts subtending the head and an involucel of entire or lobed bractlets subtending the flowers. The flowers are white to purple and sessile; the petals ovate to oblong with variously inflexed and lobed to fimbriate tips; the sepals ovate to lanceolate, acute to obtuse, entire, or rarely spinescent; the styles are shorter and do not exceed the sepals, as they lack a stylopodium; and the carpophore is absent. The fruit are globose to obovoid, scarcely flattened laterally, and variously covered with scales or tubercles; the ribs are obsolete; the commissure is broad; and the five oil-tubes are mostly inconspicuous. The seeds subterete in cross-section, and the face is plane or slightly concave . Worldwide, several species of the genus Eryngium have been used in traditional medicine, especially to treat cholesterol and diabetes problems. In Mexico there are about 50 species, of which about 27 taxa are considered for the central-west region, which includes the states of Michoacán and Jalisco, as well as areas adjacent to the neighboring states: State of Mexico, Guanajuato, and the southern zone of Nayarit . Various properties have been reported for only eight taxa since they are traditionally used as an auxiliary or complement to family health. The species reported in this review fall into the section and subsection at the infrageneric level, according to Wolff (1913) and Calviño et al. (2008) , as “American and Australian species (new world)” as follows . Some characteristics on the biology and distribution of the Eryngium species reported in the central-western region of Mexico are shown in . L. Creeping to erect, herbaceous, caulescent or acaulescent, these plants are usually glabrous biennials or perennials, which grow from stout taproots or rootstocks bearing fibrous roots. The leaves are coriaceous or membranaceous, entire, or pinnately or palmately lobed to divided, often ciliate to spinose, and the venation parallel or reticulate. The petiole’s sheathing is sometimes septate. It has an inflorescence capitate, with the heads either solitary or in cymes or racemes. It has an involucre of one or more series of entire or lobed bracts subtending the head and an involucel of entire or lobed bractlets subtending the flowers. The flowers are white to purple and sessile; the petals ovate to oblong with variously inflexed and lobed to fimbriate tips; the sepals ovate to lanceolate, acute to obtuse, entire, or rarely spinescent; the styles are shorter and do not exceed the sepals, as they lack a stylopodium; and the carpophore is absent. The fruit are globose to obovoid, scarcely flattened laterally, and variously covered with scales or tubercles; the ribs are obsolete; the commissure is broad; and the five oil-tubes are mostly inconspicuous. The seeds subterete in cross-section, and the face is plane or slightly concave . Worldwide, several species of the genus Eryngium have been used in traditional medicine, especially to treat cholesterol and diabetes problems. In Mexico there are about 50 species, of which about 27 taxa are considered for the central-west region, which includes the states of Michoacán and Jalisco, as well as areas adjacent to the neighboring states: State of Mexico, Guanajuato, and the southern zone of Nayarit . Various properties have been reported for only eight taxa since they are traditionally used as an auxiliary or complement to family health. The species reported in this review fall into the section and subsection at the infrageneric level, according to Wolff (1913) and Calviño et al. (2008) , as “American and Australian species (new world)” as follows . Some characteristics on the biology and distribution of the Eryngium species reported in the central-western region of Mexico are shown in . Many Eryngium species have traditionally been used as ornamental, edible, or medicinal plants . Various Eryngium spp. are used for the treatment of different inflammatory diseases around the world . In Mexico, the use of different Eryngium species in traditional medicine is very important, some of them are distributed in the central-western region . In general, they are known as “hierba del sapo” (toad grass) regardless of the species. Such is the case of Eryngium beecheyanum Hook. F. and Arn., a species mostly preferred by the population of the Purépecha Plateau in Michoacán, Mexico, to treat skin inflammation; consumed orally as an infusion of the aerial part of the plant, it is also applied in fomentation form to the affected area . It has been reported as an antipyretic , and the farmers of the Sierra de Huautla, Morelos, use it to treat renal inflammation . Eryngium carlinae F. Delaroche, also known as “hierba del sapo” or “mosquitas” , whose medicinal properties have been widely studied, is also one of the most used in indigenous communities in central Mexico as well as in Michoacán, Hidalgo, State of Mexico and Querétaro . It is mainly used in the treatment of kidney problems such as cystitis known as “mal de orín” in Puebla and Tlaxcala, kidney pain in Hidalgo, and as a diuretic in the State of Mexico. In general, the whole plant is used with or without the roots and taken orally as an infusion. It is also used to reduce inflammation of the stomach in the State of Mexico, and to treat biliary disease, taken on an empty stomach. In the case of inflammation caused by blows, it is topically applied to the affected area, using hot fomentations, and adding salt to the infusion. E. carlinae is also used to treat inflammatory conditions of the bowel, back pain, pain of the bones, chest, ear, and hernia, and against snake bites and fever . One of the most popular uses is in the treatment of diabetes and dyslipidemias as an infusion taken throughout the day called “agua de uso” . Additional uses have been reported, including as an aphrodisiac, an anticrotalic antidote, antispasmodic, antipodagric, antitumor, carminative, diuretic, expectorant, increased heat in the stomach, cold-caused illnesses, asystole, and gastroenteritis, among others . Eryngium comosum F. Delaroche, is used as an aphrodisiac, antigonorrheal, antipyretic, antipodagric, diuretic and oxytocic , for lipid-lowering, and to treat “mal de orín” (cysititis) related to urinary tract infections and kidney pain . Eryngium longifolium is another of the species used to treat diabetes in Hidalgo, where it is given the name “piñuela”, where the form of use is as an infusion of the dried plant (aerial part) taken throughout the day (“agua de uso”) . In addition, its use as a diuretic, emmenagogue, and alexiteric has been reported , similar to Eryngium fluitans . Another empirically well-known species is Eryngium heterophyllum Engel, also called “toad grass”, which is considered useful for the treatment of diabetes, arthritis, and hypercholesterolemia, among other diseases. The commonly recommended form of use is to prepare the aerial part of the plant in its natural state, pulverized and boiled in water, and the infusion is supplied according to the condition . It has also been reported to be useful for the control of gallstones . Eryngium nasturtiifolium is known to be used locally as a traditional medicine against type 2 diabetes mellitus and “mal de orín” , similar to other Eryngium species. The traditional medicinal uses of Eryngium species reviewed are summarized in . The use of plants in medicines ranges from crude preparations or extracts, to refined extracts and single molecular species. In terms of categories of use, these encompass food supplements, herbal medicines, botanical drugs, and prescription medicines. There is an increasing interest in plants as a source of novel pharmacophores . In this context, pharmacological studies of medicinal plants have been carried out, addressing various extract evaluation strategies in vitro or in vivo, using different extractive solvents or following traditional preparation practices. The plants of the genus Eryngium have not been the exception; thus, the evaluation of the extracts of Eryngium spp. distributed around the world, have shown multiple beneficial effects , such as anti-inflammatory , against snake and scorpion venoms , antibacterial, antioxidant , antihyperglycemic , and cytotoxic against human tumor cell lines , among others. Regarding the Eryngium spp. reported in the central-western region of Mexico, records of its use in traditional medicine were found for eight species, but reports of biological activities were found only for five of these species. 3.1. Eryngium carlinae There has been great interest in learning about its effects on diabetes control; thus, Noriega-Cisneros et al. (2012) investigated the effect of chronic administration of ethanolic extract of E. carlinae on glucose, creatinine, uric acid, total cholesterol, and triglyceride levels in the serum of streptozotocin (STZ)-induced diabetic rats. Treatment with ethanolic extract of E. carlinae prevented the increase in glucose, triglycerides, total cholesterol, and uric acid in serum; it also reduced the levels of creatinine, uric acid, total cholesterol, and triglycerides in healthy rats compared to those with diabetes. Additionally, ethanolic extract significantly decreased glycosylated hemoglobin (HbA1c) in the serum of diabetic rats. The authors concluded that administration of E. carlinae reduced cardiovascular-risk-related hyperlipidemia in diabetes mellitus. Subsequently, Noriega-Cisneros (2013) analyzed the chemical composition of the ethanolic extract of E. carlinae and studied the effect of its consumption in STZ-induced diabetic rats, and its antioxidant activity was assayed. The results showed that ethanolic extract had no hypoglycemic effect when administered orally to diabetic rats (45 mg/kg); however, it did reduce cholesterol and triglyceride levels, improving the lipid profile and reducing the cardiovascular risk index. The in vitro analysis showed antioxidant activity and a considerable amount of flavonoids and phenolic compounds related to it; however, the in vivo analysis did not have a significant effect on lipid peroxidation, and antioxidant enzymatic activity of the superoxide dismutase (SOD) and catalase (CAT) only showed an effect on reducing the nitric oxide levels. Histological analysis of the kidney showed that although the ethanolic extract of E. carlinae did not control hyperglycemia, it may offer benefits on lipid profile and progression of renal damage. Later, Noriega-Cisneros et al. (2020) investigated the mechanism of action of the hypolipidemic effect of the ethanolic extract of E. carlinae , analyzing its composition and lipid-lowering activity. The extract was administered orally to STZ-induced diabetic rats (30 mg/kg) for more than 40 days, and its effect was compared with that of atorvastatin (a drug used to lower cholesterol levels). The analyzed extract reduced total cholesterol and non-high-density lipoprotein cholesterol (C-HDL) levels and increased the C-HDL levels reduced in diabetes, decreasing the atherogenic index and, therefore, the risk of suffering cardiovascular disease risk at the same level as atorvastatin. The results demonstrated the hypolipidemic potential of ethanolic extract of E. carlinae and support its use in traditional medicine as a hypolipidemic agent. On the other hand, García-Cerrillo et al. (2018) demonstrated that the hexanic extract of E. carlinae had in vitro and in vivo antioxidant activity associated with the decrease in glucose and triacylglyceride levels during hyperglycemia and suggested that this effect could reduce the risk of developing diabetic cardiomyopathy. The authors administered hexanic extract of E. carlinae (30 mg/kg) to STZ-induced diabetic rats for seven weeks and found that serum levels of glucose, triacylglycerides, and TBARS (thiobarbituric acid reactive substances) were significantly reduced in diabetic rats supplemented with the extract. Peña-Montes et al. (2019) also evaluated the in vitro antioxidant activity of the hexanic extract of E. carlinae inflorescences in Saccharomyces cerevisiae under stress induced by hydrogen peroxide, and later, they tested the extract in STZ-induced diabetic male Wistar rats. The hexanic extract showed in vitro antioxidant activity at different concentrations compared to ascorbic acid (positive control). Oral administration (30 mg/kg) of the hexanic extract reduced blood glucose levels; lipid peroxidation in the liver, kidney, and brain; protein carbonylation; and reactive oxygen species (ROS) production in normoglycemic and hyperglycemic rats. CAT activity in the brain, kidneys, and liver also increased. These findings showed the antioxidant properties of the hexanic extract of E. carlinae inflorescences. Regarding active metabolites, Castro-Torres et al. (2017) determined the hypocholesterolemic activity of the hydroalcoholic extract of aerial parts of E. carlinae and demonstrated the presence of hexa- O -acetyl- d -mannitol and its acetylated derivatives by gas chromatography coupled with mass spectrometry (GC-MS) analysis. The authors concluded that mannitol promoted osmotic diuresis, which may favor cholesterol transport, preventing it from accumulating in enterocytes and the development of hypercholesterolemia; in this sense, mannitol-based drugs are used to promote diuresis (before irreversible renal failure) and urinary excretion of toxic substances as an antiglaucoma agent, and as an aid in the diagnosis of renal function . In the same way the diuretic effect and the excretion of toxic substances, the renoprotective activity of E. carlinae has been reported by Pérez-Ramírez et al. (2016) . The authors studied the effect of plant decoctions on renal dysfunction in high-fructose and high-fat fed rats. Decoction consumption reduced serum uric acid, urine albumin and urea, and increased creatinine clearance, which was associated with reduced hyperglycemia, renal lipid accumulation, and oxidative stress. These results suggested that E. carlinae could be used as an ingredient of functional beverages with renoprotective effects. The only clinical study of E. carlinae found during this review was reported by Montes-Moreno (2017) . The authors evaluated the effect of consuming aqueous extracts of toad grass on serum triglycerides, body composition, and anthropometric values in adults. A randomized, parallel blind clinical trial was carried out, and anthropometric measurements, body composition, and blood biochemistry were taken. Individuals with triglycerides >150 mg/dL were selected to determine the effect of drinking the aqueous extract on the baseline parameters of the participants after four weeks. The consumption of the extract reduced weight and body fat (approx. 1 kg) and triglycerides and VLDL cholesterol (21%). The results obtained suggested that drinking the infusion at a 1% concentration at least once a day could reduce and/or control high serum triglyceride levels and be an adjuvant in reducing the percentage of body fat and weight. Another reported use of Eryngium spp. is the treatment of cholelithiasis; therefore, Valdivia-Mares (2021) evaluated the effectiveness of a 50% hydroalcoholic extract of E. carlinae to treat cholelithiasis by an in vitro dissolution model using 30 stones formed by ≥70% cholesterol selected from 1597 stones obtained by cholecystectomy. To improve solubility and resemble gallbladder conditions, the test temperature was between 35 and 37 °C, and the extract was renewed every hour for 20 h. Solutions of 50% ethanol and 99% ethyl ether were used as negative and positive controls, respectively. The dissolution rate of the media was estimated as the reduction in the mass of the treated stones (g/mL/h). The extract showed a higher dissolution rate (0.00280–0.00285 g/mL/h) than that shown by ethanol (0.00255 g/mL/h) and six times lower than that shown by ethyl ether (0.00715 g/mL/h). The authors suggested that these results could contribute to the development of a safer, cheaper, and less invasive therapy, such as a product containing E. carlinae . Another of the benefits attributed to E. carlinae is its antispasmodic activity, which was confirmed in vivo by Pérez-Gutiérrez et al. (2006) . This activity was attributed to the presence of two γ-lactones from the methanol fraction isolated and characterized by the authors. Regarding the antimicrobial activity of E. carlinae , tests performed in vitro have not shown significant growth inhibition of human pathogenic bacteria . On the other hand, Galindo-Hernández (2018) evaluated the antifungal activity of the acetonic extract of E. carlinae against Candida spp. strains isolated from pediatric dental patients. The extract did not show strong antimicrobial activity against C. albicans (ATCC 90029). In contrast to the above antimicrobial studies, Espino-Garibay (2010) evaluated the antimicrobial effect of E. carlinae metabolites, identifying 21 metabolites in ethanolic extracts (leaves, peduncles, and flowers) by GC-MS. Regarding the volatile compounds, germacrene showed antifungal activity against Colletotrichum lindemuthianum (49.6%) and Botrytis cinerea (39.1%). The highest antifungal activity against C. lindemuthianum (almost 100%) was shown by spathulenol (50 mg/mL) and piperitone oxide (500 mg/mL). While spathulenol, piperitone oxide, and menthol (100 mg/mL) exerted a less inhibitory effect against B. cinerea (37.8%), only piperitone oxide (250 mg/mL) had an inhibitory effect against Fusarioum oxysporum (28.8%). On the other hand, the antimicrobial activity of E. carlinae terpenoids was lower; thus, pulegone and borneol, with a dose of 500 mg/mL, inhibited the oomycete Phytophthora cinammomis 32.1 and 30.8%, respectively. Meanwhile, spathulenol (500 mg/mL) and myrcene (250 mg/mL) exerted an inhibitory effect of 18.1 and 15.3%, respectively. The crude extracts showed higher activity against P. cinnamomi (34%). 3.2. Eryngium comosum There are few scientific reports on the biological activities of Eryngium comosum , Delaroche F.; for example, the work of Ronquillo de Jesús (2013) who determined the antioxidant activity of ethanolic, aqueous, hexanic, and ether of petroleum extracts of E. comosum using the DPPH assay. In addition, the extracts cytotoxicity was assayed in vitro in peripheral blood mononuclear cells and in vivo in Artemia salina . Ethanolic and aqueous extracts at a concentration of 1000 ppm showed IC50 values of 4.93 µg/mL and 49.52 µg/mL, respectively. None of the extracts showed toxicity in mononuclear cells, while the extract with petroleum ether did show a cytotoxic effect in A. salina (IC50 2.92 ppm). The antimicrobial activity has also been studied in E. comosum , in addition to the antioxidant activity. Díaz-Alvarado et al. (2020) evaluated the antibacterial activity by the disk diffusion method (DDM), using reference strains of equine pathogenic bacteria: Listeria monocytogenes ATCC 19115, Staphylococcus sp., Escherichia coli ATCC 25922, and Salmonella enterica serotype Enteritidis ATCC 13076. Ethanolic extract of E. comosum (50%) prepared with dried tissue (125 mg/mL) inhibited the growth of Staphylococcus sp., S. enterica , and L. monocytogenes , showing a greater effect on the latter strain. The results suggested the extract of E. comosum as a source of antimicrobial agents to treat equine infections, although further in vitro and in vivo research is required to achieve its application. In the same way, Díaz-Alvarado (2020) analyzed the bioactive compounds in aqueous and ethanolic extracts (50 and 70%) of E. comosum , and assayed antioxidant capacity and antimicrobial activity in 50% ethanolic extracts of this medicinal plant. The 50% ethanolic extract of E. comosum showed antioxidant capacity (1973.42 μM ETCA/g) and antibacterial activity against Enterococcus sp. and Salmonella sp. (inhibition zone diameter = 11.3 mm). Regarding in vivo studies, Pérez-Reyes (2016) reported that the aqueous extract of E. comosum reduced cholesterol and triglyceride levels in rats with dyslipidemia, induced with a hypercholesterolemic and hypertriglyceridemic diet. The extract was administered intragastrically for 3 weeks, testing three doses: 100, 200, and 400 mg/kg. After the treatment, the influence of the aqueous extract on the weight of adipose and muscle tissues was observed; however, a body weight reduction was not reported. On the other hand, the decrease in serum cholesterol levels was recorded at a dose of 100 mg/kg, with a serum concentration of 189.45 mg/dL in the control group and 99.16 mg/dL in the treated group; while the serum concentration of triglycerides decreased only with the 200 mg/kg dose, being 403.1 mg/dL in the control group and 337.8 mg/dL in the treated group. 3.3. Eryngium cymosum One of the most widespread traditional uses of Eryngium ssp. is the treatment of type 2 diabetes (T2D), and there are some reports about its hypoglycemic effect. For example, the study carried out by Espinoza-Hernández et al. (2021) , in which the aqueous extract of aerial parts of the E. cymosum plant was administered via gavage to Wistar rats with streptozotocin-nicotinamide-induced hypoglycemia (STZ-NA). The authors reported the antihyperglycemic effect of the extract in the pyruvate tolerance test and the significant reduction of postprandial hyperglycemia in the maltose tolerance tests. As the main mechanism of action, the extract suppressed gluconeogenesis by inhibition (almost 100%) of the enzymes glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase), which is the altered pathway that causes fasting and postprandial hyperglycemia in patients with T2D; the extract also reduced the activity of a-glucosidases by 32%. In addition, it decreased insulin levels when it was administered orally in healthy rats in both nutritional states, without affecting normoglycemia in normal curves and reducing the postprandial peak in glucose load curves. The authors concluded that the traditional form of consumption of E. cymosum is safe and regulates glucose levels both fasting and in the postprandial state. Subsequently, the same research group published a study that evaluated the chronic effects of traditional extracts on hyperglycemia and hypertriglyceridemia of some Mexican medicinal plants, including E. cymosum . The aqueous extract was administered via gavage to hyperglycemic STZ-NA Wistar rats, daily for 42 days. For the preparation of the extract, 20 g of dried and ground plant material (aerial parts) were added to 500 mL of boiling, distilled water for 15 min. Non-fasting blood glucose (NFBG), HbA1c, and blood triglycerides were determined. The authors confirmed the long-term efficacy of the extract, as E. cymosum prevented the worsening of hyperglycemia by avoiding the significant increase in glucose levels shown by the negative control group and the increase in HbA1c (2.98%). Despite its antihyperglycemic effects, the extract was less effective in controlling triglycerides. The authors generated evidence of the antihyperglycemic effect of this Mexican medicinal plant, as well as its long-term efficacy in the control of T2D. Research to reveal the mechanisms of action of the hypoglycemic effect of E. cymosum has led to the description of a new metabolite, acylated flavonol, and the isolation of known compounds both in aqueous extract and butanolic extract , whose chemical structures were elucidated using spectroscopic techniques, as described in the following section. Additionally, the role of the acylated flavonol glucoside on the inhibition of G6Pase and FBPase has been demonstrated. 3.4. Eryngium heterophyllum Some studies have been carried out in E. heterophyllum to confirm its anti-inflammatory, hypoglycemic, and hypocholesterolemic activity. Navarrete et al. (1990) reported the decrease in rat serum cholesterol when the aqueous extract of E. heterophyllum was administered orally. For his part, Miranda-Velásquez (2010) tested the hypocholesterolemic activity of crude extracts dissolved in water or in a Tween 80/saline solution at two doses of 50 and 100 mg/kg of weight administered to hypercholesterolemic mice for five days, which at the end of this period were fasted for 12 h. The results showed that only the aqueous extracts of E. heterophyllum at 100 mg/kg showed a cholesterol reduction (20.7%). Therefore, this extract was subsequently evaluated in vitro using Vero cells to determine the inhibitory effect of the HMG-CoA enzyme. The results showed that indeed the mechanism of serum cholesterol reduction was related to the inhibition of said enzyme, as in the case of statin drugs. In the same way, García-Gómez et al. (2019) , in a 1-month clinical study, showed that combined treatment of E. heterophyllum and Amphipterygium adstringens with proven hypocholesterolemic activity tested in rats, reduced triglyceride levels by an average of 20%. On the other hand, Carreón-Sánchez et al. (2013) showed that the ethanolic extract of E. heterophyllum , after being administered to mice by oral gavage in a single dose of 100 mg/kg of weight in a volume of 0.2 mL/30 g, had no hypoglycemic effect or acute or chronic anti-inflammatory effect; nor did it cause visible toxic effects in the acute poisoning model in mice. Molina-Garza et al. (2014) conducted a study to screen the trypanocidal activity of the Eryngium heterophyllum plants used in traditional Mexican medicine for the treatment of various diseases related to parasitic infections. Cultured Trypanosoma cruzi epimastigotes were incubated for 96 h with different concentrations of methanolic extract of E. heterophyllum , and the inhibitory concentration (IC50) was determined. The methanolic extracts exhibited the highest trypanocidal activity (88–100%) at a concentration of 150 µg/mL. 3.5. Eryngium longifolium A dose-independent hypoglycemic effect has been reported for E. longifolium . Andrade-Cetto et al. (2021) evaluated aqueous (30 and 310 mg/kg doses) and ethanolic (32 and 318 mg/kg doses) extracts of the aerial parts of the plant in hyperglycemic STZ-NA Wistar rats. Previously, the authors determined the basic phytochemical profiles (see next section) and acute toxicity tests, which did not show any physical problems or behavioral changes after oral administration of the maximum dose of 2000 mg/kg body weight (b.w.) of each extract; no deaths were reported and the LD50 was higher than the maximum dose used. In addition, they tested the inhibition of the G6Pase and FBPase enzymes involved in glucose metabolism. This study validated for the first time the traditional use of the aerial part of E. longifolium as a hypoglycemic agent in a hyperglycemic animal model; the results indicated that the in vitro inhibition of G6Pase and FBPase could be associated with the hypoglycemic effect in vivo. Therefore, the authors concluded that the ability to regulate hyperglycemia could involve inhibition of hepatic glucose production, which primarily controls fasting glucose levels, and that the doses traditionally consumed did not generate toxic effects. According to the previous information, the main potential of the Mexican species of Eryngium to promote health, is related to lipid metabolism, which has been proven by the capacity of its extracts to decrease cholesterol, triglycerides, and body fat levels; this has also been reported for other species of Eryngium . It is important to highlight the potential of the aqueous extracts of E. cymosum and E. longifolium for the control of diabetes, as reported for E. foetidum and E. billardieri . The information found about hypoglycemic activity shows the differences between the biological and pharmacological activity that different Mexican species of Eryngium show; in addition, such differences could be associated with the type of extract evaluated since the content and nature of the active ingredients will also vary. For instance, the acetonic and methanolic extracts of E. foetidum did not show antibacterial activity against Escherichia coli , Salmonella infantis , Listeria monocytogenes , Staphylococcus aureus , or Bacillus cereus , while the essential oil of E. maritimum showed a significant antibacterial activity against L. monocytogenes and E.coli due to its content of oxygenated sesquiterpenes , and the leaf hydromethanolic extract of E. maritimun showed antimicrobial activity against S. aureus , B. cereus , Salmonella enterica , Pseudomonas aeruginosa , P. fluorescens , P. marginalis , E. coli , and Erwinia carotovora subsp. carotovora . Although many species of Eryngium have shown antimicrobial activity against Gram-positive and Gram-negative bacteria, some species of fungi and yeasts, and viruses, it has been suggested that multi-target antimicrobial experiments should be carried out using extracts of Eymgium spp. as antimicrobial agents in order to expand the knowledge about its antimicrobial potential . Additionally, it is important to point out that the study of the anticholelithiasis and trypanocidal activity shown by E. carlinae and E. heterophyllum , respectively, could extend to other species of Eryngium ; likewise, other activities such as anticlastogenic, anticarcinogenic, antihelmintic, and larvicidal, amongst others reported for E. foetidum could be evaluated . The biological activities reported for E. carlinae , E. comosum , E. cymosum , E. heterophyllum , and E. longifolium are summarized in . There has been great interest in learning about its effects on diabetes control; thus, Noriega-Cisneros et al. (2012) investigated the effect of chronic administration of ethanolic extract of E. carlinae on glucose, creatinine, uric acid, total cholesterol, and triglyceride levels in the serum of streptozotocin (STZ)-induced diabetic rats. Treatment with ethanolic extract of E. carlinae prevented the increase in glucose, triglycerides, total cholesterol, and uric acid in serum; it also reduced the levels of creatinine, uric acid, total cholesterol, and triglycerides in healthy rats compared to those with diabetes. Additionally, ethanolic extract significantly decreased glycosylated hemoglobin (HbA1c) in the serum of diabetic rats. The authors concluded that administration of E. carlinae reduced cardiovascular-risk-related hyperlipidemia in diabetes mellitus. Subsequently, Noriega-Cisneros (2013) analyzed the chemical composition of the ethanolic extract of E. carlinae and studied the effect of its consumption in STZ-induced diabetic rats, and its antioxidant activity was assayed. The results showed that ethanolic extract had no hypoglycemic effect when administered orally to diabetic rats (45 mg/kg); however, it did reduce cholesterol and triglyceride levels, improving the lipid profile and reducing the cardiovascular risk index. The in vitro analysis showed antioxidant activity and a considerable amount of flavonoids and phenolic compounds related to it; however, the in vivo analysis did not have a significant effect on lipid peroxidation, and antioxidant enzymatic activity of the superoxide dismutase (SOD) and catalase (CAT) only showed an effect on reducing the nitric oxide levels. Histological analysis of the kidney showed that although the ethanolic extract of E. carlinae did not control hyperglycemia, it may offer benefits on lipid profile and progression of renal damage. Later, Noriega-Cisneros et al. (2020) investigated the mechanism of action of the hypolipidemic effect of the ethanolic extract of E. carlinae , analyzing its composition and lipid-lowering activity. The extract was administered orally to STZ-induced diabetic rats (30 mg/kg) for more than 40 days, and its effect was compared with that of atorvastatin (a drug used to lower cholesterol levels). The analyzed extract reduced total cholesterol and non-high-density lipoprotein cholesterol (C-HDL) levels and increased the C-HDL levels reduced in diabetes, decreasing the atherogenic index and, therefore, the risk of suffering cardiovascular disease risk at the same level as atorvastatin. The results demonstrated the hypolipidemic potential of ethanolic extract of E. carlinae and support its use in traditional medicine as a hypolipidemic agent. On the other hand, García-Cerrillo et al. (2018) demonstrated that the hexanic extract of E. carlinae had in vitro and in vivo antioxidant activity associated with the decrease in glucose and triacylglyceride levels during hyperglycemia and suggested that this effect could reduce the risk of developing diabetic cardiomyopathy. The authors administered hexanic extract of E. carlinae (30 mg/kg) to STZ-induced diabetic rats for seven weeks and found that serum levels of glucose, triacylglycerides, and TBARS (thiobarbituric acid reactive substances) were significantly reduced in diabetic rats supplemented with the extract. Peña-Montes et al. (2019) also evaluated the in vitro antioxidant activity of the hexanic extract of E. carlinae inflorescences in Saccharomyces cerevisiae under stress induced by hydrogen peroxide, and later, they tested the extract in STZ-induced diabetic male Wistar rats. The hexanic extract showed in vitro antioxidant activity at different concentrations compared to ascorbic acid (positive control). Oral administration (30 mg/kg) of the hexanic extract reduced blood glucose levels; lipid peroxidation in the liver, kidney, and brain; protein carbonylation; and reactive oxygen species (ROS) production in normoglycemic and hyperglycemic rats. CAT activity in the brain, kidneys, and liver also increased. These findings showed the antioxidant properties of the hexanic extract of E. carlinae inflorescences. Regarding active metabolites, Castro-Torres et al. (2017) determined the hypocholesterolemic activity of the hydroalcoholic extract of aerial parts of E. carlinae and demonstrated the presence of hexa- O -acetyl- d -mannitol and its acetylated derivatives by gas chromatography coupled with mass spectrometry (GC-MS) analysis. The authors concluded that mannitol promoted osmotic diuresis, which may favor cholesterol transport, preventing it from accumulating in enterocytes and the development of hypercholesterolemia; in this sense, mannitol-based drugs are used to promote diuresis (before irreversible renal failure) and urinary excretion of toxic substances as an antiglaucoma agent, and as an aid in the diagnosis of renal function . In the same way the diuretic effect and the excretion of toxic substances, the renoprotective activity of E. carlinae has been reported by Pérez-Ramírez et al. (2016) . The authors studied the effect of plant decoctions on renal dysfunction in high-fructose and high-fat fed rats. Decoction consumption reduced serum uric acid, urine albumin and urea, and increased creatinine clearance, which was associated with reduced hyperglycemia, renal lipid accumulation, and oxidative stress. These results suggested that E. carlinae could be used as an ingredient of functional beverages with renoprotective effects. The only clinical study of E. carlinae found during this review was reported by Montes-Moreno (2017) . The authors evaluated the effect of consuming aqueous extracts of toad grass on serum triglycerides, body composition, and anthropometric values in adults. A randomized, parallel blind clinical trial was carried out, and anthropometric measurements, body composition, and blood biochemistry were taken. Individuals with triglycerides >150 mg/dL were selected to determine the effect of drinking the aqueous extract on the baseline parameters of the participants after four weeks. The consumption of the extract reduced weight and body fat (approx. 1 kg) and triglycerides and VLDL cholesterol (21%). The results obtained suggested that drinking the infusion at a 1% concentration at least once a day could reduce and/or control high serum triglyceride levels and be an adjuvant in reducing the percentage of body fat and weight. Another reported use of Eryngium spp. is the treatment of cholelithiasis; therefore, Valdivia-Mares (2021) evaluated the effectiveness of a 50% hydroalcoholic extract of E. carlinae to treat cholelithiasis by an in vitro dissolution model using 30 stones formed by ≥70% cholesterol selected from 1597 stones obtained by cholecystectomy. To improve solubility and resemble gallbladder conditions, the test temperature was between 35 and 37 °C, and the extract was renewed every hour for 20 h. Solutions of 50% ethanol and 99% ethyl ether were used as negative and positive controls, respectively. The dissolution rate of the media was estimated as the reduction in the mass of the treated stones (g/mL/h). The extract showed a higher dissolution rate (0.00280–0.00285 g/mL/h) than that shown by ethanol (0.00255 g/mL/h) and six times lower than that shown by ethyl ether (0.00715 g/mL/h). The authors suggested that these results could contribute to the development of a safer, cheaper, and less invasive therapy, such as a product containing E. carlinae . Another of the benefits attributed to E. carlinae is its antispasmodic activity, which was confirmed in vivo by Pérez-Gutiérrez et al. (2006) . This activity was attributed to the presence of two γ-lactones from the methanol fraction isolated and characterized by the authors. Regarding the antimicrobial activity of E. carlinae , tests performed in vitro have not shown significant growth inhibition of human pathogenic bacteria . On the other hand, Galindo-Hernández (2018) evaluated the antifungal activity of the acetonic extract of E. carlinae against Candida spp. strains isolated from pediatric dental patients. The extract did not show strong antimicrobial activity against C. albicans (ATCC 90029). In contrast to the above antimicrobial studies, Espino-Garibay (2010) evaluated the antimicrobial effect of E. carlinae metabolites, identifying 21 metabolites in ethanolic extracts (leaves, peduncles, and flowers) by GC-MS. Regarding the volatile compounds, germacrene showed antifungal activity against Colletotrichum lindemuthianum (49.6%) and Botrytis cinerea (39.1%). The highest antifungal activity against C. lindemuthianum (almost 100%) was shown by spathulenol (50 mg/mL) and piperitone oxide (500 mg/mL). While spathulenol, piperitone oxide, and menthol (100 mg/mL) exerted a less inhibitory effect against B. cinerea (37.8%), only piperitone oxide (250 mg/mL) had an inhibitory effect against Fusarioum oxysporum (28.8%). On the other hand, the antimicrobial activity of E. carlinae terpenoids was lower; thus, pulegone and borneol, with a dose of 500 mg/mL, inhibited the oomycete Phytophthora cinammomis 32.1 and 30.8%, respectively. Meanwhile, spathulenol (500 mg/mL) and myrcene (250 mg/mL) exerted an inhibitory effect of 18.1 and 15.3%, respectively. The crude extracts showed higher activity against P. cinnamomi (34%). There are few scientific reports on the biological activities of Eryngium comosum , Delaroche F.; for example, the work of Ronquillo de Jesús (2013) who determined the antioxidant activity of ethanolic, aqueous, hexanic, and ether of petroleum extracts of E. comosum using the DPPH assay. In addition, the extracts cytotoxicity was assayed in vitro in peripheral blood mononuclear cells and in vivo in Artemia salina . Ethanolic and aqueous extracts at a concentration of 1000 ppm showed IC50 values of 4.93 µg/mL and 49.52 µg/mL, respectively. None of the extracts showed toxicity in mononuclear cells, while the extract with petroleum ether did show a cytotoxic effect in A. salina (IC50 2.92 ppm). The antimicrobial activity has also been studied in E. comosum , in addition to the antioxidant activity. Díaz-Alvarado et al. (2020) evaluated the antibacterial activity by the disk diffusion method (DDM), using reference strains of equine pathogenic bacteria: Listeria monocytogenes ATCC 19115, Staphylococcus sp., Escherichia coli ATCC 25922, and Salmonella enterica serotype Enteritidis ATCC 13076. Ethanolic extract of E. comosum (50%) prepared with dried tissue (125 mg/mL) inhibited the growth of Staphylococcus sp., S. enterica , and L. monocytogenes , showing a greater effect on the latter strain. The results suggested the extract of E. comosum as a source of antimicrobial agents to treat equine infections, although further in vitro and in vivo research is required to achieve its application. In the same way, Díaz-Alvarado (2020) analyzed the bioactive compounds in aqueous and ethanolic extracts (50 and 70%) of E. comosum , and assayed antioxidant capacity and antimicrobial activity in 50% ethanolic extracts of this medicinal plant. The 50% ethanolic extract of E. comosum showed antioxidant capacity (1973.42 μM ETCA/g) and antibacterial activity against Enterococcus sp. and Salmonella sp. (inhibition zone diameter = 11.3 mm). Regarding in vivo studies, Pérez-Reyes (2016) reported that the aqueous extract of E. comosum reduced cholesterol and triglyceride levels in rats with dyslipidemia, induced with a hypercholesterolemic and hypertriglyceridemic diet. The extract was administered intragastrically for 3 weeks, testing three doses: 100, 200, and 400 mg/kg. After the treatment, the influence of the aqueous extract on the weight of adipose and muscle tissues was observed; however, a body weight reduction was not reported. On the other hand, the decrease in serum cholesterol levels was recorded at a dose of 100 mg/kg, with a serum concentration of 189.45 mg/dL in the control group and 99.16 mg/dL in the treated group; while the serum concentration of triglycerides decreased only with the 200 mg/kg dose, being 403.1 mg/dL in the control group and 337.8 mg/dL in the treated group. One of the most widespread traditional uses of Eryngium ssp. is the treatment of type 2 diabetes (T2D), and there are some reports about its hypoglycemic effect. For example, the study carried out by Espinoza-Hernández et al. (2021) , in which the aqueous extract of aerial parts of the E. cymosum plant was administered via gavage to Wistar rats with streptozotocin-nicotinamide-induced hypoglycemia (STZ-NA). The authors reported the antihyperglycemic effect of the extract in the pyruvate tolerance test and the significant reduction of postprandial hyperglycemia in the maltose tolerance tests. As the main mechanism of action, the extract suppressed gluconeogenesis by inhibition (almost 100%) of the enzymes glucose-6-phosphatase (G6Pase) and fructose-1,6-bisphosphatase (FBPase), which is the altered pathway that causes fasting and postprandial hyperglycemia in patients with T2D; the extract also reduced the activity of a-glucosidases by 32%. In addition, it decreased insulin levels when it was administered orally in healthy rats in both nutritional states, without affecting normoglycemia in normal curves and reducing the postprandial peak in glucose load curves. The authors concluded that the traditional form of consumption of E. cymosum is safe and regulates glucose levels both fasting and in the postprandial state. Subsequently, the same research group published a study that evaluated the chronic effects of traditional extracts on hyperglycemia and hypertriglyceridemia of some Mexican medicinal plants, including E. cymosum . The aqueous extract was administered via gavage to hyperglycemic STZ-NA Wistar rats, daily for 42 days. For the preparation of the extract, 20 g of dried and ground plant material (aerial parts) were added to 500 mL of boiling, distilled water for 15 min. Non-fasting blood glucose (NFBG), HbA1c, and blood triglycerides were determined. The authors confirmed the long-term efficacy of the extract, as E. cymosum prevented the worsening of hyperglycemia by avoiding the significant increase in glucose levels shown by the negative control group and the increase in HbA1c (2.98%). Despite its antihyperglycemic effects, the extract was less effective in controlling triglycerides. The authors generated evidence of the antihyperglycemic effect of this Mexican medicinal plant, as well as its long-term efficacy in the control of T2D. Research to reveal the mechanisms of action of the hypoglycemic effect of E. cymosum has led to the description of a new metabolite, acylated flavonol, and the isolation of known compounds both in aqueous extract and butanolic extract , whose chemical structures were elucidated using spectroscopic techniques, as described in the following section. Additionally, the role of the acylated flavonol glucoside on the inhibition of G6Pase and FBPase has been demonstrated. Some studies have been carried out in E. heterophyllum to confirm its anti-inflammatory, hypoglycemic, and hypocholesterolemic activity. Navarrete et al. (1990) reported the decrease in rat serum cholesterol when the aqueous extract of E. heterophyllum was administered orally. For his part, Miranda-Velásquez (2010) tested the hypocholesterolemic activity of crude extracts dissolved in water or in a Tween 80/saline solution at two doses of 50 and 100 mg/kg of weight administered to hypercholesterolemic mice for five days, which at the end of this period were fasted for 12 h. The results showed that only the aqueous extracts of E. heterophyllum at 100 mg/kg showed a cholesterol reduction (20.7%). Therefore, this extract was subsequently evaluated in vitro using Vero cells to determine the inhibitory effect of the HMG-CoA enzyme. The results showed that indeed the mechanism of serum cholesterol reduction was related to the inhibition of said enzyme, as in the case of statin drugs. In the same way, García-Gómez et al. (2019) , in a 1-month clinical study, showed that combined treatment of E. heterophyllum and Amphipterygium adstringens with proven hypocholesterolemic activity tested in rats, reduced triglyceride levels by an average of 20%. On the other hand, Carreón-Sánchez et al. (2013) showed that the ethanolic extract of E. heterophyllum , after being administered to mice by oral gavage in a single dose of 100 mg/kg of weight in a volume of 0.2 mL/30 g, had no hypoglycemic effect or acute or chronic anti-inflammatory effect; nor did it cause visible toxic effects in the acute poisoning model in mice. Molina-Garza et al. (2014) conducted a study to screen the trypanocidal activity of the Eryngium heterophyllum plants used in traditional Mexican medicine for the treatment of various diseases related to parasitic infections. Cultured Trypanosoma cruzi epimastigotes were incubated for 96 h with different concentrations of methanolic extract of E. heterophyllum , and the inhibitory concentration (IC50) was determined. The methanolic extracts exhibited the highest trypanocidal activity (88–100%) at a concentration of 150 µg/mL. A dose-independent hypoglycemic effect has been reported for E. longifolium . Andrade-Cetto et al. (2021) evaluated aqueous (30 and 310 mg/kg doses) and ethanolic (32 and 318 mg/kg doses) extracts of the aerial parts of the plant in hyperglycemic STZ-NA Wistar rats. Previously, the authors determined the basic phytochemical profiles (see next section) and acute toxicity tests, which did not show any physical problems or behavioral changes after oral administration of the maximum dose of 2000 mg/kg body weight (b.w.) of each extract; no deaths were reported and the LD50 was higher than the maximum dose used. In addition, they tested the inhibition of the G6Pase and FBPase enzymes involved in glucose metabolism. This study validated for the first time the traditional use of the aerial part of E. longifolium as a hypoglycemic agent in a hyperglycemic animal model; the results indicated that the in vitro inhibition of G6Pase and FBPase could be associated with the hypoglycemic effect in vivo. Therefore, the authors concluded that the ability to regulate hyperglycemia could involve inhibition of hepatic glucose production, which primarily controls fasting glucose levels, and that the doses traditionally consumed did not generate toxic effects. According to the previous information, the main potential of the Mexican species of Eryngium to promote health, is related to lipid metabolism, which has been proven by the capacity of its extracts to decrease cholesterol, triglycerides, and body fat levels; this has also been reported for other species of Eryngium . It is important to highlight the potential of the aqueous extracts of E. cymosum and E. longifolium for the control of diabetes, as reported for E. foetidum and E. billardieri . The information found about hypoglycemic activity shows the differences between the biological and pharmacological activity that different Mexican species of Eryngium show; in addition, such differences could be associated with the type of extract evaluated since the content and nature of the active ingredients will also vary. For instance, the acetonic and methanolic extracts of E. foetidum did not show antibacterial activity against Escherichia coli , Salmonella infantis , Listeria monocytogenes , Staphylococcus aureus , or Bacillus cereus , while the essential oil of E. maritimum showed a significant antibacterial activity against L. monocytogenes and E.coli due to its content of oxygenated sesquiterpenes , and the leaf hydromethanolic extract of E. maritimun showed antimicrobial activity against S. aureus , B. cereus , Salmonella enterica , Pseudomonas aeruginosa , P. fluorescens , P. marginalis , E. coli , and Erwinia carotovora subsp. carotovora . Although many species of Eryngium have shown antimicrobial activity against Gram-positive and Gram-negative bacteria, some species of fungi and yeasts, and viruses, it has been suggested that multi-target antimicrobial experiments should be carried out using extracts of Eymgium spp. as antimicrobial agents in order to expand the knowledge about its antimicrobial potential . Additionally, it is important to point out that the study of the anticholelithiasis and trypanocidal activity shown by E. carlinae and E. heterophyllum , respectively, could extend to other species of Eryngium ; likewise, other activities such as anticlastogenic, anticarcinogenic, antihelmintic, and larvicidal, amongst others reported for E. foetidum could be evaluated . The biological activities reported for E. carlinae , E. comosum , E. cymosum , E. heterophyllum , and E. longifolium are summarized in . 4.1. Screening, Detection, and Identification of Metabolites Phytochemical characterization is valuable to reveal the presence and identity of secondary metabolites in a plant as well as a helpful tool in the search for bioactive compounds useful for the synthesis of new drugs and other products. The presence of various groups of metabolites has been described in plant extracts (ethanolic, aqueous, methanolic, and hexane) of aerial parts (stems, leaves, and inflorescences) qualitatively analyzed to determine the presence of flavonoids, tannins, terpenoids, and saponins. Additionally, more precise techniques such as GC-MS, nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC) are performed to detect, quantify, and identify metabolites in plant extracts. Regarding the genus Eryngium , investigations have been carried out on the phytochemical profile of some Eryngium spp. found in the central-western region of México, mainly E. carlinae , E. comosum , E. cymosum , and E. longifolium . The most widely used methods to determine the general phytochemical composition of a plant are screening and the colorimetric techniques of ultraviolet-visible spectrophotometry (UV-Vis). Phytochemical screening involves a series of chemical reactions that make it possible to qualitatively detect the groups of secondary metabolites present in plant extracts . For example, E. carlinae , one of the most common species used along the region delimited for this review, was qualitatively analyzed by Knauth et al. (2018) to determine the main groups of metabolites in methanolic extract from the aerial part of the plant. The authors reported a strong presence of tannins and saponins, as well as a slight presence of flavonoids. Galindo-Hernández (2018) evaluated the phytochemical profile of E. carlinae , confirming the presence of triterpenoids, sterols, tannins, coumarins, carboxyls, flavonoids, and carbohydrates. In the same way, Pérez-Reyes (2016) performed phytochemical screening of an aqueous extract of E. comosum , reporting the presence of alkaloids, flavonoids (flavones and xanthones), triterpenoid saponins, reducing sugars, tannins derived from catechol, phenolic compounds, and benzoquinones; in addition, the author reported in vivo hypolipidemic activity. Using UV-Vis, it is possible to determine compound concentration in a solution; this is a simple, reliable and low-cost analytical technique . In this sense, Lemus-de la Cruz-Hurtado et al. (2023) determined and quantified total phenolic compounds (TPC), total flavonoids (TF), and total terpenoids (TT) in aqueous extracts of aerial parts of E. carlinae . The authors reported concentrations of 0.0038 mg gallic acid equivalents per mL (mg GAE/mL) for TPC, 3.3032 mg quercetin equivalents per mL (mg QE/mL) for TF, and 0.0424 mg linalool equivalents per mL (mg LE/mL) for TT. Montes-Moreno (2017) reported higher TPC values in aqueous infusions and decoctions of the same species. The authors evaluated by UV-Vis, up to 2.5 mg GAE/mL of TPC and, in the case of TF, the author reported less than 0.2 mg QE/mL. Similarly, Díaz-Alvarado et al. (2020) reported the total phenolic content (4.33 mg GAE/g dry weight) and total saponins (62.2 mg/g dry weight) of the ethanolic extract of aerial parts of E. comosum . In the same sense, Díaz-Alvarado (2020) determined the phytochemical profile of ethanolic extracts (50 and 70%) of aerial parts of E. comosum by UV-Vis, confirming the presence and abundance of phenolic compounds (up to 4.3 mg GAE/mL), TPC (up to 13.33 mg/g DW), and total saponins (up to 29.33 mg/g DW), with 70% ethanol being the best extractor. Within the phenols group, phenolic acids (caffeic, rosmarinic, and chlorogenic) and their derivatives are the most abundant in the genus Eryngium ; these compounds have been analyzed and identified by more precise techniques, such as chromatography . Andrade-Cetto et al. (2021) described the chromatographic profile of the ethanolic extract of aerial parts of E. longifolium analyzed by HPLC, the authors detected three signals at wavelengths of 254 and 320 nm with retention time (RT) between 6 and 13 min, which were identified as caffeic, chlorogenic, and rosmarinic acids by comparing them to their commercial standards, rosmarinic acid being the most abundant in the analyzed extract. Likewise, the authors reported other minor intensity peaks with RT between 16 and 24 min, which were attributed to isoflavone-type compounds and glycosylated flavonoids. In a similar work, Espinoza-Hernández et al. (2021) analyzed aqueous extracts of E. cymosum by HPLC, finding caffeic, chlorogenic, and rosmarinic acids between 6 and 13 min of RT in the same UV-spectrum (320 nm). Additionally, the chromatographic profile of fractions isolated from aqueous, methanolic, and organic extracts of the same species has been analyzed by Romo-Pérez et al. (2022) , confirming that chlorogenic and rosmarinic phenolic acids were the most abundant in aqueous and methanolic extracts, while caffeic and protocatechuic acid were more abundant in the butanolic organic extract; in which, the authors reported for the first time the presence of the flavonoid kaempferol-3- O -(2,6-di- O -trans-ρ-coumaryl)-β- d -glucopyranoside in E. cymosum , associated directly with the hypoglycemic activity shown by the extracts. The identity and structure of the above compounds were corroborated by NMR. Montes-Moreno (2017) analyzed, by HPLC with a diode array detector coupled to mass spectrometry (HPLC-DAD-MS, detection wavelengths: 280, 320, and 370 nm) aqueous extract obtained by infusion and decoction at different concentrations (1 and 2%) and cooking times (5 and 10 min) of aerial parts of E. carlinae . The author reported the presence of hydroxybenzoic acids, mainly gallic acid (1300–2600 µg·mL −1 , RT = 12.5 min), and to a lesser extent 4-hydroxybenzoic and protocatechuic acids. Likewise, the next hydroxycinnamic acids were found: chlorogenic acid (400–700 µg·mL −1 , RT = 2.1 min), rosmarinic acid (40–58 µg·mL −1 , RT = 25 min), and caffeic acid (12–21 µg·mL −1 , RT = 16.3 min), while ellagic, p-coumaric, ferulic, and sinapic acids were present in the extract in smaller amounts; flavanols such as gallocatechin gallate (600–1600 µg·mL −1 , RT = 20.4 min), catechin, epicatechin, and epigallocatechin gallate; and flavonols such as quercetin (89–179 µg·mL −1 , RT = 25.5 min), rutin, and kaempferol were found in the extract as well. Additionally, the presence of flavanones and hydroxybenzaldehydes such as eriocitrin, naringenin, hesperidin, and vanillin were detected in minimal amounts. Using the same method, the author detected β-sitosterol, stigmastanol, sitosteryl-3-β-glucoside, and campesteryl-3-β-glucoside, phytosterols that could help control cholesterol levels in diseases such as hyperlipidemia, hypercholesterolemia, and atherosclerosis . Likewise, the presence of glycosylated saponins involved in anti-inflammatory and antioxidant activities such as α- l -arabinopyranoside phytolaccagenic acid and β- d -glucopyranoside hederagenin was confirmed . Pérez-Ramírez et al. (2016) evaluated the phytochemical composition of the E. carlinae decoction by HPLC with a diode array detector (HPLC-DAD), as well as its participation in the modulating activity of renal dysfunction. Ellagic acid was the most abundant phenolic compound (38.3 areas under the curve, mA), followed by caffeic (20.3 mA), protocatechuic (11.9 mA), and p-hydroxybenzoic (9.8 mA) acids; likewise, flavonoids such as rutin (14.1 mA), catechin (12.1 mA), and epicatechin (11.7 mA) were detected and quantified. Regarding the presence of sterols, the phytosterol α-7-stigmasterol (18.7 mA) was detected as the most abundant, followed by β-sitosterol (11.1 mA), β-campesterol (8.7 mA), and stigmastanol (8.4 mA). Finally, the authors also detected and quantified two major saponins, campesteryl β- d -glucopyranoside (28.9 mA) and sitosteril β- d -glucopyranoside (20.1 mA). The GC-MS technique is also a useful tool that allows the identification of volatile or semi-volatile compounds present in plant tissues. In this sense, mainly aromatic aldehydes, sesquiterpenes, and fatty acids have been detected in extracts of roots, stems, leaves, and inflorescences of different Eryngium spp. using GC-MS. Components such as 2-dodecenal, 2,4,6-trimethyl-benzaldehyde, d-elemene, a-bisabolol, α, and β-selinene, and fatty acids such as palmitic and stearic acid are common in a wide variety of Eryngium spp. found around the world, and their direct and synergistic participation in biological activities such as antiprotozoal , antibacterial , antifungal , antioxidant , and antidiabetic have been reported . In relation to the species found in Mexico, Peña-Montes et al. (2016) reported the presence and abundance of terpenes and sesquiterpenes identified in hexanic extracts of E. carlinae inflorescences. The major constituents identified were (Z)β-farnesene (38.79%), β-pinene (17.53%), calamenene (13.30%), and α-farnesene (10.38%). The authors attributed the ability to reduce lipid peroxidation and protein carbonylation to farnesene and pinene, in addition to the probable synergistic effect with other compounds present in the analyzed extract. Likewise, Espino Garibay (2010) found 21 terpenoid compounds in the ethanolic extract of leaves and inflorescences of E. carlinae . The main terpenoids that the author reported were borneol (367 mg/L), α-pinene (278 mg/L), myrcene (256 mg/L), caryophyllene (225 mg/L), and β-pinene (120 mg/L). In addition, the author reported differences in the presence and abundance through different phenological stages of the plant (before, during, and after flowering and fruiting), with the extracts of leaves and inflorescences during and after flowering being those that presented a higher content of metabolites; the isomers of pinene and farnesene were constant in both mentioned stages. Noriega-Cisneros et al. (2019) reported sesquiterpenes α-selinene (17.54%) and β-selinene (26.04%) as main components of ethanolic extract of aerial parts of E. carlinae , in addition to palmitic (14.43%) and stearic acid (14.53%), and others in proportions of less than 5%, such as humulene, stigmasterol, elemol, elemene, and α-cedrene; the authors conclude that these compounds, individually or synergistically, could be involved in the hypolipidemic and hypoglycemic response demonstrated in in vivo studies. Likewise, some oligosaccharides and polyalcohols have been detected in extracts of leaves and stems of E. carlinae . Arana-Argáez et al. (2021) described the presence of D-(−)-fructofuranose, D-(−)-fructopyranose, D-(−)-tagatofuranose, and sucrose, in addition to the polyalcohol 1,5-anhydro- d -sorbitol and cinnamic acid; the authors attributed the anti-inflammatory response, shown in vivo by the extract, to cinnamic acid. The above information is summarized in . According to the literature reviewed, the most studied species is E. carlinae , one of the most common in the central-western region of Mexico. However, research is needed on the phytochemical composition of the other species described in the region. In this sense, in the working group, a preliminary analysis of the phytochemical profile of several Eryngium species found in Michoacán and Jalisco, including E. carlinae , E. heterophyllum , E. nasturtiifolium , and E. beecheyanum , has been performed using HPTLC, and it has been possible to determine the presence of chlorogenic, rosmarinic, and caffeic acids in ethanolic extracts of inflorescences and stems. An important aspect to consider when studying or proposing the application of Eryngium spp. bioactive compounds, is the extraction methods. Eryngium extracts contain various metabolites (e.g., tannins, phenolic acids, saponins, and terpenoids) associated with biological activities that support their use in traditional medicine or reveal new therapeutic potential, serving as targets for developing novel drugs. In order to extract bioactive compounds from plants, different methods are used . Thus, multiple solvents chosen based on the polarity have been used to extract phytochemicals. While the ultrasound-assisted extraction (greater than 20 kHz) is used to disrupt plant cell walls, which helps improve the solvent’s ability to penetrate the cells and obtain a higher extraction yield, the use of a low operating temperature through processing allows the procurement of a high extract quality reducing the amount of solvent and energy used . Unfortunately, there are still some concerns related to experimental repeatability and reproducibility . Microwave-assisted extraction (MAE) has been used as an alternative to conventional techniques for the extraction of biocompounds due to its important advantages, among which are controllable and effective heating, faster energy transfer, the reduction of extraction time and use of solvents, higher selectivity, and enhanced yield . Furthermore, new nonconventional technologies are emerging offering superior extraction efficiency in terms of cost, yield, extraction time, and/or selectivity . In this sense, green synthesis of nanoparticles using plant extracts is a promising alternative to chemical methods . 4.2. Properties of Phytochemical Compounds in Species of the Genus Eryngium Rosmarinic, caffeic, and chlorogenic acids are amongst the phenolic compounds reported in the genus Eryngium , regardless of the studied species. Rosmarinic acid (RA, molecular formula C 18 H 16 O 8 ) named (R)-α-[[3-(3,4-dihydroxyphenyl)-1-oxo-2E-propenyl]oxy]-3,4-dihydroxy-enzenepropanoic acid is an ester of caffeic acid and (R)--3-(3,4-dihydroxyphenyl) lactic acid and originated from the amino acids L-phenylalanine and L-tyrosine, respectively (Hitl et al., 2020) . RA has been proven to act as (1) an anticarcinogenic, as it inhibits the gene expression, the growth, and the proliferation of tumor cells ; (2) an antidiabetic, as it prevents hyperglycemia by increasing the insulin sensitivity index and reducing the levels of blood glucose ; (3) an antimicrobial, as it inhibits the growth of Methicillin-resistant Staphylococcus aureus (MRSA), E. coli , Pseudomonas spp., and L. monocytogenes , amongst others, and in addition to the formation of biofilm, it kills planktonic cells ; and (4) an antioxidant, as it inhibits the formation of free radicals, the generation of ROS, and lipid peroxidation . In addition, RA has shown hepatoprotective, cardioprotective, antiallergic, antidepressant, anti-aging, nephroprotective, and anti-inflammatory activity . Budzianowski et al. (2023) investigated the cytotoxic effect of RA obtained from 50% ethanolic extracts of seedlings of different Eryngium species. The authors concluded that the rosmarinic acid-4′- O -β-glucopyranoside fraction did not show cytotoxic effects on cell lines involved in cancer development (MCF-7, MDA-MB-231, MCF-12A, HT-29, Caco-2, and OVCAR-3) with an IC 50 average of 400–700 µM in 24–72 h. RA is used as a bioactive ingredient in supplements, and in its isolated or semipurified form, has not shown toxicity in humans. Jia et al. (2010) concluded that the administration of the RA isolated fraction from depsides salts of Salvia miltiorrhiza in only one dose (100–200 mg/kg), did not show toxic effects in humans . Caffeic acid (CA, empirical formula C 9 H 8 O 4 , 3,4-dihydroxycinnamic acid) is a phenolic compound abundant in medicinal plants, with multiple biological activities, such as antioxidant, anti-inflammatory, anticarcinogenic, and neuroprotective . Alam et al. (2022) suggested that CA can intervene in the reduction of oxidative stress by blocking or preventing the formation of ROS molecules in the organism, having a potential antioxidant and anti-aging effect. The authors also determined that the CA could inhibit the formation and migration of tumor cells, decreasing the probability of metastasis in different types of cancer, suggesting that CA alone or in combination with other chemotherapeutic agents/drugs might be suggested to treat and manage cancer. The chlorogenic acid (CGA) is directly involved in the mitochondrial function, the reduction and prevention of oxidative stress, apoptosis, inflammation, obesity, and diabetes. Hernandes et al. (2020) investigated the cytotoxicity and genotoxicity of CA and CGA on human leukemic cell lines and determined that they showed neither cytotoxicity nor genotoxicity over the analyzed cells. However, CGA induces specific hypomethylation on Jukart cells, which can be beneficial against hematologic malignancies, since ADN altered methylation plays an important role in the pathogenic process. RA, CA, and CGA have been reported in different species of Eryngium , such as E. campestre , E. maritimum , E. plannum , E. creticum , and E. alpinum , and in some regional species addressed in this review, such as E. carlinae , E. longifolium , and E. cymosum . Kaempferol (KAE, general empirical formula C 15 H 10 O 6 , 3,4′,5,7-Tetrahydroxyflavone) is part of the frequent appearance of flavonoids in different species of Eryngium . KAE is synthesized by condensation of 4-coumaroyl-CoA (C6-C3) with three molecules of malonyl-CoA (C6). KAE and its multiple glycosylated forms have been reported as agents with antitumor, anti-inflammatory, and antioxidant activity. Wang et al. (2018) analyzed the antiproliferative activity of hepatic tumor cells of KAE and its glycosides (Kae-3- O -rha, Kae-7- O -glu and Kae-3- O -rut); the authors showed that KAE in its pure form has antiproliferative concentration-dependent activity (0–100 μM), whereas the glycosylated forms did not show such activity. Likewise, the authors reported that KAE has higher antioxidant and anti-inflammatory activity than its glycosylated forms. The safe use of KAE is being discussed since it could have antimutagenic and genotoxic activity; it also has prooxidant action since at high concentrations it can produce ROS . Additionally, in order to overcome its poor bioavailability and improve its pharmacokinetics, the use of kaempferol nanosuspensions, solid dispersions, and complexes of polysaccharides and oligosaccharides has been developed . The ellagic acid (EA, empirical formula C 14 H 6 O 8 , 4, 4′, 5, 5′, 6, 6′-hexahydrodiphenic acid 2, 6, 2′, 6′-dilactone), a flavonoid compound, has shown activity as a regulator of proinflammatory mediators and it also normalizes the lipid metabolism, in addition to having neuroprotective activity by starting several cell signaling pathways, preventing mitochondrial disfunction, and eliminating free radicals . Likewise, catechin (CAT, empirical formula C 22 H 18 O 10 , catechin 5- O -gallate) and its derivatives, ((−)-CAT), ECAT ((−)-epicatechin), ECG ((−)-epicatechingallate), EGC ((−)-epigallocatechin), and EGCG ((−)-epigallocatechin gallate), have antioxidant and anti-aging activity, eliminating free radicals and delaying the extracellular matrix degradation induced by ultraviolet radiation (UV) and skin contamination, activating the collagen synthesis and promoting the inhibition of matrix metalloproteinase . Beyond food and biomedical applications, EA and ellagitannins’ (ET) scientific relevance can be linked to advanced materials such as copolymers, chelating reagents, ion-exchange resins, and materials for electrochemical devices, among others . On its part, the rutin and its derivatives (Rut, C 27 H 30 O 16 , quercetin 3-rutinoside) are flavonoids that have antioxidant, antimicrobial, anticarcinogenic, antidiabetic, antiallergic, antidepressant, antihypertensive, and other effects . In the genus Eryngium , the flavonoids KAE, EA, CAT, and Rut have been reported in E. longifolium and E. carlinae. Quercetin and Rut could be applied in nutritional supplements and innovative complexes and formulations for pharmaceuticals . Among the terpenic compounds in the Eryngium species of the central-western region of Mexico (described in E. carlinae , ), are the borneol and α- and β-pinene, which are present in the essential oils of several aromatic plants and show activity on blood pressure regulation in addition to showing cytogenetic, gastroprotective, anxiolytic, cytoprotective, anticonvulsant, and neuroprotective effects as well as their effects against H 2 O 2 -stimulated oxidative stress, pancreatitis, stress-stimulated hyperthermia, and pulpal pain . Regarding caryophyllene (β-Caryophyllene (BCP)) and farnesene, they are natural sesquiterpenes that have several biological activities such as antimicrobial, anticarcinogenic, anti-inflammatory, antioxidant, anxiolytic-like, and local anesthetic effects. However, its volatility and poor water solubility limit its application in the pharmaceutical field . 4.3. Tocixity of Phytochemical Compounds in Species of the Genus Eryngium Some findings about the toxicity of bioactive compounds detected in the Eryngium species reviewed in this work are described below. Knauth et al. (2018) studied the cytotoxicity of extracts from various medicinal plants including E. carlinae using the Caco-2 enterocyte cell line. Most of the methanolic extracts from the tested plants showed low cytotoxicity. Agiorgiti et al. (2018) analyzed the cytotoxic and antitumor activities of five organotin complexes (1–5) with o-hydroxybenzoic or p-hydroxybenzoic acids in vitro and in vivo (in Wistar rats). All complexes exhibited strong cytotoxic activity against all cancer cell lines tested, so they could be used as potential chemotherapeutic agents. However, there is controversy about the safety of p-hydroxybenzoic acid in human health, as there is no evidence of its toxicity in humans . In this sense, Downs et al. (2023) evaluated the relation between accumulation of p-hydroxybenzoic acid and methyl, ethyl, propyl, and butyl esters and the incidence of both malignant and benign breast tumors in patients. The authors concluded that propyl and butyl paraben concentrations are higher on metastatic tissue compared to non-metastatic breast cancer tissue. Likewise, the authors suggested that factors that determine the formation of tumors in breast cancer such as age, menopause status, and family history of cancer could be controlled by changing to a paraben-free lifestyle, especially those present in cosmetics as preservatives, although more extensive studies must be carried out to confirm this association. Budzianowski et al. (2023) reported a similar effect for AR. The authors evaluated the effect of rosmarinic acid 4′- O -β-glucopyranoside (RAG4′) on five human carcinoma cell lines and one non-tumorigenic mammary epithelial cell line (MCF-12A). The highest cytotoxic activity of RAG4′ was observed in the Caco-2 cell line and RAG4′ did not show any effect on the non-tumorigenic cell line MCF-12A, indicating that it might be safe as a cosmetic and pharmaceutical ingredient. D-mannitol found in E. carlinae was evaluated in a model of hypercholesterolemia in mice, and a chronic toxicity test was performed. The mice did not show evident signs of toxicity (e.g., lack of motor coordination, piloerection, or pupil dilation) after receiving doses of 100 and 500 mg/kg of the extract for 4 weeks . Hameed et al. (2016) evaluated the cytotoxic profiles of sinapic acid (SA) belonging to the phenylpropanoid family also present in Eryngium species. SA is assumed to be therapeutically beneficial and generally non-toxic. The authors tested a wide range of concentrations in Chinese hamster lung fibroblasts (V79) and human cervical carcinoma (HeLa) cells. Concentrations up to 500 μM and 2000 μM had no effect on the viability of V79 and HeLa cells, respectively, demonstrating that SA had no cytotoxic effects in two different cell lines except at very high concentrations. The dichotomous effect of SA on concentration-dependent cell viability has also been reported: at low SA concentrations cell viability was enhanced, while at high SA concentrations cell proliferation was almost completely inhibited . Little is known about the genotoxic/antigenotoxic effects of SA, although there are several studies on the effects of different phenolic compounds . The genotoxic effect and clastogenic potential of caffeic, cinnamic, and ferulic acids was examined by Maistro et al. (2011) . The authors used in vitro comet and micronucleus (MN) assays in rat hepatoma tissue (HTC) cells at three different concentrations, for 24 h. No genotoxicity was observed by the comet assay, but clastogenic effects were found in HTC by the MN assay. The subchronic toxicity of ellagic acid, also present in Eryngium species, was analyzed by Tasaki et al. (2008) in F344 rats at five doses in a powdered basal diet for 90 days. No mortality or clinical signs related to the treatments were observed during the entire experimental period. The no-observed effect level (NOEL) was estimated to be 5% (3011 mg/kg b.w./day) for males and the no-observed-adverse-effect level (NOAEL) and NOEL in females were estimated to be 5% (3254 mg/kg b.w./day) and <1.25% (778 mg/kg b.w./day), respectively. Marcarini et al. (2011) studied the cytotoxic, apoptosis-inducing, genotoxic, and protective effects of rutin in HTC cells and demonstrated that at low concentrations (810 μM) the flavonoid decreased the viability and proliferation of cells after 72 h of treatment; however, at 24 h the authors observed induction of DNA damage, with genotoxic effects but without inducing apoptosis. The authors also observed a protective DNA effect by reducing damage induced by procarcinogenic agents such as benzo(a)pyrene suggesting an important biological activity for this compound, which can contribute to human health through diet. A subsequent report noted that rutin is considered a non-toxic and selective modulator of hypercholesterolemia . It has been reported that the administration of β-sitosterol (BS) in rats does not cause genotoxicity or cytotoxicity . A high level of BS concentrations in blood has been correlated with increased severity of heart disease in men who have previously suffered heart attacks . An extensive toxicological study of BS showed a high LD50 in rats (>2 g/kg) . A subsequent study showed that BS had no effect on the reproductive system . It has also been shown that high BS exposure is related to alteration of the G5/8 transporters of the hepatic and intestinal ATP-binding cassette and promotes potential risks to the integrity of the blood–brain barrier in diabetic rats . Several in vitro and in vivo studies have shown that CGA can protect cells against toxicities induced by chemicals of different classes (e.g., fungal/bacterial toxins, pharmaceuticals, and metals) by controlling the overproduction of nitric oxide and ROS and suppressed pro-apoptotic signaling . Cos et al. (2001) determined the kaempferol genotoxicity and attributed it mainly to its prooxidative activity. For their part, Ren et al. (2019) generated an antioxidant selectivity index (ASI), which is the maximum non-toxic dose divided by the IC50 value, which is used to assess the toxicity of flavonoids. If the ASI of the flavonoids is >100, they can be considered safe with a good antioxidant activity profile; however, further research is required to standardize it. Spagnuolo et al., 2012 carried out a study of quercetin toxicity in male F344/N rats fed 2 g per kg body weight per day of quercetin. The results showed severe chronic nephropathy, hyperplasia, and kidney tubular epithelium neoplasm after the treatment. In another study, it was reported that maternal intake of quercetin during pregnancy could increase the risk of mixed lineage leukemia (MLL) gene rearrangements, which is common in childhood leukemia, especially in the presence of compromised DNA repair . However, Utesch et al. (2008) demonstrated that orally administered quercetin (up to 2 g/kg body weight/day) did not induce unscheduled DNA synthesis in male or female rat hepatocytes, suggesting that quercetin was not genotoxic. Additionally, in the only phase I clinical trial of quercetin found, renal toxicity was detected without signs of nephritis or obstructive uropathy when a dose of 50 mg/kg (approximately 3.5 g/70 kg) was administered by intravenous infusion at three weeks or at weekly intervals . Phytochemical characterization is valuable to reveal the presence and identity of secondary metabolites in a plant as well as a helpful tool in the search for bioactive compounds useful for the synthesis of new drugs and other products. The presence of various groups of metabolites has been described in plant extracts (ethanolic, aqueous, methanolic, and hexane) of aerial parts (stems, leaves, and inflorescences) qualitatively analyzed to determine the presence of flavonoids, tannins, terpenoids, and saponins. Additionally, more precise techniques such as GC-MS, nuclear magnetic resonance (NMR), and high-performance liquid chromatography (HPLC) are performed to detect, quantify, and identify metabolites in plant extracts. Regarding the genus Eryngium , investigations have been carried out on the phytochemical profile of some Eryngium spp. found in the central-western region of México, mainly E. carlinae , E. comosum , E. cymosum , and E. longifolium . The most widely used methods to determine the general phytochemical composition of a plant are screening and the colorimetric techniques of ultraviolet-visible spectrophotometry (UV-Vis). Phytochemical screening involves a series of chemical reactions that make it possible to qualitatively detect the groups of secondary metabolites present in plant extracts . For example, E. carlinae , one of the most common species used along the region delimited for this review, was qualitatively analyzed by Knauth et al. (2018) to determine the main groups of metabolites in methanolic extract from the aerial part of the plant. The authors reported a strong presence of tannins and saponins, as well as a slight presence of flavonoids. Galindo-Hernández (2018) evaluated the phytochemical profile of E. carlinae , confirming the presence of triterpenoids, sterols, tannins, coumarins, carboxyls, flavonoids, and carbohydrates. In the same way, Pérez-Reyes (2016) performed phytochemical screening of an aqueous extract of E. comosum , reporting the presence of alkaloids, flavonoids (flavones and xanthones), triterpenoid saponins, reducing sugars, tannins derived from catechol, phenolic compounds, and benzoquinones; in addition, the author reported in vivo hypolipidemic activity. Using UV-Vis, it is possible to determine compound concentration in a solution; this is a simple, reliable and low-cost analytical technique . In this sense, Lemus-de la Cruz-Hurtado et al. (2023) determined and quantified total phenolic compounds (TPC), total flavonoids (TF), and total terpenoids (TT) in aqueous extracts of aerial parts of E. carlinae . The authors reported concentrations of 0.0038 mg gallic acid equivalents per mL (mg GAE/mL) for TPC, 3.3032 mg quercetin equivalents per mL (mg QE/mL) for TF, and 0.0424 mg linalool equivalents per mL (mg LE/mL) for TT. Montes-Moreno (2017) reported higher TPC values in aqueous infusions and decoctions of the same species. The authors evaluated by UV-Vis, up to 2.5 mg GAE/mL of TPC and, in the case of TF, the author reported less than 0.2 mg QE/mL. Similarly, Díaz-Alvarado et al. (2020) reported the total phenolic content (4.33 mg GAE/g dry weight) and total saponins (62.2 mg/g dry weight) of the ethanolic extract of aerial parts of E. comosum . In the same sense, Díaz-Alvarado (2020) determined the phytochemical profile of ethanolic extracts (50 and 70%) of aerial parts of E. comosum by UV-Vis, confirming the presence and abundance of phenolic compounds (up to 4.3 mg GAE/mL), TPC (up to 13.33 mg/g DW), and total saponins (up to 29.33 mg/g DW), with 70% ethanol being the best extractor. Within the phenols group, phenolic acids (caffeic, rosmarinic, and chlorogenic) and their derivatives are the most abundant in the genus Eryngium ; these compounds have been analyzed and identified by more precise techniques, such as chromatography . Andrade-Cetto et al. (2021) described the chromatographic profile of the ethanolic extract of aerial parts of E. longifolium analyzed by HPLC, the authors detected three signals at wavelengths of 254 and 320 nm with retention time (RT) between 6 and 13 min, which were identified as caffeic, chlorogenic, and rosmarinic acids by comparing them to their commercial standards, rosmarinic acid being the most abundant in the analyzed extract. Likewise, the authors reported other minor intensity peaks with RT between 16 and 24 min, which were attributed to isoflavone-type compounds and glycosylated flavonoids. In a similar work, Espinoza-Hernández et al. (2021) analyzed aqueous extracts of E. cymosum by HPLC, finding caffeic, chlorogenic, and rosmarinic acids between 6 and 13 min of RT in the same UV-spectrum (320 nm). Additionally, the chromatographic profile of fractions isolated from aqueous, methanolic, and organic extracts of the same species has been analyzed by Romo-Pérez et al. (2022) , confirming that chlorogenic and rosmarinic phenolic acids were the most abundant in aqueous and methanolic extracts, while caffeic and protocatechuic acid were more abundant in the butanolic organic extract; in which, the authors reported for the first time the presence of the flavonoid kaempferol-3- O -(2,6-di- O -trans-ρ-coumaryl)-β- d -glucopyranoside in E. cymosum , associated directly with the hypoglycemic activity shown by the extracts. The identity and structure of the above compounds were corroborated by NMR. Montes-Moreno (2017) analyzed, by HPLC with a diode array detector coupled to mass spectrometry (HPLC-DAD-MS, detection wavelengths: 280, 320, and 370 nm) aqueous extract obtained by infusion and decoction at different concentrations (1 and 2%) and cooking times (5 and 10 min) of aerial parts of E. carlinae . The author reported the presence of hydroxybenzoic acids, mainly gallic acid (1300–2600 µg·mL −1 , RT = 12.5 min), and to a lesser extent 4-hydroxybenzoic and protocatechuic acids. Likewise, the next hydroxycinnamic acids were found: chlorogenic acid (400–700 µg·mL −1 , RT = 2.1 min), rosmarinic acid (40–58 µg·mL −1 , RT = 25 min), and caffeic acid (12–21 µg·mL −1 , RT = 16.3 min), while ellagic, p-coumaric, ferulic, and sinapic acids were present in the extract in smaller amounts; flavanols such as gallocatechin gallate (600–1600 µg·mL −1 , RT = 20.4 min), catechin, epicatechin, and epigallocatechin gallate; and flavonols such as quercetin (89–179 µg·mL −1 , RT = 25.5 min), rutin, and kaempferol were found in the extract as well. Additionally, the presence of flavanones and hydroxybenzaldehydes such as eriocitrin, naringenin, hesperidin, and vanillin were detected in minimal amounts. Using the same method, the author detected β-sitosterol, stigmastanol, sitosteryl-3-β-glucoside, and campesteryl-3-β-glucoside, phytosterols that could help control cholesterol levels in diseases such as hyperlipidemia, hypercholesterolemia, and atherosclerosis . Likewise, the presence of glycosylated saponins involved in anti-inflammatory and antioxidant activities such as α- l -arabinopyranoside phytolaccagenic acid and β- d -glucopyranoside hederagenin was confirmed . Pérez-Ramírez et al. (2016) evaluated the phytochemical composition of the E. carlinae decoction by HPLC with a diode array detector (HPLC-DAD), as well as its participation in the modulating activity of renal dysfunction. Ellagic acid was the most abundant phenolic compound (38.3 areas under the curve, mA), followed by caffeic (20.3 mA), protocatechuic (11.9 mA), and p-hydroxybenzoic (9.8 mA) acids; likewise, flavonoids such as rutin (14.1 mA), catechin (12.1 mA), and epicatechin (11.7 mA) were detected and quantified. Regarding the presence of sterols, the phytosterol α-7-stigmasterol (18.7 mA) was detected as the most abundant, followed by β-sitosterol (11.1 mA), β-campesterol (8.7 mA), and stigmastanol (8.4 mA). Finally, the authors also detected and quantified two major saponins, campesteryl β- d -glucopyranoside (28.9 mA) and sitosteril β- d -glucopyranoside (20.1 mA). The GC-MS technique is also a useful tool that allows the identification of volatile or semi-volatile compounds present in plant tissues. In this sense, mainly aromatic aldehydes, sesquiterpenes, and fatty acids have been detected in extracts of roots, stems, leaves, and inflorescences of different Eryngium spp. using GC-MS. Components such as 2-dodecenal, 2,4,6-trimethyl-benzaldehyde, d-elemene, a-bisabolol, α, and β-selinene, and fatty acids such as palmitic and stearic acid are common in a wide variety of Eryngium spp. found around the world, and their direct and synergistic participation in biological activities such as antiprotozoal , antibacterial , antifungal , antioxidant , and antidiabetic have been reported . In relation to the species found in Mexico, Peña-Montes et al. (2016) reported the presence and abundance of terpenes and sesquiterpenes identified in hexanic extracts of E. carlinae inflorescences. The major constituents identified were (Z)β-farnesene (38.79%), β-pinene (17.53%), calamenene (13.30%), and α-farnesene (10.38%). The authors attributed the ability to reduce lipid peroxidation and protein carbonylation to farnesene and pinene, in addition to the probable synergistic effect with other compounds present in the analyzed extract. Likewise, Espino Garibay (2010) found 21 terpenoid compounds in the ethanolic extract of leaves and inflorescences of E. carlinae . The main terpenoids that the author reported were borneol (367 mg/L), α-pinene (278 mg/L), myrcene (256 mg/L), caryophyllene (225 mg/L), and β-pinene (120 mg/L). In addition, the author reported differences in the presence and abundance through different phenological stages of the plant (before, during, and after flowering and fruiting), with the extracts of leaves and inflorescences during and after flowering being those that presented a higher content of metabolites; the isomers of pinene and farnesene were constant in both mentioned stages. Noriega-Cisneros et al. (2019) reported sesquiterpenes α-selinene (17.54%) and β-selinene (26.04%) as main components of ethanolic extract of aerial parts of E. carlinae , in addition to palmitic (14.43%) and stearic acid (14.53%), and others in proportions of less than 5%, such as humulene, stigmasterol, elemol, elemene, and α-cedrene; the authors conclude that these compounds, individually or synergistically, could be involved in the hypolipidemic and hypoglycemic response demonstrated in in vivo studies. Likewise, some oligosaccharides and polyalcohols have been detected in extracts of leaves and stems of E. carlinae . Arana-Argáez et al. (2021) described the presence of D-(−)-fructofuranose, D-(−)-fructopyranose, D-(−)-tagatofuranose, and sucrose, in addition to the polyalcohol 1,5-anhydro- d -sorbitol and cinnamic acid; the authors attributed the anti-inflammatory response, shown in vivo by the extract, to cinnamic acid. The above information is summarized in . According to the literature reviewed, the most studied species is E. carlinae , one of the most common in the central-western region of Mexico. However, research is needed on the phytochemical composition of the other species described in the region. In this sense, in the working group, a preliminary analysis of the phytochemical profile of several Eryngium species found in Michoacán and Jalisco, including E. carlinae , E. heterophyllum , E. nasturtiifolium , and E. beecheyanum , has been performed using HPTLC, and it has been possible to determine the presence of chlorogenic, rosmarinic, and caffeic acids in ethanolic extracts of inflorescences and stems. An important aspect to consider when studying or proposing the application of Eryngium spp. bioactive compounds, is the extraction methods. Eryngium extracts contain various metabolites (e.g., tannins, phenolic acids, saponins, and terpenoids) associated with biological activities that support their use in traditional medicine or reveal new therapeutic potential, serving as targets for developing novel drugs. In order to extract bioactive compounds from plants, different methods are used . Thus, multiple solvents chosen based on the polarity have been used to extract phytochemicals. While the ultrasound-assisted extraction (greater than 20 kHz) is used to disrupt plant cell walls, which helps improve the solvent’s ability to penetrate the cells and obtain a higher extraction yield, the use of a low operating temperature through processing allows the procurement of a high extract quality reducing the amount of solvent and energy used . Unfortunately, there are still some concerns related to experimental repeatability and reproducibility . Microwave-assisted extraction (MAE) has been used as an alternative to conventional techniques for the extraction of biocompounds due to its important advantages, among which are controllable and effective heating, faster energy transfer, the reduction of extraction time and use of solvents, higher selectivity, and enhanced yield . Furthermore, new nonconventional technologies are emerging offering superior extraction efficiency in terms of cost, yield, extraction time, and/or selectivity . In this sense, green synthesis of nanoparticles using plant extracts is a promising alternative to chemical methods . Rosmarinic, caffeic, and chlorogenic acids are amongst the phenolic compounds reported in the genus Eryngium , regardless of the studied species. Rosmarinic acid (RA, molecular formula C 18 H 16 O 8 ) named (R)-α-[[3-(3,4-dihydroxyphenyl)-1-oxo-2E-propenyl]oxy]-3,4-dihydroxy-enzenepropanoic acid is an ester of caffeic acid and (R)--3-(3,4-dihydroxyphenyl) lactic acid and originated from the amino acids L-phenylalanine and L-tyrosine, respectively (Hitl et al., 2020) . RA has been proven to act as (1) an anticarcinogenic, as it inhibits the gene expression, the growth, and the proliferation of tumor cells ; (2) an antidiabetic, as it prevents hyperglycemia by increasing the insulin sensitivity index and reducing the levels of blood glucose ; (3) an antimicrobial, as it inhibits the growth of Methicillin-resistant Staphylococcus aureus (MRSA), E. coli , Pseudomonas spp., and L. monocytogenes , amongst others, and in addition to the formation of biofilm, it kills planktonic cells ; and (4) an antioxidant, as it inhibits the formation of free radicals, the generation of ROS, and lipid peroxidation . In addition, RA has shown hepatoprotective, cardioprotective, antiallergic, antidepressant, anti-aging, nephroprotective, and anti-inflammatory activity . Budzianowski et al. (2023) investigated the cytotoxic effect of RA obtained from 50% ethanolic extracts of seedlings of different Eryngium species. The authors concluded that the rosmarinic acid-4′- O -β-glucopyranoside fraction did not show cytotoxic effects on cell lines involved in cancer development (MCF-7, MDA-MB-231, MCF-12A, HT-29, Caco-2, and OVCAR-3) with an IC 50 average of 400–700 µM in 24–72 h. RA is used as a bioactive ingredient in supplements, and in its isolated or semipurified form, has not shown toxicity in humans. Jia et al. (2010) concluded that the administration of the RA isolated fraction from depsides salts of Salvia miltiorrhiza in only one dose (100–200 mg/kg), did not show toxic effects in humans . Caffeic acid (CA, empirical formula C 9 H 8 O 4 , 3,4-dihydroxycinnamic acid) is a phenolic compound abundant in medicinal plants, with multiple biological activities, such as antioxidant, anti-inflammatory, anticarcinogenic, and neuroprotective . Alam et al. (2022) suggested that CA can intervene in the reduction of oxidative stress by blocking or preventing the formation of ROS molecules in the organism, having a potential antioxidant and anti-aging effect. The authors also determined that the CA could inhibit the formation and migration of tumor cells, decreasing the probability of metastasis in different types of cancer, suggesting that CA alone or in combination with other chemotherapeutic agents/drugs might be suggested to treat and manage cancer. The chlorogenic acid (CGA) is directly involved in the mitochondrial function, the reduction and prevention of oxidative stress, apoptosis, inflammation, obesity, and diabetes. Hernandes et al. (2020) investigated the cytotoxicity and genotoxicity of CA and CGA on human leukemic cell lines and determined that they showed neither cytotoxicity nor genotoxicity over the analyzed cells. However, CGA induces specific hypomethylation on Jukart cells, which can be beneficial against hematologic malignancies, since ADN altered methylation plays an important role in the pathogenic process. RA, CA, and CGA have been reported in different species of Eryngium , such as E. campestre , E. maritimum , E. plannum , E. creticum , and E. alpinum , and in some regional species addressed in this review, such as E. carlinae , E. longifolium , and E. cymosum . Kaempferol (KAE, general empirical formula C 15 H 10 O 6 , 3,4′,5,7-Tetrahydroxyflavone) is part of the frequent appearance of flavonoids in different species of Eryngium . KAE is synthesized by condensation of 4-coumaroyl-CoA (C6-C3) with three molecules of malonyl-CoA (C6). KAE and its multiple glycosylated forms have been reported as agents with antitumor, anti-inflammatory, and antioxidant activity. Wang et al. (2018) analyzed the antiproliferative activity of hepatic tumor cells of KAE and its glycosides (Kae-3- O -rha, Kae-7- O -glu and Kae-3- O -rut); the authors showed that KAE in its pure form has antiproliferative concentration-dependent activity (0–100 μM), whereas the glycosylated forms did not show such activity. Likewise, the authors reported that KAE has higher antioxidant and anti-inflammatory activity than its glycosylated forms. The safe use of KAE is being discussed since it could have antimutagenic and genotoxic activity; it also has prooxidant action since at high concentrations it can produce ROS . Additionally, in order to overcome its poor bioavailability and improve its pharmacokinetics, the use of kaempferol nanosuspensions, solid dispersions, and complexes of polysaccharides and oligosaccharides has been developed . The ellagic acid (EA, empirical formula C 14 H 6 O 8 , 4, 4′, 5, 5′, 6, 6′-hexahydrodiphenic acid 2, 6, 2′, 6′-dilactone), a flavonoid compound, has shown activity as a regulator of proinflammatory mediators and it also normalizes the lipid metabolism, in addition to having neuroprotective activity by starting several cell signaling pathways, preventing mitochondrial disfunction, and eliminating free radicals . Likewise, catechin (CAT, empirical formula C 22 H 18 O 10 , catechin 5- O -gallate) and its derivatives, ((−)-CAT), ECAT ((−)-epicatechin), ECG ((−)-epicatechingallate), EGC ((−)-epigallocatechin), and EGCG ((−)-epigallocatechin gallate), have antioxidant and anti-aging activity, eliminating free radicals and delaying the extracellular matrix degradation induced by ultraviolet radiation (UV) and skin contamination, activating the collagen synthesis and promoting the inhibition of matrix metalloproteinase . Beyond food and biomedical applications, EA and ellagitannins’ (ET) scientific relevance can be linked to advanced materials such as copolymers, chelating reagents, ion-exchange resins, and materials for electrochemical devices, among others . On its part, the rutin and its derivatives (Rut, C 27 H 30 O 16 , quercetin 3-rutinoside) are flavonoids that have antioxidant, antimicrobial, anticarcinogenic, antidiabetic, antiallergic, antidepressant, antihypertensive, and other effects . In the genus Eryngium , the flavonoids KAE, EA, CAT, and Rut have been reported in E. longifolium and E. carlinae. Quercetin and Rut could be applied in nutritional supplements and innovative complexes and formulations for pharmaceuticals . Among the terpenic compounds in the Eryngium species of the central-western region of Mexico (described in E. carlinae , ), are the borneol and α- and β-pinene, which are present in the essential oils of several aromatic plants and show activity on blood pressure regulation in addition to showing cytogenetic, gastroprotective, anxiolytic, cytoprotective, anticonvulsant, and neuroprotective effects as well as their effects against H 2 O 2 -stimulated oxidative stress, pancreatitis, stress-stimulated hyperthermia, and pulpal pain . Regarding caryophyllene (β-Caryophyllene (BCP)) and farnesene, they are natural sesquiterpenes that have several biological activities such as antimicrobial, anticarcinogenic, anti-inflammatory, antioxidant, anxiolytic-like, and local anesthetic effects. However, its volatility and poor water solubility limit its application in the pharmaceutical field . Some findings about the toxicity of bioactive compounds detected in the Eryngium species reviewed in this work are described below. Knauth et al. (2018) studied the cytotoxicity of extracts from various medicinal plants including E. carlinae using the Caco-2 enterocyte cell line. Most of the methanolic extracts from the tested plants showed low cytotoxicity. Agiorgiti et al. (2018) analyzed the cytotoxic and antitumor activities of five organotin complexes (1–5) with o-hydroxybenzoic or p-hydroxybenzoic acids in vitro and in vivo (in Wistar rats). All complexes exhibited strong cytotoxic activity against all cancer cell lines tested, so they could be used as potential chemotherapeutic agents. However, there is controversy about the safety of p-hydroxybenzoic acid in human health, as there is no evidence of its toxicity in humans . In this sense, Downs et al. (2023) evaluated the relation between accumulation of p-hydroxybenzoic acid and methyl, ethyl, propyl, and butyl esters and the incidence of both malignant and benign breast tumors in patients. The authors concluded that propyl and butyl paraben concentrations are higher on metastatic tissue compared to non-metastatic breast cancer tissue. Likewise, the authors suggested that factors that determine the formation of tumors in breast cancer such as age, menopause status, and family history of cancer could be controlled by changing to a paraben-free lifestyle, especially those present in cosmetics as preservatives, although more extensive studies must be carried out to confirm this association. Budzianowski et al. (2023) reported a similar effect for AR. The authors evaluated the effect of rosmarinic acid 4′- O -β-glucopyranoside (RAG4′) on five human carcinoma cell lines and one non-tumorigenic mammary epithelial cell line (MCF-12A). The highest cytotoxic activity of RAG4′ was observed in the Caco-2 cell line and RAG4′ did not show any effect on the non-tumorigenic cell line MCF-12A, indicating that it might be safe as a cosmetic and pharmaceutical ingredient. D-mannitol found in E. carlinae was evaluated in a model of hypercholesterolemia in mice, and a chronic toxicity test was performed. The mice did not show evident signs of toxicity (e.g., lack of motor coordination, piloerection, or pupil dilation) after receiving doses of 100 and 500 mg/kg of the extract for 4 weeks . Hameed et al. (2016) evaluated the cytotoxic profiles of sinapic acid (SA) belonging to the phenylpropanoid family also present in Eryngium species. SA is assumed to be therapeutically beneficial and generally non-toxic. The authors tested a wide range of concentrations in Chinese hamster lung fibroblasts (V79) and human cervical carcinoma (HeLa) cells. Concentrations up to 500 μM and 2000 μM had no effect on the viability of V79 and HeLa cells, respectively, demonstrating that SA had no cytotoxic effects in two different cell lines except at very high concentrations. The dichotomous effect of SA on concentration-dependent cell viability has also been reported: at low SA concentrations cell viability was enhanced, while at high SA concentrations cell proliferation was almost completely inhibited . Little is known about the genotoxic/antigenotoxic effects of SA, although there are several studies on the effects of different phenolic compounds . The genotoxic effect and clastogenic potential of caffeic, cinnamic, and ferulic acids was examined by Maistro et al. (2011) . The authors used in vitro comet and micronucleus (MN) assays in rat hepatoma tissue (HTC) cells at three different concentrations, for 24 h. No genotoxicity was observed by the comet assay, but clastogenic effects were found in HTC by the MN assay. The subchronic toxicity of ellagic acid, also present in Eryngium species, was analyzed by Tasaki et al. (2008) in F344 rats at five doses in a powdered basal diet for 90 days. No mortality or clinical signs related to the treatments were observed during the entire experimental period. The no-observed effect level (NOEL) was estimated to be 5% (3011 mg/kg b.w./day) for males and the no-observed-adverse-effect level (NOAEL) and NOEL in females were estimated to be 5% (3254 mg/kg b.w./day) and <1.25% (778 mg/kg b.w./day), respectively. Marcarini et al. (2011) studied the cytotoxic, apoptosis-inducing, genotoxic, and protective effects of rutin in HTC cells and demonstrated that at low concentrations (810 μM) the flavonoid decreased the viability and proliferation of cells after 72 h of treatment; however, at 24 h the authors observed induction of DNA damage, with genotoxic effects but without inducing apoptosis. The authors also observed a protective DNA effect by reducing damage induced by procarcinogenic agents such as benzo(a)pyrene suggesting an important biological activity for this compound, which can contribute to human health through diet. A subsequent report noted that rutin is considered a non-toxic and selective modulator of hypercholesterolemia . It has been reported that the administration of β-sitosterol (BS) in rats does not cause genotoxicity or cytotoxicity . A high level of BS concentrations in blood has been correlated with increased severity of heart disease in men who have previously suffered heart attacks . An extensive toxicological study of BS showed a high LD50 in rats (>2 g/kg) . A subsequent study showed that BS had no effect on the reproductive system . It has also been shown that high BS exposure is related to alteration of the G5/8 transporters of the hepatic and intestinal ATP-binding cassette and promotes potential risks to the integrity of the blood–brain barrier in diabetic rats . Several in vitro and in vivo studies have shown that CGA can protect cells against toxicities induced by chemicals of different classes (e.g., fungal/bacterial toxins, pharmaceuticals, and metals) by controlling the overproduction of nitric oxide and ROS and suppressed pro-apoptotic signaling . Cos et al. (2001) determined the kaempferol genotoxicity and attributed it mainly to its prooxidative activity. For their part, Ren et al. (2019) generated an antioxidant selectivity index (ASI), which is the maximum non-toxic dose divided by the IC50 value, which is used to assess the toxicity of flavonoids. If the ASI of the flavonoids is >100, they can be considered safe with a good antioxidant activity profile; however, further research is required to standardize it. Spagnuolo et al., 2012 carried out a study of quercetin toxicity in male F344/N rats fed 2 g per kg body weight per day of quercetin. The results showed severe chronic nephropathy, hyperplasia, and kidney tubular epithelium neoplasm after the treatment. In another study, it was reported that maternal intake of quercetin during pregnancy could increase the risk of mixed lineage leukemia (MLL) gene rearrangements, which is common in childhood leukemia, especially in the presence of compromised DNA repair . However, Utesch et al. (2008) demonstrated that orally administered quercetin (up to 2 g/kg body weight/day) did not induce unscheduled DNA synthesis in male or female rat hepatocytes, suggesting that quercetin was not genotoxic. Additionally, in the only phase I clinical trial of quercetin found, renal toxicity was detected without signs of nephritis or obstructive uropathy when a dose of 50 mg/kg (approximately 3.5 g/70 kg) was administered by intravenous infusion at three weeks or at weekly intervals . Eryngium spp. Propagation Strategies Given the wide variety of uses that different species of the genus Eryngium have, it is important to develop strategies to cultivate them, but some limitations should be faced, such as non-synchronized or uniform seed germination, low germination rates, lack of seed availability, and the need to reach higher sowing rates . In this sense, due to the scarce reports found in literature about the propagation of the Eryngium species located in Mexico, particularly in the central-western region, some works in other species were reviewed as a starting point for future development in this matter. Mozumder et al. (2010) achieved an increase of E. foetidum germination rate, this species is of great importance in Latin America and other parts of the world. The authors reported that gibberellic acid (GA3, 1000 ppm) and kinetin (50 ppm) were effective to promote germination up to 28.54%. In a complementary work, Mozumder et al. (2017a) evaluated different storage conditions, application of growth regulators, and soaking to increase E. foetidum seeds’ germination rate. Seeds kept at low temperature (3–5 °C) had a germination percentage of 18.4% and increased to 32.3% with 12 h of soaking and the addition of growth regulators. Mozumder et al. (2017 b) evaluated seed germination and field performance of E. foetidum using soaking, growth regulators, and pesticide (0.2% copper oxychloride + 1000 ppm tetracycline) combinations. The maximum percentage of germination (74.7%) and early germination (12 days) was obtained using growth regulators (GA3 500 ppm and kinetin 50 ppm) with 96 h of soaking. Additionally, Mozumder et al. (2017c) evaluated the growth and biomass production observing that the profitability increased, and the seed production cost decreased; the authors reported the highest values of the following variables: germination percentage, number of seedlings, number of harvestable plants/m 2 , number of leaves per plant, leaf width and biomass, at 30 and 60 days with the application of growth regulators. On the other hand, Mozumder et al. (2020) used three different levels of shades and two planting methods at two locations to cultivate E. foetidum . The maximum gross yield (4944.2 thousand TK/ha) and the net yield (4438.2 thousand TK/ha) were obtained with the shade of nylon netting in broadcast planting, which was better to produce leaves and obtain higher profits. These results coincided with those reported by Kuttan (2008) who cultivated E. foetidum at three shade levels and four plant spacings. The maximum yield was obtained with a shade level of 75% (1411.04 g/plot size 120 × 150 cm). From storage studies it was concluded that at room temperature the leaves could be stored for a maximum of 5.2 days with a shade level of 75%, while under cold storage conditions and a shade level of 75% the leaves could be stored for a maximum of 109.65 days without any deterioration. The highest cost-benefit ratio (1.28) was obtained with a shade level of 75% and 15 cm × 15 cm spacing. One of the few published works reporting the propagation of Eryngium species in Mexico is that of Carrera-Quirino and Colohua-Citlahua (2014) who generated a reproduction strategy for Eryngium proteaeflorum in a nursery. The authors established recommendations for the collection of germplasm, characteristics of the mother plants, collection strategies to avoid damaging the habitat of the species and allow natural regeneration, as well as for propagation in seedbeds and transplant to planting bags. Another propagation method reported for Eryngium spp. is the vegetative tissue culture (e.g., leaf, stem, or root segments) using culture media such as Murashige and Skoog (MS). The use of basal medium supplemented or not with sucrose, macro and micronutrients has been suggested . Additionally, different explants and growth regulators, such as indole butyric acid (IBA) and indole acetic acid (IAA), have been used in various combinations , and the treatment with IBA 500 ppm and 500 ppm IAA using 7 to 10 cm shoots was the best to promote growth and obtain commercial seedlings. On the other hand, Ayuso et al. (2019) reported the use of different growth inducers, sucrose, and salt concentrations for the propagation of E. viviparum , achieving a 96% survival rate in plants. In the same way, Martin (2006) , Nagananda et al. (2012) , and Jena et al. (2020) reported protocols for the fastest and most successful clonal propagation of E. foetidum reaching an 85–90% plant survival rate using different explants, doses of inducers, and nitrogen sources. The use of plants belonging to the genus Eryngium is widely distributed around the world, especially for medicinal and culinary purposes. The species of the genus whose presence and use in phytotherapy have been reported in central-western Mexico, especially in the states of Michoacán and Jalisco, are E. cymosum , E. longifolium , E. fluitans (or mexicanum ), E. beecheyanum , E. carlinae , E. comosum , E. heterophyllum , and E. nasturtiifolium . The use of which has been recorded for the treatment of at least 27 ailments, since diverse benefits are attributed to them, such as antibacterial, anticrotalic, antispasmodic, antigonorrheal, anti-inflammatory, antioxidant, antipodagric, antitumor, carminative, digestive, hypoglycemic, hypocholesterolemic, and diuretic, among others; therefore, they are considered of great significance in the local populations’ health care. However, not all of them have been validated by scientific studies of the corresponding biological activities. Studies of biological activities have been reported only for five of them, and some of the metabolites that four of these Eryngium spp. contain, have been identified. E. carlinae is the most studied species, and its hypoglycemic and hypolipidemic properties have been confirmed both in vitro and in vivo models. In addition, it has shown antioxidant, antimicrobial, and anti-inflammatory activities. Regarding its phytochemistry, more than 30 different compounds belonging to various groups of metabolites have been identified in E. carlinae extracts : hydroxybenzoic acids, hydroxycinnamic acids, phenolic acids, flavonoids, flavanols, flavanones, hydroxybenzaldehydes, phytosterols, saponins, terpenes, and sesquiterpenes. Much less information about the other species is published, and there are fewer toxicity studies. Therefore, more research is required on its phytochemistry and in vitro and in vivo biological activities. It is recommended to carry out in vivo tests with the extracts and metabolites of the Eryngium species that have been studied in vitro, and to later carry out clinical trials on inflammatory, infectious, and chronic diseases. An important aspect to investigate is the elucidation of the mechanisms of action associated with the beneficial effects reported. Additionally, the separation, isolation, and identification of the active metabolites or bioactive compounds must be performed since the greatest advances are made only in E. carlinae , followed by E. cymosum and E. longifolium . Having this information will allow the development of effective and safe treatments, drugs, and supplements, in addition to validate and promote the use of this plant resource for medicinal purposes in the communities that possess them. However, toxicity issues need to be addressed. This literature review did not show any reports on the propagation or cultivation strategies of the Eryngium spp. addressed in this review, so it is necessary to start developing them to achieve the sustainable use of this important plant resource since they are obtained mainly by collection, causing populations to decline.
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Harnessing the Power of PAOO and Invisalign: An Interdisciplinary Approach to Orthodontic Care
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2fed6836-9c2c-494a-830b-44986efad7cd
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10221098
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Dental[mh]
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Periodontally accelerated osteogenic orthodontics (PAOO) is a dental technique that has evolved from the corticotomy-assisted orthodontic treatment (CAOT) concept . PAOO combines selective decortication bone activation with orthodontic forces, revolutionizing dentistry by providing personalized treatment options that minimize complications and improve overall outcomes. The interdisciplinary approach of PAOO reduces the risk of root resorption, relapses, decay, and infection while accelerating tooth movement and enhancing various orthodontic treatments . Considering the lack of clinical studies related to PAOO in the literature, it is important to consider well-known treatments (e.g., orthognathic surgery) before choosing this elective approach. However, the significance of PAOO lies in its ability to offer better treatment outcomes for patients. By using bone grafting materials in conjunction with corticotomy procedures, PAOO aims to prevent adverse outcomes such as bony defects, gingival recession, and potential damage to the periodontal structures [ , , ]. In addition, PAOO is an effective technique that significantly accelerates tooth movement, making it particularly advantageous for adult patients who desire to avoid lengthy ortho-surgical procedures. This method yields optimal results by stimulating the alveolar bone remodeling process and enhancing the biological response, leading to faster tooth realignment and overall shorter treatment duration. Patients highly value this expedited approach as it minimizes the impact of prolonged treatment on their personal and professional lives . PAOO’s interdisciplinary approach provides numerous patient benefits, including accelerated treatment times, improved orthodontic outcomes, and minimized complications. PAOO focuses on preserving periodontal structures and addressing potential bony defects, ensuring orthodontic treatments’ long-term success and stability . PAOO can also be combined with invisible aligners, such as Invisalign, to provide a more aesthetically pleasing and comfortable experience for the patient . Integrating advanced orthodontic technologies expedites treatment and offers a discreet solution for individuals seeking to improve their smile with minimal impact on their daily lives . This study aims to provide reliable insights into the potential benefits of combining advanced orthodontic technologies to achieve optimal treatment outcomes. The authors utilized the PAOO and Invisalign technologies to offer customized treatment options that minimized complications and improved overall outcomes. The study presents two challenging cases successfully treated using the PAOO technique in conjunction with Invisalign. The findings of these reports will contribute significantly to the advancement of orthodontic treatment, offering valuable information to clinicians seeking to provide their patients with the most effective treatment options.
The authors of this study have followed the CARE (CAse REport) guidelines to ensure a thorough and transparent reporting of their clinical and radiographic findings. By adhering to the CARE checklist and recommendations, they have collected and reported data systematically, which increases the reliability and generalizability of their findings. This approach to data collection, analysis, and reporting enhances scientific knowledge and enables future research to replicate and validate their results. The two cases followed the same procedure, but the patients differed in age—one was 26, and the other was 34. The 26-year-old woman had an anterior open bite, while the 34-year-old woman had a Class III malocclusion (according to Angle’s Classification) and a severe crossbite ( ). Both patients had previously undergone evaluations with cephalometric radiography and tomography. NemoFAB Version 2.1 (NEMOTEC software, Madrid, Spain) was used to evaluate the patients, which led to the suggestion of orthognathic surgery as the initial ortho-surgical approach. However, periodontally accelerated osteogenic orthodontics (PAOO) was also presented as an alternative treatment option. Before the start of the treatment process, the dental team made sure to inform both patients of the proposed treatment plans. They went into great detail in explaining all the possible risks, benefits, and outcomes associated with each procedure. This was to ensure that the patients had a clear understanding of what to expect every step of the way throughout their treatment. Both patients were given the opportunity to ask questions and seek clarification on any concerns they may have had. After doing so, they willingly agreed to proceed with their respective treatment plans. The dental team took all the necessary steps to obtain formal agreement from the patients, ensuring that the process was thorough and complete. This approach was taken to ensure that the patients fully understood the consequences of their decisions and were confident in their choices. The patients underwent a PAOO procedure to correct the malocclusion. Before the surgery, the patients underwent a cone beam computed tomography (CBCT) scan to assess their bone structure so a customized Invisalign treatment plan could be developed ( A). During the surgery, a piezoelectric motor (Piezosurgery ® , Piezosurgery Inc., a Mectron Company, Columbus, OH, USA) with a straight tip (OT12) was utilized to create precise cuts in the cortical bone between the teeth. The cuts extended approximately 5 mm to the middle of the palatal bone raphe ( B,C). They were followed by bone decortication using a diamond tip (OT1). After making the cuts between the teeth, an allograft bone graft (Maxxeus ® , Dayton, OH, USA) was placed over the area to enhance and strengthen the bone structure. In addition, an allograft membrane/barrier was placed on top of the bone graft to protect the area. The bone graft was prepared using leucocyte–platelet-rich fibrin (L-PRF) . In order to produce L-PRF, both red and white cap tubes were utilized to centrifuge the patient’s blood using a machine manufactured by Biohorizons ® (AL, USA). Taking into account the centrifuge’s balance and the types of tubes used, a single centrifugation was performed to simultaneously produce the L-PRF membrane and Liquid L-PRF, following the “One Spin—Double Process” (a method by C.F.M.) (L-PRF protocol: ~700 RCF-max (~400 RCF-clot) for 8 min) . After producing the L-PRF, the membranes were cut into smaller pieces and mixed with the bone graft. Subsequently, the Liquid L-PRF was incorporated into the graft, and after a waiting period of 4 to 6 min, the clot was integrated into the bone substitute, creating the “Sticky Bone” . ( D), which facilitated the handling of the graft material. Following the bone graft and membrane (Pericardium, Maxxeus ® , OH, USA) placement ( F) on top of the pericardium membrane, the L-PRF membrane was placed, and primary closure was achieved using Polytetra Fluoro Ethylene (PTFE) 4–0 sutures (Ethicon ® , Johnson & Johnson, New Brunswick, NJ, USA). Both were prescribed Augmentin 625 mg (amoxicillin and clavulanate) every 12 h for seven days and Naproxen Sodium 220 mg every 12 h for five days to manage pain and prevent infection. In addition to the medications, the patients were instructed to maintain proper oral hygiene by using a Chlorhexidine 0.12% mouthwash twice daily for 14 days. After 10 days, the patients were scheduled to have their sutures removed. Suture removal is an important step in the postoperative care process, as it ensures that the healing tissues have had ample time to knit together and regain strength. In addition, removing the sutures at the appropriate time minimizes the risk of complications such as wound dehiscence or infection, and the patients can progress toward full recovery. After the surgical procedure, they began using the first Invisalign (Invisalign ® , La Puente, CA, USA) tray and changed trays weekly for a total treatment period of 12 months. This orthodontic intervention successfully corrected the present cases, showcasing the effectiveness of the PAOO procedure in combination with Invisalign therapy ( and ). Both patients must undergo regular check-ups to ensure the effectiveness of their treatment in the long run. Dental professionals will consistently monitor their orthodontic corrections to prevent any potential relapse and assess their stability. Furthermore, the patients have been educated on the critical importance of maintaining proper oral hygiene practices and receiving regular dental cleanings to uphold optimal oral health.
Periodontally accelerated osteogenic orthodontics (PAOO) provides a major edge to patients seeking to fix dental irregularities without resorting to orthognathic surgery and modifying their facial appearance. Its primary advantage lies in its capacity to accelerate tooth movement, which proves particularly advantageous for adult patients worried about the duration of ortho-surgical procedures. The interdisciplinary approach of PAOO allows for enhanced resolution of dental issues such as crowding, impacted teeth, open bites, and molar intrusion. Combining PAOO with Invisalign provides patients with a more aesthetically pleasing and comfortable treatment option. Invisalign aligners are discreet and removable, enabling patients to maintain their self-esteem and confidence throughout the treatment . Additionally, using Invisalign aligners can make it easier for patients to maintain proper oral hygiene during treatment, reducing the risk of dental decay and gum disease. A review conducted recently has highlighted that PAOO is an attractive option for certain patients due to its numerous benefits. One significant advantage of this technique is that it enhances the volume of the alveolar bone and periodontium, allowing for the correction of dehiscences and fenestrations. Moreover, PAOO is known to reduce treatment time, providing 3–4-times-faster active orthodontic treatment when compared to traditional methods. This technique also offers better post-treatment stability and a lower risk of relapse, extending the scope of malocclusion treatment, and in some cases, avoiding orthognathic surgery and extractions. PAOO can also be utilized in some cases to improve a patient’s profile if needed and facilitate the rapid recovery of impacted teeth, such as canines. In both cases discussed in this report, the benefits of this technique were observed. Additionally, bone grafting materials are used in PAOO procedures; this helps to maintain the integrity of the periodontal structures and prevent issues such as bony defects and gingival recession, which are often seen in traditional orthodontic treatments. This approach also helps to ensure the overall health of the periodontium, which is crucial for the long-term success and stability of orthodontic treatment outcomes . While PAOO has its benefits, it may not be appropriate for all patients due to certain contraindications . Individuals with thinner mandibular cortices may have a higher risk of complications and should avoid this treatment. Patients who have active periodontal disease or gingival recession should also steer clear of PAOO as it could worsen their existing conditions. PAOO is not recommended for palatal expansion or in the treatment of severe posterior cross-bite since it may not yield the desired results. Lastly, it is not advisable to use PAOO for bimaxillary protrusion accompanied by a gummy smile. In such cases, alternative treatment options may be more effective in achieving optimal outcomes. It is crucial to consider any possible negative effects and complications when receiving treatment [ , , , , ]. An aesthetic issue that may occur is a black space between the incisors after treatment, as previously mentioned. Despite this, the patient expressed satisfaction with the outcome, highlighting the importance of keeping patients informed and addressing their concerns throughout the treatment process. In the second scenario discussed, the authors underwent palatal expansion. However, this is not the usual method used to address such cases. The authors suggest conducting further clinical studies to provide more insight into the matter. Dental professionals can ensure that patients make informed decisions about their care by thoroughly explaining the treatment plan, potential risks, and benefits. Furthermore, proper post-treatment follow-up and maintenance can help identify and address potential complications or concerns, ensuring patients achieve the most favorable results and long-term satisfaction with their treatment. The presented surgical technique aims to offer an ortho-surgical alternative in treating patients with dentofacial deformities. Nevertheless, it does not eliminate the surgical and compensatory treatments documented in the literature. It is essential to consider the patients’ preferences, as both the selection and conclusion of treatment are influenced by their opinions. Consequently, it is imperative to conduct future clinical studies (e.g., evaluation of different ortho-surgical approaches) that compare the available options while considering the individual considerations of each patient.
Combined with Invisalign, periodontally accelerated osteogenic orthodontics (PAOO) offers a viable alternative to orthognathic surgery for patients seeking to correct dental disorders. With the potential to accelerate treatment times, preserve periodontal structures, and provide a more discreet and comfortable treatment experience, PAOO and Invisalign have the potential to enhance patient satisfaction and improve overall treatment outcomes. Recognizing these promising aspects, the authors of the present article encourage further clinical studies to substantiate and refine the use of the PAOO technique in orthodontic practice. However, it is crucial for dental professionals to manage patient expectations and address potential complications to ensure the best possible results.
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Revealing the role of the rhizosphere microbiota in reproductive growth for fruit productivity when inorganic fertilizer is partially replaced by organic fertilizer in pear orchard fields
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e4d3ff60-1953-434b-beb1-5230a9b6d5b0
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10221533
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Microbiology[mh]
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The world is facing a fruit productivity crisis as a result of population explosion, environmental deterioration, and epidemic resurgence. Pear growers aim to ensure the normal operation of the global fruit supply chain by relying primarily on chemical fertilizers (CF) to boost fruit productivity, but this has adverse effects on soil biodiversity for fruit security and stable yields (Hu et al., ). The food crisis has led to the further intensification of the use of CF to improve crop yields. However, the economic cost of agrochemicals is often high, and the long‐term use of CF may lead to soil degradation, loss of biodiversity and other negative effects, this has led to strong demand from society and regulators for reduced use of chemicals in agriculture (Batista & Singh, ). The use of CF among pear farmers has increased exponentially as they chase short‐term high‐yield (Liu & Yang, ). Nearly 70% of the pears in the world are produced in China, and this high production is accompanied by a higher CF application than in other countries (Ma & Diao, ). In China, approximately 506 kg of chemical fertilizer is used to produce 17.39 tons of pears per hectare in 2018, and the contribution rate of CF to yield was estimated to be about 56.81%, which is 2.3 times the average consumption in Japan and 3.7 times that in the United States. Excessive chemical fertilization only provides the nutritious elements for soil, resulting in a deficiency of organic matter in the soil. As decomposers of organic matter, the microbiota is in a state of ‘hunger’ in environments lacking organic matter, and the soil enters a cycle of continuous fertility decline (Savci, ; Wu et al., ). Excessive application of nitrogen‐ and phosphorus‐based fertilizers would inevitably lead to the reduction of yield and fruit quality (Chen et al., ). Curtis et al. ( ) showed that the uncontrolled increase of nitrogen fertilizer would not only not be absorbed by plants, but also delayed the fruit maturity, reduced total sugar (TS) content, and aggravated the occurrence of fruit diseases for storage in pear. Vitousek et al. ( ) found that excessive CF made the colour of ripened fruit lighter than the colour observed under normal fertilization levels. Furthermore, increasing the fruit's titratable acid (TA) concentration and reducing the total soluble sugar (TSS) content makes the taste worse. Compared with soil applied with organic fertilizers in pear orchards, the decline of soil microorganisms and soil microbial biomass carbon and nitrogen, especially Actinobacia, was accompanied by the yield decline by long‐term chemical fertilizer application (Zhang et al., ). As a perennial plant, the pear ( Pyrus spp.) has undergone a slow process of transition from vegetative growth (tree canopy) to reproductive growth that is conducive to productivity improvement by absorbing soil nutrients from fertilizer. However, The demand for an increasing supply of agricultural products through agriculture is a key contributor to soil fertilizer loss (Timmis & Ramos, ). More than that, massive soil fertilizer loss every year after the pear harvesting of mature fruits and branch pruning (Moore et al., ). Excessive application of CF not only cannot remedy the soil fertility loss, but also damages the soil health, which is crucial to soil retention, plant health, agricultural productivity and biodiversity protection (Timmis & Ramos, ). Organic fertilizer is needed to mitigate soil degradation and remedy nutrient loss to improve soil organic carbon (SOC) storage (Gross et al., ; Singh & Benbi, ). It may also enhance soil microbial biomass and change microbial community structure (Torres et al., ). Shen et al. ( ) reported that organic fertilization alter the soil community structure, in particular, better organizing the potential species involved in disease suppression for plant and soil health. Manure from poultry and livestock is a form of low‐cost high‐quality organic fertilizer that can provide suitable nutrients to fruit trees (Dong & Shu, ; Hong et al., ). SOC promoted by long‐acting organic fertilizer can improve bacterial diversity and stimulate microbiota (Firmicutes, Proteobacteria, and Zygomycota), which can decompose organic compounds (Davide et al., ). Zhao et al. ( ) carried out a study of inorganic and organic fertilizer for five consecutive years in an apple orchard, and the SOC content of soils fertilized with organic fertilizer had increased by nearly 18.5% compared with single chemical fertilizer‐treated soils. Organic fertilizer has a significant effect on the promotion of microbial biomass, mainly because SOC is the limiting factor for microbial reproduction, and the addition of organic fertilizer increases the amount of carbon input into the system (Debosz et al., ). The addition of organic fertilizer from pruning compost and sheep manure to the soil led to a significant increase in the number of soil microorganisms and soil microbial biomass carbon and nitrogen (Zhang et al., ). Organic fertilizers increase the health of the soil, there is no denying the positive effect of chemical fertilizers. Reasonable application of inorganic and organic amendments can effectively increase soil nutrient content, improve soil physical and chemical conditions and stimulate the metabolic intensity of soil microorganisms. Long‐term positioning studies have shown that combined inorganic mineral and organic fertilization could improve the SOC and microbial biomass. In addition, mixed organic–inorganic fertilizer was the greatest soil fertility distributor for root growth, yield, and quality in pears (Ding et al., ; Shimada et al., ). Although the functions in the process of nutrient decomposition of many soil microorganisms in the soil ecosystem were confirmed, our understanding of the relationship between the soil microbial community in decomposing the soil nutrition, as well as the response of plant growth, is still limited. With the rapid advancement in high‐throughput molecular technologies, most previous studies have gradually shifted from focusing on the evaluation of minerals and organic matter to excavating dominant microflora that can decompose the nutrients that can be absorbed by the root system (Duan et al., ; Sun et al., ). As extensive industrial agricultural practices may cause soil degradation, there is an increasing need for research into the remedial effects of organic fertilizers such as sheep manure on soil health and soil microbiota. The bacterial community enhancement mediated by sheep manure amendment was more complex than the fungal community enhancement (Barakat & Al‐Masri, ; Huang et al., ). Researchers previously discovered that long‐term organic fertilization could alter the spatial scaling of microbial biodiversity (Liang et al., ). Huang et al. ( ) confirmed that long‐term chemical fertilization decreased the complexity of networks between plant and microbial functional communities. The novelty of this study was its emphasis on examining the potential function of fertilizer‐derived bacteria, which were beneficial to transform from vegetative growth to reproductive growth for improving fruit productivity, to address the following questions: (1) How did the soil bacterial community assemblage respond to the addition of SM? (2) What specific taxa were positively correlated with plant productivity following SM? (3) What core nutrients enhanced the networks between soil bacterial community and tree growth?
Experimental design In November 2018, a long‐term field experiment was initiated on Chongming island of Shanghai, in the eastern region of China (31°51′15″ N, 121°54′00″ E), south of the Yangtze River, in an area suitable for the growth of sand pear ( Pyrus pyrifolia ( Burm. f .) Nakai). The annual average temperature is 16°C, the average annual rainfall is 1025 mm, and the average altitude is 4 m above sea level. The soil prior to fertilization in the pear orchard was originally saline‐alkaline soil (pH = 7.70). The soil types are primarily sandy loam with good tillage. Experimental ‘Cuiguan’ pear trees were 12 years old (row spacing was 2.5 m × 4.0 m), with a density of 1005 plants/ha. The area of the pear orchards with different fertilization formulas was 1.3 ha. The test plot adopted a random arrangement design, and 20 trees were treated per experimental tests. The cultivation techniques and the pest control were traditional management approaches. Fully fermented commercial sheep manure was used as base fertilizer and applied to the tree tray every November from 2018 to 2021. The field experiment included four fertilizer treatments: For details of the different proportions of SM and CF applied to the pear orchards at 40 days and 70 days after flower blooming from 2018 to 2021, refer to Table below. Number and proportion of branches for tree canopy structure In 2021, during the fourth winter of the continuous fertilizer experiment, when the leaves had fallen from the pear tree, a single plant was used as an experimental unit with 10 replicates. The tree canopy structure included long (length longer than 20 cm), medium (length between 5–20 cm), and short branches (length less than 5 cm), their number was counted with a counter. Fruit yields and fruit qualities The fruit of ‘Cuiguan’ pears entered the mature stage on 28 July from 2019 to 2021. For each fertilization treatment, a single plant was considered as an experimental unit for statistical evaluation, five replicates for each treatment, 20 fruits from each of the five pear trees were randomly selected from the upper, middle and lower parts of the canopy, and the single fruit weight was weighed with an electronic balance (ME‐T, Thermo Fisher , US) were treated for each fertilizer treatment. The fruit‐setting number of each tree was counted with a counter. During the harvest period of the pears, the middle of the fruit‐soluble solid was measured using a handheld refractometer (Master‐20a, ATAGO, Japan). The organic acid content in the fruit was determined by acid–base titration to determine the total acid content (Thitaporn et al., ). Total acid content % = kc V3 V1 / w V2 × 100 % , where w is the weight of the sample (g), c is the concentration of NaOH (mol/L), k is the conversion factor, V1 is the total amount of sample solution during extraction (mL), V2 is the total amount of sample solution (mL) during measurement and V3 is the amount of NaOH consumed (mL). The fruiting amount of the whole tree was counted as a unit, with a single plant as a plot, with 20 replicates for each treatment. A total of 20 pear fruits of each treatment were peeled, and 5 g of the middle pulp of the fruit was taken and frozen at −40°C. In order to determine the soluble sugar content in pear fruits, the protocol previously described by Hudina and Tampar ( ) with minor modifications was used. After grinding the pulp with liquid nitrogen, 0.5 g of it was weighed, and 5 mL of 80% ethanol was added and allowed to extract at 35°C for 20 min. This was then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The extraction was repeated twice, adding 2 mL of 80% ethanol each time. The supernatant was then combined and the volume was made up to 10 mL. It was then steamed in a Concentrator plus vacuum concentrator (Eppendorf, Germany) for 3 h until completely dry and removed. The dry matter was dissolved with 1 mL of redistilled water, filtered through 0.22 μm aqueous phase filter membrane and the supernatant was collected and used for high‐performance liquid chromatography (1525, Waters, US). The Waters Sugar‐Pak 1 chromatographic column (Waters, USA) has a column temperature of 90°C; the flow rate was 0.5 mL/min, and the Waters 2414 Refractive Index Detector (Waters, USA) was used to determine the soluble sugar content. The sugar component contents (e.g. sucrose, glucose, fructose and sorbitol) were calculated by the external standard method, and the sum of the four was calculated as the total soluble sugar content. The standards used were obtained from Sigma (Sigma‐Aldrich, USA), and the components and content of fructose were analysed. Soil sample and chemical properties Considering the slow release of SM nutrients, the chemical properties of soil were evaluated in the fourth year of the test on October 17 in 2021. The vertical soil layers of 0–30 cm (main distribution layer of the root system in pear) of soil layers were selected in each test plot by the 5‐point sampling method, using a 50 mL centrifuge tube to sample approximately 20 g at each sampling point, five replicates, and placed the soil samples in liquid nitrogen to freeze for 16S rRNA gene sequencing. The rest of the soil samples grinned through a 100‐mesh sieve after drying in the shade for chemical properties determination. Soil pH and electrical conductivity values (EC‐values) were assayed with a pH meter (DZS‐706, INESA, China) at a soil: distilled water ratio of 1:2.5 (v/v) with standard electrodes. The soil organic matter (SOC) was measured using the K 2 CrO 4 oxidation method (Bao, ). The total nitrogen (TN) content was measured using the Kjeldahl method (Bremner, ). Alkaline hydrolysis nitrogen (available nitrogen, AN) was determined using a slightly modified version of the alkaline hydrolysis‐diffusion absorption method. The soil samples were first hydrolyzed with 1.8 mol/L NaOH solution, and if the soil nitrate‐nitrogen content was high, ferrous sulfate was added to reduce this to ammonium nitrogen. Since ferrous sulfate itself will neutralize part of sodium hydroxide, it is necessary to increase the concentration of alkali (1.8 mol/L, so that the concentration of alkali is kept at 1.2 mol/L). Under constant temperature conditions, the effective nitrogen is alkali‐hydrolyzed and converted into an ammonia state, and it is continuously diffused and escapes, is absorbed by H 3 BO 3 , and then titrated with standard hydrochloric acid to determine the content of soil hydrolyzable nitrogen. Available phosphorus (AVL P) was determined by the sodium bicarbonate extraction‐molybdenum antimony anti‐colourimetric method and extracted AVL P from the soil by ultrasonic wave (Karina & López, ); available potassium (AVL K) was determined to use flame photometer after NH 4 OAc extraction (Sadri et al., ). DTPA extraction was used to measure available copper (AVL Cu), available Fe (AVL Fe), available Mn (AVL Mn) and available Zn (AVL Zn), the exchangeable Ca (EB Ca) and Mg (EB Mg) were extracted by ammonium acetate‐flame photometry (Zhao et al., ). Bacterial diversity of the pear orchard soil by 16S rRNA gene sequencing Pear tree rhizosphere soil samples from the four fertilizer tests were snap frozen and stored at −80°C after collection. Bacterial DNA was isolated from the frozen four fertilizer test soil samples using a MagPure Soil DNA LQ Kit (Magen, Guangdong, China) following the protocol described by the manufacturer. DNA concentration and integrity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the V3‐V4 hypervariable regions of the bacterial 16S rRNA gene was carried out in a 25 μL reaction mix using universal primer pairs (343F: 5′‐TACGGRAGGCAGCAG‐3′; 798R: 5′‐AGGGTATCTAATCCT‐3′). The reverse primer contained a sample barcode and both primers were connected with an Illumina sequencing adapter. The amplicon quality was visualized using gel electrophoresis. The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., USA) and quantified using a Qubit dsDNA assay kit. The concentrations were then adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq6000 with two paired‐end read cycles of 250 bases each. (Illumina Inc., China). Raw sequencing data were in FASTQ format. Paired‐end reads were then processed using Cutadapt software (Martin, ) to detect and cut off the adapter. After trimming, paired‐end reads were filtered for low‐quality sequences, denoised, merged and detected, and chimera reads were cut off using DADA2 (Chao & Bunge, ) with the default parameters of QIIME2 (Hill et al., ). Finally, the software output the representative reads and the ASV abundance table. The representative read of each ASV was selected using QIIME 2 package. All representative reads were annotated and blasted against Silva database Version 138 (or Unite) using q2‐feature‐classifier with the default parameters. The 16S RNA gene amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). Data statistics method Referring to 16S rDNA gene sequencing data, each soil sample had five replicates, and a total of 20 samples. The top 30 data of genus level abundance was used to construct a stacked column chart using ggplot software (MathWorks, USA). SPSS 17.0 software (IBM USA) was used for statistical analysis of the data of branches, yields, qualities, and soil chemical properties. For each processing, we use the one‐way analysis of variance (ANOVA) with the OM, OC1, OC2 and CK fertilization treatments as fixed factors. The LSD method to analyse the difference significance data was 5%. A mean comparison of four fertilization treatments was performed based on Tukey's honestly significant difference (Duncan) test at the 0.05 probability level. The Pearson correlation coefficients (PCCs) were used to calculate linear correlations between soil chemical properties and soil microbial community by Origin 2021 software (OriginLab USA). The microbial diversity in soil samples was estimated using the alpha (α) diversity that includes the Chao1 and Shannon indexes. Beta (β) diversity was evaluated by the nonmetric multidimensional scaling (NMDS), which is a multivariate statistical analysis of non‐constrained ranking, used to show the differences and similarities between soil samples. Biomarker signatures in each group were screened by Metastats and LEfSe software for the comparison of the four soil samples comparisons to characterize differences in the organism's gene expression and fertilization‐treated samples. The db‐RDA analysis uses the corr.test function of the vegan package in R (2.15.3) to calculate the Spearman correlation coefficient values of species and environmental factors and test their significance.
In November 2018, a long‐term field experiment was initiated on Chongming island of Shanghai, in the eastern region of China (31°51′15″ N, 121°54′00″ E), south of the Yangtze River, in an area suitable for the growth of sand pear ( Pyrus pyrifolia ( Burm. f .) Nakai). The annual average temperature is 16°C, the average annual rainfall is 1025 mm, and the average altitude is 4 m above sea level. The soil prior to fertilization in the pear orchard was originally saline‐alkaline soil (pH = 7.70). The soil types are primarily sandy loam with good tillage. Experimental ‘Cuiguan’ pear trees were 12 years old (row spacing was 2.5 m × 4.0 m), with a density of 1005 plants/ha. The area of the pear orchards with different fertilization formulas was 1.3 ha. The test plot adopted a random arrangement design, and 20 trees were treated per experimental tests. The cultivation techniques and the pest control were traditional management approaches. Fully fermented commercial sheep manure was used as base fertilizer and applied to the tree tray every November from 2018 to 2021. The field experiment included four fertilizer treatments: For details of the different proportions of SM and CF applied to the pear orchards at 40 days and 70 days after flower blooming from 2018 to 2021, refer to Table below.
In 2021, during the fourth winter of the continuous fertilizer experiment, when the leaves had fallen from the pear tree, a single plant was used as an experimental unit with 10 replicates. The tree canopy structure included long (length longer than 20 cm), medium (length between 5–20 cm), and short branches (length less than 5 cm), their number was counted with a counter.
The fruit of ‘Cuiguan’ pears entered the mature stage on 28 July from 2019 to 2021. For each fertilization treatment, a single plant was considered as an experimental unit for statistical evaluation, five replicates for each treatment, 20 fruits from each of the five pear trees were randomly selected from the upper, middle and lower parts of the canopy, and the single fruit weight was weighed with an electronic balance (ME‐T, Thermo Fisher , US) were treated for each fertilizer treatment. The fruit‐setting number of each tree was counted with a counter. During the harvest period of the pears, the middle of the fruit‐soluble solid was measured using a handheld refractometer (Master‐20a, ATAGO, Japan). The organic acid content in the fruit was determined by acid–base titration to determine the total acid content (Thitaporn et al., ). Total acid content % = kc V3 V1 / w V2 × 100 % , where w is the weight of the sample (g), c is the concentration of NaOH (mol/L), k is the conversion factor, V1 is the total amount of sample solution during extraction (mL), V2 is the total amount of sample solution (mL) during measurement and V3 is the amount of NaOH consumed (mL). The fruiting amount of the whole tree was counted as a unit, with a single plant as a plot, with 20 replicates for each treatment. A total of 20 pear fruits of each treatment were peeled, and 5 g of the middle pulp of the fruit was taken and frozen at −40°C. In order to determine the soluble sugar content in pear fruits, the protocol previously described by Hudina and Tampar ( ) with minor modifications was used. After grinding the pulp with liquid nitrogen, 0.5 g of it was weighed, and 5 mL of 80% ethanol was added and allowed to extract at 35°C for 20 min. This was then centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The extraction was repeated twice, adding 2 mL of 80% ethanol each time. The supernatant was then combined and the volume was made up to 10 mL. It was then steamed in a Concentrator plus vacuum concentrator (Eppendorf, Germany) for 3 h until completely dry and removed. The dry matter was dissolved with 1 mL of redistilled water, filtered through 0.22 μm aqueous phase filter membrane and the supernatant was collected and used for high‐performance liquid chromatography (1525, Waters, US). The Waters Sugar‐Pak 1 chromatographic column (Waters, USA) has a column temperature of 90°C; the flow rate was 0.5 mL/min, and the Waters 2414 Refractive Index Detector (Waters, USA) was used to determine the soluble sugar content. The sugar component contents (e.g. sucrose, glucose, fructose and sorbitol) were calculated by the external standard method, and the sum of the four was calculated as the total soluble sugar content. The standards used were obtained from Sigma (Sigma‐Aldrich, USA), and the components and content of fructose were analysed.
Considering the slow release of SM nutrients, the chemical properties of soil were evaluated in the fourth year of the test on October 17 in 2021. The vertical soil layers of 0–30 cm (main distribution layer of the root system in pear) of soil layers were selected in each test plot by the 5‐point sampling method, using a 50 mL centrifuge tube to sample approximately 20 g at each sampling point, five replicates, and placed the soil samples in liquid nitrogen to freeze for 16S rRNA gene sequencing. The rest of the soil samples grinned through a 100‐mesh sieve after drying in the shade for chemical properties determination. Soil pH and electrical conductivity values (EC‐values) were assayed with a pH meter (DZS‐706, INESA, China) at a soil: distilled water ratio of 1:2.5 (v/v) with standard electrodes. The soil organic matter (SOC) was measured using the K 2 CrO 4 oxidation method (Bao, ). The total nitrogen (TN) content was measured using the Kjeldahl method (Bremner, ). Alkaline hydrolysis nitrogen (available nitrogen, AN) was determined using a slightly modified version of the alkaline hydrolysis‐diffusion absorption method. The soil samples were first hydrolyzed with 1.8 mol/L NaOH solution, and if the soil nitrate‐nitrogen content was high, ferrous sulfate was added to reduce this to ammonium nitrogen. Since ferrous sulfate itself will neutralize part of sodium hydroxide, it is necessary to increase the concentration of alkali (1.8 mol/L, so that the concentration of alkali is kept at 1.2 mol/L). Under constant temperature conditions, the effective nitrogen is alkali‐hydrolyzed and converted into an ammonia state, and it is continuously diffused and escapes, is absorbed by H 3 BO 3 , and then titrated with standard hydrochloric acid to determine the content of soil hydrolyzable nitrogen. Available phosphorus (AVL P) was determined by the sodium bicarbonate extraction‐molybdenum antimony anti‐colourimetric method and extracted AVL P from the soil by ultrasonic wave (Karina & López, ); available potassium (AVL K) was determined to use flame photometer after NH 4 OAc extraction (Sadri et al., ). DTPA extraction was used to measure available copper (AVL Cu), available Fe (AVL Fe), available Mn (AVL Mn) and available Zn (AVL Zn), the exchangeable Ca (EB Ca) and Mg (EB Mg) were extracted by ammonium acetate‐flame photometry (Zhao et al., ).
16S rRNA gene sequencing Pear tree rhizosphere soil samples from the four fertilizer tests were snap frozen and stored at −80°C after collection. Bacterial DNA was isolated from the frozen four fertilizer test soil samples using a MagPure Soil DNA LQ Kit (Magen, Guangdong, China) following the protocol described by the manufacturer. DNA concentration and integrity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and agarose gel electrophoresis, respectively. PCR amplification of the V3‐V4 hypervariable regions of the bacterial 16S rRNA gene was carried out in a 25 μL reaction mix using universal primer pairs (343F: 5′‐TACGGRAGGCAGCAG‐3′; 798R: 5′‐AGGGTATCTAATCCT‐3′). The reverse primer contained a sample barcode and both primers were connected with an Illumina sequencing adapter. The amplicon quality was visualized using gel electrophoresis. The PCR products were purified with Agencourt AMPure XP beads (Beckman Coulter Co., USA) and quantified using a Qubit dsDNA assay kit. The concentrations were then adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq6000 with two paired‐end read cycles of 250 bases each. (Illumina Inc., China). Raw sequencing data were in FASTQ format. Paired‐end reads were then processed using Cutadapt software (Martin, ) to detect and cut off the adapter. After trimming, paired‐end reads were filtered for low‐quality sequences, denoised, merged and detected, and chimera reads were cut off using DADA2 (Chao & Bunge, ) with the default parameters of QIIME2 (Hill et al., ). Finally, the software output the representative reads and the ASV abundance table. The representative read of each ASV was selected using QIIME 2 package. All representative reads were annotated and blasted against Silva database Version 138 (or Unite) using q2‐feature‐classifier with the default parameters. The 16S RNA gene amplicon sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China).
Referring to 16S rDNA gene sequencing data, each soil sample had five replicates, and a total of 20 samples. The top 30 data of genus level abundance was used to construct a stacked column chart using ggplot software (MathWorks, USA). SPSS 17.0 software (IBM USA) was used for statistical analysis of the data of branches, yields, qualities, and soil chemical properties. For each processing, we use the one‐way analysis of variance (ANOVA) with the OM, OC1, OC2 and CK fertilization treatments as fixed factors. The LSD method to analyse the difference significance data was 5%. A mean comparison of four fertilization treatments was performed based on Tukey's honestly significant difference (Duncan) test at the 0.05 probability level. The Pearson correlation coefficients (PCCs) were used to calculate linear correlations between soil chemical properties and soil microbial community by Origin 2021 software (OriginLab USA). The microbial diversity in soil samples was estimated using the alpha (α) diversity that includes the Chao1 and Shannon indexes. Beta (β) diversity was evaluated by the nonmetric multidimensional scaling (NMDS), which is a multivariate statistical analysis of non‐constrained ranking, used to show the differences and similarities between soil samples. Biomarker signatures in each group were screened by Metastats and LEfSe software for the comparison of the four soil samples comparisons to characterize differences in the organism's gene expression and fertilization‐treated samples. The db‐RDA analysis uses the corr.test function of the vegan package in R (2.15.3) to calculate the Spearman correlation coefficient values of species and environmental factors and test their significance.
Number and proportion of branch types for tree canopy structure The number of short, medium, and long branches and the ratio between them are important parameters to evaluate the canopy structure in pear (Wei, Wang, Dong, & Dong, ). The fruit‐bearing branch generally forms the group of short branches in the sand pear ( P. pyrifolia cv. Cuiguan) genotype (Huang et al., ). As shown in Figure , the number of short and long branches and the total branches were significantly influenced by the long‐term application of different proportions of SM and CF. The total number of branches was the largest in OC1 (112) and lowest in the CK treatment group (94). Compared with the CK, the total number of branches in OC1 was significantly improved ( p < 0.05) by 18.4%. The total number of branches ranged from 94 to 112 and ranked in the following order: OC1 > OC2 > OM > CK. There was no significant difference between OM, OC1 and OC2, however, the OM treatment of 100% SM marginally increased ( p > 0.05) the total number of branches compared with CK. In‐depth analysis of the number and the proportion of long branches in the OC1 treatment was the lowest, while the short branches in OC1 treatment were the highest; the ratios of short, medium and long branches was also highest in OC1 treatment, which was 25:1:5 (Figure ). The most dramatic response of the number of long branch was observed for tree canopy structure after the higher proportion CF (Figure ). The OC2 treatment promoted the number of long branches significantly increased ( p < 0.05). The higher proportion of long branches number (28.96%) inhibited the growth of short branches, accounting for only 66.46%. The ratio of the short, medium and long branches was only 14:1:6 (Figure ).On the contrary, compared with a high proportion of SM in the OM‐ and OC1‐treatments, the number of short fruit branches was significantly decreased by 14.7% and 21.8%, respectively, in OC2, and was significantly decreased by 26.2% and 33.9%, respectively, in CK. Fruit yield and qualities of pear trees Continuous and stable growth of yields by the long‐term application of SM and CF from 2019 to 2021 (Table ). In 2019, the yield from OM‐treated soil was the highest among all treatments due to the highest fruit‐setting number (average of 133 fruits/tree), the yield reached 54.60 ton .ha −1 . In the next 2 years, the yield of OM decreased significantly. The average single fruit weight in the OC2 treatment was the highest (429.25 g), which was much higher than the OC1 and CK treatments 16% and 10.58%. In 2020, the single fruit weight (average of 412 g) and yield (53.04 ton.ha −1 ) in the OC1 treatment were the highest. Compared with the previous 2 years (2019–2020), the yield in OM and CK with the highest proportions of SM or CF treatment began to decline to some extent in 2021. For the OC1 treatment, compared with OM and OC2 treatments, the yield was obviously superior, which is mainly attributed to the increase in average single fruit weight from 368.98 g to 434.6 g. With the increase in the proportion of SM, the soluble solid content (SSC) in the fruit showed an increased trend (Table ). In 2019, 100% SM in OM treatment had the highest content of SSC (12.46%) in pear fruits, which was significantly ( p < 0.05) higher than that of CK treatment by 12.1%. Compared with the OM treatment, the SSC content decreased linearly with the decrease of the SM proportion, and the SSC of OC1, OC2 and CK was significantly reduced by 5.28%, 6.91% and 12.11%, respectively. Acidity in flavour was contributed by the titratable acid content (TA), which was marginally decreased in OM treatment. The SSC/TA ratio of the fruit treated with OM was highest (40.31), which was 19.48% higher than that in CK, and 11.29% and 9.42% higher than in OC1 and OC2, respectively. In 2020–2021, the SSC of OC1‐treated was highest, at 12.11% and 12.17% respectively, which was significantly higher than in the other treatments. On the contrary, the TA of OC1 treatment was the lowest, resulting in the highest SSC/TA ratio, which was above 39. The average fruit SSC/TA ratio ranged from 33.8 to 38.87 and ranked in the following order: OC1 > OM > OC2 > CK. A higher proportion of SM in OM and OC1 significantly ( p < 0.05) improved the average ratio of soluble substance and acid (SSC/TA ratio), was highest in OC1 with the CF (0.3 kg/tree) instead of 50% SM from 2019 to 2021. The pear fruits gradually entered the mature stage, and the application of SM significantly ( p < 0.05) increased the TSS and fructose content (Figure ). The TSS of OM‐treatment was the highest, which is attributed to the highest content of glucose (19.12 mg/g) and fructose (28.70 mg/g) compared with the control, representing an increase of 5.38% and 5.67% respectively. The sucrose content was highest (16.98 mg/g) in OC1, compared to the CK treatment, with an increment of 29.37%. With the increase in CF proportion, the brittleness of the fruit exocarp showed a downward trend, but the adhesiveness increased and the fruit flesh became soft and fluffy. (Figure ). The brittleness of fruit exocarp (1899.61 g) in OM was highest, compared to the CK treatment and was significantly ( p < 0.05) reduced by 26.32%. Chemical properties of pear rhizosphere soil and its correlation with the intrinsic quality and yield of pear fruit The test soil was originally slightly alkaline (pH = 7.7), while the pH of CK‐tested soil with 100% CF was acidic (pH = 6.94). The pH increased consistently as a result of the increased application of SM, and the pH ranged from 7.10 to 7.44 (Table ). The EC‐value under the CK treatment was the highest (850 us/cm), which was much higher than that of OC2, OC1 and OM treatment by 13%, 36% and 30%. Compared with the CK treatment, with the increase in the proportion of SM, the content of soil organic carbon (SOC)in the soil had a significant upward trend for continuous fertilization, ranging from 43.31% to 66.90%. The SOC,C/N ratio, available nitrogen (AVL N), available K (AVL K), available Fe (AVL Fe) and available Cu (AVL Cu), were highest in OM‐treated soil. When compared to other treatments containing a certain proportion of SM, the content of TN in 100% CF‐treated soil was significantly higher, with a value of 1.78 g/kg, resulting in the lowest C/N ratio (6.38). From the spearman correlation heatmap, we observed clustering of soil chemical properties with the fruit yield and qualities (Figure ). Most significantly, including a positive correlation between the SOC, TN, C/N ratio, AVL N of the soil and pear fruit qualities included the content of fructose, the content of sucrose, brittleness, the number of short branches and the number of total branches. To conduct an integrative analysis of the significant positive correlations that existed between the soil rhizosphere nutrient indicators (SOC, TN, C/N ratio, AVL N, AVL K, AVL Fe) and the content of fructose in pears. The same significant positive correlation also existed between soil AVL Fe content and SSC ( r = 0.82, p = 0.001) (Table ); the ratio of SSC/TA ratio ( r = 0.81, p = 0.002); the fructose content ( r = 0.83, p = 0.001), both of which contribute to the sweetness of the fruit in pear. In addition, the content of sorbitol in pear fruit was significantly positively correlated with soil pH ( r = 0.71, p = 0.001), but negatively correlated with the content of AVL P ( r = −0.73, p = 0.009). The AVL Fe and SOC in soil were significantly negatively correlated with the accumulation of TA ( r Fe = −0.74, p = 0.006; r SOC = −0.58, p = 0.046) in pear fruit. Bacterial community composition and diversity of rhizosphere soil in pear orchard The V3–V4 region of bacterial 16S rRNA genes was amplified to explore the effects of soil rhizosphere microbial communities caused by four different fertilizing methods. Alpha diversity was employed to analyse the complexity of bacterial species diversity in the pear rhizosphere soil (Table ). The bacterial population diversity index of four pear rhizosphere soil samples from different fertilizer treatments was sampled to the same sequencing depth (45,421 reads per sample) and after clustering, 63,007–66,198 valid tags operated with 97% identity were obtained from operational taxonomic unit (OTU), and the number of OTUs per sample was 3728–4123. At the OTU level, the results showed that the rhizosphere soil treated with pure SM in OM‐treated soil contained 4123 OTUs, which were significantly higher than those treated with OC1, OC2 and CK fertilizers. The number of common and unique OTUs in different samples is shown in Figure . The 315 OTUs were common to soils with four different fertilizing methods, accounting for 7.6%–8.4% of the total number of OTUs. The ace index and the chao1 index in the OM treatment were significantly larger than in the OC1 and OC2 treatments, which indicated that the more bacterial OTUs contained in the rhizosphere community, the greater the richness value of the bacterial community. The numbers of OTUs in the OM and CK treatments were significantly larger than in the OC2 treatment, at 9.80% and 9.39%, respectively. Shannon's index for soils that underwent the OM treatment was the largest, while this value in OC2 treatment was the lowest, but it was not significantly different from those treatments. β (Beta) diversity was used as a comparative analysis of the microbial community composition of different soil rhizosphere samples from the four fertilization treatments. Non‐metric multidimensional scaling (NMDS) results showed that no matter how much SM replaces CF, it would significantly change the composition of the soil bacterial community With the SM ratio increasing, the inter‐group distance from CK to OC1, OC2 and SM‐treated soils was gradually closer with the increase of the SM ratio to each other, indicating that their differences in soil microbial communities were increased by SM application (Figure ). Kruskal–Wallis algorithm was used to analyse the OTUs with significant differences between fertilizer treatment groups ( p < 0.05) (Figure ), and the data showed the OTU of the top 10 diversity species abundance. Among them, OTU178 and OTU1314 belonged to Acidobacterium, and their enrichment in OM group was significantly higher than in OC1 and OC2 groups. The relative abundance of OTU642, which belongs to Deltaproteobacteria was the highest in OM treatment, significantly higher than that in OC1 and OC2 treatment. The relative abundance of OTU4063, OTU592 and OTU1554, all belonging to Gammaproteobacteria, were the highest in the soil rhizosphere of OC1 treatment, while the relative abundance of these OTUs in the OC2 treatment with the increase in CF proportion. Bacterial community composition at the phylum and genus level The relative abundance of the four fertilizer treatments had similar major predominant bacterial communities, but these predominant bacteria exhibited divergent substance preferences. Results showed that the dominant bacterial communities (96%), included Proteobacteria (55%–58%), Acidobacteria (7%–13%), Gemmatimonadetes (7%–10%), Actinobacteria (6%–8%), Bacteroidetes (6%–9%), Firmicutes (1%–4%) and Nitrospirae (1%–3%), all of which belong to azotobacter, in the four soil rhizospheres from different fertilizer treatments (Figure ). At the phylum level, compared without any SM (CK), the relative abundance of Acidobacteria, Gemmatimonadetes, Nitrospirae, Calditrichaeota and Zixibacteria were higher in soil samples than that SM were applied into pear orchard; by contrary, the relative abundance of Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Fibrobacteres were relatively low. In addition, compared to the no‐SM in CK, with the input of SM rapidly increasing, the relative abundance of Firmicutes, Elusimicrobia, Patescibacteria, Entotheonellaeota and Chlamydiae increased. Among them, it was obvious that the input of SM has significantly ( p < 0.05) increased the share of Firmicutes from 1.5% to 4.22% (Table ). With the increase in the proportion of CF in the fertilizer formula, the other bacterial communities, such as Gemmatimonadetes, Calditrichaeota, Zixibacteria, Patescibacteria and Latescibacteria, increased significantly. At the genus level, we plotted the relative abundance of heat maps at the genus taxa level of the top 30 (the most abundant species) to further compare differences in bacteria composition among fertilizer treatments (Figure ). The relative abundance of genera included Gaiella, Clostridioides, [Ruminococcus]_gnavus_group, Ruminococcus_2, MND1, IS‐44, mle1‐7 and Chryseolinea genus were higher in OM than in the OC1, OC2 and CK treatments; conversely, the relative abundance of Actinomadura , Gemmatimonas , Geobacter , Flavobacterium , Luteimonas, Rhodanobacter , Sphingomonas in OM was lower than in the OC1, OC2 and CK treatments. Compared with pure SM in the OM‐treated soil, the relative abundance of [Ruminococcus]_gnavus_group , Ruminococcus_2 ., Chryseolinea, Gaiella decreased significantly in OC1, OC2 and CK treatments, which with the increase of CF proportion into SM; the relative abundance of Pseudolabrys, Subgroup_10 , Chujaibacter , Sphingomonas , Rhodanobacter , Luteimonas , Flavobacterium , Gemmatimonas , Geobacter and Actinomadura increased significantly in OC1 and OC2 treatments; it was noteworthy that the CF ratio increased to 100% (CK), and the relative abundance of these bacterial colonies decreased significantly. Comparative assessment of soil after different proportioning of organic manure and chemical fertilizer treatment We utilized the linear discriminant analysis effect size (LEfSe) analysis to compare the bacterial affiliations (i.e. biomarkers) around the soil rhizosphere with different fertilizer treatment groups (Figure ). Abundance of different bacterial biomarkers was discriminated between fertilizer treatment groups according to linear discriminant analysis (LDA) > 2; p < 0.05. The cladogram revealed that 141 biomarkers were identified in different groups (Figure and Table ). The minimum number of discriminant clades significantly increased between OC1 (2 biomarkers) and OM (10 biomarkers) groups (Figure and Table ) indicating that the Halioglobus genus (LDA = 2.02, p = 0.005) was significantly enriched in OC1 group, that CF added into SM. Candidatus_Berkiella genus, Pseudogulbenkiania and Tagaea were significantly enriched in OM. The analysis revealed that 15 biomarkers were associated with OM and 5 were associated with CK groups (Figure and Table ). The Reyranellaceae family (LDA = 2.99, p = 0.02), the OM27 clade genus of Bdellovibrionaceae (LDA = 2.83, p = 0.04) and the Thauera genus of Rhodocyclaceae (LDA = 2.60, p = 0.009), belong to the Proteobacteria phylum were significantly enriched in OM treatment group. In the CK group, the Gemmatimonadaceae family and the Anaeromyxobacter genus, belonging to Archangiaceae, were highly abundant biomarkers that were identified. The maximum number of discriminant clades significantly increased in OC2 (32) and CK (40) groups (Figure and Table ). The Gemmatimonadaceae family (LDA = 4.19, p = 0.04), Prevotellaceae family (LDA = 3.00, p = 0.01) and Ruminococcaceae family (LDA = 2.89, p = 0.01), belong to Gemmatimonadetes, Firmicutes and Bacteroidetes phyla, respectively; and the genus Alloprevotella (LDA = 3.00, p = 0.01) and Defluviicoccus (LDA = 2.68, p = 0.01) were observed to be significantly abundant in the no‐fertilizer CK group. Compared with the soil no fertilizer CK, the cvE6 family (LDA = 3.03, p = 0.01); the Tateyamaria genus (LDA = 3.37, p = 0.005) of Proteobacteria phylum and Peptoclostridium genus of Firmicutes phylum have a significant higher advantage in OC2 soil treated group with a high proportion of CF. The number of discriminant classes was four in the OC2 group and 53 in the OM group (Figure and Table ). Acidobacteriaceae subgroup1 family (LDA = 2.22, p = 0.04), Desulfobulbaceae (LDA = 2.71, p = 0.03) family and the genus Halioglobus belonging to the Halieaceae family (LDA = 2.00, p = 0.005) showed significantly higher abundance in the OC2 group; 67_14 family (LDA = 3.14, p = 0.01), Reyranellaceae family (LDA = 3.04, p = 0.02) and Methylophilaceae (LDA = 2.97, p = 0.02) family, including the species of Defluviicoccus (LDA = 2.61, p = 0.02) have significantly higher amounts in pure SM of OM group. According to the data of OC2 vs.OC1, three biomarkers were associated with OC2 and 28 were associated with OC1 groups (Figure and Table ). The higher abundance of Micromonosporaceae family (LDA = 3.09, p = 0.04) and Reyranellales family (LDA = 3.09, p = 0.04), which belongs Actinobacteria phylum were significantly enriched in the OC1 group; with the application of chemical fertilizer into the soil, the B1_7BS.Ambiguous_taxa family (LDA = 2.72, p = 0.04) and uncultured_ Acidobacterium_ sp. family (LDA = 2.00, p = 0.03) were highly in the OC2. Compared with the OC1 (24 biomarkers) treatment group (with a low proportion of fertilizer) with no‐fertilizer CK (24 biomarkers) group, Gemmatimonadaceae family (LDA = 3.20, p = 0.04) Prevotellaceae family (LDA = 2.64, p = 0.01) and Ruminococcaceae family (LDA = 2.63, p = 0.03) were significantly abundant in the no‐fertilizer CK group. The genus Prevotellaceae UCG003 , Ruminococcaceae UCG005 and Kurthia , belonging to phylum Firmicutes , were significantly more abundant in the CK‐treated soil (Figure and Table ). The Enterobacteriaceae family (LDA = 2.94, p = 0.04), Reyanellaceae family (LDA = 2.86, p = 0.04) and Ruminococcaceae family (LDA = 2.74, p = 0.04), including the Klebsiella genus and Flavonifractor genus were identified to be significantly more abundant in the OC1 group. Redundancy analysis links soil bacterial community with soil properties Redundancy analysis (RDA) plots (Figure ), performed by fitting four soil samples from different proportions of fertilizer treatments, indicated relationships between 14 selected soil environmental chemical properties and bacterial community structure. The results denoted by the first and second axes showed that 41.54% and 13.05% of changes in the bacterial community were influenced by soil pH, EC, SOC, C:N, AVL N and AVL P (Figure ). Monte Carlo permutation test co‐occurrence data analysis showed that the interaction between soil microbial flora and soil pH, EC, C:N, AVL N characteristic values were significant (Table ). The large amount of Subgroup_11 and Blastocatellia_(Subgroup_4) under OM plots was positively correlated to pH ( r sub11 = 0.68, p = 0.015; r Bla = 0.70, p = 0.012); the AVL N had a significant effect on Gammaproteobacteria ( r = 0.77; p = 0.004), EC values were positively correlated with Bacteroidia and Longimicrobia; the C:N ratio had a significant effect on Clostridia abundance ( r = 0.73; p = 0.009). For the analysis of medium and trace mineral elements and soil bacterial community, the first two axes of the RDA explained 32.86% and 10.99% of the total changes in the major bacterial groups. In the correlation analysis of the soil medium, trace element nutrient content and the bacterial communities were also significantly increased. Soil quantitative elements AVL Cu and EB Ca had positive significant associations with the bacterial communities of Chlamydiae and Actinobacteria changed, respectively ( r Cu = 0.71; p = 0.009; r Ca = 0.71; p = 0.013) (Figure ). The large amount of Subgroup_15 and Lineage_IIa under CK plots were positively correlated with AVL Zn ( r sub‐15 = 0.69, p = 0.014; r LinIIa = 0.81, p = 0.001). Correlation analysis was used to evaluate the association between the aboveground part of the tree canopy structure and fruit productivity (yield and quality) and the underground part of the soil nutrients and microbial community in pear (Figure ). Our findings revealed that soil C, N and C/N ratio were the main factors influencing the interaction between tree canopy structure and microbial community ( r 2 = 0.58; p = 0.02).
The number of short, medium, and long branches and the ratio between them are important parameters to evaluate the canopy structure in pear (Wei, Wang, Dong, & Dong, ). The fruit‐bearing branch generally forms the group of short branches in the sand pear ( P. pyrifolia cv. Cuiguan) genotype (Huang et al., ). As shown in Figure , the number of short and long branches and the total branches were significantly influenced by the long‐term application of different proportions of SM and CF. The total number of branches was the largest in OC1 (112) and lowest in the CK treatment group (94). Compared with the CK, the total number of branches in OC1 was significantly improved ( p < 0.05) by 18.4%. The total number of branches ranged from 94 to 112 and ranked in the following order: OC1 > OC2 > OM > CK. There was no significant difference between OM, OC1 and OC2, however, the OM treatment of 100% SM marginally increased ( p > 0.05) the total number of branches compared with CK. In‐depth analysis of the number and the proportion of long branches in the OC1 treatment was the lowest, while the short branches in OC1 treatment were the highest; the ratios of short, medium and long branches was also highest in OC1 treatment, which was 25:1:5 (Figure ). The most dramatic response of the number of long branch was observed for tree canopy structure after the higher proportion CF (Figure ). The OC2 treatment promoted the number of long branches significantly increased ( p < 0.05). The higher proportion of long branches number (28.96%) inhibited the growth of short branches, accounting for only 66.46%. The ratio of the short, medium and long branches was only 14:1:6 (Figure ).On the contrary, compared with a high proportion of SM in the OM‐ and OC1‐treatments, the number of short fruit branches was significantly decreased by 14.7% and 21.8%, respectively, in OC2, and was significantly decreased by 26.2% and 33.9%, respectively, in CK.
Continuous and stable growth of yields by the long‐term application of SM and CF from 2019 to 2021 (Table ). In 2019, the yield from OM‐treated soil was the highest among all treatments due to the highest fruit‐setting number (average of 133 fruits/tree), the yield reached 54.60 ton .ha −1 . In the next 2 years, the yield of OM decreased significantly. The average single fruit weight in the OC2 treatment was the highest (429.25 g), which was much higher than the OC1 and CK treatments 16% and 10.58%. In 2020, the single fruit weight (average of 412 g) and yield (53.04 ton.ha −1 ) in the OC1 treatment were the highest. Compared with the previous 2 years (2019–2020), the yield in OM and CK with the highest proportions of SM or CF treatment began to decline to some extent in 2021. For the OC1 treatment, compared with OM and OC2 treatments, the yield was obviously superior, which is mainly attributed to the increase in average single fruit weight from 368.98 g to 434.6 g. With the increase in the proportion of SM, the soluble solid content (SSC) in the fruit showed an increased trend (Table ). In 2019, 100% SM in OM treatment had the highest content of SSC (12.46%) in pear fruits, which was significantly ( p < 0.05) higher than that of CK treatment by 12.1%. Compared with the OM treatment, the SSC content decreased linearly with the decrease of the SM proportion, and the SSC of OC1, OC2 and CK was significantly reduced by 5.28%, 6.91% and 12.11%, respectively. Acidity in flavour was contributed by the titratable acid content (TA), which was marginally decreased in OM treatment. The SSC/TA ratio of the fruit treated with OM was highest (40.31), which was 19.48% higher than that in CK, and 11.29% and 9.42% higher than in OC1 and OC2, respectively. In 2020–2021, the SSC of OC1‐treated was highest, at 12.11% and 12.17% respectively, which was significantly higher than in the other treatments. On the contrary, the TA of OC1 treatment was the lowest, resulting in the highest SSC/TA ratio, which was above 39. The average fruit SSC/TA ratio ranged from 33.8 to 38.87 and ranked in the following order: OC1 > OM > OC2 > CK. A higher proportion of SM in OM and OC1 significantly ( p < 0.05) improved the average ratio of soluble substance and acid (SSC/TA ratio), was highest in OC1 with the CF (0.3 kg/tree) instead of 50% SM from 2019 to 2021. The pear fruits gradually entered the mature stage, and the application of SM significantly ( p < 0.05) increased the TSS and fructose content (Figure ). The TSS of OM‐treatment was the highest, which is attributed to the highest content of glucose (19.12 mg/g) and fructose (28.70 mg/g) compared with the control, representing an increase of 5.38% and 5.67% respectively. The sucrose content was highest (16.98 mg/g) in OC1, compared to the CK treatment, with an increment of 29.37%. With the increase in CF proportion, the brittleness of the fruit exocarp showed a downward trend, but the adhesiveness increased and the fruit flesh became soft and fluffy. (Figure ). The brittleness of fruit exocarp (1899.61 g) in OM was highest, compared to the CK treatment and was significantly ( p < 0.05) reduced by 26.32%.
The test soil was originally slightly alkaline (pH = 7.7), while the pH of CK‐tested soil with 100% CF was acidic (pH = 6.94). The pH increased consistently as a result of the increased application of SM, and the pH ranged from 7.10 to 7.44 (Table ). The EC‐value under the CK treatment was the highest (850 us/cm), which was much higher than that of OC2, OC1 and OM treatment by 13%, 36% and 30%. Compared with the CK treatment, with the increase in the proportion of SM, the content of soil organic carbon (SOC)in the soil had a significant upward trend for continuous fertilization, ranging from 43.31% to 66.90%. The SOC,C/N ratio, available nitrogen (AVL N), available K (AVL K), available Fe (AVL Fe) and available Cu (AVL Cu), were highest in OM‐treated soil. When compared to other treatments containing a certain proportion of SM, the content of TN in 100% CF‐treated soil was significantly higher, with a value of 1.78 g/kg, resulting in the lowest C/N ratio (6.38). From the spearman correlation heatmap, we observed clustering of soil chemical properties with the fruit yield and qualities (Figure ). Most significantly, including a positive correlation between the SOC, TN, C/N ratio, AVL N of the soil and pear fruit qualities included the content of fructose, the content of sucrose, brittleness, the number of short branches and the number of total branches. To conduct an integrative analysis of the significant positive correlations that existed between the soil rhizosphere nutrient indicators (SOC, TN, C/N ratio, AVL N, AVL K, AVL Fe) and the content of fructose in pears. The same significant positive correlation also existed between soil AVL Fe content and SSC ( r = 0.82, p = 0.001) (Table ); the ratio of SSC/TA ratio ( r = 0.81, p = 0.002); the fructose content ( r = 0.83, p = 0.001), both of which contribute to the sweetness of the fruit in pear. In addition, the content of sorbitol in pear fruit was significantly positively correlated with soil pH ( r = 0.71, p = 0.001), but negatively correlated with the content of AVL P ( r = −0.73, p = 0.009). The AVL Fe and SOC in soil were significantly negatively correlated with the accumulation of TA ( r Fe = −0.74, p = 0.006; r SOC = −0.58, p = 0.046) in pear fruit.
The V3–V4 region of bacterial 16S rRNA genes was amplified to explore the effects of soil rhizosphere microbial communities caused by four different fertilizing methods. Alpha diversity was employed to analyse the complexity of bacterial species diversity in the pear rhizosphere soil (Table ). The bacterial population diversity index of four pear rhizosphere soil samples from different fertilizer treatments was sampled to the same sequencing depth (45,421 reads per sample) and after clustering, 63,007–66,198 valid tags operated with 97% identity were obtained from operational taxonomic unit (OTU), and the number of OTUs per sample was 3728–4123. At the OTU level, the results showed that the rhizosphere soil treated with pure SM in OM‐treated soil contained 4123 OTUs, which were significantly higher than those treated with OC1, OC2 and CK fertilizers. The number of common and unique OTUs in different samples is shown in Figure . The 315 OTUs were common to soils with four different fertilizing methods, accounting for 7.6%–8.4% of the total number of OTUs. The ace index and the chao1 index in the OM treatment were significantly larger than in the OC1 and OC2 treatments, which indicated that the more bacterial OTUs contained in the rhizosphere community, the greater the richness value of the bacterial community. The numbers of OTUs in the OM and CK treatments were significantly larger than in the OC2 treatment, at 9.80% and 9.39%, respectively. Shannon's index for soils that underwent the OM treatment was the largest, while this value in OC2 treatment was the lowest, but it was not significantly different from those treatments. β (Beta) diversity was used as a comparative analysis of the microbial community composition of different soil rhizosphere samples from the four fertilization treatments. Non‐metric multidimensional scaling (NMDS) results showed that no matter how much SM replaces CF, it would significantly change the composition of the soil bacterial community With the SM ratio increasing, the inter‐group distance from CK to OC1, OC2 and SM‐treated soils was gradually closer with the increase of the SM ratio to each other, indicating that their differences in soil microbial communities were increased by SM application (Figure ). Kruskal–Wallis algorithm was used to analyse the OTUs with significant differences between fertilizer treatment groups ( p < 0.05) (Figure ), and the data showed the OTU of the top 10 diversity species abundance. Among them, OTU178 and OTU1314 belonged to Acidobacterium, and their enrichment in OM group was significantly higher than in OC1 and OC2 groups. The relative abundance of OTU642, which belongs to Deltaproteobacteria was the highest in OM treatment, significantly higher than that in OC1 and OC2 treatment. The relative abundance of OTU4063, OTU592 and OTU1554, all belonging to Gammaproteobacteria, were the highest in the soil rhizosphere of OC1 treatment, while the relative abundance of these OTUs in the OC2 treatment with the increase in CF proportion.
The relative abundance of the four fertilizer treatments had similar major predominant bacterial communities, but these predominant bacteria exhibited divergent substance preferences. Results showed that the dominant bacterial communities (96%), included Proteobacteria (55%–58%), Acidobacteria (7%–13%), Gemmatimonadetes (7%–10%), Actinobacteria (6%–8%), Bacteroidetes (6%–9%), Firmicutes (1%–4%) and Nitrospirae (1%–3%), all of which belong to azotobacter, in the four soil rhizospheres from different fertilizer treatments (Figure ). At the phylum level, compared without any SM (CK), the relative abundance of Acidobacteria, Gemmatimonadetes, Nitrospirae, Calditrichaeota and Zixibacteria were higher in soil samples than that SM were applied into pear orchard; by contrary, the relative abundance of Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Fibrobacteres were relatively low. In addition, compared to the no‐SM in CK, with the input of SM rapidly increasing, the relative abundance of Firmicutes, Elusimicrobia, Patescibacteria, Entotheonellaeota and Chlamydiae increased. Among them, it was obvious that the input of SM has significantly ( p < 0.05) increased the share of Firmicutes from 1.5% to 4.22% (Table ). With the increase in the proportion of CF in the fertilizer formula, the other bacterial communities, such as Gemmatimonadetes, Calditrichaeota, Zixibacteria, Patescibacteria and Latescibacteria, increased significantly. At the genus level, we plotted the relative abundance of heat maps at the genus taxa level of the top 30 (the most abundant species) to further compare differences in bacteria composition among fertilizer treatments (Figure ). The relative abundance of genera included Gaiella, Clostridioides, [Ruminococcus]_gnavus_group, Ruminococcus_2, MND1, IS‐44, mle1‐7 and Chryseolinea genus were higher in OM than in the OC1, OC2 and CK treatments; conversely, the relative abundance of Actinomadura , Gemmatimonas , Geobacter , Flavobacterium , Luteimonas, Rhodanobacter , Sphingomonas in OM was lower than in the OC1, OC2 and CK treatments. Compared with pure SM in the OM‐treated soil, the relative abundance of [Ruminococcus]_gnavus_group , Ruminococcus_2 ., Chryseolinea, Gaiella decreased significantly in OC1, OC2 and CK treatments, which with the increase of CF proportion into SM; the relative abundance of Pseudolabrys, Subgroup_10 , Chujaibacter , Sphingomonas , Rhodanobacter , Luteimonas , Flavobacterium , Gemmatimonas , Geobacter and Actinomadura increased significantly in OC1 and OC2 treatments; it was noteworthy that the CF ratio increased to 100% (CK), and the relative abundance of these bacterial colonies decreased significantly.
We utilized the linear discriminant analysis effect size (LEfSe) analysis to compare the bacterial affiliations (i.e. biomarkers) around the soil rhizosphere with different fertilizer treatment groups (Figure ). Abundance of different bacterial biomarkers was discriminated between fertilizer treatment groups according to linear discriminant analysis (LDA) > 2; p < 0.05. The cladogram revealed that 141 biomarkers were identified in different groups (Figure and Table ). The minimum number of discriminant clades significantly increased between OC1 (2 biomarkers) and OM (10 biomarkers) groups (Figure and Table ) indicating that the Halioglobus genus (LDA = 2.02, p = 0.005) was significantly enriched in OC1 group, that CF added into SM. Candidatus_Berkiella genus, Pseudogulbenkiania and Tagaea were significantly enriched in OM. The analysis revealed that 15 biomarkers were associated with OM and 5 were associated with CK groups (Figure and Table ). The Reyranellaceae family (LDA = 2.99, p = 0.02), the OM27 clade genus of Bdellovibrionaceae (LDA = 2.83, p = 0.04) and the Thauera genus of Rhodocyclaceae (LDA = 2.60, p = 0.009), belong to the Proteobacteria phylum were significantly enriched in OM treatment group. In the CK group, the Gemmatimonadaceae family and the Anaeromyxobacter genus, belonging to Archangiaceae, were highly abundant biomarkers that were identified. The maximum number of discriminant clades significantly increased in OC2 (32) and CK (40) groups (Figure and Table ). The Gemmatimonadaceae family (LDA = 4.19, p = 0.04), Prevotellaceae family (LDA = 3.00, p = 0.01) and Ruminococcaceae family (LDA = 2.89, p = 0.01), belong to Gemmatimonadetes, Firmicutes and Bacteroidetes phyla, respectively; and the genus Alloprevotella (LDA = 3.00, p = 0.01) and Defluviicoccus (LDA = 2.68, p = 0.01) were observed to be significantly abundant in the no‐fertilizer CK group. Compared with the soil no fertilizer CK, the cvE6 family (LDA = 3.03, p = 0.01); the Tateyamaria genus (LDA = 3.37, p = 0.005) of Proteobacteria phylum and Peptoclostridium genus of Firmicutes phylum have a significant higher advantage in OC2 soil treated group with a high proportion of CF. The number of discriminant classes was four in the OC2 group and 53 in the OM group (Figure and Table ). Acidobacteriaceae subgroup1 family (LDA = 2.22, p = 0.04), Desulfobulbaceae (LDA = 2.71, p = 0.03) family and the genus Halioglobus belonging to the Halieaceae family (LDA = 2.00, p = 0.005) showed significantly higher abundance in the OC2 group; 67_14 family (LDA = 3.14, p = 0.01), Reyranellaceae family (LDA = 3.04, p = 0.02) and Methylophilaceae (LDA = 2.97, p = 0.02) family, including the species of Defluviicoccus (LDA = 2.61, p = 0.02) have significantly higher amounts in pure SM of OM group. According to the data of OC2 vs.OC1, three biomarkers were associated with OC2 and 28 were associated with OC1 groups (Figure and Table ). The higher abundance of Micromonosporaceae family (LDA = 3.09, p = 0.04) and Reyranellales family (LDA = 3.09, p = 0.04), which belongs Actinobacteria phylum were significantly enriched in the OC1 group; with the application of chemical fertilizer into the soil, the B1_7BS.Ambiguous_taxa family (LDA = 2.72, p = 0.04) and uncultured_ Acidobacterium_ sp. family (LDA = 2.00, p = 0.03) were highly in the OC2. Compared with the OC1 (24 biomarkers) treatment group (with a low proportion of fertilizer) with no‐fertilizer CK (24 biomarkers) group, Gemmatimonadaceae family (LDA = 3.20, p = 0.04) Prevotellaceae family (LDA = 2.64, p = 0.01) and Ruminococcaceae family (LDA = 2.63, p = 0.03) were significantly abundant in the no‐fertilizer CK group. The genus Prevotellaceae UCG003 , Ruminococcaceae UCG005 and Kurthia , belonging to phylum Firmicutes , were significantly more abundant in the CK‐treated soil (Figure and Table ). The Enterobacteriaceae family (LDA = 2.94, p = 0.04), Reyanellaceae family (LDA = 2.86, p = 0.04) and Ruminococcaceae family (LDA = 2.74, p = 0.04), including the Klebsiella genus and Flavonifractor genus were identified to be significantly more abundant in the OC1 group.
Redundancy analysis (RDA) plots (Figure ), performed by fitting four soil samples from different proportions of fertilizer treatments, indicated relationships between 14 selected soil environmental chemical properties and bacterial community structure. The results denoted by the first and second axes showed that 41.54% and 13.05% of changes in the bacterial community were influenced by soil pH, EC, SOC, C:N, AVL N and AVL P (Figure ). Monte Carlo permutation test co‐occurrence data analysis showed that the interaction between soil microbial flora and soil pH, EC, C:N, AVL N characteristic values were significant (Table ). The large amount of Subgroup_11 and Blastocatellia_(Subgroup_4) under OM plots was positively correlated to pH ( r sub11 = 0.68, p = 0.015; r Bla = 0.70, p = 0.012); the AVL N had a significant effect on Gammaproteobacteria ( r = 0.77; p = 0.004), EC values were positively correlated with Bacteroidia and Longimicrobia; the C:N ratio had a significant effect on Clostridia abundance ( r = 0.73; p = 0.009). For the analysis of medium and trace mineral elements and soil bacterial community, the first two axes of the RDA explained 32.86% and 10.99% of the total changes in the major bacterial groups. In the correlation analysis of the soil medium, trace element nutrient content and the bacterial communities were also significantly increased. Soil quantitative elements AVL Cu and EB Ca had positive significant associations with the bacterial communities of Chlamydiae and Actinobacteria changed, respectively ( r Cu = 0.71; p = 0.009; r Ca = 0.71; p = 0.013) (Figure ). The large amount of Subgroup_15 and Lineage_IIa under CK plots were positively correlated with AVL Zn ( r sub‐15 = 0.69, p = 0.014; r LinIIa = 0.81, p = 0.001). Correlation analysis was used to evaluate the association between the aboveground part of the tree canopy structure and fruit productivity (yield and quality) and the underground part of the soil nutrients and microbial community in pear (Figure ). Our findings revealed that soil C, N and C/N ratio were the main factors influencing the interaction between tree canopy structure and microbial community ( r 2 = 0.58; p = 0.02).
Response of soil nutrients and microorganisms diversity with organic–inorganic proportional fertilizing When applied to soil in agricultural settings, manure's rich organic components and exogenous microorganisms can alter the environmental conditions of the original plant rhizosphere microorganisms (Suleiman et al., ). Biodiversity and abundance of soil rhizosphere microbial community play a vital role in the functionality of the soil ecosystem, and it usually changes due to agricultural activities, particularly fertilizer application (Zhao et al., ). Soil microbial communities affect soil nutrient cycling and ecosystem functioning. However, the variations in microbial diversity and the community composition when SM is used as a substitute for CF remain unclear. In this study, compared with the original pear orchard soil rhizosphere bacterial community without any fertilizer, the continuous application of organic fertilizer‐sheep manure or inorganic‐fertilizer‐N‐P 2 O 5 ‐K 2 O compound chemical fertilizer over a period of 4 years had a strong impact on the microbial abundance of the bacterial community in the 0–30 cm rhizosphere soil of the pear orchard, which was consistent with our hypothesis. Alpha bacterial diversity of soil bacterial community was different among fertilization treatments, we found that the highest bacterial species richness (ACE, Chao1 indices) and the diversity of microbial communities (Shannon diversity index, p < 0.05) was seen in pure SM‐treated soil, and when the higher SMproportion was used in inorganic–organic mixed fertilizer, the soil microbial biomass and the diversity of microbial communities in the pear rhizosphere were increased. This could be explained by SM introducing some exogenous bacteria to enrich the bacterial community. Researchers have found similar results in soil after long‐term application of chemical nitrogen fertilizer (Wang et al., ). Organic amendments from treatments such as cover crop, earthworm manure, cow manure and sheep manure result in a strong change in the level of bacterial diversity, and the relative abundance and diversity of bacterial communities have different response mechanisms to the application of different types of fertilizers (Ali et al., ; Duan et al., ; Liu, Zhou, et al., ; Shang et al., ). Although the dominant phyla and class groupings of bacteria were similar among fertilizer treatments, we discovered considerable variations in the relative abundance of those bacteria. The abundance analysis of the dominant bacteria in the fertilized soil revealed that all belong to azotobacter, including Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Fibrobacteres. Awasthi et al. ( ) showed that Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes predominated in sheep manure. Our findings were similar to those indicated above when compared to the study by Xu et al. ( ), with the exception that Verrucomicrobia and Chloroflex were not in the dominant flora range. It was hypothesized that this was due to the differences in bacterial communities under local ecological and environmental conditions. Proteobacteria and Firmicutes both increased after each application of farm manure, which might lead to changes in soil community composition described by Shanks et al. ( ). Nitrospirae and Gemmatimonadetes' relative abundance increased rapidly in CK‐treated rhizospheres, with a correlation with specific nitrification rate (PNR). These findings are consistent with previous research that documented the impact of Nitrospirae and Gemmatimonadetes on PNR productivity in coastal areas where nitrogen fertilizer was applied (Yao et al., ). The main forms of inorganic nitrogen (N) fertilizer are NH 4 Cl and (NH 4 ) 2 SO4, and the pear root primarily absorbs NH 4 + . When NH 4 + is absorbed, it releases H + , NH 4 Cl and (NH 4 ) 2 SO 4 , which are selectively absorbed into HCl and H 2 SO 4 , causing soil acidification (pH: 7.44 falls to 6.94) and excess ammonium nitrogen (AN) promoted the reaction substrate of Nitrospirae, which has the ability to oxidize ammonia to nitric acid in soil. It demonstrated Nitrospirae's ability to remediate soil. In the 0–30 cm core area of the root of pear, all soil nutrients measured in the SM‐based treatment (OM, OC1, OC2) except EB Mg, AVL Mn and AVL Zn were higher than those in the soil without SM fertilization (CK), in particular, the SOC content in the soil with different proportions of SM was 1.43–1.67 times that of the CK treatment, this demonstrated that continuously spread the fertilizer contributes to the accumulation of soil SOC, thus improving soil fertility. Davide et al. ( ) noted that the organic matter content and microbial biomass carbon were significantly increased by mineral and organic fertilization. Also in the pear orchard, the content of soil organic matter, the available concentrations of nitrogen (N), phosphorus (P), potassium (K) and Fe, Mn and Zn in SM treatments were all increased to a certain degree as compared to CF treatment (Zhang et al., ). As part of the organic fertilizer was replaced by chemical fertilizer, the content of SOC, AVLN, K, Fe, Zn and Cu in the soil showed a downward trend, which also proved that the organic fertilizer as the amendments has an accumulative effect on nutrients, but the fast effect N‐P‐K compound chemical fertilizer also had a positive role in promoting the content of AVLP, EB Ca and AVL Mn. Compared with the no‐SM in CK treatment, Firmicutes, Elusimicrobia, Entotheonellaeota and Chlamydiae at the phylum level were the most representative phylum in organic fertilizer SM soil. Elusimicrobia was unique to the rare group of bacteria. Zhao et al. ( ) and Zhang et al. ( ), reported that Firmicutes and Entotheonellaeota show a high correlation with soil nutrient content, while rare groups of Elusimicrobia in bacterial communities had no correlation with soil nutrient content. It seems clear that the input of organic fertilizer has increased the portion of class Clostridia, which belongs to Firmicutes and may degrade the lignin and cellulose. The Clostridia class was also found in the intestinal flora of herbivorous cattle and cow manure (Semenova et al., ). SM had the strongest effect on SOC content increment, with primarily increasing Gaiella, MND1 and Chryseolinea genus and decreasing Pseudolabrys and Rhodanobacter . MND1 was identified as mainly involved in the soil C and N cycles and was associated with enhanced recalcitrant SOC storage (Hu et al., ). Compared with CK, chemical nitrogen fertilizer treatment significantly reduced the relative abundance of Pseudodoganella , while green manure treatment significantly increased the abundance of Chryseolinea (Bao et al., ). The C/N ratio was significantly negatively correlated with Rhodanobacter , Rhodoplanes and Bacillus in the continuous cropping of Panax quinquefolius L. (American ginseng) (Liu, Wang, et al., ). Pig manure exerted the strongest effect on SOM content and aggregation, and influenced microbial community structure more strongly than plant residues, primarily by increasing Bacillales and Gaiellales (Lin et al., ). SM and CF application increased the bacterial Proteobacteria at the phylum level, and the top 10 OTUs were detected by Kruskal–Wallis algorithm, including the classes Deltaproteobacteria and Gammaproteobacteria in all aggregate fractions. This might be attributed to adding additional C and N inputs to the soil rhizosphere, which accelerated the growth and proliferation of Deltaproteobacteria and Gammaproteobacteria through the application of SM (Chen et al., ; Huang et al., , ). Redundancy analysis (RDA) data indicated that AVL N content had a significant positive correlation with the bacterial communities of Gammaproteobacteria changed at the level of genus, the eutrophic hypothesis holding that Proteobacteria is a typical eutrophic species, Alpha‐ and Gamma‐ Proteobacteria significantly increase at high fertilization levels and higher AVL N can make the eutrophic species use more unstable carbon in the soil (Li et al., ). We also found that the relative abundance of Proteobacteria in OC2 treatment was higher than that in pure sheep manure OM‐treated soil, indicating that Proteobacteria was more significantly affected by CF than SM. Gammaproteobacteria plays an important role in the process of nitrogen metabolism in the soil rhizosphere of pear orchards. It may be that AVL N in the available fertilizer can be absorbed and utilized by pear roots, does not need to be decomposed, and significantly increases nitrification, denitrification and nitrogen fixation. In addition to soil fertility, it is also worth paying attention to soil health. The value of pH and EC are two important indicators for evaluating soil acidification and salinization (Han et al., ). The original soil prior to fertilization in the pear orchard was originally saline‐alkaline soil (pH = 7.70), with the increased proportion of chemical fertilizer, the pH value decreased continuously and soil acidification intensified. The increase of organic carbon in organic fertilizer slightly alleviated soil acidification and slightly raised again; that effect can be explained by the calcium content in organic fertilizer the resistance of soils to acidification and keeps the soil in the range of acid–base balance. This was different from N‐P‐K fertilizer, which only accelerates soil acidification (Meng et al., ; Shi et al., ). The relative abundance of class Blastocatellia_ (Subgroup_4) was positively correlated to pH. Blastocatellia (Subgroup_4) and Subgroup_6‐affiliated in accord with a positive relationship with soil pH (Poret‐Peterson et al., ), Blastocatellia (Subgroup_4) was a member of Blastocatellia that often appears in neutral and (slightly) alkaline pH soil (Huber et al., ), It was likely that Blastocatellia related to the carbon metabolism, which degraded organic matter into organic carbon and participates in the regulation of soil pH. Organic–inorganic proportional fertilizing affected the interconversion between vegetative growth and reproductive growth Pear canopy growth requires fertilization to maintain the nutrient level. Fertilizer management affects the conversion between vegetative growth and reproductive growth of the tree (Wang et al., ). Pear canopies should be treated as a population of branches. Branch (long, medium and short branches) composition and number are one of the main parameters of tree canopy structure, which was the main factor affecting vegetative growth and reproductive growth (fruit yield and quality) (Wei, Wang, Dong, & Ran, ). The genetic trait for Cuiguan ( Pyrus pyrifolia Nakai) is that flower buds mainly originate on the short branches, indicating that the shorter branches promote the turn of pear trees into reproductive growth. In contrast, the long branches would prompt vegetative growth. Results obtained in this study showed that when a proportion of CF was replaced by SM, increasing the input of C and N sources, the SOC and the range of C:N ratios increased, with the number of long branches decreased, which was an advantage for vegetative growth, and the number of short branches increased, which was an advantage for reproductive growth. Higher C/N ratio indicates that SM improved the SOC sequestration rate. The C/N ratio on a per unit SOC basis was significantly higher in the manure‐amended soils than in the CF‐treated soils (Huang et al., ). The branch quantity value of the OC1 treatment was the highest (Figure ). The application of SM as good organic amendments to pear significantly promoted the growth rhythm of the composition of branches, but the total number of branches and the short branches did not increase linearly with the increase of the proportion of organic fertilizer, which was consistent with previous findings in pears (Liu et al., ), apples (Zhao et al., ) and grapes (Qin et al., ). When the total branch and short branch number were significantly improved, the yield and fruit quality of apple trees were significantly increased (Zhang et al., ). In ‘Xinliqihao’ pear, when the short branch number was higher, the trees were more productive (Wei, Wang, Dong, & Ran, ). In ‘Le Conte’ pear trees, branch growth was obtained by application of organic farmyard manure (50 kg/tree) and (MgSO 4 ) 1.5% (Fawzi et al., ). Organic–inorganic proportional fertilizing improved pear yield and quality of reproductive growth parameters In our study, we conducted to determine the effects of organic manure alone or in combination with CF on the yield and quality of Cuiguan pear. During the first year of the fertilization experiment, the yield, SSC and SSC/TA of quality of OM were significantly higher than those of other treatments with a certain proportion of chemical fertilizer. With the fertilization experiment lasting for 3 years, OC1 treatment had significant effects on yield indicators including single fruit weight and fruit‐setting number, but the pure organic fertilizer OM treatment continued to produce 3 years, and the yield, SSC and SSC/TA slight decreased compared with the first year. Inorganic fertilizer was used in place of some organic fertilizer, which gradually showed advantages in terms of yield and quality when compared to the pure organic fertilizer that had been applied. It may be explained by the fact that the decomposition rate of mineral nutrients in inorganic fertilizer is faster than that of organic fertilizer, making it easier and quicker to keep up with the rapid growth of trees. As a result of the continuous increase in inorganic fertilizer use and the corresponding decrease in organic fertilizer use, the number and diversity of microbial flora decreased, and the reduction in soil fertility led to the gradual decline of pear productivity. The similarity results showed that 10 kg poultry manure (PM) + 300:150:250 g N‐P‐K/tree instead of pure inorganic fertilizer (600:400:400 g NPK/tree) significantly improved the yield, and fruit qualities including TSS, TS of pant pear‐18 fruits (Rathi & Bist, ). In Japanese ( Pyrus pyrifolia Nakai) pear plantation, 50% (200 kg N 2 O‐N ha −1 year −1 ) of the conventional fertilizer amount will be replaced by pig manure, which can eliminate the pollution caused by excessive nitrogen in the soil, and the yield and quality did not change significantly (Fujita et al., ). The application of organic amendments mixed with peat and chicken manure significantly increased the TSS in the fruit of Huangjin pear (Wei et al., ). Significant differences in fruit quality were found in the comparative test of the effects of CF and OM in pears. The SSC, hardness and titratable acidity were significantly higher in ‘Niitaka’ ( Pyrus pyrifolia Nakai.) pear fruits from organic manure‐treated (rice bran + coffee bran compost+ chitin‐incubated compost fermentation) than did chemical fertilized fruits (Choi et al., ). Further typical correlation analysis of important soil nutrient elements affecting fruit quality showed that total nitrogen (TN) and SOC in the soil were the main factors affecting single fruit weight and hardness, with a positive linear correlation. SSC is positively correlated with AVL K and AVL Zn, TA was linearly positively correlated with SOC and AVL Fe, and negatively correlated with TN.
When applied to soil in agricultural settings, manure's rich organic components and exogenous microorganisms can alter the environmental conditions of the original plant rhizosphere microorganisms (Suleiman et al., ). Biodiversity and abundance of soil rhizosphere microbial community play a vital role in the functionality of the soil ecosystem, and it usually changes due to agricultural activities, particularly fertilizer application (Zhao et al., ). Soil microbial communities affect soil nutrient cycling and ecosystem functioning. However, the variations in microbial diversity and the community composition when SM is used as a substitute for CF remain unclear. In this study, compared with the original pear orchard soil rhizosphere bacterial community without any fertilizer, the continuous application of organic fertilizer‐sheep manure or inorganic‐fertilizer‐N‐P 2 O 5 ‐K 2 O compound chemical fertilizer over a period of 4 years had a strong impact on the microbial abundance of the bacterial community in the 0–30 cm rhizosphere soil of the pear orchard, which was consistent with our hypothesis. Alpha bacterial diversity of soil bacterial community was different among fertilization treatments, we found that the highest bacterial species richness (ACE, Chao1 indices) and the diversity of microbial communities (Shannon diversity index, p < 0.05) was seen in pure SM‐treated soil, and when the higher SMproportion was used in inorganic–organic mixed fertilizer, the soil microbial biomass and the diversity of microbial communities in the pear rhizosphere were increased. This could be explained by SM introducing some exogenous bacteria to enrich the bacterial community. Researchers have found similar results in soil after long‐term application of chemical nitrogen fertilizer (Wang et al., ). Organic amendments from treatments such as cover crop, earthworm manure, cow manure and sheep manure result in a strong change in the level of bacterial diversity, and the relative abundance and diversity of bacterial communities have different response mechanisms to the application of different types of fertilizers (Ali et al., ; Duan et al., ; Liu, Zhou, et al., ; Shang et al., ). Although the dominant phyla and class groupings of bacteria were similar among fertilizer treatments, we discovered considerable variations in the relative abundance of those bacteria. The abundance analysis of the dominant bacteria in the fertilized soil revealed that all belong to azotobacter, including Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Fibrobacteres. Awasthi et al. ( ) showed that Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes predominated in sheep manure. Our findings were similar to those indicated above when compared to the study by Xu et al. ( ), with the exception that Verrucomicrobia and Chloroflex were not in the dominant flora range. It was hypothesized that this was due to the differences in bacterial communities under local ecological and environmental conditions. Proteobacteria and Firmicutes both increased after each application of farm manure, which might lead to changes in soil community composition described by Shanks et al. ( ). Nitrospirae and Gemmatimonadetes' relative abundance increased rapidly in CK‐treated rhizospheres, with a correlation with specific nitrification rate (PNR). These findings are consistent with previous research that documented the impact of Nitrospirae and Gemmatimonadetes on PNR productivity in coastal areas where nitrogen fertilizer was applied (Yao et al., ). The main forms of inorganic nitrogen (N) fertilizer are NH 4 Cl and (NH 4 ) 2 SO4, and the pear root primarily absorbs NH 4 + . When NH 4 + is absorbed, it releases H + , NH 4 Cl and (NH 4 ) 2 SO 4 , which are selectively absorbed into HCl and H 2 SO 4 , causing soil acidification (pH: 7.44 falls to 6.94) and excess ammonium nitrogen (AN) promoted the reaction substrate of Nitrospirae, which has the ability to oxidize ammonia to nitric acid in soil. It demonstrated Nitrospirae's ability to remediate soil. In the 0–30 cm core area of the root of pear, all soil nutrients measured in the SM‐based treatment (OM, OC1, OC2) except EB Mg, AVL Mn and AVL Zn were higher than those in the soil without SM fertilization (CK), in particular, the SOC content in the soil with different proportions of SM was 1.43–1.67 times that of the CK treatment, this demonstrated that continuously spread the fertilizer contributes to the accumulation of soil SOC, thus improving soil fertility. Davide et al. ( ) noted that the organic matter content and microbial biomass carbon were significantly increased by mineral and organic fertilization. Also in the pear orchard, the content of soil organic matter, the available concentrations of nitrogen (N), phosphorus (P), potassium (K) and Fe, Mn and Zn in SM treatments were all increased to a certain degree as compared to CF treatment (Zhang et al., ). As part of the organic fertilizer was replaced by chemical fertilizer, the content of SOC, AVLN, K, Fe, Zn and Cu in the soil showed a downward trend, which also proved that the organic fertilizer as the amendments has an accumulative effect on nutrients, but the fast effect N‐P‐K compound chemical fertilizer also had a positive role in promoting the content of AVLP, EB Ca and AVL Mn. Compared with the no‐SM in CK treatment, Firmicutes, Elusimicrobia, Entotheonellaeota and Chlamydiae at the phylum level were the most representative phylum in organic fertilizer SM soil. Elusimicrobia was unique to the rare group of bacteria. Zhao et al. ( ) and Zhang et al. ( ), reported that Firmicutes and Entotheonellaeota show a high correlation with soil nutrient content, while rare groups of Elusimicrobia in bacterial communities had no correlation with soil nutrient content. It seems clear that the input of organic fertilizer has increased the portion of class Clostridia, which belongs to Firmicutes and may degrade the lignin and cellulose. The Clostridia class was also found in the intestinal flora of herbivorous cattle and cow manure (Semenova et al., ). SM had the strongest effect on SOC content increment, with primarily increasing Gaiella, MND1 and Chryseolinea genus and decreasing Pseudolabrys and Rhodanobacter . MND1 was identified as mainly involved in the soil C and N cycles and was associated with enhanced recalcitrant SOC storage (Hu et al., ). Compared with CK, chemical nitrogen fertilizer treatment significantly reduced the relative abundance of Pseudodoganella , while green manure treatment significantly increased the abundance of Chryseolinea (Bao et al., ). The C/N ratio was significantly negatively correlated with Rhodanobacter , Rhodoplanes and Bacillus in the continuous cropping of Panax quinquefolius L. (American ginseng) (Liu, Wang, et al., ). Pig manure exerted the strongest effect on SOM content and aggregation, and influenced microbial community structure more strongly than plant residues, primarily by increasing Bacillales and Gaiellales (Lin et al., ). SM and CF application increased the bacterial Proteobacteria at the phylum level, and the top 10 OTUs were detected by Kruskal–Wallis algorithm, including the classes Deltaproteobacteria and Gammaproteobacteria in all aggregate fractions. This might be attributed to adding additional C and N inputs to the soil rhizosphere, which accelerated the growth and proliferation of Deltaproteobacteria and Gammaproteobacteria through the application of SM (Chen et al., ; Huang et al., , ). Redundancy analysis (RDA) data indicated that AVL N content had a significant positive correlation with the bacterial communities of Gammaproteobacteria changed at the level of genus, the eutrophic hypothesis holding that Proteobacteria is a typical eutrophic species, Alpha‐ and Gamma‐ Proteobacteria significantly increase at high fertilization levels and higher AVL N can make the eutrophic species use more unstable carbon in the soil (Li et al., ). We also found that the relative abundance of Proteobacteria in OC2 treatment was higher than that in pure sheep manure OM‐treated soil, indicating that Proteobacteria was more significantly affected by CF than SM. Gammaproteobacteria plays an important role in the process of nitrogen metabolism in the soil rhizosphere of pear orchards. It may be that AVL N in the available fertilizer can be absorbed and utilized by pear roots, does not need to be decomposed, and significantly increases nitrification, denitrification and nitrogen fixation. In addition to soil fertility, it is also worth paying attention to soil health. The value of pH and EC are two important indicators for evaluating soil acidification and salinization (Han et al., ). The original soil prior to fertilization in the pear orchard was originally saline‐alkaline soil (pH = 7.70), with the increased proportion of chemical fertilizer, the pH value decreased continuously and soil acidification intensified. The increase of organic carbon in organic fertilizer slightly alleviated soil acidification and slightly raised again; that effect can be explained by the calcium content in organic fertilizer the resistance of soils to acidification and keeps the soil in the range of acid–base balance. This was different from N‐P‐K fertilizer, which only accelerates soil acidification (Meng et al., ; Shi et al., ). The relative abundance of class Blastocatellia_ (Subgroup_4) was positively correlated to pH. Blastocatellia (Subgroup_4) and Subgroup_6‐affiliated in accord with a positive relationship with soil pH (Poret‐Peterson et al., ), Blastocatellia (Subgroup_4) was a member of Blastocatellia that often appears in neutral and (slightly) alkaline pH soil (Huber et al., ), It was likely that Blastocatellia related to the carbon metabolism, which degraded organic matter into organic carbon and participates in the regulation of soil pH.
Pear canopy growth requires fertilization to maintain the nutrient level. Fertilizer management affects the conversion between vegetative growth and reproductive growth of the tree (Wang et al., ). Pear canopies should be treated as a population of branches. Branch (long, medium and short branches) composition and number are one of the main parameters of tree canopy structure, which was the main factor affecting vegetative growth and reproductive growth (fruit yield and quality) (Wei, Wang, Dong, & Ran, ). The genetic trait for Cuiguan ( Pyrus pyrifolia Nakai) is that flower buds mainly originate on the short branches, indicating that the shorter branches promote the turn of pear trees into reproductive growth. In contrast, the long branches would prompt vegetative growth. Results obtained in this study showed that when a proportion of CF was replaced by SM, increasing the input of C and N sources, the SOC and the range of C:N ratios increased, with the number of long branches decreased, which was an advantage for vegetative growth, and the number of short branches increased, which was an advantage for reproductive growth. Higher C/N ratio indicates that SM improved the SOC sequestration rate. The C/N ratio on a per unit SOC basis was significantly higher in the manure‐amended soils than in the CF‐treated soils (Huang et al., ). The branch quantity value of the OC1 treatment was the highest (Figure ). The application of SM as good organic amendments to pear significantly promoted the growth rhythm of the composition of branches, but the total number of branches and the short branches did not increase linearly with the increase of the proportion of organic fertilizer, which was consistent with previous findings in pears (Liu et al., ), apples (Zhao et al., ) and grapes (Qin et al., ). When the total branch and short branch number were significantly improved, the yield and fruit quality of apple trees were significantly increased (Zhang et al., ). In ‘Xinliqihao’ pear, when the short branch number was higher, the trees were more productive (Wei, Wang, Dong, & Ran, ). In ‘Le Conte’ pear trees, branch growth was obtained by application of organic farmyard manure (50 kg/tree) and (MgSO 4 ) 1.5% (Fawzi et al., ).
In our study, we conducted to determine the effects of organic manure alone or in combination with CF on the yield and quality of Cuiguan pear. During the first year of the fertilization experiment, the yield, SSC and SSC/TA of quality of OM were significantly higher than those of other treatments with a certain proportion of chemical fertilizer. With the fertilization experiment lasting for 3 years, OC1 treatment had significant effects on yield indicators including single fruit weight and fruit‐setting number, but the pure organic fertilizer OM treatment continued to produce 3 years, and the yield, SSC and SSC/TA slight decreased compared with the first year. Inorganic fertilizer was used in place of some organic fertilizer, which gradually showed advantages in terms of yield and quality when compared to the pure organic fertilizer that had been applied. It may be explained by the fact that the decomposition rate of mineral nutrients in inorganic fertilizer is faster than that of organic fertilizer, making it easier and quicker to keep up with the rapid growth of trees. As a result of the continuous increase in inorganic fertilizer use and the corresponding decrease in organic fertilizer use, the number and diversity of microbial flora decreased, and the reduction in soil fertility led to the gradual decline of pear productivity. The similarity results showed that 10 kg poultry manure (PM) + 300:150:250 g N‐P‐K/tree instead of pure inorganic fertilizer (600:400:400 g NPK/tree) significantly improved the yield, and fruit qualities including TSS, TS of pant pear‐18 fruits (Rathi & Bist, ). In Japanese ( Pyrus pyrifolia Nakai) pear plantation, 50% (200 kg N 2 O‐N ha −1 year −1 ) of the conventional fertilizer amount will be replaced by pig manure, which can eliminate the pollution caused by excessive nitrogen in the soil, and the yield and quality did not change significantly (Fujita et al., ). The application of organic amendments mixed with peat and chicken manure significantly increased the TSS in the fruit of Huangjin pear (Wei et al., ). Significant differences in fruit quality were found in the comparative test of the effects of CF and OM in pears. The SSC, hardness and titratable acidity were significantly higher in ‘Niitaka’ ( Pyrus pyrifolia Nakai.) pear fruits from organic manure‐treated (rice bran + coffee bran compost+ chitin‐incubated compost fermentation) than did chemical fertilized fruits (Choi et al., ). Further typical correlation analysis of important soil nutrient elements affecting fruit quality showed that total nitrogen (TN) and SOC in the soil were the main factors affecting single fruit weight and hardness, with a positive linear correlation. SSC is positively correlated with AVL K and AVL Zn, TA was linearly positively correlated with SOC and AVL Fe, and negatively correlated with TN.
In general, SM is an excellent amendment that may add organic matter to the soil and lessen soil acidity for pear orchards. Its application has adjusted the C/N ratio and the effective mineral elements (AVL N, AVL Cu, AVL Zn and EB Ca) that the pear tree could absorb and utilize, as well as significantly regulating the coordination of the rhizosphere micro‐ecological (Gammaproteobacteria, Clostridia, Chlamydiae, Actinobacteria, Subgroup_15 and Lineage_IIa) environment and soil nutrients. As a result, the diversity of soil microorganisms increased, the structure of microbial communities changed, and the transition of the pear tree from vegetative to reproductive growth (increasing the number of short branches) was promoted and regulated (Figure ). It is evident that SM has a significant ecological role in regulating soil micro‐ecology; it can increase bacterial diversity and, to some extent, change microbial community structure. Synthesizing above all outcomes, the optimum additive amount was 50% SM + 25% CF.
Chun‐Hui Shi: Data curation (lead); formal analysis (lead); project administration (lead); writing – original draft (lead); writing – review and editing (lead). Xiao‐Qing Wang: Data curation (equal); formal analysis (equal); project administration (equal). Shuang Jiang: Formal analysis (supporting). Li‐Qing Zhang: Project administration (lead); supervision (lead); writing – review and editing (equal). Jun Luo: Funding acquisition (lead); investigation (lead); methodology (lead); supervision (lead).
The authors declare no conflict of interest.
Figure S1 The sugar composition and texture in pear fruit under different proportions of sheep manure (SM) and chemical fertilizer (CF). (A), The sugar composition for fruit flavour quality in pear under different proportions of SM and CF (B), The texture for fruit quality in pear under different proportions of SM and CF. Click here for additional data file. Table S1 Fruit qualities and yields under different proportioning of sheep manure (SM) and chemical fertilizer (CF) (2019–2021). a OM, 100% SM (OM); OC1, 50% SM+ 25% CF; OC2, 25% SM +50% CF; CK, 100% CF as control check. b SSC, Soluble solid content; c TA, Titratable acid content; d SSC/TA, the soluble solid content/the titratable acid content. Treatment values (mean ± standard error) within a row followed by different letters are significantly different at p ≤ 0.05 levels according to Duncan’s multiple comparison test. TABLE S2 Correlation analysis between soil properties and pear yield and quality of different proportioning of sheep manure (SM) and chemical fertilizer (CF). TABLE S3 The biomarkers were identified among treatment groups by linear discriminant analysis effect size (LEfSe) statistical method. TABLE S4 Abundance of different bacterial biomarkers was discriminated between OM and OC1. TABLE S5 Abundance of different bacterial biomarkers was discriminated between OM and CK. TABLE S6 Abundance of different bacterial biomarkers was discriminated between OC2 and CK. TABLE S7 Abundance of different bacterial biomarkers was discriminated between OM and OC2. TABLE S8 Abundance of different bacterial biomarkers was discriminated between OC1 and OC2. TABLE S9 Abundance of different bacterial biomarkers was discriminated between OC1 and CK. TABLE S10 Correlation analysis between soil properties and soil rhizosphere microbiome. Click here for additional data file.
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Weaponising microbes for peace
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8a0083c3-023e-4b84-8710-8acf0ae7c494
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10221547
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Microbiology[mh]
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Peace, and its absence – political division, aggression, invasion and warfare – have preoccupied humanity since Homo sapiens diverged from the other apes. Conflicts often result from interpersonal or otherwise local or regional tensions, but can readily become international as a result of geopolitical activities to gain/maintain power and influence, and to secure or dominate supplies of strategically important natural resources, often through proxy wars, with nations supporting one or other of the combatants, as exemplified in conflicts in the Syria (2011 onwards), Yemen (2014 onwards), Ethiopia (2020–2022) and Ukraine (2014 and 2022 onwards). The current conflict in Ukraine is a prime example of how a regional conflict can precipitate global consequences, in this case in provoking crises of global food security and energy supplies, thereby undermining national economies worldwide. And, of course, the risk of a conflict leading to a nuclear conflagration that may destroy both human society and the Earth's biosphere as we know them can never be excluded (e.g. see Timmis & Hallsworth, ). There exist many causes of human‐driven conflicts, including distrust, societal disparities, anomie (the disconnect between individual beliefs/values and social norms) accompanied by the search for simplistic explanations of complex phenomena (conspiracy theories), othering, adverse personal characteristics of powerful individuals, or extremism (be it rooted in dogma, perceived incompatibility with or inferiority of other belief systems, racism and other kinds of xenophobia, or other equally divisive worldviews). But an arguably more desperate (and some might consider potentially more justifiable and rationalisable) cause/motive is serious regional deficits in fundamental resources needed for the members of those societies to live healthy and dignified lives and that might generally be considered to constitute basic human rights. Moreover, there are often substantial asymmetries between communities, regions and nations in the availability and supply of such resources. Such asymmetries are inconsistent with sustainable development of humanity, and their elimination lies at the heart of the Sustainable Development Goals (SDGs) formulated by the United Nations ( https://sdgs.un.org/2030agenda ), such as SDG 10: Reduce inequality within and among countries . The reasons for the asymmetries can be either natural, a consequence of geography, climate, etc., or caused and perpetuated by humans, such as the (post‐)colonial redistribution of resources from the global south to the global north. Such deficits and asymmetries, and efforts to locally increase supplies to adequate levels, create competition between neighbours for limited supplies and, in turn, sometimes the potential for conflict. And, importantly, they may also be weaponised by unscrupulous leaders to demonise others and propagate self‐serving conflicts. There is an urgent need for proactive measures to reduce the causes of local conflicts. Quite apart from the humanitarian imperative to reduce the human suffering caused by deficits in basic resources, and conflicts engendered by them, the international community has a vital self‐interest in diminishing causes of conflict, and promoting social equity, harmony and peace among peoples. Worldwide elimination of deficits in basic resources must become a pivotal element of strategic policy to secure peace among nations. ‘Microbes’ and ‘peace’ are not terms that are usually associated with one another. In fact, to most people, it might well appear that we are constantly at war with microbes. We talk about killing bacteria, fighting infection, antimicrobial resistance , battles on mucosal surfaces, biocides , disinfectants , pesticides and other weapons against pathogens. Personal care products and household cleaning products, kitchen counter tops, paints, etc., often contain antimicrobial substances, as do foods (such as salt, sugar, ethanol, nitrites and other preservatives). However, most microbes and, most importantly, their associations with one another, with other organisms and with the physical components of the biosphere, provide vital ecosystem services essential to our well‐being and that of the entire biosphere. Indeed, such microbial associations, particularly the manner in which microbes cooperatively interact to maximise exploitation of available resources (e.g. see Pelz et al., ; Venkataram et al., ), can provide guidance on how societies might behave more cooperatively and harmoniously, in particular when pursuing identical goals. Here we argue that technologies based on microbes and microbial societies have unprecedented potential to serve as facilitators of peace in the world.
Clearly there are many types of deficits and asymmetries, even among High‐income countries (HICs), so the following list is neither comprehensive nor necessarily balanced, but is meant to exemplify some of the major problems: hunger: approximately 10% of the world's population suffer from hunger ( www.fao.org/state‐of‐food‐security‐nutrition/2021/en ; SDG 2). This statistic needs to be appreciated in the context of another, namely that one third of all food produced in the world is wasted ( https://www.stopfoodlosswaste.org/issue ), decreasing productive farmland, which in some cases is the direct cause of hunger. The current episode of human‐driven global climate change is causing reduced rainfall in some regions, leading to increasing desertification and loss of productive farmland, and soil losses due to wind erosion (Burrell et al., ), and increased rainfall in other regions, leading to flooding and loss of fertile soil by water erosion. The decreasing productive farmland, in turn, leads to falling agricultural productivity (SDG 15) and food security, inadequate wastewater treatment: approximately half of the world's population lack access to safe sanitation facilities ( https://www.unicef.org/stories/state‐worlds‐sanitation ), 6% practice open‐air defecation, and each year 1% die as a result of inadequate water, sanitation and hygiene ( https://www.who.int/news‐room/fact‐sheets/detail/sanitation ; SDG 6), lack of clean water: one in three have no or inadequate access to safe drinking water ( https://www.who.int/news‐room/fact‐sheets/detail/sanitation ; SDG 6), insufficient basic health coverage and access to healthcare: almost 40% of the world's population does not have access to basic health services and effective medicines ( https://www.who.int/news/item/13‐12‐2017‐world‐bank‐and‐who‐half‐the‐world‐lacks‐access‐to‐essential‐health‐services‐100‐million‐still‐pushed‐into‐extreme‐poverty‐because‐of‐health‐expenses ; SDG 3). limited living space: there is an increasing displacement of peoples from rural areas to cities, as well as to coastal regions. This trend will increase dramatically as climate change and rising sea levels, caused by global warming, squeeze habitable zones and lead to further human migrations, thereby increasing competition for living space in other parts of the world (SDGs 10, 11, 13). Moreover, poverty, hunger, disease – all of which may be exacerbated by climate change (e.g. https://www.usip.org/publications/2022/09/how‐climate‐change‐catalyzes‐more‐migration‐central‐america ) are also major factors promoting mass migrations and overcrowding in destination lands. And, of course, persecution, victimisation, discrimination, maltreatment and war itself have displaced large numbers of peoples, many of which are confined long term to refugee camps that are often horrifically overcrowded and lack many forms of basic resources. deforestation, that reduces biodiversity ecosystem services, including carbon sequestration, water retention, soil stability and fertility. It also favours an increase in the incidence and spread of zoonotic infections through increased contacts of humans with wildlife as a result of humans entering wildlife habitats (Wolfe et al., ), and the displacement of wildlife from their natural habitats into living space that humans consider their own (SDG 15), limited access to, and insufficient resources and technology to generate, energy (SDG 7), pollution of soils, waters and air, mostly but not always as a result of industrial activity (often as a result of extracting raw materials for export), which not only impacts human health but also reduces biodiversity and the services it provides, poverty: 10% of the world's population live in extreme poverty (SDG 1) with little access to decent work and income (SDG 8), limited access to education (SDG4), in the context of this discussion, to microbiology education, expertise and know‐how related to well‐being, the production of food, clean water, energy, sustainable practices, environmental protection and conservation of ecosystem services. Crucially, one basic resource deficit is usually accompanied by others, creating a polydeficit calamity, since resource deficits are by nature interdependent and to some extent mutually reinforcing, not only the ‘poverty‐hunger‐poor sanitation’ misery triangle, but also access to healthcare, education, energy and heating, etc., particularly in overcrowded environments, such as slums and refugee camps, which to some extent become parallel societies that are very likely to increase in number and scale as mass migrations increase.
While diplomacy plays a central role in the search for peace, attainment of sustainable peace requires that the root causes of conflicts be identified, addressed and resolved. And in this context, microbes have much to offer. We start with food because, as will be appreciated from regular news bulletins, starvation and malnutrition are ever‐present in many parts of the world. Food supply and security Crop yields One of the most important ecosystem services of microbes is the promotion of plant growth and health by enabling plant access to nutrients, producing biochemicals that inhibit pathogens and by parasitising diverse plant pests ( biopesticides ; Ownley et al., ; Pieterse et al., ; Roca et al., ; Ruffner et al., ; Timmis & Ramos, ; Trivedi et al., ; Verstraete et al., ). Moreover, they are key agents of disease‐suppressive soils, soils that contain plant pathogens but whose disease potential is inhibited. The microbes responsible for plant growth promotion are being technically used to increase crop yields and hence food supplies (Bakker & Berendsen, ; Hu et al., ; Trivedi et al., ), and represent agrobiochemicals or agrobiologics , which can replace costly, energy‐intensive and polluting agrochemicals (Braga et al., ; Roca et al., ; Timmis & Ramos, ). In this regard, it should be emphasised that for about a century, increases in crop yields have been to a large extent achieved through the use of relatively inexpensive and amply available fossil fuel‐based nitrogen fertilisers. But these have created a huge environmental burden and have recently become expensive, as a result of soaring energy costs, so urgently need to be replaced by non‐polluting microbial nitrogenase‐based nitrogen – green nitrogen (Isobe & Ohte, ; Matassa et al., ). Agrobiologics can increase local food production and hence contribute to the reduction of human malnutrition, unemployment and poverty (Ramos & Timmis, ; Timmis et al., ; Timmis & Ramos, ). Soil health Microbes, including the microbial partners of ecosystem engineers such as plants, earthworms and burrowing invertebrates, are key players in soil formation, health and fertility (Blouin et al., ; Hullot et al., ; Lavelle et al., ). (The concept of soil fertility in general assumes a weed‐like crop plant phenotype where the crop grows best in an open habitat, such as loamy soil with high levels of nutrients, good water‐holding capacity, and good drainage (Anderson, ). And microbe:plant partnerships are the agents of increases of soil organic carbon, which is a key driver of soil fertility, carbon sequestration and biodiversity (Gougoulias et al., ; Sykes et al., ), indeed of regenerative agriculture – agricultural practices that improve soil health (Rhodes, ). For example, such partnerships in grasslands result in an annual accumulation of about 1 ton of organic carbon per ha, which significantly enhances soil structure, water and nutrient management, and serves to counter climate change (Denman et al., ). Marginal soils Marginal soils are those that, for reasons of aridity, flooding, extremes of temperature, wind erosion, soil structure, poor nutrient content or pollution, are too stressful for plants to grow well and which produce crops whose value barely covers cultivation costs. Global warming and extreme weather events are driving the transformation of productive soils to marginal soils through desertification and erosion of topsoil. Global warming‐mediated melting of glaciers and the polar ice sheets, and the resulting rise in sea levels, are very much in focus because of displacement of human communities from coastal regions and resulting human migrations. However, another serious consequence is the increase in frequency and extent of inundation by seawater of coastal farmland and its resulting salination, a process that is exacerbated by extreme weather events. Such salination renders fertile agricultural soil inhospitable for most plants and, in turn, also contributes to a reduction in productive farmland, food production capacity and security and, for vulnerable impacted communities, the risk of insufficient nutritional resources. However, in some cases, the stresses of marginal soils for plants can be alleviated in part by microbial partners, which may either diminish the stressor and/or activate plant metabolic responses that result in elevated levels of plant‐stress tolerance. The harnessing of such microbes to alleviate plant stresses and improve plant growth on marginal soils can extend the range of cultivatable land, increase crop yields and food production, and hence reduce food insecurity in such regions (Alsharif et al., ; Egamberdieva et al., ; Gao et al., ; Maestre et al., ). Fermented food and beverages Microbes provide us with a wide diversity of fermented foodstuffs and beverages. Quite apart from contributing to cultural identity, and new and improved gustatorial and olfactory experiences, fermented foods have reduced perishability and contamination with pathogens and mycotoxins (Adebo et al., ). Moreover, fermentation adds to the food material new microbes and new compounds (Kiczorowski, et al., ) that may have health benefits, and liberates calories that would otherwise not be accessible to human digestion – this additional energy has helped feed humanity in times of food scarcity since the agricultural revolution. And ensilaging of crops grown for farm animal feed, involving lactic acid bacteria fermentations, also preserves the feed and increases its nutritional value (Guo et al., ). Non‐animal husbandry sources of meat While dietary protein for much of the world's population is provided for via the husbandry of domesticated food animals, other sources of animal meat are exploited, including wild meat (such as bushmeat; see below), insects, etc. Edible insects are generally regarded as a promising sustainable source of protein‐rich food having a much lower climate footprint and agricultural resource consumption footprint ( https://www.fao.org/3/i3253e/i3253e.pdf ). However, while the risk of zoonotic infections from insects is considered to be less than that from vertebrates, wild insects and farmed insects are known to carry diverse infectious agents that may have potential for transmission to humans (e.g. Gałęcki & Sokół, ; Percipalle et al., ). It is therefore essential that efforts to promote human consumption of insects include the development of farmed insects with reduced or no associated pathogens, for example by creating germ‐free larvae and populating them with a safe microbiota that provides all essential services for larval growth. Subsequent developments along these lines could be to evolve a microbiota that results in the production of additional/increased quantities of valuable nutrients and even health‐beneficial compounds for the consumer. Microbes as food Fungi have been a source of food perhaps even from before the times of our hunter–gatherer forebears and, more recently, through commercial mass production of fruiting bodies – the mushrooms – and of mycelium products. However, photosynthetic microbes, like the cyanobacterium Spirulina/Arthrospira , which have excellent nutritional composition, promise to become major food sources in the future and facilitate reductions in meat consumption (Garcia et al., ). This will have multiple consequences, that are both major in scale and positive in their consequences, because microbial food is relatively inexpensive to produce using sunlight and atmospheric CO 2 , can be produced locally, reducing costs and creating employment, reduces the unnecessary environmental footprint associated with current levels of animal husbandry, including greenhouse gas production, by shifting the focus away from farm animals as primary sources of protein, frees up grazing land for other uses frees up land used to produce crops for animal feed: Pikaar et al. ( ) calculated that by substituting farmed crops with microbial protein in animal feed, and thus decoupling land us from livestock on a world‐scale basis, an agricultural surface of the size of China could become available for other uses, including nature restoration. Sanitation and hygiene Wastewater treatment Microbes are the great transformers and recycle most of the waste materials produced by organisms of the biosphere, including humans. This natural activity is our most powerful system of sanitation and is technically exploited worldwide to treat human wastes in solid and wastewater treatment plants, which also serve to remove pathogens excreted in our wastes, thereby reducing disease transmission ( https://asm.org/Articles/2020/April/How‐Microbes‐Help‐Us‐Reclaim‐Our‐Wastewater ). While the primary need in human societies with insufficient WASH (drinking water, sanitation and hygiene; https://www.who.int/publications/i/item/9789240065031 ) is the destruction of pathogens, current technologies increasingly focus on the recovery and recycling of the various components of wastes to produce valuable biogas (a mixture of methane and other hydrocarbon gases produced by anaerobic digestion of organic waste materials), compost and re‐usable water (Nielsen, ). Thus, the introduction of modern wastewater treatment (WWT) systems in such communities will also extract these valuable resources for local use. Moreover, WWT will in some cases provide crucial irrigation water for agriculture in regions where this is the rate‐limiting parameter in agricultural productivity. Clean water Microbes naturally clean water – reducing faecal pathogen and industrial and agricultural pollutant loads – as it percolates through soil and sediments on its way to underlying aquifers that may serve as sources of drinking water. This natural activity is technically exploited worldwide to purify drinking water supplies and safeguard their quality (Favere et al., ; Fowler & Smets, ; Timmis et al., ). And for water supplies that are contaminated with toxic organic pollutants and/or heavy metals, there are promising emerging technologies, such as the use of melanin to bind and filter out heavy metals (Thaira et al., ). Human communities that are located in deserts and coastal regions may have limited access to fresh water. Desalination of seawater or other saline waters is an obvious solution in some instances, but traditional reverse osmosis technology is energy‐intensive and expensive, so is unaffordable by many communities (Xu et al., ). Microbial desalination cells couple wastewater treatment to desalination and thus solve two problems simultaneously. Healthcare Access to primary healthcare Disadvantaged (and frequently remote) communities often have a high incidence of diseases, particularly infectious diseases. At the same time, they have poor access to healthcare due inter alia to insufficient funds and infrastructure. There is a humanitarian imperative to provide access to healthcare and medicines to communities that lack them. In addition, infectious diseases that are otherwise treatable and containable can spread, mutate and become uncontainable, epidemic and even pandemic. While there are numerous agencies and philanthropic organisations that provide funds and medicines to impoverished communities, our focus here is the potential of microbial technologies to contribute to solutions. One concept proposed to improve access to primary healthcare is the creation of modular and independent Do‐It‐Yourself digital medical centres (dmcs; Timmis & Timmis, ), which are local and can be readily accessed (24/7). The dmcs are health professional (clinician/pharmacist/advanced practice nurse)‐supervised centres focused (initially) on simple diagnostic tests and basic routine health services that can be carried out by patients themselves, with supervision/assistance by healthcare professionals when required. The DIY dmcs would exploit a growing range of point‐of‐care‐testing kits, such as lateral flow and liquid biopsy tests, and provide corresponding decision tree patient assessment approaches and algorithm/artificial intelligence support for test result analysis, interpretation and recommendation. Such centres would also function for simple non‐DIY healthcare services, such as vaccination campaigns, the provision of tele‐health care ( https://catalyst.nejm.org/doi/full/10.1056/CAT.18.0268 ), and condition self‐management support ( https://www.hse.ie/eng/health/hl/selfmanagement/ ; https://www.hug.ch/sites/interhug/files/structures/endocrinologie_diabetologie_hypertension_et_nutrition/article_who_tpe_eng_slama_golay.pdf ). Naturally, a logical extension could include the provision and integration of mobile and wearable community/individual health monitoring and small/digital sample diagnostic devices, bearing various stakeholder needs in mind (Coert et al., ). As they would grow in number, dmcs would exert a ‘pull’ for the development of an expanding spectrum of simple diagnostic tests and frugal medical instruments (Ramadurai & Bhatia, ; Tran & Ravaud, ; https://theconversation.com/frugal‐design‐brings‐medical‐innovations‐to‐communities‐that‐lack‐resources‐during‐the‐pandemic‐147896 ). A significant fraction of the tests would involve microbial products and activities, such as reporter systems, recombinant antibodies and antigens. Such DIY centres are ideally suited to enable greater access to primary healthcare in both urban and rural settings, and serve to raise awareness of and increase knowledge about personal health. With appropriate investment and capacity building, some of the tests used in the centres could be manufactured locally, and thereby also provide both employment and income to local communities. The establishment of dmcs should obviously be integrated into the strategic planning of provision of other deficit‐reducing measures, in order to optimise access and delivery of energy requirements. For example, locating dmcs, WWT and drinking water provision facilities together at an accessible location for the community to be served would enable realisation of important synergies and exchanges of services, such as provision of a dmc with energy by an adjacent WWT plant producing biogas; monitoring by a dmc of both drinking water and wastewater streams for pathogens and other community health indicators. Focus on disease prevention Poverty, hunger and overcrowding all promote disease and – of particular concern – outbreaks and epidemics of infectious diseases. Disease prevention measures, particularly to reduce the transmission of infectious diseases, are vital in communities that do not have modern healthcare systems. Early detection of outbreaks is pivotal to transmission management, and pathogen detection in sewage may alert health authorities earlier than the emergence of the overt disease in the community (Heijnen et al., ). Disease prevention measures also require active participation of community members. Education about disease prevention and transmission of infectious diseases should, therefore, become an integral part of the strategy (see below). Microbiota modulation Microbes are at the heart of available and in development (neo‐)adjuvant/adjunct therapies, such as pro−/pre−/syn−/post−/pharmacobiotics (Vargason & Anselmo, ), faecal microbiota transplantation (El‐Salhy et al., ), specific modulation of gut microbiota composition for a variety of physical and mental clinical issues, including uptake and efficacy of oral medicines metabolised by the intestinal microbiota (Clapp et al., ), and phages and microbial predators targeting infections by antimicrobial resistant pathogens (Brüssow, , ; Gordillo Altamirano & Barr, ). Access to vaccines and medicines People in many parts of the world have limited access to vaccines and medicines. Creating local medicine‐ and vaccine‐production facilities, waiving patent and know‐how issues where necessary, and empowering local personnel and supporting capacity building, would improve such situations enormously, as well as locally providing employment, reducing poverty, and improving the local economy (Timmis et al., ). In some cases (i.e. in tropical and sub‐tropical regions), it would also help address challenges pertinent to cold chain requirements for unstable medicines. In any case, new medicines and vaccines that do not require cold chains are desperately needed to enable such communities to benefit from adequate prophylactic and therapeutic interventions, so research and development programmes to create these are needed, preferably through the development of HICs – Low‐ and Medium Income Countries (LMICs) partnerships involving newly created research and development facilities in target population countries. Discovery–creation of new vaccines and medicines Microbes provide or inspire the components of vaccines against infectious diseases, and thus play a key role in disease prevention, especially during crisis situations when devastating outbreaks such as the COVID‐19 pandemic occur (Brüssow, , ; Maeda et al., ). Moreover, as a result of their exceptional metabolic diversity, microbes produce an exceptional diversity of chemicals: over 80% of the new compounds discovered that eventually become the structural inspirations for the design of new medicines, including antibiotics, originate in microbes. The microbial world still constitutes a largely unexplored reservoir of new chemicals with different applications awaiting discovery. Many remote communities are located in regions of rich, but under‐explored microbial diversity, which may be expected to yield important new discoveries. Establishment of research and development facilities in such regions would enable exploration and prospecting for new pharmaceuticals (Timmis et al., ), with the potential for future commercial development and associated societal and financial rewards, as well as providing new avenues for employment (Timmis et al., ) and technology transfer. Pollution mitigation Many impoverished communities suffer from historical or ongoing industrial pollution with toxic chemicals that negatively impact the health of humans, their animals and/or plants and natural ecosystems (Okereafor et al., ). Moreover, there can be transmission, and in some cases bioaccumulation, of toxic chemicals within the food chain/food web ( https://www.epa.gov/salish‐sea/toxics‐food‐web ), all the way to humans. For example: fungi accumulate heavy metals – including radioactive metals – so, if grown in contaminated soils/materials and gathered/harvested for human consumption, they can transmit toxic metals to the humans who eat them rice accumulates high levels of arsenic fish accumulate toxic polychlorinated‐biphenyls and ‐dioxins and humans eat the fish per ‐ and polyfluorinated alkyl substances (PFAS) – ‘Forever Chemicals’ – are a class of more than 4700 synthetic, highly persistent chemicals that do not occur in nature, some of which are known to have adverse health effects and bioaccumulate in the food web ( https://chemtrust.org/pfas/#:~:text=PFAS%20(Per%2D%20and%20polyfluorinated%20alkyl,do'not%20occur%20in%20nature ; https://www.cdc.gov/biomonitoring/PFAS_FactSheet.html ; Lewis et al., ; Pickard et al., ). Microbes and/or microbial products (Tran‐Ly et al., ), can metabolise, bind and sequester (Cordero et al., ; Kumar et al., ), detoxify or mineralise pollutants (Boll et al., ; Qin et al., ; Varjani & Patel, ), once the appropriate bioremediation technology is implemented (Wagner‐Döbler et al., ). In some instances, plant:microbe partnerships constitute powerful phytoremediation agents (Das, ; Kumar et al., ; Yang et al., ). Bioremediation not only reduces pollutant‐associated health risks but also improves environmental health, which, in turn, counters biodiversity losses. But microbes are not all‐powerful and some pollutants, as exemplified by the super‐recalcitrant PFASs, which are found in all natural environments worldwide (Arslan & El‐Din, ). This dramatically illustrates the danger of developing and deploying new synthetic chemicals without knowing whether or not they can be degraded and recycled by microbes at rates that prevent accumulation in the environment, that is without adequate appreciation of relevant biological potentials that will naturally prevent major problems of environmental health with uncertain ecological outcomes. Energy and heating Microbes can generate biofuels through the efficient conversion of agricultural, domestic and industrial wastes to biogas, ethanol and other higher alcohols and biodiesel (Love, ; Ramos et al., ). Biofuels can reduce fossil fuel dependencies, increase energy security and contribute to an overall decrease in greenhouse gas emissions (Field et al., ; Valdivia et al., ). And, at higher latitudes, where insufficient energy for heating is problematic, new approaches involving melanin heat‐capture systems offer promise (Cordero et al., ). Reducing living space, mass migrations and overcrowding Global warming is melting the polar ice caps and glaciers, causing rises in sea levels that are triggering/will trigger the migration of human communities from coastal to inland regions. In many geographical locations, however, global warming is also reducing habitable space inland by increasing temperatures beyond that which is consistent with habitation, and/or reducing by desertification and soil erosion land productivity below that needed to support current communities that depend on it, again causing migration of peoples to other regions. Other causes of human migrations include insufficient water for drinking, irrigation, domestic use and industrial use; hunger; poverty; overcrowding; disease; discrimination; persecution; and warfare. Human migrations and displacements result in societies being increasingly squeezed into ever‐tighter habitats, often (though not always) resulting in overcrowding, often associated with significant deficits of resources and services essential to human welfare and, in worst case scenarios, human survival itself (e.g. see Calhoun, ), exemplified by the sometimes calamitous conditions that exist in some informal settlements, including refugee camps and slums. Migrations and overcrowding can create extreme stress for the migrated community, but in some cases also for the original residents of the location involved, and hence tensions that can trigger conflicts (Calhoun, ). It is in everyone's best interest to retain as much currently available living space as possible, and reduce physical and cultural displacement, so all ways and means of countering loss of living space and its sustenance potential, including microbial technologies to reduce greenhouse gas emissions, increase plant tolerance of aridity and salinity to increase crop yields and the acreage of productive farmland, and increase water availability (Table ), must be embraced. Poverty–employment–income Many of the microbial technology interventions discussed above to reduce individual deficits and resource asymmetries, and including education, technology transfer and capacity building activities, will create local employment and, in some cases, generate revenue surpluses. Local employment can, in turn, reduce poverty, which, in of itself, is prime driver of multiple deficits. But longer‐term strategies for sustained improvement should include the establishment of sustainable and integrated socio‐economic programmes that are tailored to the specific resources and opportunities afforded by local conditions. These might include natural product discovery pipelines in regions of high or unique biodiversity (see above; Timmis et al., ), sustainable biomining of natural resources (Jerez, ), and so forth. This list of microbial technologies having potential to reduce deficits in resources and services (Table ) is not, of course, comprehensive and there are a number of others that are relevant, including diverse applications of microbial melanins, such as their ability to function as sunscreens (Oh et al., ; see also Suryawanshi et al., for details about prodigiosin as a sunscreen component), capture energy (Cordero et al., ), protect against radiation (Cordero et al., ; Dadachova et al., ) and act as antioxidants, etc. (Roy & Rhim, ).
Crop yields One of the most important ecosystem services of microbes is the promotion of plant growth and health by enabling plant access to nutrients, producing biochemicals that inhibit pathogens and by parasitising diverse plant pests ( biopesticides ; Ownley et al., ; Pieterse et al., ; Roca et al., ; Ruffner et al., ; Timmis & Ramos, ; Trivedi et al., ; Verstraete et al., ). Moreover, they are key agents of disease‐suppressive soils, soils that contain plant pathogens but whose disease potential is inhibited. The microbes responsible for plant growth promotion are being technically used to increase crop yields and hence food supplies (Bakker & Berendsen, ; Hu et al., ; Trivedi et al., ), and represent agrobiochemicals or agrobiologics , which can replace costly, energy‐intensive and polluting agrochemicals (Braga et al., ; Roca et al., ; Timmis & Ramos, ). In this regard, it should be emphasised that for about a century, increases in crop yields have been to a large extent achieved through the use of relatively inexpensive and amply available fossil fuel‐based nitrogen fertilisers. But these have created a huge environmental burden and have recently become expensive, as a result of soaring energy costs, so urgently need to be replaced by non‐polluting microbial nitrogenase‐based nitrogen – green nitrogen (Isobe & Ohte, ; Matassa et al., ). Agrobiologics can increase local food production and hence contribute to the reduction of human malnutrition, unemployment and poverty (Ramos & Timmis, ; Timmis et al., ; Timmis & Ramos, ). Soil health Microbes, including the microbial partners of ecosystem engineers such as plants, earthworms and burrowing invertebrates, are key players in soil formation, health and fertility (Blouin et al., ; Hullot et al., ; Lavelle et al., ). (The concept of soil fertility in general assumes a weed‐like crop plant phenotype where the crop grows best in an open habitat, such as loamy soil with high levels of nutrients, good water‐holding capacity, and good drainage (Anderson, ). And microbe:plant partnerships are the agents of increases of soil organic carbon, which is a key driver of soil fertility, carbon sequestration and biodiversity (Gougoulias et al., ; Sykes et al., ), indeed of regenerative agriculture – agricultural practices that improve soil health (Rhodes, ). For example, such partnerships in grasslands result in an annual accumulation of about 1 ton of organic carbon per ha, which significantly enhances soil structure, water and nutrient management, and serves to counter climate change (Denman et al., ). Marginal soils Marginal soils are those that, for reasons of aridity, flooding, extremes of temperature, wind erosion, soil structure, poor nutrient content or pollution, are too stressful for plants to grow well and which produce crops whose value barely covers cultivation costs. Global warming and extreme weather events are driving the transformation of productive soils to marginal soils through desertification and erosion of topsoil. Global warming‐mediated melting of glaciers and the polar ice sheets, and the resulting rise in sea levels, are very much in focus because of displacement of human communities from coastal regions and resulting human migrations. However, another serious consequence is the increase in frequency and extent of inundation by seawater of coastal farmland and its resulting salination, a process that is exacerbated by extreme weather events. Such salination renders fertile agricultural soil inhospitable for most plants and, in turn, also contributes to a reduction in productive farmland, food production capacity and security and, for vulnerable impacted communities, the risk of insufficient nutritional resources. However, in some cases, the stresses of marginal soils for plants can be alleviated in part by microbial partners, which may either diminish the stressor and/or activate plant metabolic responses that result in elevated levels of plant‐stress tolerance. The harnessing of such microbes to alleviate plant stresses and improve plant growth on marginal soils can extend the range of cultivatable land, increase crop yields and food production, and hence reduce food insecurity in such regions (Alsharif et al., ; Egamberdieva et al., ; Gao et al., ; Maestre et al., ). Fermented food and beverages Microbes provide us with a wide diversity of fermented foodstuffs and beverages. Quite apart from contributing to cultural identity, and new and improved gustatorial and olfactory experiences, fermented foods have reduced perishability and contamination with pathogens and mycotoxins (Adebo et al., ). Moreover, fermentation adds to the food material new microbes and new compounds (Kiczorowski, et al., ) that may have health benefits, and liberates calories that would otherwise not be accessible to human digestion – this additional energy has helped feed humanity in times of food scarcity since the agricultural revolution. And ensilaging of crops grown for farm animal feed, involving lactic acid bacteria fermentations, also preserves the feed and increases its nutritional value (Guo et al., ). Non‐animal husbandry sources of meat While dietary protein for much of the world's population is provided for via the husbandry of domesticated food animals, other sources of animal meat are exploited, including wild meat (such as bushmeat; see below), insects, etc. Edible insects are generally regarded as a promising sustainable source of protein‐rich food having a much lower climate footprint and agricultural resource consumption footprint ( https://www.fao.org/3/i3253e/i3253e.pdf ). However, while the risk of zoonotic infections from insects is considered to be less than that from vertebrates, wild insects and farmed insects are known to carry diverse infectious agents that may have potential for transmission to humans (e.g. Gałęcki & Sokół, ; Percipalle et al., ). It is therefore essential that efforts to promote human consumption of insects include the development of farmed insects with reduced or no associated pathogens, for example by creating germ‐free larvae and populating them with a safe microbiota that provides all essential services for larval growth. Subsequent developments along these lines could be to evolve a microbiota that results in the production of additional/increased quantities of valuable nutrients and even health‐beneficial compounds for the consumer. Microbes as food Fungi have been a source of food perhaps even from before the times of our hunter–gatherer forebears and, more recently, through commercial mass production of fruiting bodies – the mushrooms – and of mycelium products. However, photosynthetic microbes, like the cyanobacterium Spirulina/Arthrospira , which have excellent nutritional composition, promise to become major food sources in the future and facilitate reductions in meat consumption (Garcia et al., ). This will have multiple consequences, that are both major in scale and positive in their consequences, because microbial food is relatively inexpensive to produce using sunlight and atmospheric CO 2 , can be produced locally, reducing costs and creating employment, reduces the unnecessary environmental footprint associated with current levels of animal husbandry, including greenhouse gas production, by shifting the focus away from farm animals as primary sources of protein, frees up grazing land for other uses frees up land used to produce crops for animal feed: Pikaar et al. ( ) calculated that by substituting farmed crops with microbial protein in animal feed, and thus decoupling land us from livestock on a world‐scale basis, an agricultural surface of the size of China could become available for other uses, including nature restoration.
One of the most important ecosystem services of microbes is the promotion of plant growth and health by enabling plant access to nutrients, producing biochemicals that inhibit pathogens and by parasitising diverse plant pests ( biopesticides ; Ownley et al., ; Pieterse et al., ; Roca et al., ; Ruffner et al., ; Timmis & Ramos, ; Trivedi et al., ; Verstraete et al., ). Moreover, they are key agents of disease‐suppressive soils, soils that contain plant pathogens but whose disease potential is inhibited. The microbes responsible for plant growth promotion are being technically used to increase crop yields and hence food supplies (Bakker & Berendsen, ; Hu et al., ; Trivedi et al., ), and represent agrobiochemicals or agrobiologics , which can replace costly, energy‐intensive and polluting agrochemicals (Braga et al., ; Roca et al., ; Timmis & Ramos, ). In this regard, it should be emphasised that for about a century, increases in crop yields have been to a large extent achieved through the use of relatively inexpensive and amply available fossil fuel‐based nitrogen fertilisers. But these have created a huge environmental burden and have recently become expensive, as a result of soaring energy costs, so urgently need to be replaced by non‐polluting microbial nitrogenase‐based nitrogen – green nitrogen (Isobe & Ohte, ; Matassa et al., ). Agrobiologics can increase local food production and hence contribute to the reduction of human malnutrition, unemployment and poverty (Ramos & Timmis, ; Timmis et al., ; Timmis & Ramos, ).
Microbes, including the microbial partners of ecosystem engineers such as plants, earthworms and burrowing invertebrates, are key players in soil formation, health and fertility (Blouin et al., ; Hullot et al., ; Lavelle et al., ). (The concept of soil fertility in general assumes a weed‐like crop plant phenotype where the crop grows best in an open habitat, such as loamy soil with high levels of nutrients, good water‐holding capacity, and good drainage (Anderson, ). And microbe:plant partnerships are the agents of increases of soil organic carbon, which is a key driver of soil fertility, carbon sequestration and biodiversity (Gougoulias et al., ; Sykes et al., ), indeed of regenerative agriculture – agricultural practices that improve soil health (Rhodes, ). For example, such partnerships in grasslands result in an annual accumulation of about 1 ton of organic carbon per ha, which significantly enhances soil structure, water and nutrient management, and serves to counter climate change (Denman et al., ).
Marginal soils are those that, for reasons of aridity, flooding, extremes of temperature, wind erosion, soil structure, poor nutrient content or pollution, are too stressful for plants to grow well and which produce crops whose value barely covers cultivation costs. Global warming and extreme weather events are driving the transformation of productive soils to marginal soils through desertification and erosion of topsoil. Global warming‐mediated melting of glaciers and the polar ice sheets, and the resulting rise in sea levels, are very much in focus because of displacement of human communities from coastal regions and resulting human migrations. However, another serious consequence is the increase in frequency and extent of inundation by seawater of coastal farmland and its resulting salination, a process that is exacerbated by extreme weather events. Such salination renders fertile agricultural soil inhospitable for most plants and, in turn, also contributes to a reduction in productive farmland, food production capacity and security and, for vulnerable impacted communities, the risk of insufficient nutritional resources. However, in some cases, the stresses of marginal soils for plants can be alleviated in part by microbial partners, which may either diminish the stressor and/or activate plant metabolic responses that result in elevated levels of plant‐stress tolerance. The harnessing of such microbes to alleviate plant stresses and improve plant growth on marginal soils can extend the range of cultivatable land, increase crop yields and food production, and hence reduce food insecurity in such regions (Alsharif et al., ; Egamberdieva et al., ; Gao et al., ; Maestre et al., ).
Microbes provide us with a wide diversity of fermented foodstuffs and beverages. Quite apart from contributing to cultural identity, and new and improved gustatorial and olfactory experiences, fermented foods have reduced perishability and contamination with pathogens and mycotoxins (Adebo et al., ). Moreover, fermentation adds to the food material new microbes and new compounds (Kiczorowski, et al., ) that may have health benefits, and liberates calories that would otherwise not be accessible to human digestion – this additional energy has helped feed humanity in times of food scarcity since the agricultural revolution. And ensilaging of crops grown for farm animal feed, involving lactic acid bacteria fermentations, also preserves the feed and increases its nutritional value (Guo et al., ).
While dietary protein for much of the world's population is provided for via the husbandry of domesticated food animals, other sources of animal meat are exploited, including wild meat (such as bushmeat; see below), insects, etc. Edible insects are generally regarded as a promising sustainable source of protein‐rich food having a much lower climate footprint and agricultural resource consumption footprint ( https://www.fao.org/3/i3253e/i3253e.pdf ). However, while the risk of zoonotic infections from insects is considered to be less than that from vertebrates, wild insects and farmed insects are known to carry diverse infectious agents that may have potential for transmission to humans (e.g. Gałęcki & Sokół, ; Percipalle et al., ). It is therefore essential that efforts to promote human consumption of insects include the development of farmed insects with reduced or no associated pathogens, for example by creating germ‐free larvae and populating them with a safe microbiota that provides all essential services for larval growth. Subsequent developments along these lines could be to evolve a microbiota that results in the production of additional/increased quantities of valuable nutrients and even health‐beneficial compounds for the consumer.
Fungi have been a source of food perhaps even from before the times of our hunter–gatherer forebears and, more recently, through commercial mass production of fruiting bodies – the mushrooms – and of mycelium products. However, photosynthetic microbes, like the cyanobacterium Spirulina/Arthrospira , which have excellent nutritional composition, promise to become major food sources in the future and facilitate reductions in meat consumption (Garcia et al., ). This will have multiple consequences, that are both major in scale and positive in their consequences, because microbial food is relatively inexpensive to produce using sunlight and atmospheric CO 2 , can be produced locally, reducing costs and creating employment, reduces the unnecessary environmental footprint associated with current levels of animal husbandry, including greenhouse gas production, by shifting the focus away from farm animals as primary sources of protein, frees up grazing land for other uses frees up land used to produce crops for animal feed: Pikaar et al. ( ) calculated that by substituting farmed crops with microbial protein in animal feed, and thus decoupling land us from livestock on a world‐scale basis, an agricultural surface of the size of China could become available for other uses, including nature restoration.
Wastewater treatment Microbes are the great transformers and recycle most of the waste materials produced by organisms of the biosphere, including humans. This natural activity is our most powerful system of sanitation and is technically exploited worldwide to treat human wastes in solid and wastewater treatment plants, which also serve to remove pathogens excreted in our wastes, thereby reducing disease transmission ( https://asm.org/Articles/2020/April/How‐Microbes‐Help‐Us‐Reclaim‐Our‐Wastewater ). While the primary need in human societies with insufficient WASH (drinking water, sanitation and hygiene; https://www.who.int/publications/i/item/9789240065031 ) is the destruction of pathogens, current technologies increasingly focus on the recovery and recycling of the various components of wastes to produce valuable biogas (a mixture of methane and other hydrocarbon gases produced by anaerobic digestion of organic waste materials), compost and re‐usable water (Nielsen, ). Thus, the introduction of modern wastewater treatment (WWT) systems in such communities will also extract these valuable resources for local use. Moreover, WWT will in some cases provide crucial irrigation water for agriculture in regions where this is the rate‐limiting parameter in agricultural productivity. Clean water Microbes naturally clean water – reducing faecal pathogen and industrial and agricultural pollutant loads – as it percolates through soil and sediments on its way to underlying aquifers that may serve as sources of drinking water. This natural activity is technically exploited worldwide to purify drinking water supplies and safeguard their quality (Favere et al., ; Fowler & Smets, ; Timmis et al., ). And for water supplies that are contaminated with toxic organic pollutants and/or heavy metals, there are promising emerging technologies, such as the use of melanin to bind and filter out heavy metals (Thaira et al., ). Human communities that are located in deserts and coastal regions may have limited access to fresh water. Desalination of seawater or other saline waters is an obvious solution in some instances, but traditional reverse osmosis technology is energy‐intensive and expensive, so is unaffordable by many communities (Xu et al., ). Microbial desalination cells couple wastewater treatment to desalination and thus solve two problems simultaneously.
Microbes are the great transformers and recycle most of the waste materials produced by organisms of the biosphere, including humans. This natural activity is our most powerful system of sanitation and is technically exploited worldwide to treat human wastes in solid and wastewater treatment plants, which also serve to remove pathogens excreted in our wastes, thereby reducing disease transmission ( https://asm.org/Articles/2020/April/How‐Microbes‐Help‐Us‐Reclaim‐Our‐Wastewater ). While the primary need in human societies with insufficient WASH (drinking water, sanitation and hygiene; https://www.who.int/publications/i/item/9789240065031 ) is the destruction of pathogens, current technologies increasingly focus on the recovery and recycling of the various components of wastes to produce valuable biogas (a mixture of methane and other hydrocarbon gases produced by anaerobic digestion of organic waste materials), compost and re‐usable water (Nielsen, ). Thus, the introduction of modern wastewater treatment (WWT) systems in such communities will also extract these valuable resources for local use. Moreover, WWT will in some cases provide crucial irrigation water for agriculture in regions where this is the rate‐limiting parameter in agricultural productivity.
Microbes naturally clean water – reducing faecal pathogen and industrial and agricultural pollutant loads – as it percolates through soil and sediments on its way to underlying aquifers that may serve as sources of drinking water. This natural activity is technically exploited worldwide to purify drinking water supplies and safeguard their quality (Favere et al., ; Fowler & Smets, ; Timmis et al., ). And for water supplies that are contaminated with toxic organic pollutants and/or heavy metals, there are promising emerging technologies, such as the use of melanin to bind and filter out heavy metals (Thaira et al., ). Human communities that are located in deserts and coastal regions may have limited access to fresh water. Desalination of seawater or other saline waters is an obvious solution in some instances, but traditional reverse osmosis technology is energy‐intensive and expensive, so is unaffordable by many communities (Xu et al., ). Microbial desalination cells couple wastewater treatment to desalination and thus solve two problems simultaneously.
Access to primary healthcare Disadvantaged (and frequently remote) communities often have a high incidence of diseases, particularly infectious diseases. At the same time, they have poor access to healthcare due inter alia to insufficient funds and infrastructure. There is a humanitarian imperative to provide access to healthcare and medicines to communities that lack them. In addition, infectious diseases that are otherwise treatable and containable can spread, mutate and become uncontainable, epidemic and even pandemic. While there are numerous agencies and philanthropic organisations that provide funds and medicines to impoverished communities, our focus here is the potential of microbial technologies to contribute to solutions. One concept proposed to improve access to primary healthcare is the creation of modular and independent Do‐It‐Yourself digital medical centres (dmcs; Timmis & Timmis, ), which are local and can be readily accessed (24/7). The dmcs are health professional (clinician/pharmacist/advanced practice nurse)‐supervised centres focused (initially) on simple diagnostic tests and basic routine health services that can be carried out by patients themselves, with supervision/assistance by healthcare professionals when required. The DIY dmcs would exploit a growing range of point‐of‐care‐testing kits, such as lateral flow and liquid biopsy tests, and provide corresponding decision tree patient assessment approaches and algorithm/artificial intelligence support for test result analysis, interpretation and recommendation. Such centres would also function for simple non‐DIY healthcare services, such as vaccination campaigns, the provision of tele‐health care ( https://catalyst.nejm.org/doi/full/10.1056/CAT.18.0268 ), and condition self‐management support ( https://www.hse.ie/eng/health/hl/selfmanagement/ ; https://www.hug.ch/sites/interhug/files/structures/endocrinologie_diabetologie_hypertension_et_nutrition/article_who_tpe_eng_slama_golay.pdf ). Naturally, a logical extension could include the provision and integration of mobile and wearable community/individual health monitoring and small/digital sample diagnostic devices, bearing various stakeholder needs in mind (Coert et al., ). As they would grow in number, dmcs would exert a ‘pull’ for the development of an expanding spectrum of simple diagnostic tests and frugal medical instruments (Ramadurai & Bhatia, ; Tran & Ravaud, ; https://theconversation.com/frugal‐design‐brings‐medical‐innovations‐to‐communities‐that‐lack‐resources‐during‐the‐pandemic‐147896 ). A significant fraction of the tests would involve microbial products and activities, such as reporter systems, recombinant antibodies and antigens. Such DIY centres are ideally suited to enable greater access to primary healthcare in both urban and rural settings, and serve to raise awareness of and increase knowledge about personal health. With appropriate investment and capacity building, some of the tests used in the centres could be manufactured locally, and thereby also provide both employment and income to local communities. The establishment of dmcs should obviously be integrated into the strategic planning of provision of other deficit‐reducing measures, in order to optimise access and delivery of energy requirements. For example, locating dmcs, WWT and drinking water provision facilities together at an accessible location for the community to be served would enable realisation of important synergies and exchanges of services, such as provision of a dmc with energy by an adjacent WWT plant producing biogas; monitoring by a dmc of both drinking water and wastewater streams for pathogens and other community health indicators. Focus on disease prevention Poverty, hunger and overcrowding all promote disease and – of particular concern – outbreaks and epidemics of infectious diseases. Disease prevention measures, particularly to reduce the transmission of infectious diseases, are vital in communities that do not have modern healthcare systems. Early detection of outbreaks is pivotal to transmission management, and pathogen detection in sewage may alert health authorities earlier than the emergence of the overt disease in the community (Heijnen et al., ). Disease prevention measures also require active participation of community members. Education about disease prevention and transmission of infectious diseases should, therefore, become an integral part of the strategy (see below). Microbiota modulation Microbes are at the heart of available and in development (neo‐)adjuvant/adjunct therapies, such as pro−/pre−/syn−/post−/pharmacobiotics (Vargason & Anselmo, ), faecal microbiota transplantation (El‐Salhy et al., ), specific modulation of gut microbiota composition for a variety of physical and mental clinical issues, including uptake and efficacy of oral medicines metabolised by the intestinal microbiota (Clapp et al., ), and phages and microbial predators targeting infections by antimicrobial resistant pathogens (Brüssow, , ; Gordillo Altamirano & Barr, ). Access to vaccines and medicines People in many parts of the world have limited access to vaccines and medicines. Creating local medicine‐ and vaccine‐production facilities, waiving patent and know‐how issues where necessary, and empowering local personnel and supporting capacity building, would improve such situations enormously, as well as locally providing employment, reducing poverty, and improving the local economy (Timmis et al., ). In some cases (i.e. in tropical and sub‐tropical regions), it would also help address challenges pertinent to cold chain requirements for unstable medicines. In any case, new medicines and vaccines that do not require cold chains are desperately needed to enable such communities to benefit from adequate prophylactic and therapeutic interventions, so research and development programmes to create these are needed, preferably through the development of HICs – Low‐ and Medium Income Countries (LMICs) partnerships involving newly created research and development facilities in target population countries. Discovery–creation of new vaccines and medicines Microbes provide or inspire the components of vaccines against infectious diseases, and thus play a key role in disease prevention, especially during crisis situations when devastating outbreaks such as the COVID‐19 pandemic occur (Brüssow, , ; Maeda et al., ). Moreover, as a result of their exceptional metabolic diversity, microbes produce an exceptional diversity of chemicals: over 80% of the new compounds discovered that eventually become the structural inspirations for the design of new medicines, including antibiotics, originate in microbes. The microbial world still constitutes a largely unexplored reservoir of new chemicals with different applications awaiting discovery. Many remote communities are located in regions of rich, but under‐explored microbial diversity, which may be expected to yield important new discoveries. Establishment of research and development facilities in such regions would enable exploration and prospecting for new pharmaceuticals (Timmis et al., ), with the potential for future commercial development and associated societal and financial rewards, as well as providing new avenues for employment (Timmis et al., ) and technology transfer.
Disadvantaged (and frequently remote) communities often have a high incidence of diseases, particularly infectious diseases. At the same time, they have poor access to healthcare due inter alia to insufficient funds and infrastructure. There is a humanitarian imperative to provide access to healthcare and medicines to communities that lack them. In addition, infectious diseases that are otherwise treatable and containable can spread, mutate and become uncontainable, epidemic and even pandemic. While there are numerous agencies and philanthropic organisations that provide funds and medicines to impoverished communities, our focus here is the potential of microbial technologies to contribute to solutions. One concept proposed to improve access to primary healthcare is the creation of modular and independent Do‐It‐Yourself digital medical centres (dmcs; Timmis & Timmis, ), which are local and can be readily accessed (24/7). The dmcs are health professional (clinician/pharmacist/advanced practice nurse)‐supervised centres focused (initially) on simple diagnostic tests and basic routine health services that can be carried out by patients themselves, with supervision/assistance by healthcare professionals when required. The DIY dmcs would exploit a growing range of point‐of‐care‐testing kits, such as lateral flow and liquid biopsy tests, and provide corresponding decision tree patient assessment approaches and algorithm/artificial intelligence support for test result analysis, interpretation and recommendation. Such centres would also function for simple non‐DIY healthcare services, such as vaccination campaigns, the provision of tele‐health care ( https://catalyst.nejm.org/doi/full/10.1056/CAT.18.0268 ), and condition self‐management support ( https://www.hse.ie/eng/health/hl/selfmanagement/ ; https://www.hug.ch/sites/interhug/files/structures/endocrinologie_diabetologie_hypertension_et_nutrition/article_who_tpe_eng_slama_golay.pdf ). Naturally, a logical extension could include the provision and integration of mobile and wearable community/individual health monitoring and small/digital sample diagnostic devices, bearing various stakeholder needs in mind (Coert et al., ). As they would grow in number, dmcs would exert a ‘pull’ for the development of an expanding spectrum of simple diagnostic tests and frugal medical instruments (Ramadurai & Bhatia, ; Tran & Ravaud, ; https://theconversation.com/frugal‐design‐brings‐medical‐innovations‐to‐communities‐that‐lack‐resources‐during‐the‐pandemic‐147896 ). A significant fraction of the tests would involve microbial products and activities, such as reporter systems, recombinant antibodies and antigens. Such DIY centres are ideally suited to enable greater access to primary healthcare in both urban and rural settings, and serve to raise awareness of and increase knowledge about personal health. With appropriate investment and capacity building, some of the tests used in the centres could be manufactured locally, and thereby also provide both employment and income to local communities. The establishment of dmcs should obviously be integrated into the strategic planning of provision of other deficit‐reducing measures, in order to optimise access and delivery of energy requirements. For example, locating dmcs, WWT and drinking water provision facilities together at an accessible location for the community to be served would enable realisation of important synergies and exchanges of services, such as provision of a dmc with energy by an adjacent WWT plant producing biogas; monitoring by a dmc of both drinking water and wastewater streams for pathogens and other community health indicators.
Poverty, hunger and overcrowding all promote disease and – of particular concern – outbreaks and epidemics of infectious diseases. Disease prevention measures, particularly to reduce the transmission of infectious diseases, are vital in communities that do not have modern healthcare systems. Early detection of outbreaks is pivotal to transmission management, and pathogen detection in sewage may alert health authorities earlier than the emergence of the overt disease in the community (Heijnen et al., ). Disease prevention measures also require active participation of community members. Education about disease prevention and transmission of infectious diseases should, therefore, become an integral part of the strategy (see below).
Microbes are at the heart of available and in development (neo‐)adjuvant/adjunct therapies, such as pro−/pre−/syn−/post−/pharmacobiotics (Vargason & Anselmo, ), faecal microbiota transplantation (El‐Salhy et al., ), specific modulation of gut microbiota composition for a variety of physical and mental clinical issues, including uptake and efficacy of oral medicines metabolised by the intestinal microbiota (Clapp et al., ), and phages and microbial predators targeting infections by antimicrobial resistant pathogens (Brüssow, , ; Gordillo Altamirano & Barr, ).
People in many parts of the world have limited access to vaccines and medicines. Creating local medicine‐ and vaccine‐production facilities, waiving patent and know‐how issues where necessary, and empowering local personnel and supporting capacity building, would improve such situations enormously, as well as locally providing employment, reducing poverty, and improving the local economy (Timmis et al., ). In some cases (i.e. in tropical and sub‐tropical regions), it would also help address challenges pertinent to cold chain requirements for unstable medicines. In any case, new medicines and vaccines that do not require cold chains are desperately needed to enable such communities to benefit from adequate prophylactic and therapeutic interventions, so research and development programmes to create these are needed, preferably through the development of HICs – Low‐ and Medium Income Countries (LMICs) partnerships involving newly created research and development facilities in target population countries.
Microbes provide or inspire the components of vaccines against infectious diseases, and thus play a key role in disease prevention, especially during crisis situations when devastating outbreaks such as the COVID‐19 pandemic occur (Brüssow, , ; Maeda et al., ). Moreover, as a result of their exceptional metabolic diversity, microbes produce an exceptional diversity of chemicals: over 80% of the new compounds discovered that eventually become the structural inspirations for the design of new medicines, including antibiotics, originate in microbes. The microbial world still constitutes a largely unexplored reservoir of new chemicals with different applications awaiting discovery. Many remote communities are located in regions of rich, but under‐explored microbial diversity, which may be expected to yield important new discoveries. Establishment of research and development facilities in such regions would enable exploration and prospecting for new pharmaceuticals (Timmis et al., ), with the potential for future commercial development and associated societal and financial rewards, as well as providing new avenues for employment (Timmis et al., ) and technology transfer.
Many impoverished communities suffer from historical or ongoing industrial pollution with toxic chemicals that negatively impact the health of humans, their animals and/or plants and natural ecosystems (Okereafor et al., ). Moreover, there can be transmission, and in some cases bioaccumulation, of toxic chemicals within the food chain/food web ( https://www.epa.gov/salish‐sea/toxics‐food‐web ), all the way to humans. For example: fungi accumulate heavy metals – including radioactive metals – so, if grown in contaminated soils/materials and gathered/harvested for human consumption, they can transmit toxic metals to the humans who eat them rice accumulates high levels of arsenic fish accumulate toxic polychlorinated‐biphenyls and ‐dioxins and humans eat the fish per ‐ and polyfluorinated alkyl substances (PFAS) – ‘Forever Chemicals’ – are a class of more than 4700 synthetic, highly persistent chemicals that do not occur in nature, some of which are known to have adverse health effects and bioaccumulate in the food web ( https://chemtrust.org/pfas/#:~:text=PFAS%20(Per%2D%20and%20polyfluorinated%20alkyl,do'not%20occur%20in%20nature ; https://www.cdc.gov/biomonitoring/PFAS_FactSheet.html ; Lewis et al., ; Pickard et al., ). Microbes and/or microbial products (Tran‐Ly et al., ), can metabolise, bind and sequester (Cordero et al., ; Kumar et al., ), detoxify or mineralise pollutants (Boll et al., ; Qin et al., ; Varjani & Patel, ), once the appropriate bioremediation technology is implemented (Wagner‐Döbler et al., ). In some instances, plant:microbe partnerships constitute powerful phytoremediation agents (Das, ; Kumar et al., ; Yang et al., ). Bioremediation not only reduces pollutant‐associated health risks but also improves environmental health, which, in turn, counters biodiversity losses. But microbes are not all‐powerful and some pollutants, as exemplified by the super‐recalcitrant PFASs, which are found in all natural environments worldwide (Arslan & El‐Din, ). This dramatically illustrates the danger of developing and deploying new synthetic chemicals without knowing whether or not they can be degraded and recycled by microbes at rates that prevent accumulation in the environment, that is without adequate appreciation of relevant biological potentials that will naturally prevent major problems of environmental health with uncertain ecological outcomes.
Microbes can generate biofuels through the efficient conversion of agricultural, domestic and industrial wastes to biogas, ethanol and other higher alcohols and biodiesel (Love, ; Ramos et al., ). Biofuels can reduce fossil fuel dependencies, increase energy security and contribute to an overall decrease in greenhouse gas emissions (Field et al., ; Valdivia et al., ). And, at higher latitudes, where insufficient energy for heating is problematic, new approaches involving melanin heat‐capture systems offer promise (Cordero et al., ).
Global warming is melting the polar ice caps and glaciers, causing rises in sea levels that are triggering/will trigger the migration of human communities from coastal to inland regions. In many geographical locations, however, global warming is also reducing habitable space inland by increasing temperatures beyond that which is consistent with habitation, and/or reducing by desertification and soil erosion land productivity below that needed to support current communities that depend on it, again causing migration of peoples to other regions. Other causes of human migrations include insufficient water for drinking, irrigation, domestic use and industrial use; hunger; poverty; overcrowding; disease; discrimination; persecution; and warfare. Human migrations and displacements result in societies being increasingly squeezed into ever‐tighter habitats, often (though not always) resulting in overcrowding, often associated with significant deficits of resources and services essential to human welfare and, in worst case scenarios, human survival itself (e.g. see Calhoun, ), exemplified by the sometimes calamitous conditions that exist in some informal settlements, including refugee camps and slums. Migrations and overcrowding can create extreme stress for the migrated community, but in some cases also for the original residents of the location involved, and hence tensions that can trigger conflicts (Calhoun, ). It is in everyone's best interest to retain as much currently available living space as possible, and reduce physical and cultural displacement, so all ways and means of countering loss of living space and its sustenance potential, including microbial technologies to reduce greenhouse gas emissions, increase plant tolerance of aridity and salinity to increase crop yields and the acreage of productive farmland, and increase water availability (Table ), must be embraced.
Many of the microbial technology interventions discussed above to reduce individual deficits and resource asymmetries, and including education, technology transfer and capacity building activities, will create local employment and, in some cases, generate revenue surpluses. Local employment can, in turn, reduce poverty, which, in of itself, is prime driver of multiple deficits. But longer‐term strategies for sustained improvement should include the establishment of sustainable and integrated socio‐economic programmes that are tailored to the specific resources and opportunities afforded by local conditions. These might include natural product discovery pipelines in regions of high or unique biodiversity (see above; Timmis et al., ), sustainable biomining of natural resources (Jerez, ), and so forth. This list of microbial technologies having potential to reduce deficits in resources and services (Table ) is not, of course, comprehensive and there are a number of others that are relevant, including diverse applications of microbial melanins, such as their ability to function as sunscreens (Oh et al., ; see also Suryawanshi et al., for details about prodigiosin as a sunscreen component), capture energy (Cordero et al., ), protect against radiation (Cordero et al., ; Dadachova et al., ) and act as antioxidants, etc. (Roy & Rhim, ).
The consumption of wild meat and issues of zoonotic transmission, epidemics and pandemics Many animal species consume ‘wild meat’: it is nature in action – predation – a key element of the food chain/web, and was a major source of food for our hunter–gatherer forebears. Today, wild meat may be consumed by humans for diverse reasons, including poverty, recreational hunting, opportunistic situations such as the consumption of ‘road kill’, cultural preferences for wild meat and tradition and culinary appreciation, etc. The presence of wet markets in China and Southeast Asian countries indicates that bushmeat may also be considered as a luxury food item or having medicinal value. This results in significant demand for bushmeat for traditional reasons, but also by affluent individuals in urban areas who appreciate more exotic foods. Indeed, the consumption of bushmeat is part of the culture/religion of diverse communities worldwide, including those of indigenous peoples. And the distinction between wild and domesticated can become blurred with the husbandry of animals like rabbit, deer, boar, ostrich, buffalo, etc. While the consumption of wild meat has different values for different cultures, our focus is hunger and the consequences of eating wild meat by impoverished communities whose survival depends upon it. The scale of wild meat consumption is significant. One report suggests that it accounts for about 80% of the protein intake of peoples in Central Africa ( https://www2.cifor.org/bushmeat ), which is comparable to the amount of beef production of Brazil. Other, unique dietary habits can be observed in populations of different indigenous peoples. For example, one marginalised community in India, the Musahars with a population of 2.5 million in the Bihar region, are known as ‘rat eaters’. Problematically, wild meat is known to be a significant source of infectious agents that are either human pathogens, or can mutate to become human pathogens. For example, bushmeat in Tanzania has been reported to have a high prevalence of Bacillus anthracis , Brucella and Coxiella (Katani et al., , ). The risk of zoonotic pathogen transmission associated with meat processing and consumption, which includes Ebola virus, Monkeypox virus, SARS‐CoV and even human immunodeficiency virus (HIV), is very real. The processing of wild meat is a particular risk because of the possibility of direct inoculation of meat‐associated pathogens into cuts and injuries of the handling humans. Wolfe et al. ( ) reported the emergence of zoonotic pathogens in the Cameroon–Congo Basin region between 1970–2005 that included Arboviruses, Ebola, Monkeypox, HIV‐1 and ‐2, Anthrax, Herpes B virus, Simian foamy and other viruses. It has been estimated that up to 10 distinct events of HIV‐1/2 transmission into human populations occurred in the century prior to the global emergence of HIV (Hahn et al., ). Infection from the Simian foamy viruses is frequent among hunters in the Republic of Cameroon (Wolfe et al., ). Thus, the consumption of wild meat as a necessity imposed by basic nutritional needs is accompanied by a heightened risk of zoonotic infections and an evolution of animal pathogens/commensals into human pathogens, transmission and the possibility of outbreaks of epidemics/pandemics. Communities dependent on wild meat consumption are in the front line of zoonotic transmissions, which is another element of social inequity, and their nutritional needs may constitute a driver of regional epidemics and potential global pandemics such as COVID‐19. There is therefore a compelling case for reducing the consumption of wild meat. Key to this will be adequate provision of alternative sources of food that are outlined above. However, traditional, religious and cultural norms will have to be sensitively taken into consideration, and where appropriate accompanied by supporting education and information programmes in relevant topics when offering alternative forms of nutrition. Extraction and export of valuable resources with externalisation of pollution cost burdens There are many examples of regions rich in valuable natural resources, for example gold and other metals, various minerals and petroleum oil, that are exported, but extracted by local workers who see little of the significant economic benefits of the exported resource. Moreover, in many cases, the extraction process creates local environmental pollution that often results in environmental degradation, which is prejudicial to maintenance of biodiversity and ecosystem services ( https://www.cbd.int/article/cop15‐cbd‐press‐release‐final‐19dec2022 ) and, in many cases, with serious adverse effects on the health of the community, on occasion becoming a serious cause of ill‐heath. Regrettably, such pollution is frequently not remediated or the remediation costs are externalised (i.e. the problem is not considered to be the responsibility of relevant transacting partners – the producers and customers) ( https://www.imf.org/en/Publications/fandd/issues/Series/Back‐to‐Basics/Externalities ; ostrich https://wiki.p2pfoundation.net/Externalization_of_Costs ;). Polluting industries that externalise costs are not restricted to the mining of natural resources, and include for example the clothing‐fashion industry. The practice of externalisation of costs is globally pervasive, but has a much greater impact in LMICs because affected communities have little or no power over corporations, criminals, or government actors exploiting their resources. But pollution creates an important and widespread deficit, that of a clean and healthy environment. Legacy polluted environments must be repaired and future industrial operations changed to prevent pollution. There are powerful microbially based bioremediation technologies that can contribute to the restoration of environmental health and promote biodiversity and the ecosystem services that it provides. There are also effective microbial technologies, for example waste‐gas treatment systems, that can be used to prevent or reduce pollution at source (Komang Ralebitso‐Senior et al., ). Furthermore, there exist microbial technologies that enable environmentally friendly extraction processes (Jerez, ). However, the root cause of these problems is the practice of cost externalisation and can only be corrected by a change in political will to require cost internalisation ( https://wiki.p2pfoundation.net/Externalization_of_Costs ) and proper respect of affected communities and their due rights. Microbial behaviour is instructive for us Microbes also experience deficits and engage in warfare, alliances and cooperation, so it is interesting to consider what we may learn from them in the context of this discussion. Microbial ecology is a story of constant struggle for survival, a matter of competition for limited habitat space and resources, of pioneer species and predator species, but also a matter of give and take, and – metaphorically speaking – a case of ‘market economy’ at the level of micro‐ and macro‐ecosystems. Recent insights in microbial population dynamics and ecology inform us that cooperation and moderation are the best strategies for long‐term well‐being (Tasoff et al., ). Yet, this does not mean ‘equality’ for all at all times: microbial societies may have Pareto distributions in which, for a stable set of conditions, 80% of the resources and the flow of energy appear to be captured by some 20% top performers (e.g. Pelz et al., ). While this is also true of some human resources, as proposed by Pareto ( https://en.wikipedia.org/wiki/Pareto_principle ), and indeed may be a not uncommon aspect of nature, this is not to suggest that such a distribution is a goal to which we should aspire. In any case, any significant change of conditions may result in a new Pareto distribution, that is equilibrium (Marzorati et al., ). On the other hand, drastic perturbation of specific populations or groups of populations of the microbial world, for example though use of pesticides or antibiotics, results in the emergence of certain strains having resistance traits, strains that can be or become disruptive and dangerous (Cray et al., ; Flandroy et al., ; Jain et al., ). Microbial community behaviour clearly shows that such ‘pestering’ should be avoided because it can lead to both weaponising (e.g. proliferation of antimicrobial resistance) and war (e.g. increases in dangerous infections) by the disturbed communities. This commonly occurs in disturbed habitats or other types of open habitats (Cray et al., ) and is analogous to power vacuums in human societies, which, historically, also tend to be filled by exploitative, disruptive and dangerous individuals and groups thereof. There exist many (recent) examples of this on various governance levels in all human societies and subsets thereof. This teaches us that change/behavioural modification should be achieved by more clever and balanced strategies. On the positive side, we have become aware that communication within the microbial world is of crucial importance and underpins the structure and functioning of successful microbial communities (Hughes & Sperandio, ). And, although we may learn much from observing the behaviour of microbial communities, we also need to take into account that microbes also influence our own behaviour directly. Our gut microbiota and the metabolites they create from the food we ingest profoundly influence our mental state, mood and behaviour via the gut:brain axis and the endocrine system (Clarke et al., ; Margolis et al., ; Smith & Wissel, ; https://www.leuvenmindgate.be/news/gut‐bacteria‐may‐influence‐your‐mental‐health ). And the nature of the food we eat profoundly influences the composition of our gut microbiota, and hence its influence on our mental state. In other words, thinking about peace at the level of human society needs to take into account the fact that human attitudes may to some extent be influenced by the microbial constitutions of people's guts, which, in turn, are influenced by the food they eat and, importantly, the foods they do not eat/cannot obtain. The ability to study the behaviour of millions of microbes at the single cell level, and the ecosystem services they provide, can yield important insights into microbial ecology (Wood, ) that, in constellations when interaction is primarily transactional, can help guide the design of certain parts of human ecosystems.
Many animal species consume ‘wild meat’: it is nature in action – predation – a key element of the food chain/web, and was a major source of food for our hunter–gatherer forebears. Today, wild meat may be consumed by humans for diverse reasons, including poverty, recreational hunting, opportunistic situations such as the consumption of ‘road kill’, cultural preferences for wild meat and tradition and culinary appreciation, etc. The presence of wet markets in China and Southeast Asian countries indicates that bushmeat may also be considered as a luxury food item or having medicinal value. This results in significant demand for bushmeat for traditional reasons, but also by affluent individuals in urban areas who appreciate more exotic foods. Indeed, the consumption of bushmeat is part of the culture/religion of diverse communities worldwide, including those of indigenous peoples. And the distinction between wild and domesticated can become blurred with the husbandry of animals like rabbit, deer, boar, ostrich, buffalo, etc. While the consumption of wild meat has different values for different cultures, our focus is hunger and the consequences of eating wild meat by impoverished communities whose survival depends upon it. The scale of wild meat consumption is significant. One report suggests that it accounts for about 80% of the protein intake of peoples in Central Africa ( https://www2.cifor.org/bushmeat ), which is comparable to the amount of beef production of Brazil. Other, unique dietary habits can be observed in populations of different indigenous peoples. For example, one marginalised community in India, the Musahars with a population of 2.5 million in the Bihar region, are known as ‘rat eaters’. Problematically, wild meat is known to be a significant source of infectious agents that are either human pathogens, or can mutate to become human pathogens. For example, bushmeat in Tanzania has been reported to have a high prevalence of Bacillus anthracis , Brucella and Coxiella (Katani et al., , ). The risk of zoonotic pathogen transmission associated with meat processing and consumption, which includes Ebola virus, Monkeypox virus, SARS‐CoV and even human immunodeficiency virus (HIV), is very real. The processing of wild meat is a particular risk because of the possibility of direct inoculation of meat‐associated pathogens into cuts and injuries of the handling humans. Wolfe et al. ( ) reported the emergence of zoonotic pathogens in the Cameroon–Congo Basin region between 1970–2005 that included Arboviruses, Ebola, Monkeypox, HIV‐1 and ‐2, Anthrax, Herpes B virus, Simian foamy and other viruses. It has been estimated that up to 10 distinct events of HIV‐1/2 transmission into human populations occurred in the century prior to the global emergence of HIV (Hahn et al., ). Infection from the Simian foamy viruses is frequent among hunters in the Republic of Cameroon (Wolfe et al., ). Thus, the consumption of wild meat as a necessity imposed by basic nutritional needs is accompanied by a heightened risk of zoonotic infections and an evolution of animal pathogens/commensals into human pathogens, transmission and the possibility of outbreaks of epidemics/pandemics. Communities dependent on wild meat consumption are in the front line of zoonotic transmissions, which is another element of social inequity, and their nutritional needs may constitute a driver of regional epidemics and potential global pandemics such as COVID‐19. There is therefore a compelling case for reducing the consumption of wild meat. Key to this will be adequate provision of alternative sources of food that are outlined above. However, traditional, religious and cultural norms will have to be sensitively taken into consideration, and where appropriate accompanied by supporting education and information programmes in relevant topics when offering alternative forms of nutrition.
There are many examples of regions rich in valuable natural resources, for example gold and other metals, various minerals and petroleum oil, that are exported, but extracted by local workers who see little of the significant economic benefits of the exported resource. Moreover, in many cases, the extraction process creates local environmental pollution that often results in environmental degradation, which is prejudicial to maintenance of biodiversity and ecosystem services ( https://www.cbd.int/article/cop15‐cbd‐press‐release‐final‐19dec2022 ) and, in many cases, with serious adverse effects on the health of the community, on occasion becoming a serious cause of ill‐heath. Regrettably, such pollution is frequently not remediated or the remediation costs are externalised (i.e. the problem is not considered to be the responsibility of relevant transacting partners – the producers and customers) ( https://www.imf.org/en/Publications/fandd/issues/Series/Back‐to‐Basics/Externalities ; ostrich https://wiki.p2pfoundation.net/Externalization_of_Costs ;). Polluting industries that externalise costs are not restricted to the mining of natural resources, and include for example the clothing‐fashion industry. The practice of externalisation of costs is globally pervasive, but has a much greater impact in LMICs because affected communities have little or no power over corporations, criminals, or government actors exploiting their resources. But pollution creates an important and widespread deficit, that of a clean and healthy environment. Legacy polluted environments must be repaired and future industrial operations changed to prevent pollution. There are powerful microbially based bioremediation technologies that can contribute to the restoration of environmental health and promote biodiversity and the ecosystem services that it provides. There are also effective microbial technologies, for example waste‐gas treatment systems, that can be used to prevent or reduce pollution at source (Komang Ralebitso‐Senior et al., ). Furthermore, there exist microbial technologies that enable environmentally friendly extraction processes (Jerez, ). However, the root cause of these problems is the practice of cost externalisation and can only be corrected by a change in political will to require cost internalisation ( https://wiki.p2pfoundation.net/Externalization_of_Costs ) and proper respect of affected communities and their due rights.
Microbes also experience deficits and engage in warfare, alliances and cooperation, so it is interesting to consider what we may learn from them in the context of this discussion. Microbial ecology is a story of constant struggle for survival, a matter of competition for limited habitat space and resources, of pioneer species and predator species, but also a matter of give and take, and – metaphorically speaking – a case of ‘market economy’ at the level of micro‐ and macro‐ecosystems. Recent insights in microbial population dynamics and ecology inform us that cooperation and moderation are the best strategies for long‐term well‐being (Tasoff et al., ). Yet, this does not mean ‘equality’ for all at all times: microbial societies may have Pareto distributions in which, for a stable set of conditions, 80% of the resources and the flow of energy appear to be captured by some 20% top performers (e.g. Pelz et al., ). While this is also true of some human resources, as proposed by Pareto ( https://en.wikipedia.org/wiki/Pareto_principle ), and indeed may be a not uncommon aspect of nature, this is not to suggest that such a distribution is a goal to which we should aspire. In any case, any significant change of conditions may result in a new Pareto distribution, that is equilibrium (Marzorati et al., ). On the other hand, drastic perturbation of specific populations or groups of populations of the microbial world, for example though use of pesticides or antibiotics, results in the emergence of certain strains having resistance traits, strains that can be or become disruptive and dangerous (Cray et al., ; Flandroy et al., ; Jain et al., ). Microbial community behaviour clearly shows that such ‘pestering’ should be avoided because it can lead to both weaponising (e.g. proliferation of antimicrobial resistance) and war (e.g. increases in dangerous infections) by the disturbed communities. This commonly occurs in disturbed habitats or other types of open habitats (Cray et al., ) and is analogous to power vacuums in human societies, which, historically, also tend to be filled by exploitative, disruptive and dangerous individuals and groups thereof. There exist many (recent) examples of this on various governance levels in all human societies and subsets thereof. This teaches us that change/behavioural modification should be achieved by more clever and balanced strategies. On the positive side, we have become aware that communication within the microbial world is of crucial importance and underpins the structure and functioning of successful microbial communities (Hughes & Sperandio, ). And, although we may learn much from observing the behaviour of microbial communities, we also need to take into account that microbes also influence our own behaviour directly. Our gut microbiota and the metabolites they create from the food we ingest profoundly influence our mental state, mood and behaviour via the gut:brain axis and the endocrine system (Clarke et al., ; Margolis et al., ; Smith & Wissel, ; https://www.leuvenmindgate.be/news/gut‐bacteria‐may‐influence‐your‐mental‐health ). And the nature of the food we eat profoundly influences the composition of our gut microbiota, and hence its influence on our mental state. In other words, thinking about peace at the level of human society needs to take into account the fact that human attitudes may to some extent be influenced by the microbial constitutions of people's guts, which, in turn, are influenced by the food they eat and, importantly, the foods they do not eat/cannot obtain. The ability to study the behaviour of millions of microbes at the single cell level, and the ecosystem services they provide, can yield important insights into microbial ecology (Wood, ) that, in constellations when interaction is primarily transactional, can help guide the design of certain parts of human ecosystems.
Besides political will – a proper discussion of which is beyond the scope of this contribution – one of the hindrances to effective and timely exploitation of microbial technologies that can contribute to attainment of the SDGs and improvement of the human condition is the general lack of knowledge about microbes, what they do, and how they have been and can be harnessed to carry out processes that are transformational for humanity (exemplified by vaccines, wastewater treatment). One example of this neglect is the fact that microbes are seldom considered in the context of strategies to mitigate climate change, despite being responsible for the generation and consumption of some powerful greenhouse gases such as carbon dioxide, methane and nitrous oxide (Cavicchioli et al., ; Sykes et al., ; Tiedje et al., ). In the minds of most people, microbes are to be feared, because ‘they’ are invisible actors that can have serious adverse effects by producing diseases like meningitis and pandemics such as acquired immune‐deficiency syndrome (AIDS) and COVID‐19, or cause souring of the milk/moulding of the bread and other forms of food deterioration, sometimes producing food poisoning, or red tides that ruin our fish industries and prevent us from swimming. But these ‘baddies’ are the exception and most microbes are either harmless, helpful or essential to our well‐being and that of the entire biosphere. Most of the bioeconomy is based on microbial activities. Even the ‘baddies’ can have a positive side: for example, the viruses that cause hepatitis have also provided the means to prevent hepatitis by enabling the creation of highly effective vaccines; tetanus toxin that kills us enables us to create inactive tetanus toxoid vaccine that protects us from disease. And battles with pathogens over human evolutionary time have sharpened our immune system via natural selection (Rook, ). So we must: urgently counter germophobia and help establish a more balanced and, above all, better informed perception of microbes, in order that society can form realistic, more granular opinions of microbes, their benefits and dangers (Finlay & Arrieta, ; Gupta et al., ; Timmis et al., ), convince the public and policy‐makers that the entire biosphere, and the multitude of ecosystem services it provides and upon which we depend, is sustained by the Earth's microbiome, reveal how the microbiomes of individuals provide vital services to all organisms, including humans, without which we would not be able to survive for any period of time. emphasise that microbial technologies, such as vaccine and antibiotic production, wastewater treatment and sanitation, clean drinking water provision, and fermented foods, have more than any other societal development transformationally improved life expectancy and quality for much of humanity, convey how microbial technologies, such as amino acid and vitamin production, bioremediation, seed inoculants, biocatalysis, etc., contribute greatly to human well‐being and endeavours explain that microbes are at the centre of biogeochemical cycles that on one hand, provide us with oxygen to breathe and, on the other, play crucial roles in many of the crises facing humanity, like global warming (Cavicchioli et al., ), eutrophication‐oxygen minimum zones‐loss of biodiversity, desertification, etc., and hence must receive due consideration in efforts to confront these crises, and, above all, convince society to see microbes as solutions not just problems, and to vigorously explore microbial activities for new solutions to existing and anticipated crises. All of this underscores the vital need for improved knowledge on the causes of and potential solutions to deficits, on sustainability, ecosystem services and their interconnected nature and impact on human and biosphere well‐being. For society to optimally take advantage of the products and services that microbes can deliver to improve societal well‐being, reduce inequalities and level up, and reduce conflicts, people must become microbiology literate (Timmis et al., ). Knowledge of societally relevant microbiology must be democratised and provided to all humans, albeit in forms that are catered to their needs. To quote Louis Pasteur: ‘“Science knows no country, because knowledge belongs to humanity, and is the torch which illuminates the world’. The International Microbiology Literacy Initiative (IMiLI) is creating teaching resources that will form comprehensive curricula of societally‐relevant microbiology that can be integrated into the school curricula of all countries with education authorities willing to embrace them. These resources are being created pro bono by hundreds of microbiologists worldwide and will soon be made freely available. They not only provide relevant knowledge about microbial activities that touch our lives and those of other members of the biosphere (including those of the atmosphere: see Šantl‐Temkiv, et al., ), but also explain their relevance within the context of the SDGs, and so contribute to teaching about sustainability in schools and colleges, and their relevance to decision‐making and stakeholder scrutiny of decisions, and so will teach about stewardship and stakeholder responsibilities. Most importantly, these resources aim to expose the wealth of activities of microbes that can be harnessed for the good of humanity, including the promotion of peace in the world. And, in the context of decision‐making, it is worth remembering that our gut microbes digest much of our food intake and convert it to a form we can use and, in so doing, create a plethora of metabolites, some of which are essential for us, and some of which affect our moods and, in turn, influence our decisions (Smith & Wissel, ). Without them, we would indeed be highly stressed: they are personal peace‐makers for each of us.
Humanity has a responsibility to ensure that all peoples of the world have access to resources fundamental to a basic quality of life, or if necessary provide them. This is essential to sustainable development of the human race, and peace and harmonious co‐existence of nations, and is eloquently formulated in the SDGs elaborated by the United Nations. Microbial activities are relevant to most, some would argue all, of the SDGs. But here we focus on those that can specifically contribute to peace in the world, inter alia the provision of adequate food, sanitation and clean water, the production and supply of antibiotics and vaccines, especially during the appearance of devastating outbreaks, improving soil health, plant health and crop yields, and efforts to slow loss of habitable land by reducing global warming. All these measures are microbe‐centric, as are a range of other relevant technologies (Table ). Investing in these technologies in regions of the world where they are needed should bring exceptional fruits in terms of elevating the human condition from unacceptable to acceptable and, ideally, beyond just reducing inequalities and promoting peace. And, it should be pointed out that the technologies outlined above should in some instances also be more generally exploited to increase resource security worldwide, especially but not only in times of crisis (Timmis et al., ). We started out by emphasising the polydeficit nature of impoverished communities, the fact that such deficits are interdependent and mutually re‐enforcing in nature. However, this interconnectedness is not only a characteristic of problems but also of solutions: introducing new technologies to solve problems such as hunger, poor sanitation, access to vaccines and medicines, etc., also creates synergies and facilitates demographic change and dividends by shifting the focus from risk reduction (and survival) towards mid‐ and long‐term (individual to national) planning based on improved health, education, employment (Timmis et al., ) and productivity gains. This, therefore, elevates well‐being on a number of fronts. Lifting fellow humans out of impoverishment not only advances the levelling‐up process and gain of dignity but promotes harmony among peoples and removes some root causes of conflict. Let us break the vicious circle of deficits and misery that promote conflicts, and conflicts that promote deficits and misery. Let us hinder weaponisation of deficits. Let us weaponise microbial technologies to reduce resource deficits and promote resource symmetries among nations, to reduce conflicts, and to promote societal equity, harmony and stability and cooperation between nations. Let us weaponise microbial technologies for peace!
We need to urgently supply to communities lacking adequate levels of basic resources/services the infrastructure and know‐how (capacity building), and funding for use of agrobiologics to increase crop yields, by providing green nitrogen, stimulating plant growth, and combatting pathogens and pests, exploitation of plant:microbe partnerships to improve soil health and implement regenerative agriculture, creation of nutritious fermented food from locally available crops, better use of microbes in the feed and food supply chains, production of microbial food for humans and farm animals, drinking water production and quality safeguarding, waste treatment with resource recovery, creation of modular DIY digital medical centres, production of vaccines and medicines, bioremediation and biorestoration of the environment in general and natural ecosystems in particular, to create healthier habitats and promote biodiversity (see also: https://www.cbd.int/article/cop15‐cbd‐press‐release‐final‐19dec2022 ), reduction of greenhouse gas production and capturing carbon, production of biofuels, creation of local employment opportunities associated with the above, development of transdisciplinary approaches, using chemistry‐related (Kondratov et al., ), computation technologies (Schultes et al., ), psychology‐related (Vinuesa et al., ) and other approaches that are synergistic to microbial solutions and education in societally relevant microbiology
In this Editorial, we draw the reader's attention to various challenges and potential microbial biotechnology‐based solutions (or those inspired by microbiology) that could address and reduce inequalities across the globe, which are chiefly driven by (in many cases meanwhile unnecessary) resource asymmetries that can lead to human suffering and desperation. We do not claim to be complete or prescriptive on how the thoughts and suggestions we have collated here affect, apply to or adequately reflect specific unmet needs and priorities of an overwhelmingly diverse set – across the globe – of inter alia different peoples, cultures and traditions, local and regional circumstances, interdependencies and forms of governance and policies. And we are aware that viewpoints and approaches suggested here might be adequate for one setting but not another (apparently appropriate) setting. Careful and culturally sensitive exploration and appraisal of potential solutions is of paramount importance when considering any changes to the status quo and thus must be tightly coordinated and validated with, and driven by (pull), stakeholders for which pertinent approaches are supposed to provide specific improvements. They should not be driven primarily by what facilitators (donors/funders, policy‐makers, implementing organisations, etc.) believe to be adequate or of utility; naturally, expert consultation with and by facilitators notwithstanding. Our aim is to inspire readers and diverse stakeholders to critically consider the different points we raise, the potential solutions we suggest and the notions and implications we provide, and, if appropriate, advocate for pertinent appraisal within their specific target contexts and application settings of unmet need. We also hope that this contribution will further inspire innovators (and give them confidence in their efforts) to focus on inventing and developing technologies that can help reduce resource asymmetries and inequalities – and promote peace – across the world.
Kenneth Timmis: Conceptualization (equal); writing – original draft (equal); writing – review and editing (equal). Shailly Anand: Writing – review and editing (equal). John E. Hallsworth: Writing – review and editing (equal). James Timmis: Writing – review and editing (equal). Willy Verstraete: Writing – review and editing (equal). Arturo Casadevall: Writing – review and editing (equal). Utkarsh Sood: Writing – review and editing (equal). Roshan Kumar: Writing – review and editing (equal). Princy Hira: Writing – review and editing (equal). Charu Dogra Rawat: Writing – review and editing (equal). Abhilash Kumar: Writing – review and editing (equal). Sukanya Lal: Writing – review and editing (equal). Rup Lal: Writing – review and editing (equal). Juan Luis Ramos: Writing – review and editing (equal).
Natural History Museum; Indian National Science Academy.
The authors confirm they have no conflicts of interest.
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Clinical Features of COVID-19 in Pediatric Rheumatic Diseases: 2020–2022 Survey of the Pediatric Rheumatology Association of Japan
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15dd2052-ca6c-4107-9dfd-370d9077803b
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10221643
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Internal Medicine[mh]
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The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has shown a remarkably high rate of spread since December 2019 . According to the data reported as of August 2022 by the World Health Organization, more than 598.0 million and 6.4 million people worldwide have been infected and died, respectively . From 1 August 2021 to 31 July 2022, COVID-19 was among the 10 leading causes of death in children and young people aged 0 to 19 years in the United States, ranking eighth among all causes of death, fifth in disease-related causes of death, and first in deaths caused by infectious or respiratory diseases . COVID-19 deaths constituted 2% of all causes of death in this age group during this time frame. Initially, a low prevalence rate in children with COVID-19 was reported in various countries, and it was thought that children were unlikely to be affected . Subsequently, it became clear that pediatric morbidity was underestimated. Some studies reported prevalence rates of up to approximately 22%, with half of the cases being asymptomatic . Since the Delta variant epidemic, there has been a rapid spread of infection among children through schools, indoor sports facilities, and homes, resulting in a significant increase in pediatric cases and the number of hospitalizations worldwide, including in Japan . The Omicron variant epidemic increased the number of children being hospitalized in many countries, but the hospitalization risk was reduced by a factor of three when compared with that of the Delta variant . According to the national data collected by the Japanese government using a system called HER-SYS, from 2 September 2020 to 30 August 2022, which included the epidemic seasons of the Alpha, Delta, and Omicron variants, 2,231,776 children < 10 years and 2,296,905 teenagers contracted COVID-19 . Of these, 139 children < 10 years and 64 teenagers contracted severe COVID-19. As a rule, severe cases are defined as meeting one of the following conditions: (1) being connected to a mechanical ventilator, (2) being on extracorporeal membrane oxygenation, or (3) being treated in the intensive care unit (or a similar facility) . Pediatric COVID-19 infections are associated with specific sequela. Multisystem inflammatory syndrome in children (MIS-C) is a postinfectious complication of SARS-CoV-2 infection primarily affecting children . Many patients with MIS-C had a documented history of COVID-19 within the two months before developing MIS-C during the Omicron variant epidemic . In some patients, there is no time separation between the viral-induced symptoms and MIS-C. Moreover, MIS-C shares features with pediatric rheumatic disease (PRD) and cytokine storm syndrome, frequently requiring intensive care support. Although intravenous immunoglobulin and glucocorticoids are effective therapeutics for most patients with MIS-C, patients with refractory MIS-C are treated with various biologics. Understanding the clinical features and the role of inflammatory cytokines provides a rationale for using biologics in treating severe MIS-C . Conversely, COVID-19 in children can be compounded by the presence of underlying diseases and the use of glucocorticoids, immunosuppressants, or biologics; therefore, further research in this area is required to gather comprehensive data . Additionally, these drugs may modify the symptoms of COVID-19 . Thus, this study aimed to investigate the current status of children with PRD who were infected with COVID-19 while using immunosuppressants. Here, we report the clinical features of COVID-19 in children with PRD in Japan for the first time.
2.1. Study Population and Data An online survey was conducted among members (pediatricians [majority]/physicians [minority]) of the Pediatric Rheumatology Association of Japan (PRAJ) at 318 hospitals, including 58 PRD Center Facilities in Japan. The PRD Center Facilities are base hospitals throughout Japan, certified by the PRAJ for treating patients with PRD . All PRAJ members affiliated with the PRD Center Facilities are pediatricians. We prepared 10 questions (Q1–10) for the questionnaire . Q1 was used to confirm the presence or absence of patients with PRD (age < 18 years) with COVID-19 at each hospital in Japan. Q2–Q7 revealed the number of patients with PRD and COVID-19 in the hospital in Japan. Q8–Q10 asked about the practices of pediatricians/physicians who examined patients with PRD and COVID-19 in Japan. In Q5, mild cases were defined as those that improved without the use of pharmacological agents for COVID-19 treatment; moderate cases were defined as those where pharmacological agents were used to treat COVID-19 but management in the intensive care unit was not required; and severe cases were defined as those that required intensive care unit management. The survey period was from 1 April 2020 to 31 August 2022, which included the epidemic seasons of the Alpha, Delta, and Omicron variants, and the response period was from 22 September to 4 October 2022. All members of the PRAJ were notified by email on 20 September 2022, that the survey would be conducted. The survey was only distributed during the response period. 2.2. Outcome Measurements Outcome measurements included the demographic characteristics and clinical features of COVID-19 in patients with PRD and the practices of pediatricians/physicians. Demographic characteristics included sex, age, and PRD type. Clinical features included the severity of COVID-19 and the relationship between pharmacological agents used for PRD treatment (glucocorticoids, immunosuppressants, or biologics) and COVID-19 severity. The practice of pediatricians/physicians for patients with PRD and COVID-19 included adjustments to pharmacological agents used to treat PRD (glucocorticoids, immunosuppressants, or biologics) during COVID-19 infection. 2.3. Statistical Analyses For statistical analysis of the relationship between pharmacological agents used to treat PRD and COVID-19 severity, Fisher’s exact test was used to compare variables between the two groups. Statistical significance was set at p < 0.05. Statistical analyses were performed using JMP Pro version 16 (SAS Institute, Cary, NC, USA).
An online survey was conducted among members (pediatricians [majority]/physicians [minority]) of the Pediatric Rheumatology Association of Japan (PRAJ) at 318 hospitals, including 58 PRD Center Facilities in Japan. The PRD Center Facilities are base hospitals throughout Japan, certified by the PRAJ for treating patients with PRD . All PRAJ members affiliated with the PRD Center Facilities are pediatricians. We prepared 10 questions (Q1–10) for the questionnaire . Q1 was used to confirm the presence or absence of patients with PRD (age < 18 years) with COVID-19 at each hospital in Japan. Q2–Q7 revealed the number of patients with PRD and COVID-19 in the hospital in Japan. Q8–Q10 asked about the practices of pediatricians/physicians who examined patients with PRD and COVID-19 in Japan. In Q5, mild cases were defined as those that improved without the use of pharmacological agents for COVID-19 treatment; moderate cases were defined as those where pharmacological agents were used to treat COVID-19 but management in the intensive care unit was not required; and severe cases were defined as those that required intensive care unit management. The survey period was from 1 April 2020 to 31 August 2022, which included the epidemic seasons of the Alpha, Delta, and Omicron variants, and the response period was from 22 September to 4 October 2022. All members of the PRAJ were notified by email on 20 September 2022, that the survey would be conducted. The survey was only distributed during the response period.
Outcome measurements included the demographic characteristics and clinical features of COVID-19 in patients with PRD and the practices of pediatricians/physicians. Demographic characteristics included sex, age, and PRD type. Clinical features included the severity of COVID-19 and the relationship between pharmacological agents used for PRD treatment (glucocorticoids, immunosuppressants, or biologics) and COVID-19 severity. The practice of pediatricians/physicians for patients with PRD and COVID-19 included adjustments to pharmacological agents used to treat PRD (glucocorticoids, immunosuppressants, or biologics) during COVID-19 infection.
For statistical analysis of the relationship between pharmacological agents used to treat PRD and COVID-19 severity, Fisher’s exact test was used to compare variables between the two groups. Statistical significance was set at p < 0.05. Statistical analyses were performed using JMP Pro version 16 (SAS Institute, Cary, NC, USA).
3.1. Number of Hospitals That Had Children with PRD and COVID-19 Questionnaire responses were obtained from 38 hospitals (a response rate of 12% [38/318]), including 22 PRD Center Facilities (a response rate of 38% [22/58]) and 16 non-PRD Center Facilities (a response rate of 6% [16/260]) in Japan. The responding hospitals were evenly distributed, including the northernmost, eastern, central, western, and southernmost parts of Japan. In response to Q1, 31 hospitals (82% [31/38]), including 22 PRD Center Facilities (100% [22/22]) and nine non-PRD Center Facilities (56% [9/16]), confirmed having had children with PRD and COVID-19. 3.2. Number, Sex, Age, PRD Type, and COVID-19 Severity in Children with PRD and COVID-19 presents the answers to Q2–Q5. In total, there were 165 children with COVID-19, but nine had Crohn’s disease without arthritis and were excluded from the analysis. In children with PRD and COVID-19 ( n = 156), the female-to-male ratio was 7:3, with half of the children aged 11–15 years. The next most common age groups were 6–10 years (23%) and 16–17 years (21%). In children with PRD and COVID-19, the highest proportion was accounted for by juvenile idiopathic arthritis (JIA) (52%), followed by systemic lupus erythematosus (SLE) (24%), juvenile dermatomyositis (5%), scleroderma (4%), Takayasu arteritis (3%), chronic recurrent multifocal osteomyelitis (3%), and Sjögren’s syndrome (2%). Antiphospholipid antibody syndrome, Behçet’s disease, mixed connective tissue disease (MCTD), polyarteritis nodosa, arthritis associated with Crohn’s disease, juvenile polymyositis, overlap syndrome, tubulointerstitial nephritis and uveitis syndrome, and uveitis accounted for approximately 1%. Regarding the severity of COVID-19 in children with PRD, a significant majority (97%) were either asymptomatic (10%) or presented with mild symptoms (87%), with the remaining 3% presenting with moderate symptoms. No severe cases or deaths were reported. JIA and SLE occupied the top two diseases in both PRD Center Facilities and non-PRD Center Facilities . Asymptomatic rates were 12% in the PRD Center Facilities and 0% in the non-PRD Center Facilities. Regarding the severity of COVID-19, 98% were asymptomatic/mild, and 2% were moderate cases at the PRD Center Facilities, while 92% were asymptomatic/mild, and 8% were moderate cases at the non-PRD Center Facilities. 3.3. Treatment Received by Children with PRD before and during COVID-19 The relationship between pharmacological agents used to treat PRD and COVID-19 severity was analyzed by the responses to Q6 and Q7. Of the asymptomatic/mild (n = 137) and moderate ( n = 4) cases, 77 and 3 cases were receiving glucocorticoid treatment, respectively, and the difference was not statistically significant ( p = 0.63). Of the asymptomatic/mild ( n = 137) and moderate ( n = 4) cases, 100 and 4 cases were receiving immunosuppressants, respectively, and the difference was not statistically significant ( p = 0.57). Of the asymptomatic/mild ( n = 137) and moderate ( n = 4) cases, 66 and 3 were using biologics, respectively, and the difference was not statistically significant ( p = 0.36). To summarize the above, regarding the use of glucocorticoids, immunosuppressants, or biologics for the treatment of PRD prior to COVID-19, no significant difference was found between asymptomatic/mild and moderate COVID-19 in children with PRD. shows the responses to Q8–Q10. A total of 16 (52%) of the 31 hospitals that had patients with PRD and COVID-19 suspended the administration of or reduced the dosage of pharmacological agents for PRD because of the onset of COVID-19. The hospitals that chose to suspend a treatment were in the majority: 0 (0%) for glucocorticoids, 12 (75%) for immunosuppressants, and 13 (81%) for biologics among 16 hospitals. The hospitals that chose to reduce a treatment dosage were in the minority: zero (0%) for glucocorticoids, three (100%) for immunosuppressants, and two (67%) for biologics, among three hospitals. When children with PRD contracted COVID-19, 15 of the 31 hospitals did not suspend or reduce pharmacological agents for PRD, including glucocorticoids. Of the remaining 16 hospitals, 13 suspended any pharmacological agent for PRD, but none of those 13 hospitals suspended glucocorticoids. The remaining 3 hospitals suspended or reduced any pharmacological agent for PRD, but none of those 3 hospitals suspended or reduced glucocorticoids. Thus, glucocorticoids were not suspended or reduced (although they may have been increased) in all 31 hospitals.
Questionnaire responses were obtained from 38 hospitals (a response rate of 12% [38/318]), including 22 PRD Center Facilities (a response rate of 38% [22/58]) and 16 non-PRD Center Facilities (a response rate of 6% [16/260]) in Japan. The responding hospitals were evenly distributed, including the northernmost, eastern, central, western, and southernmost parts of Japan. In response to Q1, 31 hospitals (82% [31/38]), including 22 PRD Center Facilities (100% [22/22]) and nine non-PRD Center Facilities (56% [9/16]), confirmed having had children with PRD and COVID-19.
presents the answers to Q2–Q5. In total, there were 165 children with COVID-19, but nine had Crohn’s disease without arthritis and were excluded from the analysis. In children with PRD and COVID-19 ( n = 156), the female-to-male ratio was 7:3, with half of the children aged 11–15 years. The next most common age groups were 6–10 years (23%) and 16–17 years (21%). In children with PRD and COVID-19, the highest proportion was accounted for by juvenile idiopathic arthritis (JIA) (52%), followed by systemic lupus erythematosus (SLE) (24%), juvenile dermatomyositis (5%), scleroderma (4%), Takayasu arteritis (3%), chronic recurrent multifocal osteomyelitis (3%), and Sjögren’s syndrome (2%). Antiphospholipid antibody syndrome, Behçet’s disease, mixed connective tissue disease (MCTD), polyarteritis nodosa, arthritis associated with Crohn’s disease, juvenile polymyositis, overlap syndrome, tubulointerstitial nephritis and uveitis syndrome, and uveitis accounted for approximately 1%. Regarding the severity of COVID-19 in children with PRD, a significant majority (97%) were either asymptomatic (10%) or presented with mild symptoms (87%), with the remaining 3% presenting with moderate symptoms. No severe cases or deaths were reported. JIA and SLE occupied the top two diseases in both PRD Center Facilities and non-PRD Center Facilities . Asymptomatic rates were 12% in the PRD Center Facilities and 0% in the non-PRD Center Facilities. Regarding the severity of COVID-19, 98% were asymptomatic/mild, and 2% were moderate cases at the PRD Center Facilities, while 92% were asymptomatic/mild, and 8% were moderate cases at the non-PRD Center Facilities.
The relationship between pharmacological agents used to treat PRD and COVID-19 severity was analyzed by the responses to Q6 and Q7. Of the asymptomatic/mild (n = 137) and moderate ( n = 4) cases, 77 and 3 cases were receiving glucocorticoid treatment, respectively, and the difference was not statistically significant ( p = 0.63). Of the asymptomatic/mild ( n = 137) and moderate ( n = 4) cases, 100 and 4 cases were receiving immunosuppressants, respectively, and the difference was not statistically significant ( p = 0.57). Of the asymptomatic/mild ( n = 137) and moderate ( n = 4) cases, 66 and 3 were using biologics, respectively, and the difference was not statistically significant ( p = 0.36). To summarize the above, regarding the use of glucocorticoids, immunosuppressants, or biologics for the treatment of PRD prior to COVID-19, no significant difference was found between asymptomatic/mild and moderate COVID-19 in children with PRD. shows the responses to Q8–Q10. A total of 16 (52%) of the 31 hospitals that had patients with PRD and COVID-19 suspended the administration of or reduced the dosage of pharmacological agents for PRD because of the onset of COVID-19. The hospitals that chose to suspend a treatment were in the majority: 0 (0%) for glucocorticoids, 12 (75%) for immunosuppressants, and 13 (81%) for biologics among 16 hospitals. The hospitals that chose to reduce a treatment dosage were in the minority: zero (0%) for glucocorticoids, three (100%) for immunosuppressants, and two (67%) for biologics, among three hospitals. When children with PRD contracted COVID-19, 15 of the 31 hospitals did not suspend or reduce pharmacological agents for PRD, including glucocorticoids. Of the remaining 16 hospitals, 13 suspended any pharmacological agent for PRD, but none of those 13 hospitals suspended glucocorticoids. The remaining 3 hospitals suspended or reduced any pharmacological agent for PRD, but none of those 3 hospitals suspended or reduced glucocorticoids. Thus, glucocorticoids were not suspended or reduced (although they may have been increased) in all 31 hospitals.
This study summarizes the clinical features of COVID-19 in children with PRD at 38 hospitals in Japan, contributing to the current knowledge in this area. There is a paucity of literature on the course of COVID-19 in children, particularly those with PRD. Researchers face a multitude of challenges when conducting studies in this population, including several children with asymptomatic COVID-19 and parental reluctance to enroll children in studies during the current pandemic . It is well known that children are not miniature adults. Thus, it is essential to gather and accumulate research on PRD specifically rather than relying solely on studies of adult rheumatic diseases. At this stage, the most important study on the clinical features of COVID-19 in patients with PRD is by Kearsley-Fleet et al., using data from the EULAR COVID-19 Registry, the CARRA Registry, and the COVID-19 Global Pediatric Rheumatology Database . In this study, 607 patients with PRD from 25 countries in Europe and the United States were studied, with a female-to-male ratio of 66:34, a median age of 14 years, and three-quarters having JIA, SLE, MCTD, or vasculitis. The patient demographic in our study was similar, with a female-to-male ratio of 73:26, patients primarily aged between 11–15 years, and three-quarters having JIA, SLE, MCTD, or vasculitis . Kearsley-Fleet et al. reported that 93% of patients with PRD did not require hospitalization for treatment of the COVID-19 infection . Our study showed a similar trend, where 97% of patients with COVID-19 were asymptomatic or had mild symptoms. In contrast to their report of two SLE deaths caused by COVID-19 , our study did not observe any deaths or severe cases . Although potential differences in cohort size, our findings suggest that COVID-19 is not a significant threat to patients with PRD in Japan. In many areas of Japan, medical expenses for children are free due to subsidy programs. Even hospitalization charges were free for children in the hospitals included in this study. If the patient or their family were worried about the illness, hospitalization could be suggested even in cases of mild symptoms. In addition, during the coronavirus pandemic, there were certain times or specific hospitals where all patients with COVID-19 were hospitalized, regardless of the severity of their symptoms. According to a nationwide survey by the Japan Pediatric Society, a cumulative total of 10,334 children < 20 years contracted COVID-19 from 2020 to 2022 . Of these, the number of children treated in the outpatient, hospital, or intensive care settings was as follows: 338, 995, or 10 children < 1 year (hospitalization rate 75%); 1394, 1502, or 22 children at ages 1–4 (hospitalization rate 52%); 1828, 1273, or 19 children aged 5–9 (hospitalization rate 41%); 1423, 1002, or 10 children aged 10–14 (hospitalization rate 42%); 148, 216, or 7 children aged 15–17 (hospitalization rate 60%); and 24, 120, or 3 children aged 18–19 (hospitalization rate 82%), respectively. In this survey, 5,776 children had no treatment, 4226 used acetaminophen, 234 used glucocorticoids, 114 used remdesivir, and 0 used other pharmacological agents for COVID-19, including ritonavir-boosted nirmatrelvir. Considering that 0.7% to 3.8% of children with COVID-19 require hospitalization in the United States from 2020 to 2022 , the above Japanese data suggest a high hospitalization rate among children with COVID-19 in Japan. However, most children with COVID-19 were asymptomatic or mildly symptomatic in Japan , and the treatment was mostly involved no treatment or only acetaminophen . Furthermore, the Japan Pediatric Society recommends the use of remdesivir for children with moderate to severe COVID-19 . Considering that remdesivir was actually used in 114 of the 10,334 children with COVID-19, the number of moderate-to-severe cases is low from a therapeutic point of view. The unique healthcare environment in Japan, where children can receive free treatment both as outpatients and inpatients, may have contributed to the lack of severe cases and deaths among children with concurrent PRD and COVID-19. For patients with COVID-19, our study found that 10% were asymptomatic , while Kearsley-Fleet et al. reported in their study that 23% were asymptomatic . Previous studies have found that 18% of cases in the United States , 24% in Germany , 38% in Spain , 13% in Turkey , and 21% in Portugal were asymptomatic. These differences are thought to be influenced by varying definitions of “asymptomatic” and unknown COVID-19 therapeutic interventions. Additionally, there may be other influencing factors, including the environment, SARS-CoV-2 variants, patient age, PRD type, and study design. Since our study was a questionnaire survey of pediatricians/physicians, there may have been fewer asymptomatic recruits than in cohort studies. There were more asymptomatic COVID-19 cases in children with PRD at the PRD Center Facilities than at the non-PRD Center Facilities. This could be attributed to the fact that the former group receives regular follow-ups for children with PRD, increasing awareness and detection of asymptomatic cases of COVID-19. In our study, the use of glucocorticoids, immunosuppressants, and biologics did not increase the severity of COVID-19. This finding is similar to and supports the results of a previous report . However, it would be interesting to see how Japanese pediatricians/physicians coordinated pharmacological agents for PRD treatment when patients with PRD contracted COVID-19. According to the guidance provided by the American College of Rheumatology, it is recommended that patients with PRD and COVID-19 continue taking glucocorticoids at the lowest effective dose necessary to manage their PRD. However, there may be exceptions for immunosuppressants and biologics, which are generally advised to be temporarily postponed or withheld during the COVID-19 infection . In our study, glucocorticoids were continued in all patients with COVID-19 . Some hospitals have attempted to reduce the dose of immunosuppressants and biologics, but many have discontinued these medications. The treatment plan of these doctors may have reduced the severity of COVID-19. Our results suggest that, with regard to glucocorticoids, it was common for Japanese pediatricians/physicians to continue at the same (or higher) dose when treating COVID-19 cases . On the other hand, the biologic tocilizumab is not only used for PRD treatment but also for COVID-19 treatment . It was recently suggested that the Janus kinase (JAK) inhibitor baricitinib may be effective for COVID-19 treatment . As a future prospect, when patients with PRD who are using biologics or JAK inhibitors contract COVID-19, it is desirable to present a new policy regarding the continued use of these drugs. As of 31 August 2022, tocilizumab has not been approved for use in children with COVID-19 in Japan. Baricitinib, antiviral antibody drugs, and anti-SARS-CoV-2 drugs other than remdesivir and ritonavir-boosted nirmatrelvir have not been approved for use in children in Japan. Thus, they were not available as treatment options during our survey period. Our results did not find severe cases or deaths among children with PRD with moderate COVID-19, although the choice of pharmacological agents used may have influenced these outcomes. Remdesivir has been approved by the Food and Drug Administration for use in both hospitalized and nonhospitalized children . In a trial evaluating remdesivir for use in nonhospitalized, high-risk patients with mild-to-moderate disease, a 3-day infusion was associated with an 87% reduction in the risk of hospitalization or death . Although only eight patients aged < 18 years were included in the trial, remdesivir is recommended for children with a new or increasing oxygen need . The Food and Drug Administration has issued an emergency use authorization for ritonavir-boosted nirmatrelvir for nonhospitalized children with mild-to-moderate COVID-19 who are at high risk for progression to severe disease . Although the efficacy of ritonavir-boosted nirmatrelvir has only been demonstrated in adults, with an 89% relative risk reduction compared to placebo , it is recommended for children who are at high risk of progressing to severe disease . However, drug interactions must be carefully considered when administering ritonavir-boosted nirmatrelvir, which may limit its use in certain high-risk populations, such as immunocompromised children . Compared to previous reports on COVID-19 with PRD, our study is novel because it included children infected who are during the Omicron variant epidemic. Japan experienced the pandemic of Omicron subvariant BA.1/BA.2 from January to June 2022. However, after the emergence of BA.5 in July 2022, the number of children hospitalized with COVID-19 increased dramatically. Compared with the Omicron subvariant BA.1/BA.2, BA.5 proved to be more detrimental, and the incidence of neurological complications requiring hospitalization increased . Our study likely included numerous children with PRD who were infected not only with the BA.1/BA.2 subvariant but also with the BA.5 Omicron subvariant. The results suggest that children with PRD who contract COVID-19 due to the Omicron variant are unlikely to experience severe disease. This study had several limitations. First, there was a low response rate, and some cases were omitted for answers about COVID-19 severity in this survey. The definition of COVID-19 severity used in this study was not previously reported but is original. Furthermore, in our study, we did not investigate the diagnosis method, symptoms, treatment of COVID-19, the severity of COVID-19 for each child with PRD, relapse of PRD due to COVID-19, reasons for the suspension, reduction, no suspension, or no reduction in pharmacological agents used to treat PRD during COVID-19 infection, or the clinical features of infection with each SARS-CoV-2 variant. To address these limitations, a future study will be needed.
This is the first survey on the clinical features of concurrent COVID-19 and PRD in the Japanese population. In total, 97% of the children with PRD had asymptomatic or mild COVID-19. No severe cases or deaths were reported. Thus, COVID-19, including infection with the Omicron variant, is not a threat to children with PRD in Japan. When children with PRD develop COVID-19, glucocorticoids should be continued, whereas immunosuppressants and biologics may be suspended.
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Assessment of the Adherence to ESPGHAN 2018 Guidelines in the Neonatal Intensive Care Unit of the Ghent University Hospital: A Retrospective Study
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55d73b37-2d91-43e6-a729-208dd7b69633
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10221736
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Internal Medicine[mh]
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Worldwide, nearly 1 in 10 births are estimated to be preterm, which is defined as a birth before the gestational age (GA) of 37 weeks . In Europe, 2013 preterm birth rates were estimated to have a prevalence of 6.2% . Additionally, analyses of 19 countries not only revealed a rise in preterm birth rates in most European countries, but also vast international differences; in 2008, preterm birth rates ranged from 5.5% (Finland) to 11.1% (Austria) . The adequate nutrition of preterm infants is indispensable because it establishes physical and neuronal development , reduces the risk of adverse outcomes , and it helps avoiding long-term developmental defects or death . Conversely, inadequate nutrition can lead to extrauterine growth restriction (EUGR), which refers to the inadequate growth of neonatal intensive care unit (NICU) patients during their first hospital stay . Both short- and long-term prognoses can be worsened by EUGR; for instance, EUGR increases the risk of cardiometabolic alterations during childhood . Due to gut immaturity, the oral or enteral nutrition (EN) of preterm infants is often impossible, insufficient, or contraindicated in preterm infants . As such, parenteral nutrition (PN) is considered to be the standard of care in the first postnatal days of preterm infants . In 2018, the European Society of Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) published new guidelines on pediatric PN, including recommendations for energy and macronutrient provisions . Nonetheless, several knowledge gaps in pediatric PN demand further data from clinical practice; for instance, the optimal energy or lipid intakes remain unclear . Moreover, in clinical practice, EN intakes are usually gradually introduced until the neonates are ready to be weaned off PN; however, the aforementioned 2018 ESPGHAN guidelines apply solely to PN intakes. Although a recent ESPGHAN position paper provides EN intake recommendations for preterm infants , neonatology lacks the clinical data, and thus, the guideline recommendations, concerning the combined use of PN and EN. In clinical practice, adherence to ESPGHAN PN guidelines is not always guaranteed. A 2013 survey in four European countries assessed adherence to the ESPGHAN 2005 guidelines and found a substantially high prevalence of deviations from guideline recommendations (e.g., 37% of neonatal units provided amino acids on the second, rather than the first, day of life, and 60% provided less than the recommended minimum initial dose to prevent a negative balance) . In 2015, a retrospective analysis of 29 NICUs also detected cumulative deficits in lipid and amino intakes in the first two weeks of life, and it identified the cumulative amino acid intake as an independent variable that is associated with the ∆Z-score for weight at 36 weeks . In addition to acute illnesses and metabolic complications, fluid restrictions are a major obstacle in achieving sufficient PN intakes as they limit PN infusion volumes . Especially in this critical population, non-nutritional fluids (e.g., drugs for blood pressure control) are common and can further restrict the PN infusion volumes . Although sufficient PN is crucial for adequate growth in preterm neonates, data concerning the adherence to current nutrition protocols, with regard to ESPGHAN 2018 guidelines, are sparse. Here, we address these knowledge gaps: the objective of this retrospective study was to examine how closely the macronutrient and energy provisions at the NICU at Ghent University Hospital correspond with the respective ESPGHAN 2018 guideline recommendations. As a secondary aim, the weekly growth rates for length, weight, and head circumference were assessed. The analyses were conducted for different body weight (BW) groups to ultimately determine if and how nutrient provisions should be adapted for neonates at the NICU. The results of this study will be used to further adjust the nutrition protocols at Ghent University Hospital, which aims to develop all-in-one PN admixtures, as the use of standardized PN solutions are formulated in accordance with a guideline-recommended approach due to fewer compounding errors and higher levels of patient safety .
2.1. Study Design, Patient Population, and Data Collection This retrospective cohort study analyzed 86 neonates that were admitted to the NICU at Ghent University Hospital. Data were collected from 15 December 2013 to 31 December 2014. The sole inclusion criterion was at least 5 consecutive days of PN. Patient data were excluded from analysis if they were transferred to another hospital before they were completely weaned off PN or if PN was interrupted for 14 d or longer. Data were retrieved from medical and nursing records, and lab values were obtained from electronic patient records. Data were collected in anonymized files. For subgroup analysis, patients were divided into 3 subgroups, in accordance with their BW: <1000 g, 1000 to <1500 g, and ≥1500 g. For the analysis of ESPGHAN 2018 guideline adherence, PN intakes were analyzed for up to 4 weeks. Days were excluded from the analysis if the number of available patients was below 3. Although in clinical practice, EN provisions are usually introduced as early as possible in PN-fed neonates, neither ESPGHAN PN nor EN guidelines provide recommendations on the combined energy and macronutrient intake of PN and EN. As the majority of fluid intake in the first days of life at the NICU are parenteral, we tested the adherence to ESPGHAN PN 2018 guidelines with regard to nutrient provisions, with EN intakes in g/kg/d considered to be equal to PN intakes in g/kg/d for the sake of practicality. All kinds of EN-intakes were considered, including trophic feeding. 2.2. Nutrition Protocol The PN nutrition protocol analyzed in this study was based on the ESPGHAN 2005 recommendations , which were last updated in 2013. The protocol utilizes 3 formulations composed of various PN solutions, the use of which has been well-established over the years at Ghent University Hospital: The first formulation contains glucose (available in different concentrations ranging from 6% to 30%), calcium gluconate (Mini-Plasco 10% ® , B. Braun, Diegem, Belgium), magnesium sulphate (ampoule 1 g/10 mL Sterop ® , Brussels, Belgium), water soluble vitamins (Soluvit N ® , Fresenius Kabi, Schelle, Belgium), and extra vitamin C (Vitamina C ampoule 1 g/5 mL Bayer ® , Milan, Italy). The second solution provides amino acids (Vaminolact ® , Fresenius Kabi, Schelle, Belgium), and it is complemented with Tripotassium phosphate (Kaliphos Sterop ® , Brussels, Belgium), sodium chloride (Mini-Plasco 20% ® , B. Braun, Diegem, Belgium), and trace elements (Addaven ® , Fresenius Kabi, Schelle, Belgium). The third formulation provides lipids (SMOFlipid 20% ® , Fresenius Kabi, Schelle, Belgium) and fat-soluble vitamins (Vitalipid N Infant ® , Fresenius Kabi, Schelle, Belgium) compounded in a 50 mL opaque syringe. 2.3. Variables and Data Management The primary outcome of this study was adherence to ESPGHAN 2018 guidelines, with regard to macronutrients and energy intake, for each day of PN; this was assessed by examining the fraction of patients whose nutrient/energy provision was in range of the respective minimum and maximum recommended intakes. Secondary outcomes were the weekly changes in Z-scores for weight, height, and head circumference for age. Analyzed patient demographics were sex, GA, multiple births, and reason for starting/stopping PN, as well as the weight, length, and head circumference at birth and at the start/end of PN. For each patient and day of PN, the volumes of the PN and EN provisions were recorded to calculate the volume fraction ( v / v %) of PN in terms of nutrient provisions. In order to make a comparison with the ESPGHAN 2018 guideline recommendations, the respective intakes from PN and EN were pooled. Z-scores for weight, length, and head circumference, calculated in accordance with the Fenton growth charts for preterm infants , were calculated using the PediTools app . Weekly changes in Z-scores were calculated, as shown in Equation (1): Weekly change of Z-score = (Z-score at the last day of PN − Z-score at the first day of PN)/time of PN (in weeks) (1) To test the adherence to ESPGHAN 2018 guidelines, we assessed if the provisions of macronutrients and energy were within the respective ranges, from recommended minimum to maximum. summarizes these target ranges. Although guidelines do not state a minimum lipid intake, Ghent University Hospital has a target for minimum lipid intake, which is 1.0 g/kg/d for both preterm and term neonates starting from day 2. Reaching these intake targets, using SMOFlipid 20% as the lipid source, has been well-established at Ghent University Hospital, and the ESPGHAN 2018 recommended minimum supply targets of 0.25 and 0.1 g/kg/d linoleic acid have been reached (preterm and term, respectively) . 2.4. Bias and Study Size Common confounders of single-center studies, such as preference of care or center standards, have to be considered for the interpretation of the study. No sample size calculation was performed, and thus, the study size resulted from the number of patient records available, in accordance with the inclusion and exclusion criteria. 2.5. Statistical Methods Parametric variables were summarized as the mean ± standard deviation (SD), or median, along with the first and third quartile. Categorical variables were summarized as counts and relative amounts . To check for significant differences between the respective weekly Z-score changes among the 3 BW groups, the equality of variances was first checked using Levene’s test. If the variances were significantly different, the weekly Z-score changes were compared using the Kruskal–Wallis test and pairwise Wilcoxon rank sum tests for post-hoc comparisons. If the variances were not significantly different, the Z-score changes were compared using One-Way ANOVA tests and Tukey’s test for post-hoc comparisons. For post-hoc analyses, p -values were adjusted using the Benjamini–Hochberg procedure. The significance level was 0.05.
This retrospective cohort study analyzed 86 neonates that were admitted to the NICU at Ghent University Hospital. Data were collected from 15 December 2013 to 31 December 2014. The sole inclusion criterion was at least 5 consecutive days of PN. Patient data were excluded from analysis if they were transferred to another hospital before they were completely weaned off PN or if PN was interrupted for 14 d or longer. Data were retrieved from medical and nursing records, and lab values were obtained from electronic patient records. Data were collected in anonymized files. For subgroup analysis, patients were divided into 3 subgroups, in accordance with their BW: <1000 g, 1000 to <1500 g, and ≥1500 g. For the analysis of ESPGHAN 2018 guideline adherence, PN intakes were analyzed for up to 4 weeks. Days were excluded from the analysis if the number of available patients was below 3. Although in clinical practice, EN provisions are usually introduced as early as possible in PN-fed neonates, neither ESPGHAN PN nor EN guidelines provide recommendations on the combined energy and macronutrient intake of PN and EN. As the majority of fluid intake in the first days of life at the NICU are parenteral, we tested the adherence to ESPGHAN PN 2018 guidelines with regard to nutrient provisions, with EN intakes in g/kg/d considered to be equal to PN intakes in g/kg/d for the sake of practicality. All kinds of EN-intakes were considered, including trophic feeding.
The PN nutrition protocol analyzed in this study was based on the ESPGHAN 2005 recommendations , which were last updated in 2013. The protocol utilizes 3 formulations composed of various PN solutions, the use of which has been well-established over the years at Ghent University Hospital: The first formulation contains glucose (available in different concentrations ranging from 6% to 30%), calcium gluconate (Mini-Plasco 10% ® , B. Braun, Diegem, Belgium), magnesium sulphate (ampoule 1 g/10 mL Sterop ® , Brussels, Belgium), water soluble vitamins (Soluvit N ® , Fresenius Kabi, Schelle, Belgium), and extra vitamin C (Vitamina C ampoule 1 g/5 mL Bayer ® , Milan, Italy). The second solution provides amino acids (Vaminolact ® , Fresenius Kabi, Schelle, Belgium), and it is complemented with Tripotassium phosphate (Kaliphos Sterop ® , Brussels, Belgium), sodium chloride (Mini-Plasco 20% ® , B. Braun, Diegem, Belgium), and trace elements (Addaven ® , Fresenius Kabi, Schelle, Belgium). The third formulation provides lipids (SMOFlipid 20% ® , Fresenius Kabi, Schelle, Belgium) and fat-soluble vitamins (Vitalipid N Infant ® , Fresenius Kabi, Schelle, Belgium) compounded in a 50 mL opaque syringe.
The primary outcome of this study was adherence to ESPGHAN 2018 guidelines, with regard to macronutrients and energy intake, for each day of PN; this was assessed by examining the fraction of patients whose nutrient/energy provision was in range of the respective minimum and maximum recommended intakes. Secondary outcomes were the weekly changes in Z-scores for weight, height, and head circumference for age. Analyzed patient demographics were sex, GA, multiple births, and reason for starting/stopping PN, as well as the weight, length, and head circumference at birth and at the start/end of PN. For each patient and day of PN, the volumes of the PN and EN provisions were recorded to calculate the volume fraction ( v / v %) of PN in terms of nutrient provisions. In order to make a comparison with the ESPGHAN 2018 guideline recommendations, the respective intakes from PN and EN were pooled. Z-scores for weight, length, and head circumference, calculated in accordance with the Fenton growth charts for preterm infants , were calculated using the PediTools app . Weekly changes in Z-scores were calculated, as shown in Equation (1): Weekly change of Z-score = (Z-score at the last day of PN − Z-score at the first day of PN)/time of PN (in weeks) (1) To test the adherence to ESPGHAN 2018 guidelines, we assessed if the provisions of macronutrients and energy were within the respective ranges, from recommended minimum to maximum. summarizes these target ranges. Although guidelines do not state a minimum lipid intake, Ghent University Hospital has a target for minimum lipid intake, which is 1.0 g/kg/d for both preterm and term neonates starting from day 2. Reaching these intake targets, using SMOFlipid 20% as the lipid source, has been well-established at Ghent University Hospital, and the ESPGHAN 2018 recommended minimum supply targets of 0.25 and 0.1 g/kg/d linoleic acid have been reached (preterm and term, respectively) .
Common confounders of single-center studies, such as preference of care or center standards, have to be considered for the interpretation of the study. No sample size calculation was performed, and thus, the study size resulted from the number of patient records available, in accordance with the inclusion and exclusion criteria.
Parametric variables were summarized as the mean ± standard deviation (SD), or median, along with the first and third quartile. Categorical variables were summarized as counts and relative amounts . To check for significant differences between the respective weekly Z-score changes among the 3 BW groups, the equality of variances was first checked using Levene’s test. If the variances were significantly different, the weekly Z-score changes were compared using the Kruskal–Wallis test and pairwise Wilcoxon rank sum tests for post-hoc comparisons. If the variances were not significantly different, the Z-score changes were compared using One-Way ANOVA tests and Tukey’s test for post-hoc comparisons. For post-hoc analyses, p -values were adjusted using the Benjamini–Hochberg procedure. The significance level was 0.05.
3.1. Patient Demographics summarizes the patient characteristics for the overall cohort and the different BW groups. Most patients (75, 87.2%) had a GA < 37 weeks, the mean BW was 1659.9 ± 884.3 g, and 55.8% (48) patients were male. The median time from birth to start of PN treatment was 0 d (0–0). The growth parameters at birth, and the median time from birth to PN per BW group, can be found in . 3.2. Reason for PN Initiation As shows, prematurity was the main reason for initiation of PN in the cohort (59 patients, 68.6%), followed by respiratory complications (14 patients, 16.3%). 3.3. Weekly Changes in Z-Scores summarizes the weekly changes in Z-scores for weight, length, and head circumference (see for a numeric summary). The median weekly changes were positive for all assessments. For weight and age, the weekly Z-score changes were significantly higher for neonates with a BW < 1000 g (0.21 ± 0.12), compared with those with a BW of 1000 to <1500 g (0.07 ± 0.16) or ≥1500 g (0.10 ± 0.31). Moreover, weekly changes for the other Z-scores did not differ between any of the groups. 3.4. Carbohydrate Provision shows how many neonates received a carbohydrate intake that followed ESPGHAN 2018 guideline recommendations over time (see for a summary of provided carbohydrates). Regardless of BW, the majority of carbohydrate intakes were in range of the recommendations starting from day 2, and they stayed within range afterwards. On day 1, by way of contrast, 54% of neonates with a BW < 1000 g ( A), and 50% of neonates with a BW of 1000 to <1500 g (Figure 3B), received a carbohydrate intake that fell below the recommended intake guidelines. For neonates with a BW ≥ 1500 g on day 1, approximately the same number of patients received carbohydrate intakes that exceeded (26%) and fell below (22%) guideline recommendations. 3.5. Lipid Provision shows how many neonates received a lipid intake that followed ESPGHAN 2018 guideline recommendations over time (see for a summary of provided lipids). Except for one patient on day 10 in the BW ≥ 1500 g group, all lipid intakes fulfilled the minimum intake goals. However, in all BW groups, the number of patients receiving lipid intakes above the maximum of 4 g/kg/d increased over time, although PN lipid intakes maxed out at 3.6 g/kg/d. The proportion of intakes above the maximum increased gradually in neonates with a BW < 1000 g, from 4% (day 4) to a maximum of 64% (day 24). In contrast, starting from day 5, more than half of the neonates with a BW > 1000 g ( B,C) received lipid provisions above the maximum until the end of PN. 3.6. Amino Acid Provision shows how many neonates received an amino acid intake that followed guideline recommendations over time (see for a summary of provided amino acids). Overall, most of the intakes fell below the recommended minimum, and intakes that exceeded the recommended maximum were rare. For neonates with a BW < 1000 g ( A), the amino acid provision fell below the minimum recommendation for most patients over 4 weeks. Of the patients in this group who did receive the recommended amino acid intake, as per ESPGHAN 2018 guidelines, the vast majority of patients also received the recommended amount of non-protein energy. At least half of the patients with a BW of 1000 to <1500 g ( B) received amino acids in the recommended range, starting from day 6, with the exception of day 16. Of the neonates with a BW ≥ 1500 g ( C), 63–90% received an amino acid intake that fell below the recommended minimum during the first 16 days of PN. 3.7. Energy Provision shows how many neonates received energy from all macronutrient intakes over time, in accordance with guideline recommendations (see for a summary of provided energy). All three BW groups have two distinctive features in common: first, energy intakes mostly fell below the recommended minimum for the first three days. Second, the proportion of neonates with energy intakes that adhered to the guidelines increased after this point. This increase was more pronounced for neonates with a BW > 1000 g ( B,C), as compared with neonates that had a lower BW ( A). In the period from day 5 to day 15, the proportion of energy intakes that adhered to the guidelines ranged from 50–86% for neonates with a BW of 1000 to <1500 g ( B). For neonates with a BW ≥ 1500 g, 51–90% of patients received energy intakes that adhered to the guidelines in the period from day 5 to day 15. 3.8. Volume Percentage of PN Regarding Nutrient Provisions over Time As shown in , , and , above the columns, regardless of the BW, the mean volume percentage of PN, regarding nutrient provisions, started close to 100%, and declined gradually (see for a summary). The most apparent difference between the BW groups is that the relative amount of PN declined more slowly in neonates with a BW < 1000 g; after 2 weeks, the amount declined to 69%, whereas less than 50% of the nutrient intake was parenteral in neonates with higher BWs. In neonates with a BW < 1000 g, the PN proportion in terms of nutrient provisions was at least 59% across the four weeks of observation.
summarizes the patient characteristics for the overall cohort and the different BW groups. Most patients (75, 87.2%) had a GA < 37 weeks, the mean BW was 1659.9 ± 884.3 g, and 55.8% (48) patients were male. The median time from birth to start of PN treatment was 0 d (0–0). The growth parameters at birth, and the median time from birth to PN per BW group, can be found in .
As shows, prematurity was the main reason for initiation of PN in the cohort (59 patients, 68.6%), followed by respiratory complications (14 patients, 16.3%).
summarizes the weekly changes in Z-scores for weight, length, and head circumference (see for a numeric summary). The median weekly changes were positive for all assessments. For weight and age, the weekly Z-score changes were significantly higher for neonates with a BW < 1000 g (0.21 ± 0.12), compared with those with a BW of 1000 to <1500 g (0.07 ± 0.16) or ≥1500 g (0.10 ± 0.31). Moreover, weekly changes for the other Z-scores did not differ between any of the groups.
shows how many neonates received a carbohydrate intake that followed ESPGHAN 2018 guideline recommendations over time (see for a summary of provided carbohydrates). Regardless of BW, the majority of carbohydrate intakes were in range of the recommendations starting from day 2, and they stayed within range afterwards. On day 1, by way of contrast, 54% of neonates with a BW < 1000 g ( A), and 50% of neonates with a BW of 1000 to <1500 g (Figure 3B), received a carbohydrate intake that fell below the recommended intake guidelines. For neonates with a BW ≥ 1500 g on day 1, approximately the same number of patients received carbohydrate intakes that exceeded (26%) and fell below (22%) guideline recommendations.
shows how many neonates received a lipid intake that followed ESPGHAN 2018 guideline recommendations over time (see for a summary of provided lipids). Except for one patient on day 10 in the BW ≥ 1500 g group, all lipid intakes fulfilled the minimum intake goals. However, in all BW groups, the number of patients receiving lipid intakes above the maximum of 4 g/kg/d increased over time, although PN lipid intakes maxed out at 3.6 g/kg/d. The proportion of intakes above the maximum increased gradually in neonates with a BW < 1000 g, from 4% (day 4) to a maximum of 64% (day 24). In contrast, starting from day 5, more than half of the neonates with a BW > 1000 g ( B,C) received lipid provisions above the maximum until the end of PN.
shows how many neonates received an amino acid intake that followed guideline recommendations over time (see for a summary of provided amino acids). Overall, most of the intakes fell below the recommended minimum, and intakes that exceeded the recommended maximum were rare. For neonates with a BW < 1000 g ( A), the amino acid provision fell below the minimum recommendation for most patients over 4 weeks. Of the patients in this group who did receive the recommended amino acid intake, as per ESPGHAN 2018 guidelines, the vast majority of patients also received the recommended amount of non-protein energy. At least half of the patients with a BW of 1000 to <1500 g ( B) received amino acids in the recommended range, starting from day 6, with the exception of day 16. Of the neonates with a BW ≥ 1500 g ( C), 63–90% received an amino acid intake that fell below the recommended minimum during the first 16 days of PN.
shows how many neonates received energy from all macronutrient intakes over time, in accordance with guideline recommendations (see for a summary of provided energy). All three BW groups have two distinctive features in common: first, energy intakes mostly fell below the recommended minimum for the first three days. Second, the proportion of neonates with energy intakes that adhered to the guidelines increased after this point. This increase was more pronounced for neonates with a BW > 1000 g ( B,C), as compared with neonates that had a lower BW ( A). In the period from day 5 to day 15, the proportion of energy intakes that adhered to the guidelines ranged from 50–86% for neonates with a BW of 1000 to <1500 g ( B). For neonates with a BW ≥ 1500 g, 51–90% of patients received energy intakes that adhered to the guidelines in the period from day 5 to day 15.
As shown in , , and , above the columns, regardless of the BW, the mean volume percentage of PN, regarding nutrient provisions, started close to 100%, and declined gradually (see for a summary). The most apparent difference between the BW groups is that the relative amount of PN declined more slowly in neonates with a BW < 1000 g; after 2 weeks, the amount declined to 69%, whereas less than 50% of the nutrient intake was parenteral in neonates with higher BWs. In neonates with a BW < 1000 g, the PN proportion in terms of nutrient provisions was at least 59% across the four weeks of observation.
By analyzing the proportion of patients whose macronutrient and energy intakes followed ESPGHAN 2018 recommendations, we found that guideline adherence, with regard to macronutrient and energy provisions, showed certain patterns across all three BW groups: (1) carbohydrate provisions were in range for the vast majority of patients starting from day 2, although half of the patients with a BW < 1500 g received a carbohydrate intake that fell below minimum recommendations on the first day of PN; (2) lipid provisions tended to exceed the maximum recommendations over time, and this was more pronounced in neonates with a BW ≥ 1000 g; (3) amino acid provisions generally fell below recommended levels, especially in neonates with a BW < 1000 g; and (4) energy provisions fell below the minimum recommendations for the first 3 days of PN, although, the number of patients receiving energy provisions recommended by the guidelines increased slowly, with a stronger increase in neonates with a BW ≥ 1000 g. For an appropriate interpretation of these results, certain strengths and limitations of this study should be noted. The single-center nature of this study is a double-edged sword; on the one hand, the results are not confounded by different clinical practices across centers, on the other hand, we cannot dismiss the center bias. Moreover, sample sizes are also limited, especially for neonates with a BW of 1000 to <1500 g. The small sample sizes also limit the possibility of further stratification with regard to longitudinal analyses (e.g., by pathology: during the patients’ stay, congenital heart disease occurred only in 3 patients (3.5%), and catheter-related bloodstream infections occurred in 8 patients (9.3%)). Additionally, although respiratory support was more common, occurring in 32 patient cases (37.2%), only three of those involved patients with a BW of 1000 to <1500 g (<1000 g: 16, ≥1500 g: 13). In terms of generalizability, the results mainly apply to neonates whose primary PN indication is prematurity (68.8% of patients in this study). We acknowledge that combining PN and EN intakes to analyze ESPGHAN 2018 PN guidelines is a compromise; on the one hand, current guideline recommendations lack clear recommended intakes for the combined use of EN and PN in preterm neonates, but on the other hand, EN feeds are introduced as soon as possible in clinical practice, so the time that a patient is on total PN (TPN) is often extremely short. In a real-world setting, compromises for assessing PN guideline adherence are practically unavoidable for the majority of patients. For instance, a cohort study by Boscarino et al. has defined TPN as a total PN energy intake of more than 70% during the first week of life . To account for EN intakes, and to allow readers to draw meaningful conclusions from these results, this study provides a PN fraction for each given day. Given that there is a severe lack of data on how the combined use of PN and EN relates to clinical outcomes, this is a major strength of this study. As an alternative approach to the equalization of PN and EN volume provisions, future adaptions of this approach may consider adjustments to reflect the less efficient metabolization of EN compared with PN. For instance, the ESPGHAN 2018 guidelines state that the gross energy content of 1 g of amino acids (PN) is about 10% lower than that of 1 g of protein (EN) ; therefore, to calculate the energy provisions, with regard to amino acids, volume equivalent EN intakes could be adjusted to 110% of the energy provisions of the PN amino acid intakes. Moreover, regarding the aforementioned cohort study by Boscarino et al. , one may also consider setting a certain limit on the PN intake that is required to qualify for inclusion in the dataset. Despite the differences between BW groups in terms of ESPGHAN 2018 guideline adherence, we observed positive Z-score developments across all BW groups, thus indicating that the nutrition protocols were sufficient to achieve growth. The positive growth rates contrast with the negative growth rates in the first weeks of life from other observational studies on preterm development . Differences between BW among various cohorts might explain these differences. For instance, although Izquierdo Renau et al. report negative weight developments 28 d after birth for preterm infants, the mean BW of 1200 ± 360 g in that cohort was notably lower than in our cohort (1659.9 ± 884.3 g). However, it is estimated that BW accounts for only 7% of growth variation in preterm neonates, whereas nutrition accounts for about 45% . A cohort study by Westin et al. clearly demonstrates the effect of different nutrition protocols on growth, as nutrition protocols have been continuously updated between 2004 and 2011. These include protocols that concern energy and macronutrient intakes in preterm infants, and improvements have been made, particularly when compared with the period 2004–2005; indeed, neonates in later periods received an earlier and higher provision of lipids, proteins, and energy in the first 28 d of life, thus leading to significantly higher gains in length, weight, and head circumference as per the 0.3–0.5 standard deviation scores. Additionally, linear mixed-effects model analyses by Asbury et al. have shown that early and sufficient intakes of macronutrients have positive effects on Z-scores, independent of demographics, acuity, and morbidity. Thus, we cannot exclude the possibility that a greater adherence to ESPGHAN 2018 guidelines, regarding amino acid and energy provisions, would result in higher weekly Z-score developments, or significant differences between BW groups. In terms of adherence to ESPGHAN 2018 carbohydrate recommendations , the analyses indicate sufficient carbohydrate provisions from day 2 for all BW groups. Provisions in range of the recommendations should reduce the risk of overfeeding and hyperglycemia . In fact, the latter occurred in only eight patients (9.3%) for a duration of three or more consecutive days. Regarding lipid intakes, the current protocols may be adapted to avoid the increasing number of neonates with intakes exceeding the recommended maximum of 4 g/kg/d , starting from day 4, especially for neonates with a BW > 1000 g. However, PN provisions maxed out at 3.6 g/kg/d. In a clinical practice at Ghent University Hospital, triglyceride levels are measured on a weekly basis and lipid provisions are reduced if hypertriglyceridemia is detected. In this cohort, only one patient had hypertriglyceridemia that persisted for three consecutive days, despite several patients having provisions above the recommended maximum. A multiple logistic regression model by Giretti et al. might explain this apparent lack of association; although each additional intravenous lipid intake of 1 g/kg/d increased the risk of hypertriglyceridemia by 96%, for each week of GA, and for each g/kg/d of amino acid intake, the risk of hypertriglyceridemia was reduced by 12% and 19%, respectively . Moreover, the use of fish oil emulsions was associated with a significantly lower risk of hypertriglyceridemia (a reduction of 38%), which the authors traced back to lipogenesis downregulation by fish oil emulsions. Ultimately, although our nutrition protocols may have to be adapted to not exceed ESPGHAN 2018 guideline recommendations, we can corroborate the findings of other cohort studies on the safe use of fish oil lipid emulsions in terms of hypertriglyceridemia . The analyses of amino acid administration showed a high prevalence of provisions that fell below the minimum ESPGHAN 2018 recommendations across all BW groups, especially in the BW < 1000 g and BW ≥ 1500 g groups. If the amino acid provisions can be increased, it may benefit growth rates. Linear mixed-effects model analyses showed that early and high protein intakes in the first week of life ameliorate weight decline after birth, and from day 9 onward, they are significantly associated with greater increases in weight, length, and head circumference . Although the optimal glucose and lipid intakes to safeguard protein accretion and growth are unknown , the nutrition protocol analyzed in this study showed that insufficient non-protein intakes were a rare exception (three cases on two days). However, if amino acid provision guidelines recommend an increase in the next protocol update, this finding has to be reassessed. Furthermore, we would also have to reassess how the energy intakes comply with current guideline recommendations if lipid provisions are reduced and amino acid provisions are increased. This is especially important in neonates with a BW < 1000 g, who seem to be especially susceptible to energy provisions that fall below the minimum recommended amount. Nonetheless, although insufficient energy intakes bear the risk of impaired growth , all weekly median Z-score changes were positive. Given the lack of data after patients left the NICU, it remains unclear if long-term developments differed between the BW groups. The occurrence of EUGR in this cohort might provide an estimate of how many patients had a higher risk of developing growth defects in early childhood. Although the definition of EUGR is debated , an analysis of over 800 very low birth weight or preterm neonates in an Italian cohort found that, compared with a cross-sectional definition, a longitudinal definition of EUGR was a better predictor for poorer growth outcomes 24–30 months after discharge. Furthermore, Peila et al. 2020 defined EUGR as a weight loss > 1 Z-score (Italian Neonatal Study charts) between birth and a given time (36 and 40 weeks of GA, discharge, 28 days) . However, applying this definition to the ∆Fenton Z-scores from day 1 to discharge in our cohort did not provide any indication for long-term growth deficits, as none of the patients had EUGR. A possible explanation for this observation might be that only longer durations of (T)PN are associated with EUGR occurrence. For instance, a prospective study by Wang et al. showed that the median PN duration was significantly higher for EUGR patients at 24 d compared with non-EUGR patients at 17 d , with the latter closely resembling the mean PN duration in our study. Additionally, the authors found longer PN durations to be an independent risk factor for EUGR occurrence , thus echoing the findings of similar EUGR studies . If changes in future nutrition protocols at UZ Ghent increase the (T)PN duration, the potential effect on EUGR should be assessed, including follow-ups after NICU discharge. We observed that patients in the lower BW groups showed a slower reduction in terms of the PN fraction, regarding the nutrition provision, which is indicative of the less stable condition of these patients. An observational study by Immeli et al. showed that for very low birth weight infants, the length of the transition phase is negatively correlated with the cumulative energy and macronutrient intakes at 28 d, and longer transition phases are associated with slower postnatal growth rates . This may be one more reason to increase protein and energy provisions in these patients. The results of this study may inform further adjustments to the nutrition protocols of the NICU at Ghent University Hospital, which mainly recommend increases to amino acid provisions and lower lipid provisions. Due to their well-established usage and good effect on growth, the nutrition regimen will continue to utilize standardized PN solutions. The next step will be to assess the possibilities of introducing all-in-one PN admixtures in order to reduce compounding errors and increase patient safety.
In this study, we report the results of the assessment of the current nutrition protocols at Ghent University Hospital, as per ESPGHAN 2018 PN guidelines. We found that future protocol adaptations should lower lipid provisions and increase both protein and energy provisions. In addition, our findings provide relevant insights into neonatal PN practices at a Belgian NICU on a more general level. The reported approach of considering both PN and EN intakes does not only provide real-world data on an under-researched topic of neonatal nutrition, it might also serve as a template for other centers who want to assess their PN guideline adherence pragmatically. Importantly, even with deviations from current PN guidelines, positive Z-score developments over a mean PN duration of 17.1 ± 11.4 days were possible across different BW groups. In that regard, this study also provides real-world evidence on how the use of standardized PN solutions can safeguard the development of critically ill neonates during their NICU stay. Future studies should check how adaptations of the protocol will reflect short- and long-term growth, guideline adherence, and safety outcomes.
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Medicinal Plants of the Flora of Kazakhstan Used in the Treatment of Skin Diseases
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b4c26862-288d-49a4-bbce-70c0ce4b5718
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10221907
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Pharmacology[mh]
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According to the eminent scientist, philosopher, and physician Avicenna, “a doctor has three tools: the word, the plant, the knife”. The plant kingdom is recognized as the humanity’s earliest and the most ancient healing source, which people employed to treat and prevent illnesses. Tracing back through history, the most ancient documented proof of plants’ utilization in medicine dates back to a Sumerian clay slab discovered in Nagpur about 5000 years ago. This artifact included a compilation of twelve medicinal recipes that involved over 250 diverse plant species. Sumerian healers prepared powders and therapeutic infusions from plant roots and stems. Pears and figs also possessed healing properties. Additionally, they utilized dried and ground young shoots of willow and plum trees, and pine and fir trees as components in compresses and poultices. Powders from animal and mineral sources were often mixed with ones extracted from dried and crushed plants. Notably, in addition to water, wine and beer were used as solvents. Thus, at least 80 centuries ago, people employed the most simple medicinal plant-based preparations for treatment . Regarding Kazakh folk medicine, it has not yet been fully researched. The traditional medicine of the Kazakh people covers not only the mere treatment of ailments but also rests on robust theoretical knowledge. Oteiboydak Tleukabyluly (1388–1478), a distinguished Kazakh healer who lived in the 15th century, wrote the ethnographic and medical book “Medical Narrative” between the years 1466 and 1473 with az-Zhanibek Khan’s order who held Oteiboydak Tleukabyluly in high esteem as a healer. Oteiboydak Tleukabyluly wrote in his book about the secrets of the healing art. This medical encyclopedia delineates the functions of various organs of the human body and provides a catalogue of the primary diseases associated with them. Furthermore, it includes a meticulous description of the methods used in traditional medicine at present, such as setting bones, listening to the pulse, and incantations. Through practical experimentation and experimentation conducted in the steppe laboratory, the healer formulated a total of 1108 different medicinal compounds, of which 858 were derived from medicinal plants, 318 were extracted from animal organs, and roughly 60 were sourced from metals. The moniker “Teacher without a teacher” was bestowed on Oteiboydak Tleukabyluly who created methods for treating 1050 different diseases . At present, phytotherapy is widely practiced all over the world. According to the World Health Organization’s (WHO) global review of national policies concerning traditional, complementary, and alternative medicine, as well as the regulation of herbal medicines, there is an evident growth in the European and Asian market for herbal medicines . Kazakhstan accounts for a natural flora of over 6000 plant species . The exact number of medicinal plant species present in Kazakhstan remains uncertain as the list continues to expand annually. More than 150 plant species have been employed in both official and folk medicine for various ailments. This review focuses on a selection of medicinal plants growing in the territory of the Republic of Kazakhstan that have traditionally been used to alleviate skin diseases. This study mainly discusses the plant phytochemical composition. The main components responsible for their therapeutic effects in treating dermatitis, atopic dermatitis, and eczema were analyzed. 1.1. Achillea millefolium L. Aster Family—Asteraceae Achillea millefolium L., commonly known as common yarrow, belongs to the Asteraceae family (Asteraceae Dumort.). The plant was referred to as “venus eyelashes” during the Middle Ages due to its the feathery appearance of its leaves, while the whole plant was known as “soldier’s grass” for its use in treating wounds. There are over 100 different species of Achillea millefolium L., which are found in various regions worldwide, including North America, Europe, Asia, Australia, New Zealand, and the Middle East . The plant is widespread in Kazakhstan and serves as a valuable source of nectar for honeybees . The main components of A. millefolium are essential oils and phenolic compounds, monoterpenes, sesquiterpenes, lactones , amino acids, fatty acids, salicylic and succinic acids, ascorbic acid, folic acid, caffeic acid, and flavonoids . The composition of the essential oil includes sesquiterpenoids: achillin, acetylbalquinolide, caryophyllene, proazulene (chamazulene); and monoterpenoids: camphor, thujol, cineole, pinene, borneol. In addition, alkaloids (the main one of which is achilein), flavonoids, including flavone glycosides apigenin and luteolin were found in the yarrow herb; also found were tannins (α-phylloquinone) and vitamins K, A, and B; amines: choline and stakhidrin; and esters (bornyl acetate, myrtenyl acetate), caryophyllene, organic acids, polyins (pontic epoxide, matrixar ester), cyclic alcohol viburnite (20%), menthol, and geraniol . Yarrow also contains sterols, coumarins, the biogenic amine betaine, inulin, and other polysaccharides . The bitter taste of A. millefolium can be attributed to the presence of sesquiterpene lactones in its essential oil. The quantity of essential oil produced by the plant is largely dependent on the growth stage. During the early stage of growth, the content of essential oil is 0.13%, which rises to 0.34% in the process of flowering . In traditional medicine, yarrow has been employed to alleviate a variety of ailments including respiratory diseases (such as asthma and bronchitis), dyspepsia, skin inflammation, and headaches. The aerial part of the plant, including the leaves, stems, and inflorescences, is typically collected during the flowering phase for its usage as medicinal raw material. Yarrow is often administered as infusions, extracts, and potions to treat bleeding, flatulence, and gastrointestinal diseases . A. millefolium possesses various therapeutic properties such as disinfectant, anti-inflammatory, antispasmodic, anthelmintic, antibacterial, antioxidant, and antimicrobial effects . Additionally, the herb demonstrates antiulcer and anticancer activities , while the experimental findings suggest that yarrow may stimulate thrombocytopoiesis, leading to an increase in the number of platelets in the blood . Yarrow has long been utilized in traditional medicine as an efficient remedy for various skin ailments, including acne, eczema, neurodermatitis, and urticaria. Moreover, yarrow is incorporated into medicinal preparations for vasculitis. It is administered orally to prevent the recurrence of eczema . The wound-healing and anti-inflammatory effect of yarrow is due to the content of chamazulene (12.34%) in it. It is known that chamazulene enhances regenerative processes, weakens allergic reactions, has a local anesthetic effect, adsorbs various poisons, softens the skin, promotes scarring, heals infected wounds, and restores damaged capillaries : The results of the study explain the mechanism of action of A. millefolium . Thus, the effect of the water–alcohol extract of Achillea millefolium (HEAML) on the proliferation and stimulation of the human skin fibroblast growth (HSF-PI-16) was studied. The extract selectively inhibited the proliferation of HSF-PI-16 cells at higher concentrations (>20.0 mg/mL) and were stimulated at lower concentrations (<20.0 mg/mL). After treating the HSF-PI-16 medium for 72 h with the extract, a significantly increased proliferation rate and stimulation in the scratch analysis was noted . The activity of the plant in atopic dermatitis was also investigated. The results showed that Achillea millefolium L. significantly reduces the expression of proinflammatory cytokines in mouse macrophage cells treated with lipopolysaccharide . 1.2. Acorus calamus L. Aroid Family—Araceae Acorus calamus L. (calamus) marsh is a perennial plant containing aromatic compounds and is widespread in Central Asia, India, and the Himalayas. Although its distribution has significantly diminished in Europe, it remains a common plant in the northern marshy regions with a temperate climate . It is found in Asia, Europe, and North America and is known to grow in Central Kazakhstan along the banks of rivers, swamps, and lakes, sometimes forming substantial thickets. Calamus marsh is rich in various chemical compounds, including bitter glycoside acorin, essential oil (which contains proazulene), gum, resins, ascorbic acid, tannins, starch, and mucus. The dried rhizome of Calamus marsh consists of yellow aromatic volatile oils comprising of small amounts of sesquiterpenes and their alcohols; and choline, flavone, acoradin, galangin, acolamon, and isocolamon. Furthermore, it contains cineol, limonene, terpineol, azulene, eugenol, camphene, cadinene, ethanol, galangin, magnesium, zinc, terpenes, menthol, and camphor . Calamus root is considered in traditional medicine to be a therapeutic agent for a range of ailments, such as arthritis, neuralgia, diarrhea, dyspepsia, and hair loss . The plant has been found to possess potent antioxidant, anti-inflammatory, antiulcer, antimicrobial, and wound-healing properties. It is employed in dermatology to cure pyoderma, acne vulgaris, alopecia, and eczema . The advantageous effect on the skin can be attributed to the presence of β-azarone , a phenylpropanoid class chemical compound: β-Azarone is known to contribute to the body’s natural defense against ultraviolet rays, but it has also been found to have carcinogenic properties and induce liver tumors. Calamus marsh, which includes varying amounts of β-azarone depending on the variety, has traditionally been used in Asian medicine for its anti-inflammatory properties, which can help alleviate skin itching, swelling, and redness. Meanwhile, European varieties of Calamus marsh are known to contain sesquiterpenoids, which possess psychoactive properties and display beneficial medicinal effects . Calamus rhizomes have been found to be very useful as a topical agent in skin-related problems. The rhizomes are used in the form of powder, balms, enemas, and pills and also in ghee preparations. A tub bath in the decoction of vacha, kustha (Savccera lappa) and vidanga ( Embelina ribes ) is useful in curing eczema and other skin diseases . 1.3. Agropyron repens L. Lacquer Family—Gramineae Agropyron repens L. is distributed widely across Europe, Asia, and Africa . It can be found ubiquitously throughout Kazakhstan . The chemical composition of the plant is rich in a variety of carbohydrates such as fructose, glucose, inositol, and mannitol, as well as mucous substances, pectin, triticin, thianogenic glycosides, flavonoids, saponins, essential oil, monoterpenes (such as carvacrol, carvone, transanethol, thymol, menthol), and sesquiterpenes. Moreover, the plant contains vanillin glucoside, iron, minerals, and significant quantities of silica. Among the phenolic compounds found in the plant are p-hydroxybenzoic, vanillic, and p-coumaric acids, as well as chlorogenic acid, p-hydroxycinnamic acids, and p-hydroxycinnamic acid esters. The rhizomes consist of polysaccharides, glycosides such as quercetin and luteolin, phenolic glucosides, fatty acids, and amino acids (including γ-aminobutyric acid, proline, valine, asparagine, histidine, arginine, and tryptophan) . Furthermore, the seeds of wheatgrass contain triticin, mucus, saponins, sugar alcohols (namely, mannitol, inositol, and 2–3% of the total composition), essential oil with polyacetylenes or carvone, a small amount of vanilloside (vanillin), phenol carboxylic acids, silicic acid, and silicates . Agropyron repens L. was used in folk medicine as a sedative diuretic to relieve pain and spasms in the urinary tract, and as a sedative and tonic. The traditional medicinal use of Agropyron repens L. in urolithiasis has been scientifically proved, with confirmed pharmacological effects including hypoglycemic, hypolipidemic, anti-inflammatory, and antidiabetic effects, as well as effects on motility and benefits in urinary tract infections . The presence of flavonoids, alkaloids, and coumarin in the composition of this plant evidences its potential activity in the treatment of skin diseases, such as inflammatory skin diseases, atopic dermatitis, and acne . Thus, in the paper , the effect of wheatgrass extract in a cream form on some indicators of lipid peroxidation in allergic contact dermatitis was investigated. Contact dermatitis was modeled by the double application of 0.1 mL of a 5% alcohol solution of 2.4-dinitrochlorobenzene (DNCB) on previously depilated skin areas of the lateral surface of the abdomen of experimental animals. The anti-inflammatory activity of the cream was evaluated based on the characteristics of the skin and the state of lipid peroxidation (LPO) processes, i.e., the content of oxidation products in the blood plasma—malondialdehyde (MDA), diene conjugates (DC) and the activity of the antioxidant defense enzyme catalase. According to the studies, the cream containing wheatgrass extract has anti-inflammatory activity and promotes the activation of antioxidant protection (increased catalase activity), which, in its turn, decreases the intensity of lipid peroxidation (MDA and DC levels fall). The cream accelerated recovery for 4–5 days compared to untreated rats. The anti-inflammatory effect of wheatgrass cream was comparable to that of a standard cream with glucocorticoids (Akriderm C). 1.4. Artemisia absinthium L. Aster Family—Asteraceae Artemisia absinthium L., a plant species commonly known as wormwood, is widely distributed in Asia, the Middle East, Europe, and North Africa. It grows everywhere in Kazakhstan . A. absinthium is a plant species that possesses various biologically active compounds. The grass of this plant is utilized as a source material for oil production. The oil mainly consists of thujone esters, α- and β-thujone, camphene, α-cadinene, guaiazulene, (Z)-epoxycymene, (E)-sabinyl acetate, (Z)-chrysanthenyl acetate, as well as bitter sesquiterpenoid lactones, azulene group compounds, and tannins . Moreover, it contains terpenoids (such as myrcene, germacrene D, camphor, chamazulene), flavonoids (quercetin, kaempferol, apigenin, artemetin, and rutoside), phenolic acids (chlorogenic, ferulic, gallic, coffee, syringic, vanillic, and caffeoylquinic acid derivatives), and flavonoid glycosides . The composition of the A. absinthium extract is dependent on the type of solvent utilized in the extraction process. The alcoholic extract, in particular, has a considerably higher concentration of flavonoids, phenols, and tannins in comparison to the aqueous and chloroform extracts . For many years, A. absinthium has been used in traditional medicine to cure a wide range of ailments, particularly parasitic diseases and digestive disorders, as well as fever reduction . The leaves are employed to alleviate fever, while the flowers are used to treat stomach disorders and helminthiasis. The A. absinthium tincture is highly esteemed as a tonic and digestive aid. In a published paper , the wormwood herb was noted for its efficiency in treating jaundice, constipation, obesity, splenomegaly, anemia, insomnia, bladder diseases, and non-healing wounds from traumas. Furthermore, the plant is utilized as a base for producing skin ointments and balms . A. absinthium demonstrates various biological activities, including but not limited to antibacterial, anti-inflammatory, hepatoprotective, antidepressant, antispasmodic, and antipyretic effects . Moreover, it exhibits antimicrobial, antiviral, antistress, hepatoprotective, antioxidant, and anticancer effects . In the field of dermatology, the essential oil derived from A. absinthium has been shown to expedite wound healing, diminish inflammation, and exhibit antimicrobial and wound-healing properties. This effect is due to the significant content of oxygenated components in the oil, such as camphor ( a) and tirpinene-4-ol ( b), the content of which was, respectively, 47.59% and 6.36% : Camphor induces the proliferation of primary dermal fibroblasts, maintaining or restoring collagen and elastin production in UV-exposed skin. In addition, it prevents thickening of the epidermis and subcutaneous fat layer Camphor attenuates the aging enhancement associated with β-galactosidase (SA-β-gal) activity. In addition, the oil contains chamazulene (Figure 1) (10.35%), which contributes to the manifestation of antioxidant/anti-radical activity . 1.5. Bidens tripartita L. Aster Family—Asteraceae Bidens tripartita L. is widely distributed in the European part of the CIS, Transcaucasia, Siberia, Central Asia (excluding Turkmenistan), and the southern region of the Far East. Its range also extends to North Africa and North America . In Kazakhstan, this species is ubiquitous across its regions. B. tripartita is a plant that is rich in various biologically active compounds, including essential oil, chlorophylls, flavonoids, cinnamic acid derivatives, tannins with a high polyphenol fraction content, polysaccharides, carotenoids, ascorbic acid, coumarins, chalcones, and minerals such as Zn, Sr, Se, and Mn. Flavonoids found in the plant include luteolin, butein, sulphuretin, sulphurein, cynaroside, auron, -catechin, (−)-epicatechin, rutin, myricetin, 7-hydroxyflavone, esculetin, and umbelliferone, among others . In traditional medicine, the water infusion and decoction of B. tripartita have been utilized for a considerable time period in combination with baths for the treatment of scrofula, rickets, exudative diathesis, and various pustular skin diseases such as acne and boils, as well as for the management of gout, arthritis, and articular rheumatism. They are also recommended for improving appetite and digestion, and for the treatment of liver and spleen disorders, colds, bronchitis, and diabetes mellitus . Preparations derived from B. tripartita exhibit a range of therapeutic effects, including anti-inflammatory, hemostatic, antiseptic, sedative, and wound-healing properties, lower blood pressure, and increase the amplitude of heart contractions . The antiallergic, anti-inflammatory, diuretic, and antispasmodic effects of the alcohol extract of B. tripartita have also been proved . The methanolic extract of B. tripartita manifests antioxidant activity against cancer cells and has the ability to inhibit key enzymes, such as α-amylase and α-glucosidase. In addition, according to evidence, the herb has antidiabetic activity, as well as antihyperglycemic and antioxidant effects . The broad pharmacological effects of the plant are attributed to its abundant content of various biologically active substances. Manganese ions in the plant’s enzyme systems are believed to influence hematopoiesis, blood coagulation, endocrine gland activity, liver cell function, and blood vessel and bile duct tone, and may prevent intravascular thrombus formation and enhance antimicrobial properties of the plants . Flavonoids in the plant are responsible for its antiallergic and diuretic effects by affecting metabolic processes. The presence of vitamin C can activate the function of the endocrine glands, improve metabolism, strengthen the immune system, and help treat viral infections. The essential oils present in the plant are effective in destroying pathogenic microflora and fungi . The extract of B. tripartita is used in the treatment of many skin diseases: psoriasis, seborrhea, urticaria, diathesis, acne, pimples, wounds, and ulcers, as well as small cracks. The beneficial effects on the skin can be attributed to the presence of tannins. Tannins also help to get rid of increased sweating of the armpits and legs. Thus, B. tripartita is employed for making baths, lotions, and wipes to treat microbial eczema of the feet, epidermophytosis . The mask derived from the sequence has been shown to eliminate oily sheen, tone the skin, and have a rejuvenating effect. Additionally, wiping the face with a decoction of the string has been demonstrated to reduce acne . During diathesis, an infusion of B. tripartita (from 10–30 g of herbs) is added to the bath . Khatamov et al. developed and studied a new drug: a thick extract of the sum of flavonoids in the form of ointment (1, 3 and 5%) obtained from B. tripartita , which was used to cure contact allergic dermatitis experimentally caused in guinea pigs by 2,4-dinitrochlorobenzene. The study results showed that 5% ointment had the greatest therapeutic effect compared to the antihistamines Psilo-Balsam and glucocorticoid ointment Celestoderm. At the same time, the rate of reduction in the severity of skin manifestations (Ind) was the highest compared to other studied groups—37.9%. 1.6. Capsella bursa-pastoris L. Cabbage Family—Brassicaceae Capsella bursa-pastoris L. is a wild plant with significant nutritional value that is suitable for human consumption. This plant is widely distributed across many countries, including Cyprus, Europe, Saudi Arabia, Turkey, Pakistan, India, Iraq, Iran, China, Azerbaijan, and other Asian countries . It is also commonly found in various regions of Kazakhstan. C. bursa-pastoris contains a variety of chemical components including flavonoids, polypeptides, choline, acetylcholine, histamine, tyramine, fatty acids, sterols, organic acids, amino acids, sulforaphane, vitamins , and various trace elements. In addition, it contains phenolic compounds, flavonoids, tannins, saponins, alkaloids, and phytosterols , as well as volatile fractions consisting mainly of terpenoids, alkane hydrocarbons (such as nonacosane), and fatty acids (including palmitic and linoleic acids) . In traditional medicine, C. bursa-pastoris has been used for centuries in China and Japan as a hemostatic, diuretic, and antipyretic agent . The plant has been utilized for the treatment of conditions such as edema caused by nephritis, odynuria, hemaffetia, menorrhagia, chyluria, and hypertension . The entire plant is used to make tea, which has been employed as an antiscorbutic, astringent, diuretic, emmenagogue, hemostatic, hypotensive, tonic, stimulant, vasoconstrictor, and wound-healing agent. This beverage has also been considered an excellent remedy for various types of bleeding, including those originating from the stomach, lungs, uterus, and kidneys. A homeopathic remedy for nosebleeds and urolithiasis is prepared from a fresh C. bursa-pastoris plant . Based on the literature, raw plant extracts and certain phytocomponents have been reported to exhibit various pharmacological effects, such as anti-inflammatory, antispasmodic, antimicrobial, hepatoprotective, cardiovascular, anticancer, sedative, and antioxidant effects . Furthermore, these extracts have been assumed to possess infertility-reducing properties . Extracts have also demonstrated inhibitory effects on acetylcholinesterase activity and significant antibacterial activity . C. bursa-pastoris exhibits potent antioxidant activity attributed to its flavonoid compounds, namely quercetin, chrysoeriol, kaempferol, and isorhamnetin. In vitro studies have shown that its extracts possess antioxidant activity that prevents the development of various free radicals such as DPPH radicals, peroxyl radicals, hydroxyl radicals, and hydrogen peroxide . Additionally, the plant extract has been found to have cytotoxic effects as reported by the previous studies . Furthermore, a moderate hepatoprotective activity has been observed with the extract containing specific flavonoids, including 4,7-dihydroxy-5-hydroxymethyl-6,8-diprenylflavonoid, chrysoeriol-7-O-d-glucopyranoside, sinensetin, and 6,8-diprenylgalangin . C. bursa-pastoris exhibits an excellent efficiency in the treatment of eczema in dermatology . Moreover, preparations derived from C. bursa-pastoris have been registered and recommended by the German Institute for Pharmaceuticals and Medicines for the additional treatment of skin diseases and wounds . Fumarates improve the course of psoriasis, in which both IL-12 and IL-23 promote the differentiation of pathogenic T-helpers (Th). Fumarate treatment induces IL-4-producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by an increase in heme oxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. The induced HO-1 breaks down, after which the N-terminal fragment of HO-1 is translocated into the nucleus and interacts with the AP-1 and NF-kB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19 . 1.7. Chelidonium majus L. Poppy Family—Papaveraceae Chelidonium majus L., a plant species commonly known as greater celandine, is widely distributed across Asia, North America, and northwestern Africa . The plant C. majus is known to contain a high concentration of isoquinoline alkaloids, with levels ranging from 0.27 to 2.25% in the aerial parts and 3–4% in the root. Over 70 compounds have been identified, including various alkaloids (such as chelidonin, chelerythrin, sanguinarine, berberine, protopine, allocryptopine, and koptisin), flavonoids (such as rutin, quercetin, and kaempferol), saponins, vitamins (such as vitamin A and C), mineral elements, a small amount of phytosterols (such as α-spinasterol and ergosterol), and aromatic and aliphatic acids (including chelidonic, caffeic, ferulic, polycoumaric, citric, etc.) and their derivatives. Additionally, celandine consists of polysaccharides, alcohols (1-hexocosanol, chelidoniol, and nonacosanol), choline, tyramine, histamine, and saponosides. It should be noted that a previous study provided the formulas of all organic components . The content of most mineral elements in celandine ranged from 10 to 65%, where potassium (65%) and phosphorus (54%) predominated . C. majus has a long history of traditional use in Europe, Asia, and Africa to treat various ailments, including those affecting the liver and bile ducts, as well as to cure skin conditions such as warts, calluses, and eczema. Additionally, the plant has been used to treat stomach ulcers, tuberculosis, skin rashes, and oral infections. In traditional Chinese medicine and homeopathy, C. majus is used to alleviate congestion, pain, swelling, and jaundice . Celandine extracts have been found to possess a broad spectrum of pharmacological activities including anti-inflammatory, antimicrobial, anticancer, antioxidant, hepatoprotective, natriuretic, and antidiuretic effects, corroborating some of the traditional medicinal uses of C. majus . Additionally, the plant has demonstrated immunomodulatory, lipid-lowering, and radioprotective properties . Moreover, the ethanolic extract of C. majus has been found to contain biologically active secondary metabolites with significant inhibitory effects that prevent Alzheimer’s disease . The milky juice of the celandine is rich in alkaloids among which the predominant one is chelidonine ( a). Studies have demonstrated the antimicrobial, immunomodulatory, cytostatic, and cytotoxic effects of celandine alkaloids, including their anti-keratinocyte activity. Compounds such as chelidonine ( a), sanguinarine ( b), chelerythrine, coptisine, and protopin have been found to exhibit cytotoxic activity. Sanguinarine ( b) has been shown to be particularly effective at inhibiting keratinocyte growth, indicating that celandine may have potential as an additional therapy for malignant skin diseases . 1.8. Cichorium intybus L. Aster Family—Asteraceae Cichorium intybus L., a perennial herbaceous plant belonging to the Asteraceae family, is known by various common names such as roadside grass, blue flower, roadside cornflower, bride of the sun, and sun grass. Its recognizable feature is the inflorescences-baskets that exclusively comprise reed blue flowers. However, the said baskets only open during early morning hours or in cloudy weather. The term “chicory” is derived from the Latin word, meaning “entering the fields.” Due to its therapeutic properties, this plant has earned the followings names: “king root,” “golden root,” and “cure for a hundred diseases” . C. intybus exhibits a wide geographical distribution encompassing Northern and Central Europe, Siberia, Turkey, Afghanistan, Northern and Central China, South America, South Africa, Ethiopia, Madagascar, India, Australia, and New Zealand. This herbaceous plant is capable of growing in the territories of the Commonwealth of Independent States, except the Far North region . The roots of C. intybus contain 56–65% inulin (in terms of dry matter), the maximum accumulation of which is observed in autumn. Intibin glycoside gives specific bitterness to chicory roots. Proteins, sugars, pectin, and sesquiterpene lactones were also found in the roots, as well as guayanolides: cycriosides B and C, sonchuside C, tannins and resinous substances, choline, carotene, vitamins B, B2, PP and C, from mineral elements—sodium, potassium, calcium, manganese, and phosphorus, iron. Chicory roots contain taraxasterol, phenolic acids (chlorogenic, isochlorogenic, neochlorogenic, caffeic and cicoric acids) . In the flowers of C. intybus , chicory glycoside was found, in the seeds: inulin and protocatechin aldehyde , prebiotic fructooligosaccharides, sesquiterpene lactones, caffeic acid derivatives (chicory acid, chlorogenic acid, isochlorogenic acid, dicapheoyltartaric acid), proteins, hydroxycoumarins, flavonoids, alkaloids, steroids, terpenoids, oils, volatile compounds, and vitamins . Aliphatic compounds and their derivatives make up the main fraction; terpenoids are somewhat less common in the plant. Chicory leaves contain inulin, vitamins A, B1, B2 and C, macro- and microelements (Ca, K, Mg, Na, Fe, Cu, Mn, Zn), phenolic compounds, etc. . The aerial and subterranean portions of C. intybus L. are extensively employed in traditional medicine, such as in Chinese and Mongolian practices, as an agent for modulating the immune system, promoting bile secretion, protecting the liver, and reducing blood glucose levels. The plant is documented in the Chinese Pharmacopoeia and is utilized in the formulation of homeopathic remedies in Germany. The extract of chicory herb is a constituent of the LIV-52 complex preparation from India . C. intybus exhibits antiseptic and astringent properties and produces choleretic and diuretic effects. It positively influences the nervous and cardiovascular systems. Additionally, its infusion has been employed for normalizing heart rhythm. According to the literature, preparations derived from C. intybus are efficient in treating various diseases connected with the gallbladder, liver, kidneys, and urinary system. Additionally, chicory preparations have been shown to exhibit potential therapeutic benefits in managing obesity, liver diseases, atherosclerosis, hypoacid gastritis, tachycardia, arrhythmia, and nephritis. The milky juice of the plant contains bitter substances that have been found to stimulate peristalsis of the gastrointestinal tract, increase the secretion of gastric and intestinal juice, and promote regular bowel movements and appetite. According to the published literature, C. intybus has been found to possess a notable therapeutic effect in curing and preventing diabetes mellitus and in preventing it (antidiabetic effect). This effect is attributed to the presence of inulin, a natural sugar substitute that eliminates toxins and non-nutrient substances from the body. Preparations based on C. intybus exhibit diverse pharmacological activities, including anti-inflammatory, antioxidant, antiviral, choleretic, diuretic, hepatoprotective, and antibacterial effects, making them beneficial in treating colitis, gastritis, and enteritis. Decoctions of C. intybus roots have been reported to be effective in the treatment of helminthic invasion, anemia, malaria, scurvy, eczema, and tumors of the spleen . Furthermore, some research indicates that C. intybus L. may modulate immune responses . Infusions of C. intybus flowers have been found to possess antiseptic, anti-inflammatory, moisturizing, and nourishing properties, which are beneficial in treating inflammation of the skin and eyes . A decoction of C. intybus L. is commonly applied externally (in the form of baths, applications, and lotions) for the treatment of various skin diseases, including but not limited to eczema, urticaria, psoriasis, seboroid dermatitis, neurodermatitis, atopic dermatitis, vitiligo, acne, and furunculosis. Additionally, the herb is known for its efficiency in the care of dry skin . C. intybus extract has been clinically tested on volunteers as a skin UV-protecting means. The analysis results of the microrelief control area applied with sodium lauryl sulfate and causing skin damage showed there was a significant increase in roughness after 28 days of the study, while in the areas where sodium lauryl sulfate was applied on the plant extract, the roughness of the skin did not undergo any significant changes, which indicates the plant’s protective properties . 1.9. Equisetum arvense L. Horsetail Family—Equisetaceae Equisetum arvense L., a herbaceous plant belonging to the Equisetaceae family, is widely distributed in North America, Europe, and Asia, including the territory of Kazakhstan . E. arvense contains more than 210 natural compounds distributed in various organs. These compounds include alkaloids, carbohydrates, proteins and amino acids, phytosterols, saponins, sterols, ascorbic acid, silicic acid, phenolic compounds, and their glycosides, tannins, flavonoids (such as apigenin, genquanin, luteolin, kaempferol, quercetin), triterpenoids, volatile oils, and other bioactive substances . E. arvense , a plant species from the Equisetaceae family, has been utilized in traditional medicine for its therapeutic properties. Its applications include the treatment of tuberculosis, and renal and bladder catarrh, as well as a hemostatic agent during excessive menstruation, nasal, pulmonary, and gastric bleeding, among others . The water–alcohol extract of E. arvense has demonstrated various biological activities including antioxidant , anti-inflammatory, antibacterial, and antimicrobial effects . Studies have also reported its antiproliferative activity , as well as antifungal, vasodilating, hepatoprotective , neuro- and cardioprotective, cytotoxic, and anti-cellulite properties . Additionally, E. arvense has been traditionally used for its analgesic effects on rheumatism and frostbite, as well as its anti-inflammatory properties, which can improve blood circulation. This plant has been employed as a bath agent for skin diseases and incorporated into cosmetic products as a rejuvenating, moisturizing, anti-wrinkle, anti-acne, antiperspirant, and conditioning agent . Equisetum arvense L. is recognized for its high content of silicon, a compound that is associated with promoting skin health. Silicon maintains skin firmness and elasticity, while its mild exfoliating properties allow the elimination of dead skin cells and enhancing of skin texture . The antioxidant potential of E. arvense has been attributed to the presence of flavonoids such as quercetin ( a), kaempferol ( b), and isorhamnetin ( c) . Studies show that phenolic compounds in the plant reduce the formation of ROS induced by bacterial lipopolysaccharides or fungal infections due to the direct capture of free radicals or their purification through reactions with antioxidant enzymes . Cosmetics containing the extract of this plant, which prevents early aging of the skin, have been actively introduced to the industry . Quercetin ( a) is able to reduce inflammation, accelerate reepithelialization, and stimulate cell proliferation and the formation of granulation tissue in different experimental models of skin wounds and clinical trials. These effects are associated with their ability to decrease levels of inflammatory cytokines (IL-1β, TNF-α), cell migration (neutrophils, CD68+ macrophages), mitogen-activated protein kinases (p38p, ERK-Î 2 , JNK-Î 2 ), (PEG2, leukotriene B4), inflammatory enzyme (COX-2), and transcription factor (NF-kB). In addition, quercetin promotes increased growth factors (VEGF and TGF-β1) as well as anti-inflammatory cytokines (IL-10) and antioxidant defenses (GSH, SOD, CAT). This compound also has an antifibrotic effect on second-target wounds, increasing the expression of αintergrin (a protein involved in the migration and proliferation of fibroblasts and reducing β1 integrin migration of fibroblasts and initiation of fibrosis . 1.10. Eryngium planum L. Seler Family—Apiaceae The subgenera of Eryngium are predominantly distributed throughout Europe, Africa, and Asia, with certain subgenera exhibiting a widespread presence in Australia . In Kazakhstan, Eryngium is found growing in the steppe regions of Northern Kazakhstan, as well as in the Dzungarian and Zailiyskiy Alatau mountain ranges . The aerial parts of Eryngium species are characterized by the presence of saponins, flavonoids, and essential oils, while the underground parts contain triterpene saponins, monoterpene glycosides, phenolic compounds such as flavonoids and phenolic acids, coumarin derivatives, terpene aldehyde esters, essential oils, and oligosaccharides . The isolation of eringinol from the aboveground parts of the plant was reported later . Further studies on the phytochemical constituents of the plant were conducted on leaves and roots, leading to the isolation of various aglycones and A1-barrigenol and R1-barrigenol . E. planum plays a significant role in European and Asian traditional medicine for treating various inflammatory diseases. The plant’s aboveground parts are bioactive primarily due to the presence of polyphenols and saponins . It has demonstrated potential for use in gastrointestinal diseases and exhibits antibacterial, analgesic, anthelmintic, anticonvulsant, and anticancer properties, thereby proving its crucial importance in ethnopharmacology . The aerial part of the plant collected during flowering is used for therapeutic purposes. According to the results obtained from HPLC-MS analysis, flavonoids, particularly rutin ( a) and isoquercetin ( b), are the major constituents of E. planum extracts . Rutin is known to possess skin-toning properties and to prevent the appearance of skin diseases such as rosacea and erythema. The anti-inflammatory effects of E. planum extracts may be attributed to the synergistic activity of ursolic acid ( c) and polyphenols such as chlorogenic acid ( d) and rosmarinic acid ( e), which have previously been studied for their anti-inflammatory properties . Notably, ursolic acid, which predominates in concentrated extracts of the plant, exhibits antioxidant, antimicrobial, anti-inflammatory, and hypoglycemic activities . Eryngium planum L. has potential applications in dermatology, particularly for the treatment of atrophic and purulent skin wounds when applied externally . Secondary metabolites include phenolic acids, flavonoids, coumarins, and triterpenoid. Saponins isolated from E planum L. demonstrate moderate antibacterial activity and substantial antimycotic activity. It was found that phenolic compounds inhibit microbial adhesion and inactive transport protein of a cell membrane . 1.11. Glycyrrhiza glabra L. Legume Family—Fabaceae Glycyrrhiza glabra L., commonly known as licorice, fragrant wood, or mulaiti, is a small perennial plant that grows in Eurasia, North Africa, and West Asia . This plant is found ubiquitously in Kazakhstan . The genus Glycyrrhiza is extensively distributed across the globe and has over 30 species. The root of Glycyrrhiza glabra is a significant medicinal component due to the presence of various isolated compounds. These include triterpene saponins such as the sweet saponin glycyrrhizin, flavonoids such as liquiritin which is the primary flavonoid glycoside, rhamnoliquirilin, liquiritigenin, prenillicoflavon A, glucoliquiritin apioside, 1-methoxyphaseolin, shinpterocarpin, shinflavanone, lycopyranocoumarin, glisoflavone, lycoarylcoumarin, coumarin-GU-12, isoflavonoids, and chaconne. Among these, glycyrrhizic acid is the primary biologically active component, and it is known to be 60 times as sweet as sugar cane . Licorice root has been employed as a therapeutic agent by both ancient and modern medical practitioners. Its oral administration has demonstrated efficiency in the treatment of various disorders including gastric, duodenal and esophageal ulcers, inflammation, laxatives, mouth ulcers, antispasmodic, antitussive, sedative, and expectorants. The herb’s constituents make it a promising candidate for curing respiratory diseases such as asthma, acute and chronic bronchitis, and chronic cough. Furthermore, it can be used in treating Addison’s disease. External application of licorice extracts has also been effective in treating inflammatory skin conditions, mouth ulcers, and maintaining oral hygiene . Numerous clinical and experimental studies have shown the presence of several pharmacological properties in this substance. These properties are of great advantage, including anti-inflammatory, antiviral, antimicrobial, antioxidant, anticancer, immunomodulatory, hepatoprotective, and cardioprotective effects . The ethanolic extract derived from the root of G. glabra exhibits an excellent antibacterial activity that stops the development of Propionibacterium acne and Pseudomonas aeruginosa . Due to this property G. glabra is employed in dermatology for treating skin diseases, such as dermatosis and acne . Multiple studies have demonstrated the efficacy of Glycyrrhiza glabra L., which is efficient in the treatment of skin hyperpigmentation, eczema, and psoriasis, and provides skin with antioxidant properties. Due to the presence of flavonoid compounds such as oxyresveratrol ( a), glabridin ( b) and liquiritin ( c), it has a therapeutic effect . The external application of skin care products containing licorice extract gives a healthy glow, as well as improves the overall quality and appearance of the skin . It is achieved by the oxidative abilities of these components. Flavonoids of Glycyrrhiza glabra : liquiritin ( c), glucoliquiritin apioside ( a), and glycyrrhizin ( b) have high skin permeability properties and are potential antioxidants. These components improve the histological properties of the dermis and epidermis and reduce the level of markers of inflammation and wrinkles . Glycyrrhiza glabra L. also contains licochalcone A ( c), which has anti-inflammatory and antimicrobial properties and has been found to be efficient in treating acne, inflammatory skin diseases, and other skin ailments . 1.12. Gnaphalium uliginosum L. Aster Family—Asteraceae Gnaphalium uliginosum L. is a member of the Compositae family, a group of flowering plants, and is commonly referred to as swamp cudweed. It is widely distributed, including in Kazakhstan . G. uliginosum is known to have a limited array of chemical constituents. It consists of approximately 125 compounds such as flavonoids, sesquiterpenes, diterpenes, triterpenes, phytosterols, anthraquinones, caffeylquinic and caffeylglucaric acids, flavonols, and carotenoids . Marshweed, also known as G. uliginosum , has been used in traditional medicine to alleviate a variety of ailments, including gastric disorders, edema, wounds, prostatitis, lumbago, neuritis, and angina pectoris. Additionally, it has been utilized for its antihypertensive, diuretic, antipyretic, and antimalarial properties . Pharmacological investigations on G. uliginosum extracts have revealed various beneficial effects, such as antioxidant , antibacterial, antifungal, antitussive, expectorant, antifeedant, cytotoxic, and hepatoprotective activities . Additionally, this plant has anti-inflammatory, antidiabetic, and antihyperuricemic properties . G. uliginosum is employed in medical practice as a hypotensive and wound-healing agent for treating hypertension, gastric ulcer, and difficult-to-heal wounds . Furthermore, oil extracts derived from this plant are useful for curing laryngitis, catarrh of the upper respiratory tract, and tonsillitis . In the field of dermatology, the extract derived from Gnaphalium uliginosum has been employed to treat diseases such as eczema and skin cancer . The ointment used to treat psoriasis contains an aqueous extract of “cold pressed” Gnaphalium uliginosum L. obtained immediately after harvesting . 1.13. Humulus lupulus L. Hemp Family—Cannabaceae Humulus lupulus L., commonly known as hops, is a plant species that is widely distributed in temperate regions worldwide . H. lupulus is a plant that contains many phytochemicals, with a high concentration found in the female inflorescences from which lupulin, a yellowish-brown granular powder, is obtained. Lupulin comprises bitter resins and essential oils, imparting the characteristic aroma and flavor of hops. The primary bitter acids found in hop resin are alpha acids (humulones) and beta acids (lupulones). The essential oils contain myrcene, linalool, and geraniol, which are the most important aromatic compounds. Additionally, lupulin contains polyphenols, such as quercetin ( a), kaempferol, ( b) (see above) catechins, prenylnaringenin, hydroxycinnamic acid, and condensed tannins. Ferulic acid is the most representative compound in the phenolcarboxylic acid group. Hop seeds are rich in catechins (catechin, epicatechin), which are widely used in various industries, including pharmaceuticals, cosmetics, and nutraceuticals . H. lupulus has a long history in traditional medicine, which dates back to prehistoric times. It was used to treat various ailments such as leprosy, toothache, fever, stomach issues, sleep disorders, and anxiety. Additionally, it was utilized as a bowel function enhancer and to improve the pharmaceutical properties. Due to the numerous health benefits of hop polyphenols, which include antioxidant and antimicrobial effects, they may have a therapeutic use . Hop extract has been found to possess various pharmacological properties. For instance, it exhibits antitumor and anti-inflammatory effects, as evidenced by previous studies . Moreover, the extract has been reported to possess antibacterial, anti-collagenase, and antioxidant activity . Additionally, hop extract has been found to have antiallergic, antiviral, hepatoprotective, and antithrombogenic effects . In dermatology, extracts of H. lupulus have been employed as an antipsoriatic medicine . Furthermore, they are used in the treatment of skin inflammation diseases of adolescents, and hop cones are taken orally to cure baldness, furunculosis, lichen, and scrofula . The plant is known to have an anti-collagenase effect on the skin; that is, it prevents the destruction of collagen fibers due to exposure to UV rays . Naoto et al. tested seven natural components of hop ( Humulus lupulus L.) extracts to evaluate biological activity against acne vulgaris . Five strains, Propionibacterium acnes , Staphylococcus epidermidis , Staphylococcus aureus , Kocuria rhizophila , and Staphylococcus pyogenes , were selected as the main acne-causing bacteria. Hop extracts xanthohumol ( a) and the lupulones ( b) showed strong inhibitory activities against all of the strains. Although hydrogenated derivatives did not show the same level of activity, naturally occurring xanthohumol ( a), lupulones ( b), and humulones ( c) all showed moderate to strong anti-collagenase inhibitory activities: The results of studies indicate that H. lupulus flower extract has strong antioxidant activity since it significantly reduces the production of ROS and decreases inflammation, diminishing the production of NO and the expression of COX-2 by macrophages activated by liposaccharides . 1.14. Juglans regia L. Walnut Family—Juglandaceae This plant has been observed to grow in various regions across the globe, including East Asia, Europe, North Africa, and South America . Its growth has also been documented in southern Kazakhstan and it is recognized as a protected species within the boundaries of the Sairam-Ugam State National Natural Park . The chemical composition of walnut kernels is of significant nutritional value due to the high content of polyunsaturated fatty acids (comprising up to 75% of total content), proteins, amino acids, as well as vitamins E, C, β-carotene, and essential minerals such as potassium, calcium, magnesium, sulfur, and phosphorus . Moreover, walnut is known to contain trace elements such as iron, zinc, and copper, which play a vital role in various biochemical processes within the human body . The plant is also rich in fluorine salts. Walnut partition contains trace amounts of organic substances, tannins, glycosides, alkaloids, and iodine. The chemical composition of walnut leaves is characterized by the presence of various biologically active components, including trace amounts of iodine, α- and β-hydrojuglone, polyphenols, tannins, glycosides, flavonoids, terpenoids, vitamin C, carotene, vitamin B1, essential oils, and tannins . Among the compounds contained in walnut, polyphenolic compounds are the most important ones. They include various derivatives of chlorogenic and hydroxycinnamic acids that are the major components . The study by Schwindl demonstrated that the methanolic extract derived from the leaves of Juglans regia L. includes a cumulative 40 metabolites classified under megastigmane, tetralone, phenylpropanoid, neolignan, and juglone glycosides . In traditional medicine, diverse components of Juglans regia L. are utilized to cure several ailments such as diabetes, infectious diseases, and periodontal disease . Furthermore, the plant is reputed to have antipyretic, analgesic, antidandruff, and burn-healing properties . Notably, the extract of walnut shell has demonstrated notable antibacterial and antibiofilm properties, which develop resistance to coagulase-negative staphylococci . Additionally, the lyophilized extract of the walnut septum has been reported to exhibit a marked antitussive, antioxidant, and anti-inflammatory effect . The leaves of Juglans regia L. are traditionally used to alleviate skin inflammation and excessive sweating of the hands and feet. Moreover, they are recommended for the treatment of acne, warts, eczema, and psoriasis due to the presence of flavonoids, specifically quercetin derivatives, and tannins . The high concentration of α-tocopherol in the leaves of J. regia contributes to its antioxidant effect, which promotes the repair of damaged skin and strengthens the epidermal layer . 1.15. Matricaria recutita L. Aster Family—Asteraceae Matricaria chamomilla L. is a globally distributed, well known medicinal plant . M. chamomilla contains numerous biologically active compounds, including flavonoids (such as apigenin and luteolin) and their glycosides, as well as coumarins (including gerniarin and umbelliferone) . The essential oil extracted from chamomile flowers is composed of 52 different components, with the highest concentration of terpenoids, including β-farnesene, α-farnesene, α-bisabolol, chamazulene, and germacrene, as well as spiroether . M. chamomilla has been widely employed in traditional medicine for treating a variety of ailments, including infections, neuropsychiatric disorders, respiratory tract, gastrointestinal, and liver diseases. Furthermore, the plant possesses sedative, antispasmodic, antiseptic, and antiemetic properties . Therapeutic indications for M. chamomilla encompass a diverse array of medical conditions, including inflammatory conditions, bacterial infections, and lesions of the skin and mucous membranes such as those found in the oral cavity, gastrointestinal tract, and respiratory tract. Additionally, the plant has been employed as a remedy for spasms and ulcers of the gastrointestinal tract, insomnia, and nervous breakdown . Furthermore, the plant has demonstrated pain-relieving properties and wound-healing effects , and acted as a protective agent for the kidneys and liver . M. chamomilla is regarded as a viable alternative due to a high content of bioactive secondary metabolites that can be used for the treatment of diverse skin problems, such as wounds, abscesses, and skin diseases. The plant’s therapeutic efficiency in treating skin diseases is attributed to the presence of quercetin ( a), α-bisabolol ( a), and apigenin ( b): α-Bisabolol ( a) possesses anti-inflammatory, antibacterial, and anti-irritant properties, making it suitable for use in a variety of products that protect skin from irritation caused by environmental factors. Due to its non-allergenic nature, it is widely used in hand and body lotions, aftershave creams, lipsticks, sun and after-sun care products, and baby care products . On the other hand, apigenin ( b) has been found to alleviate the symptoms of skin inflammatory diseases by protecting skin cells from oxidative-stress-induced death. Apigenin also affects the synthesis of skin barrier factors and the influx of calcium ions. Therefore, it can potentially be used to treat skin inflammatory diseases and cancer . Dos Santos et al. presented a review of 20 patents using Matricaria species as an active ingredient in skin diseases. The majority of the inventions (80.00%) contained combinations of Matricaria with other plant species, including those belonging to the genus Calendula , Salviae , Eucalyptus , Urtica , and Aloe vera . On the other hand, four patents (20.00%) reported the development of bioproducts containing only species classified as chamomile, two (10.00%) with M. parthenium , and two (10.00%), M. chamomilla . Based on the information of these extracts, externally applied pharmaceutical remedies such as creams, ointments, lotions, solutions, textile dressing, and banding were developed. Capsules, granules, and alcohol dye have been developed for oral use. Regarding the skin disease treated, the selected inventions claim to treat wounds and burns, erythema and rosacea, eczema and dyshidrosis, and spots and hyperpigmentation of the skin by UV radiation. Skin peeling and damaged stratum corneum, dermatitis, hemorrhagic incision, excoriations, hand-foot syndrome, psoriasis, and acne were also mentioned. 1.16. Ononis spinosa L. Legume Family—Fabaceae Ononis spinosa L. is widely distributed in Africa, Asia, and Europe. It is found in countries such as Algeria, Libya, Morocco, Tunisia, Afghanistan, Iran, Iraq, Palestine, Jordan, Lebanon, Syria, Turkey, Armenia, Azerbaijan, India, Denmark, Norway, Sweden, Great Britain, Austria, Belgium, Czechoslovakia, Germany, Hungary, the Netherlands, Poland, Switzerland, Estonia, Lithuania, Moldova, the European part of the Russian Federation, Albania, Bulgaria, Greece, Italy, Romania, France, Portugal, and Spain . The root of O. spinosa contains a large amount of isoflavonoids, pterocarpans, and dihydroisoflavonoids, including formononetin, calicosin, pseudobaptigenin, medicarpin, maakiain, onogenin, and sativanon, with metabolites present in the form of glucosides, glucoside malonates, glucoside acetates, and free aglycones . The roots, leaves, and flowers of O. spinosa were utilized in folk medicine for their antitussive, laxative, and diuretic properties. Infusions of the plant were employed to treat dropsy, urinary tract infections, inflammation, and rheumatism, while external applications were used to promote wound healing and alleviate skin conditions such as eczema. In Iraq, the roots were valued for their diuretic, blood purifying, laxative, and expectorant qualities . Additionally, ash derived from burned samples of O. spinosa has demonstrated resistance to various Candida species . Pharmacological investigations have demonstrated that O. spinosa exhibits noteworthy hepatoprotective and antitumor properties , and may be considered a potential therapeutic agent for treating urinary tract infections and bladder stones . O. spinosa has been utilized in dermatology for its efficiency in treating skin ailments such as dermatitis (eczema) and pruritus. It also possesses wound-healing properties beneficial in the treatment of burns . Ononis spinosa extract and glycerin have been clinically tested for facial laxity and wrinkles. The particular focus was made on immediate and delayed effects. Thirty-nine women used the product daily for an eight-week treatment period. Clinical assessment by experts and a new 2D imaging method (measuring the effect of an upper eyelid lift) were made at different time periods. The results showed an immediate and significant improvement in sagging and wrinkle parameters seven hours after the first application, in addition to significant long-term improvement. The lifting effect calculated from 2D images was 1.08 mm immediately after application and 1.80 mm after an eight-week treatment period. Ononis spinosa root extract inhibited hyaluronidases; Hyal-1 inhibition was a promising remedy for improving wound healing, tissue regeneration, and inducing diuresis. Two non-polar fractions of the roots of Ononis spinosa were the most active, causing inhibition of Hyal-1 by 86 ± 3% and 96 ± 13% at a concentration of 1 mg/mL, respectively. Chemical analysis revealed three main components, which were identified as onogenin, sativanon, and medikarpine. The percentages of inhibition for concentrations of 250 μM of these compounds were 25.3 ± 18, 61, 20 ± 20.6, and 22.4 ± 16, respectively. The IC50 of sativanone was determined to be 151 µM. Hot water and hydroalcoholic extracts of the Ononis spinosa root showed a moderate inhibitory effect on hyaluronidase-1 (Hyal-1) (IC 50 1.36, respectively, 0.73 mg/mL), while dichloromethane extract had an inhibitory effect (Hyal-1) with IC 50 190 µg/mL . 1.17. Onopordum acanthium L. Aster Family—Asteraceae Onopordum acanthium L. is a widely distributed species of plants found across Africa (Algeria), Asia (Afghanistan, Iran, Iraq, Turkey, Armenia, Azerbaijan, Georgia, Russian Federation, Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, Uzbekistan, China, India, Pakistan), throughout Europe, Australia, New Zealand, and North and South America (Argentina, Chile, Uruguay) . O. acanthium is a plant species that contains various phytochemical compounds, including saponins, alkaloids, sesquiterpene lactones, flavonoids, triterpenes, sterols, nitrogen-containing compounds, phenolic acids, coumarins, inulin, soluble sugars, proteins, and oils . The fatty acid composition of the plant includes palmitic, stearic, oleic, and linoleic acids . Additionally, phenolic, triterpene, and steroid compounds were detected in the aerial parts of O. acanthium , while the roots were found to contain sesquiterpene lactones and polyacetylenes . In traditional medicine, various preparations of O. acanthium , including its powder, juice, and decoction of the aerial part, were utilized as diuretics. This plant is known to stimulate the central nervous system and has demonstrated cardiotonic and hemostatic properties. Infusions of the leaves and inflorescences have also been employed to reduce swelling of various etiologies . Furthermore, the extract derived from this plant has exhibited bactericidal, cardiotonic, and antitumor effects . The extracts and isolated compounds from this plant have demonstrated a range of activities including anti-inflammatory, anti-radical, antiproliferative, and antibacterial effects . Additionally, this plant can produce antioxidant and anti-inflammatory effects , as well as diuretic, dermatological, tonic, sedative, anticonvulsant, cardiotonic, hemostatic, and bactericidal effects, all without causing any side effects. Eriodictyol ( a) and quercetin ( a) have been identified in the flowers of the plant, both of which possess potent antioxidant properties. Eriodictyol, in particular, has been found to protect skin cells from damage induced by UV radiation by inhibiting the MAPK signaling pathway, thereby exhibiting anti-aging effects . The antitumor activity of extracts obtained from a combination of flowers and fruits, leaves, and roots of O. acanthium resistant to A431 culture (epithelial carcinoma of the skin) was examined by the authors of . Aqueous, n-hexane, chloroform, and water–methanol extracts were utilized in the study. The results revealed that the chloroform extract of leaves and roots displayed the highest activity. A number of compounds were isolated from the roots of Onopordum acanthium L., among them 4β,14-dihydro-3-dehydrozaluzanin C ( b), which showed a general antiproliferative ability comparable to that of the reference drug cisplatin in relation to epidermoid skin carcinoma. The antiproliferative activity of compound ( b) was evaluated on epidermoid skin carcinoma cells A431 using MTT analysis . The mechanism of cytotoxicity ( b) is associated with the activation of the mitochondrial pathway of cell apoptosis through the enzymes caspase-3 and caspase-9. The term “mitochondrial pathway” refers to the initiation of the apoptosis pathway in a cell as a result of a number of internal stimuli, for example, genetic damage, oxidative stress, and hypoxia. Regulation of this pathway is carried out by a group of proteins belonging to the Bcl-2 family. Bcl-2, Bcl-W, Bcl-XL, MCL-1, and Bfl-1 proteins suppress apoptosis by blocking mitochondrial release of cytochrome-C. P53-dependent pro-apoptotic proteins Bik, Bcl-Xs, Bad, Bax, Bak, Bid, Bim, and Hrk stimulate apoptosis, increasing the permeability of mitochondria and the exit from them into the cytoplasm of cytochrome-C. The ratio of pro- and anti-apoptotic proteins determines the fate of the cell. The release of cytochrome-C into the cytoplasm leads to the activation of caspase-3 through the formation of an apoptosomal complex consisting of cytochrome-c, Apaf-1 (apoptotic protease activating factor 1) and caspase-9. A number of proteins released from mitochondria into the cytoplasm can modulate apoptosis: AIF (apoptosis-inducing factor), Smac (second mitochondria-derived activator of caspase), DIABLO (direct IAP binding protein with Lowp I) and others. They bind apoptosis suppressors, proteins of the IAP family (inhibitor of apoptosis protein), which in turn are capable of inhibiting caspases-3, -7, and -9 . O. acanthium extracts find applications in dermatology beyond skin cancer, such as in the treatment of furunculosis, purulent wounds, and lupus . 1.18. Orchis maculata L. Orchid Family—Orchidaceae Spotted orchis is indigenous to countries with a cold, temperate subtropical climate, particularly in Central and Southern Europe and Asia . Its distribution within Kazakhstan is primarily concentrated in the East Kazakhstan region . Spotted orchis comprises a mucilaginous substance that contains polysaccharide, which decomposes to mannose, in addition to dextrin, starch, proteins, bitterness, pentoses, methylpentosans, sucrose, loroglossin glycoside, and essential oil . Furthermore, the plant includes alkaloids, saponins, tannins, phenolic compounds (such as gallic acid, catechin, chlorogenic acid ( d), and syringic acid), terpenes, sterols, flavonoids, and anthocyanins . O. mascula flowers’ ethanol extracts also encompass saponins, flavonoids, anthraquinone, terpenoids, tannins, cyanogenic glycosides, and cardiac glycosides . These extracts exhibited a noteworthy antimicrobial effect against Salmonella paratyphi , Klebsiella oxytoca , and Staphylococcus aureus . The spotted orchis extract has been shown to possess anti-inflammatory, antispasmodic, diuretic, enveloping, and immunomodulatory effects, as outlined in . The enveloping effect can be attributed to the presence of loroglossin , a glycoside that protects inflamed tissues from excessive irritation . O. maculata contains anthocyanins and phenolic acids, which are potent antioxidants and have a nourishing impact. These compounds have the ability to inhibit collagenase, an enzyme that degrades collagen in the skin and hair. Catechin , for instance, influences collagen and makes it collagenase -resistant. Catechin also forms a complex with collagen, modifying its structure and making it resistant to enzyme degradation. Flavonoids, in general, contribute to scalp elasticity and nutrition, strengthen blood vessel walls, and enhance blood flow. Furthermore, polyphenols manifest antimicrobial properties, which makes them a valuable ingredient in medicines used to treat mycoses : In dermatology, the oral use of Orchis maculata L. extract is prevalent in folk medicine for senile itching, skin tuberculosis, and other dermatoses accompanied by cachexia and chronic diseases of the respiratory and gastrointestinal tracts. The extract is also employed for the speedy healing of wounds and ulcers . Additionally, cosmetic skincare products containing the extract and produced on an industrial scale are available . 1.19. Pastinaca sativa L. Seler Family—Apiaceae Parsnip ( Pastinaca sativa L.) is a plant species that is indigenous to Europe and Asia , and is also found growing in South Kazakhstan . The root of parsnip is a rich source of numerous bioactive compounds, including coumarins, furanocoumarins, polyacetylenes, essential oils, terpenes, and flavonoids . Additionally, parsnip root is rich in various minerals such as potassium, manganese, magnesium, phosphorus, zinc, and iron, as well as carotene, starch, pectin, vitamins, and sugars . Parsnip has been employed in traditional medicine since antiquity. According to Avicenna’s Canon it alleviates headache, stomatitis, ophthalmitis, dermatitis, and fever if it is used orally. Numerous studies have demonstrated the pharmacological effects of P. sativa on various bodily systems, including the central nervous, respiratory, gastrointestinal, hepatic, skin, cardiovascular, and genitourinary systems , as well as its potential in mitigating stroke, atherosclerosis, and other coronary heart diseases. Additionally, P. sativa has been shown to have positive effects on cholecystitis, constipation, anorexia, stomach pain, bladder atony, spastic enterocolitis, mild insomnia, nephritis, dysuria, renal colic, endocrine disorders such as menstrual syndrome, rheumatism, vitamin deficiency, obesity, vascular diseases, infections, loss of appetite, dysmenorrhea, fever, atherosclerosis, detoxification, anemia, and diabetes . Plants containing furanocoumarins have been used to treat leprosy and vitiligo . Furthermore, furanocoumarins extracted from parsnips have the ability to dilate peripheral vessels and coronary vessels of the heart, eliminate spasms of the bronchi and smooth muscles of the abdominal cavity, and have a moderate sedative effect. In addition, P. sativa exhibits antioxidant and anticytolytic activities . The dried seeds of P. sativa underwent steam distillation to isolate its essential oil, which was found to contain octyl acetate (78.49%) and octyl hexanoate (6.68%) as its major constituents. Remarkably, this essential oil exhibited significant antioxidant and antimicrobial activity . A large amount of vitamins A and C predominate in Pastinaca sativa . These vitamins eliminate and neutralize the free radicals responsible for body diseases (chronic diseases) and premature aging . Recent studies in dermatology have shown the effects of furanocoumarins on the skin. Heraclenol ( a) and oxypeucedanine hydrate ( b) were found to have a weak stimulatory effect on melanogenesis without affecting cell proliferation. Moreover, furanocoumarins have been employed in the treatment of vitiligo and psoriasis . The furanocoumarins xanthotoxin and bergapten are important components in leukoderma treatment . 1.20. Plantago major L. Family—Plantaginaceae Plantago major L. (plantain) is a well-known and widely used medicinal plant. The genus Plantago L. comprises approximately 300 diverse species that flourish in temperate areas all over the world, including 16 plant species that occur in Kazakhstan . In arid zones, P. major is comparatively scarce and is primarily found along riverbanks and in intensely irrigated crops. Plantain is a botanical specimen that contains diverse chemical constituents, including carbohydrates, lipids, allantoin, essential and non-essential amino acids, caffeic acid derivatives, flavonoids including baicalein, scutellarein, luteolin, baicalin, apigenin, among others; phenolcarboxylic acids and their derivatives; iridoid glycosides such as aucubin, catalpol, and aukubozid; terpenoids; and alicyclic compounds such as loliolid. Furthermore, the leaves of plantain exhibit a significant concentration of phenols and their derivatives such as ferulic acid and tyrosol, tannins, and vitamin K. The seeds of plantain contain organic acids such as succinic acid, mucus, iridoids such as aucubin, sterols such as β-sitosterol, stigmasterol, campesterol, saponins, alkaloids, tannins, flavonoids such as isoquercitrin, and fatty oil. These findings have been reported in numerous sources . For centuries plantain had been considered to possess therapeutic properties. Various parts of the plant, including mature seeds, leaves, and juice, were used for medicinal purposes. Plantain leaves were employed in the treatment of numerous diseases, including digestive, reproductive, and circulatory ailments, as well as inflammatory skin disorders and urogenital and infectious diseases . Moreover, plantain was used for pain relief and to reduce fever . The mucus, enzymes, and phytoncides present in psyllium provide an enveloping and mucolytic effect that restores the protective function of the ciliated epithelium in the respiratory tract, leading to increased secretion of bronchial mucus and liquefaction of sputum for easy expectoration. It is noted in that plantain glycoside inhibits the cough reflex, and the hemostatic properties of plantain are due to the high content of vitamin K in it, which, along with tannins, promotes blood clotting. Psyllium is also a great antioxidant and radical scavenger with immunomodulatory effects . P. major is used in various types of wound and skin diseases: deep wound, purulent wound, chronic and progressive wound, malignant wound, fire burn, erysipelas, progressive blister, pruritus, irritating urticarial, and fistula. The treatment is carried out by sprinkling the plant powder on the wound or by using a bandage covered by Plantago major together with salt or without it. It is also used for head and face skin ulcers in the same manner . This plant is noted as an effective medicinal plant in the treatment of acute urticaria . Ursolic acid, oleanolic acid, and α-linolenic acid are three P. major components that have shown inhibitory effects on cyclooxygenase (COX-2)-catalyzed prostaglandin production. Luteolin (one of the flavonoids) also has the ability to suppress leukocyte migration and inhibit mast cell degranulation, which all together can be considered as anti-urticaria treatment strategies Polysaccharides stimulate the formation of interferon, while zinc and flavonoids aid in the normalization of phagocytosis. The combination of polysaccharides with enzymes and vitamins promotes regeneration. Plantain is also used in cosmetic dermatology to treat acne scars . 1.21. Ribes nigrum L. Saxifrage Family—Saxifragaceae Ribes nigrum L. is a diminutive perennial shrub indigenous to Central Europe and North Asia that has been widely cultivated globally, including in the United States . Furthermore, it is known to thrive in the territory of Kazakhstan . Fresh blackcurrant fruits are known to contain a diverse range of functional and biologically active compounds, including soluble sugars, flavonoids, organic acids, vitamins, polyamino acids, macro- and microelements, and unsaturated fatty acids . Additionally, blackcurrants are a rich source of vitamin C . Anthocyanins, a group of biologically active compounds, are prominently found in blackcurrant berries, as well as in its seeds and leaves . Notably, blackcurrant seed oil is a valuable source of gamma-linolenic acid (γ-C18:3), stearidonic acid (C18:4), tocochromanols (primarily γ-tocopherol and α-tocopherol), and sitosterol . The fruits, leaves, and shoots of Ribes nigrum , both in fresh and dried form, have been traditionally used as a multivitamin and general tonic for hypovitaminosis and beriberi, as well as for enhancing the immune system. In folk medicine, the leaves of Ribes nigrum have been used for treating various conditions, including kidney stones, gout, cystitis, urethritis, osteochondrosis, rheumatism, muscle and joint pain, exudative diathesis, eczema, and furunculosis . Additionally, Ribes nigrum is used in homeopathy . In a study , a wide range of pharmacological actions of Ribes nigrum extract, rich in anthocyanins, were indicated. Extracts containing more than 20% anthocyanins were found to exhibit antioxidant, anti-inflammatory, and phytoestrogenic activity, anti-postprandial hyperglycemic and antidiabetic effects, and cardioprotective effects. Furthermore, the anthocyanin-rich fraction of black currant peel extract has been found to exhibit a strong cytotoxic effect on human liver cancer cells, and to have a positive effect on vision and eye health. In the field of dermatology, blackcurrant leaves have been utilized for treating skin lesions resulting from atopic dermatitis and allergic itchy dermatoses (e.g., eczema, neurodermatitis, pruritus), while leaves and fruits have been used for curing psoriasis, scleroderma, lichen planus, vasculitis, and acne vulgaris . Ribes nigrum may be helpful in treating various skin diseases, such as atopic dermatitis, psoriasis, and acne, owing to its higher anthocyanin content . The antioxidant activity of blackcurrant, attributed to the presence of flavonoids and vitamin C, has been observed to modulate cancer and inflammation signaling pathways and absorb ultraviolet radiation . Vitamin C has been shown to increase the amount of the transport protein when exposed to ultraviolet light. Furthermore, the presence of fatty acids in blackcurrant makes it therapeutically efficient for treating skin diseases . The authors of studied the effect of a polysaccharide (CAPS) isolated from blackcurrant ( Ribes nigrum ) on immunomodulation in laboratory mice. The introduction of CAPS was found out to improve the symptoms of atopic dermatitis by inhibiting the migration of mast cells into the skin of the epidermis. CAPS administration was also found to suppress immunoglobulin (IgE) overproduction and induce transcription of the IFN-γ gene in the spleen. 1.22. Rosa canina L. Rose Family—Rosaceae Rose hips have considerable economic importance and are widespread garden plants in Europe, Asia, North America, and the Middle East. The distribution of wild roses in different regions of Kazakhstan is heterogenous. In particular, a greater range of species diversity has been observed in forest and forest-steppe zones . There is a total of 21 distinct species of wild rose that grow in Kazakhstan, with five of them being present in Central Kazakhstan, including R. glabrifolia , R. laxa Retz., R. Acicularis Lindl., R. majalis Herrm. ( R. cinnamomea L.), and R. pimpinellifolia L. ( R. spinosissima L.) . The fruits of R. canina are highly valued by the food and pharmaceutical industries due to their rich content of biologically and physiologically active compounds. These include a wide range of vitamins (C, B, P, PP, E, K), flavonoids, carotenes, carbohydrates (mono- and oligosaccharides), organic acids (tartaric, citric), polyunsaturated fatty acids, trace elements, and others . The essential oil derived from rosehips is primarily composed of alcohols, monoterpenes, and sesquiterpenes . Dog rose seeds are also a valuable source of crude oil, comprising approximately 15% of their total weight. To extract oil from the seeds, various methods are employed such as pressing, solvent extraction, ultrasonic, microwave, and sub- and supercritical fluid extraction. Rosehip oil is considered particularly valuable due to its essential fatty acid content, tocopherols, phytosterols (β-sitosterol), and phenols, which contribute to its functional properties. The primary essential fatty acids present in rosehip oil are linoleic, linolenic, and oleic acids, while the γ-tocopherol isomer of tocols is the most abundant in the oil. Among the numerous health benefits of rosehip oil, its anticancer effects are particularly noteworthy. Additionally, the therapeutic effect of rosehip oil on skin diseases makes it a preferred ingredient in cosmetics . Rose hips have a well-established history in traditional medicine as a preventative and treatment remedy for colds and other infections, as well as a diuretic and therapy for various inflammatory disorders. In modern medical practice, dog rose ( Rosa canina L.) is incorporated into compositions and complexes for the treatment of inflammatory diseases, including but not limited to rheumatoid arthritis, reactive arthritis, osteoarthritis, and other types of arthritis. It is also helpful in upper respiratory tract infections and psoriasis. Its other application includes the prevention of oxidative stress in the oral cavity. The unique phytochemical composition of rose hips is of huge interest because it can be considered a promising source for making functional foods, natural medicines, and cosmo-nutraceuticals. Presently, rose hips are employed as a constituent in probiotic products . The rose hip extract’s antioxidant activity is predominantly attributable to its ascorbic acid and polyphenolic compounds. Moreover, the extract manifests antimutagenic and anticancer properties . R. canina finds a common application in cosmetology, where it is frequently utilized in conjunction with other biologically active compounds or herbal extracts. However, there are cases when it is employed as an individual ingredient; for instance, French-patented industrial technology uses dog rose extract as an active agent for curing seborrhea together with a cosmetic skincare strategy aimed at eliminating excess sebum production and dermatological manifestations caused by it . 1.23. Solanum dulcamara L. Solanaceae Family—Solanaceae Solanum dulcamara L. exhibits a wide distribution across all continents except Antarctica, with the highest concentration found in tropical and subtropical regions of Australia, Africa, and select areas of Asia, including China, India, and Japan, as well as Central and South America . Notably, the plant is found ubiquitously throughout Kazakhstan. S. dulcamara is known to contain various bioactive phytocomponents, including steroidal saponins, terpenes, flavonoids, carbohydrates (such as glucose, galactose, xylose, and rhamnose), lipids (specifically cholesterol), steroidal sapogenins (such as diosgenin, tigogenin, and yamogenin), and pigments (such as lycopene and lycoxanthin) . Notably, steroid alkaloids and glycoalkaloids are the primary chemical markers for this plant genus. Additionally, S. dulcamara has been found to contain steroidal alkaloids, including solanine in immature fruits, solasodine in flowers, and β-solamarin in roots . S. dulcamara stems have traditionally been employed in folk medicine as a narcotic agent and as a remedy for conditions such as rheumatism, migraine, and severe inflammation . An ethyl acetate extract obtained from the ripe fruits of S. dulcamara demonstrates significant anti-inflammatory and antioxidant activity . Moreover, S. dulcamara is reputed to possess a variety of therapeutic properties, including antimicrobial, analgesic, hepatoprotective, immunomodulatory, antitumor, and neurogenetic effects , as well as antioxidant , antihyperglycemic , antibacterial, and antimicrobial activity , and antirheumatic activity . The aerial part of S. dulcamara is particularly rich in alkaloids, which contribute to its antibacterial activity against Streptococcus pyogenes , Staphylococcus epidermidis , and S. aureus . S. dulcamara is a known remedy for the treatment of skin diseases and warts . This plant is particularly rich in the alkaloid solanine, which is abundant in its immature fruits and has been traditionally used in Kenya to treat skin mycotic infections and other pathological diseases . Saponins isolated from S. dulcamara possess remarkable antioxidant activity, as they are capable of absorbing free radicals. Due to their beneficial properties, saponins are often utilized in cosmetology, where they improve the rheological and foaming properties of body-washing products and reduce the risk of skin irritation . The antioxidant properties of S. dulcamara are attributed to the presence of various phenolic compounds, flavonoids, anthocyanins, and carotenoids such as lycophyll , as well as hydroxy and methoxy derivatives of coumarins . Through non-targeted LC/MS analysis, 83 metabolites have been identified in S. dulcamara fruit extracts, including 22 polyphenolic compounds comprising of 19 phenolic acid derivatives and 3 flavonoids (namely quercetin-3- O -rutinoside and kaempferol-3- O -rutinoside), 10 amides, 16 saponins, 14 steroid alkaloids, 6 lignans, and 15 other compounds . Notably, the phenolic acids in these extracts are mainly composed of chlorogenic acid ( d), caffeic acid, and p-coumaric acid. According to the investigations of the metabolites present in S. dulcamara , unripe fruits contained a higher concentration of γ-solamarin, α-solazonin, α-solanine, abutiloside H, and solanandaine compared to ripe fruits. Moreover, methanol fruit extracts were found to exhibit significant potential in eliminating DPPH and hydroxyl radicals. Interestingly, the ability of methanol extracts to remove DPPH was found to be tissue-specific, with the outer tissue (skin) of the bittersweet fruits showing a higher antioxidant activity than the inner tissues (pulp and seeds), possibly due to the higher phenol content in the peel . 1.24. Sorbus aucuparia L. Rose Family—Rosaceae Sorbus aucuparia L., a botanical species known for its nutritional and medicinal beneficial properties, is considered a valuable source of edible fruits. This plant is characterized by its ability to survive in cold and harsh environments, and is widely distributed in various regions of Northern Europe, the Caucasus, the Middle East, and East Asia . Sorbi fructus, commonly known as Rowan fruits, serve as essential medicinal resources. The berries are harvested during their complete maturation phase, from August to September, before the advent of frost. During collection, it is advisable to exercise caution and avoid damaging the branches. The stalks of harvested raw materials are cut, and then these materials are subjected to a drying process in well-ventilated rooms or dryers, employing a temperature range of 60–80 °C . This fruit, popularly referred to as a “superfruit,” contains a diverse array of phytochemicals, comprising phenolic acids: neochlorogenic and chlorogenic acids (see above), flavonoids, proanthocyanidins, iridoids, coumarins, hydrolysable tannins, carotenoids, and anthocyanins, as well as vitamins (ascorbic acid, α-tocopherol, B1, B2, P, PP, K, and folic acid) . Furthermore, it is rich in various sugars, phospholipids, pectin, organic acids, bitter substances, sorbic and parasorbic acids, essential oil, and macro- and microelements. The leaves of the plant contain vitamin C and flavonoids, while rowan seeds contain fatty oil (up to 22%) and glycoside amygdalin; the bark contains tannins . Throughout history, the fruits of S. aucuparia have been utilized in traditional medicine to alleviate ailments related to cardiovascular and digestive systems. In addition to their medicinal applications, these fruits can be eaten raw or utilized in the production of jams, syrups, and as flavoring agents in alcoholic and non-alcoholic beverages, including beer and wine . The fruit extracts derived from S. aucuparia have demonstrated antioxidant and antitumor activity . The antioxidant activity is attributed to the presence of flavonoids, vitamins C and E , and anthocyanins within their composition. Moreover, the authors of reported additional pharmacological effects of the fruit extracts, including antitumor, antiproliferative, antiviral, antibacterial, antifungal, and anti-inflammatory effects. Within the field of dermatology, Rowan berries are a valuable multivitamin raw material for the treatment of allergic diseases and other skin problems due to their wound-healing properties . Sorbic and parasorbic acids, present in the fruits of mountain ash, have an antimicrobial and antifungal effect. Based on the fruits of mountain ash, an ointment is prepared that has anti-inflammatory and wound-healing properties . 1.25. Symphytum officinale L. Borage Family—Boraginaceae Symphytum officinale L., commonly known as comfrey, is distributed across the humid meadow and lakeside regions of Asia, Europe, and America, as supported by reference . Its occurrence has also been recorded in the northwestern and eastern regions of Kazakhstan, as documented in reference . Comfrey contains various chemical compounds including phenolic compounds, flavonoids, fatty acids, polysaccharides, purine derivatives, and triterpenes . In terms of ethnopharmacology, preparations made from the roots, leaves, or entire aerial parts of comfrey have been traditionally used since ancient times to treat various internal ailments such as respiratory, gastrointestinal, and genitourinary disorders, as well as external conditions such as bruises and tumors, through the administration of tinctures, infusions, decoctions, compresses, and ointments . The literature indicates the potential therapeutic effects of Symphytum officinale . The plant has been reported to possess anti-inflammatory, anti-apoptotic, antitumor, neuroprotective, and antioxidant properties . Furthermore, comfrey has been shown to facilitate bone regeneration . Allantoin and rosmarinic acid ( e), identified as active compounds in comfrey, exhibit significant skin healing properties and have been applied in the treatment of a range of skin conditions: Allantoin has been reported to stimulate cell proliferation and tissue repair, making it a promising therapeutic agent for wound healing. Several studies have demonstrated that allantoin can accelerate the healing process of wounds, reduce inflammation, and increase skin moisture, thus exhibiting a rejuvenating effect . On the other hand, rosmarinic acid ( e) (see above) possesses antimicrobial, anti-inflammatory, and antioxidant properties, which make it an effective treatment agent for various skin diseases, such as psoriasis, acne, and eczema. Studies have shown that rosmarinic acid can reduce oxidative stress and inhibit the production of inflammatory cytokines in the skin, leading to improved skin health . Comfrey also contains caffeic acid and chlorogenic acids ( d) (see above). In vitro analyses demonstrated that caffeic acid and chlorogenic acid accelerated the proliferative response of fibroblasts, thus enhancing wound healing: Polyphenolic compounds present in hydroalcoholic extracts were shown to possess antioxidant and free radical scavenging properties, preventing the release of reactive species responsible for the oxidative stress and tissue damage in burns . Comfrey also contains tannins and pyrrolizidine alkaloids, which contribute to its anti-inflammatory and wound-healing effects . 1.26. Tanacetum vulgare L. Aster Family—Asteraceae Tanacetum vulgare L. is a well-known medicinal plant that is distributed across Northern Europe, North America, Russia, China, North Korea, Kazakhstan, and Japan . T. vulgare is rich in phenolic acids, flavonoids, and their derivatives . The plant contains surface flavonoids, such as the methyl esters of flavones scutellarin and 6-hydroxyluteolin, as well as vacuolar flavonoids, including apigenin and luteolin 7-glucorinides. Additionally, it contains caffeic acid, glycosides, sterols such as β-sitosterol, stigmasterol, cholesterol, and campesterol, and triterpenes such as α-amirin, β-amirin, and taraxasterol . In the traditional medicine of southeastern Serbia, T. vulgare flowers are commonly used to prepare tea with various therapeutic effects such as antihelminthic, carminative, antispasmodic, abdominal organ stimulant, tonic, menstruation stimulant, antidiabetic, diuretic, and antihypertensive properties . Apart from medicinal use, T. vulgare is also utilized in the production of balms, cosmetics, dyes, insecticides, drugs, and preservatives . Furthermore, T. vulgare -based preparations have been used for the treatment of several illnesses including hysteria, migraine, neuralgia, rheumatism, renal failure, and fever . The same source tells about the antibacterial, antiviral, antifungal, anti-inflammatory, and immunomodulatory activity exhibited by T. vulgare. The bioactive components of T. vulgare , including sesquiterpene lactones, volatile oils, flavonoids, and phenolic acids, have been found to possess antioxidant, anticancer, anti-inflammatory, and antiulcer properties . Taraxasterol ( a), luteolin ( b), and taraxic acid (a sesquiterpene lactone) present in T. vulgare are responsible for its anti-inflammatory and antiallergic effects, making it a potential remedy for treating skin diseases such as atopic dermatitis, eczema, and psoriasis . Inulin and chlorogenic acid ( d) (see above) have demonstrated antioxidant, prebiotic, and anti-inflammatory effects, which suggest their potential use as a therapeutic approach for managing skin disorders such as acne, rosacea, and photoaging . 1.27. Taraxacum officinale Web. Family—Compositae Taraxacum officinale Web. is a plant species commonly found in temperate climatic zones of Europe, Asia, and North America . It can also be found in Kazakhstan, where it grows in various habitats such as wetlands, meadows, and roadsides, and occasionally in the steppes . Dandelion is a plant with a rich chemical composition. Its constituents include β-carotene , chicory acid , inulin , sesquiterpene lactones, and triterpene compounds , as well as flavonoids and fatty acids . Dandelion also contains a variety of vitamins (A, C, D, E, and B), inositol, lecithin, and minerals, such as iron, magnesium, sodium, calcium, silicon, copper, phosphorus, zinc, and manganese . According to the traditional medicine specialists’ evidence it has the tonic and diuretic properties of Taraxacum officinale , as well as its anthelmintic, anti-inflammatory, and sedative effects. Dandelion has also been shown to cure metabolic disorders and leukoformula deviations, and has been used in the treatment of hepatitis, bronchitis, pneumonia, mastitis (as a local compress), and anemia. These therapeutic effects are attributed to the various phytochemical compounds present in dandelion, including sesquiterpene lactones, triterpene compounds, flavonoids, fatty acids, and vitamins and minerals such as vitamins A, C, D, E, and B, inositol, lecithin, and minerals such as iron, magnesium, sodium, calcium, silicon, copper, phosphorus, zinc, and manganese . Dandelion, being a versatile plant, has also found significant use in the field of dermatology owing to its potential in curing several skin diseases. Notably, Taraxacum officinale has been found to contain taraxasterol ( a), a compound that is helpful in curing melanoma . Caffeic acid is the predominant component of dandelion stem extract, while chlorogenic acid is predominant in dandelion leaf extract. Both extracts have the same reducing power and ability to absorb superoxide anion radical; however, the stem extract showed the strongest UVA and UVB absorption and the strongest tyrosinase inhibition. In addition, the results of molecular docking modeling have indicated that caffeic acid in the stem extract inhibits tyrosinase mainly through hydrogen bonding with its Gly165 and Pro160 residues. Thus, dandelion stem extract is a promising skin care product . Additionally, the aqueous extract of dandelion has been observed to manifest high activity in inhibiting tyrosinase . Dandelion extracts are commonly employed in the treatment of acne and warts . Furthermore, the ethyl acetate and n-butanol fractions of Taraxacum officinale Web. Have exhibited anti-inflammatory and antibacterial properties , the chloroform extract has been shown to possess anticancer properties , polyphenolic compounds in dandelion have been found to have antioxidant properties , while methanol and petroleum ether extracts have been found to have a choleretic effect . 1.28. Thymus serpyllum L. Lamiaceae Family—Lamiaceae Thymus serpyllum L., commonly known as creeping thyme, Bogorodskaya grass, and thyme, is widely distributed in countries bordering with the Mediterranean, parts of Central Europe, and Asia . The plant is found in the forest and forest-steppe zones of the European part of Russia, as well as in Western and Eastern Siberia, the Urals, Transbaikalia, and central regions of Kazakhstan, including the Ulytau mountains . The plant is a valuable source of essential oil and pharmacologically active polyphenolic compounds, as reported in the literature . Thymol is the major component of the essential oil, comprising up to 42% of the oil, alongside other constituents such as carvacrol, n-cymol, α-terpineol, and borneol. Additionally, tannins, bitterness, gum, triterpene compounds including ursolic and oleanolic acids, flavonoids, and a significant amount of mineral salts have been detected in the herb. The mature seeds of the plant have also been found to contain 33.6% fatty oil . Thyme also exhibits a high content of flavonoid phenolic and carotenoid antioxidants, such as zeaxanthin, lutein, apigenin ( b), naringenin, and luteolin ( b) (see above), as reported previously . Thymus serpyllum , a medicinal herb, is rich in oil and pharmacologically active polyphenolic compounds. It has been used in official and folk medicine to treat various ailments for many years. The herb, harvested during the flowering period, is used as a medicinal raw material after being threshed and dried in the shade or dryers at 35–40 °C. Thyme preparations have demonstrated expectorant, antimicrobial, and antifungal properties. Thyme is also used to treat a range of ailments, including sore throat, stomatitis, periodontal disease, asthma, headaches, laryngitis, and digestive system disorders . Thyme extract has been shown to possess antitumor and antioxidant activity. Additionally, thyme is utilized as an alexiteric, emmenagogue, analgesic, and sedative, and in the form of ointments and lotions for rheumatism and skin diseases . Due to its sedative and diuretic properties, medicines containing Thymus serpyllum can be employed for pruritic dermatoses . Bulgarian herbalists think that creeping thyme can be a constituent of medicines for treating eczema, neurodermatitis, and urticaria, and can be used as an external remedy to eliminate wrinkles . A study of Thymus serpyllum L. essential oil has shown that it has a strong fungistatic effect: there was almost 100% suppression of the growth of the tested fungi. Four strains of dermatomycete fungi were used in the study: Trichophyton mentagrophytes , Microsporum gypseum , Microsporum canis , and Trichophyton violaceum ; two strains of mold fungi: Scopulariopsis brevicaulis , Aspergillus niger and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and its own isolate from dog skin (IZ 1), which causes dandruff in dogs . 1.29. Vaccinium myrtillus L. Cowberry Family—Ericaceae Vaccinium myrtillus L. is a plant species that is predominantly found in forested areas in Northern Europe and North America , as well as in Europe, Asia, and North America . Its distribution in Kazakhstan is limited to the southwestern region of Altai, situated in Eastern Kazakhstan . The fruits of V. myrtillus are a rich source of bioactive compounds such as phenolic acids (chlorogenic acid being the most common), flavonoids (isoquercetin), and resveratrol in the leaf extract . In addition, they contain polyphenols, phenolic acids, and anthocyanins . Moreover, they are a rich source of trace elements and other phytochemicals such as organic acids, sugars, vitamins, fibers, and phenolic compounds (both anthocyanins and non-anthocyanins), glycosides (arbutin and myrtillin), peryl alcohol, resins, triterpene alcohol, pyrocatechin and pyrogallic tannins, free hydroquinone, ascorbic acid, carotene, and organic acids. They also contain retinol acetate, thiamine bromide, and pectin . According to traditional medicinal practices, V. myrtillus flowers are utilized as ointments to treat a lot of skin-related diseases, including but not limited to ulcers, eczema, burns, bruises, rashes, varicose veins, and acne . Moreover, this plant has demonstrated blood-glucose-lowering effects and has been shown to possess antioxidant, anti-inflammatory, and lipid-lowering properties, indicating its potential efficiency in treating chronic inflammatory diseases, including those linked to aging such as cancer and cardiovascular disease . Blueberries are regarded as a valuable source of antioxidants, which explains their utilization in treating numerous ailments (e.g., inflammation, cardiovascular disease, cancer, diabetes, and aging-related diseases) linked to an increased oxidative stress . Blueberry leaves have been found to possess hypoglycemic effects, attributed to the presence of myrtillin glycoside, which acts similarly to insulin and regulates pancreatic function. Dried blueberries are known for their astringent properties, while fresh blueberries are known to have carminative, anti-inflammatory, diuretic, hemostatic, antibiotic, and vitamin properties, and can regulate metabolism and digestive activity. In traditional medicine, blueberries have been used to treat various ailments such as bile duct and bladder stones, coughs, scurvy, and pulmonary tuberculosis. They have also been used to treat gastroenterocolitis and diarrhea, particularly in children. Due to their high content of vitamin C, blueberries have been used for scurvy treatment and external application to cure stomatitis and pharyngitis, which are accompanied by oral cavity wounds and ulcers . The high antioxidant potential of blueberry seed oil, which contains chlorogenic acid ( d), isoquercetin ( b), and resveratrol, as well as α-linolenic, linoleic, and oleic acids, has been well identified. Furthermore, a plant extract of isoquercetin ( b) has been found to have a dose-dependent inhibitory effect on edema caused by allergic contact dermatitis . The component composition of V. myrtillus species is represented by various groups of phenolic compounds, which are known to be effective exogenous factors of antioxidant protection . Currently, the great popularity of Scots blueberries is due to the high content of anthocyanins with antioxidant activity . In the case of skin diseases, long-lasting wounds, and ulcers, infusions made of fruits or leaves in the form of perfumes are applied externally. 1.30. Viscum album L. Family Beltflower—Lorantliaceae Viscum album L., commonly known as white mistletoe, is an evergreen hemiparasitic plant that grows widely in the Caucasus, Europe, and western and southern Asia . Various chemical components have been identified in mistletoe through chemical studies, including viscotoxins (a mixture of amino acids), phenylpropanes, lignans, flavonoids, amines (viscalbin, norviscalbin, tyramine, β-phenylethylamine viscamine), α-viscol (β-amirin), β-viscol (lupeol), polysaccharides, lectins, fatty acids (oleic, linoleic, and palmitic acids), alcohols (pinit, inositol, quebrachite), resinous substances, and mineral salts . Moreover, syringinin glycoside was detected in mistletoe bark . Triterpene saponins (oleanolic and ursolic acids), vitamin C, carotene, vecerin, viscol, and choline derivatives (propionylcholine and acetylcholine) have also been found in this plant, the levels of which depend on the host tree on which the mistletoe grows, according to the authors of . Viscum album L. has a rich ethnopharmacological history, with traditional uses including the treatment of various ailments such as epilepsy, anxiety, hypertension, internal bleeding, atherosclerosis, inflammation, and headaches. Additionally, it has been used as an antidote in some cultures . Mistletoe-based preparations possess hypotensive and analgesic effects. For instance, a tincture of fresh mistletoe leaves, found in the “Akofit” preparation, is utilized to treat acute radiculitis . The vasodilators “Omelen” and “Viskalen” are recommended for hypertension, while the liquid and dry extract “Reviscen” is useful for treating atherosclerosis, as it decreases blood pressure, dilates blood vessels, enhances cardiac activity, reduces nervous system excitability and intestinal atony, and acts as a hemostatic agent . The active compound viscotoxin effectively cures cancer and inhibits its progression. The lectin present in mistletoe is a natural pesticide that hinders bacterial and parasitic infiltration into the body . Additionally, Viscum album L. exhibits antioxidant , antitumor , antiviral, antibacterial, anti-inflammatory, antiepileptic, and immunostimulatory activity, and is also employed to treat neurological disorders . Preparations containing white mistletoe are utilized in obstetric and gynecological practice and are prescribed for colpitis and prolonged uterine bleeding . Studies have shown that methanol extract significantly reduces the pigmentation of primary human melanocytes. In addition, reporter promoter analysis showed that the methanol extract inhibits the transcription of microphthalmia-associated transcription factor, melanophilin, tyrosinase protein-2, and tyrosinase genes in melanoma cells . The presence of viscumneoside III and viscumneoside V in Viscum album L. extract was found to significantly inhibit the expression of monocyte chemoattractant protein 1 (MCP-1). These results imply that Viscum album L. extract, as well as its active components, viscumneoside III and viscumneoside V, can regulate the production of MCP-1 and may have the ability to reduce skin toxicity induced by erlotinib by altering the activity of macrophages, without interfering with the anticancer effect of the drug . Research of an alcoholic extract of mistletoe has indicated its efficiency in treating skin cancer. The extract is assumed to boost immune mechanisms, which in turn restrain the proliferation of cancerous cells. Additionally, when V. album extract was administered in combination with doxorubicin, it exhibited a synergistic effect, enhancing the antitumor impact on Ehrlich tumor cells . Mistletoe has been proved as a promising treating remedy in the field of dermatology, where it has been employed to manage various cutaneous conditions such as dermatitis, age-related pigmentation, moles, acne, and papillomas, as well as psoriasis and rashes . Achillea millefolium L., commonly known as common yarrow, belongs to the Asteraceae family (Asteraceae Dumort.). The plant was referred to as “venus eyelashes” during the Middle Ages due to its the feathery appearance of its leaves, while the whole plant was known as “soldier’s grass” for its use in treating wounds. There are over 100 different species of Achillea millefolium L., which are found in various regions worldwide, including North America, Europe, Asia, Australia, New Zealand, and the Middle East . The plant is widespread in Kazakhstan and serves as a valuable source of nectar for honeybees . The main components of A. millefolium are essential oils and phenolic compounds, monoterpenes, sesquiterpenes, lactones , amino acids, fatty acids, salicylic and succinic acids, ascorbic acid, folic acid, caffeic acid, and flavonoids . The composition of the essential oil includes sesquiterpenoids: achillin, acetylbalquinolide, caryophyllene, proazulene (chamazulene); and monoterpenoids: camphor, thujol, cineole, pinene, borneol. In addition, alkaloids (the main one of which is achilein), flavonoids, including flavone glycosides apigenin and luteolin were found in the yarrow herb; also found were tannins (α-phylloquinone) and vitamins K, A, and B; amines: choline and stakhidrin; and esters (bornyl acetate, myrtenyl acetate), caryophyllene, organic acids, polyins (pontic epoxide, matrixar ester), cyclic alcohol viburnite (20%), menthol, and geraniol . Yarrow also contains sterols, coumarins, the biogenic amine betaine, inulin, and other polysaccharides . The bitter taste of A. millefolium can be attributed to the presence of sesquiterpene lactones in its essential oil. The quantity of essential oil produced by the plant is largely dependent on the growth stage. During the early stage of growth, the content of essential oil is 0.13%, which rises to 0.34% in the process of flowering . In traditional medicine, yarrow has been employed to alleviate a variety of ailments including respiratory diseases (such as asthma and bronchitis), dyspepsia, skin inflammation, and headaches. The aerial part of the plant, including the leaves, stems, and inflorescences, is typically collected during the flowering phase for its usage as medicinal raw material. Yarrow is often administered as infusions, extracts, and potions to treat bleeding, flatulence, and gastrointestinal diseases . A. millefolium possesses various therapeutic properties such as disinfectant, anti-inflammatory, antispasmodic, anthelmintic, antibacterial, antioxidant, and antimicrobial effects . Additionally, the herb demonstrates antiulcer and anticancer activities , while the experimental findings suggest that yarrow may stimulate thrombocytopoiesis, leading to an increase in the number of platelets in the blood . Yarrow has long been utilized in traditional medicine as an efficient remedy for various skin ailments, including acne, eczema, neurodermatitis, and urticaria. Moreover, yarrow is incorporated into medicinal preparations for vasculitis. It is administered orally to prevent the recurrence of eczema . The wound-healing and anti-inflammatory effect of yarrow is due to the content of chamazulene (12.34%) in it. It is known that chamazulene enhances regenerative processes, weakens allergic reactions, has a local anesthetic effect, adsorbs various poisons, softens the skin, promotes scarring, heals infected wounds, and restores damaged capillaries : The results of the study explain the mechanism of action of A. millefolium . Thus, the effect of the water–alcohol extract of Achillea millefolium (HEAML) on the proliferation and stimulation of the human skin fibroblast growth (HSF-PI-16) was studied. The extract selectively inhibited the proliferation of HSF-PI-16 cells at higher concentrations (>20.0 mg/mL) and were stimulated at lower concentrations (<20.0 mg/mL). After treating the HSF-PI-16 medium for 72 h with the extract, a significantly increased proliferation rate and stimulation in the scratch analysis was noted . The activity of the plant in atopic dermatitis was also investigated. The results showed that Achillea millefolium L. significantly reduces the expression of proinflammatory cytokines in mouse macrophage cells treated with lipopolysaccharide . Acorus calamus L. (calamus) marsh is a perennial plant containing aromatic compounds and is widespread in Central Asia, India, and the Himalayas. Although its distribution has significantly diminished in Europe, it remains a common plant in the northern marshy regions with a temperate climate . It is found in Asia, Europe, and North America and is known to grow in Central Kazakhstan along the banks of rivers, swamps, and lakes, sometimes forming substantial thickets. Calamus marsh is rich in various chemical compounds, including bitter glycoside acorin, essential oil (which contains proazulene), gum, resins, ascorbic acid, tannins, starch, and mucus. The dried rhizome of Calamus marsh consists of yellow aromatic volatile oils comprising of small amounts of sesquiterpenes and their alcohols; and choline, flavone, acoradin, galangin, acolamon, and isocolamon. Furthermore, it contains cineol, limonene, terpineol, azulene, eugenol, camphene, cadinene, ethanol, galangin, magnesium, zinc, terpenes, menthol, and camphor . Calamus root is considered in traditional medicine to be a therapeutic agent for a range of ailments, such as arthritis, neuralgia, diarrhea, dyspepsia, and hair loss . The plant has been found to possess potent antioxidant, anti-inflammatory, antiulcer, antimicrobial, and wound-healing properties. It is employed in dermatology to cure pyoderma, acne vulgaris, alopecia, and eczema . The advantageous effect on the skin can be attributed to the presence of β-azarone , a phenylpropanoid class chemical compound: β-Azarone is known to contribute to the body’s natural defense against ultraviolet rays, but it has also been found to have carcinogenic properties and induce liver tumors. Calamus marsh, which includes varying amounts of β-azarone depending on the variety, has traditionally been used in Asian medicine for its anti-inflammatory properties, which can help alleviate skin itching, swelling, and redness. Meanwhile, European varieties of Calamus marsh are known to contain sesquiterpenoids, which possess psychoactive properties and display beneficial medicinal effects . Calamus rhizomes have been found to be very useful as a topical agent in skin-related problems. The rhizomes are used in the form of powder, balms, enemas, and pills and also in ghee preparations. A tub bath in the decoction of vacha, kustha (Savccera lappa) and vidanga ( Embelina ribes ) is useful in curing eczema and other skin diseases . Agropyron repens L. is distributed widely across Europe, Asia, and Africa . It can be found ubiquitously throughout Kazakhstan . The chemical composition of the plant is rich in a variety of carbohydrates such as fructose, glucose, inositol, and mannitol, as well as mucous substances, pectin, triticin, thianogenic glycosides, flavonoids, saponins, essential oil, monoterpenes (such as carvacrol, carvone, transanethol, thymol, menthol), and sesquiterpenes. Moreover, the plant contains vanillin glucoside, iron, minerals, and significant quantities of silica. Among the phenolic compounds found in the plant are p-hydroxybenzoic, vanillic, and p-coumaric acids, as well as chlorogenic acid, p-hydroxycinnamic acids, and p-hydroxycinnamic acid esters. The rhizomes consist of polysaccharides, glycosides such as quercetin and luteolin, phenolic glucosides, fatty acids, and amino acids (including γ-aminobutyric acid, proline, valine, asparagine, histidine, arginine, and tryptophan) . Furthermore, the seeds of wheatgrass contain triticin, mucus, saponins, sugar alcohols (namely, mannitol, inositol, and 2–3% of the total composition), essential oil with polyacetylenes or carvone, a small amount of vanilloside (vanillin), phenol carboxylic acids, silicic acid, and silicates . Agropyron repens L. was used in folk medicine as a sedative diuretic to relieve pain and spasms in the urinary tract, and as a sedative and tonic. The traditional medicinal use of Agropyron repens L. in urolithiasis has been scientifically proved, with confirmed pharmacological effects including hypoglycemic, hypolipidemic, anti-inflammatory, and antidiabetic effects, as well as effects on motility and benefits in urinary tract infections . The presence of flavonoids, alkaloids, and coumarin in the composition of this plant evidences its potential activity in the treatment of skin diseases, such as inflammatory skin diseases, atopic dermatitis, and acne . Thus, in the paper , the effect of wheatgrass extract in a cream form on some indicators of lipid peroxidation in allergic contact dermatitis was investigated. Contact dermatitis was modeled by the double application of 0.1 mL of a 5% alcohol solution of 2.4-dinitrochlorobenzene (DNCB) on previously depilated skin areas of the lateral surface of the abdomen of experimental animals. The anti-inflammatory activity of the cream was evaluated based on the characteristics of the skin and the state of lipid peroxidation (LPO) processes, i.e., the content of oxidation products in the blood plasma—malondialdehyde (MDA), diene conjugates (DC) and the activity of the antioxidant defense enzyme catalase. According to the studies, the cream containing wheatgrass extract has anti-inflammatory activity and promotes the activation of antioxidant protection (increased catalase activity), which, in its turn, decreases the intensity of lipid peroxidation (MDA and DC levels fall). The cream accelerated recovery for 4–5 days compared to untreated rats. The anti-inflammatory effect of wheatgrass cream was comparable to that of a standard cream with glucocorticoids (Akriderm C). Artemisia absinthium L., a plant species commonly known as wormwood, is widely distributed in Asia, the Middle East, Europe, and North Africa. It grows everywhere in Kazakhstan . A. absinthium is a plant species that possesses various biologically active compounds. The grass of this plant is utilized as a source material for oil production. The oil mainly consists of thujone esters, α- and β-thujone, camphene, α-cadinene, guaiazulene, (Z)-epoxycymene, (E)-sabinyl acetate, (Z)-chrysanthenyl acetate, as well as bitter sesquiterpenoid lactones, azulene group compounds, and tannins . Moreover, it contains terpenoids (such as myrcene, germacrene D, camphor, chamazulene), flavonoids (quercetin, kaempferol, apigenin, artemetin, and rutoside), phenolic acids (chlorogenic, ferulic, gallic, coffee, syringic, vanillic, and caffeoylquinic acid derivatives), and flavonoid glycosides . The composition of the A. absinthium extract is dependent on the type of solvent utilized in the extraction process. The alcoholic extract, in particular, has a considerably higher concentration of flavonoids, phenols, and tannins in comparison to the aqueous and chloroform extracts . For many years, A. absinthium has been used in traditional medicine to cure a wide range of ailments, particularly parasitic diseases and digestive disorders, as well as fever reduction . The leaves are employed to alleviate fever, while the flowers are used to treat stomach disorders and helminthiasis. The A. absinthium tincture is highly esteemed as a tonic and digestive aid. In a published paper , the wormwood herb was noted for its efficiency in treating jaundice, constipation, obesity, splenomegaly, anemia, insomnia, bladder diseases, and non-healing wounds from traumas. Furthermore, the plant is utilized as a base for producing skin ointments and balms . A. absinthium demonstrates various biological activities, including but not limited to antibacterial, anti-inflammatory, hepatoprotective, antidepressant, antispasmodic, and antipyretic effects . Moreover, it exhibits antimicrobial, antiviral, antistress, hepatoprotective, antioxidant, and anticancer effects . In the field of dermatology, the essential oil derived from A. absinthium has been shown to expedite wound healing, diminish inflammation, and exhibit antimicrobial and wound-healing properties. This effect is due to the significant content of oxygenated components in the oil, such as camphor ( a) and tirpinene-4-ol ( b), the content of which was, respectively, 47.59% and 6.36% : Camphor induces the proliferation of primary dermal fibroblasts, maintaining or restoring collagen and elastin production in UV-exposed skin. In addition, it prevents thickening of the epidermis and subcutaneous fat layer Camphor attenuates the aging enhancement associated with β-galactosidase (SA-β-gal) activity. In addition, the oil contains chamazulene (Figure 1) (10.35%), which contributes to the manifestation of antioxidant/anti-radical activity . Bidens tripartita L. is widely distributed in the European part of the CIS, Transcaucasia, Siberia, Central Asia (excluding Turkmenistan), and the southern region of the Far East. Its range also extends to North Africa and North America . In Kazakhstan, this species is ubiquitous across its regions. B. tripartita is a plant that is rich in various biologically active compounds, including essential oil, chlorophylls, flavonoids, cinnamic acid derivatives, tannins with a high polyphenol fraction content, polysaccharides, carotenoids, ascorbic acid, coumarins, chalcones, and minerals such as Zn, Sr, Se, and Mn. Flavonoids found in the plant include luteolin, butein, sulphuretin, sulphurein, cynaroside, auron, -catechin, (−)-epicatechin, rutin, myricetin, 7-hydroxyflavone, esculetin, and umbelliferone, among others . In traditional medicine, the water infusion and decoction of B. tripartita have been utilized for a considerable time period in combination with baths for the treatment of scrofula, rickets, exudative diathesis, and various pustular skin diseases such as acne and boils, as well as for the management of gout, arthritis, and articular rheumatism. They are also recommended for improving appetite and digestion, and for the treatment of liver and spleen disorders, colds, bronchitis, and diabetes mellitus . Preparations derived from B. tripartita exhibit a range of therapeutic effects, including anti-inflammatory, hemostatic, antiseptic, sedative, and wound-healing properties, lower blood pressure, and increase the amplitude of heart contractions . The antiallergic, anti-inflammatory, diuretic, and antispasmodic effects of the alcohol extract of B. tripartita have also been proved . The methanolic extract of B. tripartita manifests antioxidant activity against cancer cells and has the ability to inhibit key enzymes, such as α-amylase and α-glucosidase. In addition, according to evidence, the herb has antidiabetic activity, as well as antihyperglycemic and antioxidant effects . The broad pharmacological effects of the plant are attributed to its abundant content of various biologically active substances. Manganese ions in the plant’s enzyme systems are believed to influence hematopoiesis, blood coagulation, endocrine gland activity, liver cell function, and blood vessel and bile duct tone, and may prevent intravascular thrombus formation and enhance antimicrobial properties of the plants . Flavonoids in the plant are responsible for its antiallergic and diuretic effects by affecting metabolic processes. The presence of vitamin C can activate the function of the endocrine glands, improve metabolism, strengthen the immune system, and help treat viral infections. The essential oils present in the plant are effective in destroying pathogenic microflora and fungi . The extract of B. tripartita is used in the treatment of many skin diseases: psoriasis, seborrhea, urticaria, diathesis, acne, pimples, wounds, and ulcers, as well as small cracks. The beneficial effects on the skin can be attributed to the presence of tannins. Tannins also help to get rid of increased sweating of the armpits and legs. Thus, B. tripartita is employed for making baths, lotions, and wipes to treat microbial eczema of the feet, epidermophytosis . The mask derived from the sequence has been shown to eliminate oily sheen, tone the skin, and have a rejuvenating effect. Additionally, wiping the face with a decoction of the string has been demonstrated to reduce acne . During diathesis, an infusion of B. tripartita (from 10–30 g of herbs) is added to the bath . Khatamov et al. developed and studied a new drug: a thick extract of the sum of flavonoids in the form of ointment (1, 3 and 5%) obtained from B. tripartita , which was used to cure contact allergic dermatitis experimentally caused in guinea pigs by 2,4-dinitrochlorobenzene. The study results showed that 5% ointment had the greatest therapeutic effect compared to the antihistamines Psilo-Balsam and glucocorticoid ointment Celestoderm. At the same time, the rate of reduction in the severity of skin manifestations (Ind) was the highest compared to other studied groups—37.9%. Capsella bursa-pastoris L. is a wild plant with significant nutritional value that is suitable for human consumption. This plant is widely distributed across many countries, including Cyprus, Europe, Saudi Arabia, Turkey, Pakistan, India, Iraq, Iran, China, Azerbaijan, and other Asian countries . It is also commonly found in various regions of Kazakhstan. C. bursa-pastoris contains a variety of chemical components including flavonoids, polypeptides, choline, acetylcholine, histamine, tyramine, fatty acids, sterols, organic acids, amino acids, sulforaphane, vitamins , and various trace elements. In addition, it contains phenolic compounds, flavonoids, tannins, saponins, alkaloids, and phytosterols , as well as volatile fractions consisting mainly of terpenoids, alkane hydrocarbons (such as nonacosane), and fatty acids (including palmitic and linoleic acids) . In traditional medicine, C. bursa-pastoris has been used for centuries in China and Japan as a hemostatic, diuretic, and antipyretic agent . The plant has been utilized for the treatment of conditions such as edema caused by nephritis, odynuria, hemaffetia, menorrhagia, chyluria, and hypertension . The entire plant is used to make tea, which has been employed as an antiscorbutic, astringent, diuretic, emmenagogue, hemostatic, hypotensive, tonic, stimulant, vasoconstrictor, and wound-healing agent. This beverage has also been considered an excellent remedy for various types of bleeding, including those originating from the stomach, lungs, uterus, and kidneys. A homeopathic remedy for nosebleeds and urolithiasis is prepared from a fresh C. bursa-pastoris plant . Based on the literature, raw plant extracts and certain phytocomponents have been reported to exhibit various pharmacological effects, such as anti-inflammatory, antispasmodic, antimicrobial, hepatoprotective, cardiovascular, anticancer, sedative, and antioxidant effects . Furthermore, these extracts have been assumed to possess infertility-reducing properties . Extracts have also demonstrated inhibitory effects on acetylcholinesterase activity and significant antibacterial activity . C. bursa-pastoris exhibits potent antioxidant activity attributed to its flavonoid compounds, namely quercetin, chrysoeriol, kaempferol, and isorhamnetin. In vitro studies have shown that its extracts possess antioxidant activity that prevents the development of various free radicals such as DPPH radicals, peroxyl radicals, hydroxyl radicals, and hydrogen peroxide . Additionally, the plant extract has been found to have cytotoxic effects as reported by the previous studies . Furthermore, a moderate hepatoprotective activity has been observed with the extract containing specific flavonoids, including 4,7-dihydroxy-5-hydroxymethyl-6,8-diprenylflavonoid, chrysoeriol-7-O-d-glucopyranoside, sinensetin, and 6,8-diprenylgalangin . C. bursa-pastoris exhibits an excellent efficiency in the treatment of eczema in dermatology . Moreover, preparations derived from C. bursa-pastoris have been registered and recommended by the German Institute for Pharmaceuticals and Medicines for the additional treatment of skin diseases and wounds . Fumarates improve the course of psoriasis, in which both IL-12 and IL-23 promote the differentiation of pathogenic T-helpers (Th). Fumarate treatment induces IL-4-producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by an increase in heme oxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. The induced HO-1 breaks down, after which the N-terminal fragment of HO-1 is translocated into the nucleus and interacts with the AP-1 and NF-kB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19 . Chelidonium majus L., a plant species commonly known as greater celandine, is widely distributed across Asia, North America, and northwestern Africa . The plant C. majus is known to contain a high concentration of isoquinoline alkaloids, with levels ranging from 0.27 to 2.25% in the aerial parts and 3–4% in the root. Over 70 compounds have been identified, including various alkaloids (such as chelidonin, chelerythrin, sanguinarine, berberine, protopine, allocryptopine, and koptisin), flavonoids (such as rutin, quercetin, and kaempferol), saponins, vitamins (such as vitamin A and C), mineral elements, a small amount of phytosterols (such as α-spinasterol and ergosterol), and aromatic and aliphatic acids (including chelidonic, caffeic, ferulic, polycoumaric, citric, etc.) and their derivatives. Additionally, celandine consists of polysaccharides, alcohols (1-hexocosanol, chelidoniol, and nonacosanol), choline, tyramine, histamine, and saponosides. It should be noted that a previous study provided the formulas of all organic components . The content of most mineral elements in celandine ranged from 10 to 65%, where potassium (65%) and phosphorus (54%) predominated . C. majus has a long history of traditional use in Europe, Asia, and Africa to treat various ailments, including those affecting the liver and bile ducts, as well as to cure skin conditions such as warts, calluses, and eczema. Additionally, the plant has been used to treat stomach ulcers, tuberculosis, skin rashes, and oral infections. In traditional Chinese medicine and homeopathy, C. majus is used to alleviate congestion, pain, swelling, and jaundice . Celandine extracts have been found to possess a broad spectrum of pharmacological activities including anti-inflammatory, antimicrobial, anticancer, antioxidant, hepatoprotective, natriuretic, and antidiuretic effects, corroborating some of the traditional medicinal uses of C. majus . Additionally, the plant has demonstrated immunomodulatory, lipid-lowering, and radioprotective properties . Moreover, the ethanolic extract of C. majus has been found to contain biologically active secondary metabolites with significant inhibitory effects that prevent Alzheimer’s disease . The milky juice of the celandine is rich in alkaloids among which the predominant one is chelidonine ( a). Studies have demonstrated the antimicrobial, immunomodulatory, cytostatic, and cytotoxic effects of celandine alkaloids, including their anti-keratinocyte activity. Compounds such as chelidonine ( a), sanguinarine ( b), chelerythrine, coptisine, and protopin have been found to exhibit cytotoxic activity. Sanguinarine ( b) has been shown to be particularly effective at inhibiting keratinocyte growth, indicating that celandine may have potential as an additional therapy for malignant skin diseases . Cichorium intybus L., a perennial herbaceous plant belonging to the Asteraceae family, is known by various common names such as roadside grass, blue flower, roadside cornflower, bride of the sun, and sun grass. Its recognizable feature is the inflorescences-baskets that exclusively comprise reed blue flowers. However, the said baskets only open during early morning hours or in cloudy weather. The term “chicory” is derived from the Latin word, meaning “entering the fields.” Due to its therapeutic properties, this plant has earned the followings names: “king root,” “golden root,” and “cure for a hundred diseases” . C. intybus exhibits a wide geographical distribution encompassing Northern and Central Europe, Siberia, Turkey, Afghanistan, Northern and Central China, South America, South Africa, Ethiopia, Madagascar, India, Australia, and New Zealand. This herbaceous plant is capable of growing in the territories of the Commonwealth of Independent States, except the Far North region . The roots of C. intybus contain 56–65% inulin (in terms of dry matter), the maximum accumulation of which is observed in autumn. Intibin glycoside gives specific bitterness to chicory roots. Proteins, sugars, pectin, and sesquiterpene lactones were also found in the roots, as well as guayanolides: cycriosides B and C, sonchuside C, tannins and resinous substances, choline, carotene, vitamins B, B2, PP and C, from mineral elements—sodium, potassium, calcium, manganese, and phosphorus, iron. Chicory roots contain taraxasterol, phenolic acids (chlorogenic, isochlorogenic, neochlorogenic, caffeic and cicoric acids) . In the flowers of C. intybus , chicory glycoside was found, in the seeds: inulin and protocatechin aldehyde , prebiotic fructooligosaccharides, sesquiterpene lactones, caffeic acid derivatives (chicory acid, chlorogenic acid, isochlorogenic acid, dicapheoyltartaric acid), proteins, hydroxycoumarins, flavonoids, alkaloids, steroids, terpenoids, oils, volatile compounds, and vitamins . Aliphatic compounds and their derivatives make up the main fraction; terpenoids are somewhat less common in the plant. Chicory leaves contain inulin, vitamins A, B1, B2 and C, macro- and microelements (Ca, K, Mg, Na, Fe, Cu, Mn, Zn), phenolic compounds, etc. . The aerial and subterranean portions of C. intybus L. are extensively employed in traditional medicine, such as in Chinese and Mongolian practices, as an agent for modulating the immune system, promoting bile secretion, protecting the liver, and reducing blood glucose levels. The plant is documented in the Chinese Pharmacopoeia and is utilized in the formulation of homeopathic remedies in Germany. The extract of chicory herb is a constituent of the LIV-52 complex preparation from India . C. intybus exhibits antiseptic and astringent properties and produces choleretic and diuretic effects. It positively influences the nervous and cardiovascular systems. Additionally, its infusion has been employed for normalizing heart rhythm. According to the literature, preparations derived from C. intybus are efficient in treating various diseases connected with the gallbladder, liver, kidneys, and urinary system. Additionally, chicory preparations have been shown to exhibit potential therapeutic benefits in managing obesity, liver diseases, atherosclerosis, hypoacid gastritis, tachycardia, arrhythmia, and nephritis. The milky juice of the plant contains bitter substances that have been found to stimulate peristalsis of the gastrointestinal tract, increase the secretion of gastric and intestinal juice, and promote regular bowel movements and appetite. According to the published literature, C. intybus has been found to possess a notable therapeutic effect in curing and preventing diabetes mellitus and in preventing it (antidiabetic effect). This effect is attributed to the presence of inulin, a natural sugar substitute that eliminates toxins and non-nutrient substances from the body. Preparations based on C. intybus exhibit diverse pharmacological activities, including anti-inflammatory, antioxidant, antiviral, choleretic, diuretic, hepatoprotective, and antibacterial effects, making them beneficial in treating colitis, gastritis, and enteritis. Decoctions of C. intybus roots have been reported to be effective in the treatment of helminthic invasion, anemia, malaria, scurvy, eczema, and tumors of the spleen . Furthermore, some research indicates that C. intybus L. may modulate immune responses . Infusions of C. intybus flowers have been found to possess antiseptic, anti-inflammatory, moisturizing, and nourishing properties, which are beneficial in treating inflammation of the skin and eyes . A decoction of C. intybus L. is commonly applied externally (in the form of baths, applications, and lotions) for the treatment of various skin diseases, including but not limited to eczema, urticaria, psoriasis, seboroid dermatitis, neurodermatitis, atopic dermatitis, vitiligo, acne, and furunculosis. Additionally, the herb is known for its efficiency in the care of dry skin . C. intybus extract has been clinically tested on volunteers as a skin UV-protecting means. The analysis results of the microrelief control area applied with sodium lauryl sulfate and causing skin damage showed there was a significant increase in roughness after 28 days of the study, while in the areas where sodium lauryl sulfate was applied on the plant extract, the roughness of the skin did not undergo any significant changes, which indicates the plant’s protective properties . Equisetum arvense L., a herbaceous plant belonging to the Equisetaceae family, is widely distributed in North America, Europe, and Asia, including the territory of Kazakhstan . E. arvense contains more than 210 natural compounds distributed in various organs. These compounds include alkaloids, carbohydrates, proteins and amino acids, phytosterols, saponins, sterols, ascorbic acid, silicic acid, phenolic compounds, and their glycosides, tannins, flavonoids (such as apigenin, genquanin, luteolin, kaempferol, quercetin), triterpenoids, volatile oils, and other bioactive substances . E. arvense , a plant species from the Equisetaceae family, has been utilized in traditional medicine for its therapeutic properties. Its applications include the treatment of tuberculosis, and renal and bladder catarrh, as well as a hemostatic agent during excessive menstruation, nasal, pulmonary, and gastric bleeding, among others . The water–alcohol extract of E. arvense has demonstrated various biological activities including antioxidant , anti-inflammatory, antibacterial, and antimicrobial effects . Studies have also reported its antiproliferative activity , as well as antifungal, vasodilating, hepatoprotective , neuro- and cardioprotective, cytotoxic, and anti-cellulite properties . Additionally, E. arvense has been traditionally used for its analgesic effects on rheumatism and frostbite, as well as its anti-inflammatory properties, which can improve blood circulation. This plant has been employed as a bath agent for skin diseases and incorporated into cosmetic products as a rejuvenating, moisturizing, anti-wrinkle, anti-acne, antiperspirant, and conditioning agent . Equisetum arvense L. is recognized for its high content of silicon, a compound that is associated with promoting skin health. Silicon maintains skin firmness and elasticity, while its mild exfoliating properties allow the elimination of dead skin cells and enhancing of skin texture . The antioxidant potential of E. arvense has been attributed to the presence of flavonoids such as quercetin ( a), kaempferol ( b), and isorhamnetin ( c) . Studies show that phenolic compounds in the plant reduce the formation of ROS induced by bacterial lipopolysaccharides or fungal infections due to the direct capture of free radicals or their purification through reactions with antioxidant enzymes . Cosmetics containing the extract of this plant, which prevents early aging of the skin, have been actively introduced to the industry . Quercetin ( a) is able to reduce inflammation, accelerate reepithelialization, and stimulate cell proliferation and the formation of granulation tissue in different experimental models of skin wounds and clinical trials. These effects are associated with their ability to decrease levels of inflammatory cytokines (IL-1β, TNF-α), cell migration (neutrophils, CD68+ macrophages), mitogen-activated protein kinases (p38p, ERK-Î 2 , JNK-Î 2 ), (PEG2, leukotriene B4), inflammatory enzyme (COX-2), and transcription factor (NF-kB). In addition, quercetin promotes increased growth factors (VEGF and TGF-β1) as well as anti-inflammatory cytokines (IL-10) and antioxidant defenses (GSH, SOD, CAT). This compound also has an antifibrotic effect on second-target wounds, increasing the expression of αintergrin (a protein involved in the migration and proliferation of fibroblasts and reducing β1 integrin migration of fibroblasts and initiation of fibrosis . The subgenera of Eryngium are predominantly distributed throughout Europe, Africa, and Asia, with certain subgenera exhibiting a widespread presence in Australia . In Kazakhstan, Eryngium is found growing in the steppe regions of Northern Kazakhstan, as well as in the Dzungarian and Zailiyskiy Alatau mountain ranges . The aerial parts of Eryngium species are characterized by the presence of saponins, flavonoids, and essential oils, while the underground parts contain triterpene saponins, monoterpene glycosides, phenolic compounds such as flavonoids and phenolic acids, coumarin derivatives, terpene aldehyde esters, essential oils, and oligosaccharides . The isolation of eringinol from the aboveground parts of the plant was reported later . Further studies on the phytochemical constituents of the plant were conducted on leaves and roots, leading to the isolation of various aglycones and A1-barrigenol and R1-barrigenol . E. planum plays a significant role in European and Asian traditional medicine for treating various inflammatory diseases. The plant’s aboveground parts are bioactive primarily due to the presence of polyphenols and saponins . It has demonstrated potential for use in gastrointestinal diseases and exhibits antibacterial, analgesic, anthelmintic, anticonvulsant, and anticancer properties, thereby proving its crucial importance in ethnopharmacology . The aerial part of the plant collected during flowering is used for therapeutic purposes. According to the results obtained from HPLC-MS analysis, flavonoids, particularly rutin ( a) and isoquercetin ( b), are the major constituents of E. planum extracts . Rutin is known to possess skin-toning properties and to prevent the appearance of skin diseases such as rosacea and erythema. The anti-inflammatory effects of E. planum extracts may be attributed to the synergistic activity of ursolic acid ( c) and polyphenols such as chlorogenic acid ( d) and rosmarinic acid ( e), which have previously been studied for their anti-inflammatory properties . Notably, ursolic acid, which predominates in concentrated extracts of the plant, exhibits antioxidant, antimicrobial, anti-inflammatory, and hypoglycemic activities . Eryngium planum L. has potential applications in dermatology, particularly for the treatment of atrophic and purulent skin wounds when applied externally . Secondary metabolites include phenolic acids, flavonoids, coumarins, and triterpenoid. Saponins isolated from E planum L. demonstrate moderate antibacterial activity and substantial antimycotic activity. It was found that phenolic compounds inhibit microbial adhesion and inactive transport protein of a cell membrane . Glycyrrhiza glabra L., commonly known as licorice, fragrant wood, or mulaiti, is a small perennial plant that grows in Eurasia, North Africa, and West Asia . This plant is found ubiquitously in Kazakhstan . The genus Glycyrrhiza is extensively distributed across the globe and has over 30 species. The root of Glycyrrhiza glabra is a significant medicinal component due to the presence of various isolated compounds. These include triterpene saponins such as the sweet saponin glycyrrhizin, flavonoids such as liquiritin which is the primary flavonoid glycoside, rhamnoliquirilin, liquiritigenin, prenillicoflavon A, glucoliquiritin apioside, 1-methoxyphaseolin, shinpterocarpin, shinflavanone, lycopyranocoumarin, glisoflavone, lycoarylcoumarin, coumarin-GU-12, isoflavonoids, and chaconne. Among these, glycyrrhizic acid is the primary biologically active component, and it is known to be 60 times as sweet as sugar cane . Licorice root has been employed as a therapeutic agent by both ancient and modern medical practitioners. Its oral administration has demonstrated efficiency in the treatment of various disorders including gastric, duodenal and esophageal ulcers, inflammation, laxatives, mouth ulcers, antispasmodic, antitussive, sedative, and expectorants. The herb’s constituents make it a promising candidate for curing respiratory diseases such as asthma, acute and chronic bronchitis, and chronic cough. Furthermore, it can be used in treating Addison’s disease. External application of licorice extracts has also been effective in treating inflammatory skin conditions, mouth ulcers, and maintaining oral hygiene . Numerous clinical and experimental studies have shown the presence of several pharmacological properties in this substance. These properties are of great advantage, including anti-inflammatory, antiviral, antimicrobial, antioxidant, anticancer, immunomodulatory, hepatoprotective, and cardioprotective effects . The ethanolic extract derived from the root of G. glabra exhibits an excellent antibacterial activity that stops the development of Propionibacterium acne and Pseudomonas aeruginosa . Due to this property G. glabra is employed in dermatology for treating skin diseases, such as dermatosis and acne . Multiple studies have demonstrated the efficacy of Glycyrrhiza glabra L., which is efficient in the treatment of skin hyperpigmentation, eczema, and psoriasis, and provides skin with antioxidant properties. Due to the presence of flavonoid compounds such as oxyresveratrol ( a), glabridin ( b) and liquiritin ( c), it has a therapeutic effect . The external application of skin care products containing licorice extract gives a healthy glow, as well as improves the overall quality and appearance of the skin . It is achieved by the oxidative abilities of these components. Flavonoids of Glycyrrhiza glabra : liquiritin ( c), glucoliquiritin apioside ( a), and glycyrrhizin ( b) have high skin permeability properties and are potential antioxidants. These components improve the histological properties of the dermis and epidermis and reduce the level of markers of inflammation and wrinkles . Glycyrrhiza glabra L. also contains licochalcone A ( c), which has anti-inflammatory and antimicrobial properties and has been found to be efficient in treating acne, inflammatory skin diseases, and other skin ailments . Gnaphalium uliginosum L. is a member of the Compositae family, a group of flowering plants, and is commonly referred to as swamp cudweed. It is widely distributed, including in Kazakhstan . G. uliginosum is known to have a limited array of chemical constituents. It consists of approximately 125 compounds such as flavonoids, sesquiterpenes, diterpenes, triterpenes, phytosterols, anthraquinones, caffeylquinic and caffeylglucaric acids, flavonols, and carotenoids . Marshweed, also known as G. uliginosum , has been used in traditional medicine to alleviate a variety of ailments, including gastric disorders, edema, wounds, prostatitis, lumbago, neuritis, and angina pectoris. Additionally, it has been utilized for its antihypertensive, diuretic, antipyretic, and antimalarial properties . Pharmacological investigations on G. uliginosum extracts have revealed various beneficial effects, such as antioxidant , antibacterial, antifungal, antitussive, expectorant, antifeedant, cytotoxic, and hepatoprotective activities . Additionally, this plant has anti-inflammatory, antidiabetic, and antihyperuricemic properties . G. uliginosum is employed in medical practice as a hypotensive and wound-healing agent for treating hypertension, gastric ulcer, and difficult-to-heal wounds . Furthermore, oil extracts derived from this plant are useful for curing laryngitis, catarrh of the upper respiratory tract, and tonsillitis . In the field of dermatology, the extract derived from Gnaphalium uliginosum has been employed to treat diseases such as eczema and skin cancer . The ointment used to treat psoriasis contains an aqueous extract of “cold pressed” Gnaphalium uliginosum L. obtained immediately after harvesting . Humulus lupulus L., commonly known as hops, is a plant species that is widely distributed in temperate regions worldwide . H. lupulus is a plant that contains many phytochemicals, with a high concentration found in the female inflorescences from which lupulin, a yellowish-brown granular powder, is obtained. Lupulin comprises bitter resins and essential oils, imparting the characteristic aroma and flavor of hops. The primary bitter acids found in hop resin are alpha acids (humulones) and beta acids (lupulones). The essential oils contain myrcene, linalool, and geraniol, which are the most important aromatic compounds. Additionally, lupulin contains polyphenols, such as quercetin ( a), kaempferol, ( b) (see above) catechins, prenylnaringenin, hydroxycinnamic acid, and condensed tannins. Ferulic acid is the most representative compound in the phenolcarboxylic acid group. Hop seeds are rich in catechins (catechin, epicatechin), which are widely used in various industries, including pharmaceuticals, cosmetics, and nutraceuticals . H. lupulus has a long history in traditional medicine, which dates back to prehistoric times. It was used to treat various ailments such as leprosy, toothache, fever, stomach issues, sleep disorders, and anxiety. Additionally, it was utilized as a bowel function enhancer and to improve the pharmaceutical properties. Due to the numerous health benefits of hop polyphenols, which include antioxidant and antimicrobial effects, they may have a therapeutic use . Hop extract has been found to possess various pharmacological properties. For instance, it exhibits antitumor and anti-inflammatory effects, as evidenced by previous studies . Moreover, the extract has been reported to possess antibacterial, anti-collagenase, and antioxidant activity . Additionally, hop extract has been found to have antiallergic, antiviral, hepatoprotective, and antithrombogenic effects . In dermatology, extracts of H. lupulus have been employed as an antipsoriatic medicine . Furthermore, they are used in the treatment of skin inflammation diseases of adolescents, and hop cones are taken orally to cure baldness, furunculosis, lichen, and scrofula . The plant is known to have an anti-collagenase effect on the skin; that is, it prevents the destruction of collagen fibers due to exposure to UV rays . Naoto et al. tested seven natural components of hop ( Humulus lupulus L.) extracts to evaluate biological activity against acne vulgaris . Five strains, Propionibacterium acnes , Staphylococcus epidermidis , Staphylococcus aureus , Kocuria rhizophila , and Staphylococcus pyogenes , were selected as the main acne-causing bacteria. Hop extracts xanthohumol ( a) and the lupulones ( b) showed strong inhibitory activities against all of the strains. Although hydrogenated derivatives did not show the same level of activity, naturally occurring xanthohumol ( a), lupulones ( b), and humulones ( c) all showed moderate to strong anti-collagenase inhibitory activities: The results of studies indicate that H. lupulus flower extract has strong antioxidant activity since it significantly reduces the production of ROS and decreases inflammation, diminishing the production of NO and the expression of COX-2 by macrophages activated by liposaccharides . This plant has been observed to grow in various regions across the globe, including East Asia, Europe, North Africa, and South America . Its growth has also been documented in southern Kazakhstan and it is recognized as a protected species within the boundaries of the Sairam-Ugam State National Natural Park . The chemical composition of walnut kernels is of significant nutritional value due to the high content of polyunsaturated fatty acids (comprising up to 75% of total content), proteins, amino acids, as well as vitamins E, C, β-carotene, and essential minerals such as potassium, calcium, magnesium, sulfur, and phosphorus . Moreover, walnut is known to contain trace elements such as iron, zinc, and copper, which play a vital role in various biochemical processes within the human body . The plant is also rich in fluorine salts. Walnut partition contains trace amounts of organic substances, tannins, glycosides, alkaloids, and iodine. The chemical composition of walnut leaves is characterized by the presence of various biologically active components, including trace amounts of iodine, α- and β-hydrojuglone, polyphenols, tannins, glycosides, flavonoids, terpenoids, vitamin C, carotene, vitamin B1, essential oils, and tannins . Among the compounds contained in walnut, polyphenolic compounds are the most important ones. They include various derivatives of chlorogenic and hydroxycinnamic acids that are the major components . The study by Schwindl demonstrated that the methanolic extract derived from the leaves of Juglans regia L. includes a cumulative 40 metabolites classified under megastigmane, tetralone, phenylpropanoid, neolignan, and juglone glycosides . In traditional medicine, diverse components of Juglans regia L. are utilized to cure several ailments such as diabetes, infectious diseases, and periodontal disease . Furthermore, the plant is reputed to have antipyretic, analgesic, antidandruff, and burn-healing properties . Notably, the extract of walnut shell has demonstrated notable antibacterial and antibiofilm properties, which develop resistance to coagulase-negative staphylococci . Additionally, the lyophilized extract of the walnut septum has been reported to exhibit a marked antitussive, antioxidant, and anti-inflammatory effect . The leaves of Juglans regia L. are traditionally used to alleviate skin inflammation and excessive sweating of the hands and feet. Moreover, they are recommended for the treatment of acne, warts, eczema, and psoriasis due to the presence of flavonoids, specifically quercetin derivatives, and tannins . The high concentration of α-tocopherol in the leaves of J. regia contributes to its antioxidant effect, which promotes the repair of damaged skin and strengthens the epidermal layer . Matricaria chamomilla L. is a globally distributed, well known medicinal plant . M. chamomilla contains numerous biologically active compounds, including flavonoids (such as apigenin and luteolin) and their glycosides, as well as coumarins (including gerniarin and umbelliferone) . The essential oil extracted from chamomile flowers is composed of 52 different components, with the highest concentration of terpenoids, including β-farnesene, α-farnesene, α-bisabolol, chamazulene, and germacrene, as well as spiroether . M. chamomilla has been widely employed in traditional medicine for treating a variety of ailments, including infections, neuropsychiatric disorders, respiratory tract, gastrointestinal, and liver diseases. Furthermore, the plant possesses sedative, antispasmodic, antiseptic, and antiemetic properties . Therapeutic indications for M. chamomilla encompass a diverse array of medical conditions, including inflammatory conditions, bacterial infections, and lesions of the skin and mucous membranes such as those found in the oral cavity, gastrointestinal tract, and respiratory tract. Additionally, the plant has been employed as a remedy for spasms and ulcers of the gastrointestinal tract, insomnia, and nervous breakdown . Furthermore, the plant has demonstrated pain-relieving properties and wound-healing effects , and acted as a protective agent for the kidneys and liver . M. chamomilla is regarded as a viable alternative due to a high content of bioactive secondary metabolites that can be used for the treatment of diverse skin problems, such as wounds, abscesses, and skin diseases. The plant’s therapeutic efficiency in treating skin diseases is attributed to the presence of quercetin ( a), α-bisabolol ( a), and apigenin ( b): α-Bisabolol ( a) possesses anti-inflammatory, antibacterial, and anti-irritant properties, making it suitable for use in a variety of products that protect skin from irritation caused by environmental factors. Due to its non-allergenic nature, it is widely used in hand and body lotions, aftershave creams, lipsticks, sun and after-sun care products, and baby care products . On the other hand, apigenin ( b) has been found to alleviate the symptoms of skin inflammatory diseases by protecting skin cells from oxidative-stress-induced death. Apigenin also affects the synthesis of skin barrier factors and the influx of calcium ions. Therefore, it can potentially be used to treat skin inflammatory diseases and cancer . Dos Santos et al. presented a review of 20 patents using Matricaria species as an active ingredient in skin diseases. The majority of the inventions (80.00%) contained combinations of Matricaria with other plant species, including those belonging to the genus Calendula , Salviae , Eucalyptus , Urtica , and Aloe vera . On the other hand, four patents (20.00%) reported the development of bioproducts containing only species classified as chamomile, two (10.00%) with M. parthenium , and two (10.00%), M. chamomilla . Based on the information of these extracts, externally applied pharmaceutical remedies such as creams, ointments, lotions, solutions, textile dressing, and banding were developed. Capsules, granules, and alcohol dye have been developed for oral use. Regarding the skin disease treated, the selected inventions claim to treat wounds and burns, erythema and rosacea, eczema and dyshidrosis, and spots and hyperpigmentation of the skin by UV radiation. Skin peeling and damaged stratum corneum, dermatitis, hemorrhagic incision, excoriations, hand-foot syndrome, psoriasis, and acne were also mentioned. Ononis spinosa L. is widely distributed in Africa, Asia, and Europe. It is found in countries such as Algeria, Libya, Morocco, Tunisia, Afghanistan, Iran, Iraq, Palestine, Jordan, Lebanon, Syria, Turkey, Armenia, Azerbaijan, India, Denmark, Norway, Sweden, Great Britain, Austria, Belgium, Czechoslovakia, Germany, Hungary, the Netherlands, Poland, Switzerland, Estonia, Lithuania, Moldova, the European part of the Russian Federation, Albania, Bulgaria, Greece, Italy, Romania, France, Portugal, and Spain . The root of O. spinosa contains a large amount of isoflavonoids, pterocarpans, and dihydroisoflavonoids, including formononetin, calicosin, pseudobaptigenin, medicarpin, maakiain, onogenin, and sativanon, with metabolites present in the form of glucosides, glucoside malonates, glucoside acetates, and free aglycones . The roots, leaves, and flowers of O. spinosa were utilized in folk medicine for their antitussive, laxative, and diuretic properties. Infusions of the plant were employed to treat dropsy, urinary tract infections, inflammation, and rheumatism, while external applications were used to promote wound healing and alleviate skin conditions such as eczema. In Iraq, the roots were valued for their diuretic, blood purifying, laxative, and expectorant qualities . Additionally, ash derived from burned samples of O. spinosa has demonstrated resistance to various Candida species . Pharmacological investigations have demonstrated that O. spinosa exhibits noteworthy hepatoprotective and antitumor properties , and may be considered a potential therapeutic agent for treating urinary tract infections and bladder stones . O. spinosa has been utilized in dermatology for its efficiency in treating skin ailments such as dermatitis (eczema) and pruritus. It also possesses wound-healing properties beneficial in the treatment of burns . Ononis spinosa extract and glycerin have been clinically tested for facial laxity and wrinkles. The particular focus was made on immediate and delayed effects. Thirty-nine women used the product daily for an eight-week treatment period. Clinical assessment by experts and a new 2D imaging method (measuring the effect of an upper eyelid lift) were made at different time periods. The results showed an immediate and significant improvement in sagging and wrinkle parameters seven hours after the first application, in addition to significant long-term improvement. The lifting effect calculated from 2D images was 1.08 mm immediately after application and 1.80 mm after an eight-week treatment period. Ononis spinosa root extract inhibited hyaluronidases; Hyal-1 inhibition was a promising remedy for improving wound healing, tissue regeneration, and inducing diuresis. Two non-polar fractions of the roots of Ononis spinosa were the most active, causing inhibition of Hyal-1 by 86 ± 3% and 96 ± 13% at a concentration of 1 mg/mL, respectively. Chemical analysis revealed three main components, which were identified as onogenin, sativanon, and medikarpine. The percentages of inhibition for concentrations of 250 μM of these compounds were 25.3 ± 18, 61, 20 ± 20.6, and 22.4 ± 16, respectively. The IC50 of sativanone was determined to be 151 µM. Hot water and hydroalcoholic extracts of the Ononis spinosa root showed a moderate inhibitory effect on hyaluronidase-1 (Hyal-1) (IC 50 1.36, respectively, 0.73 mg/mL), while dichloromethane extract had an inhibitory effect (Hyal-1) with IC 50 190 µg/mL . Onopordum acanthium L. is a widely distributed species of plants found across Africa (Algeria), Asia (Afghanistan, Iran, Iraq, Turkey, Armenia, Azerbaijan, Georgia, Russian Federation, Kazakhstan, Kyrgyzstan, Tajikistan, Turkmenistan, Uzbekistan, China, India, Pakistan), throughout Europe, Australia, New Zealand, and North and South America (Argentina, Chile, Uruguay) . O. acanthium is a plant species that contains various phytochemical compounds, including saponins, alkaloids, sesquiterpene lactones, flavonoids, triterpenes, sterols, nitrogen-containing compounds, phenolic acids, coumarins, inulin, soluble sugars, proteins, and oils . The fatty acid composition of the plant includes palmitic, stearic, oleic, and linoleic acids . Additionally, phenolic, triterpene, and steroid compounds were detected in the aerial parts of O. acanthium , while the roots were found to contain sesquiterpene lactones and polyacetylenes . In traditional medicine, various preparations of O. acanthium , including its powder, juice, and decoction of the aerial part, were utilized as diuretics. This plant is known to stimulate the central nervous system and has demonstrated cardiotonic and hemostatic properties. Infusions of the leaves and inflorescences have also been employed to reduce swelling of various etiologies . Furthermore, the extract derived from this plant has exhibited bactericidal, cardiotonic, and antitumor effects . The extracts and isolated compounds from this plant have demonstrated a range of activities including anti-inflammatory, anti-radical, antiproliferative, and antibacterial effects . Additionally, this plant can produce antioxidant and anti-inflammatory effects , as well as diuretic, dermatological, tonic, sedative, anticonvulsant, cardiotonic, hemostatic, and bactericidal effects, all without causing any side effects. Eriodictyol ( a) and quercetin ( a) have been identified in the flowers of the plant, both of which possess potent antioxidant properties. Eriodictyol, in particular, has been found to protect skin cells from damage induced by UV radiation by inhibiting the MAPK signaling pathway, thereby exhibiting anti-aging effects . The antitumor activity of extracts obtained from a combination of flowers and fruits, leaves, and roots of O. acanthium resistant to A431 culture (epithelial carcinoma of the skin) was examined by the authors of . Aqueous, n-hexane, chloroform, and water–methanol extracts were utilized in the study. The results revealed that the chloroform extract of leaves and roots displayed the highest activity. A number of compounds were isolated from the roots of Onopordum acanthium L., among them 4β,14-dihydro-3-dehydrozaluzanin C ( b), which showed a general antiproliferative ability comparable to that of the reference drug cisplatin in relation to epidermoid skin carcinoma. The antiproliferative activity of compound ( b) was evaluated on epidermoid skin carcinoma cells A431 using MTT analysis . The mechanism of cytotoxicity ( b) is associated with the activation of the mitochondrial pathway of cell apoptosis through the enzymes caspase-3 and caspase-9. The term “mitochondrial pathway” refers to the initiation of the apoptosis pathway in a cell as a result of a number of internal stimuli, for example, genetic damage, oxidative stress, and hypoxia. Regulation of this pathway is carried out by a group of proteins belonging to the Bcl-2 family. Bcl-2, Bcl-W, Bcl-XL, MCL-1, and Bfl-1 proteins suppress apoptosis by blocking mitochondrial release of cytochrome-C. P53-dependent pro-apoptotic proteins Bik, Bcl-Xs, Bad, Bax, Bak, Bid, Bim, and Hrk stimulate apoptosis, increasing the permeability of mitochondria and the exit from them into the cytoplasm of cytochrome-C. The ratio of pro- and anti-apoptotic proteins determines the fate of the cell. The release of cytochrome-C into the cytoplasm leads to the activation of caspase-3 through the formation of an apoptosomal complex consisting of cytochrome-c, Apaf-1 (apoptotic protease activating factor 1) and caspase-9. A number of proteins released from mitochondria into the cytoplasm can modulate apoptosis: AIF (apoptosis-inducing factor), Smac (second mitochondria-derived activator of caspase), DIABLO (direct IAP binding protein with Lowp I) and others. They bind apoptosis suppressors, proteins of the IAP family (inhibitor of apoptosis protein), which in turn are capable of inhibiting caspases-3, -7, and -9 . O. acanthium extracts find applications in dermatology beyond skin cancer, such as in the treatment of furunculosis, purulent wounds, and lupus . Spotted orchis is indigenous to countries with a cold, temperate subtropical climate, particularly in Central and Southern Europe and Asia . Its distribution within Kazakhstan is primarily concentrated in the East Kazakhstan region . Spotted orchis comprises a mucilaginous substance that contains polysaccharide, which decomposes to mannose, in addition to dextrin, starch, proteins, bitterness, pentoses, methylpentosans, sucrose, loroglossin glycoside, and essential oil . Furthermore, the plant includes alkaloids, saponins, tannins, phenolic compounds (such as gallic acid, catechin, chlorogenic acid ( d), and syringic acid), terpenes, sterols, flavonoids, and anthocyanins . O. mascula flowers’ ethanol extracts also encompass saponins, flavonoids, anthraquinone, terpenoids, tannins, cyanogenic glycosides, and cardiac glycosides . These extracts exhibited a noteworthy antimicrobial effect against Salmonella paratyphi , Klebsiella oxytoca , and Staphylococcus aureus . The spotted orchis extract has been shown to possess anti-inflammatory, antispasmodic, diuretic, enveloping, and immunomodulatory effects, as outlined in . The enveloping effect can be attributed to the presence of loroglossin , a glycoside that protects inflamed tissues from excessive irritation . O. maculata contains anthocyanins and phenolic acids, which are potent antioxidants and have a nourishing impact. These compounds have the ability to inhibit collagenase, an enzyme that degrades collagen in the skin and hair. Catechin , for instance, influences collagen and makes it collagenase -resistant. Catechin also forms a complex with collagen, modifying its structure and making it resistant to enzyme degradation. Flavonoids, in general, contribute to scalp elasticity and nutrition, strengthen blood vessel walls, and enhance blood flow. Furthermore, polyphenols manifest antimicrobial properties, which makes them a valuable ingredient in medicines used to treat mycoses : In dermatology, the oral use of Orchis maculata L. extract is prevalent in folk medicine for senile itching, skin tuberculosis, and other dermatoses accompanied by cachexia and chronic diseases of the respiratory and gastrointestinal tracts. The extract is also employed for the speedy healing of wounds and ulcers . Additionally, cosmetic skincare products containing the extract and produced on an industrial scale are available . Parsnip ( Pastinaca sativa L.) is a plant species that is indigenous to Europe and Asia , and is also found growing in South Kazakhstan . The root of parsnip is a rich source of numerous bioactive compounds, including coumarins, furanocoumarins, polyacetylenes, essential oils, terpenes, and flavonoids . Additionally, parsnip root is rich in various minerals such as potassium, manganese, magnesium, phosphorus, zinc, and iron, as well as carotene, starch, pectin, vitamins, and sugars . Parsnip has been employed in traditional medicine since antiquity. According to Avicenna’s Canon it alleviates headache, stomatitis, ophthalmitis, dermatitis, and fever if it is used orally. Numerous studies have demonstrated the pharmacological effects of P. sativa on various bodily systems, including the central nervous, respiratory, gastrointestinal, hepatic, skin, cardiovascular, and genitourinary systems , as well as its potential in mitigating stroke, atherosclerosis, and other coronary heart diseases. Additionally, P. sativa has been shown to have positive effects on cholecystitis, constipation, anorexia, stomach pain, bladder atony, spastic enterocolitis, mild insomnia, nephritis, dysuria, renal colic, endocrine disorders such as menstrual syndrome, rheumatism, vitamin deficiency, obesity, vascular diseases, infections, loss of appetite, dysmenorrhea, fever, atherosclerosis, detoxification, anemia, and diabetes . Plants containing furanocoumarins have been used to treat leprosy and vitiligo . Furthermore, furanocoumarins extracted from parsnips have the ability to dilate peripheral vessels and coronary vessels of the heart, eliminate spasms of the bronchi and smooth muscles of the abdominal cavity, and have a moderate sedative effect. In addition, P. sativa exhibits antioxidant and anticytolytic activities . The dried seeds of P. sativa underwent steam distillation to isolate its essential oil, which was found to contain octyl acetate (78.49%) and octyl hexanoate (6.68%) as its major constituents. Remarkably, this essential oil exhibited significant antioxidant and antimicrobial activity . A large amount of vitamins A and C predominate in Pastinaca sativa . These vitamins eliminate and neutralize the free radicals responsible for body diseases (chronic diseases) and premature aging . Recent studies in dermatology have shown the effects of furanocoumarins on the skin. Heraclenol ( a) and oxypeucedanine hydrate ( b) were found to have a weak stimulatory effect on melanogenesis without affecting cell proliferation. Moreover, furanocoumarins have been employed in the treatment of vitiligo and psoriasis . The furanocoumarins xanthotoxin and bergapten are important components in leukoderma treatment . Plantago major L. (plantain) is a well-known and widely used medicinal plant. The genus Plantago L. comprises approximately 300 diverse species that flourish in temperate areas all over the world, including 16 plant species that occur in Kazakhstan . In arid zones, P. major is comparatively scarce and is primarily found along riverbanks and in intensely irrigated crops. Plantain is a botanical specimen that contains diverse chemical constituents, including carbohydrates, lipids, allantoin, essential and non-essential amino acids, caffeic acid derivatives, flavonoids including baicalein, scutellarein, luteolin, baicalin, apigenin, among others; phenolcarboxylic acids and their derivatives; iridoid glycosides such as aucubin, catalpol, and aukubozid; terpenoids; and alicyclic compounds such as loliolid. Furthermore, the leaves of plantain exhibit a significant concentration of phenols and their derivatives such as ferulic acid and tyrosol, tannins, and vitamin K. The seeds of plantain contain organic acids such as succinic acid, mucus, iridoids such as aucubin, sterols such as β-sitosterol, stigmasterol, campesterol, saponins, alkaloids, tannins, flavonoids such as isoquercitrin, and fatty oil. These findings have been reported in numerous sources . For centuries plantain had been considered to possess therapeutic properties. Various parts of the plant, including mature seeds, leaves, and juice, were used for medicinal purposes. Plantain leaves were employed in the treatment of numerous diseases, including digestive, reproductive, and circulatory ailments, as well as inflammatory skin disorders and urogenital and infectious diseases . Moreover, plantain was used for pain relief and to reduce fever . The mucus, enzymes, and phytoncides present in psyllium provide an enveloping and mucolytic effect that restores the protective function of the ciliated epithelium in the respiratory tract, leading to increased secretion of bronchial mucus and liquefaction of sputum for easy expectoration. It is noted in that plantain glycoside inhibits the cough reflex, and the hemostatic properties of plantain are due to the high content of vitamin K in it, which, along with tannins, promotes blood clotting. Psyllium is also a great antioxidant and radical scavenger with immunomodulatory effects . P. major is used in various types of wound and skin diseases: deep wound, purulent wound, chronic and progressive wound, malignant wound, fire burn, erysipelas, progressive blister, pruritus, irritating urticarial, and fistula. The treatment is carried out by sprinkling the plant powder on the wound or by using a bandage covered by Plantago major together with salt or without it. It is also used for head and face skin ulcers in the same manner . This plant is noted as an effective medicinal plant in the treatment of acute urticaria . Ursolic acid, oleanolic acid, and α-linolenic acid are three P. major components that have shown inhibitory effects on cyclooxygenase (COX-2)-catalyzed prostaglandin production. Luteolin (one of the flavonoids) also has the ability to suppress leukocyte migration and inhibit mast cell degranulation, which all together can be considered as anti-urticaria treatment strategies Polysaccharides stimulate the formation of interferon, while zinc and flavonoids aid in the normalization of phagocytosis. The combination of polysaccharides with enzymes and vitamins promotes regeneration. Plantain is also used in cosmetic dermatology to treat acne scars . Ribes nigrum L. is a diminutive perennial shrub indigenous to Central Europe and North Asia that has been widely cultivated globally, including in the United States . Furthermore, it is known to thrive in the territory of Kazakhstan . Fresh blackcurrant fruits are known to contain a diverse range of functional and biologically active compounds, including soluble sugars, flavonoids, organic acids, vitamins, polyamino acids, macro- and microelements, and unsaturated fatty acids . Additionally, blackcurrants are a rich source of vitamin C . Anthocyanins, a group of biologically active compounds, are prominently found in blackcurrant berries, as well as in its seeds and leaves . Notably, blackcurrant seed oil is a valuable source of gamma-linolenic acid (γ-C18:3), stearidonic acid (C18:4), tocochromanols (primarily γ-tocopherol and α-tocopherol), and sitosterol . The fruits, leaves, and shoots of Ribes nigrum , both in fresh and dried form, have been traditionally used as a multivitamin and general tonic for hypovitaminosis and beriberi, as well as for enhancing the immune system. In folk medicine, the leaves of Ribes nigrum have been used for treating various conditions, including kidney stones, gout, cystitis, urethritis, osteochondrosis, rheumatism, muscle and joint pain, exudative diathesis, eczema, and furunculosis . Additionally, Ribes nigrum is used in homeopathy . In a study , a wide range of pharmacological actions of Ribes nigrum extract, rich in anthocyanins, were indicated. Extracts containing more than 20% anthocyanins were found to exhibit antioxidant, anti-inflammatory, and phytoestrogenic activity, anti-postprandial hyperglycemic and antidiabetic effects, and cardioprotective effects. Furthermore, the anthocyanin-rich fraction of black currant peel extract has been found to exhibit a strong cytotoxic effect on human liver cancer cells, and to have a positive effect on vision and eye health. In the field of dermatology, blackcurrant leaves have been utilized for treating skin lesions resulting from atopic dermatitis and allergic itchy dermatoses (e.g., eczema, neurodermatitis, pruritus), while leaves and fruits have been used for curing psoriasis, scleroderma, lichen planus, vasculitis, and acne vulgaris . Ribes nigrum may be helpful in treating various skin diseases, such as atopic dermatitis, psoriasis, and acne, owing to its higher anthocyanin content . The antioxidant activity of blackcurrant, attributed to the presence of flavonoids and vitamin C, has been observed to modulate cancer and inflammation signaling pathways and absorb ultraviolet radiation . Vitamin C has been shown to increase the amount of the transport protein when exposed to ultraviolet light. Furthermore, the presence of fatty acids in blackcurrant makes it therapeutically efficient for treating skin diseases . The authors of studied the effect of a polysaccharide (CAPS) isolated from blackcurrant ( Ribes nigrum ) on immunomodulation in laboratory mice. The introduction of CAPS was found out to improve the symptoms of atopic dermatitis by inhibiting the migration of mast cells into the skin of the epidermis. CAPS administration was also found to suppress immunoglobulin (IgE) overproduction and induce transcription of the IFN-γ gene in the spleen. Rose hips have considerable economic importance and are widespread garden plants in Europe, Asia, North America, and the Middle East. The distribution of wild roses in different regions of Kazakhstan is heterogenous. In particular, a greater range of species diversity has been observed in forest and forest-steppe zones . There is a total of 21 distinct species of wild rose that grow in Kazakhstan, with five of them being present in Central Kazakhstan, including R. glabrifolia , R. laxa Retz., R. Acicularis Lindl., R. majalis Herrm. ( R. cinnamomea L.), and R. pimpinellifolia L. ( R. spinosissima L.) . The fruits of R. canina are highly valued by the food and pharmaceutical industries due to their rich content of biologically and physiologically active compounds. These include a wide range of vitamins (C, B, P, PP, E, K), flavonoids, carotenes, carbohydrates (mono- and oligosaccharides), organic acids (tartaric, citric), polyunsaturated fatty acids, trace elements, and others . The essential oil derived from rosehips is primarily composed of alcohols, monoterpenes, and sesquiterpenes . Dog rose seeds are also a valuable source of crude oil, comprising approximately 15% of their total weight. To extract oil from the seeds, various methods are employed such as pressing, solvent extraction, ultrasonic, microwave, and sub- and supercritical fluid extraction. Rosehip oil is considered particularly valuable due to its essential fatty acid content, tocopherols, phytosterols (β-sitosterol), and phenols, which contribute to its functional properties. The primary essential fatty acids present in rosehip oil are linoleic, linolenic, and oleic acids, while the γ-tocopherol isomer of tocols is the most abundant in the oil. Among the numerous health benefits of rosehip oil, its anticancer effects are particularly noteworthy. Additionally, the therapeutic effect of rosehip oil on skin diseases makes it a preferred ingredient in cosmetics . Rose hips have a well-established history in traditional medicine as a preventative and treatment remedy for colds and other infections, as well as a diuretic and therapy for various inflammatory disorders. In modern medical practice, dog rose ( Rosa canina L.) is incorporated into compositions and complexes for the treatment of inflammatory diseases, including but not limited to rheumatoid arthritis, reactive arthritis, osteoarthritis, and other types of arthritis. It is also helpful in upper respiratory tract infections and psoriasis. Its other application includes the prevention of oxidative stress in the oral cavity. The unique phytochemical composition of rose hips is of huge interest because it can be considered a promising source for making functional foods, natural medicines, and cosmo-nutraceuticals. Presently, rose hips are employed as a constituent in probiotic products . The rose hip extract’s antioxidant activity is predominantly attributable to its ascorbic acid and polyphenolic compounds. Moreover, the extract manifests antimutagenic and anticancer properties . R. canina finds a common application in cosmetology, where it is frequently utilized in conjunction with other biologically active compounds or herbal extracts. However, there are cases when it is employed as an individual ingredient; for instance, French-patented industrial technology uses dog rose extract as an active agent for curing seborrhea together with a cosmetic skincare strategy aimed at eliminating excess sebum production and dermatological manifestations caused by it . Solanum dulcamara L. exhibits a wide distribution across all continents except Antarctica, with the highest concentration found in tropical and subtropical regions of Australia, Africa, and select areas of Asia, including China, India, and Japan, as well as Central and South America . Notably, the plant is found ubiquitously throughout Kazakhstan. S. dulcamara is known to contain various bioactive phytocomponents, including steroidal saponins, terpenes, flavonoids, carbohydrates (such as glucose, galactose, xylose, and rhamnose), lipids (specifically cholesterol), steroidal sapogenins (such as diosgenin, tigogenin, and yamogenin), and pigments (such as lycopene and lycoxanthin) . Notably, steroid alkaloids and glycoalkaloids are the primary chemical markers for this plant genus. Additionally, S. dulcamara has been found to contain steroidal alkaloids, including solanine in immature fruits, solasodine in flowers, and β-solamarin in roots . S. dulcamara stems have traditionally been employed in folk medicine as a narcotic agent and as a remedy for conditions such as rheumatism, migraine, and severe inflammation . An ethyl acetate extract obtained from the ripe fruits of S. dulcamara demonstrates significant anti-inflammatory and antioxidant activity . Moreover, S. dulcamara is reputed to possess a variety of therapeutic properties, including antimicrobial, analgesic, hepatoprotective, immunomodulatory, antitumor, and neurogenetic effects , as well as antioxidant , antihyperglycemic , antibacterial, and antimicrobial activity , and antirheumatic activity . The aerial part of S. dulcamara is particularly rich in alkaloids, which contribute to its antibacterial activity against Streptococcus pyogenes , Staphylococcus epidermidis , and S. aureus . S. dulcamara is a known remedy for the treatment of skin diseases and warts . This plant is particularly rich in the alkaloid solanine, which is abundant in its immature fruits and has been traditionally used in Kenya to treat skin mycotic infections and other pathological diseases . Saponins isolated from S. dulcamara possess remarkable antioxidant activity, as they are capable of absorbing free radicals. Due to their beneficial properties, saponins are often utilized in cosmetology, where they improve the rheological and foaming properties of body-washing products and reduce the risk of skin irritation . The antioxidant properties of S. dulcamara are attributed to the presence of various phenolic compounds, flavonoids, anthocyanins, and carotenoids such as lycophyll , as well as hydroxy and methoxy derivatives of coumarins . Through non-targeted LC/MS analysis, 83 metabolites have been identified in S. dulcamara fruit extracts, including 22 polyphenolic compounds comprising of 19 phenolic acid derivatives and 3 flavonoids (namely quercetin-3- O -rutinoside and kaempferol-3- O -rutinoside), 10 amides, 16 saponins, 14 steroid alkaloids, 6 lignans, and 15 other compounds . Notably, the phenolic acids in these extracts are mainly composed of chlorogenic acid ( d), caffeic acid, and p-coumaric acid. According to the investigations of the metabolites present in S. dulcamara , unripe fruits contained a higher concentration of γ-solamarin, α-solazonin, α-solanine, abutiloside H, and solanandaine compared to ripe fruits. Moreover, methanol fruit extracts were found to exhibit significant potential in eliminating DPPH and hydroxyl radicals. Interestingly, the ability of methanol extracts to remove DPPH was found to be tissue-specific, with the outer tissue (skin) of the bittersweet fruits showing a higher antioxidant activity than the inner tissues (pulp and seeds), possibly due to the higher phenol content in the peel . Sorbus aucuparia L., a botanical species known for its nutritional and medicinal beneficial properties, is considered a valuable source of edible fruits. This plant is characterized by its ability to survive in cold and harsh environments, and is widely distributed in various regions of Northern Europe, the Caucasus, the Middle East, and East Asia . Sorbi fructus, commonly known as Rowan fruits, serve as essential medicinal resources. The berries are harvested during their complete maturation phase, from August to September, before the advent of frost. During collection, it is advisable to exercise caution and avoid damaging the branches. The stalks of harvested raw materials are cut, and then these materials are subjected to a drying process in well-ventilated rooms or dryers, employing a temperature range of 60–80 °C . This fruit, popularly referred to as a “superfruit,” contains a diverse array of phytochemicals, comprising phenolic acids: neochlorogenic and chlorogenic acids (see above), flavonoids, proanthocyanidins, iridoids, coumarins, hydrolysable tannins, carotenoids, and anthocyanins, as well as vitamins (ascorbic acid, α-tocopherol, B1, B2, P, PP, K, and folic acid) . Furthermore, it is rich in various sugars, phospholipids, pectin, organic acids, bitter substances, sorbic and parasorbic acids, essential oil, and macro- and microelements. The leaves of the plant contain vitamin C and flavonoids, while rowan seeds contain fatty oil (up to 22%) and glycoside amygdalin; the bark contains tannins . Throughout history, the fruits of S. aucuparia have been utilized in traditional medicine to alleviate ailments related to cardiovascular and digestive systems. In addition to their medicinal applications, these fruits can be eaten raw or utilized in the production of jams, syrups, and as flavoring agents in alcoholic and non-alcoholic beverages, including beer and wine . The fruit extracts derived from S. aucuparia have demonstrated antioxidant and antitumor activity . The antioxidant activity is attributed to the presence of flavonoids, vitamins C and E , and anthocyanins within their composition. Moreover, the authors of reported additional pharmacological effects of the fruit extracts, including antitumor, antiproliferative, antiviral, antibacterial, antifungal, and anti-inflammatory effects. Within the field of dermatology, Rowan berries are a valuable multivitamin raw material for the treatment of allergic diseases and other skin problems due to their wound-healing properties . Sorbic and parasorbic acids, present in the fruits of mountain ash, have an antimicrobial and antifungal effect. Based on the fruits of mountain ash, an ointment is prepared that has anti-inflammatory and wound-healing properties . Symphytum officinale L., commonly known as comfrey, is distributed across the humid meadow and lakeside regions of Asia, Europe, and America, as supported by reference . Its occurrence has also been recorded in the northwestern and eastern regions of Kazakhstan, as documented in reference . Comfrey contains various chemical compounds including phenolic compounds, flavonoids, fatty acids, polysaccharides, purine derivatives, and triterpenes . In terms of ethnopharmacology, preparations made from the roots, leaves, or entire aerial parts of comfrey have been traditionally used since ancient times to treat various internal ailments such as respiratory, gastrointestinal, and genitourinary disorders, as well as external conditions such as bruises and tumors, through the administration of tinctures, infusions, decoctions, compresses, and ointments . The literature indicates the potential therapeutic effects of Symphytum officinale . The plant has been reported to possess anti-inflammatory, anti-apoptotic, antitumor, neuroprotective, and antioxidant properties . Furthermore, comfrey has been shown to facilitate bone regeneration . Allantoin and rosmarinic acid ( e), identified as active compounds in comfrey, exhibit significant skin healing properties and have been applied in the treatment of a range of skin conditions: Allantoin has been reported to stimulate cell proliferation and tissue repair, making it a promising therapeutic agent for wound healing. Several studies have demonstrated that allantoin can accelerate the healing process of wounds, reduce inflammation, and increase skin moisture, thus exhibiting a rejuvenating effect . On the other hand, rosmarinic acid ( e) (see above) possesses antimicrobial, anti-inflammatory, and antioxidant properties, which make it an effective treatment agent for various skin diseases, such as psoriasis, acne, and eczema. Studies have shown that rosmarinic acid can reduce oxidative stress and inhibit the production of inflammatory cytokines in the skin, leading to improved skin health . Comfrey also contains caffeic acid and chlorogenic acids ( d) (see above). In vitro analyses demonstrated that caffeic acid and chlorogenic acid accelerated the proliferative response of fibroblasts, thus enhancing wound healing: Polyphenolic compounds present in hydroalcoholic extracts were shown to possess antioxidant and free radical scavenging properties, preventing the release of reactive species responsible for the oxidative stress and tissue damage in burns . Comfrey also contains tannins and pyrrolizidine alkaloids, which contribute to its anti-inflammatory and wound-healing effects . Tanacetum vulgare L. is a well-known medicinal plant that is distributed across Northern Europe, North America, Russia, China, North Korea, Kazakhstan, and Japan . T. vulgare is rich in phenolic acids, flavonoids, and their derivatives . The plant contains surface flavonoids, such as the methyl esters of flavones scutellarin and 6-hydroxyluteolin, as well as vacuolar flavonoids, including apigenin and luteolin 7-glucorinides. Additionally, it contains caffeic acid, glycosides, sterols such as β-sitosterol, stigmasterol, cholesterol, and campesterol, and triterpenes such as α-amirin, β-amirin, and taraxasterol . In the traditional medicine of southeastern Serbia, T. vulgare flowers are commonly used to prepare tea with various therapeutic effects such as antihelminthic, carminative, antispasmodic, abdominal organ stimulant, tonic, menstruation stimulant, antidiabetic, diuretic, and antihypertensive properties . Apart from medicinal use, T. vulgare is also utilized in the production of balms, cosmetics, dyes, insecticides, drugs, and preservatives . Furthermore, T. vulgare -based preparations have been used for the treatment of several illnesses including hysteria, migraine, neuralgia, rheumatism, renal failure, and fever . The same source tells about the antibacterial, antiviral, antifungal, anti-inflammatory, and immunomodulatory activity exhibited by T. vulgare. The bioactive components of T. vulgare , including sesquiterpene lactones, volatile oils, flavonoids, and phenolic acids, have been found to possess antioxidant, anticancer, anti-inflammatory, and antiulcer properties . Taraxasterol ( a), luteolin ( b), and taraxic acid (a sesquiterpene lactone) present in T. vulgare are responsible for its anti-inflammatory and antiallergic effects, making it a potential remedy for treating skin diseases such as atopic dermatitis, eczema, and psoriasis . Inulin and chlorogenic acid ( d) (see above) have demonstrated antioxidant, prebiotic, and anti-inflammatory effects, which suggest their potential use as a therapeutic approach for managing skin disorders such as acne, rosacea, and photoaging . Taraxacum officinale Web. is a plant species commonly found in temperate climatic zones of Europe, Asia, and North America . It can also be found in Kazakhstan, where it grows in various habitats such as wetlands, meadows, and roadsides, and occasionally in the steppes . Dandelion is a plant with a rich chemical composition. Its constituents include β-carotene , chicory acid , inulin , sesquiterpene lactones, and triterpene compounds , as well as flavonoids and fatty acids . Dandelion also contains a variety of vitamins (A, C, D, E, and B), inositol, lecithin, and minerals, such as iron, magnesium, sodium, calcium, silicon, copper, phosphorus, zinc, and manganese . According to the traditional medicine specialists’ evidence it has the tonic and diuretic properties of Taraxacum officinale , as well as its anthelmintic, anti-inflammatory, and sedative effects. Dandelion has also been shown to cure metabolic disorders and leukoformula deviations, and has been used in the treatment of hepatitis, bronchitis, pneumonia, mastitis (as a local compress), and anemia. These therapeutic effects are attributed to the various phytochemical compounds present in dandelion, including sesquiterpene lactones, triterpene compounds, flavonoids, fatty acids, and vitamins and minerals such as vitamins A, C, D, E, and B, inositol, lecithin, and minerals such as iron, magnesium, sodium, calcium, silicon, copper, phosphorus, zinc, and manganese . Dandelion, being a versatile plant, has also found significant use in the field of dermatology owing to its potential in curing several skin diseases. Notably, Taraxacum officinale has been found to contain taraxasterol ( a), a compound that is helpful in curing melanoma . Caffeic acid is the predominant component of dandelion stem extract, while chlorogenic acid is predominant in dandelion leaf extract. Both extracts have the same reducing power and ability to absorb superoxide anion radical; however, the stem extract showed the strongest UVA and UVB absorption and the strongest tyrosinase inhibition. In addition, the results of molecular docking modeling have indicated that caffeic acid in the stem extract inhibits tyrosinase mainly through hydrogen bonding with its Gly165 and Pro160 residues. Thus, dandelion stem extract is a promising skin care product . Additionally, the aqueous extract of dandelion has been observed to manifest high activity in inhibiting tyrosinase . Dandelion extracts are commonly employed in the treatment of acne and warts . Furthermore, the ethyl acetate and n-butanol fractions of Taraxacum officinale Web. Have exhibited anti-inflammatory and antibacterial properties , the chloroform extract has been shown to possess anticancer properties , polyphenolic compounds in dandelion have been found to have antioxidant properties , while methanol and petroleum ether extracts have been found to have a choleretic effect . Thymus serpyllum L., commonly known as creeping thyme, Bogorodskaya grass, and thyme, is widely distributed in countries bordering with the Mediterranean, parts of Central Europe, and Asia . The plant is found in the forest and forest-steppe zones of the European part of Russia, as well as in Western and Eastern Siberia, the Urals, Transbaikalia, and central regions of Kazakhstan, including the Ulytau mountains . The plant is a valuable source of essential oil and pharmacologically active polyphenolic compounds, as reported in the literature . Thymol is the major component of the essential oil, comprising up to 42% of the oil, alongside other constituents such as carvacrol, n-cymol, α-terpineol, and borneol. Additionally, tannins, bitterness, gum, triterpene compounds including ursolic and oleanolic acids, flavonoids, and a significant amount of mineral salts have been detected in the herb. The mature seeds of the plant have also been found to contain 33.6% fatty oil . Thyme also exhibits a high content of flavonoid phenolic and carotenoid antioxidants, such as zeaxanthin, lutein, apigenin ( b), naringenin, and luteolin ( b) (see above), as reported previously . Thymus serpyllum , a medicinal herb, is rich in oil and pharmacologically active polyphenolic compounds. It has been used in official and folk medicine to treat various ailments for many years. The herb, harvested during the flowering period, is used as a medicinal raw material after being threshed and dried in the shade or dryers at 35–40 °C. Thyme preparations have demonstrated expectorant, antimicrobial, and antifungal properties. Thyme is also used to treat a range of ailments, including sore throat, stomatitis, periodontal disease, asthma, headaches, laryngitis, and digestive system disorders . Thyme extract has been shown to possess antitumor and antioxidant activity. Additionally, thyme is utilized as an alexiteric, emmenagogue, analgesic, and sedative, and in the form of ointments and lotions for rheumatism and skin diseases . Due to its sedative and diuretic properties, medicines containing Thymus serpyllum can be employed for pruritic dermatoses . Bulgarian herbalists think that creeping thyme can be a constituent of medicines for treating eczema, neurodermatitis, and urticaria, and can be used as an external remedy to eliminate wrinkles . A study of Thymus serpyllum L. essential oil has shown that it has a strong fungistatic effect: there was almost 100% suppression of the growth of the tested fungi. Four strains of dermatomycete fungi were used in the study: Trichophyton mentagrophytes , Microsporum gypseum , Microsporum canis , and Trichophyton violaceum ; two strains of mold fungi: Scopulariopsis brevicaulis , Aspergillus niger and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and its own isolate from dog skin (IZ 1), which causes dandruff in dogs . Vaccinium myrtillus L. is a plant species that is predominantly found in forested areas in Northern Europe and North America , as well as in Europe, Asia, and North America . Its distribution in Kazakhstan is limited to the southwestern region of Altai, situated in Eastern Kazakhstan . The fruits of V. myrtillus are a rich source of bioactive compounds such as phenolic acids (chlorogenic acid being the most common), flavonoids (isoquercetin), and resveratrol in the leaf extract . In addition, they contain polyphenols, phenolic acids, and anthocyanins . Moreover, they are a rich source of trace elements and other phytochemicals such as organic acids, sugars, vitamins, fibers, and phenolic compounds (both anthocyanins and non-anthocyanins), glycosides (arbutin and myrtillin), peryl alcohol, resins, triterpene alcohol, pyrocatechin and pyrogallic tannins, free hydroquinone, ascorbic acid, carotene, and organic acids. They also contain retinol acetate, thiamine bromide, and pectin . According to traditional medicinal practices, V. myrtillus flowers are utilized as ointments to treat a lot of skin-related diseases, including but not limited to ulcers, eczema, burns, bruises, rashes, varicose veins, and acne . Moreover, this plant has demonstrated blood-glucose-lowering effects and has been shown to possess antioxidant, anti-inflammatory, and lipid-lowering properties, indicating its potential efficiency in treating chronic inflammatory diseases, including those linked to aging such as cancer and cardiovascular disease . Blueberries are regarded as a valuable source of antioxidants, which explains their utilization in treating numerous ailments (e.g., inflammation, cardiovascular disease, cancer, diabetes, and aging-related diseases) linked to an increased oxidative stress . Blueberry leaves have been found to possess hypoglycemic effects, attributed to the presence of myrtillin glycoside, which acts similarly to insulin and regulates pancreatic function. Dried blueberries are known for their astringent properties, while fresh blueberries are known to have carminative, anti-inflammatory, diuretic, hemostatic, antibiotic, and vitamin properties, and can regulate metabolism and digestive activity. In traditional medicine, blueberries have been used to treat various ailments such as bile duct and bladder stones, coughs, scurvy, and pulmonary tuberculosis. They have also been used to treat gastroenterocolitis and diarrhea, particularly in children. Due to their high content of vitamin C, blueberries have been used for scurvy treatment and external application to cure stomatitis and pharyngitis, which are accompanied by oral cavity wounds and ulcers . The high antioxidant potential of blueberry seed oil, which contains chlorogenic acid ( d), isoquercetin ( b), and resveratrol, as well as α-linolenic, linoleic, and oleic acids, has been well identified. Furthermore, a plant extract of isoquercetin ( b) has been found to have a dose-dependent inhibitory effect on edema caused by allergic contact dermatitis . The component composition of V. myrtillus species is represented by various groups of phenolic compounds, which are known to be effective exogenous factors of antioxidant protection . Currently, the great popularity of Scots blueberries is due to the high content of anthocyanins with antioxidant activity . In the case of skin diseases, long-lasting wounds, and ulcers, infusions made of fruits or leaves in the form of perfumes are applied externally. Viscum album L., commonly known as white mistletoe, is an evergreen hemiparasitic plant that grows widely in the Caucasus, Europe, and western and southern Asia . Various chemical components have been identified in mistletoe through chemical studies, including viscotoxins (a mixture of amino acids), phenylpropanes, lignans, flavonoids, amines (viscalbin, norviscalbin, tyramine, β-phenylethylamine viscamine), α-viscol (β-amirin), β-viscol (lupeol), polysaccharides, lectins, fatty acids (oleic, linoleic, and palmitic acids), alcohols (pinit, inositol, quebrachite), resinous substances, and mineral salts . Moreover, syringinin glycoside was detected in mistletoe bark . Triterpene saponins (oleanolic and ursolic acids), vitamin C, carotene, vecerin, viscol, and choline derivatives (propionylcholine and acetylcholine) have also been found in this plant, the levels of which depend on the host tree on which the mistletoe grows, according to the authors of . Viscum album L. has a rich ethnopharmacological history, with traditional uses including the treatment of various ailments such as epilepsy, anxiety, hypertension, internal bleeding, atherosclerosis, inflammation, and headaches. Additionally, it has been used as an antidote in some cultures . Mistletoe-based preparations possess hypotensive and analgesic effects. For instance, a tincture of fresh mistletoe leaves, found in the “Akofit” preparation, is utilized to treat acute radiculitis . The vasodilators “Omelen” and “Viskalen” are recommended for hypertension, while the liquid and dry extract “Reviscen” is useful for treating atherosclerosis, as it decreases blood pressure, dilates blood vessels, enhances cardiac activity, reduces nervous system excitability and intestinal atony, and acts as a hemostatic agent . The active compound viscotoxin effectively cures cancer and inhibits its progression. The lectin present in mistletoe is a natural pesticide that hinders bacterial and parasitic infiltration into the body . Additionally, Viscum album L. exhibits antioxidant , antitumor , antiviral, antibacterial, anti-inflammatory, antiepileptic, and immunostimulatory activity, and is also employed to treat neurological disorders . Preparations containing white mistletoe are utilized in obstetric and gynecological practice and are prescribed for colpitis and prolonged uterine bleeding . Studies have shown that methanol extract significantly reduces the pigmentation of primary human melanocytes. In addition, reporter promoter analysis showed that the methanol extract inhibits the transcription of microphthalmia-associated transcription factor, melanophilin, tyrosinase protein-2, and tyrosinase genes in melanoma cells . The presence of viscumneoside III and viscumneoside V in Viscum album L. extract was found to significantly inhibit the expression of monocyte chemoattractant protein 1 (MCP-1). These results imply that Viscum album L. extract, as well as its active components, viscumneoside III and viscumneoside V, can regulate the production of MCP-1 and may have the ability to reduce skin toxicity induced by erlotinib by altering the activity of macrophages, without interfering with the anticancer effect of the drug . Research of an alcoholic extract of mistletoe has indicated its efficiency in treating skin cancer. The extract is assumed to boost immune mechanisms, which in turn restrain the proliferation of cancerous cells. Additionally, when V. album extract was administered in combination with doxorubicin, it exhibited a synergistic effect, enhancing the antitumor impact on Ehrlich tumor cells . Mistletoe has been proved as a promising treating remedy in the field of dermatology, where it has been employed to manage various cutaneous conditions such as dermatitis, age-related pigmentation, moles, acne, and papillomas, as well as psoriasis and rashes . In this review, we systematically summarized 30 plants of the flora of Kazakhstan, which were used traditionally for the treatment of skin diseases. As shown by analysis of the scientific literature, in Kazakhstan, these plants have been insufficiently studied to identify the pharmacological activity in the treatment of skin diseases. Earlier works described a number of plants that were limited to the data on their use in the treatment of various types of dermatosis. While conducting a literature search, we paid attention to the phytochemical composition of plants, especially to those secondary metabolites that condition the pharmacological effect in the treatment of skin diseases. Mechanisms of action of these biologically active compounds are described for some plants. The shows medicinal plants with an indication of their main biologically active substances and biological/pharmacological activity. As we know and according to the literature data, plants Matricaria recutita , Plantago major , Chelidonium majus , Achillea millefolium , and Bidens tripartita have good potential and are used as therapeutic agents both separately and as part of phytopreparation. The high pharmacological effect is due, in most cases, to the content in plants of flavonoids (quercetin, isoquercetin, kaempferol, apigenin), phenolic acids (chlorogenic, caffeic, rosmarinic acid), tannins, and other BAS, which synergize the pharmacological action, and the pharmacological action of plant extracts is quite broad, including wound healing, antioxidant, anti-inflammatory, antimicrobial, and anticarcinogenic effects. In turn, it should be noted that there are several plant species that have been little studied for pharmacological activity in the treatment of skin diseases ( Tanacetum vulgare , Gnaphalium uliginosum , etc.). These plants can be the subject of future studies, which may add to the arsenal of phytopreparations for the treatment of dermatitis, eczema, psoriasis, lichen, and other skin inflammatory processes. In our work, we conducted a literature search, which allowed us to conclude that the medicinal plants of the flora of the Republic of Kazakhstan are rich in medicinal plants, which are widely used in medicine to create dosage forms and preparations. Most of these plants have a complex of biologically active substances that give them high biological activity. Many plants are essential for the treatment of a wide range of ailments, including skin conditions, and can be used as natural alternative medicines. In general, our results confirm the importance and value of medicinal plants of the flora of Kazakhstan for scientific and medical research.
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Synthetic Routes to Approved Drugs Containing a Spirocycle
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7f3ea6fc-620b-471f-ae55-28804bec251b
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10223694
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Pharmacology[mh]
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Spirocycles are becoming increasingly popular in drug discovery. Inherently three-dimensional, these high-F sp3 (F sp3 = number of sp 3 hybridized carbons/total carbon count) motifs are expected to rid drug candidate molecules of many liabilities which would be inevitably encountered with the formerly typical scaffolds built on flat aromatic carbo- and heterocycles. Hence it is perhaps unsurprising that the trend (articulated for the first time in the Escape from the Flatland publication by Lovering and colleagues ) has now translated itself in an increasing number of approved drugs containing spirocyclic motifs. We and these colleagues recently published a review article on the use of spirocycles in drug discovery . In this review, we partly touched upon the syntheses of approved spirocyclic drugs. However, that overview was far from exhaustive. Moreover, it only episodically touched upon approved spirocyclic drugs, also focusing on advanced, though not approved, drug candidates. Considering the apparent interest in the subject from the research community (at the time of writing this review, publication was cited over 100 times in the 1.5 years since its appearance in the literature), we thought it prudent to compile a more comprehensive compendium of synthetic routes to approved drugs containing a spirocyclic motif, with a particular emphasis on alternative synthetic approaches to the same drug molecule, if such exist. The present review is the result of realizing that intent. The review covers 23 drug molecules approved for medical use over the period of 1959 to 2021. Griseofulvin is an antifungal agent used to treat fungal infections of the fingernails and toes. It is one of the earliest spirocyclic drugs, and was approved in 1959. The exact mechanism of its action remains unclear, with putative targets including tubulin beta chain and keratin, type I cytoskeletal 12 through which griseofulvin interferes with fungal mitosis. Two total syntheses of griseofulvin were accomplished in the 1990s. Yamoto and co-workers reported the synthesis of racemic griseofulfin in 1990 . The synthesis commenced with 7-chloro-4,6-dimethoxy-3(2 H )-benzofuranone ( 1 ) which was condensed with acetaldehyde diethyl acetal in the presence of Ti(IV) chloride to give ( Z )-configured ethylidene derivative 2 . In order to bring the olefin configuration to the desired ( E ), compound 2 was subjected to somewhat low-yielding photochemical isomerization. The ( E )-configured olefin 3 was now set for a Diels–Alder reaction with diene 4 (10 equiv.), which was accomplished in refluxing toluene to furnish racemic griseofulvin in 46% yield . Pirrung and co-workers reported the first asymmetric synthesis of natural enantiomer -griseofulvin in 1991 . Commercially available phenol 5 was regiospecifically chlorinated to provide intermediate 6 . The latter underwent a Fries rearrangement in the presence of aluminum chloride and the liberated phenol was involved in the Mitsunobu coupling with non-racemic allylic alcohol 7 to give rise to pentasubstituted benzene building block 8 . The acetyl group in the latter was deprotonated and methoxycarbonylated by treatment with Mander’s reagent ( 9 ) and the resulting β-keto ester was subjected to Regitz diazo transfer to provide the diazo compound 10 . Decomposition of the latter was accomplished with the use of rhodium pivalate catalyst and the resulting rhodium carbene underwent intramolecular trapping with the nearby oxygen atom, followed by sigmatropic rearrangement to give benzofuranone 11 . Conversion of the latter to non-racemic methyl ketone 14 was accomplished via ozonolysis, the Wittig reaction with phosphonium ylide 12 , tert -butyl ester cleavage with TFA and decarboxylation on treatment with diphenylphosphoryl azide ( 13 ), followed by exposure to refluxing aqueous hydrochloric acid. Finally, methyl ketone 14 was subjected to treatment with sodium methoxide on which it underwent the Dieckmann cyclization. The resulting cyclohexane-1,3-dione (griseofulvinic acid) was O -methylated with diazomethane to furnish -griseofulvin . Spironolactone is a relatively old multi-target drug that is primarily used to treat high blood pressure and heart failure. Additional therapeutic applications of this drug include edema, cirrhosis of the liver and primary aldosteronism. The mechanism of action of spironolactone includes binding to intracellular mineralocorticoid receptors (MRs) in kidney epithelial cells, which leads to the inhibition of aldosterone binding . The first industrial synthesis of spironolactone was reported in 1957–1959 by Cella and co-workers of G. D. Searle and Co. . The synthesis commenced with previously reported ethynyl steroid building block 15 . It was carbonylated at the alkyne terminus by treatment with methyl magnesium bromide, followed by quenching with carbon dioxide. Hydrogenation of alkyne 16 over the Lindlar catalyst elaborated the propionic acid side chain in 17 required for the formation of the spirocyclic lactone at the next step. Cyclization on treatment with p -toluenesulfonic acid yielded unsaturated lactone which was hydrogenated over palladium on carbon to yield spirocyclic lactone 18 . Oppenauer oxidation of the alcohol functionality in 18 was accompanied by the migration of the double bond to give α,β-unsaturated ketone 19 . The conjugated system in 19 was extended by γ,δ-oxidation with chloranil to give compound 20 , and set the scene for the conjugate addition of thioacetic acid at the d-position, which proceeded with high diastereoselectivity and furnished spironolactone in 86% yield . Another synthetic approach of spironolactone was patented in 1980 by Ciba Geigy scientists . The synthesis stems from dehydroepiandrosterone ( 21 ), which was treated with 3-chloropropionaldehyde ethylene acetal and lithium to furnish tertiary alcohol 22 in diastereoselective fashion. The following sequence of steps (which proceeded in 75% overall yield) included bromination of the double bond, oxidation of the secondary alcohol to ketone and, finally, base-promoted double dehydrobromination, which led to α,β,γ,δ-unsaturated ketone (androstadienone) 23 . Interestingly, the conjugate addition of thioacetic acid was in this case performed prior to the elaboration of the spirocyclic lactone in the last step. Thioacetic acid also caused the deprotection of the aldehyde to furnish 24 . Oxidation of the aldehyde to the carboxylic acid in the presence of sulfuric acid brought about the closure of the lactone ring and led to the formation of spironolactone in 51% yield from 23 . A fundamentally different route to spironolactone performed in multigram scale was developed by the Chemical Process Research and Development scientists at The Upjohn Co. in the late 1980s . The synthesis relied on the readily available ethynyl steroid building block 25 , the ketone group of which was protected as ethylene glycol ketal in the first step. The key transformation which led to the formation of spirocyclic lactol intermediate 27 was rhodium-catalyzed hydroformylation at elevated temperature and pressure. Oxidation of 27 to lactone was achieved with PDC and the ketal protecting group was removed to afford 28 . The formation of α,β,γ,δ-unsaturated ketone 20 was achieved with chloranil (vide supra). and conjugate addition of thioacetic acid (catalyzed by TMSOTf) at the δ-position furnished spironolactone . Among many non-industrial syntheses of spironolactone described in the literature , the most recent one reported by Nagamitsu, Ohtawa and co-workers is quite notable . The synthesis commences with dehydroepiandrosterone ( 21 ) and is reliant on the innovative approach to γ-lactonization of homopropargyl alcohols, which involves terminal borylation of a homopropargyl moiety followed by m -CPBA oxidation. The latter sequence leads to the transformation of the alkyne moiety into a ketene intermediate which is trapped intramolecularly to give a γ-lactone. Hence, the starting material 21 was treated with propargyl magnesium bromide (under Hg(II) catalysis) to give homopropargyl alcohol 29 in nearly quantitative yield. Application of the γ-lactonization protocol described above led to the formation of spirocyclic lactone 18 . Oxidation of the secondary alcohol moiety in the latter with Dess–Martin periodinane (DMP) gave γ,δ-unsaturated ketone 28 . Transformation of the latter into spironolactone was analogous to the Upjohn Co. synthesis described above. Oxidation with chloranil installed the polyunsaturated ketone system ( 20 ) which is suitable for the conjugate addition of thioacetic acid (also TMSOTf-catalyzed in this case), resulting in the formation of spironolactone . Fluspirilene , a spirocyclic antipsychotic drug was discovered at Janssen Pharmaceutica in 1963, and by 1970 it had been approved for the treatment of schizophrenia . The primary molecular targets of fluspirilene in the central nervous system are dopamine D 2 and serotonin 5HT 2A receptors, as well as α1 subunit of voltage-dependent calcium channel . Fluspirilene is an old drug, and reviewing the vast patent literature on its synthesis would be tedious. Instead, we will focus on the most notable cases of fluspirilene assembly from the relatively recent literature. For instance, an efficient synthesis of the key spirocyclic building block 30 needed for the synthesis of the drug was published in 2018 by a team of Italian and Polish scientists . The synthesis commenced with 1-benzyl piperidin-4-one, which was involved in the Strecker reaction with aniline and trimethylsilyl cyanide to give nitrile 31 . The latter was hydrated in sulfuric acid and the resulting α-anilino carboxamide 32 was condensed with N,N -dimethylformamide dimethyl acetal to form spirocyclic imidazolinone 33 . The C-N double bond in 33 was reduced to give imidazolidinone 34 and the benzyl group was removed by hydrogenation to give the target spirocycle 30 . Interestingly, it is the synthetic assembly of the 4,4-bis(4-fluorophenyl)butyl halide building block (for the alkylation of the key spirocycle 30 ) that received much of the synthetic route development. For instance, in 2001, an Italian group reported an interesting synthesis of fluspirilene, starting from 4,4′-difluorobenzophenone ( 35 ). Acetylene bis-lithium salt was added to the keto group of 35 in the presence of ethylene diamine to give allylic alcohol 36 . The latter was hydroformylated in the presence of a rhodium catalysts cyclic hemiacetal 37 . Sodium borohydride reduction of the latter furnished diol 38 , which lost its doubly benzylic tertiary alcohol hydroxy group on hydrogenation. Alcohol 39 was brominated to alkyl halide 40 , which then was used in the completion of fluspirilene synthesis . A different approach to bromide 40 was reported in 2011 by a Shanghai Institute of Organic Chemistry group . Diol 38 was obtained by reacting γ-butyrolactone with excess 4-fluorophenyl magnesium bromide. Thereupon, it was dehydrated in refluxing aqueous hydrochloric acid to give olefinic alcohol 41 , the bromination of which gave unsaturated alkyl bromide 42 . Hydrogenation of the latter resulted in double bond saturation and went on without a loss of the bromide to give alkylating agent 40 , which was used in the preparation of fluspirilene . Fenspiride , a spirocyclic oxazolidinone, acting as an antagonist of a 1 -adrenergic receptors, was first approved in selected European countries for medical use in the 1970s for the treatment of otolaryngological or ENT (ear, nose and throat) organ diseases, as well as respiratory tract diseases . The structure of fenspiride comprises a well-established feature for binding to the hERG channel (an aromatic ring at a certain distance from a basic amine center) . Unfortunately, it does bind to this off target, and causes prolongation of the QT interval. For this reason, i.e. the increased risk of torsades de pointes, fenspiride was withdrawn from the pharmaceutical market in most markets around the world . One of the early patents, obtained by the Italian Voghera company for the synthesis of fenspiride, describes the assembly of the target molecule starting from 1-phenethyl piperidin-4-one ( 43 ), which was involved in the Reformatsky reaction with ethyl bromoacetate. After the conversion of the resulting β-hydroxy ester 44 to hydrazide 45 , the latter was diazotized, which triggered a Curtius rearrangement of the intermediate acyl azide, and the isocyanate product of the rearrangement was trapped, intramolecularly, by the nearby hydroxy group, to give fenspiride . In 1994, a Mexican group disclosed an alternative approach to elaborating the spirocyclic oxazolidinone based on 1-phenethyl piperidin-4-one, which includes the Strecker-type reaction of the ketone with trimethylsilyl cyanide. The resulting O -silylated a-hydroxy nitrile 46 was subjected to reduction with lithium alumohydride to give β-amino alcohol 47 . The latter was treated with trichloromethyl carbonate, which donated the carbonyl group and resulted in the closure of the oxazolidinone ring of fenspiride . In 2019, the Indian company Emcure Pharmaceuticals jointly with a University of Pune group published a completely novel route to fenspiride which is based on the nitro aldol reaction and is amenable to industrial-scale production of the drug substance . In fact, the researchers disclosed two possible approaches to fenspiride for comparison, and reached a conclusion that the nitro aldol route was preferred from the standpoint of pharmaceutical production. In the first approach, Corey–Chaykovsky epoxidation gave epoxide 48 , which was opened up by sodium azide to give β-azido alcohol 49 . Reduction by hydrazine (presumably to alcohol 47 ) and treatment with CDI (the donor of the carbonyl) afforded fenspiride. In the second, preferred, approach, ketone 43 was condensed with nitromethane anion and the resulting nitro compound 50 was reduced to the respective amine and Boc-protected to afford intermediate 51 . Deprotonation of the hydroxy group in the latter with potassium tert -butoxide triggered intramolecular displacement of the tert -butoxy group and ring closure to fenspiride . Additionally, the second route was performed on a large scale, starting with 1 kg of compound 50 to furnish 880 g of fenspiride. It is worth noting the effective procedure for the late-stage [ 11 C], [ 13 C] and [ 14 C] carbon isotope labeling of cyclic carbamates recently developed by Audusio and co-workers . The method allows the incorporation of labeled carbon dioxide into azidoalcohol 10 to furnish C-labeled fenspiride in a direct, cost-effective and sustainable manner. Amcinonide is a topical corticosteroid approved in 1979 for the treatment of various inflammatory dermatological conditions such as atopic dermatitis and allergic contact dermatitis. Amcinonide displayed high affinity to the glucocorticoid receptor which is thought to be the main biological target mediating its anti-inflammatory action. Binding to the receptor and its activation by amcinonide eventually leads to the induction of phospholipase A2 inhibitory proteins, which suppresses the production of arachidonic acid and the downstream production of prostaglandins as chemical mediators of inflammation . Unfortunately, synthetic routes to amcinonide are poorly described in the literature. Even the original patent issued to the American Cyanamide company features little synthetic detail. An industrial synthesis of amcinonide patented by an Indian company in 2018 is described in sufficient detail. It starts with readily available natural steroid 2-((10 S ,13 S ,14 S )-10,13-dimethyl-3-oxo-6,7,8,10,12,13,14,15-octahydro-3 H -cyclopenta[a]phenanthren-17-yl)-2-oxoethyl acetate ( 52 ). The latter is subjected to epoxidation of the non-conjugated double bond using 1,3-dibromo-5,5-dimethylhydantoin (dibromantin, 53 ) and aqueous perchloric acid, followed by the treatment of the resulting bromohydrin with potassium carbonate. Epoxide 54 is formed with high diastereoselectivity. The next step is dihydroxylation of the cyclopentene ring, which is brought about by potassium permanganate and formic acid in acetone. Finally, diol 55 is formed diastereoselectively, then subjected to the treatment with 70% hydrofluoric acid, followed by the addition of cyclopentanone. The net result of the latter step is epoxide opening with the fluoride anion and spirocyclic ketal formation leading to amcinonide . Guanadrel is an anti-hypertension drug containing a guanidine moiety which was approved in 1979 . It exerts its therapeutic action by blocking the sodium-dependent noradrenaline transporter, and thus is a typical adrenoblocker . The industrial synthesis of guanadrel was patented in 1970 by its inventors . In the first step, cyclohexanone undergoes ketalization with 3-chloro-1,2-propandiol ( 56 ), forming 2-chloromethyl-1,4-dioxyspiro[4,5]decane ( 57 ). The latter building block is used to alkylate phthalimide sodium salt to give the protected amine derivative 58 . Removal of the phthalimide protecting group with hydrazine liberates the primary amino group in 59 , which is reacted with S -methyl thiourea sulfate in aqueous medium on steam bath heating to give guanadrel as a sulfate salt . In 1999, Goodman of UC San Diego developed a novel guanidine synthesis using triflyl-diurethane protected guanidines and applied it to the synthesis of guanadrel. In this synthesis, cyclohexanone was ketalized with acetylamino-substituted diol 60 . The resulting spirocyclic building block 61 was deacetylated with hydrazine and reacted with N,N ′-di-Cbz- N ″-triflylguanidine ( 62 ). Upon the displacement of the triflic amide, which is a leaving group in 62 , bis-Cbz-protected guanidine 63 was hydrogenated over palladium on charcoal to give guanadrel in quantitative yield as a free base . Interestingly, the attempted use of Boc protecting groups in lieu of Cbz failed, since the deprotection under acidic conditions led also to the removal of the acid-prone ketal . First synthesized in 1966 and approved for medical use in 1986 buspirone , a spirocyclic serotonin 5-HT 1A receptor agonist is primarily used to treat anxiety disorders . Several patented protocols for the preparation of buspirone , including industrial synthesis, have been reported in the literature. On the plant scale, the following route was initially employed for the production of buspirone. 1-(Pyrimidin-2-yl)piperazine ( 64 ) was alkylated with 4-chlorobutyronitrile ( 65 ), and the nitrile group was hydrogenated over Raney nickel catalyst to give butylamine 66 . The latter was condensed with commercially available 3,3-tetramethylene glutaric anhydride ( 67 ) in refluxing pyridine to give, after crystallization, buspirone . Over more than three decades following the approval of buspirone for medical use, several newer synthetic routes appeared in the literature journals, some of which will be discussed below. In 2008, Wei and co-workers from China University of Mining and Technology reported a new process for the production of buspirone. In the first step, condensation of cyclopentanone with methyl isocyanoacetate catalyzed by ammonia followed by acidic ester hydrolysis afforded cyclopentane-1,1-diacetic acid ( 68 ). Heating of the latter with ammonium carbonate at 200 °C afforded 3,3-tetramethyleneglutarimide ( 69 ). The latter was alkylated with butyl bromide 70 which was prepared, in turn, from 1-(pyrimidin-2-yl)piperazine ( 64 ) by alkylation with 1,4-dibromobutane. The last step afforded buspirone in nearly quantitative yield . In 2019, Steven Ley and co-workers reported a continuous flow method to prepare buspirone which involves the direct alkylation of 1-(pyrimidin-2-yl)piperazine ( 64 ) with alcohol 71 via a Ru(II)-catalyzed hydrogen-borrowing approach . In 2022, Beller and co-workers reported a remarkable, Ni-catalyzed reductive cross-coupling of nitriles with primary and secondary amines. In order to exemplify the scope of their methodology, a novel synthesis of buspirone was presented. Nitrile 72 prepared by alkylation of 3,3-tetramethylene glutarimide ( 69 ) with 4-bromobutyronitrile reacted 1-(pyrimidin-2-yl)piperazine ( 64 ) under 40 bar of hydrogen in the presence of nickel triflate and a special triphosphine ligand to give 80% yield of buspirone . Discovered in 1975 ivermectin , an antiparasitic drug was initially approved for veterinary use in 1981. After extensive clinical research, ivermectin was approved in 1987 for use in humans, for the treatment for a wide range of parasitic infections . Ivermectin acts by binding to parasites’ glutamate-gated sodium channels and forcing them to remain open, which causes the overflow of chlorine anions and, consequently, hyperpolarization of the cell membranes. This eventually kills the parasite . Ivermectin is an approximately 80:20 mixture of two individual macrocyclic lactone (macrolide) compounds, ivermectin B 1a and B 1b . These compounds are produced by hydrogenation of the C 22 = C 23 double bond of the mixture of avermectins B 1a and B 1b which are, in turn, produced by fermentation of Streptomyces avermitilis , the bacteria discovered in 1970 by Satoshi Ōmura, for which discovery he and William Campbell of Merck were awarded the Nobel Prize in Physiology or Medicine . In one of the more recent patents, Bayer described the hydrogenation of avermectins to ivermectins performed over the RhCl 3 ∙H 2 O catalyst in the presence of triphenylphosphine . In addition, several successful total syntheses of avermectin and ivermectin by academic research groups have been reported in the literature . However, these syntheses are not economically viable for the production of the drug. Rifabutin , a macrocyclic semisynthetic drug of the rifamycin family was developed by the Italian drug company Farmitalia Carlo Erba, and received FDA approval in 1992 for the treatment of tuberculosis, as well as other bacterial infections such as Mycobacterium avium complex . The drug works via the inhibition of the DNA-dependent RNA polymerase in bacteria, leading to a suppression of RNA synthesis and cell death . The original synthesis of rifabutin was reported by Marsili and colleagues from Framitalia in 1981 . The synthesis relied on 3-aminorifamycin S ( 73 ) prepared via azidation of rifamycin S ( 74 ), which was accompanied by a spontaneous loss of nitrogen molecule . Compound 73 was treated with ammonia solution in THF to give 3-amino-4-deoxo-4-iminorifamycin S ( 75 ). The latter was isolated by crystallization and further involved in cyclocondensation with N -isobutyl piperidone, performed at ambient temperature in the presence of ammonium acetate as a mild acidic catalyst. As a result, a spirocyclic imidazoline moiety was installed, and pure rifabutin was isolated without chromatographic purification . Spirapril , a spirocyclic inhibitor of the angiotensin-converting enzyme (ACE) had been originated and developed by the Schering-Plough Corporation (now Merck & Co.) and was approved in 1995 for the treatment of hypertension. ACE is a part of the renin–angiotensin system regulating blood pressure. Inhibition of ACE suppresses the production of the potent vasoconstrictor angiotensin II from its inactive precursor pro-angiotensin and thus prevents the increase in blood pressure . Spirapril is an ethyl ester prodrug. It undergoes biotransformation to the respective carboxylic acid, spiraprilat, which is the active ACE inhibitor . Several syntheses of spirapril have been reported in the patent and periodic literature around the time of the drug’s approval. The original Schering-Plough route was patented in 1984 and later disclosed in greater detail and with a few reaction sequence variants in a 1989 research article . The synthesis commenced with Cbz-protected 4-oxoproline methyl ester ( 76 ), the carbonyl group of which was protected as spirocyclic thioketal, to give methyl ester 77 . The Cbz group was removed by 20% hydrobromic acid in glacial acetic acid, to give amino acid 78 . The ester side chain building block was prepared by reductive amination of the keto ester 79 . The resulting amino acid 80 was obtained as a mixture of diastereomers. Without separation, the mixture was activated as N -hydroxysuccinimide ester ( 81 ) and reacted with 78 in the presence of triethylamine, to give a good yield of spirapril after diastereomer separation . An alternative synthesis reported by the Squibb Corporation (now Bristol Myers Squibb) in 1988 also made use of spirocyclic amino acid 78 . The latter was Boc-protected and the carboxylic function was esterified with 2-(trimethylsilyl)ethanol in the presence of DCC and DMAP to give good yield of ester 82 . The Boc group was then removed by p -toluenesulfonic acid (PTSA) and the liberated free pyrrolidine 83 was acylated with carboxylic acid 80 activated by DCC/HOBt. This two-step sequence gave ester 84 in 70% after diastereomer separation. The removal of the 2-(trimethylsilyl)ethyl group with tetrabutylammonium fluoride trihydrate gave spiropril in quantitative yield . Irbesartan , a spirocyclic antihypertensive drug was developed by Sanofi Research (now Sanofi-Aventis) and approved for medical use in 1997. The compound is a typical representative of the sartan family of hypertensives, which act by blocking the angiotensin type 1 (AT 1 ) receptor and preventing its activation and downstream events leading the blood pressure increase . Irbesartan possesses four key elements of structure which are typical of sartans and could be traced back to losartan, the first-in-class AT 1 receptor blocker: biphenyl substituted with tetrazole in the ortho position of the distal ring, five-membered nitrogen heterocycle (in this case, imidazoline-5-one) and an n -butyl side chain . The original synthetic route to irbesartan was described in detail in 1995 publication by Bernhart and co-workers (Sanofi Research) . The synthesis relies on commercially available tetramethylene glycine ethyl ester ( 85 ) as the starting material. Amino ester 85 was condensed with imidate 86 in the presence of a catalytic amount of acetic acid in refluxing xylene to give imidazolin-5-one 87 . The latter was alkylated with 4′-(bromomethyl)-[1,1′-biphenyl]-2-carbonitrile and the resulting derivative 88 was treated with tributyl tin azide in refluxing xylene, to give irbesartan . Interestingly, the key spirocyclic imidazolin-5-one building block 87 was recently accessed by Wu and DiPoto via a novel annulation of azaoxyallyl cations. Cyclopentanoyl chloride was first transformed into O -benzyl hydroxamic acid 89 . The latter was treated with the base to generate azaoxyallyl cation 90 , which reacted with pentanenitrile to give benzyloxy-substituted imidazolin-5-one 91 . N -Deoxygenation with samarium iodide gave 87 , thereby completing the formal synthesis of irbesartan . Intermediate 87 was also key in the synthesis of irbesartan patented by a group of Israeli scientists in 2004 . The convergent route started with C -phenyl tetrazole ( 92 ), which was protected with a trityl group and borylated in the para position via lithiation to give boronic acid 93 . In parallel, intermediate 87 was N -alkylated with p -bromobenzyl bromide and the resulting intermediate 94 was involved in Suzuki coupling with boronic acid 93 , to give trityl-protected irbesartan 95 . Removal of the trityl group furnished the target molecule . Among the synthetic routes to irbesartan featured in the patent literature, the following synthesis from Dr. Reddy’s Laboratories is notable. Similarly to the Sanofi route (vide supra), the synthesis started with tetramethylene glycine ( 96 ), which was acylated with pentanoyl chloride under phase transfer conditions to give carboxylic acid 97 which, in turn, was amidated with 4′-(aminomethyl)-[1,1′-biphenyl]-2-carbonitrile to give the ‘fully decorated’ precursor 98 . The latter was cyclodehydrated in refluxing toluene in the presence of trifluoroacetic acid, to give nitrile 88 and, after [3 + 2] cycloaddition with tributyltin azide, irbesartan . An approach rather similar in spirit to the Dr. Reddy’s route was described in 2006 by ArQule scientists . It is based on the three-component, microwave-promoted cyclodehydrative synthesis of irbesartan’s key precursor 88 from ester 85 , 4′-(aminomethyl)-[1,1′-biphenyl]-2-carbonitrile and pentanoic acid, in the presence of triphenoxyphosphine. The conversion of nitrile 88 to irbesartan was achieved via hte in situ generation of tributyltin azide . Finally, a fundamentally different approach to irbesartan was developed in 2022 at Beijing University of Chemical Technology . In this route, the spirocyclic scaffold of the drug was elaborated at a more advanced stage of the synthesis, as shown in . Glycine methyl ester was acylated by pentanoyl chloride and ester 99 was amidated with 4′-(aminomethyl)-[1,1′-biphenyl]-2-carbonitrile under forcing, base-promoted conditions, to give diamide 100 . The latter was cyclodehydrated in refluxing toluene in the presence of methanesulfonic acid to give intermediate 101 , which was found to be sufficiently C–H acidic to be doubly alkylated with 1,4-dibromobutane to furnish spirocyclic imidazoline-5-one 88 . Notably, the nitrile-to-tetrazole conversion was achieved without the use of the toxic tin reagent (used in all previously described syntheses), which makes this 2022 route quite environmentally friendly and amenable to the plant-scale production of the drug. Cevimelin , a spirocyclic derivative of quinuclidine acts as an agonist of muscarinic M 1 and M 3 receptors . Originating from the Israel Institute of Biological Research, the drug was developed by the Daiichi Sankyo company and gained regulatory approval in 2000 for the treatment of dry mouth and Sjögren’s syndrome . Recently, the drug has been investigated in such therapeutic areas as cognitive impairment and neurodegenerative diseases . In the original synthesis of cevimeline patented by the Israeli group , commercially available quinuclidin-3-one was converted to epoxide 102 using the Corey–Chaykovsky reaction with trimethyloxosulfonium iodide (Me 3 S + (O)I − ) and sodium hydride. The resulting epoxide 102 was treated with hydrogen sulfide in a basic aqueous medium, which led to epoxide opening and the formation of hydroxy thiol 103 . The latter was subjected to thio/oxa acetal formation on treatment with acetaldehyde in the presence of boron trifluoride etherate. Cevimeline contained in the diastereomeric mixture of products 104 was separated by fractional crystallization . The patent presented a rather elaborate scheme for the fractional crystallization. In 2008, the Canadian company Apotex Pharmachem, Inc. filed a patent application for an improved synthesis of cevimeline which is similar in spirit to the original Israeli route. The Corey–Chaykovsky epoxidation of quinuclidine-3-one was performed with potassium tert -butoxide, and the opening of epoxide 102 was achieved with thioacetic acid in lieu of hydrogen sulfide gas. Thioacetate 105 was condensed directly with acetaldehyde dimethyl acetal, which gave an excellent yield of the diastereomeric mixture 104 , from which cevimeline was isolated by diastereomer separation . In 2013, the Active Pharmaceutical Ingredient R&D Center of the Indian company Emcure Pharmaceuticals, Ltd. published an even further improved route to cevimeline, in which safe and odorless thiourea is utilized as a thiolating reagent. Epoxide 102 was first converted to hydroxy bromide 106 , which was then refluxed with thiourea in an aqueous medium to furnish thiolactone 107 . The latter was hydrolyzed with aqueous sodium hydroxide to give the key hydroxy thiol 103 , which was converted to cevimeline using the same routine as was developed in the original synthesis (vide supra) . An intriguing, stand-alone approach to enantiomerically pure hydroxy thiol ( S )- 103 was published by F. Hoffmann-La Roche, Ltd. six years prior to the approval of cevimeline for medical use . In this synthesis, the quinuclidine nucleus was elaborated quite late into the synthetic route. The synthesis relied on olefin 108 synthesized from pyridine-4-carboxaldehyde in a few trivial steps. Sharpless epoxidation gave enantiomerically pure (ee 94%) epoxide 109 , which was opened with benzyl mercaptan to give diol 110 . Mesylation of the primary hydroxy group set the scene for the intramolecular S N 2 reaction to form the quinuclidine core. This was achieved by the removal of the Boc group from the piperidine nitrogen atom in 111 and by triggering the nucleophilic substitution by DIPEA. The bnzyl group was removed from intermediate 112 by dissolving the metal (Ca) reduction in ammonia . Eplerenone , a spirocyclic antagonist of mineralocorticoid receptors developed by Ciba-Geigy was approved in 2002 for the treatment of hypertension and heart failure . It is related to spironolactone but displays better selectivity to its primary target through the affinity to androgen, progestogen, glucocorticoid, or estrogen receptors and thus is devoid of undesired sexual side effects typical of spironolactone, which limit its use . Among industrial syntheses of eplerenone, the route patented in 1998 by Searle & Co. is quite notable . The synthesis starts with steroid intermediate 20 (also useful in the synthesis of spironolactone, vide supra) which is subjected to regiospecific and stereoselective hydroxylation by fermentation with the Aspergillus ochraceus mold. The hydroxy group in 113 serves as a kind of masked double bond which is unmasked later in the synthesis. Condensation with two equivalents of cyanide (in the form of acetone cyanohydrin) results in the formation of polycyclic enamine 114 . The latter is hydrolyzed to ketone 115 , which is then put through a sequence of steps including treatment with sodium methoxide (opening the ketone linkage as the methyl ester with simultaneous elimination of the cyanide), mesylation of the hydroxy group, and elimination of methane sulfonic acid to give the olefinic intermediate 116 . The latter is epoxidized on treatment with hydrogen peroxide in a phosphate buffered solution, to give eplerenone . In 1997, Ciba-Geigy chemists published their synthetic route to eplerenone, which basically provides another approach to the synthesis of intermediate 116 starting from the spirocyclic steroid lactone 117 . Conjugate addition of hydrogen cyanide at the δ-position proceeded stereoselectively and gave the intermediate 118 . Reduction with DIBAL-H gave aldehyde alcohol 119 , which was oxidized into keto carboxylic acid 120 . Esterification with diazomethane gave the intermediate 116 (at which point the overall yield over four steps was a remarkable 69%). Finally, epoxidation with hydrogen peroxide gave eplerenone . An interesting eplerenone synthesis was published in 2006 by Pfizer’s process chemistry team . The synthesis commences with the same spirocyclic lactone 117 as was employed by Ciba-Geigy (vide supra). 2-Methylfuran was employed as a chemical equivalent of the carboxylic group. On Lewis acid catalysis, it underwent arylation of the distal conjugated double bond. The furan moiety in 121 was hydrolyzed to enedione 122 and the latter, without isolation, was ozonolyzed to give the hemiacetal 123 . The latter was subjected to Beyer–Villiger oxidation, to give carboxylic acid 124 , which was forced to close to isolable bis-lactone 125 . The strained lactone in 125 was opened up via elimination and the intermediate carboxylic acid was esterified with methyl sulfate to give ester 116 . The latter is only one epoxidation step away from eplerenone . Intermediate 125 is clearly key to the large-scale synthesis of eplerenone. In 2011, a Chinese Academy of Sciences group published an interesting approach to it from triene 117 , which involves the conjugate addition of silyl methyl cuprate, Tamao oxidation and another intramolecular oxa-Michael addition, to give cyclic ether 126 . Curiously, methyltrifluoromethyl dioxirane was found to selectively oxidize ether 126 to lactol 127 . PDC oxidation of the latter gave a nearly quantitative yield of lactone 125 , which could now be opened and epoxidized to eplerenone as described by Pfizer (vide supra) . Drospirenone is another steroid spirocyclic lactone drug structurally related to spironolactone and eplerenone, antagonists of mineralocorticoid receptors. However, pharmacologically, drospirenone is different from the latter two drugs. In addition to having a high affinity to mineralocorticoid receptors, it binds rather tightly to progesterone receptors and less tightly to androgen receptors , which underlies its use as a birth control medication approved in 2000 . The earliest synthesis of drospirenone was published by Schering AG in 1982 . The synthesis commenced with compound 128 , obtained from 3β-hydroxy-5,15-androstadien-17-one by Corey cyclopropanation. Microbial hydroxylation of 128 gave diol 129 , which was mono-pivaloylated at the less-hindered hydroxyl group, to give allylic alcohol 130 . The olefinic portion of 130 was epoxidized with tert -butyl hydroperoxide under vanadium(IV) oxide-acetylacetonate catalysis. The resulting epoxide 131 was converted to chloride 132 by treatment with Ph 3 P/CCl 4 in pyridine. Reductive elimination of the chlorine atom on treatment with zinc in acetic acid, followed by saponification, produced the diol 133 . The latter was subjected to Simmons–Smith cyclopropanation, to establish the basic core of the target compound ( 134 ). Now the scene was set to establish the spirocyclic lactone moiety. Propargylic alcohol was doubly deprotonated and added to the ketone portion of 134 , to give propargylic tetraol 135 . The triple bond in 135 was hydrogenated over calcium carbonate. The resulting compound 136 was treated with PDC, which triggered three events: oxidation of the primary alcohol moiety to carboxylic acid and lactonization, oxidation of the secondary alcohol to ketone, and dehydration, to form the α,β-unsaturated system of drospirenone . An approach similar in spirit to the synthesis of drospirenone by Schering AG was patented in 2010 by Evestra, Inc. . The synthesis starts with bis-cyclopropane 134 , to which the C -anion of ethyl propiolate was added to the carbonyl group. Hydrogenation of the triple bond in the resulting intermediate 137 gave triol 138 , in which the secondary alcohol moiety was oxidized by TEMPO. Treatment of ketone 139 with methanolic potassium hydroxide followed by acidification with acetic acid led to the formation of spirocyclic lactone and the dehydration of the β-hydroxy cyclohexanone moiety, which resulted in drospirenone . A different approach to the construction of drospirenone’s spirocyclic lactone motif was reported in 2008 by a University of Bologna group . Ketone 134 was treated with excess vinyl magnesium bromide. The resulting tertiary allylic alcohol 140 was involved in the olefin metathesis reaction with methyl acrylate catalyzed by the second-generation Hoveyda–Grubbs catalyst. The resulting intermediate 141 , having all the requisite carbon atoms necessary for the formation of spirocyclic γ-lactone, was hydrogenated over palladium on charcoal, which resulted in the reduction of the double bond and the lactone ring closure, to give 142 . Final steps toward drospirenone were the Dess–Martin oxidation of the secondary alcohol moiety and the dehydration of the resulting ketone . Ledipasvir , an inhibitor of the hepatitis C virus non-structural protein 5A (HCV NS5A) was developed by Gilead Sciences and was approved in 2014 for the treatment of genotype 1 HCV in combination with sofosbuvir . The pertinence of this drug to the present review is determined by the spirocyclic pyrrolidine moiety in its structure. The complexity of ledipasvir’s structure mandates a lengthy synthetic route to assemble the drug molecule. The original synthesis patented by Gilead in 2013 is presented in . The synthetic scheme includes the assembly of the requisite building blocks from four different directions, thereby being remarkably convergent. From one direction, 2-bromofluorene ( 143 ) was iodinated in position 7, to give 144 . The methylene group in the latter was bis-fluorinated with N -fluorobenzenesulfonimide (NFSI) to give compound 145 , which was converted to an aryl magnesium species at the more reactive, iodo side and coupled with chloroacetic acid Weireb amide to give building block 146 . From another direction, Boc-protected 4-methylene L -proline ( 147 ) was converted to spiro dibromo cyclopropane 148 , which was dibrominated and converted to potassium carboxylate 149 . The latter was involved in a nucleophilic substitution reaction with the chloroacetyl compound 146 to give keto ester 150 , which was reacted with ammonium acetate to give the imidazole 151 . The assembly of the bicyclic pyrrolidine portion of ledipasvir started with enantiopure α-methyl benzylamine ( 152 ,) which was used to form a Schiff base with methyl glyoxylate hemiacetal. The Schiff base was involved in a Lewis acid-catalyzed Diels–Alder reaction with cyclopentadiene, and the desired bicyclic structure was established (compound 153 ). Three trivial transformations (debenzylation with double bond reduction, Boc-protection and ester hydrolysis) gave carboxylic acid 154 , which was employed in benzimidazole synthesis to give compound 155 . Now the two halves of the ledipasvir molecule ( 151 and 155 ) were brought together via a Suzuki coupling involving the in situ formation of the aryl boronic acid intermediate. The resulting intermediate 156 was deprotected and acylated at both liberated nitrogen atoms with N -methoxycarbonyl valine, to give lidepasvir. The Gilead Sciences route to ledipasvir may well be quite optimal, considering that there have not been any attempts to improve it as judged by the literature on the subject. However, in 2021, a Suzuki–Miyaura coupling of the two halves of the ledipasvir molecule was selected as a drug synthesis example to illustrate the power of the novel aryl halide to aryl boronic acid coupling protocol where no palladium catalyst is employed. The two research groups from China reported the use of the organocatalyst 157 to bring about the coupling of the aryl bromide 151 with the boronic acid pinacol ester 158 . Considering the high yield of the advanced ledipasvir precursor 156 reported for this coupling, this palladium-free approach appears much more amenable to large-scale pharmaceutical production than the original Gilead Sciences route . Rolapitant , a spirocyclic neurokinin NK1 receptor antagonist comprising three stereogenic centers originated in the laboratories of Schering-Plough, and was approved for the treatment of chemotherapy-induced nausea and vomiting in 2015 . The drug’s syntheses are primarily featured in the patent literature. The original synthesis of rolapitant patented by Schering-Plough relies on the supply of the key building block 159 . Synthetically, the latter can be traced back to ( S )-phenylglycine over eight synthetic operations, including the preparation of oxazolidinone 160 , as detailed further below. Cyclic enamine 159 was subjected to standard hydroboration–oxidation conditions, to afford alcohol 161 , which was oxidized to ketone 162 under the Swern conditions. Ketone 162 was involved in a standard hydantoin synthesis with potassium cyanide and ammonium carbonate, to afford spirocyclic compound 163 . The latter was Boc-protected, hydrolyzed and Boc-protected again to give α-amino acid 164 . The carboxylic group in 164 was reduced via a mixed anhydride to give amino alcohol 165 which was oxidized (again under the Swern conditions) and involved, without protection of the amino group, in a Horner–Wadsworth–Emmons olefination, to give the acrylate ester 166 . The latter turned out to be a direct precursor of rolapitant, and was converted to the drug by hydrogenation (accompanied by lactam formation) and protecting group removal . As to the synthesis of key building block 159 from ( S )-phenylglycine (( S )-Phg), it was accomplished as follows. ( S )-Phg was converted to diastereomerically pure oxazolidinone 160 on condensation with benzaldehyde and Cbz-protection . Oxazolidinone 160 was C -alkylated in diastereoselective fashion with chiral bromide 167 . The carbonyl group of the resulting intermediate 168 was reduced by lithium alumohydride, and the lactol 169 was obtained. The aldehyde form of the latter reacted with phosphonium ylide generated from the alkyl phosphonium bromide 170 , and the elaborate olefinic building block 171 was obtained. It was hydrogenated over platinum dioxide and the resulting acetal 172 was treated with p -toluene sulfonic acid in refluxing ethanol. This led to the removal of the acetal protecting group and the cyclization of the Cbz-protected amino group onto the liberated aldehyde carbonyl group, to give the target key intermediate 159 . In 2013, Opko Health, Inc. patented an alternative route to rolapitant which is also based on the key building block 159 . This intermediate was nitrated and reduced by lithium borohydride to give the nitro alkane 173 . Removal of the Cbz group (to give 174 ) and subsequent alkylation of the nitro alkane portion with methyl acrylate over basic alumina gave compound 175 , which was converted to the methanesulfonic acid salt 176 , for easy isolation. The reduction of 176 with zinc metal in acetic acid triggered lactamization and gave rolapitant . Thus, the Opko Health, Inc. synthesis appears to be shorter than the original Schering-Plough route and more amenable to large scale API production. In 2016, Opko Health, Inc. patented a fundamentally different route to rolapitant which relied on the chiral building block 177 (for the sake of brevity, we do not discuss its synthesis) . It was reductively alkylated with the masked aldehyde building block 178 to give the bis-olefinic compound 179 . In the latter, a scene was set for intramolecular olefin metathesis, which was successfully brought about using the Hoveyda–Grubbs second-generation ruthenium catalysts (HGII), and the resulting spirocycle 180 was isolated as hydrochloride salt. The latter advanced intermediate was free-based by aqueous sodium hydroxide and hydrogenated over palladium on charcoal, to give rolapitant . Apalutamide , a spirocyclic androgen receptor antagonist discovered by the Sawyers/Jung team at UCLA was approved for the treatment of prostate cancer in the US in 2018 and in Europe in 2019 . The original synthesis was patented in 2007 by the discovery team . It started with the benzoyl chloride 181 , which was reacted with methylamine and the resulting amide 182 was involved in an aromatic nucleophilic substitution reaction with p -methoxybenzylamine, to give 183 . Upon removal of the PMB group, aniline 184 was involved in a Strecker reaction with cyclobutanone, to give nitrile 185 . The monothiohydantoin ring of apalutamide was formed on microwave-promoted condensation with the thioisocyanate 186 , followed by acidic hydrolysis on the initially formed imine . Later, a Sloan Kettering team patented an apalutamide synthesis in which thiohydantoin formation was performed via the in situ generation of the thioisocyanate 186 from the aniline 187 by reacting the latter with Strecker nitrile 185 in the presence of thiophosgene . A number of different approaches to apalutamide, mostly focused on the synthesis of pyridine-3-amine 187 were disclosed . A somewhat different approach to apalutamide assembly which involves two copper(I)-catalysed N -arylation steps was patented in 2018 by Hangzhou Cheminspire Technologies Co., Ltd. (Hangzhou, China) . The synthesis avoided the use of thiophosgene or thiophosgene equivalents by generating the thiohydantoin using potassium thiocyanate. Cyclobutane α-amino acid 188 was used as a partner in the copper(I)-catalyzed reaction with aryl bromide 189 , to give carboxylic acid 190 . Following esterification, compound 191 was reacted with potassium thiocyanate to give the thiohydantoin portion of apalutamide ( 192 ). from which the target drug molecule was crafted via copper(I)-catalyzed arylation with 3-bromopyridine 193 . Moxidectin , a macrolide antiparasitic drug containing an ( S )-1,7-dioxaspiro[5.5]undecane motif was derived from Streptomyces bacteria discovered in the late 1980s in an Australian soil sample . It was initially employed for the treatment of helminths in house animals. However, in 2018, moxidectin received FDA approval to treat onchocerciasis (river blindness) in patients aged 12 and older . Moxidectin exerts its antiparasitic action by selectively binding to the parasite’s GABA-A and glutamate-gated chloride ion channels . Chemically, moxidectin belongs to the milbemycin class of macrocyclic lactones. It is obtained by the semisynthetic modification of nemadectin (F-29249α) which, in turn, is isolated from the fermentation broth of the bacterium Streptomyces cyanogriseus . The following four-step synthesis of moxidectin from nemadectin was disclosed in a 1991 patent . The secondary alcohol hydroxy group on the hexahydrobenzofuran moiety was chemoselectively protected by a p -nitrobenzoate (PNB) ester ( 194 ). Then the other secondary alcohol hydroxy group was subjected to Albright–Goldman oxidation (DMSO/acetic anhydride), to give ketone 195 , which was converted to O -methyl oxime 196 , and the PNB group was removed by alkaline hydrolysis (without any damage to the macrolide ester linkage) to give moxidectin . Migraine is a debilitating disease which was traditionally treated only symptomatically by serotonin receptor modulators or non-steroidal anti-inflammatory drugs. However, more recent in-depth investigation of the mechanisms underlying the onset of migraine established the calcitonin gene related peptide (CGRP) as a primary player in this pathological process. Produced in peripheral and central neurons, CGRP is a potent vasodilator and mediator of nociception. Thus, antagonizing the binding of CGRP to its receptor was established as a novel approach to the treatment of acute migraine at the fundamental level . Ubrogepant is the first-in-class drug developed by Merck and Co., and was approved in 2020 . The detailed process of the chemical route towards ubrogepant was published by the Merck team in 2017 . The final step in the assembly of the target molecule was the amidation of the enantiopure spirocyclic carboxylic acid 197 with the enantiopure amine 198 hydrochloride salt . Therefore, it is the assembly of enantiopure building blocks 197 and 198 which required most of the innovation. The synthesis of amine 198 started with a diastereomeric mixture of keto esters 199 . The latter was subjected to dynamic kinetic transaminase-mediated cyclization (using an evolved enzyme strain) to give piperidone 200 as a 3:2 mixture of epimers. N -Alkylation of the latter with 3,3,3-trifluoroethyl triflate afforded predominantly compound 201 (separated in 87% yield from the unreacted starting material and bis-alkylation product). Removal of the Boc group gave the epimeric amine 202 , which was subjected to crystallization-induced diastereoselective transformation (CIDT) with p-toluic acid and 3,5-dichlorosalicylic aldehyde to give building block 203 , which was converted to the hydrochloride salt 198 . The spirocyclic carboxylic acid 197 was assembled as follows. The 2,3-Dibromo-5-chloropyridine ( 204 ) was transformed in four steps (involving two additions of pyridine magnesium species to DMF) to hte tetrahydropyran-protected aldehyde 205 . The latter was condensed with 7-azaindolin-2-one 206 to give the olefin 207 , which was reduced by sodium borohydride, and the THP group was removed to give the alcohol 208 . Conversion of 208 into the alkyl chloride 209 set the scene for spirocyclization. On the action of aqueous sodium hydroxide, in the presence of the cinchona alkaloid-derived chiral catalyst 210 , chloride 209 underwent an efficient and enantioselective intramolecular C -alkylation to form the spirocycle 211 . Palladium-catalyzed carbonylation of the chloropyridine portion in the latter, followed by the removal of the tert -butyl group from the lactam nitrogen atom, afforded the key spirocyclic carboxylic acid 197 . An alternative approach to spirocyclic carboxylic acid 197 was reported in 2022 by Hu and co-workers . It involves the direct asymmetric spiroannulation of Boc-protected 7-azaindolin-2-one ( 213 ) with the doubly benzylic bis-bromide 212 . Thus, from cheap and readily available starting materials, spirocycle 214 was obtained in good yield and enantioselectivity. Removal of the Boc group followed by nitrile hydrolysis gave the requisite spiro acid 197 , thus completing a formal synthesis of ubrogepant . Oliceridine , a spirocyclic biased agonist of μ-opioid receptors was developed by Trevena, Inc. and was approved in 2020 for the intravenous treatment of acute moderate-to-severe post-operative pain . The only full detailed route to the oliceridine drug substance is the original Trevena, Inc. approach, patented in 2012 . The source of the spirocyclic motif was the ketone 216 , obtained by the oxidation of the perhydropyran-4-ol 215 , synthesized, in turn, by the Prins cyclization between cyclopentanone and homoallylic alcohol . Base-promoted condensation with methyl cyanoacetate afforded the olefin 217 , to which pyrid-2-yl organometallic reagent (magnesium or lithium) was added in conjugate fashion, to give the cyano ester 218 . The latter was subjected to basic hydrolysis and decarboxylation at elevated temperature to afford the racemic product, which was resolved by preparative chiral supercritical fluid chromatography to give the enantiopure nitrile 219 . This product was reduced using LAH and the resulting amine 220 was reductively alkylated with 3-methoxythiophene-2-carboxaldehyde to give oliceridine . Risdiplam is the first oral medication to treat spinal muscular atrophy, an orphan disease . The drug was developed by PTC Therapeutics and the Spinal Muscular Atrophy Foundation, and received FDA approval in 2020 for the treatment of the disease in patients aged 2 months or older . The drug acts by modifying mRNA splicing by binding to the survival of motor neuron 2 (SMN2) . It is currently marketed by Genentech, a subsidiary of Roche. The synthetic routes to risdiplam disclosed in the original chemical process patents by Roche rely on the commercial supply of the spirocylic piperazine building block, and thus do not involve the formation of the spirocyclic moiety in the course of the synthesis. In their 2015 patented route, PTC Therapeutics and Roche scientists started with 3,6-dichloro-5-methylpyridazine ( 221 ), in which the less accessible chlorine atom was substituted with ammonia and the imidazo ring was formed on condensation with bromoacetone dimethyl ketal ( 222 ). The overall yield of the resulting imidazo pyridazine 223 was obtained in relatively low yield (unsurprisingly, considering the unfavorable course of the initial nucleophilic aromatic substitution). Compound 223 was coupled, under palladium(II) catalysis, with bis(pinacolato)diboron, to give boronic acid 224 . The latter was coupled again, in Suzuki fashion, with the pyrido pyrimidone building block 225 (obtained, in turn, from 2-amino-5-fluoropyridine on condensation with dimethyl malonate). The Susuki coupling 224 + 225 resulted in the core risdiplam scaffold 226 , which was decorated with spirocyclic piperazine 227 under thermal conditions, to give risdiplam . For the more recent route following their 2019 patent application, Roche scientists started by involving 5-bromo-2-chloropyridine ( 228 ) in Buchwald–Hartwig amination with Boc-protected spirocyclic piperazine ( 229 ). The unreacted, under Pd(II)-catalyzed conditions, chlorine atom in 230 was displaced by ammonia under Pd(0) catalysis and the amino portion of the resulting intermediate 231 was converted in two steps into tosylate 232 . The latter was coupled with boronic acid 224 (prepared as described in the previous Roche patent) in Suzuki fashion, and the Boc group in the resulting coupling product 233 was removed to give risdiplam . Atogepant is a next-in-class antagonist of the calcitonin gene- related peptide (CGRP) is a direct analog of ubrogepant (the first-in-class CGRP antagonist developed by Merck, vide supra), which is different from ubrogepant since it contains a 2,3,6-trifluorophenyl ring. Discovered by Merck and developed by AbbVie, atogepant was approved in 2021 for the preventive treatment of episodic migraine in adults . The synthesis of atogepant was described in detail in a 2016 patent application by Merck , and it relies on essentially the same routine as was used in the preparation of ubrogepant. Specifically, the same enantiopure spiro acid 197 was in use. The enantiopure 3-aminopyrid-2-one building block was also assembled in a fashion similar to the amine half of ubrogepant ( 202 ). The synthesis commenced with the phenylacetic acid, which was converted to the respective acyl chloride and then the Weinreb amide 235 . The latter was treated with methylmagnesium chloride to give the methyl ketone 236 . The latter was alkylated at the most acidic, benzylic position with mesylate 237 to give keto ester 238 . The latter is a direct analog of compound 199 as a precursor to 202 —and the exact same routine, involving the use of dynamic kinetic transaminase, was used to convert compound 238 to enantiopure 3-aminopyrid-2-one 239 . Amidation of the spiro acid 197 with the amine 239 gave atogepant . Trilaciclib spirocyclic inhibitor of cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) was developed by G1 Therapeutics. The drug was indicated for cancer patients receiving certain types of chemotherapy to protect the bone marrow from chemotherapy-induced damage. As trilaciclib was the first-in-class drug developed for this important indication, it received a priority review and a breakthrough therapy designation from the FDA. It was approved for medical use in 2021 . The drug’s originator patented two synthetic routes to the compound, both involving an intramolecular N -acylation as the key step in the construction of the spirocyclic moiety . Of these two approaches, the later one is described in much greater detail, and is therefore the API production route which we chose to discuss herein. The synthesis commences with an efficient and high-yielding Strecker reaction of cyclohexanone with glycine methyl ester and trimethylsilyl cyanide, to afford the amino nitrile 240 . The latter was hydrogenated over platinum(IV) oxide. This caused the reduction of the nitrile to the aminomethyl compound, which underwent lactamization to afford the key spirocyclic building block 241 . Without protection, the sterically hindered secondary amine 241 was used to displace the labile chlorine atom in the pyrimidine template 242 . The nucleophilic aromatic substitution reaction proceeded under forcing conditions and afforded the spirocycle 243 , which was now set for the formation of the pyrrolo portion of the target molecule. As this step would require C-H deprotonation of the piperazinone moiety, the lactam nitrogen of intermediate 243 was Boc-protected to give compound 244 . Treatment of the latter with DBU at 0 ℃ in THF triggered the desired deprotonation and intramolecular cyclization, and gave the 3-hydroxypyrrolo compound 245 in quantitative yield over the last two steps (including the preceding Boc-protection). Clearly, the hydroxy group in 245 had to be removed, which was achieved by O -triflation of the compound with the subsequent palladium(0)-catalyzed reductive coupling with triethylsilane. The two steps resulted in 70% combined yield and afforded the spirocyclic pyrrolo pyrimidine 246 , which was deprotected with trifluoroacetic acid to give the advanced trilaciclib precursor 247 . To complete the synthesis, the methylthio group in 247 was oxidized to the better, leaving methylsulfonyl group with oxone in aqueous acetonitrile, to give 248 in excellent yield. The final nucleophilic aromatic substitution step between the spirocyclic template 248 and the pyridine amine 249 (prepared, in turn via nucleophilic aromatic substitution with 5-halopyridine precursor and N -methylpiperazine) required the use of a strong base (LiHMDS), and gave trilaciclib . As noted previously, the use of spirocycles in drug discovery and medicinal chemistry has seen an explosive rise . This trend is clearly reflected in the number of drugs containing a spirocyclic motif which received approval for medical use over the years. Indeed, over 50% of such drug molecules have been approved in this century. A range of synthetic methods to form a spirocyclic scaffold have been employed in the production routes towards these drugs, although at times the source of the spirocyclic moiety is the naturally occurring, biosynthesized compound or a commercially available spirocyclic building block . The trend is likely to continue, resulting in new advanced drug candidates and approved drug molecules, which mandates that the arsenal of synthetic methods applicable to spirocycle formation is expanded beyond the most popular key steps such as lactonization and lactamization, cyclocondensation (including ketalization and acetalization) and including intramolecular C -alkylation and acylation. Given the rate of development of synthetic approaches towards the construction of spirocyclic molecules employing various modern techniques (e.g., photochemistry, multicomponent reactions, diazo chemistry, asymmetric catalysis, flow synthesis, etc.) which is reflected in the high annual publication activity, we have no doubt that many of them will find application in the field of both drug design and industrial production in the near future.
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The Conundrum of Dedifferentiation in a Liposarcoma at a Peculiar Location: A Case Report and Literature Review
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15f600bd-8790-4575-a2b7-f1f91acc56e3
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10224154
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Anatomy[mh]
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Liposarcomas are rare malignant tumours which originate from the adipose tissue and can arise at any site, with a predilection for the lower extremities and retroperitoneum . According to the World Health Organization classification, liposarcomas develop as part of a heterogeneous group comprising atypical lipomatous tumour/well-differentiated liposarcoma, dedifferentiated liposarcoma, myxoid liposarcoma and pleomorphic liposarcoma . These malignant proliferations are associated with distinct genetic abnormalities and present different clinical behaviours and metastatic potentials . Myxoid liposarcoma is the most common adipocytic malignancy of the lower extremities, frequently affecting the proximal thigh . Although a rare phenomenon, dedifferentiated liposarcoma and well-differentiated liposarcoma can also occur within deep somatic adipose tissue . Dedifferentiated liposarcoma is an atypical lipomatous proliferation which usually develops within the retroperitoneum, but studies suggest that the incidence of non-peritoneal anatomic sites harbouring this malignancy may be underreported . The positive and differential diagnosis of the previously mentioned lesions is crucial, especially considering the major differences between the prognosis and therapeutic management of each pathological entity . In this matter, immunohistochemical analysis and genetic tests are additionally required for confirming and completing the histopathological diagnosis .
Our patient was referred to the Emergency University Hospital in Bucharest where the CT scan indicated a dense tumour mass in the adipose tissue of the left thigh, showing nodular enhancing areas. This radiology aspect was strongly suggestive of a liposarcoma. Therefore, surgical “en bloc” removal of the tumour mass was carried out, and the specimen was submitted to our pathology department. The tissue samples were fixed with 10% neutrally buffered formalin and then processed via conventional histopathological methods, using paraffin embedding, sectioning and Haematoxylin–Eosin (HE) staining. Afterwards, the sections were deparaffinised in toluene and alcohol, washed in PBS (phosphate saline buffer), incubated with normal serum, and then incubated with primary antibodies overnight. Later, washing in carbonate buffer and developing in 3,3′-diaminobenzidine hydrochloride/hydrogen peroxide and nuclear counterstain with Meyer’s Haematoxylin were performed. The immunohistochemical markers used were S100 antibody 4C4.9 mouse monoclonal, Biocare, Ki 67 antibody SP6 rabbit monoclonal, Biocare, CD 34 antibody QBEnd/10 mouse monoclonal, Biocare, p16 INK4a antibody BC42, mouse monoclonal, Biocare, p53 antibody DO7 rabbit monoclonal, Biocare, MDM 2 antibody ZR258 rabbit monoclonal, ZETA and CDK4 antibody ZR 394, rabbit monoclonal, ZETA. Furthermore, using the PubMed academic research engine, we reviewed all English language cases published until 2022. We included all cases of soft tissue tumours occurring in the lower extremities diagnosed as dedifferentiated liposarcoma in patients of any age and sex with any type of therapeutic management and clinical outcome of the disease. We excluded all patients diagnosed with other soft tissue tumours or benign proliferations of the adipose tissue. Furthermore, we excluded the cases of tumours reported as sarcomas with uncertain histotype.
We present a 32-year-old male patient with no significant medical history who was referred to our hospital with a slow growing, painless tumour mass located in the upper region of the left thigh. No chemotherapy or radiation therapy, nor a history of trauma, had been documented in the patient’s medical record. A CT scan was performed, revealing a 11 cm, dense tumour mass arising in the adipose tissue of the left thigh, showing nodular enhancing areas. Moreover, multiple ipsilateral adenopathies were identified. Upon further radiology investigations, no distant metastases were detected. The patient underwent a surgical procedure consisting of wide local excision of the tumour with resection margins and lymphadenectomy. Afterwards, the surgical specimens were submitted to our Pathology Department. The gross examination revealed a 11/7/2 cm well-circumscribed, multinodular tumour with a heterogeneous aspect on the cut surface. The lesion presented a course lobular contour and yellow-tan colour, firm consistency and small gelatinous areas, as well as focal nodular, solid zones. The histological examination showed a malignant mesenchymal proliferation with a lobular growth pattern, exhibiting round–oval and fusiform neoplastic cells with hyperchromatic nuclei and occasional nuclear pseudoinclusions, separated by a loose, lightly basophilic stroma, containing extracellular mucoid pools ( and ). Moreover, a prominent vasculature, formed by delicate arborizing capillaries with a plexiform arrangement, was identified. The tumour lobules were surrounded by a rim of atypical lipoblasts. The malignant tumour proliferation displayed abrupt transition towards a divergent component consisting of highly pleomorphic tumour cells with atypical mitotic figures, with associating small necrotic and haemorrhagic areas ( , and ). No lympho-vascular tumour emboli or perineural invasion were detected. Four surgically excised inguinal lymph nodes were examined, with mild reactive histiocytosis and no tumour invasion. As the histopathologic features of the lesion was highly suggestive of a dedifferentiated liposarcoma, several additional ancillary tests were carried out. First, immunohistochemical analysis was used to determine the following tumour immunophenotype: neoplastic cells of the lipogenic tumour component presented intense S100 and p16 protein expression ( and ), while CD 34 staining underlined the presence of plexiform arborizing capillary blood vessels within the lipogenic tumour component . Both components of the divergent tumour proliferation were associated with a wild type TP53 expression pattern, and approximately 10% of the tumour cells expressed the Ki 67 proliferation marker . The expression of MDM2 and CDK 4 markers within the tumour cells of the dedifferentiated component eventually established the diagnosis of a dedifferentiated liposarcoma developed on the background of a myxoid liposarcoma ( and ). The patient did not present with any metastatic lesions at the time of diagnosis. Therefore, the tumour proliferation was categorized as a stage III A lesion (pT2 pN0 M0) according to the TNM evaluation system. Genetic testing was also recommended to confirm and complete the diagnosis. The patient was later referred to the oncology department for evaluation and initiation of an adequate therapeutic scheme. After a six-month follow-up, the patient is disease-free, with no evidence of recurrence or metastases.
Dedifferentiated liposarcoma is a malignant proliferation of the adipose tissue which typically involves the retroperitoneal space and, infrequently, lower extremities, spermatic cord and structures of the head and neck . Dedifferentiated liposarcoma of the thigh is an uncommon entity, as this anatomic region is prone to develop myxoid liposarcoma or non-adipogenic malignant mesenchymal proliferations . Although there are a few cases reported, dedifferentiated liposarcoma should be taken into consideration when examining soft tissue tumour masses of the lower extremities . Myxoid liposarcoma is one of the most frequent soft tissue sarcomas and is associated with peculiar genetic mutations, implying an unfavourable prognosis . Currently, the various liposarcoma subtypes are recognised to have different biological behaviour in correlation with their distinct genetic alteration . The pathogenesis of dedifferentiated liposarcoma can be summarized by the presence of a high-level amplification in the chromosomal 12q13–15 region involving the CDK4 and MDM2 cell cycle oncogenes . The genetic abnormalities are a common feature of dedifferentiated liposarcoma and atypical lipomatous tumour/well-differentiated liposarcoma . However, recent advances in next generation sequencing show that novel genes and pathways occur in dedifferentiated liposarcoma . Whole genome sequencing disclosed various genetic alterations, including 6q23 and 1q32 coamplification, which may be harboured by dedifferentiated liposarcoma . Considering the rarity of this malignant tumour, anatomical location and histotype are infrequently mentioned throughout the results of the surveys, with no specific data upon liposarcoma of the extremities available . Although the role of MDM2 and CDK4 genes in the pathogenesis of dedifferentiated liposarcoma is well understood, the most recent studies have demonstrated that DDIT3 amplification is also identified in patients developing this malignancy . As it was mentioned, due to the uncommonness of this soft tissue neoplasm, the correlations between DDIT3 amplification and the tumour’s location and clinical behaviour are currently scarce . Moreover, numerous studies on the pathogenesis of myxoid liposarcoma have also been carried out, considering the peculiarities of this entity’s prognosis and therapeutic management. The FUS: DDIT3 fusion oncoprotein is the current genetic hallmark of myxoid liposarcoma, and it is considered that it acts like an aberrant transcription factor . Furthermore, studies reveal that overexpression of DDIT3 along with MDM2 and CDK4 is also identified in dedifferentiated liposarcoma, although considered a genetic alteration strongly associated with myxoid liposarcoma . In addition, studies suggest that the aforementioned genetic abnormality may determine the presence of myxoid-like histopathologic aspects within dedifferentiated liposarcoma . As a result, thorough correlations between the morphologic and immunohistochemical features and the genetic background of these particular soft tissue neoplasms are required for achieving the correct diagnosis. Regarding the challenging histopathologic features of some liposarcomas which may lead to a misinterpreted diagnosis, the question of heterologous differentiation and tumour metaplasia has also been put Heterologous differentiation within a myxoid liposarcoma has only rarely been reported . The main discussion in this matter refers to the morphology of the proliferated heterologous elements which may highly resemble tumour metaplasia . Up to date, five cases of myxoid liposarcoma with cartilaginous differentiation confirmed on cytogenetic analysis have been reported . In our case, the proliferation displayed an abrupt transition towards a non-lipogenic area composed of pleomorphic spindle cells with unequivocal malignant features, ruling out metaplasia. Moreover, dedifferentiated liposarcoma exhibiting a prominent myxoid stroma is an important histopathological finding which should be taken into consideration when analysing malignant lesions with adipocytic differentiation . However, this uncommon histopathologic aspect has mainly been identified during the examination of tumour masses involving the retroperitoneal space and paratesticular soft tissue . In our case, microscopic examination revealed the presence of basophilic matrix enclosing sarcomatous neoplastic cells and containing a myxoid liposarcoma-like plexiform capillary vasculature, adjacent to a non-lipogenic area with divergent differentiation. Studies provide evidence of prominent vascular capillary networks within dedifferentiated liposarcoma with myxoid stroma and suggest both myxoid liposarcoma and myxofibrosarcoma should be taken into consideration as a differential diagnosis . Apart from the differential diagnosis between myxoid liposarcoma and its dedifferentiated counterpart, there is a small focus on highly pleomorphic liposarcomas with mixed elements . Campbell et al. report a case of a mixed lesion of the axilla comprising well-differentiated, dedifferentiated elements adjacent to a myxoid liposarcoma component . In relation to this phenomenon, we highlight the importance of immunohistochemical tests and genetic analysis as reliable methods for assessing the response to neoadjuvant therapy . The evaluation of the tumour immunophenotype can facilitate the differential diagnosis of lesions with similar histomorphology, and it is also relevant in predicting the therapeutic tumour response . First, immunohistochemical analysis can be used to distinguish dedifferentiated liposarcoma from other types of malignant mesenchymal proliferations . Currently, this diagnostic procedure is guided by the use of MDM2 and CDK4 markers and confirmed using additional molecular testing of the corresponding genes amplification . Ancillary tests are necessary when dealing with tumours displaying histopathologic features suggestive of a dedifferentiated or well-differentiated liposarcoma, but with no detectable lipogenic component . The studies that were carried out performed MDM2, CDK4 and p16 staining on samples originating from tumours with the aforementioned histotype. The highest sensitivity and specificity were noted for the MDM2 marker, as an excellent correlation was obtained between the results of MDM2 fluorescence and in situ hybridization . The previous immunomarker was followed by CDK 4 and p16 which also provide sensitive and specific staining for dedifferentiated liposarcoma . Although there are a few reported cases of dedifferentiated liposarcoma of the thigh, MDM2, CDK4 and p16 were used as part of a panel to confirm the diagnosis . On the other hand, immunohistochemical study of myxoid liposarcoma is currently of limited use. As it was earlier mentioned, myxoid liposarcoma is associated with recurrent molecular alterations implying DDIT3 rearrangement; therefore, anti-DDIT3 immunoreactivity is the only specific staining for this tumour proliferation . As this immunomarker is not yet available in regular antibody panels, the ancillary study of myxoid liposarcoma of any location is limited to the examination of S100 and p16 expression and of the status of the TP53 mutation . S100 expression within neoplastic cells can be used to differentiate myxoid liposarcoma from a non-adipocytic neoplasm with myxoid stroma, while p16 has proved to be practical in distinguishing the tumour in question from its benign counterparts . Although the expression of TP53 within lesions from the pathologic spectrum of liposarcoma has been widely examined, researches do not provide evidence of a major significance of this mutation for the overall prognosis of the neoplastic disease . In our patient, the applied immunohistochemical stains provided a precise recognition of the divergent differentiation. As dedifferentiated liposarcoma of the thigh is an uncommon finding, performing ancillary tests with the tissue samples is mandatory for establishing the diagnosis. Clinical behaviour, prognosis and therapeutic management of soft tissue liposarcoma has been analysed in several studies for a notably long time. To this moment, the prognosis of patients developing dedifferentiated liposarcoma of the extremities can be estimated considering multiple prognostic factors. First, dedifferentiated liposarcoma is regarded as a malignant tumour with a better prognosis compared to myxoid and pleomorphic liposarcoma and other soft tissue sarcomas, exploiting the fact that the majority of these adipocytic tumours harbour MDM2 and CDK4 expression . Clinical trials support the benefit of CDK4 inhibitor abemaciclib for the treatment of dedifferentiated liposarcoma with CDK4 and MDM2 expression and strongly recommend the further development of anti-MDM2 agents, which, at the moment, have limited clinical use due to bone marrow toxicity resulting in thrombocytopenia . In addition, trials using immune checkpoint inhibitors as a targeted therapy for dedifferentiated liposarcomas have been proposed, as PD-1 expression has been identified within liposarcoma . Although there are several on-going trials investigating new targeted therapies for liposarcoma, patients diagnosed with this neoplasm currently receive classic cytotoxic chemotherapy agents and radiotherapy . The most widely used therapeutic schemes include doxorubicin, docetaxel and gemcitabine, which are prescribed as part of adjuvant treatment . Immunohistochemical and genetic analyses of dedifferentiated liposarcoma and the other malignant tumours with adipocytic differentiation represent an important research purpose with important new perspectives. Surveys implying new correlations between molecular biology data, genetic analysis and immunohistochemistry have constantly been performed, in order to discover advanced tools for diagnosis and customized oncological treatment. For example, there is an interest in investigating the expression of GATA proteins and other molecules within various malignant tumours. Transcription factors from the GATA protein family are involved in adipose tissue development and maturation . Their corresponding immunomarkers may be expressed within fat cells precursors and may facilitate the differential diagnosis of morphologically similar lesions . CD 70-CD27 ligand–receptor complex stimulates the development of T lymphocytes . At the moment, there is evidence of CD 70 expression in lymphomas and several sarcomas, with a significant relevance in predicting tumour response to therapy with specific inhibitory agents .
Dedifferentiation within a myxoid liposarcoma is an exceedingly rare phenomenon, although cases of myxoid liposarcoma with heterologous differentiation, as well as dedifferentiated liposarcoma with a myxoid stroma have been reported so far. The precise diagnosis of dedifferentiated liposarcoma requires the use of ancillary studies, implying immunohistochemistry and genetic analysis. Differentiating the aforementioned malignant tumour from other soft tissue sarcomas is crucial, considering the significant differences regarding the prognosis and treatment of each entity. Therapeutic procedures have constantly been improved. Therefore, a diligent assessment of the tumour histotype, immunophenotype and genetic background is mandatory for the successful management of patients developing dedifferentiated liposarcomas.
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“Evaluation of ROS1 expression and rearrangements in a large cohort of early-stage lung cancer”
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4012368f-b193-4848-80cb-df80a42fa71f
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10224579
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Anatomy[mh]
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Targeted therapy has been a game-changer in the treatment of metastatic lung cancer, providing effective treatment opportunities and improving overall survival in late-stage disease . However, for early-stage disease, the treatment opportunities have developed more slowly. There is now a focus on the potential for targeted therapy in early-stage disease, and more research is needed on the prevalence and characteristics of relevant targets in this setting. The ROS1 fusion protein is an attractive therapeutic target for patients with metastatic non-small cell lung cancer (NSCLC). Several therapies are available and recommended by the European Society for Medical Oncology (ESMO) and National Comprehensive Cancer Network (NCCN) . In studies done mostly on advanced stage disease, the prevalence of ROS1 rearrangements in NSCLC is about 1–3% . But there is now a growing interest for the prevalence of ROS1 fusions in early-stage lung cancer. For EGFR mutated resected early-stage NSCLC, adjuvant therapy with tyrosine kinase inhibitor have showed promising results . And perioperative targeted therapy can also be feasible for other oncogene addicted NSCLC. Recently, case reports have showed effect of crizotinib as neoadjuvant or adjuvant therapy in a ROS1 translocated setting , and there is also ongoing trials investigating this further . This implies that ROS1 fusions in early stage lung cancer can be a target for neoadjuvant or adjuvant therapy in the future. Two meta-analyses indicate that ROS1 fusion is more common in late-stage disease . It is also shown that the prevalence is higher in women, never-smokers, adenocarcinomas and in patients of Asian ethnicity. Two previous studies on early-stage lung cancer found a prevalence of ROS1 fusion of 0.4–1.2% in adenocarcinomas . These studies were based on one immunohistochemical (IHC) clone and fluorescence in situ hybridization (FISH). The validity of fusion detection methods, i.e. detection of clinically relevant fusions, remains challenging. Until recently, ROS1 FISH analysis was regarded as a gold standard. However, new methods like next-generation sequencing (NGS) have revealed conflicting results . An emerging strategy in fusion detection diagnostics is the reliance on a combination of several methods, including IHC, FISH, NGS and Reverse Transcription quantitative real time Polymerase Chain Reaction (RT-qPCR). NGS is now more widely available and ESMO recommends the use of NGS in patients with non-squamous NSCLC . For detection of ROS1 fusion, RNA-NGS is preferred over DNA-NGS . IHC-based methods are generally feasible, cost-effective and widely available in pathology labs. These methods are based on immunologic principles, where labeled antibodies bind to specific antigens such as cell proteins. When a part of the ROS1 gene is fused with another gene and the kinase domain is included in that fusion, the subsequent gene expression can lead to an increased ROS1 expression. IHC can therefore detect increased protein expression, but it cannot distinguish whether this is the normal/wild type ROS1 protein or a chimeric protein as a result of gene fusion . For ROS1 IHC there is still no consensus on cut-off levels, though several studies have focused on different IHC clones for ROS1-detection . Only a few studies have used NGS as part of the test algorithm . To our knowledge, there are no published studies based on a diagnostic algorithm combining different IHC-clones, FISH and comprehensive NGS-panels. This retrospective study aimed to determine the prevalence of ROS1 fusion in a cohort of Norwegian, early-stage resectable lung cancer. We used ROS1 IHC screening and performed FISH and NGS on IHC positive samples, and described relevant challenges in the interpretation of the test results from all three methods. In cases with positive FISH and negative IHC/NGS cases, we have also used RT-qPCR. In addition, we explored associations between ROS1 fusion or IHC positivity, and clinical, histopathological and comprehensive genetic characteristics.
Patients We used biobank specimens from a cohort of surgically resected lung cancer patients at Oslo University Hospital. This biobank has a connected database with histopathological-, biological- and clinical information. The surgery and sampling was done during the period 2006–2018, with a median follow-up exceeding 5 years. Mortality data were imported from the Norwegian Population Registry, which is updated monthly. Two patients were lost to follow up (emigration), and was considered negligible in the analysis. We also excluded patients with carcinoid tumour and thymoma. Written informed consent was obtained from all patients and the project was approved by the regional ethics committee (1904/2009). The staging has been done according to the latest TNM classifications of malignant tumours at the time of surgery, but for the FISH positive cases we restaged the samples to the current edition . For adenocarcinomas with ROS1 fusion, we have also used the proposed new grading system for invasive pulmonary adenocarcinoma . Specimen characteristics In this study we used full size slides from formalin-fixed paraffin embedded (FFPE) blocks, tissue micro arrays (TMA), and DNA and RNA extracted from fresh frozen material or FFPE blocks. The full size slides were taken from FFPE blocks from resections in the diagnostic biobank at Oslo University Hospital. These blocks were sliced at the Department of pathology at Vestfold hospital trust. Tissue microarray blocks were made from specimens from all the resections, each block consisting of specimens from 25–30 patients. All specimens were represented in triplicates, each core being one mm in diameter, and all tissue cylinders were harvested from the original FFPE blocks after careful selection by a trained pathologist. The TMA blocks were made and sliced at the Department of pathology at Oslo University Hospital. The majority of the TMA blocks have been used in previous projects . DNA and RNA were extracted from fresh-frozen tissue obtained at surgery. In the few cases of an insufficient number of tumour cells in the frozen tissue samples, the extraction was done from FFPE-blocks. Positive external control in ROS1-IHC can be difficult to find , because of the lack of reliable naturally ROS1 positivity in normal tissue. We used known positive FFPE tissue from a patient with a CD74-ROS1 fusion (confirmed with FISH and NGS) as a positive control. This tumour tissue was also strong and diffuse positive with the two different ROS1 IHC clones (D4D6 and SP384). Assay methods. Immunohistochemistry We performed ROS1 IHC analyses by use of two different ROS1-directed antibody clones: ROS1 (D4D6) Rabbit mAb (Cell Signaling, 3287) and the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (Roche Diagnostics, 790–6087). The slides were stained at the Department of Pathology at Vestfold Hospital Trust on a VENTANA BenchMark ULTRA system. This is a fully-automated IHC staining platform. For details about the protocols, see Additional file : Table S1. The microscope was an Axio Imager.A2 (Zeiss, item no. 490022–0009-000). We used both a qualitative and a semiquantitative scoring system. All samples were initially grouped into one of seven predefined groups; Negative, weak and focal, weak and diffuse, moderate/strong and focal, moderate/strong and diffuse, ambiguous and too few viable tumour cells (<10 viable tumour cells). Negative samples were defined by lack of tumour cell positivity under 400x (40x objective) magnification. Moderate/strong positivity was defined by dark brown staining with 25 - 200x magnification (2,5-20x objective). Weak positivity was defined by light brown staining, visible at 100-200x (10-20x objective), but requiring of 400x (40x objective) to see clearly. Diffusely positivity was defined by more than 50% of tumour cells with positive staining, including weak positivity, while focal positivity was defined by less than 50% of tumour cells with positive staining. Ambiguous samples were samples that could not be easily classified due to unspecific staining. Within the TMAs, the staining intensity and distribution were scored by assessing the cores from each case as one unit. All ambiguous samples were reexamined by an additional pathologist and were classified in a consensus meeting. To assess the inter-observer variability and ensure standardized categorization, a random sample of five of the TMA-blocks (114 cases) were examined by two pathologists individually. Any discrepancies in the interpretation were noted and discussed in a consensus meeting in order to optimize reliability. We also used a combinative semiquantitative scoring system on both the TMA slides and full size slides. In order to compare results, we used the H-score which has been used in several recent papers on ROS1 expression . We used the same formula as Huang et al. 2019 : (1x (percentage of relevant cells with 1+ staining) + (2x (percentage of relevant cells with 2+staining) + 3x (percentage of relevant cells with 3+ staining). Staining intensity was defined as absence of staining (0), weak staining (1+), moderate staining (2+) and strong staining (3+). We used the definition of 0, 1+, 2+ and 3+ from Conde et al. 2019 : Negative staining (0), which was defined as an absence of expression; weak staining (1+), which involved the use of a 40x objective; moderate staining (2+), which required the use of a 10x or 20x objective and strong cytoplasmic staining (3+), which was clearly visible with the use of a 2x or 4x objective. See Additional file : Table S2 for details about the scoring system and definitions. To assess the heterogeneity of ROS1 expression, any IHC-positive sample with more than weak and patchy staining was also reexamined with IHC on full size sections from the original FFPE blocks. Heterogeneity in staining can be due to technical issues like fixation, edge artifacts or biological heterogeneity in expression. Thereafter, the IHC-positive samples were grouped according to the percentage of tumour cells with positive IHC and staining quality (membranous/cytoplasmatic/nuclear, granular, diffuse). In addition, in order to reduce false negative IHC due to heterogenic expression, full size section IHC was also performed on a subgroup of TMA-IHC-negative cases where full size sections had already been prepared for a different study (cases with positive NTRK expression in IHC). Heterogeneity was defined as positive cases with areas of both 0 or 1 + in addition to 2 + or 3 + . Assay methods. FISH We tested IHC-positive cases (see definition of IHC-positive under variables) with FISH on full-sized slides. In addition, a sub-group of IHC-negative cases were also tested. FISH was performed at the Pathology department of Oslo University Hospital (Radiumhospitalet) with a dual break apart probe from Abbott (Vysis ROS1 Break Apart FISH Probe Kit, product number 08N29-021). The preparation was done according to the manufacturer’s recommendations . For the interpretation, we chose areas with the best preserved morphology and with clear signals. In the validation/verification of the test, the laboratory has found that a positive tumour is defined with a cut-off of at least 15% split signals, including isolated orange (5`) and green (3`) signals. Fused signals and break apart, single green /orange signals were counted in at least 50 nuclei and also at least 200 signals. Break apart split signals are green and orange signals separated by at least 1 signal diameter. An isolated 5’or 3’pattern means that an orange or green signal is present alone or together with fused or break apart signals. A fused signal is either break apart signals separated with less than 1 signal diameter or a completely fused signal that appears yellow. Assays methods. NGS NGS was performed on the same samples as FISH. Isolated DNA and RNA were extracted from fresh-frozen tissue (stored at -80 °C) with AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, 80,224) on the QIAcube (automated, spin-column-based nucleic acid extraction from Qiagen). The concentration was measured with Nanodrop (Thermo Fisher Scientific) and Qubit Fluorometer (Thermo Fisher Scientific). We used Agilent 2100 Bioanalyzer (Agilent Technologies) to analyze the RNA-quality. The tumour percentage was evaluated on frozen sections. If the percentage was below 10%, and there were no relevant findings (EGFR-, KRAS- or BRAF-mutation, or ROS1-, ALK- or RET-fusion), then NGS was repeated on slides from FFPE-blocks from the original resection. In these cases, the extraction was done from FFPE blocks using MagLead12gC (Biosystem, A1120) for DNA and Quick RNA FFPE kit (Zymo Research, R1008) for RNA. For the sequencing, we used the Oncomine Comprehensive V3-panel (OCAv3)(Thermo Fisher Scientific, A35806). This panel covers 161 genes. The library preparation was done on an Ion Chef instrument (Thermo Fisher Scientific). The sequencing was done at both Oslo University Hospital and Vestfold Hospital Trust from July 2019 – September 2021. In Oslo, the sequencing was done on an Ion Torrent S5, and in Vestfold on an Ion Torrent S5XL. For the bioinformatical analysis, we used the Ion Reporter version 5.10–5.16 with a custom filter (Oncomine Variants, 5% CI CNV ploidy > = gain of 2 over normal). The sequencing of DNA and RNA based on tissue from FFPE blocks was done in Vestfold. In these analyses, the material was more fragmented, and therefore there was more “noise” in the sequencing results. To improve interpretation, we therefore heightened the quality requirements of the original filter in these cases (coverage > 1000 reads, Phredscore > 20, allele frequency above 5%). The same quality requirements were also used in sequencing performed on fresh material, but in these cases the heightened quality requirements were not included in the filter. See Additional file : Table S3 for details about the quality requirements. Fusion transcripts with unknown gene partner, can be reported as a non-targeted fusion by a combination of primers used for different targeted isoforms . Assays method. RT-qPCR In cases with positive FISH and negative IHC/NGS, RT-qPCR was performed with Idylla GeneFusion Assay RUO/1.1 (Biocartis AO121/6). Eluat of RNA extracted for NGS was used in the procedure. The most common fusion partners (CD74, SDC4, SLC34A2, EZR, TPM3, GOPC and LRIG3) are included in the kit. In addition the method can detect if there is an expression imbalance between the 5´and 3´end of the ROS1 gene. An expression imbalance indicates that there can be a fusion with a partner other than those detected by the kit . Dependent and independent variables The main outcome of this study was ROS1 fusion positivity and ROS1 IHC-positivity. Positive ROS1-fusion was defined as samples positive in at least two of the three methods (one of the IHC-clones, FISH, NGS). Positive ROS1 IHC-staining was defined as focal moderate/strong, diffuse weak or diffuse moderate/strong staining, while negative ROS1 IHC-staining was defined as clearly negative or focal and weak staining. The independent variables included the clinical parameters like smoking status (former, current or never smoker), sex and age (continuous), stage, histopathological diagnosis and NGS-results. Study design and statistical analysis methods This was a retrospective, cohort study based on linked register data and biobank material. We examined the data by use of frequency tables. Associations between positive IHC-staining and epidemiological factors and specific mutations were tested by use of univariate logistic regression with positive IHC-staining as the dependent variable. To test for differences in heterogeneity between the two IHC-clones, we used a test for equality of proportions. We regarded two-sided P values < 0.05 as statistically significant. Differences in overall survival and time to relapse were assessed by use of Kaplan Meier plots and log rank test. STATA Release 16 was used for statistical analysis.
We used biobank specimens from a cohort of surgically resected lung cancer patients at Oslo University Hospital. This biobank has a connected database with histopathological-, biological- and clinical information. The surgery and sampling was done during the period 2006–2018, with a median follow-up exceeding 5 years. Mortality data were imported from the Norwegian Population Registry, which is updated monthly. Two patients were lost to follow up (emigration), and was considered negligible in the analysis. We also excluded patients with carcinoid tumour and thymoma. Written informed consent was obtained from all patients and the project was approved by the regional ethics committee (1904/2009). The staging has been done according to the latest TNM classifications of malignant tumours at the time of surgery, but for the FISH positive cases we restaged the samples to the current edition . For adenocarcinomas with ROS1 fusion, we have also used the proposed new grading system for invasive pulmonary adenocarcinoma .
In this study we used full size slides from formalin-fixed paraffin embedded (FFPE) blocks, tissue micro arrays (TMA), and DNA and RNA extracted from fresh frozen material or FFPE blocks. The full size slides were taken from FFPE blocks from resections in the diagnostic biobank at Oslo University Hospital. These blocks were sliced at the Department of pathology at Vestfold hospital trust. Tissue microarray blocks were made from specimens from all the resections, each block consisting of specimens from 25–30 patients. All specimens were represented in triplicates, each core being one mm in diameter, and all tissue cylinders were harvested from the original FFPE blocks after careful selection by a trained pathologist. The TMA blocks were made and sliced at the Department of pathology at Oslo University Hospital. The majority of the TMA blocks have been used in previous projects . DNA and RNA were extracted from fresh-frozen tissue obtained at surgery. In the few cases of an insufficient number of tumour cells in the frozen tissue samples, the extraction was done from FFPE-blocks. Positive external control in ROS1-IHC can be difficult to find , because of the lack of reliable naturally ROS1 positivity in normal tissue. We used known positive FFPE tissue from a patient with a CD74-ROS1 fusion (confirmed with FISH and NGS) as a positive control. This tumour tissue was also strong and diffuse positive with the two different ROS1 IHC clones (D4D6 and SP384).
We performed ROS1 IHC analyses by use of two different ROS1-directed antibody clones: ROS1 (D4D6) Rabbit mAb (Cell Signaling, 3287) and the VENTANA ROS1 (SP384) Rabbit Monoclonal Primary Antibody (Roche Diagnostics, 790–6087). The slides were stained at the Department of Pathology at Vestfold Hospital Trust on a VENTANA BenchMark ULTRA system. This is a fully-automated IHC staining platform. For details about the protocols, see Additional file : Table S1. The microscope was an Axio Imager.A2 (Zeiss, item no. 490022–0009-000). We used both a qualitative and a semiquantitative scoring system. All samples were initially grouped into one of seven predefined groups; Negative, weak and focal, weak and diffuse, moderate/strong and focal, moderate/strong and diffuse, ambiguous and too few viable tumour cells (<10 viable tumour cells). Negative samples were defined by lack of tumour cell positivity under 400x (40x objective) magnification. Moderate/strong positivity was defined by dark brown staining with 25 - 200x magnification (2,5-20x objective). Weak positivity was defined by light brown staining, visible at 100-200x (10-20x objective), but requiring of 400x (40x objective) to see clearly. Diffusely positivity was defined by more than 50% of tumour cells with positive staining, including weak positivity, while focal positivity was defined by less than 50% of tumour cells with positive staining. Ambiguous samples were samples that could not be easily classified due to unspecific staining. Within the TMAs, the staining intensity and distribution were scored by assessing the cores from each case as one unit. All ambiguous samples were reexamined by an additional pathologist and were classified in a consensus meeting. To assess the inter-observer variability and ensure standardized categorization, a random sample of five of the TMA-blocks (114 cases) were examined by two pathologists individually. Any discrepancies in the interpretation were noted and discussed in a consensus meeting in order to optimize reliability. We also used a combinative semiquantitative scoring system on both the TMA slides and full size slides. In order to compare results, we used the H-score which has been used in several recent papers on ROS1 expression . We used the same formula as Huang et al. 2019 : (1x (percentage of relevant cells with 1+ staining) + (2x (percentage of relevant cells with 2+staining) + 3x (percentage of relevant cells with 3+ staining). Staining intensity was defined as absence of staining (0), weak staining (1+), moderate staining (2+) and strong staining (3+). We used the definition of 0, 1+, 2+ and 3+ from Conde et al. 2019 : Negative staining (0), which was defined as an absence of expression; weak staining (1+), which involved the use of a 40x objective; moderate staining (2+), which required the use of a 10x or 20x objective and strong cytoplasmic staining (3+), which was clearly visible with the use of a 2x or 4x objective. See Additional file : Table S2 for details about the scoring system and definitions. To assess the heterogeneity of ROS1 expression, any IHC-positive sample with more than weak and patchy staining was also reexamined with IHC on full size sections from the original FFPE blocks. Heterogeneity in staining can be due to technical issues like fixation, edge artifacts or biological heterogeneity in expression. Thereafter, the IHC-positive samples were grouped according to the percentage of tumour cells with positive IHC and staining quality (membranous/cytoplasmatic/nuclear, granular, diffuse). In addition, in order to reduce false negative IHC due to heterogenic expression, full size section IHC was also performed on a subgroup of TMA-IHC-negative cases where full size sections had already been prepared for a different study (cases with positive NTRK expression in IHC). Heterogeneity was defined as positive cases with areas of both 0 or 1 + in addition to 2 + or 3 + .
We tested IHC-positive cases (see definition of IHC-positive under variables) with FISH on full-sized slides. In addition, a sub-group of IHC-negative cases were also tested. FISH was performed at the Pathology department of Oslo University Hospital (Radiumhospitalet) with a dual break apart probe from Abbott (Vysis ROS1 Break Apart FISH Probe Kit, product number 08N29-021). The preparation was done according to the manufacturer’s recommendations . For the interpretation, we chose areas with the best preserved morphology and with clear signals. In the validation/verification of the test, the laboratory has found that a positive tumour is defined with a cut-off of at least 15% split signals, including isolated orange (5`) and green (3`) signals. Fused signals and break apart, single green /orange signals were counted in at least 50 nuclei and also at least 200 signals. Break apart split signals are green and orange signals separated by at least 1 signal diameter. An isolated 5’or 3’pattern means that an orange or green signal is present alone or together with fused or break apart signals. A fused signal is either break apart signals separated with less than 1 signal diameter or a completely fused signal that appears yellow.
NGS was performed on the same samples as FISH. Isolated DNA and RNA were extracted from fresh-frozen tissue (stored at -80 °C) with AllPrep DNA/RNA/miRNA Universal Kit (Qiagen, 80,224) on the QIAcube (automated, spin-column-based nucleic acid extraction from Qiagen). The concentration was measured with Nanodrop (Thermo Fisher Scientific) and Qubit Fluorometer (Thermo Fisher Scientific). We used Agilent 2100 Bioanalyzer (Agilent Technologies) to analyze the RNA-quality. The tumour percentage was evaluated on frozen sections. If the percentage was below 10%, and there were no relevant findings (EGFR-, KRAS- or BRAF-mutation, or ROS1-, ALK- or RET-fusion), then NGS was repeated on slides from FFPE-blocks from the original resection. In these cases, the extraction was done from FFPE blocks using MagLead12gC (Biosystem, A1120) for DNA and Quick RNA FFPE kit (Zymo Research, R1008) for RNA. For the sequencing, we used the Oncomine Comprehensive V3-panel (OCAv3)(Thermo Fisher Scientific, A35806). This panel covers 161 genes. The library preparation was done on an Ion Chef instrument (Thermo Fisher Scientific). The sequencing was done at both Oslo University Hospital and Vestfold Hospital Trust from July 2019 – September 2021. In Oslo, the sequencing was done on an Ion Torrent S5, and in Vestfold on an Ion Torrent S5XL. For the bioinformatical analysis, we used the Ion Reporter version 5.10–5.16 with a custom filter (Oncomine Variants, 5% CI CNV ploidy > = gain of 2 over normal). The sequencing of DNA and RNA based on tissue from FFPE blocks was done in Vestfold. In these analyses, the material was more fragmented, and therefore there was more “noise” in the sequencing results. To improve interpretation, we therefore heightened the quality requirements of the original filter in these cases (coverage > 1000 reads, Phredscore > 20, allele frequency above 5%). The same quality requirements were also used in sequencing performed on fresh material, but in these cases the heightened quality requirements were not included in the filter. See Additional file : Table S3 for details about the quality requirements. Fusion transcripts with unknown gene partner, can be reported as a non-targeted fusion by a combination of primers used for different targeted isoforms .
In cases with positive FISH and negative IHC/NGS, RT-qPCR was performed with Idylla GeneFusion Assay RUO/1.1 (Biocartis AO121/6). Eluat of RNA extracted for NGS was used in the procedure. The most common fusion partners (CD74, SDC4, SLC34A2, EZR, TPM3, GOPC and LRIG3) are included in the kit. In addition the method can detect if there is an expression imbalance between the 5´and 3´end of the ROS1 gene. An expression imbalance indicates that there can be a fusion with a partner other than those detected by the kit .
The main outcome of this study was ROS1 fusion positivity and ROS1 IHC-positivity. Positive ROS1-fusion was defined as samples positive in at least two of the three methods (one of the IHC-clones, FISH, NGS). Positive ROS1 IHC-staining was defined as focal moderate/strong, diffuse weak or diffuse moderate/strong staining, while negative ROS1 IHC-staining was defined as clearly negative or focal and weak staining. The independent variables included the clinical parameters like smoking status (former, current or never smoker), sex and age (continuous), stage, histopathological diagnosis and NGS-results.
This was a retrospective, cohort study based on linked register data and biobank material. We examined the data by use of frequency tables. Associations between positive IHC-staining and epidemiological factors and specific mutations were tested by use of univariate logistic regression with positive IHC-staining as the dependent variable. To test for differences in heterogeneity between the two IHC-clones, we used a test for equality of proportions. We regarded two-sided P values < 0.05 as statistically significant. Differences in overall survival and time to relapse were assessed by use of Kaplan Meier plots and log rank test. STATA Release 16 was used for statistical analysis.
The biobank contained tissue from 921 resection specimens. The median age was 67.5 years (range 39.2 to 87). The majority (93.1%) were current or former smokers with a median pack-year of 32.9. 83.7% were in stage I and II and 16.1% were in stage III and IV. The three most common histology subtypes were adenocarcinoma (58.8%), squamous cell carcinoma (32.6%) and large cell carcinoma (3.0%). See Table . Distribution of tumour markers We made 36 TMAs of the samples. Some of the cores in the TMA contained too few tumour cells to interpret the immunohistochemical analyses (32 for D4D6 and 21 for SP384). Twenty eight (D4D6) and forty (SP384) cases showed more than weak and focal staining and were defined as positive IHC. These cases went forward to further testing with FISH, NGS and full size section IHC. In addition, several negative and weak and focal cases were analyzed with this expanded testing (Fig. ). In total, 50 of the 921 cases were identified as positive by at least one of the IHC clones. Among these, 18 cases were identified as positive by both clones, whereas 10 of the D4D6-positive cases were negative with SP384 and 22 of the SP384-positive cases were negative with D4D6. SP384 had a higher number of IHC-positive cases than D4D6. In each scoring group except in the group with diffuse, moderate/strong staining, the mean H-score was higher with SP384 (Table ). The interobserver agreement was high in determining whether the cases were either negative/focal and weak/not enough tumour cells or strong/diffusely weak, with an observed agreement of 0.99 for D4D6 and 0.97 for SP384. The discrepant cases were mostly due to different interpretation of reactive pneumocytes and macrophages. We performed 101 FISH analyses, with 27 and 37 of the D4D6 and SP384 positive cases respectively. FISH found five cases of ROS1-fusion. The percentage of positive FISH signals ranged from 16–64%. Surprisingly, two cases that were negative in IHC with both clones showed a split pattern (above 15%) with FISH. These two cases (case number 2 and 3, Table ), were also completely IHC-negative (H-score 0) on the full slide sections. One of the cases had a percentage of isolated/split signals of 16% (just above the threshold). This case was also negative with RNA-NGS. The other case had a percentage of isolated/split signals of 28%, which is low, but considered clearly positive. In this last case the NGS failed on FFPE material and fresh frozen tissue was not available. RT-qPCR was negative on these two discrepant cases, and there were no 3´-5´ ROS1 expression imbalance. The cases with positive FISH were in stage IIb-IIIa . The adenocarcinomas are all grad 3/poorly differentiated tumours with predominantly (> 20%) high grade pattern (solid and trabecular/complex glandular patterns) . With NGS we detected three cases with ROS1-fusion. These cases (case number 1, 4 and 5, Table ) showed a strong, granular, cytoplasmatic staining without membranous attenuation and no heterogeneity with both clones and they were all FISH positive. All three cases had a TP53 mutation, the CD74 –gene was the fusion partner and they were all adenocarcinomas in stage IIb or IIIa. As shown in Fig. , 76 of the D4D6 and 64 of the SP384 negative samples were also analyzed with NGS, but we found no ROS1 fusion in this group. Three cases were positive in at least two of the three methods (IHC, FISH, NGS). These were considered confirmed cases with ROS1-fusions according to our definition of ROS1-fusion positive (samples positive in at least two of the three methods). All of these cases were positive in all three methods. They showed diffuse and strong staining intensity, and all of them were adenocarcinomas (Fig. ). Thus, the prevalence of ROS1 fusion was 0.6% in adenocarcinomas and 0.3% in the whole cohort. There was no significant differences in heterogeneity between D4D6 and SP384 ( p = 0.967). The proportion of positive cases with heterogenic staining was 48.3% and 48.8% for D4D6 and SP384 respectively. None of the three ROS1-fusion confirmed cases showed heterogenic staining. Eight cases showed strong and diffuse staining with D4D6. Three of five cases where we could not confirm a ROS1 rearrangement showed a partly lepidic or acinar/tubular growth pattern in contrast to the confirmed cases that had solid or trabecular growth patterns. The non-confirmed cases also showed more heterogenic staining on the full size slides and H-score on these slides were from 100–240. We found the same pattern with SP384 where ten of twelve non-confirmed cases showed a lepidic or acinar/tubular growth pattern, and more heterogenic staining on full size slides. Expression and ROS1 fusion related to standard prognostic variables, genetics, relapse and overall survival Positive ROS1 IHC-staining was strongly associated with adenocarcinomas. The estimated ORs for positive IHC-staining was 20.1 (95% CI 2.7–148.4, p = 0.003) and 9.3 (95% CI 2.8–30.1, p < 0.001) for the D4D6 clone and the SP384 clone respectively. Positive IHC-staining with the SP384 clone was statistically less frequent among former and current smokers than never-smokers (OR 0.2, 95% CI 0.08–0.36, p < 0.001). The same association was not found for the D4D6 clone. For both IHC-clones, there were no associations between positive staining and pack-year of smoking, stage, sex or age. We found TP53 mutation in all three cases with confirmed ROS1 fusions, and they had no other driver mutations like KRAS, BRAF or EGFR. Among the 104 NGS analyzed cases, the two most frequent mutations were TP53 ( n = 49) and KRAS ( n = 20). IHC-positivity was associated with KRAS mutation, both in D4D6 (OR 6.5, 95% CI 2.3–18.7, p = 0.001) and SP384 (OR 2.9, 95% CI 1.1–8.1, p = 0.04), but when we adjusted for adenocarcinoma histology there was no significant association. There were no statistically significant associations between positive IHC and TP53. There was no statistically significant association between IHC expression and overall survival or time to relapse. Two of the patients with ROS1 fusion (case number 4 and 5, Table ) received adjuvant chemotherapy. Patient number 1 relapsed after 3 years, and died of lung cancer one month later. Patient number 5 relapsed after only 3 months, and died of lung cancer shortly after that. While patient number 4 has still not relapsed and is still alive eleven years after surgery. One of the two patients with positive FISH and negative IHC/RT-qPCR/NGS (case number 2, Table ) never relapsed and died after 14 years (other cause). Patient number 3 relapsed after almost 14 months and died 6 months later. None of these two were tested for ROS1 fusion.
We made 36 TMAs of the samples. Some of the cores in the TMA contained too few tumour cells to interpret the immunohistochemical analyses (32 for D4D6 and 21 for SP384). Twenty eight (D4D6) and forty (SP384) cases showed more than weak and focal staining and were defined as positive IHC. These cases went forward to further testing with FISH, NGS and full size section IHC. In addition, several negative and weak and focal cases were analyzed with this expanded testing (Fig. ). In total, 50 of the 921 cases were identified as positive by at least one of the IHC clones. Among these, 18 cases were identified as positive by both clones, whereas 10 of the D4D6-positive cases were negative with SP384 and 22 of the SP384-positive cases were negative with D4D6. SP384 had a higher number of IHC-positive cases than D4D6. In each scoring group except in the group with diffuse, moderate/strong staining, the mean H-score was higher with SP384 (Table ). The interobserver agreement was high in determining whether the cases were either negative/focal and weak/not enough tumour cells or strong/diffusely weak, with an observed agreement of 0.99 for D4D6 and 0.97 for SP384. The discrepant cases were mostly due to different interpretation of reactive pneumocytes and macrophages. We performed 101 FISH analyses, with 27 and 37 of the D4D6 and SP384 positive cases respectively. FISH found five cases of ROS1-fusion. The percentage of positive FISH signals ranged from 16–64%. Surprisingly, two cases that were negative in IHC with both clones showed a split pattern (above 15%) with FISH. These two cases (case number 2 and 3, Table ), were also completely IHC-negative (H-score 0) on the full slide sections. One of the cases had a percentage of isolated/split signals of 16% (just above the threshold). This case was also negative with RNA-NGS. The other case had a percentage of isolated/split signals of 28%, which is low, but considered clearly positive. In this last case the NGS failed on FFPE material and fresh frozen tissue was not available. RT-qPCR was negative on these two discrepant cases, and there were no 3´-5´ ROS1 expression imbalance. The cases with positive FISH were in stage IIb-IIIa . The adenocarcinomas are all grad 3/poorly differentiated tumours with predominantly (> 20%) high grade pattern (solid and trabecular/complex glandular patterns) . With NGS we detected three cases with ROS1-fusion. These cases (case number 1, 4 and 5, Table ) showed a strong, granular, cytoplasmatic staining without membranous attenuation and no heterogeneity with both clones and they were all FISH positive. All three cases had a TP53 mutation, the CD74 –gene was the fusion partner and they were all adenocarcinomas in stage IIb or IIIa. As shown in Fig. , 76 of the D4D6 and 64 of the SP384 negative samples were also analyzed with NGS, but we found no ROS1 fusion in this group. Three cases were positive in at least two of the three methods (IHC, FISH, NGS). These were considered confirmed cases with ROS1-fusions according to our definition of ROS1-fusion positive (samples positive in at least two of the three methods). All of these cases were positive in all three methods. They showed diffuse and strong staining intensity, and all of them were adenocarcinomas (Fig. ). Thus, the prevalence of ROS1 fusion was 0.6% in adenocarcinomas and 0.3% in the whole cohort. There was no significant differences in heterogeneity between D4D6 and SP384 ( p = 0.967). The proportion of positive cases with heterogenic staining was 48.3% and 48.8% for D4D6 and SP384 respectively. None of the three ROS1-fusion confirmed cases showed heterogenic staining. Eight cases showed strong and diffuse staining with D4D6. Three of five cases where we could not confirm a ROS1 rearrangement showed a partly lepidic or acinar/tubular growth pattern in contrast to the confirmed cases that had solid or trabecular growth patterns. The non-confirmed cases also showed more heterogenic staining on the full size slides and H-score on these slides were from 100–240. We found the same pattern with SP384 where ten of twelve non-confirmed cases showed a lepidic or acinar/tubular growth pattern, and more heterogenic staining on full size slides.
Positive ROS1 IHC-staining was strongly associated with adenocarcinomas. The estimated ORs for positive IHC-staining was 20.1 (95% CI 2.7–148.4, p = 0.003) and 9.3 (95% CI 2.8–30.1, p < 0.001) for the D4D6 clone and the SP384 clone respectively. Positive IHC-staining with the SP384 clone was statistically less frequent among former and current smokers than never-smokers (OR 0.2, 95% CI 0.08–0.36, p < 0.001). The same association was not found for the D4D6 clone. For both IHC-clones, there were no associations between positive staining and pack-year of smoking, stage, sex or age. We found TP53 mutation in all three cases with confirmed ROS1 fusions, and they had no other driver mutations like KRAS, BRAF or EGFR. Among the 104 NGS analyzed cases, the two most frequent mutations were TP53 ( n = 49) and KRAS ( n = 20). IHC-positivity was associated with KRAS mutation, both in D4D6 (OR 6.5, 95% CI 2.3–18.7, p = 0.001) and SP384 (OR 2.9, 95% CI 1.1–8.1, p = 0.04), but when we adjusted for adenocarcinoma histology there was no significant association. There were no statistically significant associations between positive IHC and TP53. There was no statistically significant association between IHC expression and overall survival or time to relapse. Two of the patients with ROS1 fusion (case number 4 and 5, Table ) received adjuvant chemotherapy. Patient number 1 relapsed after 3 years, and died of lung cancer one month later. Patient number 5 relapsed after only 3 months, and died of lung cancer shortly after that. While patient number 4 has still not relapsed and is still alive eleven years after surgery. One of the two patients with positive FISH and negative IHC/RT-qPCR/NGS (case number 2, Table ) never relapsed and died after 14 years (other cause). Patient number 3 relapsed after almost 14 months and died 6 months later. None of these two were tested for ROS1 fusion.
In this study we aimed to map the prevalence of ROS1 rearrangement in a Norwegian cohort of early-stage resectable lung cancer, and see whether the tumours with ROS1 fusion or ROS1 protein expression were associated with specific epidemiological, histological or genetic characteristics. The prevalence of ROS1 fusion in resected adenocarcinomas in this cohort was 0.6%. The SP384 was in general more often positive and therefore less specific, but both clones identified the three cases that were positive with both FISH and NGS. Positive staining was strongly associated with adenocarcinomas. Most of the cases with strong and diffuse ROS1 expression where we could not confirm a ROS1 fusion had an acinar or lepidic growth pattern, in contrast to the confirmed cases that showed solid or trabecular growth pattern. In SP384, positive ROS1 IHC-staining was more common among never-smokers than among former- and current smokers, but this association was not found when using the D4D6 clone. There were no associations between IHC based ROS1 expression and other prognostic markers like age, sex, stage or pack-year of smoking. Strengths and limitations This study was based on comprehensive cohort data from a large number of resected lung cancer patients, with the use of multimodal testing by use of two different IHC-clones, FISH, NGS and RT-qPCR. We believe these features are strengths of the study. There are several limitations in this study, including the use of TMA and IHC as a screening method, the interpretation of IHC and FISH and detection of fusions with novel/uncommon partners. First, we used TMA in the IHC-based ROS1-screening process. TMA is a cost effective method for IHC-based mass screening. However, morphology assessment can be challenging on IHC-slides and even more challenging on TMAs. Morphology assessment is particularly important as macrophages and reactive pneumocytes can stain positive for ROS1. To reduce the risk of false positive staining, we included a slide from the TMA block with principal staining (hematoxylin and eosin stain), so that the morphology could be examined together with the IHC. Second, since we only had a small area of the tumour in a TMA, tumour heterogeneity could give a wrong impression of the general protein expression in the entire tumour. The degree and direction of such heterogeneity related sampling bias in our data are unknown. However, previous studies have shown substantial correlation between TMA and full size slides . In our study, the correlation between TMA and full size slides was also good (see Additional file : S7). Furthermore, we found that the NGS and FISH positive cases had a homogenous staining pattern, and this is consistent with other studies, especially with the SP384 . Third, we performed manual IHC scoring. Several conditions may affect the subjective interpretation of the intensity of staining. As highlighted by Giltnane et al., background staining, stromal staining and the order in which a slide is observed may be of importance . In our experience, the calibration will also differ from day to day. The subjective assessment of IHC-staining represents a risk of misclassification bias. We attempted to reduce the impact of misclassification bias by using a precise definition in addition to having two pathologists independently calibrate the interpretation. One of the pathologists scored all the TMA twice to reduce the impact of day to day differences in calibrations. In the FISH interpretation, we used the number of split apart signals/isolated signals and not the number of positive cells which has been more commonly used in previous studies . For FFPE slides, tangentially cut nuclei can give both false negative (split signals not presented on the slide) and false positive results (fused signals are separated in the cutting giving an impression of a single signal). In the validation of the method, the laboratory found that this could be compensated for by counting signals instead of positive cells, an approach for which they have long experience. For cytological specimens, the other formula with a score based on the number of positive cells can be used, as whole nuclei can be evaluated. Finally, as mentioned in the introduction, for sensitive fusion detection RNA-NGS is preferred over DNA-NGS . However, for targeted RNA-panels like OCAv3, the sensitivity is best if the partner gene is known. We can therefore not totally exclude false negative NGS in the cases with uncommon/novel fusion partners. Nevertheless, the OCAv3 can potentially detect novel/uncommon fusion partners by combinations of primers in the panel, and they are then reported as a non-targeted fusions. We did not find any non-targeted fusions. In addition, in cases with positive FISH and negative NGS/IHC, we performed RT-qPCR with the ability to find evidence of uncommon fusion partners with the 3´and 5´ expression imbalance. No cases with expression imbalance were detected. Unexpected findings Two cases were positive with FISH, but negative with IHC and RT-qPCR. One of them was also negative by NGS and the other one failed. It is known that IHC can give false positive results, and that ROS1 IHC always has to be confirmed with a supplementary test such as RT-qPCR, FISH or NGS. Even though FISH is considered to be the gold standard, other studies also found that positive FISH results are not always reproducible . In cases with isolated 3`pattern there is a biological explanation for this, because this may represent a deletion of the segment of the gene containing the binding site for the 5`probe . In cases with positive FISH and negative IHC, the fusion may have been inactivated after posttranslational modification or it might be a non-functioning fusion where the kinase domain is not included in the fusion . Since FISH break apart probes can detect fusions independent of the fusion partner, the discrepant cases might also be a result of false negative RT-qPCR and NGS. As mentioned earlier, novel/uncommon fusion partners can give rise to false negative RT-qPCR and NGS. However, both the Idylla GeneFusion assay (RT-qPCR) and OCAv3 (targeted RNA-NGS) may manage to detect non-targeted fusions were the fusions partner is not included in the kit . Thus, the negative NGS-finding and in addition negative RT-qPCR may imply that there are true false positive results from the FISH-analysis. None of these two patients have been treated with ROS1 inhibitors. In total, 50 of the 921 cases were positive by IHC, but only three cases could be confirmed with NGS and FISH. Thus, positive IHC staining is much more common than the presumably clinically relevant ROS1 fusions in this study. In addition to the 47 FISH- and NGS negative cases with IHC-positive staining, we noted unspecific staining in macrophages, osteoclasts and reactive pneumocytes. As discussed, there might be several reasons for unspecific staining . The different clones react to different epitopes on the antigen. The antigen, in this case, is the ROS1 protein, either wild type or chimeric/fused. Chromosomal rearrangement leads to activation of the ROS1-gene with sub-sequent overexpression of the chimeric ROS1 protein. Thus, IHC positive cases in wild type tissue/tumour cells may be due to either preanalytical conditions (too long or short fixation, crushed tissue, etc.), cross reactivity to other epitopes, or biological factors (necrosis, phagocytosis of antigens by macrophages, etc.) . In addition, wild type ROS1 protein overexpression could also explain IHC-positivity, and ROS1 seems to be particularly enriched in lung tissue . Lung cancer seems to be even more enriched, perhaps through genetic or epigenetic mechanisms . Contrary to what is seen in ERBB2 amplification and Her-2 overexpression , ROS1 amplification does not seem to affect the ROS1 protein expression . Based on our definition of fusion positivity (2 of 3 tests positive), we found no false negative FISH or NGS. False negative FISH might be because of complex fusions where signals appear normal. Biological reasons for false negative FISH are mostly due to intrachromosomal fusions like GOPC, also called FIG . False negative RNA-NGS is mainly seen in specimens with poor RNA quality, and it is important to evaluate the quality metrics in each sample . The reasons for discrepancies in our two cases with FISH positivity are not clear. In both cases, we found a mix of isolated 3’signals and split signals, but in one of the cases, the percentage was just above the threshold. Discussion on how the results from the study integrate with the existing evidence In this Norwegian cohort of early-stage lung cancer, the prevalence of ROS1-fusion in adenocarcinomas was 0.6%. This is consistent with other studies on early-stage disease. In the study by Selinger et al., 0.4% (1/278) of resected stage I-III adenocarcinomas had a ROS1 fusion and Warth et al. (both used ROS1 IHC and FISH) found a prevalence of 1.2% (5/405) in resected adenocarcinomas . In Warth`s study the majority of patients were in stage I-III, but 2.5% were in stage IV. Bergethon et al. (Reverse Transcription PCR /Sanger sequencing and FISH) found a prevalence of 0.6% in stages I and II and 2.8% in stage III and IV . The three cases with confirmed ROS1 fusion had an H-score between 200 and 300 in both clones, and there was no heterogenic staining. Other studies that have used the two different ROS1 clones also found that the clones had good sensitivity, but the specificity was lower depending on the cut-off level . The average H-score is higher for SP384 in both studies, which is consistent with our findings. The two studies have used the same dilution, detection system and staining platform as we have. While Conde et al. also found that the D4D6 showed a more heterogenic staining pattern in ROS1-positive tumours, statistical difference in heterogenic staining was not found in our material. Both Conde et al. and Hofman et al. have reported a moderate or good interobserver agreement and that positive staining can be found in normal lung tissue as well, which is also consistent with our findings . In ROS1 FISH negative cases they also found some degree of staining in 13–32.1% and 3–20.3% cases analysed with SP384 and D4D6, respectively. Although mostly weak or focal, this emphasizes that ROS1 IHC can show of unspecific staining in non-rearranged cases. We found a strong association between adenocarcinoma histology and IHC positivity, and for SP384 an association with never smoking. There was no association between positive IHC and overall survival, time to relaps, age, sex, stage or pack-year of smoking. Warth et al. also found an association with adenocarcinoma histology. They also found that ROS1 positivity was associated with longer overall survival and female sex . One reason for this discrepancy may be that they defined positive IHC as cases showing any positivity, while we defined positive as more than focal weak staining. All three cases with ROS1 fusion had a TP53 co-mutation and the CD74-gene as fusion partner. TP53 is known to be the most frequent co-mutation in ROS1 fusion and is associated with a shorter progression free survival with firstline crizotinib therapy . CD74 is the most common ROS1 fusion partner .
This study was based on comprehensive cohort data from a large number of resected lung cancer patients, with the use of multimodal testing by use of two different IHC-clones, FISH, NGS and RT-qPCR. We believe these features are strengths of the study. There are several limitations in this study, including the use of TMA and IHC as a screening method, the interpretation of IHC and FISH and detection of fusions with novel/uncommon partners. First, we used TMA in the IHC-based ROS1-screening process. TMA is a cost effective method for IHC-based mass screening. However, morphology assessment can be challenging on IHC-slides and even more challenging on TMAs. Morphology assessment is particularly important as macrophages and reactive pneumocytes can stain positive for ROS1. To reduce the risk of false positive staining, we included a slide from the TMA block with principal staining (hematoxylin and eosin stain), so that the morphology could be examined together with the IHC. Second, since we only had a small area of the tumour in a TMA, tumour heterogeneity could give a wrong impression of the general protein expression in the entire tumour. The degree and direction of such heterogeneity related sampling bias in our data are unknown. However, previous studies have shown substantial correlation between TMA and full size slides . In our study, the correlation between TMA and full size slides was also good (see Additional file : S7). Furthermore, we found that the NGS and FISH positive cases had a homogenous staining pattern, and this is consistent with other studies, especially with the SP384 . Third, we performed manual IHC scoring. Several conditions may affect the subjective interpretation of the intensity of staining. As highlighted by Giltnane et al., background staining, stromal staining and the order in which a slide is observed may be of importance . In our experience, the calibration will also differ from day to day. The subjective assessment of IHC-staining represents a risk of misclassification bias. We attempted to reduce the impact of misclassification bias by using a precise definition in addition to having two pathologists independently calibrate the interpretation. One of the pathologists scored all the TMA twice to reduce the impact of day to day differences in calibrations. In the FISH interpretation, we used the number of split apart signals/isolated signals and not the number of positive cells which has been more commonly used in previous studies . For FFPE slides, tangentially cut nuclei can give both false negative (split signals not presented on the slide) and false positive results (fused signals are separated in the cutting giving an impression of a single signal). In the validation of the method, the laboratory found that this could be compensated for by counting signals instead of positive cells, an approach for which they have long experience. For cytological specimens, the other formula with a score based on the number of positive cells can be used, as whole nuclei can be evaluated. Finally, as mentioned in the introduction, for sensitive fusion detection RNA-NGS is preferred over DNA-NGS . However, for targeted RNA-panels like OCAv3, the sensitivity is best if the partner gene is known. We can therefore not totally exclude false negative NGS in the cases with uncommon/novel fusion partners. Nevertheless, the OCAv3 can potentially detect novel/uncommon fusion partners by combinations of primers in the panel, and they are then reported as a non-targeted fusions. We did not find any non-targeted fusions. In addition, in cases with positive FISH and negative NGS/IHC, we performed RT-qPCR with the ability to find evidence of uncommon fusion partners with the 3´and 5´ expression imbalance. No cases with expression imbalance were detected.
Two cases were positive with FISH, but negative with IHC and RT-qPCR. One of them was also negative by NGS and the other one failed. It is known that IHC can give false positive results, and that ROS1 IHC always has to be confirmed with a supplementary test such as RT-qPCR, FISH or NGS. Even though FISH is considered to be the gold standard, other studies also found that positive FISH results are not always reproducible . In cases with isolated 3`pattern there is a biological explanation for this, because this may represent a deletion of the segment of the gene containing the binding site for the 5`probe . In cases with positive FISH and negative IHC, the fusion may have been inactivated after posttranslational modification or it might be a non-functioning fusion where the kinase domain is not included in the fusion . Since FISH break apart probes can detect fusions independent of the fusion partner, the discrepant cases might also be a result of false negative RT-qPCR and NGS. As mentioned earlier, novel/uncommon fusion partners can give rise to false negative RT-qPCR and NGS. However, both the Idylla GeneFusion assay (RT-qPCR) and OCAv3 (targeted RNA-NGS) may manage to detect non-targeted fusions were the fusions partner is not included in the kit . Thus, the negative NGS-finding and in addition negative RT-qPCR may imply that there are true false positive results from the FISH-analysis. None of these two patients have been treated with ROS1 inhibitors. In total, 50 of the 921 cases were positive by IHC, but only three cases could be confirmed with NGS and FISH. Thus, positive IHC staining is much more common than the presumably clinically relevant ROS1 fusions in this study. In addition to the 47 FISH- and NGS negative cases with IHC-positive staining, we noted unspecific staining in macrophages, osteoclasts and reactive pneumocytes. As discussed, there might be several reasons for unspecific staining . The different clones react to different epitopes on the antigen. The antigen, in this case, is the ROS1 protein, either wild type or chimeric/fused. Chromosomal rearrangement leads to activation of the ROS1-gene with sub-sequent overexpression of the chimeric ROS1 protein. Thus, IHC positive cases in wild type tissue/tumour cells may be due to either preanalytical conditions (too long or short fixation, crushed tissue, etc.), cross reactivity to other epitopes, or biological factors (necrosis, phagocytosis of antigens by macrophages, etc.) . In addition, wild type ROS1 protein overexpression could also explain IHC-positivity, and ROS1 seems to be particularly enriched in lung tissue . Lung cancer seems to be even more enriched, perhaps through genetic or epigenetic mechanisms . Contrary to what is seen in ERBB2 amplification and Her-2 overexpression , ROS1 amplification does not seem to affect the ROS1 protein expression . Based on our definition of fusion positivity (2 of 3 tests positive), we found no false negative FISH or NGS. False negative FISH might be because of complex fusions where signals appear normal. Biological reasons for false negative FISH are mostly due to intrachromosomal fusions like GOPC, also called FIG . False negative RNA-NGS is mainly seen in specimens with poor RNA quality, and it is important to evaluate the quality metrics in each sample . The reasons for discrepancies in our two cases with FISH positivity are not clear. In both cases, we found a mix of isolated 3’signals and split signals, but in one of the cases, the percentage was just above the threshold.
In this Norwegian cohort of early-stage lung cancer, the prevalence of ROS1-fusion in adenocarcinomas was 0.6%. This is consistent with other studies on early-stage disease. In the study by Selinger et al., 0.4% (1/278) of resected stage I-III adenocarcinomas had a ROS1 fusion and Warth et al. (both used ROS1 IHC and FISH) found a prevalence of 1.2% (5/405) in resected adenocarcinomas . In Warth`s study the majority of patients were in stage I-III, but 2.5% were in stage IV. Bergethon et al. (Reverse Transcription PCR /Sanger sequencing and FISH) found a prevalence of 0.6% in stages I and II and 2.8% in stage III and IV . The three cases with confirmed ROS1 fusion had an H-score between 200 and 300 in both clones, and there was no heterogenic staining. Other studies that have used the two different ROS1 clones also found that the clones had good sensitivity, but the specificity was lower depending on the cut-off level . The average H-score is higher for SP384 in both studies, which is consistent with our findings. The two studies have used the same dilution, detection system and staining platform as we have. While Conde et al. also found that the D4D6 showed a more heterogenic staining pattern in ROS1-positive tumours, statistical difference in heterogenic staining was not found in our material. Both Conde et al. and Hofman et al. have reported a moderate or good interobserver agreement and that positive staining can be found in normal lung tissue as well, which is also consistent with our findings . In ROS1 FISH negative cases they also found some degree of staining in 13–32.1% and 3–20.3% cases analysed with SP384 and D4D6, respectively. Although mostly weak or focal, this emphasizes that ROS1 IHC can show of unspecific staining in non-rearranged cases. We found a strong association between adenocarcinoma histology and IHC positivity, and for SP384 an association with never smoking. There was no association between positive IHC and overall survival, time to relaps, age, sex, stage or pack-year of smoking. Warth et al. also found an association with adenocarcinoma histology. They also found that ROS1 positivity was associated with longer overall survival and female sex . One reason for this discrepancy may be that they defined positive IHC as cases showing any positivity, while we defined positive as more than focal weak staining. All three cases with ROS1 fusion had a TP53 co-mutation and the CD74-gene as fusion partner. TP53 is known to be the most frequent co-mutation in ROS1 fusion and is associated with a shorter progression free survival with firstline crizotinib therapy . CD74 is the most common ROS1 fusion partner .
The ROS1 prevalence in adenocarcinomas was 0.6% with our algorithm. Both IHC-clones showed strong and homogenous staining with an H-score above 200 in the three cases where both FISH and NGS confirmed the presence of ROS1 fusion. 50 of 921 cases were positive with IHC, but only three of these cases were confirmed with NGS/FISH. The cases with confirmed fusion showed a solid/trabecular growth pattern, in contrast to most of the non-confirmed cases that showed a lepidic or acinar/tubular growth pattern. We also found ROS1-FISH positive cases that could not be confirmed with IHC, NGS or RT-qPCR. This illustrates that we might need dual testing with two different modalities. Currently, the prevalence of ROS1 fusion in late-stage disease in the Norwegian population is unknown, but as ROS1 testing has become mandatory in adenocarcinomas we will know more about this in the coming years. Relevant tests should be able to identify treatment responsive tumours. FISH ROS1 has been considered to be the “gold standard”, however we need more data from treatment studies to determine which test and algorithm identifies the clinically relevant ROS1 positive tumours. Hopefully, more trials will lead to broader therapy options for both early and late-stage lung cancer .
Additional file 1: Table S1. Details on immunohistochemical protocols. Table S2. Details about the scoring system and definitions. Equation for a positive/rearranged ROS1 tumour with FISH. Table S3. Quality requirements NGS. Table S4. Comparing D4D6 and SP384. Table S5. Table including DNA NGS with CNV and hotspot mutation. Table S6. IHC and stage, sex and smoking. Table S7. TMA versus full size slides.
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Improved species level bacterial characterization from rhizosphere soil of wilt infected
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3c63ca04-9c11-4be6-8bce-f106a1057560
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10224976
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Microbiology[mh]
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Pomegranate ( Punica granatum , Family: Punicaceae) is an excellent source of a variety of nutrients and minerals, dietary fibre, phenolic compounds, alkaloids and sterols. The pomegranate peel contains abundant polyphenols such as ellagitannins, gallotannins, proanthocyanidins, anthocyanins, and ellagic acid derivatives, while the seed oil is composed of unsaturated fatty acids, notably omega 5 punicic acid , . Due to the presence of these constituents the pomegranate extract and its juice have been extensively studied for their nutritive value, medicinal properties and prebiotic effects . With about 2.62 lakh hectares of land dedicated to pomegranate cultivation and a production yield of 30.34 lakh tonnes, India currently holds the largest share of the global pomegranate market, accounting for over 50% of the total global production. In addition, the cultivation of pomegranate provides a source of livelihood to over 2.5 lakh families in India (NRCP Annual Report, 2020). Although persistent efforts are being made in India to improve, promote and market the crop, various factors such as abiotic and biotic stressors, physiological limitations, genetic constraints, excessive growth rate, nutrient unavailability, as well as pest and disease infestations, have been identified as severe impediments to its growth , . Diseases such as wilt complex , anthracnose , bacterial blight , Coniella fruit rot , , foliar diseases such as leaf spot and fruit spot disease are some of the major diseases that affect the pomegranate crop. For decades, Wilt disease in pomegranate from various parts of India has reported heavy crop loss . Despite the fact that bacterial-host associations and their adaptations are complex, early and accurate detection of pathogens could prevent further crop and yield loss. 16S rRNA gene sequencing has proven to be an excellent approach for identifying bacterial pathogens with higher accuracy as there are signature specific sequences in bacterial species . The MinION platform (Oxford Nanopore Technologies Ltd. MinION) offers a unique possibility to perform soil microbial characterization. While the use of this platform has been established in major epidemiological, laboratory-based experiments, community studies or even samples collected from remote microbiomes such as glaciers , building-dust , fresh water monitoring , environmental metagenomes , in situ bioprospecting at desert locations , International Space Station (ISS) , irrigation water , study of ribosomal operons , ebola surveillance , the use of MinION in soil studies to address plant–microbe associations is limited. Sequenced data is subject to a number of quality checks before being analysed with reference sequenced from 16S rRNA data repositories for species identification. Currently, there are a number of publicly accessible 16S reference databases such as Ribosomal Database Project (RDP, http://rdp.cme.msu.edu/ ), Genome Taxonomy Database (GTDB, https://gtdb.ecogenomic.org/ ), SILVA database ( https://www.arb-silva.de/ ), Greengenes 16S rRNA database ( https://greengenes.secondgenome.com/ ), and 16S-UDb . Additionally, there are a few commercial solutions like EZBioCloud ( https://www.ezbiocloud.net/resources ) and SmartGene ( https://www.smartgene.com/services/modules/16s-microbiome ). The size, scope, curation methods, and frequency of updates across these databases vary greatly, as do the types of data they contain (partial sequences vs. whole genomes vs. type strains, etc.). The success of microbiome studies relies on the completeness and consistency of the existing 16S rRNA sequence repositories , . Therefore, benchmarking of multiple databases was performed to assess their taxonomy assignment potential from phylum to genus level against the gold standard NCBI’s 16S reference database. This could in turn enhance the exploratory potential, effectiveness and accuracy in identification of the pathogens. In the present study, 16S rRNA sequencing is implemented using the MinION platform to screen for bacterial communities from wilt affected pomegranate rhizosphere soil samples. An improved approach benchmarking various 16S rRNA databases has been performed in this study. It is observed that this approach could enhance the accuracy of detection significantly minimizing false positives and negatives. Using the approach, variations in abundance of growth promoting bacteria are observed along with predicted enriched pathways. The study's findings have important implications for agriculture and crop management, and it can be inferred that identifying and promoting growth-promoting bacterial communities could be an effective strategy for improving crop yield and combating diseases. By providing insights into the microbial communities present in pomegranate crops and their potential roles in promoting growth and preventing wilt disease, the study could inform the development of targeted treatment strategies.
Site description, sampling and physicochemical characterization Rhizosphere soil samples were collected from an orchard close to Chikkaballapur region of Karnataka, India with coordinates of 13.3907° N, 77.6880° E. The farmer had experienced a streak of losses for five consecutive years at the time of this study, with no sign of abatement, and the losses appeared to be escalating. The soil samples were processed and the wilt infected samples were physically examined for disease symptoms confirming the presence of wilt like symptoms. The plants were identified as wilt infected with Intermediate Stage Infection (ISI) and Advanced Stage Infection (ASI) on the basis of physical examination of the leaves, stem, fruits and roots. In the ISI sample, the fruits had dark coloured irregular spots with cracking, whereas in the severely infected plants the fruits were completely dry with dark brown pigmentation. Leaves showed yellowing, presence of moisture, dark-coloured irregular spots in the infected plants, and complete defoliation in the ASI or severely infected samples. The root systems of the infected plants were dry and reduced with elongated galls. Dark brown colouration of the stem which had turned completely dry was observed. Severely infected plants resulted in the production of infected fruits with no recovery. Soil samples of ISI and ASI were collected from four corners and one from the center of the orchard, each taken from plants showing similar symptoms. The samples were collected in triplicates, and then pooled. The samples were submitted under the BioProject name PRJNA540763 with the accession numbers infected sample ISI (SAMN11555162; SRR9002407) and severely infected sample ASI (SAMN11555163; SRR9002406). As a control HSC, sequence data of a healthy plant sample was used from a separate study (BioProject PRJNA540834; SRR9003394). The sample was collected from the same orchard under identical conditions . Whole metagenome analysis of the samples ISI and ASI has been performed and published in a separate study and the presence of Fusarium oxysporum has been ascertained followed by further assessment of its adaptations . All the necessary permissions to carry out this study have been obtained in accordance with the local state regulations. An overview of the entire protocol is depicted in Fig. . Physicochemical characterization and total microbial count estimation of the samples were carried out similar to the protocol outlined in our previous study employing whole metagenomics . Sample preparation, microbial community DNA extraction and sequencing DNA extraction and quality control DNA from the soil samples was extracted using the commercially available DNeasy Powersoil kit (Catalog No. 12888-50) as per the manufacturer's recommendations. The soil sample was first prepared by adding it to the powerbead tube, where C1 solution was added and vortexed. Soil sample preparation was followed by cell lysis, wherein C2 solution was added and the sample was incubated at 2–8 °C. Inhibitors were removed at this stage by adding solution C3 and incubated at 2–8 °C. Binding of DNA was carried out by adding solution C4 in the MB spin column and washed with solution C5. Elution was performed by adding solution C6. Extracted DNA from the samples were quantified using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and GEL Check before being taken for PCR amplification. The NanoDrop readings of 260/280 at an approximate value of 1.8 to 2 was used to determine the quality of DNA. Thereafter, the PCR Amplicon QC was performed, which included amplification of the 16S PCR product, which was then purified and subjected to GEL Check and NanoDrop QC. The NanoDrop readings of 260/280 with ~ value of 1.8 to 2 were inferred to be purified and used for further downstream processing. PCR amplification of 16S gene Composition of TAQ Master mix included a High-Fidelity DNA Polymerase, 0.5 mM dNTPs, 3.2 mM MgCl 2 , PCR Enzyme Buffer, and primers (16F: 5′ AGAGTTTGATCMTGGCTCAG 3′,16R: 5′ TACGGYTACCTTGTTACGACTT 3′). Extracted DNA (40 ng) was used for amplification along with 10 pM of each primer. The samples were subjected to 25 cycles of initial denaturation at 95 °C for 15 s, followed by annealing at 60 °C for 15 s, elongation at 72 °C for 2 min, and final extension at 72 °C for 10 min. The samples were finally kept at 4 °C. Sequencing protocol Nanopore sequencing was performed using 1 μg of DNA template, followed by end repair/dA tailing ligation of barcode adapter and barcoding PCR, end repair/dA tailing, blunt end adapter ligation. Thereafter purification was done using AMPure XP bead binding. Finally, the priming was carried and loaded on the SpotON flow cell. Metagenome sequence analysis Preparation of databases Bacterial 16S refseq nucleotide sequences (n = 22,423) were obtained from NCBI RefSeq Targeted Loci Project ( https://www.ncbi.nlm.nih.gov/refseq/targetedloci/ ). The corresponding taxonomy in 7 lineage level hierarchy in Quantitative Insight Into Microbial Ecology (QIIME) compatible format was generated using the python script entrez_qiime.py. ( https://github.com/bakerccm/entrez_qiime ). Ribosomal database project (RDP) Unaligned bacterial 16S rRNA sequences (n = 31,96,041) from RDP project ( https://rdp.cme.msu.edu/download/current_Bacteria_unaligned.fa.gz ) were downloaded, and made QIIME compatible by sequentially removing all sequences containing < 1200, > 2000 or any ambiguous nucleotides (N). The filtered data set (n = 12,89,001 sequences) was subjected to clustering at 99% threshold using VSEARCH (2.21.1). The final dataset contained 167,789 sequences. In addition, a taxonomy mapping file in QIIME compatible format was created by linking RDP sequence identifiers of the representative sequences with 7-level (domain, phylum, class, order, family, genus, and species) lineage hierarchy. GTDB Sativa curated 16S sequences (gtdb-sbdi-sativa.r06rs202.fna; n = 46,126) from the GTDB database release R06-RS202 ( https://gtdb.ecogenomic.org ), were obtained from the figshare repository ( https://scilifelab.figshare.com/articles/dataset/SBDI_Sativa_curated_16S_GTDB_database/14869077 ). The sequence and corresponding taxonomy mapping file was generated in QIIME compatible format. EzBioCloud 16S rRNA sequences with their corresponding taxonomy in QIIME compatible format (n = 64,660) were obtained from EzBioCloud server. SILVA Silva 138 SSURef NR99 full-length sequences (n = 4,36,680) ( https://data.qiime2.org/2020.6/common/silva-138-99-seqs.qza ) and taxonomy ( https://data.qiime2.org/2020.6/common/silva-138-99-tax.qza ) in QIIME2 compatible format was obtained from QIIME data resource repository. Greengenes Greengenes 16S OTUs (n = 2,03,452) and corresponding taxonomy were obtained from Greengenes FTP. ftp://greengenes.microbio.me/greengenes_release/gg_13_5/gg_13_8_otus.tar.gz . 16S unified database 16S rRNA sequences with their corresponding taxonomy in QIIME compatible format were obtained from 16sUDB github repository ( https://github.com/sarangian/16S-UDb ). Preparation of test dataset Bacterial 16S refseq nucleotide sequences (n = 22,423) were obtained from NCBI RefSeq Targeted Loci Project ( https://www.ncbi.nlm.nih.gov/refseq/targetedloci/ ). The corresponding taxonomy in 7 lineage level hierarchy in QIIME compatible format was generated using the python script entrez_qiime.py. ( https://github.com/bakerccm/entrez_qiime ). Comparison of the 16S rRNA sequence databases The performances of the five 16S rRNA databases (Greengenes, SILVA, RDP, GTDB, EzBioCloud) in correctly classifying the Bacterial 16S RefSeq nucleotide sequences (NCBI RefSeq Targeted Loci Project; test dataset) were determined up to the genus levels. This comparison was done using the “classify-consensus-blast” utility of QIIME2 feature-classifier program with parameters—p-perc-identity 0.8—p-query-cov 0.8—p-maxaccepts 10. The resultant classification output (taxonomy.qza) of individual 16S databases (n = 5) were converted to tsv format and compared against the known taxonomy mapping file of the test dataset. Performance of each database was calculated as the proportion of correctly classified sequences in the test dataset. ONT data analysis in QIIME2 framework The FASTQ files were processed using the MetONTIIME pipeline ( https://github.com/MaestSi/MetONTIIME ), a framework based on QIIME2 using Silva V138 (Silva 138 SSURef NR99) database and BLAST classifier. Parameters used were [-n 32 -c blast -m 10 -q 0.8 -i 0.8] . The resulting BIOM file and obtained representative sequences were subjected to downstream functional analysis. Functional profiling predictions and statistical analysis PICRUSt2 was employed for predictive functional profiling analysis and functional annotation of the sequences were based on Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO; www.kegg.jp/kegg/kegg1.html ). Pathways significance differentiation was further analyzed using statistical tests. All statistical tests including differential abundance analysis was performed using STAMP 2.1.3 ( http://kiwi.cs.dal.ca/Software/STAMP ) for each of the samples. Two-sided Fisher’s exact test was used to compare samples Storey’s false discovery rate method of multiple test correction (p value ≤ 0.05) using DP at 95% confidence intervals . Ethical approval All the necessary permissions to carry out this study have been obtained in accordance with the state regulations.
Rhizosphere soil samples were collected from an orchard close to Chikkaballapur region of Karnataka, India with coordinates of 13.3907° N, 77.6880° E. The farmer had experienced a streak of losses for five consecutive years at the time of this study, with no sign of abatement, and the losses appeared to be escalating. The soil samples were processed and the wilt infected samples were physically examined for disease symptoms confirming the presence of wilt like symptoms. The plants were identified as wilt infected with Intermediate Stage Infection (ISI) and Advanced Stage Infection (ASI) on the basis of physical examination of the leaves, stem, fruits and roots. In the ISI sample, the fruits had dark coloured irregular spots with cracking, whereas in the severely infected plants the fruits were completely dry with dark brown pigmentation. Leaves showed yellowing, presence of moisture, dark-coloured irregular spots in the infected plants, and complete defoliation in the ASI or severely infected samples. The root systems of the infected plants were dry and reduced with elongated galls. Dark brown colouration of the stem which had turned completely dry was observed. Severely infected plants resulted in the production of infected fruits with no recovery. Soil samples of ISI and ASI were collected from four corners and one from the center of the orchard, each taken from plants showing similar symptoms. The samples were collected in triplicates, and then pooled. The samples were submitted under the BioProject name PRJNA540763 with the accession numbers infected sample ISI (SAMN11555162; SRR9002407) and severely infected sample ASI (SAMN11555163; SRR9002406). As a control HSC, sequence data of a healthy plant sample was used from a separate study (BioProject PRJNA540834; SRR9003394). The sample was collected from the same orchard under identical conditions . Whole metagenome analysis of the samples ISI and ASI has been performed and published in a separate study and the presence of Fusarium oxysporum has been ascertained followed by further assessment of its adaptations . All the necessary permissions to carry out this study have been obtained in accordance with the local state regulations. An overview of the entire protocol is depicted in Fig. . Physicochemical characterization and total microbial count estimation of the samples were carried out similar to the protocol outlined in our previous study employing whole metagenomics .
DNA extraction and quality control DNA from the soil samples was extracted using the commercially available DNeasy Powersoil kit (Catalog No. 12888-50) as per the manufacturer's recommendations. The soil sample was first prepared by adding it to the powerbead tube, where C1 solution was added and vortexed. Soil sample preparation was followed by cell lysis, wherein C2 solution was added and the sample was incubated at 2–8 °C. Inhibitors were removed at this stage by adding solution C3 and incubated at 2–8 °C. Binding of DNA was carried out by adding solution C4 in the MB spin column and washed with solution C5. Elution was performed by adding solution C6. Extracted DNA from the samples were quantified using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and GEL Check before being taken for PCR amplification. The NanoDrop readings of 260/280 at an approximate value of 1.8 to 2 was used to determine the quality of DNA. Thereafter, the PCR Amplicon QC was performed, which included amplification of the 16S PCR product, which was then purified and subjected to GEL Check and NanoDrop QC. The NanoDrop readings of 260/280 with ~ value of 1.8 to 2 were inferred to be purified and used for further downstream processing. PCR amplification of 16S gene Composition of TAQ Master mix included a High-Fidelity DNA Polymerase, 0.5 mM dNTPs, 3.2 mM MgCl 2 , PCR Enzyme Buffer, and primers (16F: 5′ AGAGTTTGATCMTGGCTCAG 3′,16R: 5′ TACGGYTACCTTGTTACGACTT 3′). Extracted DNA (40 ng) was used for amplification along with 10 pM of each primer. The samples were subjected to 25 cycles of initial denaturation at 95 °C for 15 s, followed by annealing at 60 °C for 15 s, elongation at 72 °C for 2 min, and final extension at 72 °C for 10 min. The samples were finally kept at 4 °C. Sequencing protocol Nanopore sequencing was performed using 1 μg of DNA template, followed by end repair/dA tailing ligation of barcode adapter and barcoding PCR, end repair/dA tailing, blunt end adapter ligation. Thereafter purification was done using AMPure XP bead binding. Finally, the priming was carried and loaded on the SpotON flow cell.
DNA from the soil samples was extracted using the commercially available DNeasy Powersoil kit (Catalog No. 12888-50) as per the manufacturer's recommendations. The soil sample was first prepared by adding it to the powerbead tube, where C1 solution was added and vortexed. Soil sample preparation was followed by cell lysis, wherein C2 solution was added and the sample was incubated at 2–8 °C. Inhibitors were removed at this stage by adding solution C3 and incubated at 2–8 °C. Binding of DNA was carried out by adding solution C4 in the MB spin column and washed with solution C5. Elution was performed by adding solution C6. Extracted DNA from the samples were quantified using the NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and GEL Check before being taken for PCR amplification. The NanoDrop readings of 260/280 at an approximate value of 1.8 to 2 was used to determine the quality of DNA. Thereafter, the PCR Amplicon QC was performed, which included amplification of the 16S PCR product, which was then purified and subjected to GEL Check and NanoDrop QC. The NanoDrop readings of 260/280 with ~ value of 1.8 to 2 were inferred to be purified and used for further downstream processing.
Composition of TAQ Master mix included a High-Fidelity DNA Polymerase, 0.5 mM dNTPs, 3.2 mM MgCl 2 , PCR Enzyme Buffer, and primers (16F: 5′ AGAGTTTGATCMTGGCTCAG 3′,16R: 5′ TACGGYTACCTTGTTACGACTT 3′). Extracted DNA (40 ng) was used for amplification along with 10 pM of each primer. The samples were subjected to 25 cycles of initial denaturation at 95 °C for 15 s, followed by annealing at 60 °C for 15 s, elongation at 72 °C for 2 min, and final extension at 72 °C for 10 min. The samples were finally kept at 4 °C.
Nanopore sequencing was performed using 1 μg of DNA template, followed by end repair/dA tailing ligation of barcode adapter and barcoding PCR, end repair/dA tailing, blunt end adapter ligation. Thereafter purification was done using AMPure XP bead binding. Finally, the priming was carried and loaded on the SpotON flow cell.
Preparation of databases Bacterial 16S refseq nucleotide sequences (n = 22,423) were obtained from NCBI RefSeq Targeted Loci Project ( https://www.ncbi.nlm.nih.gov/refseq/targetedloci/ ). The corresponding taxonomy in 7 lineage level hierarchy in Quantitative Insight Into Microbial Ecology (QIIME) compatible format was generated using the python script entrez_qiime.py. ( https://github.com/bakerccm/entrez_qiime ). Ribosomal database project (RDP) Unaligned bacterial 16S rRNA sequences (n = 31,96,041) from RDP project ( https://rdp.cme.msu.edu/download/current_Bacteria_unaligned.fa.gz ) were downloaded, and made QIIME compatible by sequentially removing all sequences containing < 1200, > 2000 or any ambiguous nucleotides (N). The filtered data set (n = 12,89,001 sequences) was subjected to clustering at 99% threshold using VSEARCH (2.21.1). The final dataset contained 167,789 sequences. In addition, a taxonomy mapping file in QIIME compatible format was created by linking RDP sequence identifiers of the representative sequences with 7-level (domain, phylum, class, order, family, genus, and species) lineage hierarchy. GTDB Sativa curated 16S sequences (gtdb-sbdi-sativa.r06rs202.fna; n = 46,126) from the GTDB database release R06-RS202 ( https://gtdb.ecogenomic.org ), were obtained from the figshare repository ( https://scilifelab.figshare.com/articles/dataset/SBDI_Sativa_curated_16S_GTDB_database/14869077 ). The sequence and corresponding taxonomy mapping file was generated in QIIME compatible format. EzBioCloud 16S rRNA sequences with their corresponding taxonomy in QIIME compatible format (n = 64,660) were obtained from EzBioCloud server. SILVA Silva 138 SSURef NR99 full-length sequences (n = 4,36,680) ( https://data.qiime2.org/2020.6/common/silva-138-99-seqs.qza ) and taxonomy ( https://data.qiime2.org/2020.6/common/silva-138-99-tax.qza ) in QIIME2 compatible format was obtained from QIIME data resource repository.
Bacterial 16S refseq nucleotide sequences (n = 22,423) were obtained from NCBI RefSeq Targeted Loci Project ( https://www.ncbi.nlm.nih.gov/refseq/targetedloci/ ). The corresponding taxonomy in 7 lineage level hierarchy in Quantitative Insight Into Microbial Ecology (QIIME) compatible format was generated using the python script entrez_qiime.py. ( https://github.com/bakerccm/entrez_qiime ).
Unaligned bacterial 16S rRNA sequences (n = 31,96,041) from RDP project ( https://rdp.cme.msu.edu/download/current_Bacteria_unaligned.fa.gz ) were downloaded, and made QIIME compatible by sequentially removing all sequences containing < 1200, > 2000 or any ambiguous nucleotides (N). The filtered data set (n = 12,89,001 sequences) was subjected to clustering at 99% threshold using VSEARCH (2.21.1). The final dataset contained 167,789 sequences. In addition, a taxonomy mapping file in QIIME compatible format was created by linking RDP sequence identifiers of the representative sequences with 7-level (domain, phylum, class, order, family, genus, and species) lineage hierarchy.
Sativa curated 16S sequences (gtdb-sbdi-sativa.r06rs202.fna; n = 46,126) from the GTDB database release R06-RS202 ( https://gtdb.ecogenomic.org ), were obtained from the figshare repository ( https://scilifelab.figshare.com/articles/dataset/SBDI_Sativa_curated_16S_GTDB_database/14869077 ). The sequence and corresponding taxonomy mapping file was generated in QIIME compatible format.
16S rRNA sequences with their corresponding taxonomy in QIIME compatible format (n = 64,660) were obtained from EzBioCloud server.
Silva 138 SSURef NR99 full-length sequences (n = 4,36,680) ( https://data.qiime2.org/2020.6/common/silva-138-99-seqs.qza ) and taxonomy ( https://data.qiime2.org/2020.6/common/silva-138-99-tax.qza ) in QIIME2 compatible format was obtained from QIIME data resource repository.
Greengenes 16S OTUs (n = 2,03,452) and corresponding taxonomy were obtained from Greengenes FTP. ftp://greengenes.microbio.me/greengenes_release/gg_13_5/gg_13_8_otus.tar.gz .
16S rRNA sequences with their corresponding taxonomy in QIIME compatible format were obtained from 16sUDB github repository ( https://github.com/sarangian/16S-UDb ).
Bacterial 16S refseq nucleotide sequences (n = 22,423) were obtained from NCBI RefSeq Targeted Loci Project ( https://www.ncbi.nlm.nih.gov/refseq/targetedloci/ ). The corresponding taxonomy in 7 lineage level hierarchy in QIIME compatible format was generated using the python script entrez_qiime.py. ( https://github.com/bakerccm/entrez_qiime ).
The performances of the five 16S rRNA databases (Greengenes, SILVA, RDP, GTDB, EzBioCloud) in correctly classifying the Bacterial 16S RefSeq nucleotide sequences (NCBI RefSeq Targeted Loci Project; test dataset) were determined up to the genus levels. This comparison was done using the “classify-consensus-blast” utility of QIIME2 feature-classifier program with parameters—p-perc-identity 0.8—p-query-cov 0.8—p-maxaccepts 10. The resultant classification output (taxonomy.qza) of individual 16S databases (n = 5) were converted to tsv format and compared against the known taxonomy mapping file of the test dataset. Performance of each database was calculated as the proportion of correctly classified sequences in the test dataset. ONT data analysis in QIIME2 framework The FASTQ files were processed using the MetONTIIME pipeline ( https://github.com/MaestSi/MetONTIIME ), a framework based on QIIME2 using Silva V138 (Silva 138 SSURef NR99) database and BLAST classifier. Parameters used were [-n 32 -c blast -m 10 -q 0.8 -i 0.8] . The resulting BIOM file and obtained representative sequences were subjected to downstream functional analysis. Functional profiling predictions and statistical analysis PICRUSt2 was employed for predictive functional profiling analysis and functional annotation of the sequences were based on Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO; www.kegg.jp/kegg/kegg1.html ). Pathways significance differentiation was further analyzed using statistical tests. All statistical tests including differential abundance analysis was performed using STAMP 2.1.3 ( http://kiwi.cs.dal.ca/Software/STAMP ) for each of the samples. Two-sided Fisher’s exact test was used to compare samples Storey’s false discovery rate method of multiple test correction (p value ≤ 0.05) using DP at 95% confidence intervals .
The FASTQ files were processed using the MetONTIIME pipeline ( https://github.com/MaestSi/MetONTIIME ), a framework based on QIIME2 using Silva V138 (Silva 138 SSURef NR99) database and BLAST classifier. Parameters used were [-n 32 -c blast -m 10 -q 0.8 -i 0.8] . The resulting BIOM file and obtained representative sequences were subjected to downstream functional analysis.
PICRUSt2 was employed for predictive functional profiling analysis and functional annotation of the sequences were based on Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology (KO; www.kegg.jp/kegg/kegg1.html ). Pathways significance differentiation was further analyzed using statistical tests. All statistical tests including differential abundance analysis was performed using STAMP 2.1.3 ( http://kiwi.cs.dal.ca/Software/STAMP ) for each of the samples. Two-sided Fisher’s exact test was used to compare samples Storey’s false discovery rate method of multiple test correction (p value ≤ 0.05) using DP at 95% confidence intervals .
All the necessary permissions to carry out this study have been obtained in accordance with the state regulations.
The soil samples were collected from an orchard situated in Karnataka, India, which were categorized based on their stage of infection (ISI, ASI) and compared with a healthy sample (HSC). Physical examination of the plants, with respect to their roots, leaves, stem and fruits, revealed the presence of root knots in the wilt infected plants, which could be attributed to the root knot nematode, Meloidogyne. The soil from infected samples had a significantly lower pH compared to the healthy sample. The pH of ISI and ASI samples were 6.35 and 6.63, respectively, as compared to a pH of 7.66 in the healthy rhizosphere soil sample. Electrical conductivity (EC) was estimated to be 139.5 µS/cm in ISI soil, and significantly higher in the ASI soil (180 µS/cm) as compared to HSC soil sample (123.33 µS/cm). An estimated total N (0.191%), P (0.01%), K (0.01%), organic carbon (OC)(0.85%), Cl(18%), Fe (0.93%), Cu (26.33 ppm), Mn (9.10 ppm), Zn (30.9 ppm), B (4.1 ppm) were reported for the ISI soil. Cl and B were significantly higher in the ISI soil as compared to the HSC soil. On the other hand, for the ASI sample, the estimated total N (0.20%), P (0.11%), K (0.014%), OC (0.97%), Cl (21%), Fe (0.98%), Cu (31.4 ppm), Mn (9.6 ppm), Zn (33.2 ppm) and B (4.3 ppm) were reported. Cl, Cu, Zn and B were found to significantly higher in the ASI as compared to the HSC soil. No significant variations in the total bacterial and total fungal counts could be found within the samples. The HSC soil had a total bacterial and total fungal counts of 176 cfu/g and 2249.3 cfu/g, respectively. Whereas the corresponding total bacterial and total fungal counts for ISI and ASI soil were 2240 cfu/g and 170 cfu/g, and 2126 cfu/g and 154 cfu/g, respectively (Table ). In this study, the sequencing data generated from Nanopore sequencing platform was used for taxonomic profiling of microbial communities based on 16S rRNA sequencing. Sequence metrics of the samples from the MinION sequencing were estimated to be 36,000 sequence counts (ISI) and 31,868 (ASI) samples (Table ). Comparison of databases Accuracy in assigning bacterial lineages from Phylum to Genus level using classify-consensus-blast algorithm as furnished in Table . At the phylum level, the GreenGenes database revealed the maximum number of hits 22,276 (99.34%), followed by EzBioCloud 22,290 (95.17%), SILVA 20,625 (91.98%), RDP 20,491 (91.38%) and GTDB 19,033 (84.88%). The results from the comparative assessment revealed SILVA returned the maximum number of correct hits at the genus level, which was 17,149 (76.48%). EzBioCloud returned 15,229 (67.91%), followed by RDP 15,173 (67.67%), GTDB 13,370 (59.63%) and GreenGenes 13,034 (58.13%) (Table ). Out of the 22,423 sequences in the test dataset, 17,149 sequences (76.48%) were correctly classified at genus level based on the SILVA database. The unique list of genera correctly identified by each of the database were subjected to JVenn for the generation of the Venn diagram (Fig. ). The highest number of correctly identified unique genera were returned from the SILVA database (n = 1681). Based on these results, the SILVA database was selected for further species level analysis of the samples. Relative abundance estimates Relative abundance of bacterial species level resolution showed predominance of Staphylococcus epidermidis , Bacillus megatarium , Cutibacterium acnes , Micrococcus luteus , Pseudomonas aeruginosa , Pseudomonas putida , Pseudomonas stutzeri in the ISI sample (Fig. ; Table ). Results show significant variations in the growth promoting bacterial species in the ISI soil sample as compared to the HSC soil. Bacillus species are known to produce several compounds such as antibiotics, siderophores, cell wall hydrolases and induced systemic resistance (ISR) that make them promising biocontrol agents , . Bacillus subtilis is a known biocontrol agent against wilt caused by C. fimbriata . While significantly lower numbers of Bacillus subtilis were observed in the ISI soil, Bacillus megaterium was found to be significantly dominant in the ISI soil. Bacillus megaterium is known for its plant growth promoting properties and employed as biocontrol agent against pathogens such as Alternaria japonica and Brassica oleracea var . italica . Furthermore, Cutibacterium acnes , an opportunistic human pathogen has been often reported as part of the skin flora or gastrointestinal tract. They are known to form biofilms in the skin-gland regions leading to inflammation and skin diseases. Additionally, a reduction in the number of Pseudomonas aeruginosa was observed. While, Pseudomonas aeruginosa has been reported to cause Bacterial root rot disease in Ginseng , there are certain strains of Pseudomonas aeruginosa that are plant growth-promoting rhizobacteria (PGPR) . Staphylococcus epidermidis , although a known human pathogen, has been reported in prior studies for their plant growth promoting properties. A study on tomato bacterial wilt disease reported the presence of endophytic bacteria Staphylococcus epidermidis indicating their effectiveness as biocontrol agents against R. solanacearum . On the other hand, an abundance of Micrococcus luteus has been reported in the ISI sample. Micrococcus luteus is a gram-positive bacterium that has been reported to exhibit antifungal activity , and its growth promoting properties, biocontrol properties , biotic and abiotic stress tolerance . Another study has reported the growth promoting properties of Micrococcus luteus against F.oxysporum in chickpea . However, there are reports of Micrococcus luteus causing plant diseases. A study reported the role of Micrococcus luteus in leafspot disease in Mangifera indica . More experimental evidence is required to validate the role of the Micrococcus luteus in the pathogenesis of the wilt disease in pomegranate. Predictive pathway profiling Pathway predictions performed using PICRUSt2 and subsequently with STAMP for statistical analysis of the results revealed a significant increase in the transporter protein families involved in signalling and cellular processes (Table ). K02015 (Iron complex transport system substrate binding protein), K02016 (Iron complex transport system substrate binding protein), K05846 osmoprotectant transport system permease protein, K03293 Amino acid transporter, K07024 sucrose-6-phosphatase and K02013 Iron-complex transport system showed significant increase in the ISI soil. The hits that showed significant increase in the ISI sample were K07498 putative transposases, K07497 putative transposase and K07052 uncharacterized protein (Fig. ). It is noteworthy that iron complex transport system proteins are differentially abundant in the pathways predicted in the ISI soil sample. On the other hand, the most abundant pathways predicted in comparison to the ASI soil sample were transporter proteins involved in signalling and cellular processes, K07114 Ca-activated chlorine channel (CaCC), K02004 putative ABC transport system permease protein and K03088 RNA polymerase sigma-70 factor from ECF family (Fig. ). Peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers) were significantly enriched in ISI soil sample. In the ASI soil sample, aerobic respiration I (cytochrome c) and Kdo transfer to lipid IVA III (Chlamydia) pathways were found to be significantly enriched (Fig. ). Furthermore, correlating the findings, abiotic factors such as acidic pH, along with availability of iron (Fe) and manganese (Mn) have been reported to facilitate the growth of F. oxysporum , one that has a higher requirement for micronutrients . The oxidation state of metals such as Fe and Mn determines their bioavailability, which is reportedly driven by the soil pH along with redox potential . The present study demonstrates the capabilities of the 16S rRNA sequencing platform in identifying potential key players involved in disease pathogenesis from soil samples collected from different pomegranate plants ranging from healthy to severely infected within the same orchard. Although various reports have described the disease symptoms in detail, the access to the diversity of the bacterial population can be facilitated through 16S rRNA sequencing using MinION. Examining the soil microbiome using 16S rRNA sequencing provides a platform for pathogenomics studies. These studies include exploring the microbial diversity and the key regulators that could provide valuable insights into the disease-causing pathogens, their adaptations and factors that influence their existence. A limitation to consider here is the amplicon-based prediction being less capable of strain-level identification. In a separate study, the microbiome of the infected soil samples the collection site have been explored extensively using the shotgun metagenomics approach . This method offers improved sensitivity, resolution, and detailed characterization of microbial communities compared to traditional methods , . The study delved into the fungal communities and their adaptations, with a focus on Fusarium oxysporum , a known causative organism of wilt in pomegranate. The adaptations of this pathogen were also investigated. It is worth noting that wilt disease in pomegranate is caused by multiple pathogens and is often referred to as wilt complex. Furthermore, a number of beneficial bacterial communities Staphylococcus epidermidis , Bacillus subtilis , Bacillus megatarium , Micrococcus luteus , Pseudomonas aeruginosa were found in this study. In particular, the present study revealed the prevalence or co-dominance of bacterial communities, which could be essential in establishing effective biocontrol strategies against wilt in pomegranate. Significant variations in the number of beneficial bacterial communities have been observed in this study. Current findings are consistent with our previous report on whole metagenome studies of infected samples and other reports from literature. The results suggest that abiotic factors, such as an acidic pH and the availability of Fe and Mn, may be contributing to the growth of Fusarium oxysporum , as previously observed. Past reports have recommended that limiting bioavailable micronutrients such as Fe and Mn can serve as a biocontrol strategy. However, this finding has not been validated in the present study . Nonetheless, a methodology is proposed for better characterization of bacterial species through 16S metagenome analysis. Furthermore, new knowledge and significant insights into the beneficial bacterial communities and enriched pathways have been revealed that may represent functional adaptations. As mentioned earlier, the accuracy and effectiveness of 16S metagenomics studies depends on the completeness and consistency of existing 16S rRNA sequence repositories. The information derived from such repositories plays a central role in identifying key players among both beneficial and pathogenic bacterial communities, which is demonstrated in the present study by exploring the complex multi-pathogen-host systems such as the Wilt complex. In conclusion, this study reveals the complex interactions between bacteria, soil physicochemical properties, and the wilt complex disease affecting pomegranate crops. The proposed approach has the potential to improve the utilization of 16S metagenomics sequencing data for accurate microbial identification and functional profiling predictions. Overall, the study emphasises the significance of utilizing advanced approaches and technologies to precisely detect and characterize microbial communities in agricultural settings, taking into account abiotic factors such as soil physicochemical characteristics. Further investigation could result in substantial enhancements in the management and productivity of pomegranate crops.
Accuracy in assigning bacterial lineages from Phylum to Genus level using classify-consensus-blast algorithm as furnished in Table . At the phylum level, the GreenGenes database revealed the maximum number of hits 22,276 (99.34%), followed by EzBioCloud 22,290 (95.17%), SILVA 20,625 (91.98%), RDP 20,491 (91.38%) and GTDB 19,033 (84.88%). The results from the comparative assessment revealed SILVA returned the maximum number of correct hits at the genus level, which was 17,149 (76.48%). EzBioCloud returned 15,229 (67.91%), followed by RDP 15,173 (67.67%), GTDB 13,370 (59.63%) and GreenGenes 13,034 (58.13%) (Table ). Out of the 22,423 sequences in the test dataset, 17,149 sequences (76.48%) were correctly classified at genus level based on the SILVA database. The unique list of genera correctly identified by each of the database were subjected to JVenn for the generation of the Venn diagram (Fig. ). The highest number of correctly identified unique genera were returned from the SILVA database (n = 1681). Based on these results, the SILVA database was selected for further species level analysis of the samples. Relative abundance estimates Relative abundance of bacterial species level resolution showed predominance of Staphylococcus epidermidis , Bacillus megatarium , Cutibacterium acnes , Micrococcus luteus , Pseudomonas aeruginosa , Pseudomonas putida , Pseudomonas stutzeri in the ISI sample (Fig. ; Table ). Results show significant variations in the growth promoting bacterial species in the ISI soil sample as compared to the HSC soil. Bacillus species are known to produce several compounds such as antibiotics, siderophores, cell wall hydrolases and induced systemic resistance (ISR) that make them promising biocontrol agents , . Bacillus subtilis is a known biocontrol agent against wilt caused by C. fimbriata . While significantly lower numbers of Bacillus subtilis were observed in the ISI soil, Bacillus megaterium was found to be significantly dominant in the ISI soil. Bacillus megaterium is known for its plant growth promoting properties and employed as biocontrol agent against pathogens such as Alternaria japonica and Brassica oleracea var . italica . Furthermore, Cutibacterium acnes , an opportunistic human pathogen has been often reported as part of the skin flora or gastrointestinal tract. They are known to form biofilms in the skin-gland regions leading to inflammation and skin diseases. Additionally, a reduction in the number of Pseudomonas aeruginosa was observed. While, Pseudomonas aeruginosa has been reported to cause Bacterial root rot disease in Ginseng , there are certain strains of Pseudomonas aeruginosa that are plant growth-promoting rhizobacteria (PGPR) . Staphylococcus epidermidis , although a known human pathogen, has been reported in prior studies for their plant growth promoting properties. A study on tomato bacterial wilt disease reported the presence of endophytic bacteria Staphylococcus epidermidis indicating their effectiveness as biocontrol agents against R. solanacearum . On the other hand, an abundance of Micrococcus luteus has been reported in the ISI sample. Micrococcus luteus is a gram-positive bacterium that has been reported to exhibit antifungal activity , and its growth promoting properties, biocontrol properties , biotic and abiotic stress tolerance . Another study has reported the growth promoting properties of Micrococcus luteus against F.oxysporum in chickpea . However, there are reports of Micrococcus luteus causing plant diseases. A study reported the role of Micrococcus luteus in leafspot disease in Mangifera indica . More experimental evidence is required to validate the role of the Micrococcus luteus in the pathogenesis of the wilt disease in pomegranate. Predictive pathway profiling Pathway predictions performed using PICRUSt2 and subsequently with STAMP for statistical analysis of the results revealed a significant increase in the transporter protein families involved in signalling and cellular processes (Table ). K02015 (Iron complex transport system substrate binding protein), K02016 (Iron complex transport system substrate binding protein), K05846 osmoprotectant transport system permease protein, K03293 Amino acid transporter, K07024 sucrose-6-phosphatase and K02013 Iron-complex transport system showed significant increase in the ISI soil. The hits that showed significant increase in the ISI sample were K07498 putative transposases, K07497 putative transposase and K07052 uncharacterized protein (Fig. ). It is noteworthy that iron complex transport system proteins are differentially abundant in the pathways predicted in the ISI soil sample. On the other hand, the most abundant pathways predicted in comparison to the ASI soil sample were transporter proteins involved in signalling and cellular processes, K07114 Ca-activated chlorine channel (CaCC), K02004 putative ABC transport system permease protein and K03088 RNA polymerase sigma-70 factor from ECF family (Fig. ). Peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers) were significantly enriched in ISI soil sample. In the ASI soil sample, aerobic respiration I (cytochrome c) and Kdo transfer to lipid IVA III (Chlamydia) pathways were found to be significantly enriched (Fig. ). Furthermore, correlating the findings, abiotic factors such as acidic pH, along with availability of iron (Fe) and manganese (Mn) have been reported to facilitate the growth of F. oxysporum , one that has a higher requirement for micronutrients . The oxidation state of metals such as Fe and Mn determines their bioavailability, which is reportedly driven by the soil pH along with redox potential . The present study demonstrates the capabilities of the 16S rRNA sequencing platform in identifying potential key players involved in disease pathogenesis from soil samples collected from different pomegranate plants ranging from healthy to severely infected within the same orchard. Although various reports have described the disease symptoms in detail, the access to the diversity of the bacterial population can be facilitated through 16S rRNA sequencing using MinION. Examining the soil microbiome using 16S rRNA sequencing provides a platform for pathogenomics studies. These studies include exploring the microbial diversity and the key regulators that could provide valuable insights into the disease-causing pathogens, their adaptations and factors that influence their existence. A limitation to consider here is the amplicon-based prediction being less capable of strain-level identification. In a separate study, the microbiome of the infected soil samples the collection site have been explored extensively using the shotgun metagenomics approach . This method offers improved sensitivity, resolution, and detailed characterization of microbial communities compared to traditional methods , . The study delved into the fungal communities and their adaptations, with a focus on Fusarium oxysporum , a known causative organism of wilt in pomegranate. The adaptations of this pathogen were also investigated. It is worth noting that wilt disease in pomegranate is caused by multiple pathogens and is often referred to as wilt complex. Furthermore, a number of beneficial bacterial communities Staphylococcus epidermidis , Bacillus subtilis , Bacillus megatarium , Micrococcus luteus , Pseudomonas aeruginosa were found in this study. In particular, the present study revealed the prevalence or co-dominance of bacterial communities, which could be essential in establishing effective biocontrol strategies against wilt in pomegranate. Significant variations in the number of beneficial bacterial communities have been observed in this study. Current findings are consistent with our previous report on whole metagenome studies of infected samples and other reports from literature. The results suggest that abiotic factors, such as an acidic pH and the availability of Fe and Mn, may be contributing to the growth of Fusarium oxysporum , as previously observed. Past reports have recommended that limiting bioavailable micronutrients such as Fe and Mn can serve as a biocontrol strategy. However, this finding has not been validated in the present study . Nonetheless, a methodology is proposed for better characterization of bacterial species through 16S metagenome analysis. Furthermore, new knowledge and significant insights into the beneficial bacterial communities and enriched pathways have been revealed that may represent functional adaptations. As mentioned earlier, the accuracy and effectiveness of 16S metagenomics studies depends on the completeness and consistency of existing 16S rRNA sequence repositories. The information derived from such repositories plays a central role in identifying key players among both beneficial and pathogenic bacterial communities, which is demonstrated in the present study by exploring the complex multi-pathogen-host systems such as the Wilt complex. In conclusion, this study reveals the complex interactions between bacteria, soil physicochemical properties, and the wilt complex disease affecting pomegranate crops. The proposed approach has the potential to improve the utilization of 16S metagenomics sequencing data for accurate microbial identification and functional profiling predictions. Overall, the study emphasises the significance of utilizing advanced approaches and technologies to precisely detect and characterize microbial communities in agricultural settings, taking into account abiotic factors such as soil physicochemical characteristics. Further investigation could result in substantial enhancements in the management and productivity of pomegranate crops.
Relative abundance of bacterial species level resolution showed predominance of Staphylococcus epidermidis , Bacillus megatarium , Cutibacterium acnes , Micrococcus luteus , Pseudomonas aeruginosa , Pseudomonas putida , Pseudomonas stutzeri in the ISI sample (Fig. ; Table ). Results show significant variations in the growth promoting bacterial species in the ISI soil sample as compared to the HSC soil. Bacillus species are known to produce several compounds such as antibiotics, siderophores, cell wall hydrolases and induced systemic resistance (ISR) that make them promising biocontrol agents , . Bacillus subtilis is a known biocontrol agent against wilt caused by C. fimbriata . While significantly lower numbers of Bacillus subtilis were observed in the ISI soil, Bacillus megaterium was found to be significantly dominant in the ISI soil. Bacillus megaterium is known for its plant growth promoting properties and employed as biocontrol agent against pathogens such as Alternaria japonica and Brassica oleracea var . italica . Furthermore, Cutibacterium acnes , an opportunistic human pathogen has been often reported as part of the skin flora or gastrointestinal tract. They are known to form biofilms in the skin-gland regions leading to inflammation and skin diseases. Additionally, a reduction in the number of Pseudomonas aeruginosa was observed. While, Pseudomonas aeruginosa has been reported to cause Bacterial root rot disease in Ginseng , there are certain strains of Pseudomonas aeruginosa that are plant growth-promoting rhizobacteria (PGPR) . Staphylococcus epidermidis , although a known human pathogen, has been reported in prior studies for their plant growth promoting properties. A study on tomato bacterial wilt disease reported the presence of endophytic bacteria Staphylococcus epidermidis indicating their effectiveness as biocontrol agents against R. solanacearum . On the other hand, an abundance of Micrococcus luteus has been reported in the ISI sample. Micrococcus luteus is a gram-positive bacterium that has been reported to exhibit antifungal activity , and its growth promoting properties, biocontrol properties , biotic and abiotic stress tolerance . Another study has reported the growth promoting properties of Micrococcus luteus against F.oxysporum in chickpea . However, there are reports of Micrococcus luteus causing plant diseases. A study reported the role of Micrococcus luteus in leafspot disease in Mangifera indica . More experimental evidence is required to validate the role of the Micrococcus luteus in the pathogenesis of the wilt disease in pomegranate.
Pathway predictions performed using PICRUSt2 and subsequently with STAMP for statistical analysis of the results revealed a significant increase in the transporter protein families involved in signalling and cellular processes (Table ). K02015 (Iron complex transport system substrate binding protein), K02016 (Iron complex transport system substrate binding protein), K05846 osmoprotectant transport system permease protein, K03293 Amino acid transporter, K07024 sucrose-6-phosphatase and K02013 Iron-complex transport system showed significant increase in the ISI soil. The hits that showed significant increase in the ISI sample were K07498 putative transposases, K07497 putative transposase and K07052 uncharacterized protein (Fig. ). It is noteworthy that iron complex transport system proteins are differentially abundant in the pathways predicted in the ISI soil sample. On the other hand, the most abundant pathways predicted in comparison to the ASI soil sample were transporter proteins involved in signalling and cellular processes, K07114 Ca-activated chlorine channel (CaCC), K02004 putative ABC transport system permease protein and K03088 RNA polymerase sigma-70 factor from ECF family (Fig. ). Peptidoglycan biosynthesis II (staphylococci) and TCA cycle VII (acetate-producers) were significantly enriched in ISI soil sample. In the ASI soil sample, aerobic respiration I (cytochrome c) and Kdo transfer to lipid IVA III (Chlamydia) pathways were found to be significantly enriched (Fig. ). Furthermore, correlating the findings, abiotic factors such as acidic pH, along with availability of iron (Fe) and manganese (Mn) have been reported to facilitate the growth of F. oxysporum , one that has a higher requirement for micronutrients . The oxidation state of metals such as Fe and Mn determines their bioavailability, which is reportedly driven by the soil pH along with redox potential . The present study demonstrates the capabilities of the 16S rRNA sequencing platform in identifying potential key players involved in disease pathogenesis from soil samples collected from different pomegranate plants ranging from healthy to severely infected within the same orchard. Although various reports have described the disease symptoms in detail, the access to the diversity of the bacterial population can be facilitated through 16S rRNA sequencing using MinION. Examining the soil microbiome using 16S rRNA sequencing provides a platform for pathogenomics studies. These studies include exploring the microbial diversity and the key regulators that could provide valuable insights into the disease-causing pathogens, their adaptations and factors that influence their existence. A limitation to consider here is the amplicon-based prediction being less capable of strain-level identification. In a separate study, the microbiome of the infected soil samples the collection site have been explored extensively using the shotgun metagenomics approach . This method offers improved sensitivity, resolution, and detailed characterization of microbial communities compared to traditional methods , . The study delved into the fungal communities and their adaptations, with a focus on Fusarium oxysporum , a known causative organism of wilt in pomegranate. The adaptations of this pathogen were also investigated. It is worth noting that wilt disease in pomegranate is caused by multiple pathogens and is often referred to as wilt complex. Furthermore, a number of beneficial bacterial communities Staphylococcus epidermidis , Bacillus subtilis , Bacillus megatarium , Micrococcus luteus , Pseudomonas aeruginosa were found in this study. In particular, the present study revealed the prevalence or co-dominance of bacterial communities, which could be essential in establishing effective biocontrol strategies against wilt in pomegranate. Significant variations in the number of beneficial bacterial communities have been observed in this study. Current findings are consistent with our previous report on whole metagenome studies of infected samples and other reports from literature. The results suggest that abiotic factors, such as an acidic pH and the availability of Fe and Mn, may be contributing to the growth of Fusarium oxysporum , as previously observed. Past reports have recommended that limiting bioavailable micronutrients such as Fe and Mn can serve as a biocontrol strategy. However, this finding has not been validated in the present study . Nonetheless, a methodology is proposed for better characterization of bacterial species through 16S metagenome analysis. Furthermore, new knowledge and significant insights into the beneficial bacterial communities and enriched pathways have been revealed that may represent functional adaptations. As mentioned earlier, the accuracy and effectiveness of 16S metagenomics studies depends on the completeness and consistency of existing 16S rRNA sequence repositories. The information derived from such repositories plays a central role in identifying key players among both beneficial and pathogenic bacterial communities, which is demonstrated in the present study by exploring the complex multi-pathogen-host systems such as the Wilt complex. In conclusion, this study reveals the complex interactions between bacteria, soil physicochemical properties, and the wilt complex disease affecting pomegranate crops. The proposed approach has the potential to improve the utilization of 16S metagenomics sequencing data for accurate microbial identification and functional profiling predictions. Overall, the study emphasises the significance of utilizing advanced approaches and technologies to precisely detect and characterize microbial communities in agricultural settings, taking into account abiotic factors such as soil physicochemical characteristics. Further investigation could result in substantial enhancements in the management and productivity of pomegranate crops.
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Linguistic analysis of plain language summaries and corresponding scientific summaries of Cochrane systematic reviews about oncology interventions
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981e795b-68c3-40e4-a00e-e41b762f9897
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10225178
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Internal Medicine[mh]
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INTRODUCTION Health literacy is defined as the degree to which individuals have the capacity to obtain, process and understand basic health information and services needed to make appropriate health decisions. Its importance has been confirmed through numerous studies, not only related to the health care of individuals but also in public health care system. , , , In the field of oncology, it is especially important for patients to have high level of health literacy, as well as the access to health information presented in a way understandable to the lay public. Cancer treatment options have very much advanced from conventional treatment approaches, and patients and their families are taking more active roles in treatment decisions. Once faced with a life‐changing diagnosis of cancer, most of them tend to seek further information from the Internet, forums, acquaintances, other patients, social support groups, articles, books and other sources. , However, studies have shown that nearly half of the patients with cancer have difficulty understanding information about the treatment for their particular type of cancer. Moreover, the readability of cancer information on the Internet is too demanding and most of the information does not meet the criteria for readability and understandability by lay population. , , , , Despite the evidence that the readability levels regarding oncology information for patients are unacceptably high, the readability of newly created materials from professional societies has not improved to an adequate level and there is still room for advancement in all oncology fields, across information providers. , One of the organizations that aims to improve the quality of health information for the public is Cochrane, whose Database of Systematic Reviews has emerged as one of the most trustworthy sources for the effectiveness of health interventions, due to its high‐quality systematic reviews. As a way of presenting the results and conclusions of systematic reviews, Cochrane Systematic Reviews include not only a scientific abstract (SA), but also a plain language summary (PLS). Plain language summaries are intended as a form of presenting the information from systematic reviews to the lay public and should be written in a way understandable to most readers. The level of readability and comprehension of the text can be influenced by different factors: emotional charge of the text, text characteristics, subjective expressions of attitudes or format of the presentation. , , , , , PLSs are written by the review authors. To ensure that PLSs are written in a standard format, the Cochrane has created the Standards for the Reporting of Plain Language Summaries in New Cochrane Intervention Reviews (PLEACS). It has been shown that these standards are often not followed. To improve the quality of the PLSs, several studies explored the influence of the factors such as the use of infographics, numerical presentation and framing of the direction of the results, as well as different summary formats. It was shown that there is still an effort to be made to produce content and format that will meet the need of health care users. PLSs and other sources of oncology information for lay public have been analysed in various ways. , , , Cochrane Systematic Review's PLSs of oncology interventions are an important format of reliable and understandable information to cancer patients and their families in their decision‐making, but their characteristics have not been studied. The aim of this study was to assess the language characteristics of PLS of Cochrane systematic reviews of oncology interventions in comparison with corresponding SAs.
METHODS 2.1 Study design and data sources In this cross‐sectional study, we analysed the linguistic characteristics of PLSs and SAs of Cochrane systematic reviews of oncology interventions. Both types of summaries are written by review authors. 2.2 Inclusion and exclusion criteria We included all PLSs and corresponding SAs available in the Cochrane Library up to February 2019. Summaries that were not intervention studies were excluded. We included abstracts from Cochrane Review Groups clearly focussed on oncology: Breast Cancer; Childhood Cancer; Colorectal Cancer; Gynaecological, Neuro‐oncology and Orphan Cancer; Haematological Malignancies; and Lung Cancer. These groups were taken as a proxy for different clinical types of cancer. Although there are systematic reviews in other Cochrane groups which can be placed in the field of oncology, we included summaries from groups that explicitly and only work on oncological systematic reviews. Two authors (J.Š. and I.B.) analysed all included PLSs and corresponding SAs, discussing each abstract separately. 2.3 Data extraction Data extraction spreadsheet was tested by two authors (J.Š. and I.B.). One author extracted the data, and the other one independently reviewed the data in a 10% random sample of PLSs and corresponding SAs. Then, it was checked whether the entry in the table was correct. The third author (A.M.) checked the entries from the extraction of the two authors in a sample of 20 PLSs and SAs at the beginning of the study. Interobserver agreement was high (kappa range 0.80–1.00, 95% confidence interval [CI] = 0.84–1.00). We resolved the differences in rating through the discussion with the third author (A.M.) before full data extraction. The extracted data included the name of the Cochrane Review Group, number of authors, year of publication, readability score, linguistic characteristics and categories of conclusiveness. 2.4 Simple measure of Gobbledygook index The readability of summary formats in English was assessed using the Simple Measure of Gobbledygook (SMOG) index. SMOG index assesses the readability of certain content by counting polysyllabic words, and the result is presented as the number of years of education required to understand a given text. It is considered to be suitable for health information. , SMOG index for PLSs in English was calculated using an online tool: https://readable.com/ . Regarding SMOG index interpretation, the official recommendation of American Medical Association and National Health Institute is that health information should be written at the reading level of the sixth grade in the US education system. , , 2.5 Linguistic characteristics Plain language summaries and corresponding scientific summaries were analysed regarding their linguistic characteristics. To analyse the number of words and the length of the sentences, we used the Linguistic Inquiry and Word Count—LIWC ( www.liwc.app/demo ). This analysis is dictionary‐based and categorizes text words into four main variables: analytical, clout, authenticity and emotionality share in the tone of the text. These variables are presented as the percentage of words from the text in a particular category. Analytical thinking category is based on recognizing words associated with logic or with connecting concepts and putting them into a relationship. Greater use of words related to analytical thinking is related to cognitive complexity and to abstract thinking. Clout speech is a variable that refers to the use of terms that denote self‐confidence, leadership or social status. A higher proportion of such words suggest that the author speaks from a position of expertise and certainty in what is stated, and a lower proportion suggests a style of presenting information that is humbler. Authenticity is determined by the percentage of words related to personality, such as the use of personal nouns in the first person (‘I’, ‘my’ and ‘mine’), present tense and relative adverbs (near, now). Use of these words is connected to a writing that is more personal and honest. , Emotional share relates to how positive the tone is according to the words used. A score of 100 in emotional tone would mean the tone is maximally positive; a score of 50 means an even balance of positive and negative emotion words. 2.6 Conclusiveness Conclusiveness of statements about efficacy and safety was categorized into nine categories : Positive—there is (moderate/high‐quality) evidence of effectiveness/safety, that is the drug was proven effective/safe, Positive inconclusive (there is evidence of effectiveness/safety, yet it is of a low quality/inconclusive or authors state that more research is required), No evidence (there is no evidence from randomized controlled trials [RCTs] because the literature search did not result in any eligible studies, i.e. empty reviews), No opinion (authors provided no opinion), Negative—there is (moderate/high‐quality) evidence of no effect or evidence of harm (ineffectiveness/harmful) or authors advised against the intervention/comparison or it is not recommended, Negative inconclusive (there is evidence of ineffectiveness/harm [evidence show that there was no effect or the intervention was not safe] or authors advised against the intervention/comparison or it is not recommended; yet evidence is of a low quality/ inconclusive or authors state that more research is required), Unclear, more research is needed (authors state that more research is required), Equal—analysed interventions were of equal effectiveness/safety, and Equal inconclusive—interventions are equally effective/safe; yet evidence is of a low quality/inconclusive, or authors state that more research is required. 2.7 Statistical analysis The number of PLSs per Cochrane Review Group and categories of conclusiveness were described as frequencies and percentages. The number of words and sentences, readability score and linguistic variables were described as median with interquartile range (IQR) or 95% CI, due to the uneven distribution of data, as determined by Kolmogorov Smirnov test. For comparison of summaries of different oncology groups, we used Kruskal–Wallis test and Conover Iman post hoc test. Conover Iman test was used for post hoc comparisons to account for multiple comparisons. All analysis was done in JASP version 0.12.1.0. (JASP Team, 2020, https://jasp‐stats.org/JASP ).
Study design and data sources In this cross‐sectional study, we analysed the linguistic characteristics of PLSs and SAs of Cochrane systematic reviews of oncology interventions. Both types of summaries are written by review authors.
Inclusion and exclusion criteria We included all PLSs and corresponding SAs available in the Cochrane Library up to February 2019. Summaries that were not intervention studies were excluded. We included abstracts from Cochrane Review Groups clearly focussed on oncology: Breast Cancer; Childhood Cancer; Colorectal Cancer; Gynaecological, Neuro‐oncology and Orphan Cancer; Haematological Malignancies; and Lung Cancer. These groups were taken as a proxy for different clinical types of cancer. Although there are systematic reviews in other Cochrane groups which can be placed in the field of oncology, we included summaries from groups that explicitly and only work on oncological systematic reviews. Two authors (J.Š. and I.B.) analysed all included PLSs and corresponding SAs, discussing each abstract separately.
Data extraction Data extraction spreadsheet was tested by two authors (J.Š. and I.B.). One author extracted the data, and the other one independently reviewed the data in a 10% random sample of PLSs and corresponding SAs. Then, it was checked whether the entry in the table was correct. The third author (A.M.) checked the entries from the extraction of the two authors in a sample of 20 PLSs and SAs at the beginning of the study. Interobserver agreement was high (kappa range 0.80–1.00, 95% confidence interval [CI] = 0.84–1.00). We resolved the differences in rating through the discussion with the third author (A.M.) before full data extraction. The extracted data included the name of the Cochrane Review Group, number of authors, year of publication, readability score, linguistic characteristics and categories of conclusiveness.
Simple measure of Gobbledygook index The readability of summary formats in English was assessed using the Simple Measure of Gobbledygook (SMOG) index. SMOG index assesses the readability of certain content by counting polysyllabic words, and the result is presented as the number of years of education required to understand a given text. It is considered to be suitable for health information. , SMOG index for PLSs in English was calculated using an online tool: https://readable.com/ . Regarding SMOG index interpretation, the official recommendation of American Medical Association and National Health Institute is that health information should be written at the reading level of the sixth grade in the US education system. , ,
Linguistic characteristics Plain language summaries and corresponding scientific summaries were analysed regarding their linguistic characteristics. To analyse the number of words and the length of the sentences, we used the Linguistic Inquiry and Word Count—LIWC ( www.liwc.app/demo ). This analysis is dictionary‐based and categorizes text words into four main variables: analytical, clout, authenticity and emotionality share in the tone of the text. These variables are presented as the percentage of words from the text in a particular category. Analytical thinking category is based on recognizing words associated with logic or with connecting concepts and putting them into a relationship. Greater use of words related to analytical thinking is related to cognitive complexity and to abstract thinking. Clout speech is a variable that refers to the use of terms that denote self‐confidence, leadership or social status. A higher proportion of such words suggest that the author speaks from a position of expertise and certainty in what is stated, and a lower proportion suggests a style of presenting information that is humbler. Authenticity is determined by the percentage of words related to personality, such as the use of personal nouns in the first person (‘I’, ‘my’ and ‘mine’), present tense and relative adverbs (near, now). Use of these words is connected to a writing that is more personal and honest. , Emotional share relates to how positive the tone is according to the words used. A score of 100 in emotional tone would mean the tone is maximally positive; a score of 50 means an even balance of positive and negative emotion words.
Conclusiveness Conclusiveness of statements about efficacy and safety was categorized into nine categories : Positive—there is (moderate/high‐quality) evidence of effectiveness/safety, that is the drug was proven effective/safe, Positive inconclusive (there is evidence of effectiveness/safety, yet it is of a low quality/inconclusive or authors state that more research is required), No evidence (there is no evidence from randomized controlled trials [RCTs] because the literature search did not result in any eligible studies, i.e. empty reviews), No opinion (authors provided no opinion), Negative—there is (moderate/high‐quality) evidence of no effect or evidence of harm (ineffectiveness/harmful) or authors advised against the intervention/comparison or it is not recommended, Negative inconclusive (there is evidence of ineffectiveness/harm [evidence show that there was no effect or the intervention was not safe] or authors advised against the intervention/comparison or it is not recommended; yet evidence is of a low quality/ inconclusive or authors state that more research is required), Unclear, more research is needed (authors state that more research is required), Equal—analysed interventions were of equal effectiveness/safety, and Equal inconclusive—interventions are equally effective/safe; yet evidence is of a low quality/inconclusive, or authors state that more research is required.
Statistical analysis The number of PLSs per Cochrane Review Group and categories of conclusiveness were described as frequencies and percentages. The number of words and sentences, readability score and linguistic variables were described as median with interquartile range (IQR) or 95% CI, due to the uneven distribution of data, as determined by Kolmogorov Smirnov test. For comparison of summaries of different oncology groups, we used Kruskal–Wallis test and Conover Iman post hoc test. Conover Iman test was used for post hoc comparisons to account for multiple comparisons. All analysis was done in JASP version 0.12.1.0. (JASP Team, 2020, https://jasp‐stats.org/JASP ).
RESULTS In total, we collected 275 Cochrane PLSs and corresponding SAs of systematic reviews of oncology interventions (Figure ). The median number of authors for systematic review was 5 (IQR = 4–6). The analysis of the summaries in different Cochrane Cancer Review Groups showed that the fewest number of authors was in the Childhood Cancer Group and Colorectal Cancer Group (Table ). 3.1 Readability analysis In general, PLSs were shorter and easier to read than SAs. SMOG index for PLSs was double the recommended 6 years of education (median = 13.0, 95% CI = 12.8–13.3), compared to 16.6 (95% CI = 16.4–16.8) for SAs ( p < 0.001; Wilcoxon nonparametric paired samples test). SAs were also significantly longer than PLSs: median word count 604 (95% CI = 566–653) versus 364 (95% CI = 339–388), respectively ( p < 0.001; Wilcoxon nonparametric paired samples test). Readability scores did not differ among different Cochrane Cancer Review Groups, both for PLSs and for SAs (Table ). The Groups differed in the text length of the summaries, with both PLSs and SAs being the shortest for the Colorectal Cancer Group (Table ). 3.2 Linguistic characteristics The use of the analytical tone was most prominent in both PLSs and SAs (Table ). Half of the words in both PLSs and SAs were categorized as clout speaking. Authentic and emotional tones were used less frequently in both types of summaries. In general, PLSs used language with significantly more authenticity and emotional tones, but fewer words related to analytical expressions than SAs. With regard to different Cochrane Cancer Review Groups, there was no statistically significant difference in the use of the analytical tone in PLSs (Table ). The clout tone was more dominant in PLSs from Gynaecological, Neuro‐oncology and Orphan Cancer Groups. In the Childhood Cancer Group, we observed the least use of emotional tone, while the authentic tone was least used in Haematological Malignancies Group (Table ). As in PLSs, the predominant use of the clout tone was found in SAs from Gynaecological, Neuro‐oncology and Orphan Cancer Groups. The analytical tone was predominant in SAs in Childhood Cancer Group. SAs from the Lung Cancer Group had the greatest proportion of words related to authenticity, as well as the greatest use of emotional tone (Table ). 3.3 Conclusiveness Categories of conclusiveness differed between summaries from different Cochrane Review Groups. PLSs from the Lung Cancer Group there had the greatest proportion of negative conclusion compared with the other groups. In Childhood Cancer Group, we found no PLSs with positive conclusions (Figure ; Table ). In Gynaecological, Neuro‐oncology and Orphan Cancer Group and Breast Cancer Group, we found no summaries where the authors did not provide an opinion (Figure ; Table ). SAs followed a similar pattern (Figure ; Table ). Seven (2.5%) out of 275 pairs of SAs and PLSs did not agree in conclusiveness category (one in Childhood Cancer Group, two in Colorectal Cancer Group, one in Gynaecological, Neuro‐oncology and Orphan Cancer Group, two in Haematological Malignancies Group and one in Lung Cancer Group). We also analysed the readability of PLSs regarding their conclusion. Among PLSs, those summaries that provided ‘No evidence’ conclusion were significantly easier to read than the summaries with other conclusions (Table ). There was no statistically significant difference in the readability of SAs with different conclusions (Table ). Regarding the readability of PLSs versus SAs with different conclusiveness, seven pairs did not match in conclusiveness, so it was not possible to analyse these differences on the whole sample. Plain language summaries with a ‘Positive’ conclusion had significantly greater use of analytic tone. There was no significant difference in other linguistic characteristics, that is use of clout, authentic and emotional tones. Furthermore, there was no statistically significant difference for the length of the summaries with different conclusions (Table ). In contrast, SAs with ‘No evidence’ or ‘Negative’ conclusions were significantly shorter than the abstracts with other conclusions. There was no significant difference in the linguistic characteristics of SAs with different conclusion types (Table ).
Readability analysis In general, PLSs were shorter and easier to read than SAs. SMOG index for PLSs was double the recommended 6 years of education (median = 13.0, 95% CI = 12.8–13.3), compared to 16.6 (95% CI = 16.4–16.8) for SAs ( p < 0.001; Wilcoxon nonparametric paired samples test). SAs were also significantly longer than PLSs: median word count 604 (95% CI = 566–653) versus 364 (95% CI = 339–388), respectively ( p < 0.001; Wilcoxon nonparametric paired samples test). Readability scores did not differ among different Cochrane Cancer Review Groups, both for PLSs and for SAs (Table ). The Groups differed in the text length of the summaries, with both PLSs and SAs being the shortest for the Colorectal Cancer Group (Table ).
Linguistic characteristics The use of the analytical tone was most prominent in both PLSs and SAs (Table ). Half of the words in both PLSs and SAs were categorized as clout speaking. Authentic and emotional tones were used less frequently in both types of summaries. In general, PLSs used language with significantly more authenticity and emotional tones, but fewer words related to analytical expressions than SAs. With regard to different Cochrane Cancer Review Groups, there was no statistically significant difference in the use of the analytical tone in PLSs (Table ). The clout tone was more dominant in PLSs from Gynaecological, Neuro‐oncology and Orphan Cancer Groups. In the Childhood Cancer Group, we observed the least use of emotional tone, while the authentic tone was least used in Haematological Malignancies Group (Table ). As in PLSs, the predominant use of the clout tone was found in SAs from Gynaecological, Neuro‐oncology and Orphan Cancer Groups. The analytical tone was predominant in SAs in Childhood Cancer Group. SAs from the Lung Cancer Group had the greatest proportion of words related to authenticity, as well as the greatest use of emotional tone (Table ).
Conclusiveness Categories of conclusiveness differed between summaries from different Cochrane Review Groups. PLSs from the Lung Cancer Group there had the greatest proportion of negative conclusion compared with the other groups. In Childhood Cancer Group, we found no PLSs with positive conclusions (Figure ; Table ). In Gynaecological, Neuro‐oncology and Orphan Cancer Group and Breast Cancer Group, we found no summaries where the authors did not provide an opinion (Figure ; Table ). SAs followed a similar pattern (Figure ; Table ). Seven (2.5%) out of 275 pairs of SAs and PLSs did not agree in conclusiveness category (one in Childhood Cancer Group, two in Colorectal Cancer Group, one in Gynaecological, Neuro‐oncology and Orphan Cancer Group, two in Haematological Malignancies Group and one in Lung Cancer Group). We also analysed the readability of PLSs regarding their conclusion. Among PLSs, those summaries that provided ‘No evidence’ conclusion were significantly easier to read than the summaries with other conclusions (Table ). There was no statistically significant difference in the readability of SAs with different conclusions (Table ). Regarding the readability of PLSs versus SAs with different conclusiveness, seven pairs did not match in conclusiveness, so it was not possible to analyse these differences on the whole sample. Plain language summaries with a ‘Positive’ conclusion had significantly greater use of analytic tone. There was no significant difference in other linguistic characteristics, that is use of clout, authentic and emotional tones. Furthermore, there was no statistically significant difference for the length of the summaries with different conclusions (Table ). In contrast, SAs with ‘No evidence’ or ‘Negative’ conclusions were significantly shorter than the abstracts with other conclusions. There was no significant difference in the linguistic characteristics of SAs with different conclusion types (Table ).
DISCUSSION In this study of a cohort of PLSs and corresponding SAs from six Cochrane Review Groups in oncology, PLSs were easier to read than the corresponding SAs, but the required literacy level was still too high for a general population. PLSs were shorter than SAs and had less use of analytical tone, with more use of authenticity and emotional tones. These results should be interpreted with several limitations in mind. First, we used only the Cochrane Library as a source of summaries for scientific papers. However, summaries from the Cochrane Library had the same format of presenting health information, making them comparable. Second, we only analysed the summaries in English, since it was the only common language for the summaries of oncology. On average, 13 years of education was needed to read a PLS for the systematic reviews in oncology from our study. The American Medical Association and National Health Institute recommended that the reading grade for the texts with health information intended for the lay public should be 6, , meaning that 6 years of education are needed for the comprehension of a given text. When writing a PLS for a scientific article, it is not easy to achieve that reading level because complex scientific expressions need to be simplified; at the same time, the information must be translated to the reader in an accurate way. Our study showed that there was no significant difference in readability across Cochrane Review Groups related to oncology, so there is a clear need for an overall improvement of PLSs in the field of oncology. Improving PLSs is only a part of the solution—we must bear in mind the current state of health literacy in the general public, as it has been shown that about a third of US citizens have low health literacy. , Moreover, it has been demonstrated that lay public rarely uses health information based on evidence‐based medicine, like scientific articles or websites of professional organizations. , , Instead, most of them tend to use commercial websites. , Both the PLSs and SAs are written by the same authors, who are scientists and are not trained in writing for nonscientific audiences. It has been shown that press releases for Cochrane systematic reviews, written by communication specialists, have a more engaging language in comparison with PLSs and SAs. A part of the solution to make a bridge between low health literacy of the lay public on one side and complex health information on the other side would be a coordinated work of health journalists alongside with oncology professionals. The need for more input from professional communicators is supported by our finding that a small number of PLSs and SAs pairs differed in the category of conclusiveness, although they were written by the same authors. Possible option for translating SAs into health‐related material for lay public could be use of artificial intelligence‐using algorithms that will be instructed to achieve the recommended readability levels. Different media channels available to the public, like radio, TV or social media networks could be used for consistent communication, although the simplicity of sharing information in these ways could be a double‐edged sword because it is as easy to share a misinformation as it is to share correct information. The importance of health literacy and its impact on social and economic determinants of health has already been recognized by some government agencies, which led to forming national plans for improving health literacy. , , Regarding the linguistic characteristics of the textual summaries of Cochrane systematic reviews, we found them to be similar in both PLSs and SAs: the most of the words were related to analytical tone, about half of the words were related to clout speaking, while approximately one quarter was categorized as authentic and emotional speaking. This similarity can be explained by the same authorship of the two types of texts. There still were some differences between PLSs and SAs: in general, PLSs were shorter and used less analytical and more authentic and emotional tones compared with SAs. As the aim of an SA is an accurate presentation of facts, it can explain the higher analytical tone compared with a PLS. The analytical tone relates to heightened abstract thinking and cognitive complexity. On the contrary, the use of authentic tone is connected to a writing that is personal, honest and less filtered, , which suits PLSs and their intention to bring the subject closer to the lay public. The emotional tone relates to how positive the tone is. Apparently, scientists tend to look on the bright side of research results in general, and it appears that, when writing PLSs, authors tend to write them in a more positive tone, maybe due to their inexperience as writers for general public engaging materials. Most of the systematic reviews gave unclear conclusions about the efficacy of intervention and most of them concluded that further evidence is needed for definitive decision. This can be explained by the lack of sufficient evidence for new treatments. For the development of a new cancer drug, the essential part of evaluating the efficacy of a new therapeutic option are clinical trials. However, it has been estimated that less than 5% of adult cancer patients enrol in cancer clinical trials. , On one hand, there is a need for more clinical trials, but on the other hand, small number of patients participate in them, even though 70% of cancer patients in the United States seem to be willing to participate in clinical trials. Unclear conclusion at the end of the summary can be the source of confusion for the lay public. To prevent that, authors could state that the summary does not provide a clear answer and suggest consultation with a medical oncologist, especially because every treatment decision should be made upon a thorough assessment of the oncologic value of treatment. Childhood Cancer Review Group was the one where we found no summaries with positive conclusions. The rarity of childhood cancer, combined with safety and regulatory requirements, makes it very demanding to conduct RCTs. We still mostly rely on indirect evidence from adult population, early phase clinical trials and case series, where it is challenging to make conclusions on the effectiveness and safety of an intervention. More randomized evidence and individual patient data meta‐analyses are needed to increase certainty and precision in the care of paediatric cancer patients. Plain language summaries from Breast Cancer Review Group were the PLSs that had the most positive conclusions. Breast cancer is the most common cancer in women. Over the last two decades, we have witnessed an enhancement of the conventional oncology treatments with new options such as immunotherapy, conjugated antibodies and targeting other metabolic pathways. Conducting randomized trials is relatively simpler for this cancer than for other, less common types of cancer; and there is also a strong commercial interest for creating new therapies. On the other side, PLSs from the Lung Cancer Review Group had the greatest proportion of negative conclusions. As mentioned before, new therapeutic options are available, especially for nonsmall lung cancer. Yet, despite these options and new clinical trials, lung cancer remains a significant challenge and contributes to most cases of cancer‐related death worldwide. The part of the problem, aside from tumour resistance to therapeutic options, is that lung cancer is mostly diagnosed in stages III and IV, unlike breast cancer, where screening programme shifted to the earlier stage at the time of diagnosis. We found that the summaries that provided ‘No evidence’ conclusion were significantly easier to read than the summaries with other conclusions. This is easy to explain since there was nothing to discuss or further elaborate in a summary if there was no evidence. However, we found no statistically significant difference in the readability of the PLSs with different conclusions, so the conclusion about the efficacy of given therapy or intervention does not seem to influence the readability of a PLS. This finding is in line with previous studies , that have shown that scientific literature is becoming harder to read in general and that scientists tend to use scientific jargon when writing, so further simplification for PLSs is still a translational challenge.
CONCLUSION Plain language summaries of Cochrane systematic reviews of oncology interventions have low readability and most give unclear conclusion about the effectiveness of therapy and procedures. To enhance the quality of the PLSs, authors and Cochrane Review Groups should be more attentive to follow the standards for writing summaries intended for general public. An input from the professionals who write public engaging materials, such as journalists, may help create health information material that will satisfy different health users yet provide sufficient scientific information and dissemination of evidence. Further research should focus on the improvement of PLSs and deeper analysis of reasons for unclear evidence in oncology trials.
Jelena Šuto: Investigation (equal); writing – original draft (lead). Ivan Buljan: Methodology (equal); writing – review and editing (equal). Ana Marušić: Supervision (lead); writing – review and editing (equal).
This study was funded by the Croatian Science Foundation (‘Professionalism in Health—Decision making in practice and research, ProDeM’) under Grant agreement No. IP‐2019‐04‐4882. The funder had no role in the design of this study, its execution, analyses, interpretation of the data or decision to submit results.
The authors declare no conflict of interest except for the membership in Cochrane.
Table S1. Table S2. Table S3. Table S4. Table S5. Table S6. Click here for additional data file.
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Clinical application of the anti‐human telomerase reverse transcriptase (h
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a8c3256a-f14c-4612-bf4f-e9d9a0cec938
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10225183
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Anatomy[mh]
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INTRODUCTION Urine cytology is a widely used, first‐line screening tool for the diagnosis of urothelial carcinoma. Even though the diagnostic accuracy of this test has been relatively high for detecting high‐grade urothelial carcinoma, reported sensitivity ranges from 26.3% to 88%, depending on the type of sample collection. , Especially, urine cytology has a lower accuracy rate and interobserver variability , for the indeterminate diagnosis of “atypical urothelial cells” (AUC), which encompasses a wide range of urothelial lesions from non‐neoplastic conditions to malignancy. As the atypical category could subsequently reveal urothelial carcinoma in follow‐up cytology or surgical pathology, this leads to confusion of clinicians on how to interpret this result and manage patients with atypical urine cytology interpretations. , , Therefore, urine‐based molecular tests, such as the UroVysion fluorescence in situ hybridization (FISH) assay (Vysis), Nuclear matrix protein (NMP) 22 test (Matritech), and ImmunoCyt test (DiagnoCure), have been approved as ancillary tests in the diagnosis and surveillance of urothelial malignancies. However, the overall sensitivity and specificity of these tests are suboptimal, especially when they lack a simultaneous cytological assessment, while the latter is still considered the gold standard in these settings. For that reason, immunostaining of cells has been introduced in urine cytology to identify potential biomarkers, with the goal to enhance its overall diagnostic performance. , Recently, immunocytochemistry (ICC) with the anti‐hTERT (human telomerase reverse transcriptase) monoclonal antibody (SCD‐A7, INOVIQ Ltd.) has been approved by the US Food and Drug Administration (FDA) for the detection and surveillance of bladder cancer in urine cytology. hTERT is a highly conserved catalytic subunit of the telomerase holoenzyme, which is responsible for maintaining the DNA sequence at the end of the chromosome, reextending it to its normal length after each cell division. Telomerase is known to be activated, at a low level, only in a small subset of normal stem cells, whereas it has been shown to be augmented in many cancer cell types, maintaining their telomere length and inducing their immortality. , , Being closely related to telomerase, hTERT high expression is a sufficient surrogate marker of enhanced telomerase activity. Increased transcription of hTERT has been detected in primary and recurred bladder cancer. In this context, there have been a few previous attempts to detect hTERT in urine or urinary bladder tissue samples. , , In this article, we describe a prospective validation study to evaluate the reliability of hTERT ICC in urine cytology. Also, we aimed to determine whether this biomarker could effectively triage the atypical urine cytology cases, by identifying the ones harboring urothelial carcinoma in follow‐up histology.
MATERIALS AND METHODS 2.1 Case selection This prospective study initially reviewed 561 urine cytology cases collected at Seoul National University Hospital (SNUH) between December 2019 and May 2022. The urine samples were mainly voided (531 out of 561), whereas 5.3% of them (30 out of 561) were catheterized and washings. The cytologic diagnosis was rendered by two experienced cytopathologists (J.H.M and H.S.R). Among the reviewed cases, a total of 413 were cytologically atypical, suspicious, or malignant. The rest 148 contained normal urothelium without any evidence of carcinoma from both cytologic evaluation and the clinical information from each patient's electronic medical records. The original cytology diagnoses were reclassified for this study according to the Paris system for Reporting Urinary Cytology as: (1) negative for high‐grade urothelial carcinoma (NHGUC), (2) atypical urothelial cells (AUC), (3) suspicious for high‐grade urothelial carcinoma (SHGUC), (4) high‐grade urothelial carcinoma (HGUC), and (5) low‐grade urothelial neoplasm (LGUN). The study protocol was reviewed and approved by the institutional review board of the Seoul National University Hospital (IRB no. 1907‐183‐1051). The waiver of informed consent was granted by the IRB. 2.2 Cytologic preparations and ICC for h TERT Samples were prepared with the BD SurePath (Becton, Dickinson and Company) liquid‐based cytology (LBC) protocol, followed by Papanicolaou staining. Following the routine diagnostic procedure, residual SurePath urine samples were processed on a separate slide for hTERT ICC. The latter was performed according to the manufacturer's guidelines. Briefly, slides were fixed in 1:1 acetone and methanol for 10 min at room temperature, and then placed into 10% neutral buffered formalin for 2 min at room temperature. They were then rinsed into a Ventana reaction buffer and kept “wet” during transfer to the ICC lab. Antigen retrieval was carried out at 95°C for 32 min using the Benchmark Ultra CC1 (Roche Diagnostics). Anti‐hTERT Antibody (SCD‐A7) was diluted (1:30) in Ventana Antibody Diluent with Casein (Roche Diagnostics) and incubated at 37°C for 32 min. The OptiView DAB detection system was used. Hematoxylin II was incubated for 16 min, while the bluing reagent was incubated for 4 min. The anti‐hTERT immunostaining was localized into the nucleus. For ICC assessment, samples containing less than 15 urothelial cells (either isolated and/or within clusters) were deemed insufficient cellularity and excluded from subsequent analysis. The lymphocytes, common in patients presenting with hematuria or tumor, were used as an internal positive control. Immunoreaction of hTERT in two or more cells, with an increased nucleus‐to‐cytoplasmic (N/C) ratio (>0.5) and/or hyperchromatic nucleus, was considered as a positive ICC result. 2.3 Statistical analysis The receiver‐operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy of hTERT in differentiating malignant from benign urinary cytology cases. The sensitivity (SN), specificity (SP), diagnostic accuracy, and area under the ROC curve (AUROC) of the hTERT ICC with 95% confidence interval (CI) were also evaluated. The analysis was performed with R, version 4.1.2 (R Foundation for Statistical Computing) with the packages “caret” and “pROC.”
Case selection This prospective study initially reviewed 561 urine cytology cases collected at Seoul National University Hospital (SNUH) between December 2019 and May 2022. The urine samples were mainly voided (531 out of 561), whereas 5.3% of them (30 out of 561) were catheterized and washings. The cytologic diagnosis was rendered by two experienced cytopathologists (J.H.M and H.S.R). Among the reviewed cases, a total of 413 were cytologically atypical, suspicious, or malignant. The rest 148 contained normal urothelium without any evidence of carcinoma from both cytologic evaluation and the clinical information from each patient's electronic medical records. The original cytology diagnoses were reclassified for this study according to the Paris system for Reporting Urinary Cytology as: (1) negative for high‐grade urothelial carcinoma (NHGUC), (2) atypical urothelial cells (AUC), (3) suspicious for high‐grade urothelial carcinoma (SHGUC), (4) high‐grade urothelial carcinoma (HGUC), and (5) low‐grade urothelial neoplasm (LGUN). The study protocol was reviewed and approved by the institutional review board of the Seoul National University Hospital (IRB no. 1907‐183‐1051). The waiver of informed consent was granted by the IRB.
Cytologic preparations and ICC for h TERT Samples were prepared with the BD SurePath (Becton, Dickinson and Company) liquid‐based cytology (LBC) protocol, followed by Papanicolaou staining. Following the routine diagnostic procedure, residual SurePath urine samples were processed on a separate slide for hTERT ICC. The latter was performed according to the manufacturer's guidelines. Briefly, slides were fixed in 1:1 acetone and methanol for 10 min at room temperature, and then placed into 10% neutral buffered formalin for 2 min at room temperature. They were then rinsed into a Ventana reaction buffer and kept “wet” during transfer to the ICC lab. Antigen retrieval was carried out at 95°C for 32 min using the Benchmark Ultra CC1 (Roche Diagnostics). Anti‐hTERT Antibody (SCD‐A7) was diluted (1:30) in Ventana Antibody Diluent with Casein (Roche Diagnostics) and incubated at 37°C for 32 min. The OptiView DAB detection system was used. Hematoxylin II was incubated for 16 min, while the bluing reagent was incubated for 4 min. The anti‐hTERT immunostaining was localized into the nucleus. For ICC assessment, samples containing less than 15 urothelial cells (either isolated and/or within clusters) were deemed insufficient cellularity and excluded from subsequent analysis. The lymphocytes, common in patients presenting with hematuria or tumor, were used as an internal positive control. Immunoreaction of hTERT in two or more cells, with an increased nucleus‐to‐cytoplasmic (N/C) ratio (>0.5) and/or hyperchromatic nucleus, was considered as a positive ICC result.
Statistical analysis The receiver‐operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy of hTERT in differentiating malignant from benign urinary cytology cases. The sensitivity (SN), specificity (SP), diagnostic accuracy, and area under the ROC curve (AUROC) of the hTERT ICC with 95% confidence interval (CI) were also evaluated. The analysis was performed with R, version 4.1.2 (R Foundation for Statistical Computing) with the packages “caret” and “pROC.”
RESULTS 3.1 Patient characteristics A total of 561 samples were collected during the study period. In the first screening of hTERT ICC slides, 190 and 34 samples were excluded due to insufficient cellularity and the absence of follow‐up results, respectively. Finally, a cohort of 337 samples composed of HGUCs and SHGUCs ( n = 181), AUCs ( n = 66), LGUNs ( n = 3), and NHGUCs ( n = 87) was enrolled in the study (Figure ). All 337 samples analyzed had sufficient cellularity and available follow‐up result for comparison. The demographic findings and histologic diagnoses of these cases are described in Table . The mean age of the patients included was 71.8 years (range, 45–91 years), while the cohort consisted of 266 males and 71 females (male:female ratio = 3.7:1). The follow‐up histological diagnoses were as follows: 99 (29.4%) noninvasive papillary urothelial carcinomas; 25 (7.4%) carcinomas in situ (CIS); 86 (25.5%) invasive urothelial carcinomas, submucosal invasive; 36 (10.7%) invasive urothelial carcinomas, muscle invasive; and 1 (0.3%) adenocarcinoma of the urinary bladder. In addition, 90 cases (26.7%) were found to be benign (Table ). 3.2 Negative immunoreactivity of h TERT in most NHGUC cases Eighty‐seven urine cytology cases interpreted as NHGUC and without a clinical history of urinary disease were selected to be stained with hTERT (Table ). Lymphocytes were regarded as a positive internal control. Whereas hTERT was strongly stained in lymphocytes, most benign urothelial cells (including umbrella cells) showed no or weak nuclear immunoreactivity (Figure ). As a result, just 2/87 NHGUC cases (2.3%) exhibited strong hTERT immunopositivity. Similarly, 96.7% ( n = 87/90) of the cases confirmed to be benign during follow‐up were hTERT‐negative (Table ). 3.3 Positive immunoreactivity of h TERT in urothelial carcinoma We enrolled 181 SHGUCs and HGUCs with sufficient cellularity for hTERT ICC, 180 of which were also histologically confirmed as urothelial carcinomas. Among these 180 cases, 175 (97.2%) showed strong positive hTERT immunoreactivity in carcinoma cells (Figure ). By contrast, adjacent normal urothelial cells were hTERT‐negative or weakly positive (Figure ). We further evaluated the hTERT ICC expression of our cases in relation to their follow‐up histologic diagnosis, according to the 4th edition of the WHO classification. From the 247 cases diagnosed as malignant during follow‐up histology, 243 were classified as high‐grade (98.4%), including 217 high‐grade urothelial carcinomas, 25 CIS, and one bladder adenocarcinoma (Tables and ). hTERT protein was overexpressed in 233/243 (95.9%, Figure and ) of these neoplasms. The rest four cases (1.6%), which were classified as low‐grade urothelial carcinomas, showed 100% positive hTERT immunoreactivity (4/4, Figure ). 3.4 h TERT protein expression pattern in AUC To evaluate the diagnostic efficacy of hTERT ICC in detecting urothelial carcinoma in the subgroup comprising atypical cytology samples, 187 urine LBC samples with an AUC interpretation were examined; 98 with inadequate cellularity were excluded. Of the remaining 89 cases, matched histologic diagnosis was available in 66 cases, which were included in the final analysis (Table ); 64 of them were histologically confirmed as urothelial carcinoma, whereas two as benign. Among the 64 AUC cases confirmed as malignant in follow‐up histology, 59 (92.2%) were hTERT‐positive (Figure ), whereas five (7.8%) were hTERT‐negative (Table ). Both AUC samples, eventually turned out to be benign in their matched histology, were found to be hTERT‐negative. 3.5 Correlation of hTERT expression between malignant and benign urine cytology The diagnostic performance of hTERT ICC to detect the presence of cancer was analyzed (Table ). The assessment of the overall diagnostic accuracy of hTERT ICC in our LBC cohort showed the following results: 96.3% SN, 98.8% SP, and 97.0% (95% CI: 94.5–98.5) accuracy. The hTERT SN in the subgroup composed of SHGUC and HGUC cytology cases was 97.2%, while the accuracy was 96.7% (95% CI: 93.0–98.7). The SN of hTERT to detect cancer in the subgroup comprising AUC cases was 92.2%. SP could not be calculated, as all samples with benign histological follow‐up were hTERT‐negative in the subgroup. The ROC curve of hTERT ICC in total of 337 cases showed an AUROC value of 0.963 (95% confidence interval [CI: 0.960–0.967], Figure ).
Patient characteristics A total of 561 samples were collected during the study period. In the first screening of hTERT ICC slides, 190 and 34 samples were excluded due to insufficient cellularity and the absence of follow‐up results, respectively. Finally, a cohort of 337 samples composed of HGUCs and SHGUCs ( n = 181), AUCs ( n = 66), LGUNs ( n = 3), and NHGUCs ( n = 87) was enrolled in the study (Figure ). All 337 samples analyzed had sufficient cellularity and available follow‐up result for comparison. The demographic findings and histologic diagnoses of these cases are described in Table . The mean age of the patients included was 71.8 years (range, 45–91 years), while the cohort consisted of 266 males and 71 females (male:female ratio = 3.7:1). The follow‐up histological diagnoses were as follows: 99 (29.4%) noninvasive papillary urothelial carcinomas; 25 (7.4%) carcinomas in situ (CIS); 86 (25.5%) invasive urothelial carcinomas, submucosal invasive; 36 (10.7%) invasive urothelial carcinomas, muscle invasive; and 1 (0.3%) adenocarcinoma of the urinary bladder. In addition, 90 cases (26.7%) were found to be benign (Table ).
Negative immunoreactivity of h TERT in most NHGUC cases Eighty‐seven urine cytology cases interpreted as NHGUC and without a clinical history of urinary disease were selected to be stained with hTERT (Table ). Lymphocytes were regarded as a positive internal control. Whereas hTERT was strongly stained in lymphocytes, most benign urothelial cells (including umbrella cells) showed no or weak nuclear immunoreactivity (Figure ). As a result, just 2/87 NHGUC cases (2.3%) exhibited strong hTERT immunopositivity. Similarly, 96.7% ( n = 87/90) of the cases confirmed to be benign during follow‐up were hTERT‐negative (Table ).
Positive immunoreactivity of h TERT in urothelial carcinoma We enrolled 181 SHGUCs and HGUCs with sufficient cellularity for hTERT ICC, 180 of which were also histologically confirmed as urothelial carcinomas. Among these 180 cases, 175 (97.2%) showed strong positive hTERT immunoreactivity in carcinoma cells (Figure ). By contrast, adjacent normal urothelial cells were hTERT‐negative or weakly positive (Figure ). We further evaluated the hTERT ICC expression of our cases in relation to their follow‐up histologic diagnosis, according to the 4th edition of the WHO classification. From the 247 cases diagnosed as malignant during follow‐up histology, 243 were classified as high‐grade (98.4%), including 217 high‐grade urothelial carcinomas, 25 CIS, and one bladder adenocarcinoma (Tables and ). hTERT protein was overexpressed in 233/243 (95.9%, Figure and ) of these neoplasms. The rest four cases (1.6%), which were classified as low‐grade urothelial carcinomas, showed 100% positive hTERT immunoreactivity (4/4, Figure ).
h TERT protein expression pattern in AUC To evaluate the diagnostic efficacy of hTERT ICC in detecting urothelial carcinoma in the subgroup comprising atypical cytology samples, 187 urine LBC samples with an AUC interpretation were examined; 98 with inadequate cellularity were excluded. Of the remaining 89 cases, matched histologic diagnosis was available in 66 cases, which were included in the final analysis (Table ); 64 of them were histologically confirmed as urothelial carcinoma, whereas two as benign. Among the 64 AUC cases confirmed as malignant in follow‐up histology, 59 (92.2%) were hTERT‐positive (Figure ), whereas five (7.8%) were hTERT‐negative (Table ). Both AUC samples, eventually turned out to be benign in their matched histology, were found to be hTERT‐negative.
Correlation of hTERT expression between malignant and benign urine cytology The diagnostic performance of hTERT ICC to detect the presence of cancer was analyzed (Table ). The assessment of the overall diagnostic accuracy of hTERT ICC in our LBC cohort showed the following results: 96.3% SN, 98.8% SP, and 97.0% (95% CI: 94.5–98.5) accuracy. The hTERT SN in the subgroup composed of SHGUC and HGUC cytology cases was 97.2%, while the accuracy was 96.7% (95% CI: 93.0–98.7). The SN of hTERT to detect cancer in the subgroup comprising AUC cases was 92.2%. SP could not be calculated, as all samples with benign histological follow‐up were hTERT‐negative in the subgroup. The ROC curve of hTERT ICC in total of 337 cases showed an AUROC value of 0.963 (95% confidence interval [CI: 0.960–0.967], Figure ).
DISCUSSION In this study, we demonstrated the value of hTERT as a novel diagnostic biomarker in liquid‐based urine cytology. hTERT was exclusively overexpressed in urothelial carcinoma including SHGUC and HGUC samples ( n = 176/181, 97.2%), as opposed to its low expression in NHGUC samples ( n = 2/87, 2.3%). Since an increased activity of hTERT, which is a catalytic component of the human telomerase, is detected in human cancer but not in most normal cells, this enzyme has been considered as a promising tumor marker in various cancers, including urothelial carcinoma. , We adopted a specific hTERT antibody, SCD‐A7, which is the first FDA‐approved in vitro diagnostic test for assessing hTERT protein expression in urine cytology. Two previously published studies using the SCD‐A7 antibody on urine cytology also demonstrated increased hTERT expression rates in urothelial carcinoma. Allison et al. demonstrated that nine of the 11 HGUC samples showed hTERT positivity, while in another study by Xing et al. six of the nine SGHUC and HGUC samples showed strong staining with hTERT. These consistent findings support hTERT ICC utility in detecting malignant urine cytology samples. Our study enrolled a large cohort of urothelial malignancies, as 247/337 cases were histologically confirmed as carcinomas. hTERT ICC showed strong immunoreactivity not only in the cytology cases proved to be high‐grade urothelial carcinomas ( n = 208/217, 95.9%) or CIS ( n = 24/25, 96%) but also in the ones subsequently diagnosed as low‐grade urothelial carcinomas ( n = 4/4, 100%) during follow‐up. Of interest, 92.2% ( n = 59/64) of the AUC cytology cases histologically diagnosed as urothelial carcinomas also showed positive hTERT ICC expression. These findings suggest SCD‐A7 ICC could be applied to triage indeterminant cytology cases, by identifying the ones harboring urothelial carcinoma. In the initial application of ICC, however, there was a quality issue related to the immunoreactivity of hTERT. We noticed severe background staining in addition to overstaining of epithelial cells, regardless of several staining protocol alterations, including antibody incubation or retrieval time. After several trials, we found this technical issue was possibly caused due to the unsuitable type of glass slides we initially applied. Subsequently, the aforementioned background staining was significantly reduced by performing ICC on the New Silane III slides (Muto Pure Chemicals Co., Ltd.), compared with other types of coated slides. New Silane III slides exhibit stronger adhesive strength and fewer dyeing spots during immunostaining. Therefore, we believe these slides are appropriate for ICC, at least for the samples prepared using the SurePath technique. In our study, hTERT nuclear or nonspecific cytoplasmic staining was also identified in benign urothelial cells, such as umbrella cells or parabasal cells, which confused interpretation in as many as 29 cases (29/337, 8.6%). However, the immunoreactivity for hTERT was weaker in benign cells compared with urothelial carcinoma, which is concordant with the findings of previous studies. , For instance, Xing et al., who performed ICC on ThinPrep slides, recognized hTERT positivity in normal or nonurothelial cells, yet more often a stronger staining pattern in urothelial carcinoma. More specifically, whereas 37/60 (62%) of their NHGUC cases showed hTERT immunopositivity, just 8/37 (22%) exhibited a strong signal intensity. In our cohort, only 2/87 NHGUC cases (2.3%) exhibited strong hTERT immunopositivity. Therefore, when evaluating hTERT ICC, it is necessary to combine the intensity of hTERT immunoreactivity with cytomorphology. However, further studies would be necessary for a prospective validation of these findings, using different preparation methods (e.g., ThinPrep vs. SurePath) and sample types. The current study is limited by its small number of atypical cytology samples that proved to be benign in histological follow‐up, also the exclusion of some cases due to insufficient cellularity or the lack of positive internal control during hTERT ICC. Therefore, future prospective studies should primarily focus on the AUC reporting category, including higher numbers of AUC cases associated with benign histology, to reflect better routine clinical practice and thus evaluate more efficiently the value of hTERT ICC in urine cytology. Nevertheless, our findings suggest that hTERT ICC using the SCD‐A7 antibody might be able to overcome the limitations of other methods, including genetic tests detecting hTERT promoter mutations or mRNA expression. , , , These genetic tests are costly and time‐consuming, while genetic expression is not necessarily associated with protein expression. Furthermore, the translated proteins themselves are the functional units within each cell in both physiological and pathological conditions. , In conclusion, the results of this study demonstrated that hTERT ICC appears to be a reliable biomarker for detecting malignancy in liquid‐based urine cytology. In addition, it might help triage cases showing AUC by identifying the ones harboring urothelial carcinoma.
Ji Hye Moon: Conceptualization (equal); formal analysis (lead); investigation (equal); writing – original draft (lead). Ilias P. Nikas: Investigation (equal); writing – review and editing (equal). Kyung Chul Moon: Supervision (equal). Bohyun Kim: Investigation (equal). Han Suk Ryu: Conceptualization (equal); supervision (lead); writing – review and editing (lead).
The authors declare no conflicts of interest.
The study protocol was reviewed and approved by the institutional review board of the Seoul National University Hospital (IRB no. 1907–183‐1051). The IRB also waived the requirement to obtain written informed consent from the patients.
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Can primary care physicians recognize type 1 diabetes in children on time?
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824e3acd-57f9-42e1-bcfb-b35d20b4d612
|
10226347
|
Family Medicine[mh]
|
Type 1 diabetes (DM1) is a chronic metabolic disorder caused by insufficient insulin secretion due to pancreatic β-cells’ destruction, which is mainly due to the autoimmune process. It is the most common form of diabetes in children and adolescents worldwide. Nevertheless, evidence shows that trends in its incidence vary between different countries, within countries, and between different ethnic populations, with the highest incidence rates observed in Finland and Sardinia (40.2 and 38.8 per 100,000, respectively), and the lowest in Japan (approximately 2 per 100,000) . The higher incidence in European Caucasians is associated with unique HLA susceptibility genes . It is somehow more difficult to explain the systematic increase in the incidence rate of DM1 in children from Central and Eastern Europe observed in recent decades. The EURODIAB study revealed that the incidence of DM1 is increasing by 0.6–15% per year, totalling approximately 96,000 new cases in children under 15 years old annually . Although diagnostic criteria for diabetes in children and adolescents based on plasma blood glucose levels and the presence or absence of symptoms should be widely known to all health professionals, there are still many cases of DM1 not being diagnosed on time [ , , ]. Delayed diagnosis is the most important risk factor for the development of diabetic ketoacidosis (DKA), which is the most severe, life-threatening, first manifestation of DM1 . The prevalence of DKA as the first manifestation of DM1 varies between countries from 15% to 70% in Europe and North America . The EURODIAB study conducted between 1989 and 1994 revealed a DKA incidence of 33% among patients with newly diagnosed DM1 in Europe . Our previous study indicated that DKA was diagnosed in 22.4% of patients admitted to hospital between 2006 and 2011 . That information is particularly worrying because DKA and its complications are the most frequent cause of death in the diabetic paediatric population, responsible for at least 50% of deaths [ – ]. Many authors have in the past attempted to identify the major risk factors for delayed diagnosis of DM1. Some of them identified, among other factors, the lesser amount of experience and inferior professional qualifications of healthcare professionals . For this reason, we decided to compare our own experiences of the diagnosis of DM1 by doctors at the beginning of their careers with those who have been practicing as family doctors and paediatricians for many years. The purpose of the study was to answer the question of whether general practitioners know the criteria for diagnosing diabetes in children.
The voluntary survey was completed by 50 doctors, specialists in paediatrics or family medicine, and 50 doctors without specialization, up to 4 years after completing their medical studies. All were living and working in Poland and were questioned by way of face-to-face or e-mail interviews during November 2019 and November 2020. The survey list consisted of 3 parts. The first part concerned years of experience as a physician, specialization, years of experience in family medicine and the actual place of work. The second part included questions about their own experience with newly diagnosed diabetes: How many times did you diagnose DM1? How often do you use a glucometer in your practice? The third part checked the knowledge of the diagnostic criteria for diabetes: What is the fasting plasma glucose concentration for the diagnosis of DM1? What is the value of random plasma glucose concentration for the diagnosis of DM1? What is the value of two-hour post-load glucose concentration during OGTT for the diagnosis of DM1? Is the OGTT obligatory to diagnose DM1? What kind of fluid is used in rehydration with newly diagnosed DM1? and What are the typical early diabetes symptoms? Statistical analysis Data were analysed using Statistica 13.0 PL for Windows. The mean and standard deviations (SD) were calculated for continuous variables. The categorical data sets were analysed using Pearson’s χ 2 test. A P-value of less than 0.05 was considered to be statistically significant.
Data were analysed using Statistica 13.0 PL for Windows. The mean and standard deviations (SD) were calculated for continuous variables. The categorical data sets were analysed using Pearson’s χ 2 test. A P-value of less than 0.05 was considered to be statistically significant.
The mean professional experience of a family doctor (primary care paediatrician) among specialists was 14.6 years (SD 11.8), and 1.6 years (SD 1.4) among doctors without specialization. During that time, medical specialists diagnosed 76 new cases of DM1 – each one from 0 to 12. The highest result was obtained by a medical specialist who worked also as an emergency care unit physician. Doctors without specialization diagnosed 13 new cases of DM1 – each from 0 to 5 new DM1 cases. Medical specialists more frequently confirmed using a glucometer in their daily practice (mean 6 vs. 1, p < 0.05), but after taking into account the years of work, the difference was insignificant. Most of the study participants from both groups correctly answered questions about the diagnostic criteria of diabetes and the latter’s management in the event of its diagnosis ( ). The most problematic question seems to be the one regarding diagnostic criteria for diabetes in oral glucose tolerance test. The correct answers varied from 96% to 72% among medical specialists and physicians during the training, respectively ( ).
According to worldwide data, being a younger child or an ethnic minority, lower socioeconomic status, a lack of health insurance, lower BMI, a previous infection, living in equatorial areas, and in a country with a low prevalence of type 1 diabetes mellitus are risk factors for delayed diagnosis and development of DKA. On the other hand, higher parental educational status and a positive diabetes family history seem to be protective factors . The occurrence of DKA varies between countries. According to the EURODIAB study, DKA presentation at the time of DM1 diagnosis negatively correlates with the DM1 incidence rate . The latter may confirm that the higher the diabetes incidence, the more aware healthcare professionals are of the initial symptoms present. According to a paper by Muñoz et al ., patients with a missed diagnosis of DM1 are at a 17.6% increased risk for DKA compared to those who are correctly diagnosed at onset . In the past, researchers in several countries have tried to establish if better medical education would reduce the risk of DKA. One of the most extensive intervention studies was the Parma campaign performed by Vanelli et al . The project was based on a general, public information campaign. The aim of the study was to check whether education about the symptoms of diabetes among doctors, teachers, and parents may have an influence on earlier diagnosis of DM1 and therefore avoiding DKA and its consequences . The Parma campaign obtained a reduction in DKA incidence at diabetes diagnosis unfound before, from 78% to 12.5%, while in neighbouring provinces it remained unchanged . Similar studies were launched in Europe and Australia, but without such spectacular effect [ – ]. Nevertheless, the authors of all the relevant studies agree that healthcare professionals, especially family doctors and primary care paediatricians, are the most important targets for education . According to our survey data, most Polish medical specialists and young physicians during training have some general knowledge about DM1, its symptoms, and diagnosis. However, to our dismay, this is rather theoretical knowledge. Analysing the prevalence of type 1 diabetes among Polish children, and the amount of general advice provided by general practitioners, the low number of newly diagnosed patients with DM1 is worrying. Even more astonishing is the very rare use of such an accessible tool as the glucometer. The results obtained must be interpreted within the context of the study design. Eligible participants were all members of a single healthcare system, potentially limiting the generalizability of the findings to other populations. Nevertheless, similar observations were published recently for the US population of adult patients with type 2 diabetes, also indicating insufficient engagement of doctors despite having the appropriate tools . Patients may be misdiagnosed or overlooked at disease onset because the initial symptoms of type 1 diabetes may be nonspecific . As stated previously, many children with newly diagnosed DM1 had seen a paediatrician or family doctor within the previous 4 weeks . That proves that hyperglycaemia is initially missed in some patients. The most common alternate diagnoses in children and adolescents are infectious diseases, common in these age groups . Many symptoms of viral illnesses are nonspecific and may overlap with DM1. As was shown in the past, awareness campaigns focusing on symptoms more specific to DM1 should be performed. However, as our research shows, knowledge alone is not sufficient. Practical education of physicians is also necessary. In the Polish healthcare system, the primary care physician is responsible for conducting the interview and examining the patient. Procedures such as measuring blood glucose with a glucometer are performed by a nurse, while some doctors are not even able to operate such equipment. This can raise an objective difficulty in some situations. It also seems that, as in the case of the Parma campaign, closer cooperation between primary care physicians and diabetes departments, and greater availability of consultations would be beneficial .
It is important to increase awareness and promote early diagnosis for DM1 in primary care physicians, despite their professional experience and qualifications. Even more attention should be paid to the practical aspects of education.
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Autopsy Findings of Severe COVID-19 Pneumonia Combined with Pulmonary Aspergillosis, Pneumothorax, and Pulmonary Thromboembolisms
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619f6aba-561c-4394-8c5d-c3d6a5a98371
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10226672
|
Forensic Medicine[mh]
|
Coronavirus disease 2019 (COVID-19) is accompanied by many complications. Pneumonia is most frequently observed and can cause acute respiratory distress syndrome (ARDS). Pulmonary thromboembolism (PTE) is also a well-known complication. A pooled analysis of 3342 COVID-19 patients revealed that the incidence of PTE was 16.5% in total, and reached 24.7% in patients admitted to the Intensive Care Unit (ICU) . In a study of mechanically ventilated COVID-19 patients in an ICU, 20 of 168 patients (11.9%) developed COVID-19-associated pulmonary aspergillosis (CAPA), 13 of whom (65%) died . A review of 34 CAPA cases also reported high mortality (64.7%) . However, autopsy cases with multiple complications associated with COVID-19 have not been well reported. Here, we describe an autopsy case with severe COVID-19 pneumonia, PTE, CAPA, and CAPA-induced pneumothorax to help in the understanding of severe COVID-19 and the future development of its treatment strategy.
A 64-year-old Japanese man presented with fever and a cough (day 0) and was diagnosed with COVID-19 by positivity for SARS-CoV-2 PCR in the delta variant spread period in Japan (July to September, 2021). Six days later (day 6), COVID-19 pneumonia was recognized by computed tomography showing a wide area of ground-glass opacities ( ). He was admitted to a hospital because of hypoxemia and started to receive oxygen inhalation, remdesivir (200 mg on the first day and then 100 mg per day), dexamethasone (6.6 mg per day), baricitinib (4 mg per day), and edoxaban (60 mg per day). However, his respiratory condition worsened, noninvasive positive-pressure ventilation (NPPV) management was initiated on day 15, and he was transferred to our hospital on day 17. He had never received SARS-CoV-2 vaccination. Although he had type 2 diabetes mellitus, he did not receive any treatment. He had no other comorbidities. SARS-CoV-2 was again positive by PCR. His height was 163 cm, and his body weight was 73 kg (body mass index 27.5). The respiratory rate was 28 per minute. Fine crackles were auscultated. SpO 2 was 90% under NPPV with FiO 2 100%. Blood tests revealed WBC 24 740/mL (neutrophils 95.9%), hemoglobin 13.6 g/dL, platelets 59 000/mL, C-reactive protein 2.94 mg/dL, lactate dehydrogenase 985 U/L, glucose 318 mg/dL, HbA1c 8.5%, KL-6 5671 U/mL, ferritin 1318 ng/mL, D-dimer 12.2 mg/mL, and b-D-glucan 9.1 pg/mL. Chest CT showed extremely extended ground-glass opacities in both lung fields with partial consolidation posteriorly ( ). Arterial blood gas analysis (NPPV, FiO 2 100%) showed pH 7.359, PaCO 2 42.9 Torr, and PaO2 58.7 Torr. Invasive mechanical ventilation was started. Management by prone positioning could be implemented for only 1 day due to manpower issues. Methylprednisolone (1000 mg per day) instead of dexamethasone was administered because of progressive respiratory failure during dexamethasone and doses were reduced by half every 3 days. Intravenous unfractionated heparin was started continuously based on its short half-time and adjusted according to activated partial thromboplastin time. A foot pump was also used to combat risk of thromboembolism. Because Aspergillus fumigatus was detected from intratracheal samples when the patient began invasive mechanical ventilation management prior to the start of high-dose methylprednisolone, voriconazole was started (800 mg on the first day and then 400 mg per day). Despite these treatments, the platelet count further decreased to 13 000/mL, and platelet transfusions were needed. Hypoxemia deteriorated and bilateral consolidations on chest X-ray increased on day 28 ( ). At this time, SARS-CoV-2 PCR was negative. On day 31, right pneumothorax occurred, and a drainage tube was inserted. On day 32, the patient died of respiratory failure. Autopsy was performed. Macroscopically, there was a hole in the right upper lobe ( ). Microscopically, there were several cavities accompanied by hemorrhage and necrosis dominantly in the right upper lobe ( ). One cavity formation adjacent to the pleural membrane was broken, which was the cause of the pneumothorax ( ). In and out of cavities, agglomeration of aspergillus was recognized, consistent with invasive pulmonary aspergillosis ( ). Inflammatory cells infiltrated around aspergillus were predominantly neutrophils, with some lymphocytes and histiocytes, accompanied by nuclear waste materials. Aspergillus was also detected in the left upper lobe. In a wider area of both lungs than pulmonary aspergillosis, alveolar structures were destroyed with fibrotic changes and infiltration of inflammatory cells and fibroblasts ( ), suggesting diffuse alveolar damage (DAD) had occurred. The formation of hyaline membranes was also observed ( ), suggesting a relatively new DAD. Pulmonary edema was shown in some parts (not shown). Many thromboembolisms were recognized in peripheral pulmonary arteries ( ). Organized thromboses were also observed ( ). There were no obvious thromboses in organs other than the lungs.
Pathological findings of autopsy are summarized as follows: (1) old and newly developed DAD in a wide area of the lungs, which is consistent with ARDS due to COVID-19 pneumonia; (2) PTEs in peripheral pulmonary arteries; (3) CAPA; and (4) pneumothorax by CAPA. The autopsy clarified that these pathological changes led to respiratory failure. DAD is the most common pathological finding of ARDS due to COVID-19 pneumonia . DAD was recognized in 127 (80.9%) of 157 COVID-19 patients in whom postmortem autopsy was performed . In the present case, various time phases of DAD were recognized: the exudative phase, represented by the formation of hyaline membranes; the proliferative phase, in which many inflammatory cells and fibroblasts are present; and the fibrotic phase, in which fibrosis is dominant. Although over 1 month had passed from COVID-19 onset, and SARS-CoV-2 PCR had already turned negative, findings of not only the late phase but also the early phase of ARDS were observed. This might be suggestive of insufficiency of the dose or period of corticosteroids or non-response to corticosteroids. Corticosteroids are now recommended for severe COVID-19 pneumonia . In the RECOVERY trial, dexamethasone (6 mg/day for 10 days) decreased mortality for COVID-19 patients receiving either oxygen or invasive mechanical ventilation . A recent prospective randomized trial showed that the methylprednisolone (2 mg/kg/day) group (n=47) demonstrated better clinical status, including a lower admission rate, improvement in the WHO ordinal scale, and a lower rate of mechanical ventilation use, than the dexamethasone (6 mg/day) group (n=46) . There is no evidence of efficacy of high-dose corticosteroids such as methylprednisolone pulse therapy for COVID-19 pneumonia. In the present case, ARDS progressed even after treatment with a high dose of methylprednisolone. On the other hand, there are COVID-19 cases that improve without corticosteroid. There is no uniform dose of corticosteroid suitable for all cases. Thus, the quantity of necessary corticosteroids should be decided for each case depending on the clinical situation, such as infection. Venous thromboembolism, including PTE, is well-recognized as a complication of COVID-19. Intensified doses of heparin are recommended for antithrombotic prophylaxis, depending on the clinical or biological severity of COVID-19 . Although low-molecular-weight heparin, such as enoxaparin, would also be effective , standard drugs and their doses have not yet been determined for COVID-19. In the present case, an intensified dose of unfractionated heparin was intravenously administered. However, thrombocytopenia continued throughout the course, and autopsy revealed many newly formed PTEs. This suggests that cases exist in which even continuous heparin is insufficient for antithrombotic prophylaxis. Several autopsies of CAPA cases have been reported [ , , ]. Several patterns have been reported, including airway colonization , focal pulmonary aspergillosis , and invasive pulmonary aspergillosis (IPA) . The current case was classified as IPA, and the existence of ARDS would be a characteristic finding of CAPA. A study reported that the incidence of CAPA increased in the second wave of the pandemic, in which corticosteroids were regularly used for moderate-to-severe COVID-19 pneumonia, compared with the first wave . As characteristics of patient with CAPA, hypertension, diabetes mellitus, obese, use of mechanical ventilation, and use of corticosteroids have been reported . Interleukin-6 is secreted in severe COVID-19 patients and can play an important role in protective immunity against Aspergillus . In this regard, blockade of interleukin-6 may potentially progress Aspergillus infection. Although the efficacy of corticosteroids in severe COVID-19 pneumonia is established, the pros and cons of corticosteroids in the development of CAPA are not clear. In the present case, risk factors for CAPA development could be uncontrolled diabetes mellitus, dexamethasone (6.6 mg per day for 10 days), baricitinib, management with mechanical ventilation, and no SARS-CoV-2 vaccination history. However, the reason for such medication and respiratory management is severe COVID-19, and we already know some ways to prevent COVID-19 from becoming severe, including vaccination and appropriate blood glucose control. Pneumothorax is a complication of severe COVID-19 and occurs in approximately 10% of critically ill patients . Several causes of pneumothorax in ARDS have been illustrated, including increased alveolar pressure caused by mechanical ventilation, changes in alveolar structure and function, increased negative pressure of the pleural cavity by severe cough or forced inhalation, and shear stress . The present case revealed that CAPA can cause pneumothorax, which is the first report of its kind. The autopsy of the present case showed that the findings observed in the severe COVID-19 case were gathered in 1 patient. In addition, ARDS, PTE, and CAPA were all still active states; in other words, their treatments were insufficient. The treatment strategy, including the dose of corticosteroids for such severe COVID-19 cases, is an unresolved problem.
Active states of combined ARDS, PTEs, CAPA, and CAPA-induced pneumothorax were observed in 1 patient with severe COVID-19. It is difficult to improve these conditions simultaneously because their treatments can induce antagonizing biological actions. Severe COVID-19 can be prevented by the use of vaccinations and reduction of risk factors.
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An artificial neural network-based radiomics model for predicting the radiotherapy response of advanced esophageal squamous cell carcinoma patients: a multicenter study
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0c8f22db-373a-4429-b89f-242ea961885a
|
10226996
|
Internal Medicine[mh]
|
Esophageal cancer (EC) is one of the fatal subtypes of malignant tumors and has the seventh-highest mortality rate among all subtypes . For Asia, squamous cell carcinoma is the primary pathological subtype of EC. Radical surgery and chemoradiotherapy are crucial treatments for esophageal squamous cell carcinoma (ESCC) patients . Radical radiotherapy is recommended as a preferred treatment for cervical and middle thoracic esophageal carcinoma located at a higher position that is difficult to completely resect by surgery. For unresectable advanced ESCC, chemotherapy and radiotherapy are still needed to relieve symptoms and extend survival – . Nevertheless, sensitivity to radiotherapy varies among different patients , leading to significant differences in treatment response. Adverse events and side effects are more likely to be observed in patients with radiation-resistant ESCC , . To this end, a practical and noninvasive approach that can estimate radiotherapy precisely before treatment implementation needs to be explored in ESCC patients. In recent decades, the general classification of esophageal contrast (medullary type, fungating type, constrictive type, and ulcerative type) to predict the radiotherapy response has been widely used in clinical work , . However, this prediction is entirely based on empirical evaluation by radiologists, which causes differences among actual treatment responses. Otherwise, the molecular biomarkers related to radiotherapy sensitivity have not been prospectively validated for routine clinical usage. Recent studies have indicated that radiomics based on artificial intelligence (AI) can extract noninvasive radiographic virtual biopsy biomarkers, effectively providing predictive information for treatment response , . Lu et al. found that the deep learning-based model showed high accuracy in identifying the origins of cancers of unknown primary. Zhong indicated that multiparametric magnetic resonance imaging (mp-MRI)-based radiomics features could be considered prognostic factors in patients with localized prostate cancer after radiotherapy. Gao showed that radiomics signatures based on longitudinal diffusion-weighted MRIs could be used to estimate radiotherapy effects preoperatively. Zhu reported that a nomogram model based on computed tomography (CT) imaging radiomic signatures and clinical factors showed proper sensitivity and specificity in estimating the risk of local recurrence in nasopharyngeal carcinoma (NPC) after intensity-modulated radiotherapy (IMRT). Previous radiomics studies reported that radiomics features significantly improved the evaluation of the complete pathological response after neoadjuvant chemoradiation in EC patients . However, few relevant studies based on radiomics to predict the response of radiotherapy in ESCC have been reported. Herein, in this study, a large cohort of 248 patients with ESCC was used to develop a novel baseline CT-based radiomics signature model by a deep learning algorithm to validate their performance in predicting response to radiotherapy.
Patients Baseline information and imaging data, including demographic data, clinical data, pathological findings of biopsies before treatment, pre- and postsurgical imaging data, and surgical records, of patients with ESCC who underwent radiotherapy at Institution 1 (The First Affiliated Hospital of Xi'an Jiaotong University) from 2013 to 2019 were collected and analyzed. Moreover, we also retrieved and collected the same related data from patients with ESCC who received radiotherapy at Institution 2 (The Second Affiliated Hospital of Xi'an Jiaotong University) from 2017 to 2019. All patients underwent CT examination at the time of positioning before the beginning of their radiotherapy, and the CT data were collected retrospectively from 2021 to 2022. The main inclusion criteria were as follows: (1) biopsy-diagnosed ESCC; (2) clinically diagnosed advanced ESCC by CT and contrast imaging; (3) underwent complete radical radiotherapy (and did not drop out during the treatment); and (4) pre- and postradiotherapy imaging data were recorded after the same institution. The main exclusion criteria were as follows: (1) no biopsy or pathological confirmation; (2) no surgery or more than two weeks of adjuvant chemotherapy before radiotherapy; and (3) no reexamination of imaging data after radiotherapy. The general classifications of all patients were based on the Chinese CSCO guidelines . We used the standard of clinical staging for nonoperative esophageal cancer (Draft) for tumor grading . Written informed consent was obtained from all patients in this study. All methods undertaken in this work were carried out in accordance with the relevant guidelines and regulations. All patients' clinical information was consecutively enrolled, and the Ethics Committee of Xi'an Jiaotong University approved this study. Twenty percent of the patients from Institution 1 were randomly selected for the internal validation cohort, and the rest of the patients from Institution 1 were grouped into the training cohort. The patients from Institution 2 were chosen as the external validation cohort. The detailed experimental flow is illustrated in Fig. . Radiotherapy treatment and response assessment All patients in our research accepted the localizing by Brilliance CT locator (Philps, UK) before radiotherapy. The main operating parameters: tube voltage: 140 kV; tube electric current: 500 mA; beam spacing: 0.625 layer thickness; rotation time: 0.5 s; matrix: 512 × 512; detector size: 24 mm. The scanning parameters: layer thickness ≤ 5 mm; interval ≤ 5 mm. Before the scanning of CT, two radiotherapy technologists and doctors were required to accompany with the patients. All patients were required to fast for 4 to 6 h before CT scanning and drink 0.5 L of water during the scanning to make the esophagus dilated as much as possible. All patients were asked to hold their breath during using the multi-layer spiral CT machine to continuously scan the chest. The scanning range started from the upper edge of the supraclavicular fossa 5 cm above, down to the level of the 1st lumbar vertebra. Then the data of CT scanning was passed to the workstation for reconstruction. All patients in this study received radical radiotherapy, with a dose of radiation ranging from 60 to 66 Gy at an energy of 6 MV, 1.8–2.0 Gy/fraction, 5 times per week. Organs at risk (OAR), including the bilateral lungs, spinal cord, gastric duct and heart, were outlined for protection. The maximum tolerated doses for key normal structures were as follows: spinal cord: < 40 Gy; heart V40 ≤ 30%; bilateral lungs: V20 ≤ 28% and V30 ≤ 20%; and stomach: V40 ≤ 40%. However, because the data were retrieved retrospectively, the actual dose of radiotherapy for each patient was slightly different. Despite this, each radiotherapy plan fell within the scope of the radical dose recommended by the NCCN guidelines . Imaging specialists assessed patients' imaging data to measure the tumor's maximum diameter in each plane recommended by RECIST 1.1 guidelines . Then, the maximum diameter shrinkage rate of the tumor could be calculated (preradiotherapy maximum diameter/postradiotherapy maximum diameter), which is popularly used in clinical work to evaluate the treatment response. According to the maximum tumor diameter shrinkage rate for all patients, we selected 0.5 as the potential threshold to divide patients into two categories. After experiments, we chose the optimal cutoff value, which was 0.5 as the threshold to divide patients (tumor reduction rate greater than 50% and tumor reduction rate less than 50%). Delineation of regions of interest (ROIs) The continuous planes of CT images in all patients were outlined by two radiologists with more than eight years of experience in radiotherapy target area delineation using Monaco 5.2. Furthermore, another radiologist with more than 15 years of experience checked the target area and obtained the final radiotherapy target area, which guaranteed that the whole tumor of each patient was reflected by the target area from continuous planes of CT images. In this trial, we used the outlined target area images for the construction of a radiotherapy response model. To minimize the influence of the tumor margin on the model, for each patient, we chose the images that best described the tumor site. Dataset pre-processing The patient CT and target delineation data collected in this experiment are stored in DICOM file format, and the CT data in the DICOM files are converted into 2D PNG format images through Python. In this way, we can extract 10 to 60 slices of 2D images outlining the tumor target area from each patient. Afterwards, professional radiologists selects the most representative 2D CT images of the tumor (usually images around the maximum diameter of the tumor). The dataset collected from institution 1 was randomly divided into training set validation sets on a patient basis. The data collected from Institution 2 is distributed to the external validation group. In machine learning experiments, 2D CT images are transformed into feature vectors through feature extraction and subsequent experiments are conducted. In artificial neural networks, we applied data augmentation methods such as rotation, flipping, zooming, and distortion to the CT images. Through data augmentation, the dataset of CT images increased to 6000. And the 2D CT images were the direct input to the artificial neural network models. Prediction using machine learning In this study, patients from institution one (training cohort and internal cohort) were used to construct and verify the classification model. Radiomic features of the CT images were extracted using PyRadiomics image extraction software version 3.0. A total of 102 2D features were extracted from each patient, including first-order, shape 2D, gray level cooccurrence matrix (GLCM), gray level run length matrix (GLRLM), gray level size zone matrix (GLSZM), neighboring gray-tone difference matrix (NGTDM), and gray level dependence matrix (GLDM) features . Moreover, we grouped the correlated features (> 0.8) by the Pearson correlation coefficient algorithm, and the less predictive features in the same group were ignored in the feature selection algorithm (Supplementary Fig. ). The random forest algorithm was used to decrease the data dimensions and select the most predictive features . The formula and explanation of the feature selection algorithm are as follows: 1 [12pt]{minimal}
$${}( {} ) = {0.7ex} = 1}}^{{}} {( {{}_{{{}}} - {}_{{{}}} } )} }$} \!} = 1}}^{{}} {( {{}_{{{}}} - {}_{{{}}} } )} } N}}.-0pt} \!0.7ex}$$ Importance X = ∑ i = 1 N err OOB2 - err OOB1 / N in which X represents the feature, [12pt]{minimal}
$${}_{2}$$ err OOB 2 represents the out-of-bag error when we add noise to feature X and [12pt]{minimal}
$${}_{1}$$ err OOB 1 represents the out-of-bag error without adding noise. Ten sets of experiments with the number of features ranging from one to ten were applied to determine the optimal number of features for the model. Hence, the five most predictive features were selected to train the classifier and achieved the best performance. Meanwhile, multiple classifiers were tested in this study, including support vector machine (SVM) with linear kernel, SVM with radial basis kernel linear regression model , , linear regression model and random forest. After comparing the AUCs of the classifiers, we selected the random forest algorithm because it outperformed all other classifiers. Prediction using deep learning In this study, patients from institution one were used to build the training cohort and internal validation cohort, and patients from institution two were used to build the external validation cohort to verify the efficiency of generalization. The CNN model has been proven to be very effective in the field of image classification – . End-to-end CNN models provide precise prediction results without additional image feature extractions, which significantly improves the model's efficiency. In this study, multiple neural networks were used to classify the CT images of the patients. The model's learning rate was set to 0.0005, and the root mean square prop (RMSprop) optimizer was used. Meanwhile, we used binary cross entropy as the loss function of the mode; the formula is as follows: 2 [12pt]{minimal}
$$H_{p} ( q ) = - _{i = 1}^{N} {y_{i} ( {p( {y_{i} } )} ) + ( {1 - y_{i} } ) ( {1 - p( {y_{i} } )} )}$$ H p q = - 1 N ∑ i = 1 N y i · log p y i + 1 - y i · log 1 - p y i in which y i represents the label for each image, [12pt]{minimal}
$$({}_{})$$ p ( y i ) represents the probability of the image being positive and [12pt]{minimal}
$$$$ q represents the real distribution. In addition, to prevent overfitting problems, we applied a dropout layer and set the dropout rate to 0.2 (randomly ignoring 20% of the neurons). Finally, a sigmoid layer was applied to the model before the output layer to normalize the outputs. The sigmoid function is defined as follows: 3 [12pt]{minimal}
$$S({x}_{i})=^{-{x}_{i}}}.$$ S x i = 1 1 + e - x i . The batch size was set to 16 and achieved the best performance among other sizes. By training the neural network model, the probability statistics of patients' radiotherapy sensitivity were finally obtained (a probability of more than 0.5 is sensitive, and a probability of less than 0.5 is resistant). The detailed structure of the neural network models is illustrated in Fig. a. Diverse research on pretrained neural networks has shown state-of-the-art performance in image classification tasks , . Meanwhile, the channel attention mechanism has been proven to be efficient in improving the performance of deep learning models. In this study, a pretrained ResNet50 (trained on ImageNet from Keras) with a channel attention layer was applied – . The channel attention layer was applied to capture the most critical channels from the model's output; the formula of the function is as follows: 4 [12pt]{minimal}
$${M}_{c}(F)= (MLP(AvgPool(F)))+MLP(MaxPool(F)),$$ M c F = σ M L P A v g P o o l F + M L P M a x P o o l F , in which [12pt]{minimal}
$$$$ MLP represents the multilayer perceptron and [12pt]{minimal}
$$$$ F represents the inputs. The learning rate was set to 0.01, and the Adam optimizer was used. The loss function in these experiments was also binary cross entropy. Meanwhile, the dropout rate of the dropout layer was set to 0.5. After multiple experiments, the batch size was set to 32 and achieved the best performance. The pretrained model outperformed all the other methods we applied in this study. The optimal classifier with the best AUC was used for further exploration. The detailed structure of the neural network models is illustrated in Fig. b. When evaluating the predictive performance of each models, we use the area under the ROC curve (AUC value) to measure the accuracy of each predictive models. However, the area under the ROC curve can not take into account the clinical practicability of the prediction model. Therefore, we use the decision curve to further evaluate each models. The decision curve integrates the preferences of decision makers into the analysis, and can actually evaluate the benefit in clinical practice after using this method. Thus, it meets the actual needs of clinical decision-making and is increasingly widely used in clinical analysis Statistical analysis We used SPSS statistical software version 18 to calculate the significant differences by the X2 test or Fisher’s exact test for categorical variables. A 2-tailed P < 0.05 was considered statistically significant. The association analysis of univariate logistic was also calculated by SPSS statistical software version 18. The calibration curve and decision curve were performed to test the calibration performance and clinical utility . Python software version 3.8 (Python) was used for graphic depiction.
Baseline information and imaging data, including demographic data, clinical data, pathological findings of biopsies before treatment, pre- and postsurgical imaging data, and surgical records, of patients with ESCC who underwent radiotherapy at Institution 1 (The First Affiliated Hospital of Xi'an Jiaotong University) from 2013 to 2019 were collected and analyzed. Moreover, we also retrieved and collected the same related data from patients with ESCC who received radiotherapy at Institution 2 (The Second Affiliated Hospital of Xi'an Jiaotong University) from 2017 to 2019. All patients underwent CT examination at the time of positioning before the beginning of their radiotherapy, and the CT data were collected retrospectively from 2021 to 2022. The main inclusion criteria were as follows: (1) biopsy-diagnosed ESCC; (2) clinically diagnosed advanced ESCC by CT and contrast imaging; (3) underwent complete radical radiotherapy (and did not drop out during the treatment); and (4) pre- and postradiotherapy imaging data were recorded after the same institution. The main exclusion criteria were as follows: (1) no biopsy or pathological confirmation; (2) no surgery or more than two weeks of adjuvant chemotherapy before radiotherapy; and (3) no reexamination of imaging data after radiotherapy. The general classifications of all patients were based on the Chinese CSCO guidelines . We used the standard of clinical staging for nonoperative esophageal cancer (Draft) for tumor grading . Written informed consent was obtained from all patients in this study. All methods undertaken in this work were carried out in accordance with the relevant guidelines and regulations. All patients' clinical information was consecutively enrolled, and the Ethics Committee of Xi'an Jiaotong University approved this study. Twenty percent of the patients from Institution 1 were randomly selected for the internal validation cohort, and the rest of the patients from Institution 1 were grouped into the training cohort. The patients from Institution 2 were chosen as the external validation cohort. The detailed experimental flow is illustrated in Fig. .
All patients in our research accepted the localizing by Brilliance CT locator (Philps, UK) before radiotherapy. The main operating parameters: tube voltage: 140 kV; tube electric current: 500 mA; beam spacing: 0.625 layer thickness; rotation time: 0.5 s; matrix: 512 × 512; detector size: 24 mm. The scanning parameters: layer thickness ≤ 5 mm; interval ≤ 5 mm. Before the scanning of CT, two radiotherapy technologists and doctors were required to accompany with the patients. All patients were required to fast for 4 to 6 h before CT scanning and drink 0.5 L of water during the scanning to make the esophagus dilated as much as possible. All patients were asked to hold their breath during using the multi-layer spiral CT machine to continuously scan the chest. The scanning range started from the upper edge of the supraclavicular fossa 5 cm above, down to the level of the 1st lumbar vertebra. Then the data of CT scanning was passed to the workstation for reconstruction. All patients in this study received radical radiotherapy, with a dose of radiation ranging from 60 to 66 Gy at an energy of 6 MV, 1.8–2.0 Gy/fraction, 5 times per week. Organs at risk (OAR), including the bilateral lungs, spinal cord, gastric duct and heart, were outlined for protection. The maximum tolerated doses for key normal structures were as follows: spinal cord: < 40 Gy; heart V40 ≤ 30%; bilateral lungs: V20 ≤ 28% and V30 ≤ 20%; and stomach: V40 ≤ 40%. However, because the data were retrieved retrospectively, the actual dose of radiotherapy for each patient was slightly different. Despite this, each radiotherapy plan fell within the scope of the radical dose recommended by the NCCN guidelines . Imaging specialists assessed patients' imaging data to measure the tumor's maximum diameter in each plane recommended by RECIST 1.1 guidelines . Then, the maximum diameter shrinkage rate of the tumor could be calculated (preradiotherapy maximum diameter/postradiotherapy maximum diameter), which is popularly used in clinical work to evaluate the treatment response. According to the maximum tumor diameter shrinkage rate for all patients, we selected 0.5 as the potential threshold to divide patients into two categories. After experiments, we chose the optimal cutoff value, which was 0.5 as the threshold to divide patients (tumor reduction rate greater than 50% and tumor reduction rate less than 50%).
The continuous planes of CT images in all patients were outlined by two radiologists with more than eight years of experience in radiotherapy target area delineation using Monaco 5.2. Furthermore, another radiologist with more than 15 years of experience checked the target area and obtained the final radiotherapy target area, which guaranteed that the whole tumor of each patient was reflected by the target area from continuous planes of CT images. In this trial, we used the outlined target area images for the construction of a radiotherapy response model. To minimize the influence of the tumor margin on the model, for each patient, we chose the images that best described the tumor site.
The patient CT and target delineation data collected in this experiment are stored in DICOM file format, and the CT data in the DICOM files are converted into 2D PNG format images through Python. In this way, we can extract 10 to 60 slices of 2D images outlining the tumor target area from each patient. Afterwards, professional radiologists selects the most representative 2D CT images of the tumor (usually images around the maximum diameter of the tumor). The dataset collected from institution 1 was randomly divided into training set validation sets on a patient basis. The data collected from Institution 2 is distributed to the external validation group. In machine learning experiments, 2D CT images are transformed into feature vectors through feature extraction and subsequent experiments are conducted. In artificial neural networks, we applied data augmentation methods such as rotation, flipping, zooming, and distortion to the CT images. Through data augmentation, the dataset of CT images increased to 6000. And the 2D CT images were the direct input to the artificial neural network models.
In this study, patients from institution one (training cohort and internal cohort) were used to construct and verify the classification model. Radiomic features of the CT images were extracted using PyRadiomics image extraction software version 3.0. A total of 102 2D features were extracted from each patient, including first-order, shape 2D, gray level cooccurrence matrix (GLCM), gray level run length matrix (GLRLM), gray level size zone matrix (GLSZM), neighboring gray-tone difference matrix (NGTDM), and gray level dependence matrix (GLDM) features . Moreover, we grouped the correlated features (> 0.8) by the Pearson correlation coefficient algorithm, and the less predictive features in the same group were ignored in the feature selection algorithm (Supplementary Fig. ). The random forest algorithm was used to decrease the data dimensions and select the most predictive features . The formula and explanation of the feature selection algorithm are as follows: 1 [12pt]{minimal}
$${}( {} ) = {0.7ex} = 1}}^{{}} {( {{}_{{{}}} - {}_{{{}}} } )} }$} \!} = 1}}^{{}} {( {{}_{{{}}} - {}_{{{}}} } )} } N}}.-0pt} \!0.7ex}$$ Importance X = ∑ i = 1 N err OOB2 - err OOB1 / N in which X represents the feature, [12pt]{minimal}
$${}_{2}$$ err OOB 2 represents the out-of-bag error when we add noise to feature X and [12pt]{minimal}
$${}_{1}$$ err OOB 1 represents the out-of-bag error without adding noise. Ten sets of experiments with the number of features ranging from one to ten were applied to determine the optimal number of features for the model. Hence, the five most predictive features were selected to train the classifier and achieved the best performance. Meanwhile, multiple classifiers were tested in this study, including support vector machine (SVM) with linear kernel, SVM with radial basis kernel linear regression model , , linear regression model and random forest. After comparing the AUCs of the classifiers, we selected the random forest algorithm because it outperformed all other classifiers.
In this study, patients from institution one were used to build the training cohort and internal validation cohort, and patients from institution two were used to build the external validation cohort to verify the efficiency of generalization. The CNN model has been proven to be very effective in the field of image classification – . End-to-end CNN models provide precise prediction results without additional image feature extractions, which significantly improves the model's efficiency. In this study, multiple neural networks were used to classify the CT images of the patients. The model's learning rate was set to 0.0005, and the root mean square prop (RMSprop) optimizer was used. Meanwhile, we used binary cross entropy as the loss function of the mode; the formula is as follows: 2 [12pt]{minimal}
$$H_{p} ( q ) = - _{i = 1}^{N} {y_{i} ( {p( {y_{i} } )} ) + ( {1 - y_{i} } ) ( {1 - p( {y_{i} } )} )}$$ H p q = - 1 N ∑ i = 1 N y i · log p y i + 1 - y i · log 1 - p y i in which y i represents the label for each image, [12pt]{minimal}
$$({}_{})$$ p ( y i ) represents the probability of the image being positive and [12pt]{minimal}
$$$$ q represents the real distribution. In addition, to prevent overfitting problems, we applied a dropout layer and set the dropout rate to 0.2 (randomly ignoring 20% of the neurons). Finally, a sigmoid layer was applied to the model before the output layer to normalize the outputs. The sigmoid function is defined as follows: 3 [12pt]{minimal}
$$S({x}_{i})=^{-{x}_{i}}}.$$ S x i = 1 1 + e - x i . The batch size was set to 16 and achieved the best performance among other sizes. By training the neural network model, the probability statistics of patients' radiotherapy sensitivity were finally obtained (a probability of more than 0.5 is sensitive, and a probability of less than 0.5 is resistant). The detailed structure of the neural network models is illustrated in Fig. a. Diverse research on pretrained neural networks has shown state-of-the-art performance in image classification tasks , . Meanwhile, the channel attention mechanism has been proven to be efficient in improving the performance of deep learning models. In this study, a pretrained ResNet50 (trained on ImageNet from Keras) with a channel attention layer was applied – . The channel attention layer was applied to capture the most critical channels from the model's output; the formula of the function is as follows: 4 [12pt]{minimal}
$${M}_{c}(F)= (MLP(AvgPool(F)))+MLP(MaxPool(F)),$$ M c F = σ M L P A v g P o o l F + M L P M a x P o o l F , in which [12pt]{minimal}
$$$$ MLP represents the multilayer perceptron and [12pt]{minimal}
$$$$ F represents the inputs. The learning rate was set to 0.01, and the Adam optimizer was used. The loss function in these experiments was also binary cross entropy. Meanwhile, the dropout rate of the dropout layer was set to 0.5. After multiple experiments, the batch size was set to 32 and achieved the best performance. The pretrained model outperformed all the other methods we applied in this study. The optimal classifier with the best AUC was used for further exploration. The detailed structure of the neural network models is illustrated in Fig. b. When evaluating the predictive performance of each models, we use the area under the ROC curve (AUC value) to measure the accuracy of each predictive models. However, the area under the ROC curve can not take into account the clinical practicability of the prediction model. Therefore, we use the decision curve to further evaluate each models. The decision curve integrates the preferences of decision makers into the analysis, and can actually evaluate the benefit in clinical practice after using this method. Thus, it meets the actual needs of clinical decision-making and is increasingly widely used in clinical analysis
We used SPSS statistical software version 18 to calculate the significant differences by the X2 test or Fisher’s exact test for categorical variables. A 2-tailed P < 0.05 was considered statistically significant. The association analysis of univariate logistic was also calculated by SPSS statistical software version 18. The calibration curve and decision curve were performed to test the calibration performance and clinical utility . Python software version 3.8 (Python) was used for graphic depiction.
The baseline clinical characteristics of patients and association analysis with radiotherapy Our study enrolled 248 ESCC patients, including 154 (62.1%) patients in the training cohort, 45 (18.1%) in the internal validation cohort and 49 (19.8%) in the external validation cohort. The clinical characteristics of patients in the three cohorts are summarized in Table . The mean (SD) ages of the training, internal validation, and external validation cohorts were 69.36. The entire cohort comprised 245 patients (98.8%) who were diagnosed with clinical stage III ESCC at the time of treatment. The training cohort comprised 62 responders and 92 nonresponders, the internal validation cohort comprised 14 responders and 31 nonresponders, and the external validation cohort comprised 18 responders and 31 nonresponders. There were no statistically significant differences in the sex ratio, clinical stage, tumor location, general type, or tobacco use during radiotherapy between responders and nonresponders in the training, internal validation, and external validation cohorts (Table ). Analyzing the patients' characteristics by univariate logistic regression showed no associations with radiotherapy response, including sex, age, maximum diameter of tumor before radiotherapy, clinical tumor stage, clinical node stage, clinical metastatic stage, clinical stage, general type, tumor location, alcohol use, and tobacco use in each cohort ( P > 0.05 ) (Supplementary Table ). Machine learning radiomics models using random forest for predicting response to radiotherapy Four machine learning classifiers were used to construct radiomics models, including linear regression, SVM with linear kernel, SVM with radial basis kernel, and random forest models. The ten most predictive features identified by the random forest algorithm were selected to train the classifier. Compared with the performance outcomes of each classifier, the random forest model showed the highest AUCs in the training and internal validation cohorts, which were 0.767 (95% CI, 0.734–0.790) and 0.594 (95% CI, 0.562–0.631), respectively. The SVM with a linear kernel achieved an AUC of 0.561 (95% CI, 0.530–0.594) in the internal validation cohort, while the SVM with a radial basis kernel model achieved an AUC of 0.539 (95% CI, 0.510–0.564) in the internal cohort. The AUC of the linear regression model in the internal validation cohort was 0.589 (95% CI, 0.561–0.646) (Supplementary Fig. a–c). Then, to compare the performance of combined features model with independent feature model, we used the five most predictive features of combined model to train the model individually by random forest. The performance of each independent feature in the radiomics model showed a lower AUC than that of the combined features in the same radiomics model (Fig. a and b, Supplementary Table ). A deep learning radiomics model using pretrained ResNet50 and a channel attention mechanism for predicting response to radiotherapy The deep learning radiomics model constructed of pretrained ResNet50 and the channel attention mechanism outperformed all other methods in predicting radiotherapy response. The model achieved AUCs of 0 0.876 (95% CI 0.853–0.895), 0.802 (95% CI 0.775–0.837), and 0.732 (95% CI 0.672–0.797) in primary, internal and external cohorts, while the model trained by a CNN from scratch achieved AUCs of 0.805(95% CI 0.774–0.830), 0.770(95% CI 0.729–0.802), and 0.678(95% CI 0.619–0.738), respectively (Fig. a–c). This result indicated that the features of CT were feasible for constructing a reliable prognostic radiomics model. In comparing the performance of the machine learning radiomics models, the CNN radiomics models showed higher AUCs in all three cohorts, which revealed that the deep learning radiomics models, without decreasing data dimensions and removing redundant features, improved the performance of the radiomics model. The process of dimensionality reduction of radiomics features may lead to a lack of perspective information. To estimate the predictive stability of the radiomics models and evaluate the benefits in clinical applications, decision curves and calibration curve were used to evaluate the performance of two neural network models. The decision curves showed that with the consideration of preferences of decision, using Att. Pretrained model will obtain more benefits than using CNN model in clinical practice (Fig. d, e). A calibration curve was applied to the pretrained ResNet50 model in the primary and internal cohorts (Fig. f), which showed a high degree of agreement between the predicted and actual results of the model.
Our study enrolled 248 ESCC patients, including 154 (62.1%) patients in the training cohort, 45 (18.1%) in the internal validation cohort and 49 (19.8%) in the external validation cohort. The clinical characteristics of patients in the three cohorts are summarized in Table . The mean (SD) ages of the training, internal validation, and external validation cohorts were 69.36. The entire cohort comprised 245 patients (98.8%) who were diagnosed with clinical stage III ESCC at the time of treatment. The training cohort comprised 62 responders and 92 nonresponders, the internal validation cohort comprised 14 responders and 31 nonresponders, and the external validation cohort comprised 18 responders and 31 nonresponders. There were no statistically significant differences in the sex ratio, clinical stage, tumor location, general type, or tobacco use during radiotherapy between responders and nonresponders in the training, internal validation, and external validation cohorts (Table ). Analyzing the patients' characteristics by univariate logistic regression showed no associations with radiotherapy response, including sex, age, maximum diameter of tumor before radiotherapy, clinical tumor stage, clinical node stage, clinical metastatic stage, clinical stage, general type, tumor location, alcohol use, and tobacco use in each cohort ( P > 0.05 ) (Supplementary Table ).
Four machine learning classifiers were used to construct radiomics models, including linear regression, SVM with linear kernel, SVM with radial basis kernel, and random forest models. The ten most predictive features identified by the random forest algorithm were selected to train the classifier. Compared with the performance outcomes of each classifier, the random forest model showed the highest AUCs in the training and internal validation cohorts, which were 0.767 (95% CI, 0.734–0.790) and 0.594 (95% CI, 0.562–0.631), respectively. The SVM with a linear kernel achieved an AUC of 0.561 (95% CI, 0.530–0.594) in the internal validation cohort, while the SVM with a radial basis kernel model achieved an AUC of 0.539 (95% CI, 0.510–0.564) in the internal cohort. The AUC of the linear regression model in the internal validation cohort was 0.589 (95% CI, 0.561–0.646) (Supplementary Fig. a–c). Then, to compare the performance of combined features model with independent feature model, we used the five most predictive features of combined model to train the model individually by random forest. The performance of each independent feature in the radiomics model showed a lower AUC than that of the combined features in the same radiomics model (Fig. a and b, Supplementary Table ).
The deep learning radiomics model constructed of pretrained ResNet50 and the channel attention mechanism outperformed all other methods in predicting radiotherapy response. The model achieved AUCs of 0 0.876 (95% CI 0.853–0.895), 0.802 (95% CI 0.775–0.837), and 0.732 (95% CI 0.672–0.797) in primary, internal and external cohorts, while the model trained by a CNN from scratch achieved AUCs of 0.805(95% CI 0.774–0.830), 0.770(95% CI 0.729–0.802), and 0.678(95% CI 0.619–0.738), respectively (Fig. a–c). This result indicated that the features of CT were feasible for constructing a reliable prognostic radiomics model. In comparing the performance of the machine learning radiomics models, the CNN radiomics models showed higher AUCs in all three cohorts, which revealed that the deep learning radiomics models, without decreasing data dimensions and removing redundant features, improved the performance of the radiomics model. The process of dimensionality reduction of radiomics features may lead to a lack of perspective information. To estimate the predictive stability of the radiomics models and evaluate the benefits in clinical applications, decision curves and calibration curve were used to evaluate the performance of two neural network models. The decision curves showed that with the consideration of preferences of decision, using Att. Pretrained model will obtain more benefits than using CNN model in clinical practice (Fig. d, e). A calibration curve was applied to the pretrained ResNet50 model in the primary and internal cohorts (Fig. f), which showed a high degree of agreement between the predicted and actual results of the model.
Radiotherapy is considered one of the most crucial treatments for ESCC patients . The construction of models for predicting the response to radiotherapy is significantly instructive for individualized precision treatment . What makes radiation response prediction difficult in ESCC is the lack of predictive molecular markers of radiation sensitivity . Moreover, the traditional clinical characteristics and general type of ESCC showed limited correlation with the response to radiotherapy. Thus, an available model that can efficiently predict the response to radiotherapy in patients with ESCC needs to be developed. Recently, radiomics signature models based on AI have been applied to different areas and have shown incredible performance in predicting the response to radiotherapy – . Here, we aimed to design a pretreatment CT-based radiomics model for radiotherapy response prediction in patients with ESCC, which can cover the shortage of predictive molecular markers. Machining learning and deep learning models have been widely used in radiomics research – . In a previous study, traditional machining learning algorithms, such as random forest and SVM, were mentioned more frequently than deep learning algorithms due to the limitation of the population of cohorts , . Moreover, end-to-end algorithms in deep learning have begun to be used in cancer research in recent years, not only using radiographic images but also using histopathological images , . Screening the reproducibility of features, which is considered an indispensable part of reducing the overfitting of traditional machine learning radiomic models, seems to improve the performance of the radiomics model. In contrast, end-to-end algorithms do not need to reduce the dimensionality and aim to make full use of all image information to draw conclusions . However, the superiority of these two types of algorithms has not been compared to predict radiotherapy response. To determine which of these two algorithms can construct a more effective radiomics model, in our study, both algorithms were used to construct radiomics models in the same ESCC patient cohorts. The results implied that whether in the training, internal validation or external validation cohorts, the radiomics model constructed by the end-to-end deep learning algorithm showed better performance. This suggests that end-to-end deep learning algorithms should receive more attention in subsequent radiomics studies. Moreover, the CNN model from scratch and the CNN pretrained model were also compared in our studies. Although the use of pretrained neural network models has become an increasingly mainstream choice in recent research, pretrained and models from scratch have rarely been compared in radiomics studies . Our studies showed some evidence regarding pretrained models. Meanwhile, recent studies reported that the channel attention mechanism could significantly improve the performance of neural network models. In our study, the channel attention layer significantly improved the convergence difficulty caused by too many channels in the pretrained model and improved the performance of the model by nearly 3% in the external validation cohort. Recently, advances in radiogenomics have shown that radiomics signatures have distinct correlations with gene expression patterns . The radiomics signatures driven by different pathways involved in immune regulation, tumor proliferation, treatment responses and cellular functions further explain the biological basis of radiomics . This result suggests that we can reflect intratumoral heterogeneity, to a certain extent, by constructing a radiomics model. Compared with traditional clinical characteristics, radiomics features better predict the treatment response to radiotherapy. Therefore, we hypothesize that end-to-end algorithms' overall utilization of CT image information may reflect tumor heterogeneity. The difference in extracting image information may cause an apparent discrepancy between these two different algorithms. However, these theories have not been elucidated by radiogenomics and multiomics studies. Our retrospective study was limited to temporal discontinuities in the included patients. Although our study minimized differences in imaging data by using a standardizing process, the differences in CT equipment between each period and institution may lead to bias in collecting imaging data. To our knowledge, this study is the first multicenter study of radiomics in nonsurgical ESCC patients. However, the two institutions of our study are located in the same province, and the number of patients is limited. A large patient population of other regions is still needed to evaluate the extrapolation of the model. Finally, our study only analyzed the 2D radiomics phenotype and clinical characteristics due to the limitation of cohorts, and the 3D radiomics phenotype did not show good performance in our model. Therefore, the 3D radiomics phenomenon still needs to be explored in the next step. It is also necessary to combine other omics to further reveal the biological significance of radiomics. This is the first multicenter radiomics study to develop an Att. Resnet50 pretrained network radiomics model in patients with advanced ESCC. It enables clinical decision-making, relying not only on the clinical doctors' experience but also on an objective basis. The effective prediction of radiotherapy provides these patients with reasonable individualized and precise treatment options, as well as timely alternative curative-intent treatment approaches to prevent any unnecessary side effects of radiotherapy and improve the quality of life and survival outcomes of advanced ESCC patients. In addition, our study uses existing routine diagnostic CT imaging, which does not add additional financial burden to patients. At the same time, the Att. Resnet50 pretrained network radiomics model does not require standardized extraction of radiomics signatures, which can be more convenient in clinical use for oncologists to predict the radiotherapy response during diagnosis.
Developing an Att. Resnet50 pretrained network radiomics model for predicting the response to radiotherapy in patients with advanced ESCC can not only help oncologists formulate effective individualized radiotherapy plans promptly and guide clinical decision-making but also complement the lack of molecular markers for predicting radiosensitivity. It is hoped that our study can be included in the radiomics database of ESCC and be considered a baseline study of radiomics in advanced ESCC. The model has the potential to apply to other medical image classification tasks.
Supplementary Information.
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Diagnostic Value of the International Society of Cardio‐Oncology Definition for Suspected Immune Checkpoint Inhibitor–Associated Myocarditis
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313f7d48-8879-4ceb-b07f-b5100a906255
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10227269
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Internal Medicine[mh]
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This work was supported by the Fondation Coeur et Recheche, Fédération Française de Cardiologie and Assistance Publique–Hôpitaux de Marseille.
F.T. has received support for attending meetings from Astra‐Zeneca and Boerhinger‐Ingelheim. C.D. has received grants and consulting fees from Bioserenity. J.A. has received grants and consulting fees from Bioserenity, Bayer, and Biotronik; consulting fees from Bayer, Novartis, Bristol‐Myers Squibb, Astra‐Zenzca, Servier, Boerhinger‐Ingelheim, Biotronik, and Bioserenity; honoraria for lectures from Bayer, Novartis, and Astra‐Zeneca; and support for attending meetings from Bayer and Bristol‐Myers Squibb. J.C. has received honoraria for lectures from Janssen, Novartis, Pfizer, and Bayer; consulting fees from Boerhinger‐Ingelheim and Janssen; and support for attending meetings from Bayer and Pfizer. The remaining authors have no disclosures to report.
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Comprehensive use of cardiopulmonary exercise testing in pediatrics
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fed26e63-caff-415b-8bca-d54cb127c365
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10227478
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Pediatrics[mh]
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The cardiopulmonary exercise test (CPET) allows the possibility to appoint the pathophysiological limitations of exercise and also the significance of functional impairment. It began to be use as a “gold standard” to evaluate the result of surgical, medical and rehabilitative treatment on cardiopulmonary function and to investigate the integrated physiological reactions to exercise in paediatric medicine. It is widely use in paediatric patients and adults and significantly improved the understanding of cardiopulmonary development in children and adolescents . A large number of research tools are used in clinical practice to assess physical fitness. These tools have a lot of advantages and disadvantages . Cardiopulmonary exercise test has become an chief clinical non-invasive tool to assess and predict the capacity of exercise in patients with heart failure and in different cardiac conditions. It supplies estimation of the exercise responses, affecting the cardiovascular, pulmonary, skeletal muscle, metabolism and the cellular system, which are not well reflected in individual organ systems by measuring function . Cardiopulmonary exercise test carry physiological parameters at rest and during progressive exercise. It determines the ability to produce energy at metabolically relevant time points as anaerobic threshold and the body’s cardiorespiratory fitness . Resting pulmonary and cardiac function cannot reliably estimate physical performance and functional capacity . Energetic human capacity is the most significant factor that sets the limits of physical capacity . The cardiopulmonary exercise test allows to assess body’s response during sub and maximal exercise. Mainly measurements include gas exchange parameters such as: oxygen consumption, carbon dioxide production, minute ventilation, ECG monitoring, blood pressure and pulse oximetry . In the latest years of CPET exploitation, the test has been appreciably identified by medical interest and as a physiological bases of different variables, which were before unknown and by accentuation proof for a multivariable approach. Most of problems with ventilation and its control were taken into consideration . An obstacle in performing the CPET test was mostly described as the financial barrier. Hospitals and institutions trying to initiate the action mentioned lack of funding as the most common reason for not being able to test .
We performed a literature search at Google Scholar, PubMed, Science Direct, available literature from the book from 2006 to 2019 and internet sources. The bibliography search was reviewed and performed using selected keywords. This study is based on analysis of literature about cardiopulmonary exercise test and cardiorespiratory fitness.
Physical effort requires coordinated actions of physiological mechanisms related to the functioning of the nervous system, circulatory system, respiratory system and internal function to cover the escalated energy demand of working muscles . Features which condition physical performance are: efficiency of aerobic muscle supply and activation of biochemical processes determining the use of oxygen energy sources, removal of catabolite, efficiency of thermoregulation and size and efficiency of energy substrates use. Considering on the subject about efficiency, we can not forget about the tolerance of fatigue changes during CPET test, which it can affect: aversion to effort or fear of effort. It can also occur pain, dyspnoea, palpitation, or excessive sweating . In the study of children and adolescents, we must also consider the race of subjects. Studies conducted on Caucasian race in the United Kingdom have shown that, English children have higher cardiovascular fitness than Indian children . Social, religious, linguistic and cultural traditions exclude involvement in physical activity, therefore their ability and approach to sport or recreation may be different from other children . The surveys on male and female have shown that in swimmers of age from 9 to 20 years from West Bengal, had importantly lower than norm value of VO 2max , than international athletes, which practiced endurance type of sport, however they had significantly higher VO 2max parameter than sedentary girls of West Bengal . Cardiorespiratory fitness, is a solid parameter to estimate the capacity of the cardiovascular system to overcome extended physical work. It has been depicted to be the most dominant predictor of death rate and morbidity, besides of classical cardiovascular disease, risk factors such as cholesterol, smoking cigarettes, hypertension, and diabetes mellitus type 1 (T1DM) and diabetes mellitus type 2 (T2DM) . In recent years, studies have documented the health benefits of regular physical activity. Nowadays it is highly appreciated that higher cardiorespiratory fitness and physical activity standards are beneficial for the diseases prophylaxis and prevention . Physical activity is essential for human health in every period of life, and it gains special value during the time of the fastest and most intense development, i.e. childhood . It has a positive effect on dealing with civilization diseases such as diabetes type 2 (T2DM), improves bone health, reduces the incidence of cancer, reduces signs of disability and extends life . Cardiopulmonary exercise test variables The aim of spirometry is the continuous survey of respiration (spirography) and respiratory gas metabolism . The tests are performed on a treadmill, cycle-ergometer or outdoor. Portable Ergospirometers are very often used to study physiological ventilation variables in field tests . There are 2 basic types of ergospirometers. The first one is an ergospirometer with a mixing chamber. The principle of its operation, is that during breathing, samples of exhaled gas are taken and collected in a reservoir (chamber), where they are mixed. The size of a single sample is proportional to the current tidal volume (VT). In every constant period of time, measurements are made of the gas composition in the chamber, which is a mixture of taken samples. Measurements of average O 2 and CO 2 concentrations are obtained, e.g. another set of measurement data every 10 seconds. The action of the second one is based on continuous sampling of breathing air with a constant gas sample volume. In this method, measurements of O 2 and CO 2 concentrations require the use of fast gas analysers, usually with a response time of less than 120 ms. In addition, synchronization of the flow, O 2 and CO 2 concentrations is required due to delays in the sample drain and in the gas sensor itself. Measurements of temporary O 2 and CO 2 concentrations, are obtained after each breath. The advantage of the “breath-by-breath” type ergospirometers compared to devices with a mixing chamber is the high accuracy of the measurement regardless of changing environmental conditions, because the concentration of O 2 and CO 2 is measured both during the inspiration and exhalation phases . The major results are schedule in the following order: maximal oxygen uptake (VO2max/peak), carbon dioxide emission (VCO 2 ), ventilatory threshold (VT), minute ventilation (VE), ventilatory equivalents for oxygen (VE/VO 2 ) and carbon dioxide (VE/VCO 2 ), respiratory exchange ratio (RER/R, VCO 2 /VO 2 ), heart rate (HR), saturation (SatO 2 ), ECG, blood pressure (BP) The most important parameter examined in the assessment of physical fitness is VO 2max , which we describe as the maximum integrated capacity of the pulmonary system, cardiovascular system and muscular system to uptake, transport and utilize O 2 . Through the value of the oxygen uptake kinetic reaction its survey is complex by the large “inter-breath” change in oxygen uptake in children during the test. It cuts the reliance in which kinetic variables can be asses and necessitates the measurement of variety identic transitions . VO2peak is highest speed attained at the end of the test . Ventilatory threshold is described as a the level at which, sudden growth in blood lactate is noticed. Output of lactic acid in the muscle rise curvilinear with increasing work load . We also pay attention to the carbon dioxide that it is the sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The amount of carbo-dioxide exhaled in oxidative metabolism for each litre of oxygen consumed is named (RER/R) the respiratory exchange ratio . This parameter nearing to 0.7 if the dominant fuel is fat to 1.0 if the prevalent fuel is carbohydrate. During dynamic exercise, the heart rate (HR) increases in order to respond to higher oxygen demand. It is accompanied by an increase in the stroke volume of the heart, which reaches its maximum value already at 30–50% VO 2max . Enhanced work of the heart causes an augmentation in blood flow mainly in working skeletal muscles, heart and skin at the expense of a decrease in flow through the kidneys, liver and visceral organs. During physical effort, the body increases its oxygen demand, so the lung ventilation process potentiates. After beginning of training, there is an increase in VE (minute ventilation), the breathing cycle speeds up and gets deeper. The rapid increase in VE lasts a few seconds after initiation of activity, then this trend slows down until it reaches a level of stabilization. The transition phase occurs when you stop exercising operations. In the case of intense effort, the VE value enhance constantly, the steady state phase does not occur. During low intensity exercise, VE increases proportionally to VO 2 until it reaches 50–75% VO 2max . Parameters related to cardiopulmonary exercise test were divided into this, which characterize circulatory system, lung ventilation, metabolic changes and those which are enters into gas exchange in the lungs . Contraindications and savouireship Each patient should receive instructions and basic information on how the laboratory equipment works and what the test procedure consists of. The patient should avoid eating meals, smoking cigarettes and drinking alcohol at least 2 hours before the test. Patient should wear comfortable clothing and footwear. It should also be also follow the history of medications and perform resting supine ECG to identify individual for whom the test could be contraindicated or should be performed with special safety features . The basis that we can modify is the protocol with increasing linear load. It is able to choose Ramp or stepwise protocol. During the measurement process, the child should achieve a constant speed of 60 to 80 rpm. The load is gradually increased, depending on the chosen linear protocol. It is set to 1 W/1 kg of body weight as the basic load and increase the resistance every 10 seconds by 1 W. The load is heightened by increasing the resistance of the cycle-ergometer pedals. After reaching the desired parameters or when indicators to stop the examination appear, the doctor or paramedic decides to finish the survey. The test can also be interrupted at any time at the patient’s request or when disturbing symptoms appear. After the effort, a rest phase follows, then the patient is disconnected from the device and the electrodes are peeled off and discarded. The duration of the test lasts from 30 to 60 minutes . We increase the effort load to: Obtain the maximum rhythm frequency (220-age), occurrence of symptoms indicating need to end the test (maximum stress test limited by symptoms), achieving 85% of the maximum frequency rhythm (submaximal exercise test) . Absolute contraindications and exclusion criteria for children and adolescents are described in detail by American Heart Association (AHA). We can include among them: disagreement of person being examined/guardian, severe respiratory failure, congestive heart failure, active rheumatic fever with carditis, significant aortic stenosis, significant mitral valve stenosis, uncontrolled cardiac arrhythmias causing clinical symptoms or disadvantaging hemodynamics, severe arterial hypertension (systolic pressure & gt: 200 mm Hg and/or diastolic pressure & gt: 120 mm Hg), hypertrophic cardiomyopathy with former cases of collapse, diabetic children hypoglycaemia, hypoglycaemia above 250 mg/dl, severe disorders of other organs which may impact on the course of the effort or increase under their influence (e.g. infection, kidney failure, thyrotoxicosis), lower extremity phlebitis, physical disability which may prevent to perform safe and adequate test, mental disability preventing cooperation . However, some children, adolescents and adults noticed discomfort with the mouthpiece, facemask, or with nose clip. Consequently, all these inconvenience, should be reported before starting the CPET test. They serve to show the need for versatile initial patient assessment, and precise monitoring during the survey . Cardiopulmonary exercise test should be interpreted and controlled by a consultant with an experience in conducting the cardiopulmonary exercise testing. Furthermore, the individual performing the CPET test should be experienced in working on cardiopulmonary tests like also interpreting the outcomes . However, despite their precision and reproducibility, cardiopulmonary exercise testing physicians (cardiologists, pulmonologists, and physiologists) must be well trained to avoid misinterpretation pitfalls and above all, highly experienced in clinical practice and pathological conditions .
The aim of spirometry is the continuous survey of respiration (spirography) and respiratory gas metabolism . The tests are performed on a treadmill, cycle-ergometer or outdoor. Portable Ergospirometers are very often used to study physiological ventilation variables in field tests . There are 2 basic types of ergospirometers. The first one is an ergospirometer with a mixing chamber. The principle of its operation, is that during breathing, samples of exhaled gas are taken and collected in a reservoir (chamber), where they are mixed. The size of a single sample is proportional to the current tidal volume (VT). In every constant period of time, measurements are made of the gas composition in the chamber, which is a mixture of taken samples. Measurements of average O 2 and CO 2 concentrations are obtained, e.g. another set of measurement data every 10 seconds. The action of the second one is based on continuous sampling of breathing air with a constant gas sample volume. In this method, measurements of O 2 and CO 2 concentrations require the use of fast gas analysers, usually with a response time of less than 120 ms. In addition, synchronization of the flow, O 2 and CO 2 concentrations is required due to delays in the sample drain and in the gas sensor itself. Measurements of temporary O 2 and CO 2 concentrations, are obtained after each breath. The advantage of the “breath-by-breath” type ergospirometers compared to devices with a mixing chamber is the high accuracy of the measurement regardless of changing environmental conditions, because the concentration of O 2 and CO 2 is measured both during the inspiration and exhalation phases . The major results are schedule in the following order: maximal oxygen uptake (VO2max/peak), carbon dioxide emission (VCO 2 ), ventilatory threshold (VT), minute ventilation (VE), ventilatory equivalents for oxygen (VE/VO 2 ) and carbon dioxide (VE/VCO 2 ), respiratory exchange ratio (RER/R, VCO 2 /VO 2 ), heart rate (HR), saturation (SatO 2 ), ECG, blood pressure (BP) The most important parameter examined in the assessment of physical fitness is VO 2max , which we describe as the maximum integrated capacity of the pulmonary system, cardiovascular system and muscular system to uptake, transport and utilize O 2 . Through the value of the oxygen uptake kinetic reaction its survey is complex by the large “inter-breath” change in oxygen uptake in children during the test. It cuts the reliance in which kinetic variables can be asses and necessitates the measurement of variety identic transitions . VO2peak is highest speed attained at the end of the test . Ventilatory threshold is described as a the level at which, sudden growth in blood lactate is noticed. Output of lactic acid in the muscle rise curvilinear with increasing work load . We also pay attention to the carbon dioxide that it is the sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The sum of exhaled CO 2 by a examined patient is an act of the substrate and metabolic rate utilized in oxidative metabolism. The amount of carbo-dioxide exhaled in oxidative metabolism for each litre of oxygen consumed is named (RER/R) the respiratory exchange ratio . This parameter nearing to 0.7 if the dominant fuel is fat to 1.0 if the prevalent fuel is carbohydrate. During dynamic exercise, the heart rate (HR) increases in order to respond to higher oxygen demand. It is accompanied by an increase in the stroke volume of the heart, which reaches its maximum value already at 30–50% VO 2max . Enhanced work of the heart causes an augmentation in blood flow mainly in working skeletal muscles, heart and skin at the expense of a decrease in flow through the kidneys, liver and visceral organs. During physical effort, the body increases its oxygen demand, so the lung ventilation process potentiates. After beginning of training, there is an increase in VE (minute ventilation), the breathing cycle speeds up and gets deeper. The rapid increase in VE lasts a few seconds after initiation of activity, then this trend slows down until it reaches a level of stabilization. The transition phase occurs when you stop exercising operations. In the case of intense effort, the VE value enhance constantly, the steady state phase does not occur. During low intensity exercise, VE increases proportionally to VO 2 until it reaches 50–75% VO 2max . Parameters related to cardiopulmonary exercise test were divided into this, which characterize circulatory system, lung ventilation, metabolic changes and those which are enters into gas exchange in the lungs .
Each patient should receive instructions and basic information on how the laboratory equipment works and what the test procedure consists of. The patient should avoid eating meals, smoking cigarettes and drinking alcohol at least 2 hours before the test. Patient should wear comfortable clothing and footwear. It should also be also follow the history of medications and perform resting supine ECG to identify individual for whom the test could be contraindicated or should be performed with special safety features . The basis that we can modify is the protocol with increasing linear load. It is able to choose Ramp or stepwise protocol. During the measurement process, the child should achieve a constant speed of 60 to 80 rpm. The load is gradually increased, depending on the chosen linear protocol. It is set to 1 W/1 kg of body weight as the basic load and increase the resistance every 10 seconds by 1 W. The load is heightened by increasing the resistance of the cycle-ergometer pedals. After reaching the desired parameters or when indicators to stop the examination appear, the doctor or paramedic decides to finish the survey. The test can also be interrupted at any time at the patient’s request or when disturbing symptoms appear. After the effort, a rest phase follows, then the patient is disconnected from the device and the electrodes are peeled off and discarded. The duration of the test lasts from 30 to 60 minutes . We increase the effort load to: Obtain the maximum rhythm frequency (220-age), occurrence of symptoms indicating need to end the test (maximum stress test limited by symptoms), achieving 85% of the maximum frequency rhythm (submaximal exercise test) . Absolute contraindications and exclusion criteria for children and adolescents are described in detail by American Heart Association (AHA). We can include among them: disagreement of person being examined/guardian, severe respiratory failure, congestive heart failure, active rheumatic fever with carditis, significant aortic stenosis, significant mitral valve stenosis, uncontrolled cardiac arrhythmias causing clinical symptoms or disadvantaging hemodynamics, severe arterial hypertension (systolic pressure & gt: 200 mm Hg and/or diastolic pressure & gt: 120 mm Hg), hypertrophic cardiomyopathy with former cases of collapse, diabetic children hypoglycaemia, hypoglycaemia above 250 mg/dl, severe disorders of other organs which may impact on the course of the effort or increase under their influence (e.g. infection, kidney failure, thyrotoxicosis), lower extremity phlebitis, physical disability which may prevent to perform safe and adequate test, mental disability preventing cooperation . However, some children, adolescents and adults noticed discomfort with the mouthpiece, facemask, or with nose clip. Consequently, all these inconvenience, should be reported before starting the CPET test. They serve to show the need for versatile initial patient assessment, and precise monitoring during the survey . Cardiopulmonary exercise test should be interpreted and controlled by a consultant with an experience in conducting the cardiopulmonary exercise testing. Furthermore, the individual performing the CPET test should be experienced in working on cardiopulmonary tests like also interpreting the outcomes . However, despite their precision and reproducibility, cardiopulmonary exercise testing physicians (cardiologists, pulmonologists, and physiologists) must be well trained to avoid misinterpretation pitfalls and above all, highly experienced in clinical practice and pathological conditions .
Cardiopulmonary exercise test in clinical praxis is very useful and has potential indication for use in assessing the functional capacity of young people with moderate to severe valvular defects to evaluate for possible surgical intervention and to determine whether early fatigue is due to defect or deconditioning . Cardiopulmonary exercise test contains estimation of tolerance and intolerance during exercise, rating of patients with cardiovascular like: (heart failure, transplantation, cardiac rehabilitation, and exercise individualization) and respiratory diseases as: (chronic obstructive pulmonary disease (COPD), cystic fibrosis, interstitial lung diseases, pulmonary vascular disease and exercise-induced bronchospasm) and different clinical applicabilities like exercise rehabilitation, preoperative risk evaluation and exercise prescription to overall health improvement . The cardiopulmonary exercise test with survey of metabolic parameters, such as peak myocardial oxygen consumption and also exercise ventilation, may help in the clinical assessment of hypertrophic cardiomyopathy (HCM) patients in their functional capacity . Measurements of gas exchange are taking place more and more often in sports medicine. . It is a useful tool for assessing limitations during daily activities, that have a physiological basis on individual with chronic organ failure Cardiopulmonary exercise test is one of the most important diagnostic methods used in cardiology and sports medicine. Measurements, including gas exchange parameters during exercise, are characterized by a high prognostic value in patients not only with heart failure, but also with respiratory diseases . It would seems that it is impossible to perform a test on people with mucoviscoidosis. With the right approach and load dosing, Urquhart and Vendrusculo conducted a study on a group of 4 children from the age of 14 to 15. The measurement of performance and efficiency in cooperation with the musculoskeletal system and the cardiovascular system provided by CPET test adds more information to individualize exercise programmes for patients with highest risk suffering on cystic fibrosis . Also in patients with chronic obstructive pulmonary disease (COPD), VO2max/peak is the best indicator of aerobic fitness, as long as patients are able to exercise more than their limits . In studies conducted by Hunt et al . cardiorespiratory fitness on children was measured by FitnessGram assessment protocol. This is a good comparative method to the cardiopulmo-nary exercise test, because of the cost and the possibility of conducting it in the field. FitnesGram is usually use to estimate cardiorespiratory fitness and improve health and physical activity in children and adolescents .
Measurement of expiratory gas exchange during the test, physical activity is a repeatable and objective method, which enables accurate measurement of functional capacity. In this way, it is possible to detect the causes of reduced tolerance of effort, to notice the severity of many diseases, to monitor the effects of treatment and rehabilitation, but also to confirm the complete health and ability to exercise intensively.
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Recent Medicinal Chemistry Studies against Neurodegenerative Diseases
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c38248b0-1af4-457f-83bf-ad8a7cb44957
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10227922
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Pharmacology[mh]
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GPs’ and pharmacists’ views of integrating pharmacists into general practices: a qualitative study
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9c95a373-e1e6-4ae1-b563-4f6003a6259a
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10227995
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Family Medicine[mh]
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Health workforce shortages and the increasing complexity of caring for older people with chronic conditions have increased pressure on primary care services. – Pharmacists were introduced into general practices to alleviate some of the pressures within primary care (known as practice-based pharmacists; PBPs) by delivering a range of activities. , – Five-year PBP pilot schemes have been launched in both England and Northern Ireland (NI) to integrate PBPs into general practice. , Most emerging literature has focused on determining barriers and facilitators to the PBP role in general practice; however, there was little detail in relation to the context of the PBP role. Additional research was recommended to assess healthcare professionals’ (HCPs) experiences of this new role in general practice and the impact on GP workload. Exploring such views on PBPs will enhance the understanding of their impact in primary care and help inform the development of the role. Furthermore, the effectiveness of the primary healthcare team is limited by what HCPs know about each other and each other’s roles. – Little is known about the views of key HCPs, particularly GPs and community pharmacists (CPs), regarding the introduction and integration of the PBP role. A previous cross-sectional study conducted by the authors exploring NI GPs’ views on PBPs highlighted some issues in relation to integration and development of the PBP role that merit further examination. Therefore, this research aimed to contribute to a comprehensive exploration of GPs’, PBPs’, and CPs’ experiences of working with PBPs, and their views on the integration of PBPs into general practice and their impact on primary healthcare delivery in NI.
A qualitative approach to data collection was adopted using semi-structured individual interviews with GPs, PBPs, and CPs across NI. The study was reported according to the Consolidated Criteria for Reporting Qualitative Research (COREQ) checklist. Study population and setting The perspectives of three key HCPs (GPs, PBPs, and CPs) practising throughout NI were sought . As a result of the COVID- 19 pandemic, interviews were conducted individually, via telephone or a virtual meeting platform such as Microsoft Teams. Sampling and recruitment Purposive and snowball sampling were used to obtain GPs’, PBPs’, and CPs’ perspectives, and to enhance recruitment. Previous studies have found that, if a general practice agrees to participate in a study, associated community pharmacies will also agree. , Therefore, sampling was initiated by contacting three GPs (known to two of the authors), to ask them to identify potential GPs and PBPs whom they knew and who might be interested in the study. These GPs asked permission from these potential participants to share their contact details with the researcher (the first author). In turn, during the interviews, a recruited GP and PBP were asked to nominate and suggest the name of a potential CP participant with whom the practice had most contact. The researcher contacted that pharmacist using publicly available contact details. Therefore, triads were recruited, each consisting of a GP, PBP, and CP, from across the five Health and Social Care Trust areas in NI (that is, administrative healthcare areas in NI) with a view to recruiting up to three triads per area and one triad per practice. The researcher invited potential HCP participants by telephone/email. If interested, the researcher provided a formal invitation letter and information sheet. Interviews were conducted with the GP and PBP within the potential triad when both the GP and PBP agreed to participate. If a CP within a triad did not agree to participate, the GP and PBP interviews were retained for analysis and it was noted that a CP could not be recruited. Data collection Interviews were conducted by the researcher between August 2020 and October 2021. Participants provided written informed consent and were offered £50 for participation. Separate topic guides were developed for each HCP group (Supplementary Information S1–S3), based on published literature within the field, and piloted. , , – All interviews were audiorecorded with the permission of the participant. Data management and analysis All interviews were recorded, transcribed verbatim, and checked for accuracy. To ensure anonymity and confidentiality, each participant was assigned a two-digit identification number reflecting the triad with which the participant was associated (for example Triad 1: GP01, PBP01, CP01). Transcripts were imported and managed in NVivo 12 Pro. Data analysis, using thematic analysis, was performed in parallel with data collection by two independent researchers. Data from interviews with each type of HCP were analysed separately to monitor data saturation within each HCP category. Themes were reviewed and refined by the research team. The quality and rigour of research reporting was closely monitored (Supplementary Table S1).
The perspectives of three key HCPs (GPs, PBPs, and CPs) practising throughout NI were sought . As a result of the COVID- 19 pandemic, interviews were conducted individually, via telephone or a virtual meeting platform such as Microsoft Teams.
Purposive and snowball sampling were used to obtain GPs’, PBPs’, and CPs’ perspectives, and to enhance recruitment. Previous studies have found that, if a general practice agrees to participate in a study, associated community pharmacies will also agree. , Therefore, sampling was initiated by contacting three GPs (known to two of the authors), to ask them to identify potential GPs and PBPs whom they knew and who might be interested in the study. These GPs asked permission from these potential participants to share their contact details with the researcher (the first author). In turn, during the interviews, a recruited GP and PBP were asked to nominate and suggest the name of a potential CP participant with whom the practice had most contact. The researcher contacted that pharmacist using publicly available contact details. Therefore, triads were recruited, each consisting of a GP, PBP, and CP, from across the five Health and Social Care Trust areas in NI (that is, administrative healthcare areas in NI) with a view to recruiting up to three triads per area and one triad per practice. The researcher invited potential HCP participants by telephone/email. If interested, the researcher provided a formal invitation letter and information sheet. Interviews were conducted with the GP and PBP within the potential triad when both the GP and PBP agreed to participate. If a CP within a triad did not agree to participate, the GP and PBP interviews were retained for analysis and it was noted that a CP could not be recruited.
Interviews were conducted by the researcher between August 2020 and October 2021. Participants provided written informed consent and were offered £50 for participation. Separate topic guides were developed for each HCP group (Supplementary Information S1–S3), based on published literature within the field, and piloted. , , – All interviews were audiorecorded with the permission of the participant.
All interviews were recorded, transcribed verbatim, and checked for accuracy. To ensure anonymity and confidentiality, each participant was assigned a two-digit identification number reflecting the triad with which the participant was associated (for example Triad 1: GP01, PBP01, CP01). Transcripts were imported and managed in NVivo 12 Pro. Data analysis, using thematic analysis, was performed in parallel with data collection by two independent researchers. Data from interviews with each type of HCP were analysed separately to monitor data saturation within each HCP category. Themes were reviewed and refined by the research team. The quality and rigour of research reporting was closely monitored (Supplementary Table S1).
Eleven triads were recruited from across the five administrative areas , at which point data saturation was deemed to have occurred. A CP in one triad from the Western Trust was unable to participate because of COVID-19 vaccination workload; however, the GP and PBP interviews were retained for analysis. Of the 32 interviews conducted, most were undertaken by telephone ( n = 22), with the remainder using Microsoft Teams. Demographic data In total, 11 GPs, 11 PBPs, and 10 CPs participated . The participants’ demographics demonstrated variety in terms of key characteristics relevant to the research as they were recruited from a range of geographical areas (that is, five administrative areas; see Supplementary Information S4 for more details about demographic data). At the time of the study, most GPs ( n = 8, 73%) had two PBPs working part time in their practices. More than half of participating PBPs ( n = 6, 55%) had been working as a PBP for >2 years. CPs had been in community practice for an average of 18.1 (standard deviation 11.1) years. Main themes from thematic analysis Thematic analysis revealed four main themes in relation to PBPs’ integration into general practices. This study found that the evolution of the role; PBP attributes; collaboration and communication; and impact on care contributed to integration of PBPs into general practice. Furthermore, integration and impact on care were interrelated (as indicated by the double-headed arrow in ) in that integration led to an impact on care, and impact on care contributed to better integration. Theme 1: evolution of the role All GPs and PBPs described the wide range of activities undertaken by PBPs that could also differ between practices. They emphasised that the PBP role largely consisted of two main activities: medicines reconciliation of hospital discharge letters and outpatient letters, and medication reviews (see Supplementary Information S5 for definition). The role evolved with time and PBPs began to engage with other tasks, such as independent prescribing and conducting chronic disease review clinics: ‘They have a wide range of roles … they do the medicine reconciliation from the discharge letters, outpatient letters … do medication reviews … so that would be kind of core work in addition to that they do some of the repeat dispensing prescriptions and also some appropriate acute requests … they would do hypertension clinic, diabetes clinics … so it’s a very busy role and I think it’s a very expanding role as well.’ (GP11) PBPs and GPs reported that conducting chronic disease review clinics (see Supplementary Information S5 for definition) in general practice would continue to be an expanding and evolving role for PBPs, with better integration as they undertook more clinical roles. However, there were workload pressures and time constraints that sometimes prevented this happening, leading to prioritisation of medication reconciliation and medication reviews: ‘I feel practice pharmacists have a very good knowledge base and very good capability and skills to be able to contribute to the long-term management review clinics but sometimes that is not possible because their time is needed elsewhere in the medication reconciliation process and medication reviews in general.’ (PBP05) PBPs reported that time was wasted doing some administrative tasks that could be passed to pharmacy technicians to allow the PBP to do more clinical work and ultimately achieve better integration: ‘… get away from the hospital letters, start the technician role, get more clinics, see more patients that would be for me a better integration.’ (PBP10) In addition, GPs revealed that having more full-time PBPs in general practice would allow more activities to be undertaken: ‘Our practice-based pharmacists are busy … I suppose if we could have more full- time pharmacists that would be fantastic … there’s a huge amount of work that could be done.’ (GP06) Practices used different approaches to decide which activities the PBP would undertake. As PBPs were employed by a federation, this could influence what PBPs did. Participants described that there were ‘two bosses’, ‘a blended approach’, or ‘dual function’ to decide the activities of PBPs within general practices (that is, through the key performance indicators set by the federation and the needs of the practice): ‘Sometimes it can be quite difficult because you nearly have two bosses; you have your federation boss and then you have your practice manager and your lead GP in your doctor surgery.’ (PBP01) However, some GPs and PBPs believed that practices should have had more input into how they wanted the PBP to work within the practice: ‘Full integration for me would really be where the role of the pharmacist is very much driven by the individual practice … so driving on the structure of the practice.’ (GP05) Many participants highlighted a lack of awareness of the PBP role by others, including patients as summarised below. GPs’ awareness of the PBP role GPs and PBPs emphasised that the PBP role had not been clearly defined at the beginning of the initiative. However, they reported this had evolved and was now much clearer: ‘I think it has become much clearer over time … initially when the practice-based pharmacist was first introduced into the practice, we probably thought [of] some of the things that they could do and then after being with us, we were able to see more and more the value of them.’ (GP08) Despite this clarity, GPs and PBPs said that the role was not clear to all GPs: ‘Not to all GPs. I think there are some GPs who … do not let them [PBPs] use their skills and their training to their full potential.’ (GP02) CPs’ awareness of the PBP role Most PBPs perceived that their role was not entirely clear to CPs; CPs had similar views, but it largely depended on the relationship between the CP and the PBP. CPs’ limited awareness of the role was attributed to the lack of information about a PBP joining a general practice; they only became more aware while working with PBPs on a day-to-day basis: ‘I think sometimes they [CPs] think we don’t do anything … but in a word “no”, I don’t think they know the role.’ (PBP11) ‘I’d say it’s not entirely clear … I’m not entirely sure of the role.’ (CP11) ‘… we actually built relationships … I think that helped community pharmacists realise what we do.’ (PBP10) ‘I don’t think we have been like that for example given any further information regarding what practice pharmacists work where and when … now we can hear what they do from day to day and their roles certainly but probably not initially.’ (CP04) Patients’ awareness of the PBP role Most participants believed that patients were largely unaware of the PBP role and did not understand the difference between CPs and PBPs (that is, patients might think that the PBP worked in community pharmacy and did not realise that there was a pharmacist based in the practice): ‘A lot of patients are not familiar that we have maybe [a] pharmacist in the practice … I think a lot of them would not really differentiate between practice pharmacists and community pharmacists.’ (GP02) ‘A lot of patients are unaware that practices have practice-based pharmacists, and they think sometimes the practice pharmacist is just someone that works in community pharmacy and provides a prescription …’ (PBP07) ‘I would say probably not … you would still have patients that maybe don’t realise that there is a pharmacist based in the surgery …’ (CP05) Most GPs and PBPs felt that full integration of PBPs into general practice and promotion of the role would lead to greater awareness so that, in the future, patients could contact the practice to speak to a pharmacist about an acute problem or the practice team could refer patients to the PBP when appropriate rather than to the GP: ‘So full integration to me would be … they [patients] know what we exactly do, what our role is, and that we’re there to help them and they don’t necessarily need to always speak to GP about an issue.’ (PBP03) Theme 2: PBP attributes Participating GPs and PBPs reported that communication skills were essential for PBPs to undertake their role. Other useful skills related to: consultations, teamwork and leadership, independent working, time management, and information technology functions: ‘They need good communication skills as they are quite a link between everybody … skills would be team working skills, leadership skills, and independent working skills … consultation skills with dealing with the huge variety of the general public …’ (GP06) Furthermore, a number of GPs and PBPs revealed that PBPs would need clinical skills to allow them to undertake their role and would need the confidence to be able to use these skills: ‘… they need to have basic clinical skills such as being able to take blood pressure …’ (GP07) ‘You need like clinical skills to make sure that you’re doing things safely and we need to have the confidence to be able to check hospital medications as well as community medications …’ (PBP06) All participating PBPs had previous experience of working in community pharmacy, which was beneficial in understanding how community pharmacy operated and the importance of having good communication skills: ‘Because I have experience in community pharmacy, I felt confident with what I do now, so I felt confident coming into that role, so certainly having … good communications with both patients and the multidisciplinary team.’ (PBP08) However, all PBPs indicated that there were more opportunities to develop and acquire new skills, including practice-based learning compared with when working in a community pharmacy, and, therefore, they were more satisfied with the PBP role: ‘I think a lot of the people in the post really enjoy it … there are more training opportunities, more development opportunities … a lot of them have moved from community pharmacy and wanted a change … or better work–life balance.’ (PBP09) Several GPs and PBPs believed that it would be very beneficial for the PBP to be an independent prescriber. It was noted that the PBPs who were independent prescribers saved GP time because of reduced duplication of work. Moreover, this qualification enhanced PBPs’ confidence and experience: ‘… that saves us [GPs] a lot of time … there was a lot of duplication of work when he [PBP] wasn’t able to prescribe …’ (GP01) ‘Everyone needs independent prescribing … it’s about building up the level of confidence and experience …’ (PBP06) Theme 3: collaboration and communication Participants emphasised that teamwork, supported by clear communication and strong relationships, would be key to achieving collaboration. Trust in, and mutual respect for, each HCP’s expertise and individual skills were also reported as factors to facilitate relationship building and, thus, achieving and enhancing collaborative work: ‘Interprofessional collaboration would mean communication, having a good relationship … developing trust in the people that you’re working with. I do think that’s the way we work here, it’s very much respectful … so that helps enhance team spirit; feeling that everyone is working within a team.’ (GP06) Participants reported several benefits of collaboration between GPs, PBPs, and CPs: streamlining healthcare delivery, reduction in duplication of effort, rapid response to each HCP, and improved patient outcomes and safety: ‘Ultimately collaboration will seek to reduce duplication of effort and ultimately, if we’re working together synergistically for the patient, that will improve patient outcomes … it streamlines everything … and things are done so much quicker …’ (PBP10) Most GPs and PBPs identified that having more formal meetings, involving the PBP in practice meetings and social activities, having full-time PBP positions in the general practice, and having designated times to communicate and to meet regularly would enhance communication, working relationships, and consequently achieve better integration: ‘It [full integration of PBPs] is very much focused on regular meetings and to make that protected time for the pharmacist to meet with all members of the team.’ (GP03) ‘… as long as pharmacists are involved in practice meetings and decisions and have those relationships, I think there’s not much more that could be done.’ (PBP09) Further details on the impact of full- time PBP positions on integration are given below. Development of relationships across the practice team and with patients Many GPs and PBPs described the importance of having full-time PBP positions in the general practice to develop relationships across the practice team as well as between the PBP and patients: ‘… each time that a practice pharmacist has come to our practice, we have spent time developing these relationships and then, unfortunately, sometimes they’ve had to move on … and sometimes there’s a reluctance to invest time unless you’re sure that the pharmacist that you’re getting is going to be there for long term.’ (GP07) ‘… being permanently employed within a practice is key … it [PBP role] needs to be a permanent role … so that GPs get what they want from practice-based pharmacists … and practice-based pharmacists can develop relationships with GPs and patients within the practice as well …’ (PBP02) Completion of PBPs’ tasks A number of PBPs highlighted the difficulties of covering too many practices and the effect on some tasks, for example, following up hospital letters: ‘… you would find it kind of hard to sort of go to one surgery one day and then go to another surgery the next day, we need to be able to build up relationship with other workers in the primary care team, but also be able to build it up with patients, and also some following up things, it just kind of sort of the hospital letters when you are and then that’s you move on to the next surgery …’ (PBP03) The response to CPs’ queries Some CPs described occasional delays in response if a PBP was not in the practice on a full-time basis: ‘… I have noticed that you know a surgery with a practice-based pharmacist, you get [a] response much quicker, whereas compared to a surgery that wouldn’t have one or the practice-based pharmacist doesn’t work that day, there will be delay sometimes.’ (CP04) PBPs were considered to be in a perfect position to link between different professionals and to act as the first point of contact in general practice for CPs. Many saw PBPs as a ‘central hub–middleman’ between general practice and community pharmacies and between primary and secondary care. CPs felt that it was easier to communicate with the PBP as opposed to GPs who had historically dealt with queries from CPs: ‘I’m sort of the middleman … I pass all that information into our community pharmacy to make sure that they can order the right items for the patient …’ (PBP01) ‘We don’t speak to the GP as much … but they’re [PBPs] more the middleman now …’ (CP10) ‘Practice-based pharmacists can respond to queries often quicker and more efficient …’ (CP05) Participants emphasised the need to have direct access to the PBP (for example, direct telephone line/email) to improve communication: ‘We don’t have direct access to speak with the practice pharmacist … the best way for them [practices] to do this would [be to] have a dedicated phone line for the practice-based pharmacist.’ (CP07) Participants noted that having shared education, training, and meetings among the multidisciplinary team and involving CPs would be essential to ensure that all HCPs would adhere to the same principles and procedures in patient care. Participants indicated that these activities would contribute to good working relationships, improve collaboration and communication, and increase HCPs’ awareness of the PBP role: ‘I suppose if we have time to maybe do teaching or learning together, I suppose that would be really helpful … we could all know that we’re all doing the same thing …’ (GP10) ‘I think it’s a fantastic idea where you have multidisciplinary groups in the same table … I might bring to the table something that the others don’t, and the others obviously bring something there I don’t see.’ (CP07) PBPs emphasised that better collaboration and communication would be achieved by increasing primary care team members’ awareness and patients’ awareness of the PBP role: ‘I think education is number one there … I think as a first and foremost educating the main people about the role for example GPs. I think that improves the interprofessional sort of collaboration and communication.’ (PBP01) Theme 4: impact on care GPs and PBPs reported that PBPs reduced GPs’ workload and saved time by conducting activities previously performed by GPs. As a result, this provided the GP with more time to see patients, resulting in better patient care: ‘They [PBPs] have given us more time … we’re not trying to terminate the consultation in order to do the huge pile of paperwork so we can do more work directly with the patient … and that the patient will get the benefit of that.’ (GP06) Furthermore, GPs indicated that PBPs were an invaluable source of information, ensuring that GPs were aware of the most current guidelines. ‘They [PBPs] are a great source of information … they are able to sit down and look at the drugs and really take the time to go through them properly … they are very up to date as well on the guidelines, as well quite a lot of time they are keeping us up to date with the guidelines …’ (GP10) GPs and PBPs also reported that PBPs’ activities enhanced patient safety as they had more time to focus on hospital discharge letters, chronic disease review clinics, and queries from community pharmacies and secondary care. These activities reduced interruptions experienced by GPs during their work and ultimately reducing the risk of errors. GPs also reported that the PBP was detail oriented: ‘It has made our life better and safer and more pleasant … it has done that for me when you’re coming into a busy day and they’re at the end of it, you have maybe got sixty letters to process, you know, mistakes are going to be made with tiredness and with just too much volume of stuff … so there’s attention to detail and having a bit more time …’ (GP03) Participants indicated that the PBP had a critical role in medicines optimisation, particularly for the older population who were at higher risk of adverse drug events and those with polypharmacy: ‘I feel like medicines optimisation is kind of our main role … our medication review specifically wants to focus on those patients with polypharmacy … those that are frail and elderly …’ (PBP06)
In total, 11 GPs, 11 PBPs, and 10 CPs participated . The participants’ demographics demonstrated variety in terms of key characteristics relevant to the research as they were recruited from a range of geographical areas (that is, five administrative areas; see Supplementary Information S4 for more details about demographic data). At the time of the study, most GPs ( n = 8, 73%) had two PBPs working part time in their practices. More than half of participating PBPs ( n = 6, 55%) had been working as a PBP for >2 years. CPs had been in community practice for an average of 18.1 (standard deviation 11.1) years.
Thematic analysis revealed four main themes in relation to PBPs’ integration into general practices. This study found that the evolution of the role; PBP attributes; collaboration and communication; and impact on care contributed to integration of PBPs into general practice. Furthermore, integration and impact on care were interrelated (as indicated by the double-headed arrow in ) in that integration led to an impact on care, and impact on care contributed to better integration.
All GPs and PBPs described the wide range of activities undertaken by PBPs that could also differ between practices. They emphasised that the PBP role largely consisted of two main activities: medicines reconciliation of hospital discharge letters and outpatient letters, and medication reviews (see Supplementary Information S5 for definition). The role evolved with time and PBPs began to engage with other tasks, such as independent prescribing and conducting chronic disease review clinics: ‘They have a wide range of roles … they do the medicine reconciliation from the discharge letters, outpatient letters … do medication reviews … so that would be kind of core work in addition to that they do some of the repeat dispensing prescriptions and also some appropriate acute requests … they would do hypertension clinic, diabetes clinics … so it’s a very busy role and I think it’s a very expanding role as well.’ (GP11) PBPs and GPs reported that conducting chronic disease review clinics (see Supplementary Information S5 for definition) in general practice would continue to be an expanding and evolving role for PBPs, with better integration as they undertook more clinical roles. However, there were workload pressures and time constraints that sometimes prevented this happening, leading to prioritisation of medication reconciliation and medication reviews: ‘I feel practice pharmacists have a very good knowledge base and very good capability and skills to be able to contribute to the long-term management review clinics but sometimes that is not possible because their time is needed elsewhere in the medication reconciliation process and medication reviews in general.’ (PBP05) PBPs reported that time was wasted doing some administrative tasks that could be passed to pharmacy technicians to allow the PBP to do more clinical work and ultimately achieve better integration: ‘… get away from the hospital letters, start the technician role, get more clinics, see more patients that would be for me a better integration.’ (PBP10) In addition, GPs revealed that having more full-time PBPs in general practice would allow more activities to be undertaken: ‘Our practice-based pharmacists are busy … I suppose if we could have more full- time pharmacists that would be fantastic … there’s a huge amount of work that could be done.’ (GP06) Practices used different approaches to decide which activities the PBP would undertake. As PBPs were employed by a federation, this could influence what PBPs did. Participants described that there were ‘two bosses’, ‘a blended approach’, or ‘dual function’ to decide the activities of PBPs within general practices (that is, through the key performance indicators set by the federation and the needs of the practice): ‘Sometimes it can be quite difficult because you nearly have two bosses; you have your federation boss and then you have your practice manager and your lead GP in your doctor surgery.’ (PBP01) However, some GPs and PBPs believed that practices should have had more input into how they wanted the PBP to work within the practice: ‘Full integration for me would really be where the role of the pharmacist is very much driven by the individual practice … so driving on the structure of the practice.’ (GP05) Many participants highlighted a lack of awareness of the PBP role by others, including patients as summarised below. GPs’ awareness of the PBP role GPs and PBPs emphasised that the PBP role had not been clearly defined at the beginning of the initiative. However, they reported this had evolved and was now much clearer: ‘I think it has become much clearer over time … initially when the practice-based pharmacist was first introduced into the practice, we probably thought [of] some of the things that they could do and then after being with us, we were able to see more and more the value of them.’ (GP08) Despite this clarity, GPs and PBPs said that the role was not clear to all GPs: ‘Not to all GPs. I think there are some GPs who … do not let them [PBPs] use their skills and their training to their full potential.’ (GP02) CPs’ awareness of the PBP role Most PBPs perceived that their role was not entirely clear to CPs; CPs had similar views, but it largely depended on the relationship between the CP and the PBP. CPs’ limited awareness of the role was attributed to the lack of information about a PBP joining a general practice; they only became more aware while working with PBPs on a day-to-day basis: ‘I think sometimes they [CPs] think we don’t do anything … but in a word “no”, I don’t think they know the role.’ (PBP11) ‘I’d say it’s not entirely clear … I’m not entirely sure of the role.’ (CP11) ‘… we actually built relationships … I think that helped community pharmacists realise what we do.’ (PBP10) ‘I don’t think we have been like that for example given any further information regarding what practice pharmacists work where and when … now we can hear what they do from day to day and their roles certainly but probably not initially.’ (CP04) Patients’ awareness of the PBP role Most participants believed that patients were largely unaware of the PBP role and did not understand the difference between CPs and PBPs (that is, patients might think that the PBP worked in community pharmacy and did not realise that there was a pharmacist based in the practice): ‘A lot of patients are not familiar that we have maybe [a] pharmacist in the practice … I think a lot of them would not really differentiate between practice pharmacists and community pharmacists.’ (GP02) ‘A lot of patients are unaware that practices have practice-based pharmacists, and they think sometimes the practice pharmacist is just someone that works in community pharmacy and provides a prescription …’ (PBP07) ‘I would say probably not … you would still have patients that maybe don’t realise that there is a pharmacist based in the surgery …’ (CP05) Most GPs and PBPs felt that full integration of PBPs into general practice and promotion of the role would lead to greater awareness so that, in the future, patients could contact the practice to speak to a pharmacist about an acute problem or the practice team could refer patients to the PBP when appropriate rather than to the GP: ‘So full integration to me would be … they [patients] know what we exactly do, what our role is, and that we’re there to help them and they don’t necessarily need to always speak to GP about an issue.’ (PBP03)
GPs and PBPs emphasised that the PBP role had not been clearly defined at the beginning of the initiative. However, they reported this had evolved and was now much clearer: ‘I think it has become much clearer over time … initially when the practice-based pharmacist was first introduced into the practice, we probably thought [of] some of the things that they could do and then after being with us, we were able to see more and more the value of them.’ (GP08) Despite this clarity, GPs and PBPs said that the role was not clear to all GPs: ‘Not to all GPs. I think there are some GPs who … do not let them [PBPs] use their skills and their training to their full potential.’ (GP02)
Most PBPs perceived that their role was not entirely clear to CPs; CPs had similar views, but it largely depended on the relationship between the CP and the PBP. CPs’ limited awareness of the role was attributed to the lack of information about a PBP joining a general practice; they only became more aware while working with PBPs on a day-to-day basis: ‘I think sometimes they [CPs] think we don’t do anything … but in a word “no”, I don’t think they know the role.’ (PBP11) ‘I’d say it’s not entirely clear … I’m not entirely sure of the role.’ (CP11) ‘… we actually built relationships … I think that helped community pharmacists realise what we do.’ (PBP10) ‘I don’t think we have been like that for example given any further information regarding what practice pharmacists work where and when … now we can hear what they do from day to day and their roles certainly but probably not initially.’ (CP04)
Most participants believed that patients were largely unaware of the PBP role and did not understand the difference between CPs and PBPs (that is, patients might think that the PBP worked in community pharmacy and did not realise that there was a pharmacist based in the practice): ‘A lot of patients are not familiar that we have maybe [a] pharmacist in the practice … I think a lot of them would not really differentiate between practice pharmacists and community pharmacists.’ (GP02) ‘A lot of patients are unaware that practices have practice-based pharmacists, and they think sometimes the practice pharmacist is just someone that works in community pharmacy and provides a prescription …’ (PBP07) ‘I would say probably not … you would still have patients that maybe don’t realise that there is a pharmacist based in the surgery …’ (CP05) Most GPs and PBPs felt that full integration of PBPs into general practice and promotion of the role would lead to greater awareness so that, in the future, patients could contact the practice to speak to a pharmacist about an acute problem or the practice team could refer patients to the PBP when appropriate rather than to the GP: ‘So full integration to me would be … they [patients] know what we exactly do, what our role is, and that we’re there to help them and they don’t necessarily need to always speak to GP about an issue.’ (PBP03)
Participating GPs and PBPs reported that communication skills were essential for PBPs to undertake their role. Other useful skills related to: consultations, teamwork and leadership, independent working, time management, and information technology functions: ‘They need good communication skills as they are quite a link between everybody … skills would be team working skills, leadership skills, and independent working skills … consultation skills with dealing with the huge variety of the general public …’ (GP06) Furthermore, a number of GPs and PBPs revealed that PBPs would need clinical skills to allow them to undertake their role and would need the confidence to be able to use these skills: ‘… they need to have basic clinical skills such as being able to take blood pressure …’ (GP07) ‘You need like clinical skills to make sure that you’re doing things safely and we need to have the confidence to be able to check hospital medications as well as community medications …’ (PBP06) All participating PBPs had previous experience of working in community pharmacy, which was beneficial in understanding how community pharmacy operated and the importance of having good communication skills: ‘Because I have experience in community pharmacy, I felt confident with what I do now, so I felt confident coming into that role, so certainly having … good communications with both patients and the multidisciplinary team.’ (PBP08) However, all PBPs indicated that there were more opportunities to develop and acquire new skills, including practice-based learning compared with when working in a community pharmacy, and, therefore, they were more satisfied with the PBP role: ‘I think a lot of the people in the post really enjoy it … there are more training opportunities, more development opportunities … a lot of them have moved from community pharmacy and wanted a change … or better work–life balance.’ (PBP09) Several GPs and PBPs believed that it would be very beneficial for the PBP to be an independent prescriber. It was noted that the PBPs who were independent prescribers saved GP time because of reduced duplication of work. Moreover, this qualification enhanced PBPs’ confidence and experience: ‘… that saves us [GPs] a lot of time … there was a lot of duplication of work when he [PBP] wasn’t able to prescribe …’ (GP01) ‘Everyone needs independent prescribing … it’s about building up the level of confidence and experience …’ (PBP06)
Participants emphasised that teamwork, supported by clear communication and strong relationships, would be key to achieving collaboration. Trust in, and mutual respect for, each HCP’s expertise and individual skills were also reported as factors to facilitate relationship building and, thus, achieving and enhancing collaborative work: ‘Interprofessional collaboration would mean communication, having a good relationship … developing trust in the people that you’re working with. I do think that’s the way we work here, it’s very much respectful … so that helps enhance team spirit; feeling that everyone is working within a team.’ (GP06) Participants reported several benefits of collaboration between GPs, PBPs, and CPs: streamlining healthcare delivery, reduction in duplication of effort, rapid response to each HCP, and improved patient outcomes and safety: ‘Ultimately collaboration will seek to reduce duplication of effort and ultimately, if we’re working together synergistically for the patient, that will improve patient outcomes … it streamlines everything … and things are done so much quicker …’ (PBP10) Most GPs and PBPs identified that having more formal meetings, involving the PBP in practice meetings and social activities, having full-time PBP positions in the general practice, and having designated times to communicate and to meet regularly would enhance communication, working relationships, and consequently achieve better integration: ‘It [full integration of PBPs] is very much focused on regular meetings and to make that protected time for the pharmacist to meet with all members of the team.’ (GP03) ‘… as long as pharmacists are involved in practice meetings and decisions and have those relationships, I think there’s not much more that could be done.’ (PBP09) Further details on the impact of full- time PBP positions on integration are given below. Development of relationships across the practice team and with patients Many GPs and PBPs described the importance of having full-time PBP positions in the general practice to develop relationships across the practice team as well as between the PBP and patients: ‘… each time that a practice pharmacist has come to our practice, we have spent time developing these relationships and then, unfortunately, sometimes they’ve had to move on … and sometimes there’s a reluctance to invest time unless you’re sure that the pharmacist that you’re getting is going to be there for long term.’ (GP07) ‘… being permanently employed within a practice is key … it [PBP role] needs to be a permanent role … so that GPs get what they want from practice-based pharmacists … and practice-based pharmacists can develop relationships with GPs and patients within the practice as well …’ (PBP02) Completion of PBPs’ tasks A number of PBPs highlighted the difficulties of covering too many practices and the effect on some tasks, for example, following up hospital letters: ‘… you would find it kind of hard to sort of go to one surgery one day and then go to another surgery the next day, we need to be able to build up relationship with other workers in the primary care team, but also be able to build it up with patients, and also some following up things, it just kind of sort of the hospital letters when you are and then that’s you move on to the next surgery …’ (PBP03) The response to CPs’ queries Some CPs described occasional delays in response if a PBP was not in the practice on a full-time basis: ‘… I have noticed that you know a surgery with a practice-based pharmacist, you get [a] response much quicker, whereas compared to a surgery that wouldn’t have one or the practice-based pharmacist doesn’t work that day, there will be delay sometimes.’ (CP04) PBPs were considered to be in a perfect position to link between different professionals and to act as the first point of contact in general practice for CPs. Many saw PBPs as a ‘central hub–middleman’ between general practice and community pharmacies and between primary and secondary care. CPs felt that it was easier to communicate with the PBP as opposed to GPs who had historically dealt with queries from CPs: ‘I’m sort of the middleman … I pass all that information into our community pharmacy to make sure that they can order the right items for the patient …’ (PBP01) ‘We don’t speak to the GP as much … but they’re [PBPs] more the middleman now …’ (CP10) ‘Practice-based pharmacists can respond to queries often quicker and more efficient …’ (CP05) Participants emphasised the need to have direct access to the PBP (for example, direct telephone line/email) to improve communication: ‘We don’t have direct access to speak with the practice pharmacist … the best way for them [practices] to do this would [be to] have a dedicated phone line for the practice-based pharmacist.’ (CP07) Participants noted that having shared education, training, and meetings among the multidisciplinary team and involving CPs would be essential to ensure that all HCPs would adhere to the same principles and procedures in patient care. Participants indicated that these activities would contribute to good working relationships, improve collaboration and communication, and increase HCPs’ awareness of the PBP role: ‘I suppose if we have time to maybe do teaching or learning together, I suppose that would be really helpful … we could all know that we’re all doing the same thing …’ (GP10) ‘I think it’s a fantastic idea where you have multidisciplinary groups in the same table … I might bring to the table something that the others don’t, and the others obviously bring something there I don’t see.’ (CP07) PBPs emphasised that better collaboration and communication would be achieved by increasing primary care team members’ awareness and patients’ awareness of the PBP role: ‘I think education is number one there … I think as a first and foremost educating the main people about the role for example GPs. I think that improves the interprofessional sort of collaboration and communication.’ (PBP01)
Many GPs and PBPs described the importance of having full-time PBP positions in the general practice to develop relationships across the practice team as well as between the PBP and patients: ‘… each time that a practice pharmacist has come to our practice, we have spent time developing these relationships and then, unfortunately, sometimes they’ve had to move on … and sometimes there’s a reluctance to invest time unless you’re sure that the pharmacist that you’re getting is going to be there for long term.’ (GP07) ‘… being permanently employed within a practice is key … it [PBP role] needs to be a permanent role … so that GPs get what they want from practice-based pharmacists … and practice-based pharmacists can develop relationships with GPs and patients within the practice as well …’ (PBP02)
A number of PBPs highlighted the difficulties of covering too many practices and the effect on some tasks, for example, following up hospital letters: ‘… you would find it kind of hard to sort of go to one surgery one day and then go to another surgery the next day, we need to be able to build up relationship with other workers in the primary care team, but also be able to build it up with patients, and also some following up things, it just kind of sort of the hospital letters when you are and then that’s you move on to the next surgery …’ (PBP03)
Some CPs described occasional delays in response if a PBP was not in the practice on a full-time basis: ‘… I have noticed that you know a surgery with a practice-based pharmacist, you get [a] response much quicker, whereas compared to a surgery that wouldn’t have one or the practice-based pharmacist doesn’t work that day, there will be delay sometimes.’ (CP04) PBPs were considered to be in a perfect position to link between different professionals and to act as the first point of contact in general practice for CPs. Many saw PBPs as a ‘central hub–middleman’ between general practice and community pharmacies and between primary and secondary care. CPs felt that it was easier to communicate with the PBP as opposed to GPs who had historically dealt with queries from CPs: ‘I’m sort of the middleman … I pass all that information into our community pharmacy to make sure that they can order the right items for the patient …’ (PBP01) ‘We don’t speak to the GP as much … but they’re [PBPs] more the middleman now …’ (CP10) ‘Practice-based pharmacists can respond to queries often quicker and more efficient …’ (CP05) Participants emphasised the need to have direct access to the PBP (for example, direct telephone line/email) to improve communication: ‘We don’t have direct access to speak with the practice pharmacist … the best way for them [practices] to do this would [be to] have a dedicated phone line for the practice-based pharmacist.’ (CP07) Participants noted that having shared education, training, and meetings among the multidisciplinary team and involving CPs would be essential to ensure that all HCPs would adhere to the same principles and procedures in patient care. Participants indicated that these activities would contribute to good working relationships, improve collaboration and communication, and increase HCPs’ awareness of the PBP role: ‘I suppose if we have time to maybe do teaching or learning together, I suppose that would be really helpful … we could all know that we’re all doing the same thing …’ (GP10) ‘I think it’s a fantastic idea where you have multidisciplinary groups in the same table … I might bring to the table something that the others don’t, and the others obviously bring something there I don’t see.’ (CP07) PBPs emphasised that better collaboration and communication would be achieved by increasing primary care team members’ awareness and patients’ awareness of the PBP role: ‘I think education is number one there … I think as a first and foremost educating the main people about the role for example GPs. I think that improves the interprofessional sort of collaboration and communication.’ (PBP01)
GPs and PBPs reported that PBPs reduced GPs’ workload and saved time by conducting activities previously performed by GPs. As a result, this provided the GP with more time to see patients, resulting in better patient care: ‘They [PBPs] have given us more time … we’re not trying to terminate the consultation in order to do the huge pile of paperwork so we can do more work directly with the patient … and that the patient will get the benefit of that.’ (GP06) Furthermore, GPs indicated that PBPs were an invaluable source of information, ensuring that GPs were aware of the most current guidelines. ‘They [PBPs] are a great source of information … they are able to sit down and look at the drugs and really take the time to go through them properly … they are very up to date as well on the guidelines, as well quite a lot of time they are keeping us up to date with the guidelines …’ (GP10) GPs and PBPs also reported that PBPs’ activities enhanced patient safety as they had more time to focus on hospital discharge letters, chronic disease review clinics, and queries from community pharmacies and secondary care. These activities reduced interruptions experienced by GPs during their work and ultimately reducing the risk of errors. GPs also reported that the PBP was detail oriented: ‘It has made our life better and safer and more pleasant … it has done that for me when you’re coming into a busy day and they’re at the end of it, you have maybe got sixty letters to process, you know, mistakes are going to be made with tiredness and with just too much volume of stuff … so there’s attention to detail and having a bit more time …’ (GP03) Participants indicated that the PBP had a critical role in medicines optimisation, particularly for the older population who were at higher risk of adverse drug events and those with polypharmacy: ‘I feel like medicines optimisation is kind of our main role … our medication review specifically wants to focus on those patients with polypharmacy … those that are frail and elderly …’ (PBP06)
Summary This qualitative study highlighted that the PBP role had evolved since its introduction across general practice in NI and the PBP role has had a positive impact on GPs, CPs, and patients. Insights were provided into participants’ views on what had contributed to integration (for example, a good GP–PBP collaboration) as well as aspects that required further attention (for example, patients’ awareness of the role) to ensure better and continuing integration of PBPs into general practice. Strengths and limitations The qualitative design and the triad approach provided a more comprehensive overview of the working relationships between the three HCP groups and allowed for an in-depth and thorough understanding of participants’ views. Data were analysed independently by two researchers and decisions were made on the themes through a consensus approach, enhancing the trustworthiness of the findings and in accordance with the COREQ checklist. A number of limitations should be noted. First, recruitment was limited to one UK geographical region. Most participants were recruited through snowball sampling that may have introduced potential bias. No PBPs were interviewed who had previously worked in hospital pharmacy (that is, all had a community pharmacy experience), which could affect the transferability of the findings. However, despite this, three key HCPs from different NI trust areas were included and their perspectives are broadly similar to those reported in the literature, thereby reinforcing the transferability of these findings. Comparison with existing literature In this study, it was apparent that the PBP role varied between practices and there were several activities undertaken by PBPs that have previously been identified. , , , – However, insufficient time and current workload were perceived as barriers to undertaking some activities and have been noted in other research. , , , Integrating pharmacy technicians into general practice was suggested as a way to save PBP time to conduct more clinical work and thus make better use of PBP skills. Previous studies have shown that there is the potential to expand the role of pharmacy technicians in the UK to become more involved in the future delivery of medication reviews. – Lack of patient awareness of the role was highlighted by nearly all participants. As the PBP role was relatively new and varied between GP practices, participants believed that patients did not understand the difference between CPs and PBPs, which is consistent with other studies. , , – Nearly all participants in the current study emphasised the need to properly inform patients and primary care team members about PBPs, including roles and responsibilities, to raise patients’ awareness and therefore encourage the uptake of PBPs’ services, ultimately leading to better integration of the PBP. , , , , , Further work to explore patients’ understanding and views of the PBP role in general practice is necessary to corroborate the participants’ concerns in the current study. Awareness of the PBP role was one of the key aspects that requires further attention to achieve better integration. This current study indicates that, although most GPs were aware of the role, participants reported that a minority were not. Furthermore, the PBP role was not entirely clear to CPs, which has been previously reported. , Clearly defined roles improve collaboration and decrease misunderstandings about responsibilities and authority. – Consistent with the findings in this study, previous studies highlighted that scheduling meetings with individual team members was a common approach to informing the team about the role. , Having full-time PBPs was described as a way to enhance communication. Many highlighted the advantage of having a full-time PBP to build and develop strong relationships and ultimately better integration into a team. This has been reported elsewhere. , , , Working across many practices could possibly lead to a lack of continuity, thus hindering integration, , while also having an impact on tasks such as managing repeat medication re-authorisation. A previous study found that patients reported difficulties in arranging appointments with PBPs who covered multiple practices. , The findings of this current study have indicated that PBPs are ideally placed to use their skills and knowledge to help the GP practices and liaise with CPs. Evidence has shown that PBPs provide valuable services to ease the burden on the GP and reduce patient waiting times. – In the current study, it was perceived that PBPs had an impact not only on GP workload but also on patient safety and care, and medicines optimisation. These are reassuring findings as, for example, 20% of hospital admissions among older people are the result of adverse effects of prescribed medications. Implications for practice There are a number of recommendations for practices based on the results of this study. As the role of the PBP is expected to expand to include more clinical patient-facing roles, pharmacists must be prepared for this. New standards for the initial education and training of pharmacists across the UK have been recently introduced, for example, qualified to undertake independent prescribing from the point of registration. Furthermore, this study revealed that PBPs would need clinical skills to allow them to undertake their role and would need the confidence to be able to use these skills. A recently published Delphi study has produced a core set of clinical skills required for pharmacist prescribers working in general practice that could inform training. However, expansion of the role and increasing patient awareness of the role may exacerbate workload issues. Therefore, having more PBPs and pharmacy technicians in general practice may help to address these demands. A number of policies have been launched in the UK to expand the current integration of PBPs in general practice. – Therefore, the findings from this study provide valuable insights for policymakers, practice managers, and service commissioners into what is required to ensure better, efficient, and smooth integration of PBPs. The findings from this study may be useful in countries where consideration is being given to the development of pharmacist services in general practice.
This qualitative study highlighted that the PBP role had evolved since its introduction across general practice in NI and the PBP role has had a positive impact on GPs, CPs, and patients. Insights were provided into participants’ views on what had contributed to integration (for example, a good GP–PBP collaboration) as well as aspects that required further attention (for example, patients’ awareness of the role) to ensure better and continuing integration of PBPs into general practice.
The qualitative design and the triad approach provided a more comprehensive overview of the working relationships between the three HCP groups and allowed for an in-depth and thorough understanding of participants’ views. Data were analysed independently by two researchers and decisions were made on the themes through a consensus approach, enhancing the trustworthiness of the findings and in accordance with the COREQ checklist. A number of limitations should be noted. First, recruitment was limited to one UK geographical region. Most participants were recruited through snowball sampling that may have introduced potential bias. No PBPs were interviewed who had previously worked in hospital pharmacy (that is, all had a community pharmacy experience), which could affect the transferability of the findings. However, despite this, three key HCPs from different NI trust areas were included and their perspectives are broadly similar to those reported in the literature, thereby reinforcing the transferability of these findings.
In this study, it was apparent that the PBP role varied between practices and there were several activities undertaken by PBPs that have previously been identified. , , , – However, insufficient time and current workload were perceived as barriers to undertaking some activities and have been noted in other research. , , , Integrating pharmacy technicians into general practice was suggested as a way to save PBP time to conduct more clinical work and thus make better use of PBP skills. Previous studies have shown that there is the potential to expand the role of pharmacy technicians in the UK to become more involved in the future delivery of medication reviews. – Lack of patient awareness of the role was highlighted by nearly all participants. As the PBP role was relatively new and varied between GP practices, participants believed that patients did not understand the difference between CPs and PBPs, which is consistent with other studies. , , – Nearly all participants in the current study emphasised the need to properly inform patients and primary care team members about PBPs, including roles and responsibilities, to raise patients’ awareness and therefore encourage the uptake of PBPs’ services, ultimately leading to better integration of the PBP. , , , , , Further work to explore patients’ understanding and views of the PBP role in general practice is necessary to corroborate the participants’ concerns in the current study. Awareness of the PBP role was one of the key aspects that requires further attention to achieve better integration. This current study indicates that, although most GPs were aware of the role, participants reported that a minority were not. Furthermore, the PBP role was not entirely clear to CPs, which has been previously reported. , Clearly defined roles improve collaboration and decrease misunderstandings about responsibilities and authority. – Consistent with the findings in this study, previous studies highlighted that scheduling meetings with individual team members was a common approach to informing the team about the role. , Having full-time PBPs was described as a way to enhance communication. Many highlighted the advantage of having a full-time PBP to build and develop strong relationships and ultimately better integration into a team. This has been reported elsewhere. , , , Working across many practices could possibly lead to a lack of continuity, thus hindering integration, , while also having an impact on tasks such as managing repeat medication re-authorisation. A previous study found that patients reported difficulties in arranging appointments with PBPs who covered multiple practices. , The findings of this current study have indicated that PBPs are ideally placed to use their skills and knowledge to help the GP practices and liaise with CPs. Evidence has shown that PBPs provide valuable services to ease the burden on the GP and reduce patient waiting times. – In the current study, it was perceived that PBPs had an impact not only on GP workload but also on patient safety and care, and medicines optimisation. These are reassuring findings as, for example, 20% of hospital admissions among older people are the result of adverse effects of prescribed medications.
There are a number of recommendations for practices based on the results of this study. As the role of the PBP is expected to expand to include more clinical patient-facing roles, pharmacists must be prepared for this. New standards for the initial education and training of pharmacists across the UK have been recently introduced, for example, qualified to undertake independent prescribing from the point of registration. Furthermore, this study revealed that PBPs would need clinical skills to allow them to undertake their role and would need the confidence to be able to use these skills. A recently published Delphi study has produced a core set of clinical skills required for pharmacist prescribers working in general practice that could inform training. However, expansion of the role and increasing patient awareness of the role may exacerbate workload issues. Therefore, having more PBPs and pharmacy technicians in general practice may help to address these demands. A number of policies have been launched in the UK to expand the current integration of PBPs in general practice. – Therefore, the findings from this study provide valuable insights for policymakers, practice managers, and service commissioners into what is required to ensure better, efficient, and smooth integration of PBPs. The findings from this study may be useful in countries where consideration is being given to the development of pharmacist services in general practice.
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Association between the person-centered maternity care experience and mental health after delivery in urban and rural Dhading, Nepal: a cross-sectional study
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ef6e545f-ed15-4757-80a3-afd084d6ebd7
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10228024
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Patient-Centered Care[mh]
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Improving the overall quality of maternal healthcare is the key to ensuring positive health outcomes . Most preventable deaths and disabilities during childbirth can be reduced by health services providing quality care . However, the utilization of quality maternity services is restricted in some resource-limited settings, especially in sub-Saharan Africa and South Asia . In these settings, women reported the barriers to using quality services at a facility, including socio-cultural norms, limited access, financial problems, and a negative perception of quality care . Several women were reluctant to undergo a facility delivery due to mistreatment, verbal abuse, rudeness, and neglect . To overcome these barriers, person-centered maternity care (PCMC) has been introduced in several countries. PCMC is the component of quality care, which is provided according to the preferences and aspirations of users . Such care includes dignity, autonomy, privacy/confidentiality, communication, social support, supportive care, and trust . PCMC allows women to make decisions for treatment, maintain their dignity, and improve their capabilities . To implement PCMC, several supportive care or dignity interventions have been conducted. They include consultations with trained midwives or therapy groups; such care showed positive effect on women’s perinatal experience . Those effects decreased women’s anxiety and birth-related concerns . However, some women experience psychological birth trauma, and one of the risk factors is poor interaction with healthcare providers . Approximately 20% of women experience mental difficulties after childbirth in low- and middle-income countries, which is higher than in high-income countries . Therefore, maternal mental health management is required after delivery, especially in resource-limited settings . Nepal, one of the lower-middle-income countries in South Asia, is now focusing on high-quality health systems . Although maternity care usage has increased in the past 20 years, the facility delivery rate still has room for improvement . One barrier to facility delivery was women’s perception of healthcare providers’ disrespectful care and the quality of health services . Women’s intentions for future maternity care utilization were influenced by the waiting time, received information, and overall care at the facility . Despite this, women were dissatisfied with the physical resources and interpersonal aspects, such as compassion, respect, and honesty at public hospitals in Nepal . Thus, a better quality of maternity care that considers women’s needs and satisfaction is required. PCMC is also necessary to prevent mothers’ mental problems and improve their well-being. Nowadays, postpartum depressive symptoms are gradually gaining attention in Nepal . However, how the childbirth experiences at healthcare facilities, particularly with PCMC, are associated with women’s mental health after delivery in Nepal remains unknown. Moreover, only a few studies were conducted on the difference in maternal mental health between urban and rural settings in Nepal. Different risks were reported for postpartum depression, depending on the place and circumstances of residence . Therefore, this study was conducted to examine the association between PCMC experiences at healthcare facilities and maternal mental health after delivery among women living in Dhading, Nepal. This study also explored the difference between urban and rural communities in the association of PCMC with maternal mental health after delivery.
Study design and settings We conducted a cross-sectional study in Dhading District, Nepal. Dhading belongs to Bagmati Province and is adjacent to Kathmandu, the capital of Nepal. According to the preliminary Population Census 2021 result, the population of Dhading is 336,067 . Its population density is 168 per square kilometer, ranked 38th among all 77 districts in Nepal . As the study site, we covered all 2 municipalities and 5 out of 11 rural municipalities. These municipalities and rural municipalities had both urban and rural areas at the ward level based on the classification of the Central Bureau of Statistics in Nepal. Ward is the smallest local unit in Nepal. This classification was also used in the Demographic Health Survey in 2016 . Rural areas consisted of smaller wards with an average of 104 households per ward. Urban areas consisted of larger wards with an average of 800 households per ward . Participants We recruited mothers who were 15–49 years old, had a child aged 1–12 months, and gave birth at public healthcare facilities located in Dhading. The child’s age was set at 1–12 months because many women experience postpartum blues or mental disorders immediately after childbirth . We excluded women who were referred to a tertiary referral hospital in another district or could not speak the Nepali language. We also excluded women living in remote areas, where access was difficult. We calculated the sample size using Open Epi version 3.01 for the cross-sectional study. We calculated the number based on the previous study with a similar study design conducted by Abbott et al. , with 80% power and the significance level set at 5%. The required sample size was 310 for each urban and rural setting; thus, the total expected sample size was 620. We recruited women through the following procedure. First, we selected 5 out of 13 healthcare facilities in urban areas and then 6 out of 49 facilities in rural areas. These were purposively selected based on the number of deliveries in the previous fiscal year. Many healthcare facilities had fewer than 30 deliveries within the previous fiscal year, so we excluded these facilities. Hence, the included health facilities had more deliveries than others. Also, we considered the location of healthcare facilities and excluded the regions that were difficult to visit due to the feasibility of data collection; therefore, 10 healthcare facilities were excluded even though they had more than 30 deliveries within the previous fiscal year. Consequently, we included different types of public healthcare facilities: district hospitals, primary health centers, and health posts. The health post is the primary level public health center, which provides basic health services . Thereafter, we estimated the number of eligible women for each ward where the healthcare facilities were located to determine the required sample size per healthcare facility. Healthcare facility staff assisted us in obtaining the list of eligible women and their living wards. Finally, we visited wards to find these eligible women by asking local people about the areas of residence for the eligible women on the list. We finished the recruitment at the ward after enlisting the required number of eligible women purposively. Variables and assessment Exposure variable The exposure variable was the mothers’ perception of PCMC experience during childbirth. We used the PCMC scale for the assessment . This scale is validated and has been used in several low- and middle-income countries . It measures mothers’ experience in a healthcare facility during their latest delivery, and especially how they were treated at the facility. The PCMC scale consists of 30 items with 3 sub-scales: dignity and respect, communication and autonomy, and supportive care. Each item was coded from 0 to 3, and the total scores ranged from 0 to 90 points. For the following questions, reverse coding was conducted so that high scores equaled high PCMC: 1, 21, 22, and 26. When the participant answered “not applicable (N/A)”, it was scored with the highest point as suggested in the guideline of the questionnaire. A higher score indicates women who experienced better PCMC. Also, the Cronbach’s alpha was calculated for each scale to assess internal consistency. The Cronbach’s alpha was 0.93 for the PCMC scale. Outcome variables The outcome variables were maternal depressive symptoms and mental well-being after delivery. We set two outcome variables to measure the negative and positive sides of maternal mental health. The Edinburgh Postnatal Depression Scale (EPDS) was used to measure depressive symptoms . EPDS is a validated scale for screening postnatal depression, and we used the validated Nepali version of EPDS . EPDS has 10 items, each of which scored from 0 to 3; therefore, its total score ranges from 0 to 30. Reverse coding was conducted for the following questions: 3, 5–10. The cut-off point for the possibility of having depressive symptoms was EPDS score > 12, according to previous studies . In this study, the Cronbach’s alpha was 0.85 for EPDS. The Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) was used to assess mental well-being . WEMWBS is a validated scale that is used in a variety of settings, including low- and middle-income countries . Although the target population was not mothers, WEMWBS was validated in Nepal previously . WEMWBS consists of 14 positively-worded items with 5 response categories. The score ranges from 14 to 70. A higher score of WEMWBS represents higher mental well-being. The Cronbach’s alpha was 0.86 for WEMWBS. Other variables Mental status before delivery and support from family or social members were included as potential confounders. Previous mental status may confound because it is related to the perception of care during childbirth; it also becomes a risk factor for postpartum depression . Support from family or social members may confound because practical and emotional support from birth companions has been shown to positively affect the childbirth experience . A low level of social support was one of the risk factors for postpartum depression . We measured “support from family” and “support from social members” based on women’s perception toward the support, and the responses were Yes, No, or I don’t know. Socio-demographic factors and perinatal characteristics were also measured. These variables were used based on the questionnaire of the Nepal Demographic Health Survey in 2016 . Questionnaire Questions for the PCMC scale, WEMWBS, and other variables were translated from English into Nepali by two Nepali researchers and then back-translated into English by two different Nepali researchers. All researchers had a background in health sciences. We conducted a pilot study to test all scales and questions among 20 women living in the Kathmandu district. These data were not included in the main study. After the pilot study, we revised several phrases. For example, for greater specificity, we modified the possible responses regarding baby’s birth weight from “1. being very large to 5. being very small” to “1. being very large (>4,500 g) to 5. being very small (<1,500 g)”. Additional file shows the English version of the questionnaire used in this study. Data collection We collected data from July to August 2019 using a structured questionnaire. We visited households of eligible women and conducted face-to-face interviews. We hired eight research assistants with public health backgrounds for the data collection. They received a one-day training session for data collection, research, and ethical considerations. Data were collected using a paper-based questionnaire and then entered into EpiData 3.1. The first author double-checked the entered data. Participants with missing data for PCMC, EPDS, or WEMWBS were excluded from the analyses. Missing data on socio-demographic factors and perinatal characteristics were replaced with “I don’t know” or “N/A.” Data analyses We measured the differences in women’s characteristics between urban and rural areas using the Mann-Whitney test for age, chi-squared tests for other socio-demographic factors, and women’s perinatal characteristics. We performed the principal component analysis to estimate the household’s wealth score based on household wealth variables. We used the score to categorize women into five wealth quintiles from the 1st (poorest) to the 5th (richest). We calculated the means and standard deviations (SD) of the PCMC scale, EPDS, and WEMWBS. We compared the scores of each scale between urban and rural areas by using a t-test to observe whether there were any differences. We performed a multiple regression analysis to analyze the association of the PCMC experience with the levels of maternal depressive symptoms and mental well-being, adjusting for other variables. The following variables were controlled for in the analysis: mother’s age, religion, caste/ethnicity, education, occupation, wealth quintile, baby’s age, the number of antenatal care (ANC) visits, baby’s sex, baby’s birth weight, parity, length of stay at the healthcare facility after delivery, husband/partner living elsewhere, delivery (vaginal delivery or cesarean section), complication during pregnancy or delivery, diagnosis of previous mental problems, anxiety/stress/depressive symptoms during pregnancy, unwanted pregnancy, and family support after delivery. We have excluded “friends/relatives/community support during pregnancy” and “friends/relatives/community support after delivery” because the variance inflation factors were 10 or higher to avoid possible multi-collinearity. We conducted bootstrapping for the multiple regression analysis considering a cluster effect in each ward. Moreover, we performed a simple regression analysis to show the unadjusted results. The significance level was set at 5%. All data analyses were performed using Stata/SE 15. Ethical considerations In this study, we obtained ethical approval from the University of Tokyo Research Ethics Committee (Serial number: 2019088NI) and the Nepal Health Research Council (Reference number: 51). We obtained written informed consent before the interview. All the women voluntarily participated in this study, and we assured their confidentiality.
We conducted a cross-sectional study in Dhading District, Nepal. Dhading belongs to Bagmati Province and is adjacent to Kathmandu, the capital of Nepal. According to the preliminary Population Census 2021 result, the population of Dhading is 336,067 . Its population density is 168 per square kilometer, ranked 38th among all 77 districts in Nepal . As the study site, we covered all 2 municipalities and 5 out of 11 rural municipalities. These municipalities and rural municipalities had both urban and rural areas at the ward level based on the classification of the Central Bureau of Statistics in Nepal. Ward is the smallest local unit in Nepal. This classification was also used in the Demographic Health Survey in 2016 . Rural areas consisted of smaller wards with an average of 104 households per ward. Urban areas consisted of larger wards with an average of 800 households per ward .
We recruited mothers who were 15–49 years old, had a child aged 1–12 months, and gave birth at public healthcare facilities located in Dhading. The child’s age was set at 1–12 months because many women experience postpartum blues or mental disorders immediately after childbirth . We excluded women who were referred to a tertiary referral hospital in another district or could not speak the Nepali language. We also excluded women living in remote areas, where access was difficult. We calculated the sample size using Open Epi version 3.01 for the cross-sectional study. We calculated the number based on the previous study with a similar study design conducted by Abbott et al. , with 80% power and the significance level set at 5%. The required sample size was 310 for each urban and rural setting; thus, the total expected sample size was 620. We recruited women through the following procedure. First, we selected 5 out of 13 healthcare facilities in urban areas and then 6 out of 49 facilities in rural areas. These were purposively selected based on the number of deliveries in the previous fiscal year. Many healthcare facilities had fewer than 30 deliveries within the previous fiscal year, so we excluded these facilities. Hence, the included health facilities had more deliveries than others. Also, we considered the location of healthcare facilities and excluded the regions that were difficult to visit due to the feasibility of data collection; therefore, 10 healthcare facilities were excluded even though they had more than 30 deliveries within the previous fiscal year. Consequently, we included different types of public healthcare facilities: district hospitals, primary health centers, and health posts. The health post is the primary level public health center, which provides basic health services . Thereafter, we estimated the number of eligible women for each ward where the healthcare facilities were located to determine the required sample size per healthcare facility. Healthcare facility staff assisted us in obtaining the list of eligible women and their living wards. Finally, we visited wards to find these eligible women by asking local people about the areas of residence for the eligible women on the list. We finished the recruitment at the ward after enlisting the required number of eligible women purposively.
Exposure variable The exposure variable was the mothers’ perception of PCMC experience during childbirth. We used the PCMC scale for the assessment . This scale is validated and has been used in several low- and middle-income countries . It measures mothers’ experience in a healthcare facility during their latest delivery, and especially how they were treated at the facility. The PCMC scale consists of 30 items with 3 sub-scales: dignity and respect, communication and autonomy, and supportive care. Each item was coded from 0 to 3, and the total scores ranged from 0 to 90 points. For the following questions, reverse coding was conducted so that high scores equaled high PCMC: 1, 21, 22, and 26. When the participant answered “not applicable (N/A)”, it was scored with the highest point as suggested in the guideline of the questionnaire. A higher score indicates women who experienced better PCMC. Also, the Cronbach’s alpha was calculated for each scale to assess internal consistency. The Cronbach’s alpha was 0.93 for the PCMC scale. Outcome variables The outcome variables were maternal depressive symptoms and mental well-being after delivery. We set two outcome variables to measure the negative and positive sides of maternal mental health. The Edinburgh Postnatal Depression Scale (EPDS) was used to measure depressive symptoms . EPDS is a validated scale for screening postnatal depression, and we used the validated Nepali version of EPDS . EPDS has 10 items, each of which scored from 0 to 3; therefore, its total score ranges from 0 to 30. Reverse coding was conducted for the following questions: 3, 5–10. The cut-off point for the possibility of having depressive symptoms was EPDS score > 12, according to previous studies . In this study, the Cronbach’s alpha was 0.85 for EPDS. The Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) was used to assess mental well-being . WEMWBS is a validated scale that is used in a variety of settings, including low- and middle-income countries . Although the target population was not mothers, WEMWBS was validated in Nepal previously . WEMWBS consists of 14 positively-worded items with 5 response categories. The score ranges from 14 to 70. A higher score of WEMWBS represents higher mental well-being. The Cronbach’s alpha was 0.86 for WEMWBS. Other variables Mental status before delivery and support from family or social members were included as potential confounders. Previous mental status may confound because it is related to the perception of care during childbirth; it also becomes a risk factor for postpartum depression . Support from family or social members may confound because practical and emotional support from birth companions has been shown to positively affect the childbirth experience . A low level of social support was one of the risk factors for postpartum depression . We measured “support from family” and “support from social members” based on women’s perception toward the support, and the responses were Yes, No, or I don’t know. Socio-demographic factors and perinatal characteristics were also measured. These variables were used based on the questionnaire of the Nepal Demographic Health Survey in 2016 .
The exposure variable was the mothers’ perception of PCMC experience during childbirth. We used the PCMC scale for the assessment . This scale is validated and has been used in several low- and middle-income countries . It measures mothers’ experience in a healthcare facility during their latest delivery, and especially how they were treated at the facility. The PCMC scale consists of 30 items with 3 sub-scales: dignity and respect, communication and autonomy, and supportive care. Each item was coded from 0 to 3, and the total scores ranged from 0 to 90 points. For the following questions, reverse coding was conducted so that high scores equaled high PCMC: 1, 21, 22, and 26. When the participant answered “not applicable (N/A)”, it was scored with the highest point as suggested in the guideline of the questionnaire. A higher score indicates women who experienced better PCMC. Also, the Cronbach’s alpha was calculated for each scale to assess internal consistency. The Cronbach’s alpha was 0.93 for the PCMC scale.
The outcome variables were maternal depressive symptoms and mental well-being after delivery. We set two outcome variables to measure the negative and positive sides of maternal mental health. The Edinburgh Postnatal Depression Scale (EPDS) was used to measure depressive symptoms . EPDS is a validated scale for screening postnatal depression, and we used the validated Nepali version of EPDS . EPDS has 10 items, each of which scored from 0 to 3; therefore, its total score ranges from 0 to 30. Reverse coding was conducted for the following questions: 3, 5–10. The cut-off point for the possibility of having depressive symptoms was EPDS score > 12, according to previous studies . In this study, the Cronbach’s alpha was 0.85 for EPDS. The Warwick-Edinburgh Mental Wellbeing Scale (WEMWBS) was used to assess mental well-being . WEMWBS is a validated scale that is used in a variety of settings, including low- and middle-income countries . Although the target population was not mothers, WEMWBS was validated in Nepal previously . WEMWBS consists of 14 positively-worded items with 5 response categories. The score ranges from 14 to 70. A higher score of WEMWBS represents higher mental well-being. The Cronbach’s alpha was 0.86 for WEMWBS.
Mental status before delivery and support from family or social members were included as potential confounders. Previous mental status may confound because it is related to the perception of care during childbirth; it also becomes a risk factor for postpartum depression . Support from family or social members may confound because practical and emotional support from birth companions has been shown to positively affect the childbirth experience . A low level of social support was one of the risk factors for postpartum depression . We measured “support from family” and “support from social members” based on women’s perception toward the support, and the responses were Yes, No, or I don’t know. Socio-demographic factors and perinatal characteristics were also measured. These variables were used based on the questionnaire of the Nepal Demographic Health Survey in 2016 .
Questions for the PCMC scale, WEMWBS, and other variables were translated from English into Nepali by two Nepali researchers and then back-translated into English by two different Nepali researchers. All researchers had a background in health sciences. We conducted a pilot study to test all scales and questions among 20 women living in the Kathmandu district. These data were not included in the main study. After the pilot study, we revised several phrases. For example, for greater specificity, we modified the possible responses regarding baby’s birth weight from “1. being very large to 5. being very small” to “1. being very large (>4,500 g) to 5. being very small (<1,500 g)”. Additional file shows the English version of the questionnaire used in this study.
We collected data from July to August 2019 using a structured questionnaire. We visited households of eligible women and conducted face-to-face interviews. We hired eight research assistants with public health backgrounds for the data collection. They received a one-day training session for data collection, research, and ethical considerations. Data were collected using a paper-based questionnaire and then entered into EpiData 3.1. The first author double-checked the entered data. Participants with missing data for PCMC, EPDS, or WEMWBS were excluded from the analyses. Missing data on socio-demographic factors and perinatal characteristics were replaced with “I don’t know” or “N/A.”
We measured the differences in women’s characteristics between urban and rural areas using the Mann-Whitney test for age, chi-squared tests for other socio-demographic factors, and women’s perinatal characteristics. We performed the principal component analysis to estimate the household’s wealth score based on household wealth variables. We used the score to categorize women into five wealth quintiles from the 1st (poorest) to the 5th (richest). We calculated the means and standard deviations (SD) of the PCMC scale, EPDS, and WEMWBS. We compared the scores of each scale between urban and rural areas by using a t-test to observe whether there were any differences. We performed a multiple regression analysis to analyze the association of the PCMC experience with the levels of maternal depressive symptoms and mental well-being, adjusting for other variables. The following variables were controlled for in the analysis: mother’s age, religion, caste/ethnicity, education, occupation, wealth quintile, baby’s age, the number of antenatal care (ANC) visits, baby’s sex, baby’s birth weight, parity, length of stay at the healthcare facility after delivery, husband/partner living elsewhere, delivery (vaginal delivery or cesarean section), complication during pregnancy or delivery, diagnosis of previous mental problems, anxiety/stress/depressive symptoms during pregnancy, unwanted pregnancy, and family support after delivery. We have excluded “friends/relatives/community support during pregnancy” and “friends/relatives/community support after delivery” because the variance inflation factors were 10 or higher to avoid possible multi-collinearity. We conducted bootstrapping for the multiple regression analysis considering a cluster effect in each ward. Moreover, we performed a simple regression analysis to show the unadjusted results. The significance level was set at 5%. All data analyses were performed using Stata/SE 15.
In this study, we obtained ethical approval from the University of Tokyo Research Ethics Committee (Serial number: 2019088NI) and the Nepal Health Research Council (Reference number: 51). We obtained written informed consent before the interview. All the women voluntarily participated in this study, and we assured their confidentiality.
Socio-demographic characteristics of women In this study, we recruited 314 women from urban areas and 308 women from rural areas. Due to incomplete or missing data, we excluded 18 women from urban areas and 9 women from rural areas. In total, we included 595 (95.7%) women for data analyses. Table shows the socio-demographic characteristics of the women included in this study. The mean age was 24.8 years (SD 4.5) in urban areas and 23.2 years (SD 4.2) in rural areas. While 25.3% of women completed secondary education or higher in urban areas, only 16.7% completed it in rural areas. Regarding the wealth quintile, women who were categorized in the 4th (richer) or 5th (richest) quintile were 44.6% in urban areas and 35.5% in rural areas. Perinatal characteristics of women Table shows women’s perinatal characteristics. More than 80% of women received ANC four times or more (83.8% in urban areas and 87.0% in rural areas) and had a vaginal delivery (94.3% in urban areas and 99.3% in rural areas). In urban areas, 79.4% of women lived with their husbands, while this was 88.6% in rural areas. Less than 5% of women experienced complications during the last delivery in both areas (4.7% in urban areas and 3.7% in rural areas). Scores of each scale The mean score of the PCMC scale was higher in rural areas (66.7, SD 12.1) than in urban areas (63.3, SD 13.5) ( p = 0.002). In EPDS, the mean score was 5.25 (SD 5.42) in urban areas, and was 5.04 (SD 4.59) in rural areas. The proportion of women with scores over the EPDS cut-off point was 12.2% in urban areas and 8.4% in rural areas ( p = 0.126). The mean score of WEMWBS was 56.1 (SD 7.52) in urban areas and 58.1 (SD 7.27) in rural areas ( p = 0.008). Additional file shows the histogram for the scores of each scale. Association between PCMC experience and depressive symptoms Table shows the factors associated with depressive symptoms among women living in Dhading. In urban areas, the experience of better PCMC was associated with lower depressive symptom scores (unstandardized coefficient [B]= − 0.09, 95% CI: − 0.13, − 0.04). It means for each additional score increased in PCMC, the score of depressive symptoms decreased by an average of − 0.09 points, holding the other exposure variables constant. Among other variables, the poorest wealth quintile was associated with lower depressive symptom scores (B= − 2.55, 95% CI: − 3.81, − 1.28), compared to the middle quintile. It means the score of depressive symptoms was − 2.55 points lower for women with the poorest wealth quintile than for the reference group (middle wealth quintile). The following factors were significantly associated with higher depressive symptom scores: working as a farmer (B= 3.77, 95% CI: 1.47, 6.08, compared with housewife), staying at the healthcare facility after delivery for 48–72 h (B= 4.99, 95% CI: 2.54, 7.43), and longer than 72 h (B= 2.76, 95% CI: 0.11, 5.40) compared to staying for 24–48 h. In addition, having no formal education (B= 2.03, 95% CI: 0.10, 3.96, compared with secondary education), having visited ANC less than 4 times (B= 2.00, 95% CI: 0.04, 3.97), and having been diagnosed with mental problems previously (B= 4.41, 95% CI: 2.20, 6.60) were associated with higher depressive symptom scores, which was unique in urban areas. Likewise, the experience of better PCMC was associated with lower depressive symptom scores in rural areas (B= − 0.10, 95% CI: − 0.15, − 0.04). The following factors were associated with higher depressive symptom scores: working as a farmer (B= 2.33, 95% CI: 0.88, 3.78, compared to housewife), staying at the healthcare facility after delivery for 48–72 h (B= 1.75, 95% CI: 0.35, 3.15) and longer than 72 h (B= 3.83, 95% CI: 1.87, 5.80) compared to staying for 24–48 h. In addition, having a husband living elsewhere (B= 2.15, 95% CI: 0.21, 4.09), feeling anxious during pregnancy (B= 2.89, 95% CI: 1.20, 4.59), and delivering a baby with cesarean section (B= − 5.68, 95% CI: − 8.60, − 2.76, compared with vaginal delivery) were associated with higher depressive symptom scores, which was unique in rural areas. Association between PCMC experience and mental well-being Table shows the factors associated with mental well-being among women living in Dhading. In urban areas, the experience of better PCMC was associated with higher mental well-being scores (B= 0.30, 95% CI: 0.25, 0.35). Increased age was associated with higher mental well-being scores (B= 1.07, 95% CI: 0.14, 2.00), which was unique in urban areas. In rural areas, the experience of better PCMC was associated with higher mental well-being scores (B= 0.15, 95% CI: 0.03, 0.27). However, the following factors were also associated with lower mental well-being scores: delivering a baby girl (B= − 1.78, 95% CI: − 3.51, − 0.06), having a husband living elsewhere (B= − 2.58, 95% CI: − 4.60, − 0.56), and unwanted pregnancy (B= − 3.65, 95% CI: − 5.53, − 1.77).
In this study, we recruited 314 women from urban areas and 308 women from rural areas. Due to incomplete or missing data, we excluded 18 women from urban areas and 9 women from rural areas. In total, we included 595 (95.7%) women for data analyses. Table shows the socio-demographic characteristics of the women included in this study. The mean age was 24.8 years (SD 4.5) in urban areas and 23.2 years (SD 4.2) in rural areas. While 25.3% of women completed secondary education or higher in urban areas, only 16.7% completed it in rural areas. Regarding the wealth quintile, women who were categorized in the 4th (richer) or 5th (richest) quintile were 44.6% in urban areas and 35.5% in rural areas.
Table shows women’s perinatal characteristics. More than 80% of women received ANC four times or more (83.8% in urban areas and 87.0% in rural areas) and had a vaginal delivery (94.3% in urban areas and 99.3% in rural areas). In urban areas, 79.4% of women lived with their husbands, while this was 88.6% in rural areas. Less than 5% of women experienced complications during the last delivery in both areas (4.7% in urban areas and 3.7% in rural areas).
The mean score of the PCMC scale was higher in rural areas (66.7, SD 12.1) than in urban areas (63.3, SD 13.5) ( p = 0.002). In EPDS, the mean score was 5.25 (SD 5.42) in urban areas, and was 5.04 (SD 4.59) in rural areas. The proportion of women with scores over the EPDS cut-off point was 12.2% in urban areas and 8.4% in rural areas ( p = 0.126). The mean score of WEMWBS was 56.1 (SD 7.52) in urban areas and 58.1 (SD 7.27) in rural areas ( p = 0.008). Additional file shows the histogram for the scores of each scale.
Table shows the factors associated with depressive symptoms among women living in Dhading. In urban areas, the experience of better PCMC was associated with lower depressive symptom scores (unstandardized coefficient [B]= − 0.09, 95% CI: − 0.13, − 0.04). It means for each additional score increased in PCMC, the score of depressive symptoms decreased by an average of − 0.09 points, holding the other exposure variables constant. Among other variables, the poorest wealth quintile was associated with lower depressive symptom scores (B= − 2.55, 95% CI: − 3.81, − 1.28), compared to the middle quintile. It means the score of depressive symptoms was − 2.55 points lower for women with the poorest wealth quintile than for the reference group (middle wealth quintile). The following factors were significantly associated with higher depressive symptom scores: working as a farmer (B= 3.77, 95% CI: 1.47, 6.08, compared with housewife), staying at the healthcare facility after delivery for 48–72 h (B= 4.99, 95% CI: 2.54, 7.43), and longer than 72 h (B= 2.76, 95% CI: 0.11, 5.40) compared to staying for 24–48 h. In addition, having no formal education (B= 2.03, 95% CI: 0.10, 3.96, compared with secondary education), having visited ANC less than 4 times (B= 2.00, 95% CI: 0.04, 3.97), and having been diagnosed with mental problems previously (B= 4.41, 95% CI: 2.20, 6.60) were associated with higher depressive symptom scores, which was unique in urban areas. Likewise, the experience of better PCMC was associated with lower depressive symptom scores in rural areas (B= − 0.10, 95% CI: − 0.15, − 0.04). The following factors were associated with higher depressive symptom scores: working as a farmer (B= 2.33, 95% CI: 0.88, 3.78, compared to housewife), staying at the healthcare facility after delivery for 48–72 h (B= 1.75, 95% CI: 0.35, 3.15) and longer than 72 h (B= 3.83, 95% CI: 1.87, 5.80) compared to staying for 24–48 h. In addition, having a husband living elsewhere (B= 2.15, 95% CI: 0.21, 4.09), feeling anxious during pregnancy (B= 2.89, 95% CI: 1.20, 4.59), and delivering a baby with cesarean section (B= − 5.68, 95% CI: − 8.60, − 2.76, compared with vaginal delivery) were associated with higher depressive symptom scores, which was unique in rural areas.
Table shows the factors associated with mental well-being among women living in Dhading. In urban areas, the experience of better PCMC was associated with higher mental well-being scores (B= 0.30, 95% CI: 0.25, 0.35). Increased age was associated with higher mental well-being scores (B= 1.07, 95% CI: 0.14, 2.00), which was unique in urban areas. In rural areas, the experience of better PCMC was associated with higher mental well-being scores (B= 0.15, 95% CI: 0.03, 0.27). However, the following factors were also associated with lower mental well-being scores: delivering a baby girl (B= − 1.78, 95% CI: − 3.51, − 0.06), having a husband living elsewhere (B= − 2.58, 95% CI: − 4.60, − 0.56), and unwanted pregnancy (B= − 3.65, 95% CI: − 5.53, − 1.77).
In this study, women who received better PCMC had lower depressive symptom scores and were more likely to have higher mental well-being after delivery, regardless of urban or rural settings. Through the analysis, other factors were also found to be associated with mental health status. Although we expected to see differences in the association of PCMC with mental health outcomes between the urban and rural areas, we found similar results in both areas. In this study, women who experienced better PCMC had lower depressive symptom scores, both in urban and rural areas. Women’s satisfaction with the medical staff members’ attitudes during childbirth was negatively associated with postpartum post-traumatic stress in Turkey . In Kenya, PCMC improved women’s self-efficacy and positively affected maternal and child health . PCMC during delivery decreases anxiety or negative feeling toward childbirth. However, disrespect and abusive care experiences during childbirth were associated with women’s postpartum depression in Brazil . These findings are consistent with the results of the current study. Though the association between PCMC and depressive symptoms was statistically significant, the effect size was relatively small. One reason could be that depressive symptoms are complex, and various factors influence them. Another reason might be that we assessed PCMC through self-reported scores, which could underestimate the inappropriateness of care. Nevertheless, when creating a favorable delivery environment, it is required that healthcare providers can provide adequate PCMC. This study also found that women who experienced better PCMC were more likely to have higher mental well-being after delivery in urban and rural settings. Building a favorable relationship between mothers and midwives during childbirth was essential for mothers to have a positive birth experience . In Iran, positive childbirth experiences improved women’s self-efficacy and self-esteem after delivery . Furthermore, in Rwanda, sufficient support and adequate maternity care increased the health-related quality of life for women who delivered within 1–13 months . Women who trust medical staff members might seek healthcare when they encounter health problems after delivery and thus receive support. Therefore, PCMC during delivery contributes to improving women’s well-being after delivery by providing positive childbirth experiences and promoting sufficient support after delivery. Initially, we expected that the associations of PCMC with depressive symptoms and mental well-being in urban areas would be different from those in rural areas. Previous studies in Nepal reported that 17.1–30.3% of mothers had depressive symptoms in urban areas, while 9.8% of women were distressed in rural areas [ , , , ]. The mean PCMC scores also varied depending on the settings. For example, one study reported that urban Kenya presented the highest mean score, while rural Ghana showed the lowest mean score . Additionally, a previous systematic review showed that the mismatch between birth expectation and experience can decrease birth satisfaction and possibly increase postnatal post-traumatic stress disorder . Since urban areas have more facility type options, such as private hospitals vs. public facilities, this might increase expectations and consequently, the association between mothers’ delivery experience and maternal mental health would be stronger in urban areas than in rural areas. Since Nepal has a multiethnic and multicultural society , we hypothesized that the setting might influence the association between PCMC and mental health. However, we attained similar results for both urban and rural areas, that is, although the magnitudes were not the same, there was a negative association between PCMC and depressive symptoms and a positive association between PCMC and mental well-being in both urban and rural areas. This may be because we compared urban and rural areas within the same district. We classified areas called “municipalities” as urban areas and areas classified “rural municipalities” as rural areas based on the Nepalese administrative categorization. Therefore, those areas included in this study may not necessarily represent urban or rural settings. Another reason could be that urban-rural settings may not matter regarding PCMC, as it is based on women’s customs and culture . Therefore, PCMC can improve maternal mental health regardless of the settings. Further research is required to determine how the living settings influence PCMC and maternal mental health. Along with PCMC, factors such as being a farmer, a longer stay in a healthcare facility after delivery, and previous mental problems were associated with depressive symptoms in both urban and rural areas. Working women were more likely to have postnatal depression in India . Women working as farmers might have difficulties when working and parenting concurrently. Clinical problems may extend the length of stay . Adverse reproductive health was associated with depressive symptoms . Furthermore, socio-psychological problems were risk factors for developing depressive symptoms after delivery . In urban areas, lower education levels, fewer ANC visits, and the poorest wealth quintile were factors associated with depressive symptoms, while increased age was the unique factor associated with higher mental well-being. Women’s education level was one of the barriers to using adequate ANC in India . Fewer ANC visits increased the risk of depressive symptoms due to a lower chance of consulting and receiving support . However, women in the poorest wealth quintile had lower depressive symptom scores in this study. This could be because the wealth quintile did not include household income and overseas remittances or because the women got special support. In addition, increased age was one of the determinants of decision-making toward sexual and reproductive health in Nepal . In rural areas, delivering a baby girl, having a husband living elsewhere, an unwanted pregnancy, and cesarean section were factors associated with mental well-being. Women reported some pressure to have sons, which was related to virilocal or patrilineal inheritance in Nepal . In Vietnam, negative family responses to the baby reduced mental well-being . Poor relationships with husbands or a lack of partner support were also risk factors for depressive symptoms . Husband’s involvement was important for maternal health and safe childbirth . Experience of unwanted pregnancy was also associated with maternal complications in rural India and reported as a risk factor for postpartum depressive symptoms . Cesarean section was shown to increase the risk of depressive symptoms in Nepal in a previous study . However, we could not verify the association between depressive symptoms and cesarean section from our results since only a few women delivered through a cesarean section in rural areas. This study had several limitations: First, participating women were not randomly recruited from all the eligible women in the selected areas. Due to our sampling method, we might have missed certain eligible women who were not introduced to us by local people. Second, this is a cross-sectional study, and the possibility of reverse causality exists for women’s delivery care experience and maternal mental health. Many factors are associated with childbirth care experiences and mental health, and unknown confounders should be considered. Furthermore, recall of childbirth experiences or mental status can change over time. Third, we conducted a one-day training for the research but did not measure inter-rater reliability. Despite some limitations, this is one of the few studies on the association between PCMC experiences and maternal mental health after delivery in Nepal. Healthcare facilities require further improvement regarding PCMC aspects, especially during childbirth care. For example, healthcare staff is required to have more effective communication, pay attention to women’s needs and feelings, and maintain safe facilities. Such improvements in PCMC may contribute to better maternal mental health. The findings may have implications for constructing better healthcare systems in Nepal and in other resource-limited settings.
This study showed that PCMC during childbirth benefits women’s maternal mental health after delivery. Healthcare facilities are required to improve quality care by increasing the awareness of PCMC aspects. Providing suitable and equitable maternity care is important and can be done by considering the various backgrounds of care users. Further research is required to determine better PCMC for women to build better quality care.
Below is the link to the electronic supplementary material. Additional File 1: Questionnaire Additional File 2: Histogram for each scale Additional File 3: Datasets
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The impact of COVID-19 on the interpretation of psycho-oncological support trial results: a quasi-experimental approach using the data from the new form of care “Integrated cross-sectoral psycho-oncology (nFC-isPO)”
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cc9ad3ed-4502-4e4c-9938-9541adae6fe0
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10228457
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Internal Medicine[mh]
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COVID-19 has taken a toll on people and health systems around the world, and many studies have examined this novel disease and its effects . In addition to the general impact and the impact on the health system, COVID-19 also affected ongoing trials, e.g. in form of changes in the shape of care supply. These challenges were thus in addition to the usual challenges such as slow recruitment or managing multi-site. However, these problems with trials did not only affect providers, but especially vulnerable patient groups who had a higher risk of infection due to pre-existing conditions and a correspondingly weakened immune system . One of these vulnerable groups was patients with cancer, especially those who received their first diagnosis during the pandemic. A cancer diagnosis is already a stressful and drastic event and often leads to stress, depression and especially anxiety. When patients receive such a diagnosis in an exceptional situation such as the COVID-19 pandemic, psychological stress is compounded in many cases . For this reason, holistic psycho-oncological treatment, i.e. in addition to treating the cause of the cancer, the inclusion of mental health and its treatment is an important component . The project and the new form of care (nFC) “Integrated cross-sectoral psycho-oncology” (isPO) started in 2017 with precisely this intention, namely holistic psycho-oncological care and above all with the aim of creating structured needs-oriented psycho-oncological care in Germany, and was then challenged by the consequences of the pandemic during the project period . isPO was conducted at four sites in North-Rhein Westphalia using a quasi-experimental study design, the regression discontinuity design (RDD) in combination with the Hospital Anxiety and Depression Scale (HADS) to assess individual psycho-oncological needs . The new form of care aims to successfully reduce anxiety and depression within the first year after cancer diagnosis in patients with cancer through psychological, psychotherapeutical and psychosocial measures. Given the study design and psychological treatment, the aim is to determine whether HADS scores decrease in the intervention group. During the study, after the COVID-19 pandemic onset (March 2020) , the four isPO care networks provided feedback that higher HADS values were observed in many patients, and the potential for anxiety and depression increased . Therefore, the question arose as to how the influence of the pandemic can be taken into account in the evaluation of the study. This should be considered with regard to effectiveness in terms of an increase in HADS scores, but also with regard to necessary adjustments in care, such as switching from face-to-face care to care via telephone and video telephony. The aim is to discuss whether the higher HADS values observed by caregivers are reflected in the study results by performing stratified analyses (descriptive and using RDD) according to the time of enrolment in the study programme (T1).
General study information and study design In isPO, newly diagnosed patients with cancer were offered psycho-oncological care at different levels of care according to their individual needs which are assessed using the HADS. Patients were requested to complete the HADS questionnaire at three different time points (Baseline (T1), after 4 months of care (T2) and after 12 months of care (T3)). T1-HADS scores are used for assignment to the different levels of care and thus for classification into control and intervention groups. This is done as a quasi-randomization using the RDD, which uses a threshold criterion to divide patients into control and treatment groups and measure treatment effect by comparing the intercepts of regression equations above and below a predefined threshold and within a bandwidth . In isPO patients with baseline HADS values ≤ 14 were allocated to the control group (psychosocial intervention). Patients with higher values (≥ 15) received psycho-oncological, psychotherapeutical treatment (intervention group). T2 and T3 (primary endpoint) are used to compare the allocated patients around the threshold and measure their psychological needs after receiving the individual treatment. Therefore, patients with less distress at the first time point were compared to those with higher distress. This procedure aims to record and review the effectiveness of psycho-oncological psychotherapeutical care . Data collection and analysis population N = 1,757 newly diagnosed patients with cancer were recruited in the period 01/2019-03/2021. Data were collected at four isPO care networks (Cologne, Troisdorf, Mönchengladbach and Neuss) in North Rhine-Westphalia (NRW) and recorded in a computer-assisted assistance system (CAPSYS 2020 ) developed for the isPO project. For proper analysis of stratified data and primary end point at T3, only cases followed for 12 months were considered (N = 1,140). The main analysis (RDD) compares the HADS scores of N = 203 patients within the two strata (T1 pre-pandemic (N = 96) vs. T1 post-pandemic (N = 107)) within the pre defined bandwidth of 13–16 points at baseline. All patients provided written informed consent. A detailed overview of the study and analysis population is shown in Fig. . Instruments The main instrument of the isPO study was the Hospital Anxiety and Depression Scale (HADS). The HADS consists of 14 items, 7 of which are assigned to the depression subscale and 7 to the anxiety subscale with a score from 0 to 3 for each response. The added values of the individual items result in the subscales, whereby values between 0 and 7 are to be interpreted as inconspicuous, values from 8 to 10 as suspicious and values > 10 as conspicuous. The total score ranges from 0 to 42. Based on the interpretation of the subscales, 14.5 was chosen as the threshold for group assignment for the regression discontinuity design within the nFC-isPO . Statistics Simple stratification by time intervals was used to account for the pandemic influence. Therefore, an additional categorical variable in the database was built. The variable considers at what point in the study a patient completed the HADS questionnaire at timepoint T1 (baseline). This variable was then used to stratify patients into a pre- and post-pandemic groups (T1 in Q1/19-Q1/20 vs. T1 in Q2/20-Q2/21), to identify differences between patients´ anxiety and depression levels over time and to explore possible differences during the chronological trend. First, descriptive analyses were conducted to provide an overview of the study population. Therefore, categorical variables are reported as absolute and relative frequencies in percentage. For continuous variables mean with standard deviation and median with interquartile range are given. Next, RDD analysis were conducted in the two strata to measure the effect of the psycho-oncological treatment. An RDD analysis is based on simple linear regression in which two regression equations are compared below and above a threshold . The difference between the two regression lines or the difference between the two axis intercepts at the pre defined threshold then gives the treatment effect. This is also called the local average treatment effect (LATE) and is graphically represented as a discontinuity (jump) of the regression lines at the threshold . In addition, mean baseline HADS scores were compared with HADS scores after 12 months of care to show changes over time. All analyses were performed using IBM SPSS Version 28 with available R extensions, e.g. regression discontinuity analysis and figures were generated with R Version 4.1.2. Results were considered statistically significant for p ≤ 0.05 with α = 0.05 as the significance level.
In isPO, newly diagnosed patients with cancer were offered psycho-oncological care at different levels of care according to their individual needs which are assessed using the HADS. Patients were requested to complete the HADS questionnaire at three different time points (Baseline (T1), after 4 months of care (T2) and after 12 months of care (T3)). T1-HADS scores are used for assignment to the different levels of care and thus for classification into control and intervention groups. This is done as a quasi-randomization using the RDD, which uses a threshold criterion to divide patients into control and treatment groups and measure treatment effect by comparing the intercepts of regression equations above and below a predefined threshold and within a bandwidth . In isPO patients with baseline HADS values ≤ 14 were allocated to the control group (psychosocial intervention). Patients with higher values (≥ 15) received psycho-oncological, psychotherapeutical treatment (intervention group). T2 and T3 (primary endpoint) are used to compare the allocated patients around the threshold and measure their psychological needs after receiving the individual treatment. Therefore, patients with less distress at the first time point were compared to those with higher distress. This procedure aims to record and review the effectiveness of psycho-oncological psychotherapeutical care .
N = 1,757 newly diagnosed patients with cancer were recruited in the period 01/2019-03/2021. Data were collected at four isPO care networks (Cologne, Troisdorf, Mönchengladbach and Neuss) in North Rhine-Westphalia (NRW) and recorded in a computer-assisted assistance system (CAPSYS 2020 ) developed for the isPO project. For proper analysis of stratified data and primary end point at T3, only cases followed for 12 months were considered (N = 1,140). The main analysis (RDD) compares the HADS scores of N = 203 patients within the two strata (T1 pre-pandemic (N = 96) vs. T1 post-pandemic (N = 107)) within the pre defined bandwidth of 13–16 points at baseline. All patients provided written informed consent. A detailed overview of the study and analysis population is shown in Fig. .
The main instrument of the isPO study was the Hospital Anxiety and Depression Scale (HADS). The HADS consists of 14 items, 7 of which are assigned to the depression subscale and 7 to the anxiety subscale with a score from 0 to 3 for each response. The added values of the individual items result in the subscales, whereby values between 0 and 7 are to be interpreted as inconspicuous, values from 8 to 10 as suspicious and values > 10 as conspicuous. The total score ranges from 0 to 42. Based on the interpretation of the subscales, 14.5 was chosen as the threshold for group assignment for the regression discontinuity design within the nFC-isPO .
Simple stratification by time intervals was used to account for the pandemic influence. Therefore, an additional categorical variable in the database was built. The variable considers at what point in the study a patient completed the HADS questionnaire at timepoint T1 (baseline). This variable was then used to stratify patients into a pre- and post-pandemic groups (T1 in Q1/19-Q1/20 vs. T1 in Q2/20-Q2/21), to identify differences between patients´ anxiety and depression levels over time and to explore possible differences during the chronological trend. First, descriptive analyses were conducted to provide an overview of the study population. Therefore, categorical variables are reported as absolute and relative frequencies in percentage. For continuous variables mean with standard deviation and median with interquartile range are given. Next, RDD analysis were conducted in the two strata to measure the effect of the psycho-oncological treatment. An RDD analysis is based on simple linear regression in which two regression equations are compared below and above a threshold . The difference between the two regression lines or the difference between the two axis intercepts at the pre defined threshold then gives the treatment effect. This is also called the local average treatment effect (LATE) and is graphically represented as a discontinuity (jump) of the regression lines at the threshold . In addition, mean baseline HADS scores were compared with HADS scores after 12 months of care to show changes over time. All analyses were performed using IBM SPSS Version 28 with available R extensions, e.g. regression discontinuity analysis and figures were generated with R Version 4.1.2. Results were considered statistically significant for p ≤ 0.05 with α = 0.05 as the significance level.
Characteristics of the patients A total of 1,140 patients completed the HADS questionnaire after 12 months. Of these, 575 completed the HADS for the first time between Q1/19-Q1/20, before the pandemic, and 565 between Q2/20-Q2/21, after the pandemic outbreak. The gender ratio was about two-thirds women to one-third men with a median age of about 58 years (IQR=[50–66], mean = 56.8, SD = 13.1). The most common diagnoses were breast cancer (30%) and malignant neoplasms of the digestive organs (13.3%). An overview of all sociodemographic and clinical patient characteristics is given in Table . Modification of HADS The mean and median baseline (T1) HADS scores of the anxiety subscale are minimally higher in the group during the pandemic (9.0 ± 4.4 and 9 [6;12]) than in the group before the pandemic (8.8 ± 4.6 and 9 [5;12]). The same applies to the depression subscale (7.1 ± 4.6 and 6 [4;10] vs. 6.5 ± 4.5 and 6 [3;9]). However, for both groups (before and during the pandemic), it is visible that the T3 HADS decreases to a similar extent, by about 2 points each for the anxiety subscale and by about 1 point each for the depression subscale (see Table ). Primary outcome pre- vs. post-pandemic Scatterplots with all cases of the two groups according to time within the pandemic are shown (Fig. 2). In both groups, i.e. in the control group below the threshold and in the treatment group above the threshold, the higher the T1-HADS score, the higher the T3-HADS score. The second row of Fig. 2 then shows the applied RDD analysis in the previously defined relevant section (bandwidth) of the entire collective. The area of interest was the bandwidth from 13 to 16 points on the HADS scale. The pre-pandemic group consisted in total of N = 575 patients (50.4%), of which N = 96 patients lie within the pre defined bandwidth. The post-pandemic group had a total N of 565 patients (49.6%) and N = 107 patients within the bandwidth. Both groups show a discontinuity of the regression lines in the area around the threshold. However, the local treatment effect (LATE) measured in each case is not significant. In addition, only the local treatment effect of the post-pandemic group points in the right direction (LATE=-0.2933), as it is negative and thus indicates an improvement (reduction) in the HADS values. Mean HADS over time In addition to the primary outcome, mean baseline and HADS scores after 12 months of treatment are compared in Fig. . Across all quarters, the overall mean HADS baseline scores are found to be in a very similar range between 15 and 16 points. 12 months after treatment, the HADS scores are also in a very similar range between 12 and 14 points. A direct comparison of the mean HADS scores at baseline and 12 months after treatment shows a reduction of about 2–3 points. Comparing the control and intervention groups, the mean pre-treatment HADS scores in the intervention group at baseline (T1) are around 22 points across all quarters, well above the chosen threshold. The mean HADS scores of the control group, on the other hand, were around 8 to 9 points and thus, as with the intervention group, not in the range of 13–16 points relevant for RDD analysis. Comparing the mean HADS scores over time after 12 months of treatment between the two groups (intervention vs. control), it is visible that in the intervention group there was a reduction of the scores from over 20 points at the beginning of treatment to about 15 to 17 points after 12 months. In the control group, on the other hand, a slight deterioration, i.e. an increase in the scores, can be seen.
A total of 1,140 patients completed the HADS questionnaire after 12 months. Of these, 575 completed the HADS for the first time between Q1/19-Q1/20, before the pandemic, and 565 between Q2/20-Q2/21, after the pandemic outbreak. The gender ratio was about two-thirds women to one-third men with a median age of about 58 years (IQR=[50–66], mean = 56.8, SD = 13.1). The most common diagnoses were breast cancer (30%) and malignant neoplasms of the digestive organs (13.3%). An overview of all sociodemographic and clinical patient characteristics is given in Table .
The mean and median baseline (T1) HADS scores of the anxiety subscale are minimally higher in the group during the pandemic (9.0 ± 4.4 and 9 [6;12]) than in the group before the pandemic (8.8 ± 4.6 and 9 [5;12]). The same applies to the depression subscale (7.1 ± 4.6 and 6 [4;10] vs. 6.5 ± 4.5 and 6 [3;9]). However, for both groups (before and during the pandemic), it is visible that the T3 HADS decreases to a similar extent, by about 2 points each for the anxiety subscale and by about 1 point each for the depression subscale (see Table ).
Scatterplots with all cases of the two groups according to time within the pandemic are shown (Fig. 2). In both groups, i.e. in the control group below the threshold and in the treatment group above the threshold, the higher the T1-HADS score, the higher the T3-HADS score. The second row of Fig. 2 then shows the applied RDD analysis in the previously defined relevant section (bandwidth) of the entire collective. The area of interest was the bandwidth from 13 to 16 points on the HADS scale. The pre-pandemic group consisted in total of N = 575 patients (50.4%), of which N = 96 patients lie within the pre defined bandwidth. The post-pandemic group had a total N of 565 patients (49.6%) and N = 107 patients within the bandwidth. Both groups show a discontinuity of the regression lines in the area around the threshold. However, the local treatment effect (LATE) measured in each case is not significant. In addition, only the local treatment effect of the post-pandemic group points in the right direction (LATE=-0.2933), as it is negative and thus indicates an improvement (reduction) in the HADS values.
In addition to the primary outcome, mean baseline and HADS scores after 12 months of treatment are compared in Fig. . Across all quarters, the overall mean HADS baseline scores are found to be in a very similar range between 15 and 16 points. 12 months after treatment, the HADS scores are also in a very similar range between 12 and 14 points. A direct comparison of the mean HADS scores at baseline and 12 months after treatment shows a reduction of about 2–3 points. Comparing the control and intervention groups, the mean pre-treatment HADS scores in the intervention group at baseline (T1) are around 22 points across all quarters, well above the chosen threshold. The mean HADS scores of the control group, on the other hand, were around 8 to 9 points and thus, as with the intervention group, not in the range of 13–16 points relevant for RDD analysis. Comparing the mean HADS scores over time after 12 months of treatment between the two groups (intervention vs. control), it is visible that in the intervention group there was a reduction of the scores from over 20 points at the beginning of treatment to about 15 to 17 points after 12 months. In the control group, on the other hand, a slight deterioration, i.e. an increase in the scores, can be seen.
The nFC-isPO aimed to reduce anxiety and depression in patients with cancer as measured by HADS. However, due to the pandemic, it was suspected that patients would be exposed to additional psychological distress on top of the already high psychological distress caused by cancer, which could prejudice the treatment effect. Therefore, stratified analyses for primary outcome- (T3, 12 months of care) were performed in this article to examine the effects of COVID-19 in the isPO trial. In contrast to the initial feedback from the four isPO care networks, the results of our descriptive analyses over time showed that the HADS scores in the post-pandemic group were only slightly higher on average or median overall than in the pre-pandemic group. This is also shown in other studies, such as Bargon et al. being one of the few studies that examines the association between mental health in relation to cancer and the impact of the pandemic. Many other studies investigated COVID-19 and the resulting deterioration in mental health . isPO, tried to reduce anxiety and depression in newly diagnosed patients with cancer with a stepped care approach. It was started before and carried on during the pandemic while facing additional challenges as a result of the pandemic. Even before the pandemic, supporting and counseling vulnerable groups, e.g. patients with cancer, was considered as very important . In the face of additional sudden factors, like the pandemic, it is even more important to avoid additional stress. This is also reflected in the results of isPO, as the average anxiety and depression scores before the outbreak of the pandemic hardly changed compared to after. On the other hand, cancer studies without psycho-oncology stepped care approach, show significantly higher anxiety and depression scores due to an external factor such as COVID-19 . It can be concluded that needs-oriented psycho-oncological support has a positive impact and reduces anxiety not only with regard to the cancer diagnosis, but also with regard to other medical factors/problems that have a negative impact on anxiety and depression, such as COVID-19 . This implies that because the isPO-patients already received personalized psycho-oncological care during the pandemic, the HADS scores did not increase significantly compared to patients who received a cancer diagnosis during the pandemic without additional psycho-oncological care (no nFC-isPO) . Regarding the results of the RDD analysis, the comparison of the pandemic groups showed that the discontinuity, i.e. treatment effect, increased in the pre-pandemic intervention group instead of decreasing as expected. In comparison, the post-pandemic group showed the expected decrease in HADS scores in the intervention group. This outcome might be caused by the fact that the patients who had completed the HADS questionnaire at T1 pre-pandemic almost all only completed the T3 survey during, many also right at the beginning, of the pandemic. That is, the higher HADS scores could be due to additional stress from the pandemic. The group during the pandemic, on the other hand, filled out all questionnaires after the start of the pandemic, so that the pandemic situation was still tense, but no longer completely new. Moreover, by the time the pandemic started, nFC-isPO was already more advanced and matured within the four sites. Therefore, working routines saved the ongoing of the intervention with high quality . Limitations A major limitation of the study was the high number of cases required for conducting RDD analysis. Originally, a case number of over 3,500 patients was planned in order to have sufficient cases for the RDD analyses (power calculation). After adjustments to the sample size due to poor enrollment, including the pandemic and the complexity of the nFC-isPO, only about half (N = 1,757) of the originally planned sample size could be enrolled. Although this was the minimum number of cases for the relevant RDD analyses, RDDs still benefits from the highest case numbers possible. Another limitation for the proof of effectiveness was the patient group selected for ethical reasons. In the range between 13 and 16 points of the HADS, the treatment group showed a rather low need for psycho-oncological care. A more targeted examination of more severely stressed cases showed that the care offers were taken up and also showed corresponding effects (see summative study report ). Furthermore, the effectiveness of a psychotherapeutic intervention cannot be presented without the effect of time (in this case 12 months) and the effect of individual dose (number of therapeutical talks). Finally, the analyses conducted here cannot conclusively clarify whether the short-term reported increase in HADS scores by isPO-staff during pandemic reflects fear of additional illness and the impact on cancer treatment, or whether scores increased in the short term due to fears of poor accessibility to doctors or other cancer support services.
A major limitation of the study was the high number of cases required for conducting RDD analysis. Originally, a case number of over 3,500 patients was planned in order to have sufficient cases for the RDD analyses (power calculation). After adjustments to the sample size due to poor enrollment, including the pandemic and the complexity of the nFC-isPO, only about half (N = 1,757) of the originally planned sample size could be enrolled. Although this was the minimum number of cases for the relevant RDD analyses, RDDs still benefits from the highest case numbers possible. Another limitation for the proof of effectiveness was the patient group selected for ethical reasons. In the range between 13 and 16 points of the HADS, the treatment group showed a rather low need for psycho-oncological care. A more targeted examination of more severely stressed cases showed that the care offers were taken up and also showed corresponding effects (see summative study report ). Furthermore, the effectiveness of a psychotherapeutic intervention cannot be presented without the effect of time (in this case 12 months) and the effect of individual dose (number of therapeutical talks). Finally, the analyses conducted here cannot conclusively clarify whether the short-term reported increase in HADS scores by isPO-staff during pandemic reflects fear of additional illness and the impact on cancer treatment, or whether scores increased in the short term due to fears of poor accessibility to doctors or other cancer support services.
Patients with cancer are considered as vulnerable group who suffer from anxiety and stress very suddenly , regardless of other external factors such as the COVID-19 pandemic presented here. Our results underline how reliable and resilient the nFC-isPO is to external influencing factors in everyday hospital practice. On the one hand, it turned out that HADS scores were not permanently higher, although this was perceived by the isPO care networks at the beginning of the pandemic. For another, despite the pandemic, the nFC-isPO led to a decrease in HADS scores on average over time. Interestingly, this was despite the need to switch from face-to-face care to sometimes exclusively telephone or video-based care . Therefore, the results underline the importance of early and personalized psycho-oncological support in order to reduce stress, anxiety and depression in patients with cancer.
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Volume imaging to interrogate cancer cell-tumor microenvironment interactions in space and time
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cc7a0208-ed15-4874-9abb-c8ca2abc5ccc
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10228654
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Internal Medicine[mh]
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Introduction In the past decade, cancer research and clinical oncology have experienced a paradigm shift driven by the development of immunotherapies to cure patients with aggressive cancers . Ever since, cancer research around the world has begun to untangle the diverse functions of the tumor microenvironment (TME), the non-transformed cells and extracellular matrix which constitute the niche in which cancers arise and proliferate. Whilst cancer research today appreciates an underlying spatial organization of solid tumors , this organization is too complex to be unraveled by a single two-dimensional tissue section, the historic platform for cancer staging, calling for volumetric analyses that capture larger tumor regions if not the entire lesion at cellular resolution . This is particularly true for the TME due to the heterogeneity of immune and stromal infiltrates within the complex architecture of solid tumors and the existence of rare components of the TME that despite their sparsity may have important implications in tumor biology. To visualize the full complexity of the TME, high-resolution volume imaging techniques are required . Two powerful technologies for volumetric analyses are ex vivo spatial microscopy of tissue-cleared whole organs and intravital microscopy (IVM) of cancer cells in the context of a living organism. Optical clearing to visualize tissues in three dimensions was first developed at the beginning of the 20 th century and has dramatically been refined and exploited in the past decade. Originally rediscovered by neurobiology laboratories to achieve 3D maps of the central nervous system, tissue clearing has now been widely applied to study tumor complexity . The TME is a dynamic ensemble of cells and molecules that can change dramatically throughout tumor progression . By imaging live tissue of animal models, IVM meets the need to scrutinize such dynamics and visualize immune and stromal infiltration and ECM remodeling over time. This review discusses the recent advances in tissue clearing and IVM to visualize the interaction between cancer cells and cells and molecules in their microenvironment. In addition, we discuss the utilization of these two platforms to investigate the crosstalk between tumor and TME, and to advance our understanding of cancer cell biology in the context of the complex architecture and dynamic organization of cancers.
Applying tissue clearing to inspect tumors and their TME in 3D Around seventy tissue clearing techniques have been developed to perform volume fluorescence imaging of tissue resections, whole organs, and entire organisms. This ex vivo platform allows for the visualization of cells and macromolecules within the native architecture of their tissue. The first application of clearing in the context of fluorescence microscopy of whole-mount tissue was developed to study fish and Drosophila embryos and was later widely applied in neurobiology to study the architecture of the mammalian brain . To this day, tissue clearing has been applied to myriad tissues, organs, and organisms including tumors from animal models and human patients . Technically, tissue clearing consists of sample permeabilization and clarification in refractive index matching solutions to render specimens transparent and visualize their entire architecture and internal complexity. The available techniques for tissue permeabilization and refractive index (RI) matching in cancer and other samples have been reviewed extensively . Briefly, permeabilization is achieved by lipid elimination using alcohols or detergents, with zwitterionic detergents showing the highest efficacy for delipidation . Electrophoresis can be used to drive the infiltration of antibodies and dyes into the tissue . Clearing is achieved by immersing the sample in an organic solvent or aqueous solution that matches the RI of the tissue. Organic solvents include ethyl cinnamate (ECi), benzyl alcohol/benzyl benzoate (BABB), tetrahydrofuran and dichloromethane (used in DISCO-derived techniques). Aqueous solutions include fructose gradients (as in FRUIT), fructose combined with glycerol and urea (for example, for FUnGI), more complex chemical combinations as in CUBIC (antipyrine, nicotinamide and N-butyldiethanolamine, and other chemicals), as well as commercial solutions like RapiClear® . The selection of 3D imaging modalities for cleared tissue depends on the sample size and desired optical resolution. Light-sheet fluorescence microscopes provide cellular resolution and can be used to image centimeter-scale samples. Confocal microscopes offer a higher resolution in the xy plane and have been widely utilized to image whole rodent organs and cancer resections. For image rendering and analysis, the free image processing package Fiji can be used , and specific software for volume images such as Imaris and Aivia is commercially available . 2.1 Clearing cancer organotypic cultures Organoids and organotypic cultures can pose barriers to antibody labeling and microscopic analysis due to their high cellularity and embedded growth in thick culture matrices that prevent access of labeling agents and cause extensive light scattering. Consequently, permeabilization and RI matching have proven useful for complex organoid 3D cultures in which antibody access and light penetration can be limited . Tissue clearing has enabled the visualization of glioma and GBM cells in brain organoids using ECi and FRUIT clearing . Complex cocultures comprising lung cancer cell organoids with fibroblasts and endothelial cells were cleared with a mixture of polyglycerols, DMSO and urea (HyClear). Each cell line was engineered to express a different, fluorescent protein marker allowing spatial analyses by 3D fluorescent microscopy . 2.2 Clearing approaches to interrogate the interaction between cancer cells and their epithelial neighbors Carcinomas originate from epithelial cells and interact and compete with their surrounding healthy epithelium . Tissue clearing has been used to study how the epithelial architecture changes upon transformation, and how healthy epithelial cells respond to neighboring tumors. Cytokeratins have been widely used as markers to distinguish epithelia from nonepithelial tissues by 3D imaging. Keratin-8/18 (K8/18) was used to label luminal epithelial cells, and K5 to visualize myoepithelial cells in human prostate biopsies with intraductal carcinoma cleared by iDISCO . In prostate cancer samples from FFPE patient biopsies, BABB clearing showed that the K5-expressing myoepithelial cells co-localize with K8-positive tumor regions with a differentiated ductal architecture . Both keratins are also expressed in the epithelium of the mammary ductal tree and were visualized together with genetically encoded fluorescent proteins using FUnGI . Similarly, fructose glycerol clearing was shown to be applicable to mammary glands with transplanted organoids . In the MMTV-PyMT breast cancer model, K8 staining was used to detect spontaneous metastases in lungs cleared by FLASH and to demonstrate their interaction with the airway and alveolar epithelia labelled with podoplanin and surfactant protein C (SFTPC) . K7 is a marker of luminal and glandular epithelia in the endometrium, and has been used together with CUBIC clearing to investigate the architecture of occluded glands and adenomyosis in humans, associated to cervical cancer . Genetically KrasG12D transformed cells in the pancreas, liver and lung were visualized through a combination of K19 immunolabelling with antibody staining for red and green fluorescent protein (RFP, GFP) lineage tracers . 2.3 Tissue clearing techniques for brain cancer models Many clearing techniques were established to clear the brain and study its anatomy in pathophysiology . Hence, the interaction of different brain tumors with cells of the normal brain parenchyma, including neurons and glia, could be readily investigated in 3D. The astrocyte marker glial fibrillary acidic protein (GFAP) has been used to visualize the interaction of glial cells with GBM models in mouse brains cleared in tetrahydrofuran, urea, azide and nycodenz , and with CUBIC and iDISCO . Co-invasion of human glioma cell lines and astrocytes has been investigated in brain slices labelled with anti-GFAP antibody and imaged after clearing in α-thioglycerol, fructose and RapiClear . These studies also labelled laminin to image the interaction between tumor cells and the surrounding extracellular matrix, and vimentin, which marks processes of glioma cells reaching into the surrounding environment . Astrocyte interaction with breast cancer tumor metastases in the brain administered by intracarotid or intracranial injection was visualized with CUBIC and PACT clearing . The interaction between tumors and axons in 3D has been achieved through lipid staining and clearing with urea and triton , and staining for myelin basic protein (MBP) in GBM cleared with CUBIC and iDISCO . 2.4 Clearing the tumor immune microenvironment Tissue clearing constitutes a useful approach to accurately assess the infiltration of immune cells into the tumor. A general visualization of immune infiltration can be achieved with the pan-immune cell marker CD45, as demonstrated in a mouse glioblastoma (GBM) model cleared with iDISCO and CUBIC . The infiltration of fluorescently labelled tumor-associated macrophages (TAMs) has been visualized in murine lung cancer models cleared with CUBIC . Macrophage infiltration in mouse lung adenocarcinoma tumors generated by intravenous injection of A549 cells was also achieved using an Iba-1 antibody and CUBIC clearing . Primary and secondary tumors from anaplastic thyroid tumors achieved by orthotopic and intravenous injection into mice respectively were cleared with CUBIC, and TAMs visualized using a Macrin-VT680XL nanoparticle . Clearing and imaging of infiltrating T cells has also been achieved. CD8 + cells and their contribution within the heterogeneous expression landscape of immune checkpoint molecule PD-L1 was performed in mouse mammary tumor models cleared in fructose . CD3 staining was used to evaluate the infiltration of T cells in formalin fixed paraffin-embedded (FFPE) breast cancer patient biopsies cleared with CLARITY , and surgical resections of patients with head and neck squamous cell carcinoma (HNSCC) were stained with antibodies against CD3 for T cells and CD66b for neutrophils before clearing with ECi . Recent work using FLASH has revealed differences in T and B cell infiltration in whole tumour-bearing lungs, and has proved useful in the identification of tumour-adjacent immune structures such as tertiary lymphoid structures which are defined by their histology . The study of other immune cell types in different types of tumors remains to be performed, but specialized techniques such as EMOVI have been used to detect immune cells in non-cancerous samples, including antigen presenting cells using MHC-II antibodies, T cells with a CD3 antibody, and B cells and dendritic cells with antibodies against CD21 and CD35 in lungs, lymph nodes, adipose tissue, kidney, brain, liver and heart , giving a promising outlook for the future study of these infiltrates and their interaction with cancer cells. However, fluorescence imaging of large volumes is usually restricted to 4-5 markers, which does not meet the requirements for an in-depth characterization of immune infiltrates. The development of multiplex imaging technologies compatible with 3D imaging brings promise to the spatial characterization of immune infiltration within the tumor volume (See section 6). 2.5 Visualizing the tumor vasculature with tissue clearing Tissue clearing has been used extensively to map the vasculature of biological samples including tumors. In addition to antibodies that specifically label the endothelium or perivascular cells, vessels can be easily stained in animal models through intravenous injection of fluorescent dextran or lectin. Lectin dyes bind to the luminal side of endothelial cells and have been applied to image the architecture of the vascular network in mouse models of glioma using 3DISCO and FluoClearBABB , HNSCC xenografts from human cell lines using the commercial Binaree clearing kit , human hepatoma xenografts and human pancreatic ductal adenocarcinoma (PDAC) xenografts in mice cleared with CUBIC , and a genetic PDAC mouse model cleared with FLASH . Lectins have also been used to label the vasculature of entire mice with primary and metastatic orthotopic tumor models for breast cancer, PDAC, and lung cancer cleared with vDISCO . Lectins can be used in combination with antibody labelling as shown in a mouse model for GBM, in which the vasculature was stained both with lectin and a CD31 antibody and the tumors cleared in 80% glycerol . Tumor perfusion and drug penetration throughout the tumor can also be evaluated using lectin labelling. HER-2 overexpressing breast cancer xenografts have been imaged to evaluate the penetration of a fluorescent HER-2-targeting antibody (trastuzumab) in tumors cleared with BABB . Similarly, in HeLa subcutaneous xenografts, lectin was used to map the distribution of nanoparticles in tumors cleared with CUBIC . Dextran can also be used to label blood vasculature, as shown in A549 lung tumors cleared with CUBIC-plus . Dextran permeates through fenestrated vessels and can hence be used to evaluate the integrity and permeability of the vasculature, as demonstrated in GBM xenografts cleared in ECi . Widely-used epitopes to label vasculature include α-smooth muscle actin (α-SMA, labelling pericytes), used in an orthotopic model of lung cancer cleared with CUBIC , in a breast cancer model cleared with fructose , and in a renal carcinoma brain metastasis model visualized in whole mice cleared with CUBIC . CD31 to label the endothelium has also been widely applied, for example in murine cutaneous squamous cell carcinoma tumors cleared in ECi , mammary tumors cleared in fructose , melanoma xenografts cleared in BABB, CUBIC and TDE, and primary human cancer biopsies cleared with TDE , human colon adenocarcinoma biopsies cleared in FocusClear . CD31 labelling was compared to CD34 in a study that used tissue clearing on murine GBM with CUBIC and iDISCO , and concluded higher specificity of CD34 antibody for labelling endothelium. CD34 was also used to image the vasculature of mouse and human kidney , indicating that it labels endothelium specifically in different tissues. Other markers used in the study of tumor vascularization are VEGFR3, a marker of angiogenesis and CD42c, a platelet marker, used in CUBIC-cleared lung cancer xenografts , coagulation factor VIII which was detected in melanoma xenografts and MECA-79, used to visualize high endothelial venules in fibrosarcoma and 4T1 xenografts . The lymphatic system, especially tumor draining lymph nodes, is a site of particular interest in cancer research since it is a key hub for cancer cell dissemination and immune education. Lymphangiogenesis and the architecture and function of the lymphatic vasculature plays an important role in maintaining the niche of stem cells in healthy tissues and potentially the niche of cancer stem cells as well. Lymphatics can be labelled using an anti-Lyve1 antibody as demonstrated in the liver and in DCM and DBE cleared bladder cancer resections . Lymphatics of PanIN and PDAC lesions were visualized in human and mouse samples with antibodies against Lyve-1 and podoplanin and clearing in RapiClear 1.52 . Tissue clearing of lymph nodes has been achieved with techniques such as Ce3D for imaging immune cell populations and vasculature , and has enabled to track the population dynamics of cells involved in adaptive immunity such as CD8 + T cell differentiation . 2.6 Tissue clearing to image tumor innervation Cancer innervation is a timely field of research, as depending on the context, nerves can promote or inhibit cancer growth and cancer cells can invade perineurally . Neurons can readily be mapped by many clearing protocols, and have been imaged in normal tissues of the human colon , pancreas , and in a mouse model for pancreatic neoplasia where the GFAP-positive glia and TUJ1-positive neurons were found to be enriched around lesions. This study visualized both innervation and vascularization in the same samples which suggests that angiogenesis and innervation occur in parallel during neoplastic hyperproliferation and early stages of transformation in the pancreas .
Clearing cancer organotypic cultures Organoids and organotypic cultures can pose barriers to antibody labeling and microscopic analysis due to their high cellularity and embedded growth in thick culture matrices that prevent access of labeling agents and cause extensive light scattering. Consequently, permeabilization and RI matching have proven useful for complex organoid 3D cultures in which antibody access and light penetration can be limited . Tissue clearing has enabled the visualization of glioma and GBM cells in brain organoids using ECi and FRUIT clearing . Complex cocultures comprising lung cancer cell organoids with fibroblasts and endothelial cells were cleared with a mixture of polyglycerols, DMSO and urea (HyClear). Each cell line was engineered to express a different, fluorescent protein marker allowing spatial analyses by 3D fluorescent microscopy .
Clearing approaches to interrogate the interaction between cancer cells and their epithelial neighbors Carcinomas originate from epithelial cells and interact and compete with their surrounding healthy epithelium . Tissue clearing has been used to study how the epithelial architecture changes upon transformation, and how healthy epithelial cells respond to neighboring tumors. Cytokeratins have been widely used as markers to distinguish epithelia from nonepithelial tissues by 3D imaging. Keratin-8/18 (K8/18) was used to label luminal epithelial cells, and K5 to visualize myoepithelial cells in human prostate biopsies with intraductal carcinoma cleared by iDISCO . In prostate cancer samples from FFPE patient biopsies, BABB clearing showed that the K5-expressing myoepithelial cells co-localize with K8-positive tumor regions with a differentiated ductal architecture . Both keratins are also expressed in the epithelium of the mammary ductal tree and were visualized together with genetically encoded fluorescent proteins using FUnGI . Similarly, fructose glycerol clearing was shown to be applicable to mammary glands with transplanted organoids . In the MMTV-PyMT breast cancer model, K8 staining was used to detect spontaneous metastases in lungs cleared by FLASH and to demonstrate their interaction with the airway and alveolar epithelia labelled with podoplanin and surfactant protein C (SFTPC) . K7 is a marker of luminal and glandular epithelia in the endometrium, and has been used together with CUBIC clearing to investigate the architecture of occluded glands and adenomyosis in humans, associated to cervical cancer . Genetically KrasG12D transformed cells in the pancreas, liver and lung were visualized through a combination of K19 immunolabelling with antibody staining for red and green fluorescent protein (RFP, GFP) lineage tracers .
Tissue clearing techniques for brain cancer models Many clearing techniques were established to clear the brain and study its anatomy in pathophysiology . Hence, the interaction of different brain tumors with cells of the normal brain parenchyma, including neurons and glia, could be readily investigated in 3D. The astrocyte marker glial fibrillary acidic protein (GFAP) has been used to visualize the interaction of glial cells with GBM models in mouse brains cleared in tetrahydrofuran, urea, azide and nycodenz , and with CUBIC and iDISCO . Co-invasion of human glioma cell lines and astrocytes has been investigated in brain slices labelled with anti-GFAP antibody and imaged after clearing in α-thioglycerol, fructose and RapiClear . These studies also labelled laminin to image the interaction between tumor cells and the surrounding extracellular matrix, and vimentin, which marks processes of glioma cells reaching into the surrounding environment . Astrocyte interaction with breast cancer tumor metastases in the brain administered by intracarotid or intracranial injection was visualized with CUBIC and PACT clearing . The interaction between tumors and axons in 3D has been achieved through lipid staining and clearing with urea and triton , and staining for myelin basic protein (MBP) in GBM cleared with CUBIC and iDISCO .
Clearing the tumor immune microenvironment Tissue clearing constitutes a useful approach to accurately assess the infiltration of immune cells into the tumor. A general visualization of immune infiltration can be achieved with the pan-immune cell marker CD45, as demonstrated in a mouse glioblastoma (GBM) model cleared with iDISCO and CUBIC . The infiltration of fluorescently labelled tumor-associated macrophages (TAMs) has been visualized in murine lung cancer models cleared with CUBIC . Macrophage infiltration in mouse lung adenocarcinoma tumors generated by intravenous injection of A549 cells was also achieved using an Iba-1 antibody and CUBIC clearing . Primary and secondary tumors from anaplastic thyroid tumors achieved by orthotopic and intravenous injection into mice respectively were cleared with CUBIC, and TAMs visualized using a Macrin-VT680XL nanoparticle . Clearing and imaging of infiltrating T cells has also been achieved. CD8 + cells and their contribution within the heterogeneous expression landscape of immune checkpoint molecule PD-L1 was performed in mouse mammary tumor models cleared in fructose . CD3 staining was used to evaluate the infiltration of T cells in formalin fixed paraffin-embedded (FFPE) breast cancer patient biopsies cleared with CLARITY , and surgical resections of patients with head and neck squamous cell carcinoma (HNSCC) were stained with antibodies against CD3 for T cells and CD66b for neutrophils before clearing with ECi . Recent work using FLASH has revealed differences in T and B cell infiltration in whole tumour-bearing lungs, and has proved useful in the identification of tumour-adjacent immune structures such as tertiary lymphoid structures which are defined by their histology . The study of other immune cell types in different types of tumors remains to be performed, but specialized techniques such as EMOVI have been used to detect immune cells in non-cancerous samples, including antigen presenting cells using MHC-II antibodies, T cells with a CD3 antibody, and B cells and dendritic cells with antibodies against CD21 and CD35 in lungs, lymph nodes, adipose tissue, kidney, brain, liver and heart , giving a promising outlook for the future study of these infiltrates and their interaction with cancer cells. However, fluorescence imaging of large volumes is usually restricted to 4-5 markers, which does not meet the requirements for an in-depth characterization of immune infiltrates. The development of multiplex imaging technologies compatible with 3D imaging brings promise to the spatial characterization of immune infiltration within the tumor volume (See section 6).
Visualizing the tumor vasculature with tissue clearing Tissue clearing has been used extensively to map the vasculature of biological samples including tumors. In addition to antibodies that specifically label the endothelium or perivascular cells, vessels can be easily stained in animal models through intravenous injection of fluorescent dextran or lectin. Lectin dyes bind to the luminal side of endothelial cells and have been applied to image the architecture of the vascular network in mouse models of glioma using 3DISCO and FluoClearBABB , HNSCC xenografts from human cell lines using the commercial Binaree clearing kit , human hepatoma xenografts and human pancreatic ductal adenocarcinoma (PDAC) xenografts in mice cleared with CUBIC , and a genetic PDAC mouse model cleared with FLASH . Lectins have also been used to label the vasculature of entire mice with primary and metastatic orthotopic tumor models for breast cancer, PDAC, and lung cancer cleared with vDISCO . Lectins can be used in combination with antibody labelling as shown in a mouse model for GBM, in which the vasculature was stained both with lectin and a CD31 antibody and the tumors cleared in 80% glycerol . Tumor perfusion and drug penetration throughout the tumor can also be evaluated using lectin labelling. HER-2 overexpressing breast cancer xenografts have been imaged to evaluate the penetration of a fluorescent HER-2-targeting antibody (trastuzumab) in tumors cleared with BABB . Similarly, in HeLa subcutaneous xenografts, lectin was used to map the distribution of nanoparticles in tumors cleared with CUBIC . Dextran can also be used to label blood vasculature, as shown in A549 lung tumors cleared with CUBIC-plus . Dextran permeates through fenestrated vessels and can hence be used to evaluate the integrity and permeability of the vasculature, as demonstrated in GBM xenografts cleared in ECi . Widely-used epitopes to label vasculature include α-smooth muscle actin (α-SMA, labelling pericytes), used in an orthotopic model of lung cancer cleared with CUBIC , in a breast cancer model cleared with fructose , and in a renal carcinoma brain metastasis model visualized in whole mice cleared with CUBIC . CD31 to label the endothelium has also been widely applied, for example in murine cutaneous squamous cell carcinoma tumors cleared in ECi , mammary tumors cleared in fructose , melanoma xenografts cleared in BABB, CUBIC and TDE, and primary human cancer biopsies cleared with TDE , human colon adenocarcinoma biopsies cleared in FocusClear . CD31 labelling was compared to CD34 in a study that used tissue clearing on murine GBM with CUBIC and iDISCO , and concluded higher specificity of CD34 antibody for labelling endothelium. CD34 was also used to image the vasculature of mouse and human kidney , indicating that it labels endothelium specifically in different tissues. Other markers used in the study of tumor vascularization are VEGFR3, a marker of angiogenesis and CD42c, a platelet marker, used in CUBIC-cleared lung cancer xenografts , coagulation factor VIII which was detected in melanoma xenografts and MECA-79, used to visualize high endothelial venules in fibrosarcoma and 4T1 xenografts . The lymphatic system, especially tumor draining lymph nodes, is a site of particular interest in cancer research since it is a key hub for cancer cell dissemination and immune education. Lymphangiogenesis and the architecture and function of the lymphatic vasculature plays an important role in maintaining the niche of stem cells in healthy tissues and potentially the niche of cancer stem cells as well. Lymphatics can be labelled using an anti-Lyve1 antibody as demonstrated in the liver and in DCM and DBE cleared bladder cancer resections . Lymphatics of PanIN and PDAC lesions were visualized in human and mouse samples with antibodies against Lyve-1 and podoplanin and clearing in RapiClear 1.52 . Tissue clearing of lymph nodes has been achieved with techniques such as Ce3D for imaging immune cell populations and vasculature , and has enabled to track the population dynamics of cells involved in adaptive immunity such as CD8 + T cell differentiation .
Tissue clearing to image tumor innervation Cancer innervation is a timely field of research, as depending on the context, nerves can promote or inhibit cancer growth and cancer cells can invade perineurally . Neurons can readily be mapped by many clearing protocols, and have been imaged in normal tissues of the human colon , pancreas , and in a mouse model for pancreatic neoplasia where the GFAP-positive glia and TUJ1-positive neurons were found to be enriched around lesions. This study visualized both innervation and vascularization in the same samples which suggests that angiogenesis and innervation occur in parallel during neoplastic hyperproliferation and early stages of transformation in the pancreas .
IVM: a window to visualize TME-cancer cell interaction dynamics in 4D Static volume imaging allows the molecular phenotyping in the 3D cancer space but represents a snapshot in time and lacks insight into the dynamics of the same cancer cells over time. The combination of IVM with subsequent 3D molecular analysis by tissue clearing poses an attractive solution combining the strengths of both technologies . Recently, IVM has proven extremely useful to shed light on dynamic processes at play in tumor formation and progression, immune escape and metastasis . 3.1 Recent protocols and window optimizations for IVM In recent years the field of intravital microscopy has been broadened and specialized by developments that adapted the technology for different organs. Each organ casts unique demands due to its location that dictates optical accessibility, and due to its organ architecture and underlying physical (and optical) properties. Today an abundance of IVM techniques exists tailored to specific organs. For acute imaging, most organs can be accessed surgically. On the other hand, longitudinal imaging necessitates technologies to grant long-term access to the same organ and region over multiple days to weeks . This has for long been accomplished through the help of imaging windows, small sterile rings of solid inorganic material with a microscopy cover-glass inset. Such windows can be surgically implanted in the mouse skin and peritoneum, thereby rendering inner organs optically accessible. In recent years the general window design has been considerably modified to meet unique demands of different implantation sites. Most recent optimizations include windows for the pancreas and thyroid , and flexible silicon-based imaging windows for expanding tissues and tumors . Several approaches also exist to image cancerous processes in the bone marrow, ranging from the use of endoscopic devices for the femur , to the surgical exposure of tibia and calvarium . Some organs, such as those of the respiratory system and the gastrointestinal tract, are under constant movement and need to be stabilized for high resolution image acquisition. For the colon, a window with a stabilizing ferromagnetic scaffold minimizes such motion artifacts specifically during the imaging whilst granting the organ free movement in the imaging breaks to prevent obstruction and maintain intestinal function . Using a specialized lung window for longitudinal imaging, Entenberg and colleagues could visualize all stages of the metastatic process, including tumor cell arrival, extravasation, metastatic growth and progression to micrometastases . The dorsal skinfold window represents a widely used alternative to organ specific imaging windows. In this model cancer cells are engrafted subcutaneously in a window chamber implanted in the back skin of the mouse. This approach presents a robust platform to visualize cell-cell interactions in the living organism and may be seen as a straight forward alternative to test hypotheses in vivo that were gained in in vitro experiments, albeit restricted to questions where the role of the cancer-surrounding normal organ is neglected. Alternative xenograft-based approaches have visualized tumor cell behavior through cancer cell injection in the tongue , and engraftment in the eye which allow noninvasive longitudinal IVM without the need of imaging windows. Once the organ of interest can be optically accessed, different microscopy setups enable to address a multitude of questions. Optical access into deeper tissue layers is hampered by the tissue inherent light scattering and most studies employ 2-photon microscopes to visualize several hundred μm of tissue depth. The recent development of IVM protocols for 3-photon microscopy further increases the imaging depth . In addition, fast acquisition speed, for example using a spinning disk or a resonant scanner confocal microscope, can be of advantage for moving organs and allows the capture of highly dynamic events as for example the movement of leukocytes in the blood stream and tumor interstitium . The best combination of strategies for optical access and image acquisition will depend on the cancer type and biological question asked. Dedicated protocols for the imaging of primary cancers, metastases and the tumor microenvironment in different sites help identify the optimal strategy . Whilst the majority of previous IVM studies focused their attention on cellular behavior, the coming years are poised to see a broadening of IVM utilization with ongoing developments that multiplex bioluminescent and fluorescent reporters and molecular probes to film sub-cellular processes, and approaches that enable the in vivo visualization of nanoscale structures by 3D-stimulated emission depletion (3D-STED) microscopy , thereby opening the window to view molecular biology in the living organism. 3.2 Tools and strategies to visualize cell-cell interactions in vivo Given the ability to access and visualize the anatomic site of interest, a range of fluorescent labels and detection strategies enable the identification of cancer cells and structural and cellular components of the TME in the context of the organ architecture . The recent development of a photoactivatable Cre-recombinase mouse allows to target cancerous mutations to specific regions and cells in the organ landscape . Cancer cells can be labelled genetically through the expression of fluorescent proteins, and thus traced over sequential imaging sessions of several days to weeks. Proteins that can be activated, or photo-converted to change the fluorescent protein emission spectrum, allow the tracking of individual groups of cells based on their localization . With this approach, the colonization of distant organs from breast tumors was mapped by following the dissemination of cancer cells converted in the mammary gland in comparison to cancer cells converted in the lymph node . Proliferation of cancer and TME cells can be visualized indirectly by targeting individual cells with fluorescent proteins for lineage tracing, or directly through genetically encoded fluorescent cell cycle reporters . Fluorescent dyes like the recently developed apotracker-red are an option to identify apoptotic cells . Today the toolbox of IVM-ready fluorescent labels also includes a range of probes that allow to monitor the activity of the ERK, FAK, TGF-β/SMAD3 and nuclear factor kB (NF-kB) signaling pathways . A fluorescent reporter of nuclear factor of activated T cell (NFAT) activation was recently used to directly visualize how Treg cells receive TCR signals in the TME . The tumor-surrounding matrix can be readily visualized through second harmonic generations (SHG) detection of fibrillar collagen. Furthermore, recent efforts have targeted individual matrix components with fluorescent proteins. Fluorescently tagged fibronectin (FN) was used to track T cell interaction with FN in the inflamed ear dermis, where Th1 cells were found to migrate along FN fibers . The development of a laminin beta1-Dendra2 reporter mouse allows to visualize basement-membrane dynamics in vivo and can shed light on how cancer cells invade normal tissues . Tumor vasculature is commonly visualized through intravenous injection of fluorescent dextrans, lectins or quantum dots. The choice of dextran molecular weight allows to fine tune this detection to measure vascular permeability , and to label phagocytic immune cells . IVM has been instrumental to describe a wide range of fundamental immunological principles and immune cell behaviors . Genetic reporters and intravenously injected fluorophore-conjugated antibodies are commonly used to detect immune cells . Recent developments for multiplexed in vivo antibody labelling through click chemistry offer the opportunity to drastically expand the cell types that can be visualized in the same mouse . Nevertheless, it remains challenging to quantify rare cell populations in circulation. Recent developments enable real time imaging of individual fast circulating cells in the blood stream through quantum dots , a promising step in the direction of visualizing aggregation dynamics of circulating tumor cells (CTCs), or the interplay between lymphocytes, platelets, and CTCs in circulation in vivo .
Recent protocols and window optimizations for IVM In recent years the field of intravital microscopy has been broadened and specialized by developments that adapted the technology for different organs. Each organ casts unique demands due to its location that dictates optical accessibility, and due to its organ architecture and underlying physical (and optical) properties. Today an abundance of IVM techniques exists tailored to specific organs. For acute imaging, most organs can be accessed surgically. On the other hand, longitudinal imaging necessitates technologies to grant long-term access to the same organ and region over multiple days to weeks . This has for long been accomplished through the help of imaging windows, small sterile rings of solid inorganic material with a microscopy cover-glass inset. Such windows can be surgically implanted in the mouse skin and peritoneum, thereby rendering inner organs optically accessible. In recent years the general window design has been considerably modified to meet unique demands of different implantation sites. Most recent optimizations include windows for the pancreas and thyroid , and flexible silicon-based imaging windows for expanding tissues and tumors . Several approaches also exist to image cancerous processes in the bone marrow, ranging from the use of endoscopic devices for the femur , to the surgical exposure of tibia and calvarium . Some organs, such as those of the respiratory system and the gastrointestinal tract, are under constant movement and need to be stabilized for high resolution image acquisition. For the colon, a window with a stabilizing ferromagnetic scaffold minimizes such motion artifacts specifically during the imaging whilst granting the organ free movement in the imaging breaks to prevent obstruction and maintain intestinal function . Using a specialized lung window for longitudinal imaging, Entenberg and colleagues could visualize all stages of the metastatic process, including tumor cell arrival, extravasation, metastatic growth and progression to micrometastases . The dorsal skinfold window represents a widely used alternative to organ specific imaging windows. In this model cancer cells are engrafted subcutaneously in a window chamber implanted in the back skin of the mouse. This approach presents a robust platform to visualize cell-cell interactions in the living organism and may be seen as a straight forward alternative to test hypotheses in vivo that were gained in in vitro experiments, albeit restricted to questions where the role of the cancer-surrounding normal organ is neglected. Alternative xenograft-based approaches have visualized tumor cell behavior through cancer cell injection in the tongue , and engraftment in the eye which allow noninvasive longitudinal IVM without the need of imaging windows. Once the organ of interest can be optically accessed, different microscopy setups enable to address a multitude of questions. Optical access into deeper tissue layers is hampered by the tissue inherent light scattering and most studies employ 2-photon microscopes to visualize several hundred μm of tissue depth. The recent development of IVM protocols for 3-photon microscopy further increases the imaging depth . In addition, fast acquisition speed, for example using a spinning disk or a resonant scanner confocal microscope, can be of advantage for moving organs and allows the capture of highly dynamic events as for example the movement of leukocytes in the blood stream and tumor interstitium . The best combination of strategies for optical access and image acquisition will depend on the cancer type and biological question asked. Dedicated protocols for the imaging of primary cancers, metastases and the tumor microenvironment in different sites help identify the optimal strategy . Whilst the majority of previous IVM studies focused their attention on cellular behavior, the coming years are poised to see a broadening of IVM utilization with ongoing developments that multiplex bioluminescent and fluorescent reporters and molecular probes to film sub-cellular processes, and approaches that enable the in vivo visualization of nanoscale structures by 3D-stimulated emission depletion (3D-STED) microscopy , thereby opening the window to view molecular biology in the living organism.
Tools and strategies to visualize cell-cell interactions in vivo Given the ability to access and visualize the anatomic site of interest, a range of fluorescent labels and detection strategies enable the identification of cancer cells and structural and cellular components of the TME in the context of the organ architecture . The recent development of a photoactivatable Cre-recombinase mouse allows to target cancerous mutations to specific regions and cells in the organ landscape . Cancer cells can be labelled genetically through the expression of fluorescent proteins, and thus traced over sequential imaging sessions of several days to weeks. Proteins that can be activated, or photo-converted to change the fluorescent protein emission spectrum, allow the tracking of individual groups of cells based on their localization . With this approach, the colonization of distant organs from breast tumors was mapped by following the dissemination of cancer cells converted in the mammary gland in comparison to cancer cells converted in the lymph node . Proliferation of cancer and TME cells can be visualized indirectly by targeting individual cells with fluorescent proteins for lineage tracing, or directly through genetically encoded fluorescent cell cycle reporters . Fluorescent dyes like the recently developed apotracker-red are an option to identify apoptotic cells . Today the toolbox of IVM-ready fluorescent labels also includes a range of probes that allow to monitor the activity of the ERK, FAK, TGF-β/SMAD3 and nuclear factor kB (NF-kB) signaling pathways . A fluorescent reporter of nuclear factor of activated T cell (NFAT) activation was recently used to directly visualize how Treg cells receive TCR signals in the TME . The tumor-surrounding matrix can be readily visualized through second harmonic generations (SHG) detection of fibrillar collagen. Furthermore, recent efforts have targeted individual matrix components with fluorescent proteins. Fluorescently tagged fibronectin (FN) was used to track T cell interaction with FN in the inflamed ear dermis, where Th1 cells were found to migrate along FN fibers . The development of a laminin beta1-Dendra2 reporter mouse allows to visualize basement-membrane dynamics in vivo and can shed light on how cancer cells invade normal tissues . Tumor vasculature is commonly visualized through intravenous injection of fluorescent dextrans, lectins or quantum dots. The choice of dextran molecular weight allows to fine tune this detection to measure vascular permeability , and to label phagocytic immune cells . IVM has been instrumental to describe a wide range of fundamental immunological principles and immune cell behaviors . Genetic reporters and intravenously injected fluorophore-conjugated antibodies are commonly used to detect immune cells . Recent developments for multiplexed in vivo antibody labelling through click chemistry offer the opportunity to drastically expand the cell types that can be visualized in the same mouse . Nevertheless, it remains challenging to quantify rare cell populations in circulation. Recent developments enable real time imaging of individual fast circulating cells in the blood stream through quantum dots , a promising step in the direction of visualizing aggregation dynamics of circulating tumor cells (CTCs), or the interplay between lymphocytes, platelets, and CTCs in circulation in vivo .
Applications of volume imaging to visualize cell-TME interactions in cancer 4.1 Cellular relationships between epithelial cells The crosstalk between cancer cells and their neighboring normal epithelium dictates the biology of an arising lesion already at the earliest stages when only a couple of oncogenic cells start to proliferate. IVM experiments demonstrated how the crosstalk between skin cells bearing oncogenic mutations and their wildtype neighbors can correct aberrant growths . In the ear skin, hair follicle stem cells carrying an activating Hras mutation outcompeted their wildtype neighbors, yet were integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium led to benign outgrowths . Tissue clearing of skin, lung, liver and pancreas showed that the morphological appearance of early lesions is governed by an interplay between the architecture of the host tissue and deregulation of cancer cell actomyosin activity . Importantly, the mechanical crosstalk extends to the nuclear level where it causes dynamic changes in nuclear morphology and may threaten genetic integrity . Genomic cancer cell evolution and the dynamic TME instruct single cell biology and behavior and lead to diversification in the tumor bulk. IVM demonstrated that this heterogeneity extends to the metabolic level in ER + breast cancer. Using fluorescent biosensors, Kondo and colleagues demonstrated how actin remodeling, phosphatidylinositol 3-kinase (PI3K) and bromodomain activity modulate glycolysis resulting in metabolically distinct tumor cell populations that reside in different areas of a lesion . How this regional diversification affects the cellular interactions amongst cancer cells, as well as the interplay of cancer cells with the TME is subject to ongoing investigation. Alongside tumor expansion cancer cells actively remodel their microenvironment, and the TME is understood to be spatially patterned with differing constituents and function in different tumor areas . 4.2 Interaction of cancer cells and the tumor vasculature In the study of the TME, vascular remodeling has received particular attention as it impacts tumor nutrition, oxygenation, dissemination, and the recruitment of immune components. Quantitative assessments of the extent of tumor vascularization can be readily achieved by both tissue clearing and IVM, and have shown that several tumor types share a tendency to increase the vascular density within their mass . In some cancers the angiogenic capabilities may extend to lymphangiogenesis, as seen in pancreatic cancer . The cellular interactions between cancer cells and the tumor vasculature are multifold and reciprocal. IVM experiments on an orthotopic HNSCC model suggest that angiogenesis is specifically induced by a subpopulation of CD44 + cancer cells . On the other hand, 3D imaging of the tumor-stroma interface in cutaneous SCC showed that the blood vasculature regionally expresses TGF-β, thereby sustaining the cancer stem cell niche and contributing to cancer cell heterogeneity . In an HRAS-driven murine model of cutaneous SCC, angiogenesis was shown through tissue clearing to be triggered during the transition from papilloma to malignant carcinoma. This de novo vascularization in late stages of lesion progression facilitates leptin infiltration into the tumor, triggering Pi3k-Akt-mTor signaling, further contributing to the benign to malignant transition . Cancer progression through vascular remodeling is also seen in the bone marrow in acute myeloid leukemia (AML), where IVM experiments visualized how the remodeling of endosteal blood vessels abrogates hematopoietic stem cells (HSCs), HSC niches and osteoblasts through physical dislodgment via the damaged vasculature. Pharmacological MMP inhibition reduced vascular permeability and healthy cell loss, thereby limiting AML infiltration, proliferation, and cell migration . 4.3 Cancer cell crosstalk with the immune microenvironment The tumor vasculature is a significant route of tumor entry for immune cells that may clear cancer cells or be recruited for their tumor protective behavior. IVM successfully captured the critical steps in the cascade of lymphocyte adhesion to tumor-associated high endothelial venules (TA-HEVs), from initial lymphocyte tethering to the vessel wall to lymphocyte rolling and sticking, and extravasation . Tumor-infiltrating lymphocyte (TIL) motility changes both longitudinally during tumor progression , as well as regionally. Cytotoxic T lymphocytes (CTLs) show reduced motility and activity in avascular tumor areas but were found to be highly motile in vicinity to peripheral blood vessels and cleared tumor cells around them. Interestingly, blood flow is essential for CTL migration . Concomitantly, CTLs were shown to first accumulate in the tumor periphery and subsequently redistribute towards the invasive tumor front . Based on their migration patterns, CTLs have been understood to display a tumor cell searching behavior in the tumor periphery, which abates in velocity and changes into a “probing” behavior in tumor cell proximity indicating target engagement . IVM imaging showed that in the process of tumor cell killing, multiple CTLs undergo temporary sublethal contacts with individual tumor cells to induce killing in a process of additive cytotoxicity . On the other hand, CTL recognition of a few tumor cells can elicit sufficient IFNγ secretion to cover distances of several hundred micrometers and inhibit tumor growth even in regions not frequented by CTLs . Cancers hijack various cellular pathways to evade the immune response. T regulatory (Treg) cells are inherently poised to recognize antigens in the TME, and auto-regulate through short-lived interactions with conventional dendritic cells (cDCs) . Neutrophils stimulate T cells in a type I interferon (IFN) signaling dependent manner . However, tumor cells can elicit the production of neutrophil extracellular traps (NETs) from neutrophils and granulocytic myeloid-derived suppressor cells (MDSCs), and these NETs enwrap cancer cells, thereby reducing interfaces with CTLs and shielding against cytotoxicity . Neutrophils can promote tumor growth by activating MDSC that in turn inhibit the immune response orchestrated by effector T cells against cancer cells, as observed in cleared murine HNSCC tumors . Peritumoral neutrophils are more motile than intratumoral neutrophils , suggesting a different interactive behavior in specific tumor areas. TAMs play a multifold role in cancer progression. As for lymphocytes and neutrophils, volume imaging data suggest that TAM behavior differs drastically between tumor subtypes, stage and among different areas of the same lesion. IVM showed that the migratory behavior of TAMs is different in genetically distinct GBMs . In an orthotopic model of anaplastic thyroid cancer, tissue clearing suggested that the infiltration of tumor-associated macrophages is significantly higher in the primary tumor than in the metastases, indicating that the microenvironment in which the tumor develops conditions the infiltration of tumor-promoting TAMs . Using the dorsal skinfold chamber to track the crosstalk between macrophages and cancer grafts by IVM identified macrophage subpopulations based on their localization in the tumor core or periphery, and found that those populations differed in Arg1 gene expression and motility. This study also demonstrated the powerful combination of IVM with single cell RNA sequencing to gain molecular insight from a single cell phenotype that can only be observed in the native organ context . The cell-cell interaction between TAMs and cancer cells is a driving factor of tumor aggression. Macrophages were found to induce cancer cell stemness though Notch-Jagged signaling , and enable cancer cell dissemination through dynamic multicellular interactions . In an orthotopic breast cancer model, monocytes were shown to arrive at the tumor as motile streaming TAMs that then differentiated into sessile perivascular macrophages. This differentiation occurred through cancer cell induced TGFβ-dependent upregulation of CXCR4 in monocytes while CXCL12 expressed by perivascular fibroblasts attracted these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiated into perivascular macrophages, promoting vascular leakiness and intravasation . Together volume imaging experiments of the recent years demonstrated that tumor- supportive and tumor-inhibiting activities of immune cells crucially depend on the regional microenvironment, and are modulated in a highly dynamic way through complex multi-cellular interactions. 4.4 The basement membrane and cancer cell invasion The basement membrane constitutes a key barrier to tumor cell migration into the surrounding parenchyma. Recent IVM experiments, using a fluorescent laminin beta1 reporter mouse, show that basement membrane components are synthesized by cancer cells as well as endothelial cells in a dynamic and regionally heterogeneous manner. Interestingly, laminin beta1 turnover is higher in cancerous areas compared to normal epithelium, suggesting a local pre-conditioning of the basement membrane preceding tumor cell invasion . IVM imaging of cancer cell invasion has been instrumental to understand the cellular processes and interaction of cancer cell dissemination. Myosin 10 filopodia support the basement membrane in early lesions, but contribute to a proinvasive phenotype later in disease . Interestingly, cells that are able to breach the basement membrane, due to experimentally induced loss of E-cadherin which increases cancer cell motility, require further signals in order to disseminate into the surrounding parenchyma . Together, 3D cellular confinement, cell density and cell-cell junction stability orchestrate epithelial fluidization and single cell release , and set out the paths of migration and leader-follower cell relationships in collective movements. Cancer cells were found to migrate along collagen fibrils and blood vessels in several tumor models . Leader cells undergo significant deformations, potentially due to the confinement by the ECM, but create paths for follower cells that have more normalized cell and nuclear shapes . This synchronous migration along common paths may reflect an expansion along newly generated space in the ECM confinement, but could also be instructed directly from leader to follower cells through extracellular vesicle exchange . Thereby, the migratory behavior differs throughout the tumor landscape. GBM comprises an invasive margin with slow directed invasion and a diffuse infiltration margin with fast but less directed cell movements. Migration along blood vessels correlated with higher velocity and direction away from the tumor core whereas intraparenchymal migration along white matter tracts was measured to be slower and less directed . In glioma, IVM showed that GFAP splice variants fine‐tune invasion by modulating migration persistence. Whilst depletion of either isoform increased the migratory capacity of glioma cells, GFAPδ‐depleted cells migrated randomly through the brain tissue, whereas GFAPα‐depleted cells displayed a directionally persistent invasion into the brain parenchyma . 4.5 Cancer cell-TME interactions in transit to distant organs Tumor cell intravasation is a multicellular process in the TME which in breast cancer models was shown to require direct contacts between a proangiogenic perivascular macrophage, a tumor cell overexpressing the actin regulator protein Mena, and an endothelial cell that together create the “tumor microenvironment of metastasis” (TMEM), allowing cancer cell intravasation through transient vascular permeability . Vascular travel is a bottle neck in tumor cell dissemination as only a portion of the cells entering the blood stream are able to seed and colonize distant organs. Real-time visualization of circulating tumor cell (CTC) subpopulations through conjugated quantum dots showed that PDAC CTCs expressing the surface protein CD24 have the ability to migrate along vessel walls to distant organs . CTC packing in cell clusters can provide support for traveling cancer cells and has been debated to occur through collective intravasation or de novo clustering in the circulation. Intravital imaging showed that breast cancer CTC clusters form in circulation through CD44 protein mediated cell aggregation . Tissue clearing showed that breast cancer cells extravasate with the help of actin-rich protrusions . CTC extravasation depends on reciprocal interactions between CTCs and endothelial cells of distant organs that can prohibit or permit CTC organ entry. The bone endothelium expresses ephrin-B2 which activates ephrin-B4 in circulating melanoma cells thereby repulsing CTCs from attaching to the bone endothelial cells and barricading against metastasis formation . In the mouse lung, extravasation of breast cancer cells is dependent on physical interaction between tumor cells and macrophages in a IL4-CXCR2 dependent manner . 4.6 Cell-cell crosstalk at metastatic sites Metastasizing cancer cells can disseminate and establish tumors in organs with disparate cellular and physicochemical characteristics, or can present strong tropism and invade only one secondary organ . Tissue clearing of entire mice has shown the diverse tropism of different cancer cell lines such as mammary MDA-231 and pancreatic OS-RC-2 cells, and their colocalization with the vasculature across the mouse body . Volume imaging uncovered a widespread heterogeneity in the seeding potential, and the likelihood for successful metastatic outgrowth of different tumor cell subpopulations. These factors are now understood to be modulated by cell intrinsic properties and CSC traits on the one hand, as well as by dynamic multi-cellular contacts at the metastatic site on the other hand. Multicellular interactions influence the survival and outgrowth of metastasis seeding cancer cells. In the brain, early extravasating cells may be cleared by phagocytosis from TAM and microglia, although these effects abated in later stages of metastasis formation . As in primary tumors, the multi-cellular arrangements of cancer cells and TME components in metastatic sites show spatial and longitudinal heterogeneity. There is a highly heterogeneous cellular interplay between metastases even from the same tumor, as illustrated in breast cancer brain metastases that involve glial cells and astrocytes of the brain parenchyma to very different extents . The communication between cancer cells and the TME at different organs may amplify the heterogeneity seen in metastatic lesions. Indeed, the proliferation dynamics of cancer cells were found to depend on the organ colonized as shown with A549 lung adenocarcinoma cells after intracardiac administration into mice . One environmental mechanism may involve the access of pro-and antitumorigenic immune cells. Neutrophils are highly motile in lung capillaries and change their behavior to slow patrolling and stationary upon recruitment to lung metastasis where they were filmed to correlate with tumor cell proliferation .
Cellular relationships between epithelial cells The crosstalk between cancer cells and their neighboring normal epithelium dictates the biology of an arising lesion already at the earliest stages when only a couple of oncogenic cells start to proliferate. IVM experiments demonstrated how the crosstalk between skin cells bearing oncogenic mutations and their wildtype neighbors can correct aberrant growths . In the ear skin, hair follicle stem cells carrying an activating Hras mutation outcompeted their wildtype neighbors, yet were integrated into clinically normal skin hair follicles. In contrast, targeting the Hras mutation to the upper noncycling region of the skin epithelium led to benign outgrowths . Tissue clearing of skin, lung, liver and pancreas showed that the morphological appearance of early lesions is governed by an interplay between the architecture of the host tissue and deregulation of cancer cell actomyosin activity . Importantly, the mechanical crosstalk extends to the nuclear level where it causes dynamic changes in nuclear morphology and may threaten genetic integrity . Genomic cancer cell evolution and the dynamic TME instruct single cell biology and behavior and lead to diversification in the tumor bulk. IVM demonstrated that this heterogeneity extends to the metabolic level in ER + breast cancer. Using fluorescent biosensors, Kondo and colleagues demonstrated how actin remodeling, phosphatidylinositol 3-kinase (PI3K) and bromodomain activity modulate glycolysis resulting in metabolically distinct tumor cell populations that reside in different areas of a lesion . How this regional diversification affects the cellular interactions amongst cancer cells, as well as the interplay of cancer cells with the TME is subject to ongoing investigation. Alongside tumor expansion cancer cells actively remodel their microenvironment, and the TME is understood to be spatially patterned with differing constituents and function in different tumor areas .
Interaction of cancer cells and the tumor vasculature In the study of the TME, vascular remodeling has received particular attention as it impacts tumor nutrition, oxygenation, dissemination, and the recruitment of immune components. Quantitative assessments of the extent of tumor vascularization can be readily achieved by both tissue clearing and IVM, and have shown that several tumor types share a tendency to increase the vascular density within their mass . In some cancers the angiogenic capabilities may extend to lymphangiogenesis, as seen in pancreatic cancer . The cellular interactions between cancer cells and the tumor vasculature are multifold and reciprocal. IVM experiments on an orthotopic HNSCC model suggest that angiogenesis is specifically induced by a subpopulation of CD44 + cancer cells . On the other hand, 3D imaging of the tumor-stroma interface in cutaneous SCC showed that the blood vasculature regionally expresses TGF-β, thereby sustaining the cancer stem cell niche and contributing to cancer cell heterogeneity . In an HRAS-driven murine model of cutaneous SCC, angiogenesis was shown through tissue clearing to be triggered during the transition from papilloma to malignant carcinoma. This de novo vascularization in late stages of lesion progression facilitates leptin infiltration into the tumor, triggering Pi3k-Akt-mTor signaling, further contributing to the benign to malignant transition . Cancer progression through vascular remodeling is also seen in the bone marrow in acute myeloid leukemia (AML), where IVM experiments visualized how the remodeling of endosteal blood vessels abrogates hematopoietic stem cells (HSCs), HSC niches and osteoblasts through physical dislodgment via the damaged vasculature. Pharmacological MMP inhibition reduced vascular permeability and healthy cell loss, thereby limiting AML infiltration, proliferation, and cell migration .
Cancer cell crosstalk with the immune microenvironment The tumor vasculature is a significant route of tumor entry for immune cells that may clear cancer cells or be recruited for their tumor protective behavior. IVM successfully captured the critical steps in the cascade of lymphocyte adhesion to tumor-associated high endothelial venules (TA-HEVs), from initial lymphocyte tethering to the vessel wall to lymphocyte rolling and sticking, and extravasation . Tumor-infiltrating lymphocyte (TIL) motility changes both longitudinally during tumor progression , as well as regionally. Cytotoxic T lymphocytes (CTLs) show reduced motility and activity in avascular tumor areas but were found to be highly motile in vicinity to peripheral blood vessels and cleared tumor cells around them. Interestingly, blood flow is essential for CTL migration . Concomitantly, CTLs were shown to first accumulate in the tumor periphery and subsequently redistribute towards the invasive tumor front . Based on their migration patterns, CTLs have been understood to display a tumor cell searching behavior in the tumor periphery, which abates in velocity and changes into a “probing” behavior in tumor cell proximity indicating target engagement . IVM imaging showed that in the process of tumor cell killing, multiple CTLs undergo temporary sublethal contacts with individual tumor cells to induce killing in a process of additive cytotoxicity . On the other hand, CTL recognition of a few tumor cells can elicit sufficient IFNγ secretion to cover distances of several hundred micrometers and inhibit tumor growth even in regions not frequented by CTLs . Cancers hijack various cellular pathways to evade the immune response. T regulatory (Treg) cells are inherently poised to recognize antigens in the TME, and auto-regulate through short-lived interactions with conventional dendritic cells (cDCs) . Neutrophils stimulate T cells in a type I interferon (IFN) signaling dependent manner . However, tumor cells can elicit the production of neutrophil extracellular traps (NETs) from neutrophils and granulocytic myeloid-derived suppressor cells (MDSCs), and these NETs enwrap cancer cells, thereby reducing interfaces with CTLs and shielding against cytotoxicity . Neutrophils can promote tumor growth by activating MDSC that in turn inhibit the immune response orchestrated by effector T cells against cancer cells, as observed in cleared murine HNSCC tumors . Peritumoral neutrophils are more motile than intratumoral neutrophils , suggesting a different interactive behavior in specific tumor areas. TAMs play a multifold role in cancer progression. As for lymphocytes and neutrophils, volume imaging data suggest that TAM behavior differs drastically between tumor subtypes, stage and among different areas of the same lesion. IVM showed that the migratory behavior of TAMs is different in genetically distinct GBMs . In an orthotopic model of anaplastic thyroid cancer, tissue clearing suggested that the infiltration of tumor-associated macrophages is significantly higher in the primary tumor than in the metastases, indicating that the microenvironment in which the tumor develops conditions the infiltration of tumor-promoting TAMs . Using the dorsal skinfold chamber to track the crosstalk between macrophages and cancer grafts by IVM identified macrophage subpopulations based on their localization in the tumor core or periphery, and found that those populations differed in Arg1 gene expression and motility. This study also demonstrated the powerful combination of IVM with single cell RNA sequencing to gain molecular insight from a single cell phenotype that can only be observed in the native organ context . The cell-cell interaction between TAMs and cancer cells is a driving factor of tumor aggression. Macrophages were found to induce cancer cell stemness though Notch-Jagged signaling , and enable cancer cell dissemination through dynamic multicellular interactions . In an orthotopic breast cancer model, monocytes were shown to arrive at the tumor as motile streaming TAMs that then differentiated into sessile perivascular macrophages. This differentiation occurred through cancer cell induced TGFβ-dependent upregulation of CXCR4 in monocytes while CXCL12 expressed by perivascular fibroblasts attracted these motile TAMs toward the blood vessels, bringing motile cancer cells with them. Once on the blood vessel, the migratory TAMs differentiated into perivascular macrophages, promoting vascular leakiness and intravasation . Together volume imaging experiments of the recent years demonstrated that tumor- supportive and tumor-inhibiting activities of immune cells crucially depend on the regional microenvironment, and are modulated in a highly dynamic way through complex multi-cellular interactions.
The basement membrane and cancer cell invasion The basement membrane constitutes a key barrier to tumor cell migration into the surrounding parenchyma. Recent IVM experiments, using a fluorescent laminin beta1 reporter mouse, show that basement membrane components are synthesized by cancer cells as well as endothelial cells in a dynamic and regionally heterogeneous manner. Interestingly, laminin beta1 turnover is higher in cancerous areas compared to normal epithelium, suggesting a local pre-conditioning of the basement membrane preceding tumor cell invasion . IVM imaging of cancer cell invasion has been instrumental to understand the cellular processes and interaction of cancer cell dissemination. Myosin 10 filopodia support the basement membrane in early lesions, but contribute to a proinvasive phenotype later in disease . Interestingly, cells that are able to breach the basement membrane, due to experimentally induced loss of E-cadherin which increases cancer cell motility, require further signals in order to disseminate into the surrounding parenchyma . Together, 3D cellular confinement, cell density and cell-cell junction stability orchestrate epithelial fluidization and single cell release , and set out the paths of migration and leader-follower cell relationships in collective movements. Cancer cells were found to migrate along collagen fibrils and blood vessels in several tumor models . Leader cells undergo significant deformations, potentially due to the confinement by the ECM, but create paths for follower cells that have more normalized cell and nuclear shapes . This synchronous migration along common paths may reflect an expansion along newly generated space in the ECM confinement, but could also be instructed directly from leader to follower cells through extracellular vesicle exchange . Thereby, the migratory behavior differs throughout the tumor landscape. GBM comprises an invasive margin with slow directed invasion and a diffuse infiltration margin with fast but less directed cell movements. Migration along blood vessels correlated with higher velocity and direction away from the tumor core whereas intraparenchymal migration along white matter tracts was measured to be slower and less directed . In glioma, IVM showed that GFAP splice variants fine‐tune invasion by modulating migration persistence. Whilst depletion of either isoform increased the migratory capacity of glioma cells, GFAPδ‐depleted cells migrated randomly through the brain tissue, whereas GFAPα‐depleted cells displayed a directionally persistent invasion into the brain parenchyma .
Cancer cell-TME interactions in transit to distant organs Tumor cell intravasation is a multicellular process in the TME which in breast cancer models was shown to require direct contacts between a proangiogenic perivascular macrophage, a tumor cell overexpressing the actin regulator protein Mena, and an endothelial cell that together create the “tumor microenvironment of metastasis” (TMEM), allowing cancer cell intravasation through transient vascular permeability . Vascular travel is a bottle neck in tumor cell dissemination as only a portion of the cells entering the blood stream are able to seed and colonize distant organs. Real-time visualization of circulating tumor cell (CTC) subpopulations through conjugated quantum dots showed that PDAC CTCs expressing the surface protein CD24 have the ability to migrate along vessel walls to distant organs . CTC packing in cell clusters can provide support for traveling cancer cells and has been debated to occur through collective intravasation or de novo clustering in the circulation. Intravital imaging showed that breast cancer CTC clusters form in circulation through CD44 protein mediated cell aggregation . Tissue clearing showed that breast cancer cells extravasate with the help of actin-rich protrusions . CTC extravasation depends on reciprocal interactions between CTCs and endothelial cells of distant organs that can prohibit or permit CTC organ entry. The bone endothelium expresses ephrin-B2 which activates ephrin-B4 in circulating melanoma cells thereby repulsing CTCs from attaching to the bone endothelial cells and barricading against metastasis formation . In the mouse lung, extravasation of breast cancer cells is dependent on physical interaction between tumor cells and macrophages in a IL4-CXCR2 dependent manner .
Cell-cell crosstalk at metastatic sites Metastasizing cancer cells can disseminate and establish tumors in organs with disparate cellular and physicochemical characteristics, or can present strong tropism and invade only one secondary organ . Tissue clearing of entire mice has shown the diverse tropism of different cancer cell lines such as mammary MDA-231 and pancreatic OS-RC-2 cells, and their colocalization with the vasculature across the mouse body . Volume imaging uncovered a widespread heterogeneity in the seeding potential, and the likelihood for successful metastatic outgrowth of different tumor cell subpopulations. These factors are now understood to be modulated by cell intrinsic properties and CSC traits on the one hand, as well as by dynamic multi-cellular contacts at the metastatic site on the other hand. Multicellular interactions influence the survival and outgrowth of metastasis seeding cancer cells. In the brain, early extravasating cells may be cleared by phagocytosis from TAM and microglia, although these effects abated in later stages of metastasis formation . As in primary tumors, the multi-cellular arrangements of cancer cells and TME components in metastatic sites show spatial and longitudinal heterogeneity. There is a highly heterogeneous cellular interplay between metastases even from the same tumor, as illustrated in breast cancer brain metastases that involve glial cells and astrocytes of the brain parenchyma to very different extents . The communication between cancer cells and the TME at different organs may amplify the heterogeneity seen in metastatic lesions. Indeed, the proliferation dynamics of cancer cells were found to depend on the organ colonized as shown with A549 lung adenocarcinoma cells after intracardiac administration into mice . One environmental mechanism may involve the access of pro-and antitumorigenic immune cells. Neutrophils are highly motile in lung capillaries and change their behavior to slow patrolling and stationary upon recruitment to lung metastasis where they were filmed to correlate with tumor cell proliferation .
Volume imaging as a platform for therapy development Whilst historically tissue culture assays have been used to carry out cancer drug screens, an ever-growing body of literature highlights limitations in the predictive value of cell culture assays to extrapolate the effect of drugs from in vitro to an entire organism. This may be attributed to a difference in signals that cells cultured in vitro lack in comparison to cancer cells in vivo . Cancer cells receive stimuli from neighboring cancer cells, non-tumor cells, architectural constraints and systemic physical factors that shape tumor biology in vivo . Recent studies using IVM showed that even cell-intrinsic molecular characteristics may not be adequately reflected in 2D/3D culture systems . In fact, when transplanted in vivo , a panel of breast cancer cell lines showed remarkable cellular heterogeneity, even in the same nodule that was not observed in vitro . Hence, preclinical models remain essential to evaluate the effect of new therapeutic agents . Combining preclinical models with fluorescence volume imaging constitutes a powerful tool to assess potential anti-cancer treatments, many of which directly targeted against components of the TME, and to study how they affect the interaction between cancer cells and the TME. 5.1 Drug delivery is dependent on tumor vascularization and perfusion Volume imaging technologies pose an attractive platform to evaluate a new drug, map off-target effects and identify the mechanisms underlying acquired therapy resistance. Several studies used tissue clearing to assess the therapeutic delivery of drugs and drug vehicles in vivo . The distribution of nanoparticles and PEGylated liposomes, and their spatial relation with the vasculature was used to evaluate efficiency of drug delivery into the tumor . Similarly, tissue clearing visualized tumor perfusion by fluorescently tagged anti-tumor antibodies, such as the HER-2-targetting antibody trastuzumab, and a fluorescent analogue of the chemotherapeutic agent docetaxel . In cases where the therapeutic agent cannot be traced directly, drug interference with fluorescent signals from the target cells may serve as proxy for measuring drug engagement. In an elegant example, doxorubicin uptake was measured in vivo on a single cell level by exploiting the changes in fluorescence lifetime of histone-GFP fusions upon doxorubicin binding to chromatin. This uncovered drastic differences in doxorubicin binding between different peritoneal metastases in the same mouse, as well as different regions and cells in the same lesion . In a murine model of non-Hodgkin lymphoma, tissue clearing revealed that the delivery of therapeutic CD20 fragment antigen-binding (FAB) antibody regions was dependent on tumor perfusion . IVM showed that preserving endosteal vessels in AML improves the efficacy of chemotherapy . IVM of melanoma and PDAC xenografts implanted under a dorsal skinfold window was used as a platform to quantify the vascular effects of acute cyclic hypoxia and antiangiogenic treatment . A key goal of treatment development has been to educate and exploit the immune system to reject cancer progression. Immune infiltration into the tumor is also dependent on its vascularization. IVM assays are an elegant tool to measure the biodistribution and efficacy of CAR T cells, as demonstrated for primary central nervous system lymphoma (PCNSL) and GBM . Volume imaging identified tumor associated high endothelial venules (TA-HEVs) as the main sites of lymphocyte arrest and extravasation into tumors that received anti-PD-1/anti-CTLA-4 immunotherapy. Increasing TA-HEV endothelial cells (TA-HEC) frequency and maturation improved CTL recruitment and therapy response . IVM experiments showed that carboplatin chemotherapy profoundly alters the TME to promote lymphocyte adhesive interactions with the tumor vasculature resulting in improveed lymphocyte trafficking . Another IVM study in a GBM model suggested that radiotherapy may aid CAR T cell extravasation and expansion . CTLs engineered to overexpress the chemokine receptor CXCR4 displayed increased homing to the bone marrow and engraftment, and acquired greater anti-tumor immunity . Cancer-derived adenosine weakens the physical interaction between activated effector CTLs and target cells, providing cancer cells with an escape way from immunotherapy. IVM showed that antagonization of adenosine A2a receptor (ADORA2a) signaling improved CTL-target engagement and cancer cell killing . 5.2 Measuring drug efficacy A key aspect of therapy development is the measure of in vivo drug efficacy. IVM of calvarium bone marrow tracked the migratory behavior of acute myeloid leukemia (AML) cells in comparison to T cell acute lymphoblastic leukemia (T-ALL), and demonstrated distinct traits of the lineages in their response to chemotherapy and CXCR4 antagonism, highlighting the importance of carefully evaluating cancer traits in vivo for each tumor type . Tissue clearing has been beneficial to evaluate therapy efficiency in various preclinical cancer models, such as HepG2 xenografts to assess the severing of the vasculature induced by antiangiogenic therapeutic siRNAs , and HER-2-expressing breast cancer xenografts treated with colony-stimulation factor 1 receptor inhibitor PLX3397, which reduced tumor burden without impairing the infiltration of macrophages . Tissue clearing for whole organ quantifications of metastatic burden showed that targeting macrophages with clodronate liposomes reduced lung colonization by intravenously injected A549 cancer cells . KPL-4 breast cancer xenografts were found to present a normalized vasculature when treating mice with anti-VEGF antiangiogenic therapy , indicating an improvement of tumor irrigation and of the penetration of nutrients and oxygen, but potentially also of other therapeutic agents that may hence more efficiently find and eliminate cancer cells. 5.3 Immunotherapy Immunotherapy is efficient in immunogenic tumors that express immune checkpoint molecules, whether in the cancer cells or in other infiltrating cells of the TME. Tissue clearing has been used to map the expression of immune checkpoint molecule PD-L1 and immune cells throughout tumors such as MMTV-HER2/Neu and NSCLC patient resections . Such 3D quantifications are more precise than 2D analyses of tissue sections and could be applied to patient biopsies as a prognostic marker that predicts the response to immunotherapy. In addition to tissue clearing, IVM has been successfully applied to evaluate the biodistribution of immunotherapies . Furthermore, such in vivo assays have proven useful to pinpoint the reasons for immunotherapy failure, and could demonstrate for example how a fluorophore-conjugated PD-1 targeting antibody shown to target PD-1 + tumor infiltrating CD8 T-cells was immediately cleared by PD-1 - tumor associated macrophages, preventing tumor regression . 5.4 Cancer therapy side effects One key advantage of tissue clearing is the power to detect rare events in large tissue volumes in a quantitative manner and at little time expenditure. These traits make tissue clearing an attractive solution to study side effects of cancer therapies. Once organ sites suffering under off-target effects and drug toxicity are identified, IVM can add to this informative value by revealing the dynamic interplay of cell populations. Liver toxicity is a common side effect of viral therapies and Naumenko and colleagues demonstrated how IVM can be used to track the interactions between hepatocytes, Kupffer cells, CD8 T-cells and neutrophils to study oncolytic adenovirus associated liver toxicity . Apart from insights that educate drug refinement, IVM may also uncover new treatment options. For example, IVM experiments uncovered evidence that bisphosphonate drugs, which were designed to target bone, readily accumulate in microcalcifications in breast cancers where they have been suggested to stimulate TAMs and improve cancer patient survival independent of the drug’s antiresorptive effects on bone tissue . 5.5 Therapy resistance A common trait of many cancers is the ability to acquire therapy resistance over the course of a drug treatment. The underlying mechanisms can be partially attributed to genomic and epigenomic evolution and molecular adaptation of tumor cell subpopulations. The process of epithelial-to-mesenchymal transition (EMT) has been under intense debate to play a key role in cancer aggression and IVM experiments proposed that cancer cells that have undergone EMT may be a source for cancer recurrence upon chemotherapy . Volume imaging suggests that the acquisition of therapy resistance, coming from subpopulations in the tumor bulk, may be linked to specific architectural regions of a tumor lesion. Indeed, cells in the invasive front were found to display a higher degree of radiotherapy resistance and survival. Combinatorial integrin targeting sensitized these cells and abrogated tumor relapse . Apart from tumor cell intrinsic molecular paths towards drug resistance, IVM experiments also demonstrated the role of direct interactions and crosstalk between tumor cells and the TME in this context. Matrix stiffness was found to dictate PDAC chemosensitivity and IVM showed that therapy priming through focal adhesion kinase (FAK) inhibition pulses could drastically increase PDAC cell sensitivity to gemcitabine/abraxane .
Drug delivery is dependent on tumor vascularization and perfusion Volume imaging technologies pose an attractive platform to evaluate a new drug, map off-target effects and identify the mechanisms underlying acquired therapy resistance. Several studies used tissue clearing to assess the therapeutic delivery of drugs and drug vehicles in vivo . The distribution of nanoparticles and PEGylated liposomes, and their spatial relation with the vasculature was used to evaluate efficiency of drug delivery into the tumor . Similarly, tissue clearing visualized tumor perfusion by fluorescently tagged anti-tumor antibodies, such as the HER-2-targetting antibody trastuzumab, and a fluorescent analogue of the chemotherapeutic agent docetaxel . In cases where the therapeutic agent cannot be traced directly, drug interference with fluorescent signals from the target cells may serve as proxy for measuring drug engagement. In an elegant example, doxorubicin uptake was measured in vivo on a single cell level by exploiting the changes in fluorescence lifetime of histone-GFP fusions upon doxorubicin binding to chromatin. This uncovered drastic differences in doxorubicin binding between different peritoneal metastases in the same mouse, as well as different regions and cells in the same lesion . In a murine model of non-Hodgkin lymphoma, tissue clearing revealed that the delivery of therapeutic CD20 fragment antigen-binding (FAB) antibody regions was dependent on tumor perfusion . IVM showed that preserving endosteal vessels in AML improves the efficacy of chemotherapy . IVM of melanoma and PDAC xenografts implanted under a dorsal skinfold window was used as a platform to quantify the vascular effects of acute cyclic hypoxia and antiangiogenic treatment . A key goal of treatment development has been to educate and exploit the immune system to reject cancer progression. Immune infiltration into the tumor is also dependent on its vascularization. IVM assays are an elegant tool to measure the biodistribution and efficacy of CAR T cells, as demonstrated for primary central nervous system lymphoma (PCNSL) and GBM . Volume imaging identified tumor associated high endothelial venules (TA-HEVs) as the main sites of lymphocyte arrest and extravasation into tumors that received anti-PD-1/anti-CTLA-4 immunotherapy. Increasing TA-HEV endothelial cells (TA-HEC) frequency and maturation improved CTL recruitment and therapy response . IVM experiments showed that carboplatin chemotherapy profoundly alters the TME to promote lymphocyte adhesive interactions with the tumor vasculature resulting in improveed lymphocyte trafficking . Another IVM study in a GBM model suggested that radiotherapy may aid CAR T cell extravasation and expansion . CTLs engineered to overexpress the chemokine receptor CXCR4 displayed increased homing to the bone marrow and engraftment, and acquired greater anti-tumor immunity . Cancer-derived adenosine weakens the physical interaction between activated effector CTLs and target cells, providing cancer cells with an escape way from immunotherapy. IVM showed that antagonization of adenosine A2a receptor (ADORA2a) signaling improved CTL-target engagement and cancer cell killing .
Measuring drug efficacy A key aspect of therapy development is the measure of in vivo drug efficacy. IVM of calvarium bone marrow tracked the migratory behavior of acute myeloid leukemia (AML) cells in comparison to T cell acute lymphoblastic leukemia (T-ALL), and demonstrated distinct traits of the lineages in their response to chemotherapy and CXCR4 antagonism, highlighting the importance of carefully evaluating cancer traits in vivo for each tumor type . Tissue clearing has been beneficial to evaluate therapy efficiency in various preclinical cancer models, such as HepG2 xenografts to assess the severing of the vasculature induced by antiangiogenic therapeutic siRNAs , and HER-2-expressing breast cancer xenografts treated with colony-stimulation factor 1 receptor inhibitor PLX3397, which reduced tumor burden without impairing the infiltration of macrophages . Tissue clearing for whole organ quantifications of metastatic burden showed that targeting macrophages with clodronate liposomes reduced lung colonization by intravenously injected A549 cancer cells . KPL-4 breast cancer xenografts were found to present a normalized vasculature when treating mice with anti-VEGF antiangiogenic therapy , indicating an improvement of tumor irrigation and of the penetration of nutrients and oxygen, but potentially also of other therapeutic agents that may hence more efficiently find and eliminate cancer cells.
Immunotherapy Immunotherapy is efficient in immunogenic tumors that express immune checkpoint molecules, whether in the cancer cells or in other infiltrating cells of the TME. Tissue clearing has been used to map the expression of immune checkpoint molecule PD-L1 and immune cells throughout tumors such as MMTV-HER2/Neu and NSCLC patient resections . Such 3D quantifications are more precise than 2D analyses of tissue sections and could be applied to patient biopsies as a prognostic marker that predicts the response to immunotherapy. In addition to tissue clearing, IVM has been successfully applied to evaluate the biodistribution of immunotherapies . Furthermore, such in vivo assays have proven useful to pinpoint the reasons for immunotherapy failure, and could demonstrate for example how a fluorophore-conjugated PD-1 targeting antibody shown to target PD-1 + tumor infiltrating CD8 T-cells was immediately cleared by PD-1 - tumor associated macrophages, preventing tumor regression .
Cancer therapy side effects One key advantage of tissue clearing is the power to detect rare events in large tissue volumes in a quantitative manner and at little time expenditure. These traits make tissue clearing an attractive solution to study side effects of cancer therapies. Once organ sites suffering under off-target effects and drug toxicity are identified, IVM can add to this informative value by revealing the dynamic interplay of cell populations. Liver toxicity is a common side effect of viral therapies and Naumenko and colleagues demonstrated how IVM can be used to track the interactions between hepatocytes, Kupffer cells, CD8 T-cells and neutrophils to study oncolytic adenovirus associated liver toxicity . Apart from insights that educate drug refinement, IVM may also uncover new treatment options. For example, IVM experiments uncovered evidence that bisphosphonate drugs, which were designed to target bone, readily accumulate in microcalcifications in breast cancers where they have been suggested to stimulate TAMs and improve cancer patient survival independent of the drug’s antiresorptive effects on bone tissue .
Therapy resistance A common trait of many cancers is the ability to acquire therapy resistance over the course of a drug treatment. The underlying mechanisms can be partially attributed to genomic and epigenomic evolution and molecular adaptation of tumor cell subpopulations. The process of epithelial-to-mesenchymal transition (EMT) has been under intense debate to play a key role in cancer aggression and IVM experiments proposed that cancer cells that have undergone EMT may be a source for cancer recurrence upon chemotherapy . Volume imaging suggests that the acquisition of therapy resistance, coming from subpopulations in the tumor bulk, may be linked to specific architectural regions of a tumor lesion. Indeed, cells in the invasive front were found to display a higher degree of radiotherapy resistance and survival. Combinatorial integrin targeting sensitized these cells and abrogated tumor relapse . Apart from tumor cell intrinsic molecular paths towards drug resistance, IVM experiments also demonstrated the role of direct interactions and crosstalk between tumor cells and the TME in this context. Matrix stiffness was found to dictate PDAC chemosensitivity and IVM showed that therapy priming through focal adhesion kinase (FAK) inhibition pulses could drastically increase PDAC cell sensitivity to gemcitabine/abraxane .
Future perspectives in volume imaging of the TME Since its rediscovery about a decade ago, tissue clearing has been drastically refined to meet the most diverse problems in cancer research, from the study of cancer cell interactions with the infiltrating TME to the mapping of tumor vascularization and innervation, and quantifications of whole-body metastatic load. A wealth of excellent techniques exists tailored to different organs, cancers, and fluorescent readouts. The recent development of accessible whole mouse clearing techniques and antibody labelling of cells spread throughout the entire organism allows to study the differential TMEs at both the primary tumor and metastatic sites in the same organism. Today most of the tissues and architectural structures can be visualized in 3D. Open opportunities remain in the detection of pathogens, such as whole organ quantifications of the distribution of microbiota in tumors . One bottleneck of tissue clearing remains in the limited detection of multiple cell types in the same sample. However, technical solutions may not be far as pioneering studies on 2D tissue sections have pushed the boundaries in multiplexing for fluorescent microscopy. Imaging mass cytometry detects up to 32 markers , and 3D mass cytometry has recently been achieved for thick sections of around 300 um depth . Whilst tissue clearing constitutes a powerful tool to examine entire tissues, organs and organisms, refractive index matching can be combined with expansion microscopy to visualize subcellular details . With this technology, super-resolution information of the molecular cancer cell-TME interactions can be unveiled. In order to connect 3D information with an understanding of cellular dynamics, IVM today offers several platforms, such as the dorsal skin fold chamber, tumor grafts in the eye or tissue-engineered bone constructs with a skin window for longitudinal imaging. Together they enable cancer research to evaluate potential therapeutics in a more relevant setting in vivo without the expenditure of developing strategies to study cancers in their native organ site . Tumor cell grafts readily remodel their surrounding vasculature and stand in intense crosstalk with the immune system, providing ample opportunity to understand and interfere with fundamental cancer traits such as tumor angiogenesis and immune evasion. However, these strategies may give little opportunity to study the crosstalk between cancer cells and their neighboring normal epithelial cells. Furthermore, the tumor graft origin from in vitro cultures may fundamentally alter cancer cell properties. Indeed, 3D imaging showed that the angiogenic capacity of xenograft models depends on the culture conditions prior to injection, suggesting that epigenetic changes in vitro condition the communication between cancer cells and the vascular microenvironment in vivo . Furthermore, intravital imaging of lung metastases showed that spontaneously disseminating tumor cells are more capable to form distant metastasis than experimentally metastasized tumor cells . Whilst the majority of volume imaging studies have used these platforms for fundamental and preclinical research, 3D imaging also poses considerable attractiveness for cancer staging. Tissue clearing could be exploited for the analysis of cancer patient resections in a diagnostic setting. 3D mapping of vasculature and lymphatics was shown to improve the prognostic evaluation of bladder cancer . The presence of high endothelial venules in models of fibrosarcoma and mammary carcinoma was found to correlate with tumor regression upon Treg cell depletion , and to predict survival and αPD-1/αCTLA-4 immunotherapy response of melanoma patients . Pathways for 3D image automation and standardization are already being laid out, as demonstrated in the successful computational segmentation of prostate cancer patient samples . A 3D high-resolution view into a clinical cancer resection holds great informational value and can uncover unexpected subtleties in the cellular composition and spatial arrangements . For example, tissue clearing showed that luminal prostate cancer, widely expressing K8 (marker of luminal epithelium) contains regions enriched in K5 + myoepithelium which acquire a more differentiated, ductal conformation . Nevertheless, clinical adoption and utilization will require careful evaluation of 3D imaging data and side by side comparisons with conventional histopathological assessment to educate data interpretation towards the best treatment decisions and to avoid patient overtreatment.
JA and HM researched literature for the review, discussed content, wrote, reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.
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Paternity through use of assisted reproduction technology in male adult and childhood cancer survivors: a nationwide register study
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c2c16b0e-0d07-4088-b25a-d08480de3f64
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10228784
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Forensic Medicine[mh]
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It is well known that male cancer survivors are more likely to suffer from infertility than other males ( ). This is mainly due to utilization of various surgical procedures and cytostatic and radiotherapy regimens which can have a negative impact on semen quality, by impairing ejaculation or hypothalamic, pituitary, and/or testicular function ( ). Over the last few decades, major advances in the field of oncology have led to average 10-year survival rate reaching nearly 80% in patients with childhood, adolescence, or young adulthood cancer ( ). With a globally growing population of young male cancer survivors, more patients are expected to experience reduced fertility. For men with impaired semen quality following cancer treatment ( ; ; ), the most obvious way of achieving fatherhood would be by using ART, such as IVF or ICSI. Conception through IVF is most common in cases of less severe forms of male subfertility, impaired female fertility, or a combination of both. ICSI treatment, on the other hand, is recommended for men with more severe impairment of fertility ( ; ). It is estimated that in 2013, up to 3.6% of offspring born in Sweden were conceived through ART ( ). Multiple studies have analyzed the use of fertility preservation and outcomes of assisted reproduction in men with history of cancer ( ; ; ). However, studies exploring the paternity rates through ART after cancer treatment and identifying subgroups of male cancer survivors having the highest proportion of children conceived by ARTs are few ( ; ), and no analysis discriminating between use of IVF and ICSI, has been performed. Such studies require access to data with many cancer-treated men and an adequate follow-up regarding their reproductive life. According to the recently launched European Atlas of Fertility Treatment Policies ( ), Sweden has been classified as ‘very good’ when it comes to providing access to a well-funded, fertility treatment evenly across the country. Therefore, we could assume that since it is nearly independent of socioeconomic status, ART in Sweden is provided to the majority of male cancer survivors with a desire for fatherhood but with the inability to conceive a child naturally. The objective of this study was, using national Swedish population-based registries, to estimate the proportion of ART-conceived children among male cancer survivors, and to identify diagnostic subgroups most likely to conceive their child using ART.
Data extraction from national registers The cohort was created by combining information from the Swedish Medical Birth Register, the Swedish Multi-generation Register, the Swedish Register of Education, the Swedish Cancer Register, and the Swedish National Quality Register of Assisted Reproduction (Q-IVF). Ethical approval was granted by the ethics committee at Lund University (LU 2016/104). Information on all children born alive in Sweden between 1994 and 2014 (2 108 569) and their fathers, was retrieved from the Swedish Medical Birth Register and the Swedish Multi-generation Register, respectively. Subsequently, non-first born offspring during the study period (907 109) and cases of missing paternal (19 970) or maternal (2) identification numbers, were excluded. Furthermore, using data on gestational length from the Swedish Medical Birth Register, we could estimate dates of conception. Among fathers with a cancer diagnosis, in order to ensure that the malignancy was diagnosed prior to conception, we excluded men who were diagnosed with cancer after or <12 months before offspring conception, which resulted in 1 159 959 fathers and the same number of children being included in the present analysis. The Swedish Medical Birth Register provided data on the mode of offspring conception for live births before 2007, and Q-IVF for those born in 2007 or later. The Swedish Cancer Register and the Swedish Register of Education provided data on any possible cancer diagnoses and information concerning paternal education, respectively. Information regarding any cancer diagnoses was available from 1 January 1958 until 31 December 2014 ( ). Paternal cancer groups Based on the age at cancer diagnosis, cancer patients included in the cohort were grouped into three main groups: (i) childhood cancer survivors (diagnosis of cancer at age <15), (ii) teenage and young adult cancer survivors (diagnosis of cancer at age ≥15 and <24), and (iii) adult cancer survivors (diagnosis of cancer at age ≥24). With the aim of subgroup analyses, fathers with a history of cancer were further divided into eight categories based on the information regarding cancer localization. The groups were defined according to the International Classification of Diseases, 7th revision (ICD-7), and were as follows: (i) skin cancer (ICD-7: 140.0–140.9, 190.0–191.9); (ii) prostate cancer (ICD-7: 177.0–177.9); (iii) testicular cancer (ICD-7: 178.0–178.9); (iv) digestive, respiratory, and urogenital tract cancers (ICD-7: 141.0–163.9, 179.0–181.9); (v) central nervous system and eye cancers (ICD-7: 192.0–193.1); (vi) soft tissue and bone cancers (ICD-7: 193.3, 193.8, 193.9, 196.0–197.9); (vii) hematological and lymphatic cancers (ICD-7: 200.0–207.9); and (viii) all other cancer diagnoses (ICD-7: 164.0–164.9, 170.1, 170.2, 194.0–194.9, 195.0–195.9, 199.1–199.9). Statistical analysis Associations between age at onset of cancer, as well as type of cancer diagnosis, and conception through either ICSI or IVF were evaluated using univariate and multivariate logistic regression models, which yielded an unadjusted and adjusted odds ratio (aOR) with a 95% CI. Information on use of donated spermatozoa was only available from Q-IVF, and thus, the analysis of likelihood of fathering a child through ART with donated gametes was restricted to children born between 2007 and 2014. The length of the time interval since completion of cancer treatment could affect the utilization of ART, both due to the degree of recovery of spermatogenesis but also other factors, including the number of attempts to achieve pregnancy and age-related changes in fertility of the partner. Therefore, we also examined the association between the number of years between cancer diagnosis and offspring conception (1–3, 3–5, and >5) and likelihood of using ART, when compared to controls. This analysis was restricted to only adult cancer survivors and controls were matched on year of childbirth, with a 1:1 ratio. Variables used in the adjusted model were fathers’ age at childbirth (continuous) and paternal years of formal education (<10, 10–14, ≥15, or missing data), factors known to be associated with use of assisted reproduction. The first case of successful birth through ICSI treatment was reported in 1992 ( ), which is why access to this type of ART was limited in the beginning of the study period. Therefore, we also decided to adjust for the year of offspring conception (continuous) in the adjusted model. Fathers without a cancer diagnosis were used as controls in all analyses performed. A P -value <0.05 (two-sided) was considered as statistically significant. All statistical analyses were conducted using IBM SPSS Statistics version 28 (IBM Corp.).
The cohort was created by combining information from the Swedish Medical Birth Register, the Swedish Multi-generation Register, the Swedish Register of Education, the Swedish Cancer Register, and the Swedish National Quality Register of Assisted Reproduction (Q-IVF). Ethical approval was granted by the ethics committee at Lund University (LU 2016/104). Information on all children born alive in Sweden between 1994 and 2014 (2 108 569) and their fathers, was retrieved from the Swedish Medical Birth Register and the Swedish Multi-generation Register, respectively. Subsequently, non-first born offspring during the study period (907 109) and cases of missing paternal (19 970) or maternal (2) identification numbers, were excluded. Furthermore, using data on gestational length from the Swedish Medical Birth Register, we could estimate dates of conception. Among fathers with a cancer diagnosis, in order to ensure that the malignancy was diagnosed prior to conception, we excluded men who were diagnosed with cancer after or <12 months before offspring conception, which resulted in 1 159 959 fathers and the same number of children being included in the present analysis. The Swedish Medical Birth Register provided data on the mode of offspring conception for live births before 2007, and Q-IVF for those born in 2007 or later. The Swedish Cancer Register and the Swedish Register of Education provided data on any possible cancer diagnoses and information concerning paternal education, respectively. Information regarding any cancer diagnoses was available from 1 January 1958 until 31 December 2014 ( ).
Based on the age at cancer diagnosis, cancer patients included in the cohort were grouped into three main groups: (i) childhood cancer survivors (diagnosis of cancer at age <15), (ii) teenage and young adult cancer survivors (diagnosis of cancer at age ≥15 and <24), and (iii) adult cancer survivors (diagnosis of cancer at age ≥24). With the aim of subgroup analyses, fathers with a history of cancer were further divided into eight categories based on the information regarding cancer localization. The groups were defined according to the International Classification of Diseases, 7th revision (ICD-7), and were as follows: (i) skin cancer (ICD-7: 140.0–140.9, 190.0–191.9); (ii) prostate cancer (ICD-7: 177.0–177.9); (iii) testicular cancer (ICD-7: 178.0–178.9); (iv) digestive, respiratory, and urogenital tract cancers (ICD-7: 141.0–163.9, 179.0–181.9); (v) central nervous system and eye cancers (ICD-7: 192.0–193.1); (vi) soft tissue and bone cancers (ICD-7: 193.3, 193.8, 193.9, 196.0–197.9); (vii) hematological and lymphatic cancers (ICD-7: 200.0–207.9); and (viii) all other cancer diagnoses (ICD-7: 164.0–164.9, 170.1, 170.2, 194.0–194.9, 195.0–195.9, 199.1–199.9).
Associations between age at onset of cancer, as well as type of cancer diagnosis, and conception through either ICSI or IVF were evaluated using univariate and multivariate logistic regression models, which yielded an unadjusted and adjusted odds ratio (aOR) with a 95% CI. Information on use of donated spermatozoa was only available from Q-IVF, and thus, the analysis of likelihood of fathering a child through ART with donated gametes was restricted to children born between 2007 and 2014. The length of the time interval since completion of cancer treatment could affect the utilization of ART, both due to the degree of recovery of spermatogenesis but also other factors, including the number of attempts to achieve pregnancy and age-related changes in fertility of the partner. Therefore, we also examined the association between the number of years between cancer diagnosis and offspring conception (1–3, 3–5, and >5) and likelihood of using ART, when compared to controls. This analysis was restricted to only adult cancer survivors and controls were matched on year of childbirth, with a 1:1 ratio. Variables used in the adjusted model were fathers’ age at childbirth (continuous) and paternal years of formal education (<10, 10–14, ≥15, or missing data), factors known to be associated with use of assisted reproduction. The first case of successful birth through ICSI treatment was reported in 1992 ( ), which is why access to this type of ART was limited in the beginning of the study period. Therefore, we also decided to adjust for the year of offspring conception (continuous) in the adjusted model. Fathers without a cancer diagnosis were used as controls in all analyses performed. A P -value <0.05 (two-sided) was considered as statistically significant. All statistical analyses were conducted using IBM SPSS Statistics version 28 (IBM Corp.).
Study population Among a total of 1 159 959 fathers included in the cohort, 1 125 431 (97%), 20 220 (1.7%), and 14 560 (1.3%) of their first-born children were conceived naturally, or through IVF and through ICSI, respectively. Among fathers with history of cancer, 861 (16.0%) fathers were diagnosed with childhood cancer, 1509 (28.1%) with teenage and young adulthood cancer, and 3002 (55.9%) with adult cancer. Among childhood cancer survivors, 6.0% conceived by ART, while this proportion was 10.1% for teenage and young adult cancer survivors, 13.3% for the adult cancer survivors, and 3.0% for the controls. Further background characteristics of the fathers are shown in . Conceiving through IVF and ICSI in male cancer survivors When compared to controls, the entire group of cancer survivors was statistically significantly more likely to father through ART (aOR 3.18, 95% CI 2.91–3.48; P < 0.001). Adult cancer survivors were more likely to father through both ICSI (aOR 5.52, 95% CI 4.86–6.27; P < 0.001) and IVF treatment (aOR 1.32, 95% CI 1.09–1.60; P = 0.004), while teenage and young adult cancer survivors only had a statistically significant increased likelihood of conceiving through ICSI (aOR 6.84, 95% CI 5.64–8.30; P < 0.001) and not IVF treatment (aOR 1.27, 95% CI 0.90–1.80; P = 0.17). Similarly, childhood cancer survivors were only more likely to conceive a child through ICSI (aOR 3.52, 95% CI 2.52–4.93; P < 0.001) but not through IVF (aOR 1.02, 95% CI 0.61–1.70; P = 0.955). When compared to the general population, childhood cancer survivors (aOR 8.84, 95% CI 4.41–17.7; P < 0.001), teenage and young adult cancer survivors (aOR 8.28, 95% CI 5.02–13.6; P < 0.001), and adult cancer survivors (aOR 5.46, 95% CI 3.78–7.90; P < 0.001) were all more likely to father a child through ART using donated spermatozoa. The proportions of men utilizing donated spermatozoa for conception of their offspring were 33.3%, 7.6%, and 8.3% for childhood, teenage and young adulthood, and adult cancer groups, respectively. The adjusted ORs for ICSI conception according to the cancer diagnosis are given in . Childhood survivors of testicular cancer (aOR 5.15, 95% CI 1.20–22.0; P = 0.027), soft tissue and bone cancers (aOR 4.70, 2.13–10.40; P < 0.001), hematological and lymphatic cancers (aOR 4.49, 95% CI 2.72–7.40; P < 0.001), or central nervous system (CNS) and eye cancers (aOR 2.64, 95% CI 1.23–5.67; P = 0.012), were at an increased likelihood of fathering through ICSI. Teenage and young adult survivors of testicular cancer (aOR 15.4, 95% CI 11.5–20.7; P < 0.001), hematological and lymphatic cancers (aOR 9.84, 95% CI 6.93–14.0; P < 0.001), or soft tissue and bone cancers (aOR 6.83, 95% CI 3.53–13.2; P < 0.001) were more likely to father through ICSI treatment. Adult survivors of prostate cancer (aOR 15.7, 95% CI 6.70–36.9; P < 0.001), testicular cancer (aOR 9.54, 95% CI 7.81–11.7; P < 0.001), hematological and lymphatic cancers (aOR 11.3, 95% CI 8.63–14.9; P < 0.001), digestive, respiratory, and urogenital tract cancers (aOR 2.62, 95% CI 1.75–3.92; P < 0.001), CNS and eye cancers (aOR 2.74, 95% CI 1.48–5.08; P = 0.001), or skin cancer (aOR 1.68, 95% CI 1.08–2.62; P = 0.022) were more likely to father through ICSI treatment. The adjusted ORs for IVF conception according to the cancer diagnosis are also given in . Among teenage and young adult cancer survivors, a history of testicular cancer (aOR 1.77, 95% CI 0.98–3.18; P = 0.057) and hematological and lymphatic cancers (aOR 1.98, 95% CI 1.10–3.56; P = 0.022) was associated with an increased likelihood of achieving paternity through IVF treatment. Adult survivors of testicular cancer (aOR 1.88, 95% CI 1.37–2.58; P < 0.001) and hematological and lymphatic cancers (aOR 1.53, 95% CI 0.94–2.47; P = 0.088) ( ), were more likely to conceive through IVF, compared to the background population. No cancer types among childhood cancer survivors were shown to be significantly associated with fathering through IVF treatment ( ). The likelihood of fathering a child through ICSI in adult cancer survivors conceiving 1–3 years (aOR 4.55, 95% CI 3.16–6.56; P < 0.001), 3–5 years (aOR 3.75, 95% CI 2.49–5.66; P < 0.001) and more than 5 years (aOR 6.31, 95% CI 4.49–8.87; P < 0.001) after cancer diagnosis was approximately equally elevated. However, the aORs for achieving paternity through IVF in adult cancer survivors was only slightly elevated during all three time periods, with this increase reaching level of statistical significance only for those becoming fathers 3–5 years post-cancer diagnosis ( ).
Among a total of 1 159 959 fathers included in the cohort, 1 125 431 (97%), 20 220 (1.7%), and 14 560 (1.3%) of their first-born children were conceived naturally, or through IVF and through ICSI, respectively. Among fathers with history of cancer, 861 (16.0%) fathers were diagnosed with childhood cancer, 1509 (28.1%) with teenage and young adulthood cancer, and 3002 (55.9%) with adult cancer. Among childhood cancer survivors, 6.0% conceived by ART, while this proportion was 10.1% for teenage and young adult cancer survivors, 13.3% for the adult cancer survivors, and 3.0% for the controls. Further background characteristics of the fathers are shown in .
When compared to controls, the entire group of cancer survivors was statistically significantly more likely to father through ART (aOR 3.18, 95% CI 2.91–3.48; P < 0.001). Adult cancer survivors were more likely to father through both ICSI (aOR 5.52, 95% CI 4.86–6.27; P < 0.001) and IVF treatment (aOR 1.32, 95% CI 1.09–1.60; P = 0.004), while teenage and young adult cancer survivors only had a statistically significant increased likelihood of conceiving through ICSI (aOR 6.84, 95% CI 5.64–8.30; P < 0.001) and not IVF treatment (aOR 1.27, 95% CI 0.90–1.80; P = 0.17). Similarly, childhood cancer survivors were only more likely to conceive a child through ICSI (aOR 3.52, 95% CI 2.52–4.93; P < 0.001) but not through IVF (aOR 1.02, 95% CI 0.61–1.70; P = 0.955). When compared to the general population, childhood cancer survivors (aOR 8.84, 95% CI 4.41–17.7; P < 0.001), teenage and young adult cancer survivors (aOR 8.28, 95% CI 5.02–13.6; P < 0.001), and adult cancer survivors (aOR 5.46, 95% CI 3.78–7.90; P < 0.001) were all more likely to father a child through ART using donated spermatozoa. The proportions of men utilizing donated spermatozoa for conception of their offspring were 33.3%, 7.6%, and 8.3% for childhood, teenage and young adulthood, and adult cancer groups, respectively. The adjusted ORs for ICSI conception according to the cancer diagnosis are given in . Childhood survivors of testicular cancer (aOR 5.15, 95% CI 1.20–22.0; P = 0.027), soft tissue and bone cancers (aOR 4.70, 2.13–10.40; P < 0.001), hematological and lymphatic cancers (aOR 4.49, 95% CI 2.72–7.40; P < 0.001), or central nervous system (CNS) and eye cancers (aOR 2.64, 95% CI 1.23–5.67; P = 0.012), were at an increased likelihood of fathering through ICSI. Teenage and young adult survivors of testicular cancer (aOR 15.4, 95% CI 11.5–20.7; P < 0.001), hematological and lymphatic cancers (aOR 9.84, 95% CI 6.93–14.0; P < 0.001), or soft tissue and bone cancers (aOR 6.83, 95% CI 3.53–13.2; P < 0.001) were more likely to father through ICSI treatment. Adult survivors of prostate cancer (aOR 15.7, 95% CI 6.70–36.9; P < 0.001), testicular cancer (aOR 9.54, 95% CI 7.81–11.7; P < 0.001), hematological and lymphatic cancers (aOR 11.3, 95% CI 8.63–14.9; P < 0.001), digestive, respiratory, and urogenital tract cancers (aOR 2.62, 95% CI 1.75–3.92; P < 0.001), CNS and eye cancers (aOR 2.74, 95% CI 1.48–5.08; P = 0.001), or skin cancer (aOR 1.68, 95% CI 1.08–2.62; P = 0.022) were more likely to father through ICSI treatment. The adjusted ORs for IVF conception according to the cancer diagnosis are also given in . Among teenage and young adult cancer survivors, a history of testicular cancer (aOR 1.77, 95% CI 0.98–3.18; P = 0.057) and hematological and lymphatic cancers (aOR 1.98, 95% CI 1.10–3.56; P = 0.022) was associated with an increased likelihood of achieving paternity through IVF treatment. Adult survivors of testicular cancer (aOR 1.88, 95% CI 1.37–2.58; P < 0.001) and hematological and lymphatic cancers (aOR 1.53, 95% CI 0.94–2.47; P = 0.088) ( ), were more likely to conceive through IVF, compared to the background population. No cancer types among childhood cancer survivors were shown to be significantly associated with fathering through IVF treatment ( ). The likelihood of fathering a child through ICSI in adult cancer survivors conceiving 1–3 years (aOR 4.55, 95% CI 3.16–6.56; P < 0.001), 3–5 years (aOR 3.75, 95% CI 2.49–5.66; P < 0.001) and more than 5 years (aOR 6.31, 95% CI 4.49–8.87; P < 0.001) after cancer diagnosis was approximately equally elevated. However, the aORs for achieving paternity through IVF in adult cancer survivors was only slightly elevated during all three time periods, with this increase reaching level of statistical significance only for those becoming fathers 3–5 years post-cancer diagnosis ( ).
In this study, we found that, as compared to fathers without history of cancer, a statistically significant higher proportion of male cancer survivors achieve paternity through ICSI treatment. Offspring conception through IVF was slightly, but statistically significant increased, among adult but not among teenage and young adult, or childhood male cancer survivors. Our findings are in accordance with previously published data which implies that young male cancer survivors are at a 3-fold increased risk of utilizing ART ( ). A Norwegian study found that the probability of first-time paternity at the age of 35 years was of the same magnitude in male cancer survivors as in men from the general population ( ). However, the reported post-treatment hazard ratio of paternity in males treated for cancer was decreased in another report ( ). In Sweden, reduced male fertility is still the major indication for undergoing ICSI treatment ( ). Thus, in our study, undergoing ICSI treatment can be used as a proxy for severe male subfertility, and higher utilization of ICSI within a patient group can be attributed to more severe impairment of male fertility. It is by law mandatory to report all newly diagnosed cancers to the Swedish Cancer Register, which thus has a completeness of 96% ( ). Similar rules apply to the remaining registers applied in this study, hence ensuring a near complete assessment of patient records ( ; ). Information about conception through intrauterine insemination, which is a rarely used assisted reproduction technique in Sweden, was not available for the entire cohort. Therefore, for the purpose of this study, ART only refers to conception through either IVF or ICSI. As compared to controls, all three groups of cancer survivors were more likely to conceive through ICSI. The probability of using IVF was statistically significantly increased for the adult group, with the risk estimate being similar for teenage and young adult cancer survivors, without reaching the level of statistical significance. No increase in IVF use was seen for those diagnosed with childhood cancer. This finding could be due to more severe forms of infertility in the latter group as compared to those treated during adulthood. This theory is further supported when considering that childhood cancer survivors was the cancer group with the highest utilization of donated spermatozoa, which could be used as a proxy for azoospermia. From the point of view of planning for future offspring, our results imply that adult cancer survivors can be informed that their likelihood of requiring ICSI to achieve paternity is of approximately the same magnitude, regardless of the time since completion of cancer treatment. In Sweden, cryopreservation of semen prior to cancer treatment is widely used. In cases where the cancer treatment has led to permanent sterility, cryopreserved gametes can be used for fertility treatment, with the preferred method being ICSI. Therefore, the higher rates of ICSI use among almost all cancer groups could be partly attributed to the use of sperm cryopreserved prior to gonadotoxic treatment. However, as has been previously shown, only a minority of male cancer survivors decide to use their cryopreserved spermatozoa ( ), and this is usually done in cases of post-cancer azoospermia or severe oligozoospermia. Therefore, a lack of information on use of cryopreserved gametes has limited the impact of the estimates regarding consumption of ART in this cohort. As expected, men diagnosed with testicular and prostate cancer were substantially more likely to conceive through ART, when compared to the own fathers, as these malignancies directly affect the reproductive system. For patients diagnosed with cancer, a reduced fertility status caused by the cancer itself, even before any cancer therapy, has been observed by numerous studies ( ; ; ; ). This effect is most noticeable in men with testicular cancer. This is suspected as being due to not only its localization but also may be related to testicular dysgenesis syndrome, where the development of testicular germ cell cancer and decreased testicular function are pathogenetically linked ( ). Both orchidectomy as well as the cytostatic and irradiation regimes have been shown to significantly contribute to the reduced semen quality in survivors of testicular cancer ( ; ). However, it cannot be disregarded that sexual dysfunction could also contribute to the inability to conceive a child spontaneously. Prostatectomy is usually applied in the treatment of prostate cancer and leads to anejaculation. In such men, post-cancer treatment gametes can be obtained using testicular sperm extraction (TESE) ( ). TESE retrieved gametes can only be used for fertilization through ICSI treatment which might explain why fathers with history of prostate cancer were statistically more likely to utilize ICSI but not IVF. Our data showed that paternity rates achieved by use of ART in survivors of hematological and lymphatic cancers was similar to those in survivors treated for malignancies in reproductive organs. It is reported that the mixed cytostatic cancer therapies, especially the ones used in advanced stages of hematologic malignancies, have a particularly negative impact on gonadal function ( ; ). However, some studies have indicated decreased fertility in those patients already before initiation of cancer therapy ( ; ; ), thus it is unclear as to what extent the impairment of testicular function is related to the chemotherapy and/or radiotherapy applied in treating those men. Another pathogenic mechanism which could contribute to a higher utilization of ART in such men is sexual dysfunction, which is estimated to affect between 20% and 54% of lymphoma survivors ( ). This could be related to a higher prevalence of psychosocial disorders following cancer diagnosis, but also may be partially linked to the use of chemotherapy leading to impairment of testosterone production, and thereby, to a diminished libido and reduced sexual function ( ; ). Our study provides further insight into which groups of patients could post-treatment be the most likely to use ART. Such information is useful for physicians and other healthcare workers for the purpose of counseling young male cancer survivors concerning the risk of infertility, and even for policy makers in order to provide equal access to fertility treatment. Accessibility to ART across Europe varies with only 3 out of 43 countries (7.0%) offering full funding for up to 6 cycles of IVF/ICSI treatment ( ). Income level has been linked to incidence rates of many common cancers ( ; ), and thus the population with a lower socioeconomic status is more likely to struggle financially with the inability to afford expensive fertility treatments, if they suffer from cancer-treatment induced subfertility. In this sense, in countries with no or poor funding of ART, the social status of the cancer survivors might be a main determining factor in family planning. Our results demonstrate that ever since the implementation of ART in Sweden, 6% of childhood cancer survivors and 13% of adult cancer survivor who fathered a child, needed to use ART to produce offspring after cancer treatment. Our findings would be useful for targeting populations which could mostly benefit from improvements in public funding and/or lowered costs of ART, when improving fertility treatment policies. Furthermore, the finding of almost all categories of male cancer survivors being more likely to father through using ART, underlines the need for developing new cancer therapies with lower gonadotoxicity. Maintaining fertility or achieving paternity after being cured for cancer is a major contributor to sustained quality of life in those men ( ; ), and as the number of cancer survivors will keep increasing, the importance of this issue will be growing. A major strength of this study is the use of nationwide Swedish registers, which resulted in the inclusion of over 1.1 million men in the cohort. Moreover, as mentioned earlier, owing to the high completeness of the compulsory datasets ( ; ; ), information on use of ART in practically all Swedish male cancer survivors was available. However, inclusion of only livebirths can be considered as a limitation since the cases where the pregnancy has ended in miscarriage or stillbirth are not reported. Furthermore, the most severe cases of male infertility could have been missed since there is a lack of data on men for whom ART was not applied or was not successful. Hence no definite conclusions can be made concerning the extent of infertility among different groups of cancer survivors. Additionally, some severe cases of male infertility might have been managed by insemination with donated spermatozoa which would have led to those men being categorized as conceiving naturally. However, such misclassifications would more likely strengthen than weaken the statistically significant associations reported by us. Another weakness is the lack of treatment data, which invalidates our ability to provide risk estimates adjusted for the intensity and type of the cancer treatment given. However, even such a refinement of our analyses would not eliminate the need for individualized counseling even taking into consideration other factors, such as the pre-treatment fertility status of the patient. In conclusion, men with a history of cancer had two to three times higher odds of using of IVF/ICSI treatments to father a child, when compared to those not being treated for cancer. Although almost all male cancer survivors were more likely to use ART, patients treated for malignancies in male reproductive organs or in hematological or lymphatic system, particularly those diagnosed during teenage years or young adulthood and later, were most likely to conceive through ART. Information concerning the potential post-cancer fertility treatment, fertility issues, and fertility preservations options should be made easily accessible for male cancer patients.
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Commentary: Deciphering the code of machine learning – An ophthalmologist’s step by step guide to posterity
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9f44a0b7-34b7-4666-8ecd-9b7e435a3372
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10228912
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Ophthalmology[mh]
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Machine learning is a subset of the field of computer science that involves creating and using algorithms that can learn from data and then make predictions or decisions based on newly acquired knowledge. Though machine learning has become an integral part of medicine, there are still some challenges doctors may face when implementing these technologies into their practices, as optimal understanding is required for the successful integration of AI in the clinical setup. The key problem here is the “black box dilemma,” which is the inability to provide an explainable AI model. Though the AI model produces an outcome, the rationale behind it is unclear, as it does not answer the question of why it produced that result. So, the step toward the future should be to provide an explainable and interpretable AI model.
The process of machine learning and data handling can be complex and time-consuming, which has often been a hindering factor for many people to enter the field of AI. However, some solutions can make it easier for doctors to incorporate machine learning into their practices. To start with, we need to acquire a massive dataset. Also, it is a known fact that a huge dataset is required to create a successful model. This is the advantage of “big data,” which is not available to the masses, and its scope of handling is beyond an individual’s reach. Alternatively, you can use a cloud-based solution system with blockchain technology to handle the data for you, allowing you to focus on building your model without worrying about infrastructure and hardware. So, every “small data” that is available to an individual may prove useful on a larger scale by integrating with blockchain technology.
Once we have our dataset, we must choose an algorithm depending on the requirements. There are many different types of machine learning algorithms available to us today, but some of the most common ones include decision trees, support vector machines (SVM), random forests, and neural networks, each having its own set of benefits and uses. The authors of the accompanying article have utilized open computer vision and deep learning models to train their data. Deep learning is a subset of machine learning, which is essentially a neural network with three or more layers. These neural networks attempt to simulate the behavior of the human brain – albeit far from matching its ability – allowing it to “learn” from large amounts of data.
The next basic step would be annotations, which means data labeling. Several software are available for this process, both free and paid applications. One such tool that we use is the Microsoft visual object training tool (VoTT). The VoTT is a user-friendly tool that offers customizable bounding boxes for labeling the data. This is crucial for the model to be able to learn from the data and predict with accuracy. This method utilizes human-in-the-loop training model. This method of machine learning requires human input and feedback to help create the algorithm. This process pairs the medical personnel with the AI throughout the entire process, thereby acquiring the much-needed human input. The feedback process is repeated until the model reaches a level of accuracy that can be used in a real-world scenario.
Currently, we have many working algorithms in place, the majority of which are for single-source imaging systems and not multimodal AI. Multimodal AI is imperative to provide a more comprehensive analysis and judgment, providing a more accurate diagnosis. It is just at the budding stage in ophthalmology and has yet to reach widespread usage due to its complex nature. A recent concept of utilizing multimodal imaging of angle structures and the anterior chamber is being developed to create a quantitative AI model. Here, the anterior segment optical coherence tomography (AS-OCT), ultrasound biomicroscopy (UBM), and Scheimpflug imaging are used to study the anterior chamber and angle, along with Lenstar (Haag-Streit, Ohio, USA) values to quantify the anterior chamber. We have utilized a digital protractor to quantify the angles, followed by the VoTT annotation tool to label the angles’ diagnosis . Categorical classification was done for Lenstar values, which will then be trained using deep learning techniques utilizing convolutional neural networks. AS-OCT, high-frequency UBM, and Scheimpflug imaging serve as effective tools for challenging gonioscopic circumstances. This technology will provide an objective noncontact method of assessing the angle structure along with the information provided by gonioscopy. This novel concept of multimodal imaging can help conglomerate the various modalities and bring forth an infallible system. Looking forward, it is important to use all available avenues to start working on machine learning to create a system that can help revolutionize healthcare and reduce the disease burden.
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Commentary: Artificial intelligence and machine learning in ocular oncology: Retinoblastoma
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82362364-21af-46dd-9d2a-78daeea32ee6
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10228960
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Internal Medicine[mh]
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Long-term retinal changes after strabismus surgery, suspected signs of past scleral perforations
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f1b9343f-8c46-454a-94c8-11808c13f26d
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10228968
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Ophthalmology[mh]
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This retrospective study included patients who underwent strabismus surgery in our facility and received follow-up care afterward. The [redacted for review] Eye Institute is a tertiary university-affiliated referral center that treats patients in a large service area in [redacted for review]. All surgeries were performed or supervised by one of two experienced fellowship-trained strabismus surgeons in our facility. The study was approved by the medical center’s institutional review board. All participants signed an informed consent form. Patients who had strabismus surgery between 1998 and 2008 were contacted for a follow-up appointment. A long postoperative timeframe window (>10 years) was defined with the intention that patients would be at an age to allow thorough peripheral retinal examination. Additionally, we excluded all patients who had undergone other ocular posterior segment operations before strabismus surgery as well as those who had any other known retinal disease. Patient information and surgical data were obtained from the patients’ medical records, including demographics, primary strabismic pattern, dates of surgeries, muscles involved, and surgical techniques. Each examination was carried out after pupil dilation with topical tropicamide 0.5% and phenylephrine 10% to achieve maximal dilation. Patients whose pupils would not dilate to at least 8 mm were excluded. Meticulous slit-lamp retinal examinations focusing on the periphery were performed while documenting any retinal changes in the potential suture sites, which we assumed to be signs of scleral perforation from the past surgeries. Potential suture locations were estimated based on the surgery title and procedure description as documented in the medical records. In all cases of a positive retinal finding, we attempted to document each finding with a color fundus camera. Statistical analysis was carried out with Microsoft Excel ™ 2019 Version 16.23 for Mac (Microsoft ® Corporation, Redmond, WA, USA).
A total of 71 eyes of 43 patients met the inclusion criteria and were examined. Fifty-three percent of the patients were male. The mean age at the time of examination was 27 years (range 13–70), and the mean age at the first surgery was 11 years (range 1–54). The total number of strabismus surgeries in all patients was 78, with a mean of 1.8 surgeries per patient. displays the patients’ demographic data. A total of five patients were excluded after completing the follow-up appointments: two due to insufficient pupil dilation, one due to corneal opacity inhibiting an adequate view of the retina to exclude mild retinal changes, one due to poor patient cooperation, and one due to limitations in complete duction movement inhibiting adequate examination of the peripheral retina. Of the 71 eyes operated on, 61 had horizontal muscle procedures, 14 had vertical muscle procedures, and 15 had oblique muscle procedures (as some eyes had more than one procedure or more than one muscle type per procedure). A total of 80 recessions, 30 resections, 12 transpositions, and nine myectomies/tenectomies were performed. A spatulated needle was used during surgery for all procedures. Four eyes had irregular retinal findings in the dilated examination, three of which were found at the suture site and therefore suspected to be signs of previous scleral perforation . The first patient with suspicious retinal findings was a 17-year-old male who underwent two surgical corrections of infantile esotropia in early childhood: bilateral medial rectus recessions (each 5.5 mm with hang-back sutures) at age 2 and bilateral lateral rectus resections (each 5.5 mm) at age 6. His finding was a retinal hole with surrounding pigment in the temporal periphery of his right eye . The second patient with retinal findings was a 53-year-old man who suffered from left hypertropia and underwent two surgeries: left inferior rectus resection (of 5 mm) and superior rectus recession (of 6 mm) at age 40 and, 3 years later, right inferior rectus recession (of 5 mm on adjustable sutures) and superior rectus resection (of 4 mm). The finding in his examination was an atrophic retinal hole with surrounding pigmentary changes at the 6 o’clock position of the right eye. This retinal finding had been detected during previous examinations after the second surgery and was evaluated by a retinal specialist who did not feel that any further treatment was necessary; moreover, the patient was always asymptomatic. Notably, we did not find any mention of this retinal hole in the examinations before both surgeries. Due to the far peripheral location of the hole, we could not capture it successfully with the fundus camera. The third patient with retinal findings was a 31-year-old woman who had undergone bilateral medial rectus recessions (of 6.5 mm) at age 1.5 for esotropia correction and a second surgery of bilateral lateral rectus resections (of 8 mm) at age 2 for residual esotropia. She underwent a third surgery at age 15 for right dissociated vertical deviation – right superior rectus recession (of 3 mm on adjustable sutures). The finding in her examination was retinal pigmentary and atrophic changes in the temporal periphery of her right eye . A fourth patient with abnormal retinal findings had an atrophic hole at the 6 o’clock position in the inferior periphery of her right eye; she was post bilateral medial rectus recessions as well as bilateral lateral rectus recessions. Due to the inconsistency between the location of the abnormal retinal finding and the potential suture locations from her surgeries, we did not include this case as a suspected past surgical scleral perforation. The total incidence of suspected scleral perforations in relation to eyes operated on was 4.2% (and 3.6% per surgery). None of the patients had a serious sight-threatening complication, such as retinal detachment or endophthalmitis. Of note, a review of the original surgical reports revealed that no scleral perforation was suspected during the surgical procedure in all cases.
Scleral perforation during strabismus surgery is a rare but serious complication that must always be considered before, during, and after surgery. During most muscle surgeries, the involved muscles are cut, recessed, or resected according to the deviation being corrected and then sutured to the sclera directly or alternatively to the muscle stump. Scleral thickness varies in different locations (ranging between 0.3 and 1.0 mm); the thinnest area, which is directly behind the rectus muscles’ insertions, is known to be the most frequent location of inadvertent scleral perforations. The potential sequelae of such perforations include intraocular hemorrhage, retinal detachment, and endophthalmitis, all of which may result in severe vision loss. In reviewing the recent literature from the past two decades, we found a wide variance of scleral perforation incidence rates reported during strabismus surgery. In particular, two large retrospective studies reported a very low incidence of recognized perforation cases. Awad et al . described 15 scleral perforations out of 4886 strabismus correction surgeries, with an overall incidence of 0.3%. In 2013, Bradbury and Taylor published the results of a national survey conducted in the UK to gather data on all severe complications reported by strabismus surgeons. The most common complication reported was globe perforation, with an incidence of 0.08% (19 out of 24,000 surgeries). Recent prospective studies that evaluated unrecognized scleral perforations and penetrations by postsurgical indirect ophthalmoscopy reported perforation rates to be between 1.4% and 2.8% per eye and the rate of scleral penetrations as up to 5.1% per eye (this referenced study defined scleral penetration as a full-thickness scleral pass without retinal break). It is a common assumption that the reported scleral perforation rates during strabismus surgery, whether in retrospective or prospective studies, represent underestimates of the true rates, especially in the young patient community. While most studies rely on the surgeon’s report of perforation as observed during surgery, conceivably, many microperforations remain undiscovered during surgery and in the short follow-up period. The low detection rate may be partly due to the asymptomatic nature of most cases and/or the young age of patients causing them to be unable to withstand peripheral retinal examinations. Another factor to be considered is the thick gel-like consistency of the vitreous in children, which immediately tamponades the aperture and prevents the seepage of vitreous outside or detachment of the retina inside. The sole remaining indicator would be the retinal or choroidal scar or pigmentary changes that develop in the area of local tissue damage. In theory, such changes may increase the risk of future retinal tears in these locations. Our aim in this study was to evaluate any long-term retinal changes found in patients at least a decade after strabismus surgery as possible signs of occult scleral perforations during surgery. All patients underwent a meticulous dilated slit-lamp fundus examination in search of suspicious chorioretinal changes, especially in areas that had experienced previous muscle manipulation. Out of 43 examined patients (71 eyes), three were determined to have retinal findings at the operated muscle area suggestive of past surgical scleral perforations, with an overall incidence of 4.2% per eye. Several risk factors have been identified as contributing to higher rates of scleral perforation. In a large review of the literature by Wan and Hunter, myopia, muscle recessions, younger age, and surgeons’ inexperience were reported to be related to scleral perforation. In myopic patients, axial elongation creates a thinner sclera, which might be more at risk of perforation. Similarly, Park et al . showed that myopic refractive error had a relatively weak yet significant correlation with scleral perforation. Muscle recession and surgical intervention on horizontal rectus muscles were found to relate to scleral perforation in a prospective study. Apparently, the possibility of perforation increases when recession is made using scleral passes as opposed to the hang-back technique (in which the scleral passes are avoided by passing the needle through the muscle insertion). In our study, two of the three patients with suspicious retinal findings at the suture sites had undergone horizontal muscle surgery at that location. The first was after a recession and the second after a resection, in one of which adjustable sutures were used. It is possible that a scleral insult happened during a deep pass in the insertion area or perhaps during a different surgical step (theoretically, when using a locking forceps on the sclera to stabilize the globe during surgery or when passing a full-thickness bite through the central part of the muscle while it is still attached to the sclera). Younger age at surgery was also reported as a risk factor for scleral perforation, most likely due to lower scleral rigidity in younger patients. In our study, two of the patients who were found to have retinal changes were aged 3 and 6 years during their surgery (significantly younger than the mean age of 11 years that characterized these patients during surgery), consistent with previous findings of higher risk at a young age. The surgeon’s experience level has also been suggested to be related to higher perforation rates, though reports have varied. For example, although Simon et al . reported that residents made twice as many scleral perforations as skilled ophthalmologists, Cibis reported no difference related to the skill level of the surgeon. The present study findings strengthen the notion that scleral perforation rates during strabismus operations are higher than those previously reported in retrospective studies and are comparable to or slightly higher than previous prospective works have indicated. As for retrospective studies, this outcome was to be expected since reported cases mainly involve large perforations that have resulted in vitreous or choroidal protrusion through the sclera, retinal detachment, or intraocular hemorrhage. Such cases constitute the minority of iatrogenic perforations during surgical procedures involving scleral needling; meanwhile, small occult perforations can go unnoticed and unreported. Previous prospective papers have relied on the evaluation of retinal scars observed during indirect fundoscopy at the time of short-term follow-up after surgery. In our study, we maximized our detection rate by purposefully designing a long postoperative time frame for fundoscopic examination (>10 years) in order to increase patients’ cooperation during a thorough peripheral retinal examination (mean age at examination was 27 years). In addition, all patients’ pupils were maximally dilated during examination. Presumably, some chorioretinal scarring might take longer to develop and would not yet be visible in the short-term post-op examination. Lastly, we acknowledge that the retinal changes we detected were indicative of full-thickness scleral perforations as well as penetrations to the suprachoroidal or subretinal space, which might account for the higher rate. The main limitation of our study is the relatively small sample size, which is highly likely to have amplified the incidence rate calculated. Our patient count was further decreased by the study’s exclusion criteria, as well as the patients whose pupils did not dilate sufficiently, which might have introduced another form of bias. The surgical procedures were conducted by different surgeons and spanned a decade (1998–2008). Additionally, the retinal and choroidal changes observed and assumed to be signs of past iatrogenic scleral perforations might be coincidental findings that were unrelated to any previous procedures in those locations.
The rate of scleral small occult perforations during strabismus surgery are probably higher than those previously reported. In our study, which was designed to maximize the detection rate of these events, we found an overall incidence of 4.2% per eye. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Nil.
There are no conflicts of interest.
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Commentary: Scleral penetration or perforation during strabismus surgery
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fbf26100-878f-4fb7-abab-c1734f31b3cb
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10228971
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Ophthalmology[mh]
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Akzeptanz einer telemetrischen Versorgung bei Patienten mit sICD-Sicherheitshinweis
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4243ddeb-37ce-41a7-9de3-9b3282355e54
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10229693
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Internal Medicine[mh]
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Patienten können nach Implantation eines Kardioverter-Defibrillators (ICD) mittels Telemonitoring nachgesorgt werden . Trotz wissenschaftlich bewiesener Vorteile, wie Effektivität und Sicherheit, gibt es Patientengruppen, die eine direkte Nachsorge in Präsenz durch die Ärzte bevorzugen . Da die Nutzung des kardiologischen Telemonitorings in Zukunft zunehmen wird , ist es von Bedeutung, die Versorgung patientenorientiert zu gestalten sowie die Akzeptanz des Telemonitorings zu steigern . Ziel dieser Studie ist daher die Untersuchung der Einstellungen zum Telemonitoring von Patienten mit einem subkutanen Defibrillator und einem Sicherheitshinweis. Telemonitoring Das Telemonitoring stellt eine Möglichkeit dar, mittels digitaler Technik, sowohl patientenspezifische Parameter als auch gerätespezifische Daten kontinuierlich zu erheben und an ein Überwachungszentrum zu übermittelt . Dabei kann der Zeitraum zwischen dem Auftreten technischer oder medizinischer Störungen und dem Ergreifen entsprechender Maßnahmen verringert werden . Studienkontext Im Rahmen der Studie wurde die Akzeptanz zum Telemonitoring von subkutanen ICD (sICD) der Firma Boston Scientific, Marlborough, Massachusetts, United States of America untersucht. Diese Firma stellt die telemetrische Nachsorge mittels des LATITUDE™ NXT WAVE Communicator zur Verfügung . Hierfür aktiviert der Patient den Communicator und führt die Datenübertragung zu einem festgelegten Zeitraum eigenständig zuhause durch. Der Communicator überprüft den sICD und sammelt die Daten. Diese werden dann automatisch an eine gesicherte Internetseite gesendet, worauf ausschließlich das medizinische Versorgungsteam Zugriff hat. Der Vorgang umfasst lediglich eine Datenabfrage, es werden weder Eingriffe in die Funktion noch Änderungen an dem Implantat vorgenommen . Im Jahr 2020 gab die Firma Boston Scientific einen Sicherheitshinweis bekannt . Dieser Sicherheitshinweis umfasste den sICD vom Typ EMBLEM A209 oder A2019 sowie die sICD-Elektrode vom TYP EMBLEM Modell 3501 und die Empfehlung zur Anbindung der Patienten an das Telemonitoring.
Das Telemonitoring stellt eine Möglichkeit dar, mittels digitaler Technik, sowohl patientenspezifische Parameter als auch gerätespezifische Daten kontinuierlich zu erheben und an ein Überwachungszentrum zu übermittelt . Dabei kann der Zeitraum zwischen dem Auftreten technischer oder medizinischer Störungen und dem Ergreifen entsprechender Maßnahmen verringert werden .
Im Rahmen der Studie wurde die Akzeptanz zum Telemonitoring von subkutanen ICD (sICD) der Firma Boston Scientific, Marlborough, Massachusetts, United States of America untersucht. Diese Firma stellt die telemetrische Nachsorge mittels des LATITUDE™ NXT WAVE Communicator zur Verfügung . Hierfür aktiviert der Patient den Communicator und führt die Datenübertragung zu einem festgelegten Zeitraum eigenständig zuhause durch. Der Communicator überprüft den sICD und sammelt die Daten. Diese werden dann automatisch an eine gesicherte Internetseite gesendet, worauf ausschließlich das medizinische Versorgungsteam Zugriff hat. Der Vorgang umfasst lediglich eine Datenabfrage, es werden weder Eingriffe in die Funktion noch Änderungen an dem Implantat vorgenommen . Im Jahr 2020 gab die Firma Boston Scientific einen Sicherheitshinweis bekannt . Dieser Sicherheitshinweis umfasste den sICD vom Typ EMBLEM A209 oder A2019 sowie die sICD-Elektrode vom TYP EMBLEM Modell 3501 und die Empfehlung zur Anbindung der Patienten an das Telemonitoring.
Als Leitlinie für den Forschungsprozess wurde ein qualitatives Studiendesign gewählt. Darüber hinaus lässt sich die Studie als eine nichtinterventionelle, nichtkontrollierte und nichtrandomisierte monozentrische Querschnittstudie charakterisieren. Die Patienten wurden anhand von teilstandardisierten leitfadengestützten Einzelinterviews befragt. Für die Strukturierung in der Datenerhebung und Konzeption des Leitfadens wurde auf aktuelle Modelle der Technikakzeptanzforschung und praxisorientierten Vorannahmen zurückgegriffen. Zusätzlich wurde eine Literaturrecherche von Januar bis Ende März in medizinischen Datenbanken wie PubMed (Medline) durchgeführt. Hierbei dienten zuvor definierte Schlüsselbegriffe („telemonitoring“, „implantable cardioverter-defibrillator“, „acceptance digital technologies“) als Hilfsmittel. Der Leitfaden beinhaltet folgende Fragedimensionen: Erfahrungen mit dem telemetrischen Angebot, Erfahrungen mit der Nutzung des Telemonitorings, Einfluss von sozialen Ressourcen und Erwartungen sowie Haltung der Patienten. Zur Beschreibung der Stichprobe wurden zusätzlich noch soziodemografische Daten erhoben. Die Interviews wurden zwischen August und September 2021 face-to-face durchgeführt. Zuvor lag eine Zustimmung der Ethikkommission anhand eines positiven Ethikvotums der Universität Heidelberg vor (S-573/2021). Die Transkription und Auswertung der Tonaufnahmen der Interviews erfolgten anhand der computergestützten qualitativen Daten- und Textanalyse-Software MAXQDA (Version 20.3). Das Auswertungsverfahren orientiert sich an der inhaltlich strukturierenden qualitativen Inhaltsanalyse nach Udo Kuckartz (2018). Hierbei wird das transkribierte Datenmaterial anhand von Kategorien und Subkategorien inhaltlich strukturiert . Rekrutierung und Sampling Der Rekrutierungszeitraum erstreckte sich von Juli bis September 2021. Ein Einschlusskriterium war die Implantation eines sICD aufgrund von primär- oder sekundärprophylaktischer Indikation. Zusätzlich wurden ausschließlich Patienten des Herstellers Boston Scientific eingeschlossen, die von dem Sicherheitshinweis betroffen sind. Um mögliche Unterschiede in der Einstellung der Studienteilnehmenden aufdecken zu können, wurden in dieser Arbeit zwei Gruppen, eine mit Telemonitoring und eine das Telemonitoring ablehnende Gruppe, untersucht. Für die Patienten mit Telemonitoring wurde eine Mindestdauer der Nutzung von 3 Monaten festgelegt. Darüber hinaus waren alle Personen mindestens 18 Jahre alt und einwilligungsfähig. Demgegenüber wurden Patienten mit schwerwiegenden psychischen Erkrankungen sowie unzureichenden Deutschkenntnissen von der Studienteilnahme ausgeschlossen .
Der Rekrutierungszeitraum erstreckte sich von Juli bis September 2021. Ein Einschlusskriterium war die Implantation eines sICD aufgrund von primär- oder sekundärprophylaktischer Indikation. Zusätzlich wurden ausschließlich Patienten des Herstellers Boston Scientific eingeschlossen, die von dem Sicherheitshinweis betroffen sind. Um mögliche Unterschiede in der Einstellung der Studienteilnehmenden aufdecken zu können, wurden in dieser Arbeit zwei Gruppen, eine mit Telemonitoring und eine das Telemonitoring ablehnende Gruppe, untersucht. Für die Patienten mit Telemonitoring wurde eine Mindestdauer der Nutzung von 3 Monaten festgelegt. Darüber hinaus waren alle Personen mindestens 18 Jahre alt und einwilligungsfähig. Demgegenüber wurden Patienten mit schwerwiegenden psychischen Erkrankungen sowie unzureichenden Deutschkenntnissen von der Studienteilnahme ausgeschlossen .
Patientenpopulation Insgesamt konnten 14 Patienten (7 = w; 7 = m) rekrutiert werden. Die Gruppe ohne Telemonitoring umfasste 5 Patienten (2 = w; 3 = m). Die Gruppe mit Telemonitoring enthielt 9 Patienten (5 = w; 4 = m). Das durchschnittliche Alter aller Teilnehmenden betrug zum Zeitpunkt der Interviews 53 Jahre (Min. = 25 Jahre; Max. = 70 Jahre). Die Zeitspanne zwischen der Defibrillator-Implantation bis zum Telemonitoring-Angebot lag im Durchschnitt in der Gruppe mit Telemonitoring bei ca. 20 Monaten (Min. = 2; Max. = 39) und in der Gruppe ohne Telemonitoring bei ca. 38 Monaten (Min. = 4; Max. = 71). In der Gruppe mit Telemonitoring lag die Dauer der einzelnen Interviews im Mittel bei 41 min (Min. = 21 min; Max. = 64 min). In der Gruppe ohne Telemonitoring umfassten die Interviews eine Spanne von 21 min (Min. = 13 min; Max. = 32 min; Tab. ). Wahrnehmung krankheitsbedingter (Vor‑)Erfahrungen Es stellt sich heraus, dass die Indikation und die Erfahrungen bezüglich des sICD sowie die Informationen über den Sicherheitshinweis des Herstellers Boston Scientific bei den Patienten Verunsicherung bezüglich des sICD auslösen. Als Indikation wurde überwiegend von einem plötzlichen Herzstillstand (Sekundärprophylaxe) sowie von Herzrhythmusstörungen (Primärprophylaxe) berichtet. Es wird deutlich, dass die Patientengruppe mit Telemonitoring und überwiegender sekundärprophylaktischer Indikation emotionaler auf die Defibrillator-Implantation reagierte als die Gruppe ohne Telemonitoring. Die Patientengruppe mit Telemonitoring berichtet hierbei eher von einem unerwarteten und schockierenden Ereignis, welches sie überfordert hat. Hierbei äußern sich die Interviewten beispielhaft wie folgt: „Ich war überfordert. … Ich war vorher noch nie im Krankenhaus. Dann war ich da halt mit dem Herzstillstand und dann Defi-Operation, ach Gott, was kommt da auf mich zu …“ (B_11 w, Pos. 64) . Patienten ohne Telemonitoring äußern sich demgegenüber eher nüchtern, wie an folgender Aussage deutlich wird: „… Gut fand ich es nicht, aber wie gesagt, so als, als Lebensversicherung hab’ ich’s halt dann doch machen lassen“ (B_10 mN, Pos. 16). Wahrnehmung des Informationsangebots Im Hinblick auf die Informationsvermittlung bezüglich des Telemonitorings wird deutlich, dass die Patienten aus beiden Gruppen über wenige Kenntnisse zum Telemonitoring verfügen. In der Gruppe ohne Telemonitoring erinnern sich die Patienten nur noch teilweise an das Aufklärungsgespräch. Dabei stellt das Aufklärungsgespräch die Hauptinformationsquelle zum Telemonitoring für beide Patientengruppen dar. Zusätzlich lässt sich festhalten, dass das Angebot an Informationen während des Aufklärungsgesprächs in beiden Patientenkollektiven gering ist. „…, die … Ärztin, die diese erste Untersuchung … vornahm, ... sagte das nebenbei, dass es eben auch diese Möglichkeit gibt ..., aber großartig viel mehr Informationen habe ich da eigentlich nicht bekommen“ (B_10 mN, Pos. 62). Einflussfaktoren auf die Entscheidung In der Patientengruppe ohne Telemonitoring waren die Technikskepsis und der Datenschutz wesentliche Einflussfaktoren für eine Ablehnung des Telemonitorings. Es zeigt sich, dass Patienten aus dieser Gruppe weniger Vertrauen in technische Geräte haben. „… Ich trau dem Kram nicht so“ (B_12 mN, Pos. 20). Im Hinblick auf den Datenschutz äußert sich ein Patient dahingehend, dass ihm keine hundertprozentige Sicherheit hinsichtlich der Weiterverarbeitung seiner Daten gewährleistet werden konnte. „… kann man sich immer sicher sein, wo die Daten von einem hingelangen? Wo ist da die hundertprozentige Sicherheit? …“ (B_1 mN, Pos. 34). In der Gruppe mit Telemonitoring konnte eine offene Haltung gegenüber digitalen Technologien sowie ein persönlicher Nutzen als relevante Einflussfaktoren für den Entscheidungsprozess festgestellt werden. Im Hinblick auf die Technikeinschätzung sehen die Patienten das Telemonitoring als Möglichkeit, die mit vielen Vorteilen verbunden ist. Hierbei ist der vorhandene Sicherheitshinweis ein zentraler Aspekt. Sie erwarten, dass sie sich sicherer fühlen durch die telemetrische Überwachung und der damit verbundenen Möglichkeit frühzeitige Fehlfunktionen zu erkennen. „Also ich erhoffe mir einfach, dass das ein Vorteil ist, wenn wirklich was wäre und man vielleicht das nicht mitkriegt … Ich glaub das ist … einfach so ein Sicherheitsaspekt“ (B_3 w, Pos. 92–94). Misstrauen gegenüber dem Datenschutz wird von den Patienten nicht geäußert. Das Thema wird von diesen Patienten kaum angesprochen. Im Einzelfall wird dieser Aspekt in dem Kontext als weniger relevant erachtet. Erfahrungen mit Telemonitoring In diesem Punkt kann festgehalten werden, dass die Patienten überwiegend über positive Erfahrungen mit dem Telemonitoring berichten. Vor allem die Erwartungen hinsichtlich eines höheren Sicherheitsgefühls bestätigten sich. Der einfache Umgang und die leichte Installation des Communicators wurden von den Patienten dahingehend betont, dass die Nutzung der telemetrischen Versorgung sowie die Bedienung des Communicators auch für ältere Menschen geeignet sei. Darüber hinaus wird festgestellt, dass bei keinem der Patienten zum Zeitpunkt der Interviews bereits Auffälligkeiten durch die telemetrische Überwachung identifiziert worden sind (Abb. ).
Insgesamt konnten 14 Patienten (7 = w; 7 = m) rekrutiert werden. Die Gruppe ohne Telemonitoring umfasste 5 Patienten (2 = w; 3 = m). Die Gruppe mit Telemonitoring enthielt 9 Patienten (5 = w; 4 = m). Das durchschnittliche Alter aller Teilnehmenden betrug zum Zeitpunkt der Interviews 53 Jahre (Min. = 25 Jahre; Max. = 70 Jahre). Die Zeitspanne zwischen der Defibrillator-Implantation bis zum Telemonitoring-Angebot lag im Durchschnitt in der Gruppe mit Telemonitoring bei ca. 20 Monaten (Min. = 2; Max. = 39) und in der Gruppe ohne Telemonitoring bei ca. 38 Monaten (Min. = 4; Max. = 71). In der Gruppe mit Telemonitoring lag die Dauer der einzelnen Interviews im Mittel bei 41 min (Min. = 21 min; Max. = 64 min). In der Gruppe ohne Telemonitoring umfassten die Interviews eine Spanne von 21 min (Min. = 13 min; Max. = 32 min; Tab. ).
Es stellt sich heraus, dass die Indikation und die Erfahrungen bezüglich des sICD sowie die Informationen über den Sicherheitshinweis des Herstellers Boston Scientific bei den Patienten Verunsicherung bezüglich des sICD auslösen. Als Indikation wurde überwiegend von einem plötzlichen Herzstillstand (Sekundärprophylaxe) sowie von Herzrhythmusstörungen (Primärprophylaxe) berichtet. Es wird deutlich, dass die Patientengruppe mit Telemonitoring und überwiegender sekundärprophylaktischer Indikation emotionaler auf die Defibrillator-Implantation reagierte als die Gruppe ohne Telemonitoring. Die Patientengruppe mit Telemonitoring berichtet hierbei eher von einem unerwarteten und schockierenden Ereignis, welches sie überfordert hat. Hierbei äußern sich die Interviewten beispielhaft wie folgt: „Ich war überfordert. … Ich war vorher noch nie im Krankenhaus. Dann war ich da halt mit dem Herzstillstand und dann Defi-Operation, ach Gott, was kommt da auf mich zu …“ (B_11 w, Pos. 64) . Patienten ohne Telemonitoring äußern sich demgegenüber eher nüchtern, wie an folgender Aussage deutlich wird: „… Gut fand ich es nicht, aber wie gesagt, so als, als Lebensversicherung hab’ ich’s halt dann doch machen lassen“ (B_10 mN, Pos. 16).
Im Hinblick auf die Informationsvermittlung bezüglich des Telemonitorings wird deutlich, dass die Patienten aus beiden Gruppen über wenige Kenntnisse zum Telemonitoring verfügen. In der Gruppe ohne Telemonitoring erinnern sich die Patienten nur noch teilweise an das Aufklärungsgespräch. Dabei stellt das Aufklärungsgespräch die Hauptinformationsquelle zum Telemonitoring für beide Patientengruppen dar. Zusätzlich lässt sich festhalten, dass das Angebot an Informationen während des Aufklärungsgesprächs in beiden Patientenkollektiven gering ist. „…, die … Ärztin, die diese erste Untersuchung … vornahm, ... sagte das nebenbei, dass es eben auch diese Möglichkeit gibt ..., aber großartig viel mehr Informationen habe ich da eigentlich nicht bekommen“ (B_10 mN, Pos. 62).
In der Patientengruppe ohne Telemonitoring waren die Technikskepsis und der Datenschutz wesentliche Einflussfaktoren für eine Ablehnung des Telemonitorings. Es zeigt sich, dass Patienten aus dieser Gruppe weniger Vertrauen in technische Geräte haben. „… Ich trau dem Kram nicht so“ (B_12 mN, Pos. 20). Im Hinblick auf den Datenschutz äußert sich ein Patient dahingehend, dass ihm keine hundertprozentige Sicherheit hinsichtlich der Weiterverarbeitung seiner Daten gewährleistet werden konnte. „… kann man sich immer sicher sein, wo die Daten von einem hingelangen? Wo ist da die hundertprozentige Sicherheit? …“ (B_1 mN, Pos. 34). In der Gruppe mit Telemonitoring konnte eine offene Haltung gegenüber digitalen Technologien sowie ein persönlicher Nutzen als relevante Einflussfaktoren für den Entscheidungsprozess festgestellt werden. Im Hinblick auf die Technikeinschätzung sehen die Patienten das Telemonitoring als Möglichkeit, die mit vielen Vorteilen verbunden ist. Hierbei ist der vorhandene Sicherheitshinweis ein zentraler Aspekt. Sie erwarten, dass sie sich sicherer fühlen durch die telemetrische Überwachung und der damit verbundenen Möglichkeit frühzeitige Fehlfunktionen zu erkennen. „Also ich erhoffe mir einfach, dass das ein Vorteil ist, wenn wirklich was wäre und man vielleicht das nicht mitkriegt … Ich glaub das ist … einfach so ein Sicherheitsaspekt“ (B_3 w, Pos. 92–94). Misstrauen gegenüber dem Datenschutz wird von den Patienten nicht geäußert. Das Thema wird von diesen Patienten kaum angesprochen. Im Einzelfall wird dieser Aspekt in dem Kontext als weniger relevant erachtet.
In diesem Punkt kann festgehalten werden, dass die Patienten überwiegend über positive Erfahrungen mit dem Telemonitoring berichten. Vor allem die Erwartungen hinsichtlich eines höheren Sicherheitsgefühls bestätigten sich. Der einfache Umgang und die leichte Installation des Communicators wurden von den Patienten dahingehend betont, dass die Nutzung der telemetrischen Versorgung sowie die Bedienung des Communicators auch für ältere Menschen geeignet sei. Darüber hinaus wird festgestellt, dass bei keinem der Patienten zum Zeitpunkt der Interviews bereits Auffälligkeiten durch die telemetrische Überwachung identifiziert worden sind (Abb. ).
Ein wesentlicher Aspekt für die Entscheidungsfindung ist die Indikation für die ICD-Implantation. Die Patienten mit vorwiegend sekundärprophylaktischer Indikation entschieden sich für das Telemonitoring. Diese Patienten verspüren eine größere Verunsicherung hinsichtlich ihres Gesundheitszustands als die Patienten mit primärprophylaktischer Indikation, die sich gegen das Telemonitoring entschieden . Dies lässt vermuten, dass den Patienten mit Telemonitoring die Wichtigkeit der Funktionsweise des sICD bewusster ist als der Gruppe ohne Telemonitoring. Die Patienten ohne Telemonitoring scheinen die Notwendigkeit ihres Defibrillators als geringer einzuschätzen. Es scheint, dass die Patienten ohne Telemonitoring durch die geringere Verunsicherung keinen persönlichen Nutzen in der telemetrischen Überwachung sehen. Im Gegensatz dazu zeigen die Patienten mit Telemonitoring ein höheres Maß an Verunsicherung hinsichtlich ihres sICD. Sie haben die Erwartung, dass sie durch die telemetrische Versorgung und die kontinuierliche Überwachung ihres sICD ein stetigeres Sicherheitsgefühl im Alltag bekommen. Es könnte geschlussfolgert werden, dass die Krankheitsvorerfahrungen die Einstellung zum implantierten Defibrillator prägen. Die Einstellung zum sICD könnte wiederum die Haltung und Akzeptanz zum Telemonitoring nachhaltig beeinflussen. Zusätzlich zeigt sich, dass beide Patientengruppen Wissenslücken und Verständnisprobleme hinsichtlich der Funktionsweise des Telemonitorings haben. Vor allem bei den Patienten mit Telemonitoring scheint dies der Fall zu sein. Dabei zeigte sich, dass die Patienten sich weniger für die detaillierten technischen Aspekte der Funktionsweise des Telemonitorings interessieren, sondern vielmehr für die Auswirkungen des allgemeinen Erhalts bzw. der Verbesserung ihrer Lebensqualität. Dies steht im Einklang mit den Studienergebnissen der Untersuchung von Senn et al. (2020) . Die Teilnehmenden sind sich mehrheitlich einig, dass es sich hierbei um eine einfache Nutzung handelt. Die Praktikabilität wird dahingehend betont, dass die Patienten der Meinung sind, dass die telemetrische Abfrage auch für ältere Menschen geeignet und bewältigbar sei. Da der ältere Bevölkerungsanteil den Großteil an Patienten für eine ICD-Implantation ausmacht , scheint das Telemonitoring auch für die Hauptanwendergruppe eine mögliche Versorgungsform zu sein. Es wird angenommen, dass die technischen Anforderungen für die Nutzung des Telemonitorings kein Hindernis für die Akzeptanz dieser Patientengruppe und somit für die Verbreitung in der kardiologischen Versorgung darstellen. Dies bestätigt auch den Forschungsstand zur Technikakzeptanz bei älteren Menschen . Zusätzlich stimmen die positiven Erfahrungswerte der Patienten mit den Ergebnissen der REMOTE-Studie zur hohen Patientenzufriedenheit hinsichtlich der Fernüberwachung überein .
Ein längerer Erhebungszeitraum sowie eine multizentrische Analyse und andere Erhebungszeitpunkte könnten noch differenziertere Ergebnisse hervorbringen. Bei allen eingeschlossenen Patienten liegt ein Sicherheitshinweis vor. Zusätzlich umfasst die Stichprobe ausschließlich sICD-Patienten des Herstellers Boston Scientific.
Im Aufklärungsgespräch sollte den Patienten mit primärprophylaktischer Indikation besondere Aufmerksamkeiten geschenkt werden. Auf Grund der mitunter geringer wahrgenommenen Notwendigkeit sollte die Nutzung der telemetrischen Versorgung umfassend erläutert werden. Ein standardisiertes, gestuftes Aufklärungsgespräch ggf. mit Anwendung von Kurzvideos zur Gerätedemonstration kann für ein qualitativ und quantitativ verbessertes Informationsangebot sorgen. Zudem kann die Demonstration des Communicators Unsicherheiten verringern.
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Adoption, implementation, definitions, and future of blockchain technology in ophthalmology
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fe1eda9c-d99b-4cfc-9b17-84a304248f9b
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10229909
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Ophthalmology[mh]
|
A lot of ophthalmologists depend on ASCRS intraocular lens (IOL) calculators for the calculation of IOL power. Having such data on the server can be a potential target of cybercrime and malware and may result in data modification without the users’ knowledge. To avoid this problem, blockchain technology has emerged as a possible solution. This technology could also assist in maintaining an extensive database of A constants worldwide. The pre and postoperative refractive surgery data may not always be available for IOL power calculation. By using blockchain technology, this data can be stored and later can be retrieved in the future. The stored data will also be protected from cyber-attack or criminal corruption. A patient’s genome can be secured with the help of blockchain technology, which can be of academic support in future applications. Genecoin is a company that helps store a patient’s genome with blockchain technology. Blockchain technology could also revolutionize international data collection of various private and government institutes aimed at better patient care and isolating epidemiological data. The prime examples include endophthalmitis, toxic anterior segment syndrome, and cataract surgical outcomes. Blockchain technology has been utilized in bitcoins and cryptocurrencies as a method of payment. They may be used to accept payments for elective surgeries like LASIK, cataracts, pterygium, etc. A large number of retinal AI clinical trials are being conducted across the world. These trials require automated analysis of retinal images and optical coherence tomography (OCT) scans, database preservation, data security, confidentiality, application of deep learning, a firm belief among different organizations for data transfer, and building relationships with a collaborative effort. This has been possible only through the concept of blockchain technology. The global coronavirus pandemic gave birth to multiple epidemics and pandemics, such as mucormycosis, mask-associated dry eyes, and digital eye strain, leading to the global myopia pandemic. Tan et al . designed a blockchain-enabled platform using hyperledger fabric to tackle the global myopia pandemic by developing deep learning algorithms for the same. They proved that blockchain technology helps secure data transfer, sharing, and validation of models, testing for three separate sites in two countries. They developed a deep learning module for the automated detection of myopic changes in the retina by analyzing the retinal changes. COVID-19 vaccines for ophthalmology patients, total parenteral nutrition for Xerophthalmia, intravitreal anti- Vascular Endothelial Growth Factor (VEGF), autologous serum, blood products, and biologics require stringent storage conditions. They are expensive products mandating good supply chain management. Wu et al . proposed the concept of online pharmacy for ophthalmology patients using blockchain technology. They suggested that prescriptions can be monitored, drugs can be delivered to remote locations, and, if needed, can also be renewed. Blockchain technology can also monitor and increase patient compliance with drugs and treatment. This will revolutionize patient treatment with costly medicines. To conclude, the adoption and implementation of blockchain technology have revolutionized the ophthalmological network ensuring transparent, large-volume, efficient data-sharing across digital platforms and maintaining integrity. Knowledge of AI and blockchain technology has helped maintain data integrity, an international collection and sharing of data along various platforms with accountability, validity, and transparency. Novel uses like IOL power calculation and refractive surgery have aroused interest in data annotators for rapid implementation of this technology. The characteristics of blockchain networking, such as consensus, provenance, immutability, and finality, have ensured the smooth flow of data, sharing of the patient database, and use by the patient themselves. This has also paved the way for future research for the development of newer protocols and algorithms for the widespread implementation of AI in ophthalmology. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest.
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Comments on: Clinical and diagnostic color-coding in ophthalmology - An indispensable educational tool for ophthalmologists
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35d17e99-c4d2-4c94-8868-4abe759e9d7b
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10229910
|
Ophthalmology[mh]
|
Nil. There are no conflicts of interest.
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The epidemiology and disease pattern of pediatric ocular morbidities in Western India: The National Institute of OphthalMology AmBlyopia StUdy in Indian Paediatric EyeS (NIMBUS) study report 1
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4ba8a196-9c59-4b2a-8ef9-e9c5b8c3b8b2
|
10229925
|
Ophthalmology[mh]
|
A retrospective longitudinal study was conducted at the outpatient department of a tertiary eye hospital in western India. The study was performed between January 2016 and December 2019 and included all consecutive children aged £15 years who presented to the outpatient department for the first time. The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethics committee of the institute. Written informed consent was taken from the parent/guardian of the study participants. A detailed retrospective chart analysis was performed, and the following data were collected for each patient: demographics, data pertaining to detailed ocular and systemic history, uncorrected visual acuity (UCVA), best-corrected visual acuity (BCVA, when available), refractive error, intraocular pressure (IOP, when available), diagnosis, and management. All patients were evaluated by a team of experienced optometrists and a single pediatric ophthalmologist (JK) with over 17 years of experience. The patients had undergone a comprehensive ocular evaluation including BCVA, ocular motility, IOP, cycloplegic refraction, and anterior and posterior segment examination. The BCVA assessment was based on the Snellen chart or Kay picture test for children under 5 years of age. A slit lamp and torch were used to examine the anterior segment. An air puff noncontact tonometer was used to measure the IOP. After dilating the pupil, a posterior segment examination was conducted with an indirect ophthalmoscope. The final diagnosis was based upon a comprehensive clinical examination by an experienced pediatric ophthalmologist. The BCVA classification was as per the WHO recommendations: ³20/60, <20/60–20/200, <20/200–20/400, <20/400, and indeterminable. Additionally, the patients were also stratified based upon their age: £5 years, 5–10 years, and >10–15 years. The dataset was entered and analyzed using the Statistical Package for the Social Sciences (SPSS) v23 (IBM SPSS Statistics for Windows, version 23.0; IBM Corp., Armonk, NY, USA). Continuous variables were described as mean ± standard deviation (SD), whereas the categorical variables were described as numbers and percentages. Demographics In this study, 5563 children aged up to 15 years were examined during the 4 years. The mean age of the study population was 5.15 (±3.32) years. Approximately half of the patient population (2792, 50.19%) was under the age of 5, followed by those between the ages of 5 and 10 (2509, 45.1%) and those up to 15 years of age (262, 4.71%). Out of the 5563 children, 3175 (57.07%) were males and 2388 (42.93%) were females. The gender distribution was found to be consistent across all age groups. shows the age and gender distribution in the study population. Visual acuity Eleven thousand one hundred and twenty-six eyes of 5563 children were evaluated in the study. A majority of the study eyes had a BCVA of ³20/60 (6515, 58.57%), followed by indeterminable BCVA (3912, 35.16%). A small proportion of eyes exhibited lower BCVA, including <20/60–20/200 (581, 5.22%), <20/200–20/400 (38, 0.34%), and <20/400 (80, 0.71%). After stratifying by age, a greater proportion of patients up to 5 years of age had interminable visual acuity (3811/5572 eyes, 68.4%). At the same time, a vast majority of the children aged 5–10 and >10–15 years had a BCVA ³20/60 (5–10 years: 4398/5017 eyes, 87.66%; >10–15 years: 488/537 eyes, 90.8%). summarizes the BCVA of the study population stratified by age. Ocular morbidities Nearly half of the study eyes (5125 eyes, 46.06%) were emmetropes with normal ocular examination. Refractive errors were the most common ocular morbidity affecting more than one-fourth of the study eyes (3223 eyes, 28.97%). Allergic conjunctivitis was the second commonest ocular morbidity (850 eyes, 7.64%), followed by strabismus (551 eyes, 4.95%), and nasolacrimal duct obstruction (NLDO; 376 eyes, 3.38%). The other causes of ocular diseases included infectious conjunctivitis (197 eyes, 1.77%), blepharitis (132 eyes, 1.19%), congenital eye diseases (123 eyes, 1.11%), cataract (other than congenital; 187 eyes, 0.78%), ptosis (72 eyes, 0.65%), chalazion (72 eyes, 0.65%), stye (69 eyes, 0.62%), congenital cataract (63 eyes, 0.57%), trauma (53 eyes, 0.48%), glaucoma (32 eyes, 0.29%), retinopathy of prematurity (ROP; 19 eyes, 0.17%), keratitis (12 eyes, 0.1%), retinoblastoma (12 eyes, 0.1%), and other miscellaneous conditions (58 eyes, 0.52%). According to the age stratification, refractive errors, allergic conjunctivitis, and strabismus were the most common causes of ocular morbidity across all age groups. presents the distribution of eye conditions across the total study population as well as age groups. In this study, 5563 children aged up to 15 years were examined during the 4 years. The mean age of the study population was 5.15 (±3.32) years. Approximately half of the patient population (2792, 50.19%) was under the age of 5, followed by those between the ages of 5 and 10 (2509, 45.1%) and those up to 15 years of age (262, 4.71%). Out of the 5563 children, 3175 (57.07%) were males and 2388 (42.93%) were females. The gender distribution was found to be consistent across all age groups. shows the age and gender distribution in the study population. Eleven thousand one hundred and twenty-six eyes of 5563 children were evaluated in the study. A majority of the study eyes had a BCVA of ³20/60 (6515, 58.57%), followed by indeterminable BCVA (3912, 35.16%). A small proportion of eyes exhibited lower BCVA, including <20/60–20/200 (581, 5.22%), <20/200–20/400 (38, 0.34%), and <20/400 (80, 0.71%). After stratifying by age, a greater proportion of patients up to 5 years of age had interminable visual acuity (3811/5572 eyes, 68.4%). At the same time, a vast majority of the children aged 5–10 and >10–15 years had a BCVA ³20/60 (5–10 years: 4398/5017 eyes, 87.66%; >10–15 years: 488/537 eyes, 90.8%). summarizes the BCVA of the study population stratified by age. Nearly half of the study eyes (5125 eyes, 46.06%) were emmetropes with normal ocular examination. Refractive errors were the most common ocular morbidity affecting more than one-fourth of the study eyes (3223 eyes, 28.97%). Allergic conjunctivitis was the second commonest ocular morbidity (850 eyes, 7.64%), followed by strabismus (551 eyes, 4.95%), and nasolacrimal duct obstruction (NLDO; 376 eyes, 3.38%). The other causes of ocular diseases included infectious conjunctivitis (197 eyes, 1.77%), blepharitis (132 eyes, 1.19%), congenital eye diseases (123 eyes, 1.11%), cataract (other than congenital; 187 eyes, 0.78%), ptosis (72 eyes, 0.65%), chalazion (72 eyes, 0.65%), stye (69 eyes, 0.62%), congenital cataract (63 eyes, 0.57%), trauma (53 eyes, 0.48%), glaucoma (32 eyes, 0.29%), retinopathy of prematurity (ROP; 19 eyes, 0.17%), keratitis (12 eyes, 0.1%), retinoblastoma (12 eyes, 0.1%), and other miscellaneous conditions (58 eyes, 0.52%). According to the age stratification, refractive errors, allergic conjunctivitis, and strabismus were the most common causes of ocular morbidity across all age groups. presents the distribution of eye conditions across the total study population as well as age groups. In the first study report of the NIMBUS project, we describe the rationale, methodology, and spectrum of ocular disorders affecting 11,126 eyes of 5563 children aged up to 15 years at a tertiary eye care center in western India. In our study population, the most prevalent ocular morbidity was refractive errors, followed by allergic conjunctivitis and strabismus. More than half of the study eyes had visual acuity of 20/60 or better, with a small minority of less than one-tenth of the eyes having BCVA poorer than 20/60. In the present study, more than half of the patients were below the age of 5 years. This finding is consistent with those reported from similar hospital-based studies in Nigeria and southern India. On the other hand, multiple other studies have demonstrated that children between 10 and 15 years of age are more frequently affected by some form of ocular disease. It has been proposed that the prevalence of ocular abnormalities is higher in older children due to the chronic nature of many eye disorders and their capability to communicate their symptoms more clearly. However, less than 5% of our study population were older than 10 years. The current study was conducted at a tertiary eye care facility in urban India that received a sizable number of referral patients from all across the country. A majority of the parents/guardians were educated, and therefore were sensitized to eye-related problems through some medium. Thus, even though younger children could have a difficult time expressing their issues, they accounted for the majority of the study’s participants. Our study had a male preponderance of 57.07%, with females accounting for 42.93%. This was similar to another Ethiopian study in which 53% of the participants were males and 47% were females. Male predominance was also found in another hospital-based study from South India. This gender bias in health-seeking practices may be brought on by social, cultural, economic, and behavioral practices that place boys and girls differently in terms of values and preferences. This is particularly true in developing countries such as India. More than half of the study eyes had visual acuity better than 20/60, while it was indeterminable in another one-third of them. Only around 6.27% of the eyes had vision poorer than 20/60. This is greater than the 1.67% reported in the meta-analysis by Yekta et al . that included 769,720 eyes from 80 studies. This higher rate in our study is most likely because it used hospital-based data, whereas the meta-analysis mostly used population-based data. Refractive error was the commonest cause of ocular morbidity in our study population, accounting for more than one-fourth of cases (28.97%). Even after age categorization, this was still the case. This is consistent with other studies from North India that have shown the highest prevalence rates for refractive errors, ranging from 20% to 25%. Refractive errors were the primary cause of visual impairment in a meta-analysis of 80 publications from 28 different countries. The rates of refractive errors varied between studies, ranging from 48.3% to 96.8%. Numerous studies failed to follow the Refractive Error Study in Children (RESC) Protocol, which mandates the use of cycloplegic refraction, while many of them had different age groups, and these may account for some of the significant variances among publications. Refractive errors are more likely to cause vision impairment in children over 7 years of age than in those under 7 years, according to the literature. Indeed, the authors found that the prevalence of refractive errors was higher in children aged 5–10 years (36.04%) than in those aged under 5 years (23.47%). One of the key contributors to the high prevalence of refractive errors in studies that sampled older children is the age-related increase in the prevalence of myopia. A significant age-related increase in the prevalence of myopia was noted in a meta-analysis from the Middle East region. The authors observed that the rates rose from 3.5% for children under the age of 5 years to more than 47% for those over the age of 18 years. A comprehensive analysis of the type and magnitude of refractive error was beyond the scope of this manuscript, which was primarily focused on the spectrum of ocular disorders in children. The details of the pattern of refractive errors in our NIMBUS project will be addressed in the subsequent study reports, which are scheduled to be completed in the near future. Allergic conjunctivitis is one the most common eye diseases to affect the quality of life. It is also one of the most commonly underdiagnosed and undertreated eye conditions. In our study population, allergic conjunctivitis affected about 7.64% of the eyes and was the second most frequent cause of ocular morbidity. Several more studies from India have found a comparable higher frequency ranging from 3% to 17.5%. Collaborations across international scientific networks formed the International Study of Asthma and Allergies in Childhood (ISAAC) to investigate the global prevalence, distribution, and temporal trends of allergic diseases. The findings of the ISSAC study provide the first global overview of allergy diseases in children. In its third phase, the ISAAC tested 1,059,053 kids from 98 nations between 2002 and 2003. It reported that the average prevalence of rhinoconjunctivitis in children aged 13–14 years was 14.6%, whereas it was 11.7% in children aged 6–7 years. This research also included prevalence data from several places throughout the world. The prevalence of childhood allergy diseases was lower in Northern and Eastern Europe (9.2%), Africa (18.0%), and Latin America (17.3%). Seasonal variations along with differences in socioeconomic conditions and hygiene levels can account for the wide range of prevalence rates of allergic conjunctivitis. Multiple factors, such as the dusty environment, higher temperatures, significant air pollution, and pollen and other allergens that are commonly present in the western part of India, may be associated with the increased preponderance of allergic conjunctivitis noted in our study. In our study, strabismus was the third commonest ocular morbidity and was seen in 4.95% of the study eyes. Previous studies performed in India have shown prevalence rates ranging from 0.2% to 7.4%. In a hospital-based study conducted in North India, 6.9% of the study population of 24,475 patients had strabismus. Following age stratification, the authors noted that 14.8% of patients aged up to 12 years had strabismus, which was greater than in our study (4.95%). Previous hospital-based studies from other parts of the world have reported a higher prevalence of strabismus, including 17.9% in Ethiopia and 22% in Cameroon. The prevalence is marginally higher than the rates reported in other multi-ethnic population-based studies such as the MultiEthnic Pediatric Eye Disease Study (MEPEDS) from the USA (African Americans and Hispanics: 2.5%; Asians and non-Hispanic whites: 3.24%), the Strabismus, Amblyopia, and Refractive Error in Singaporean Children Study (STARS) from Singapore (0.8%), the Baltimore Pediatric Eye Disease Study (BPEDS) from the USA (Whites: 3.3%; African Americans: 2.1%) and from the UK (2.4%), Australia (2.8%), Brazil (1.4%), and Japan (1.28%). The scope of this paper, which was largely concerned with the spectrum of pediatric ocular diseases, did not allow for a detailed review of the specific form of strabismus. The specifics of the strabismus pattern in our NIMBUS project will be discussed in upcoming study reports. In our large series, we found a wide range of other ocular abnormalities, including NLDO (3.56%), infectious conjunctivitis (1.77%), blepharitis (1.19%), congenital eye diseases (1.11%), cataract (other than congenital, 0.78%), ptosis (0.65%), chalazion (0.65%), stye (0.62%), congenital cataract (0.57%), trauma (0.48%), glaucoma (0.29%), ROP (0.17%), keratitis (0.1%), retinoblastoma (0.1%), and other miscellaneous conditions (0.52%). A similar spectrum has also been observed with other hospital-based studies and population-based studies. Overall, hospital-based data show greater rates of morbidity than population-based studies. Because most population-based studies reported in the literature are conducted among the general population, they may provide reliable information about disease prevalence in the real world. Simultaneously, the higher rates reported in the hospital-based study indicate increased referrals for a variety of ocular disorders. This is especially true for tertiary care facilities, as was the case in our study. The major limitation of the present study is its retrospective design. Also, because it was a medical record analysis, there is the possibility of minor data inaccuracies due to incomplete or missing data. Nonetheless, this is a very remote possibility, and because the study sample is quite large, the errors caused by such inconsistencies in the final results are negligible. Despite these limitations, the data give very useful information on the entire spectrum of ocular diseases at a tertiary eye care hospital in India. The large sample size of 5563 children provides very robust data regarding the spectrum as well as the burden of various ocular morbidities. Such data are crucial for planning and providing eye care, as well as for developing a targeted approach to address the problem of pediatric ocular morbidity. In conclusion, our data from a large cohort of eyes (11,126) demonstrate that refractive error remains the single most important cause of ocular morbidity in children up to 15 years of age. Allergic conjunctivitis and strabismus are the other important ocular diseases seen in pediatric eyes. In light of this study, it is vital to plan screening programs at the community and national levels to reduce the burden of ocular diseases. Furthermore, these programs must be seamlessly linked to primary and secondary health-care facilities, in addition to having a proper referral system developed. This would help ensure quality eye care delivery, while also decreasing the workload of overburdened tertiary centers. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest.
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Clinical features of early-onset pediatric traumatic glaucoma and predictive factors for the need of early glaucoma surgery
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a59b94b2-14d1-46ef-9984-9fadcac7026b
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10229941
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Ophthalmology[mh]
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This was a single-center, retrospective, and analytical study conducted at a tertiary eye care hospital in India. The study was conducted after getting clearance from the institutional review board. Informed written consent was obtained from the parents of patients before any investigative or therapeutic procedures. The study was conducted over a duration of seven years from January 2014 to December 2020. Case records of pediatric patients (<18 years) who developed glaucoma secondary to CGI were retrospectively reviewed. Several stringent criteria were used to selectively identify early glaucoma/ocular hypertension (OHT) cases. Firstly, only treatment-naïve CGI cases were scrutinized for possible inclusion, which means no previous use of steroid or antiglaucoma medication (AGM) which can potentially affect clinical findings. Among such cases, those who developed raised IOP within a month of trauma, “early-onset traumatic glaucoma,” were identified and recruited. Patients with previous congenital or developmental glaucoma and with a history of any ocular surgery done before trauma were excluded. Patients who took treatment from elsewhere were excluded to avoid clouding of baseline findings. Patients who were suspected of having steroid-induced OHT were also excluded. Such patients were identified easily as IOP normalized once steroids were tapered. Patients having a follow-up of less than 6 months were also excluded from the study. Parameters such as age, sex, object causing trauma, time lapse between injury and hospital consultation (in days), and other relevant demographic details were recorded. Injuries were classified according to the Birmingham eye trauma terminology system (BETTS). Vision was evaluated with Snellen or any other age-appropriate chart. IOP was evaluated using Goldmann applanation tonometer. Gonioscopy (single-mirror goniolens; Ocular Instruments, Bellevue, Washington, USA) was done in cooperative children with good corneal clarity. Posterior segment evaluation was performed using indirect ophthalmoscopy. In eyes with poor visualization, B-scan ultrasonography was performed. Visual fields were also evaluated using Humphrey field analyzer whenever possible in cooperative children. All the treatment details and surgery data were also recorded. OHT was diagnosed whenever the IOP values were found above 21 mmHg. Glaucoma was diagnosed clinically when the OHT was associated with increased optic disc cupping from the previous values or there was an inter-eye difference of more than 0.2, as the visual field details were not available for all patients. Treatment protocol Patients diagnosed as having traumatic glaucoma/OHT were initially given a trial of AGMs. Prostaglandin analogs were avoided in the trauma setting as they can worsen inflammation. Injection mannitol was given to patients who presented with an IOP of >40 mmHg. Other AGMs were also prescribed singly or in combination, depending upon the clinical profile: tablet dorzolamide (250 mg, 8–30 mg/day divided four times/day), timolol eyedrop (0.5%, twice a day), brimonidine eyedrop (0.2%, thrice a day), and dorzolamide eyedrop (2%, thrice a day). Often, such patients were prescribed topical steroids (prednisolone acetate 1% eyedrop, frequency varied according to the severity of anterior chamber [AC] inflammation) and cycloplegics (cyclopentolate 0.5% eyedrop, twice a day). Eyes with an IOP of >30 mmHg for 7 days or >50 for 5 days indicated the need of invasive approach as such high levels are a threat to the sensitive nerve fibers and eventually to vision. In such cases, AC wash was carried out in the presence of hyphema or corneal endothelial blood staining. Uncontrolled IOP for 10–14 days despite maximum AGMs + AC wash along with disc changes was an indication for glaucoma surgery. Trabeculectomy with mitomycin C (TMMC; 0.4 mg/ml) was the choice of filtration surgery if needed. A simplified treatment algorithm has been presented in . Statistical analysis Data analysis was done among all early glaucoma cases and they were followed till 6 months. Patients were categorized into two groups based on the need of TMMC to control IOP. Those who underwent TMMC within 6 months were included in group A and the remaining patients were included in group B. Patients who underwent AC hyphema wash alone were also included in group B. Data entry was done in the Microsoft Excel worksheet (Microsoft Corporation, Redmond, Washington, USA), version 14. Continuous variables like age, IOP, and so on were presented as mean ± standard deviation (SD) and range. Mann–Whitney U test was used to compare nonparametric continuous variables. On the other hand, categorical variables were presented in the form of frequency and percentages. Chi-square test and Fischer’s exact test were used to identify statistical significance depending upon the frequency. Extensive bivariate analysis was performed to find the predictors of early surgery, which included presenting best corrected visual acuity (BCVA) of <3/60, high IOP (>50 mmHg) at presentation, corneal diffuse microcystic edema, hyphema, severe AC inflammation of grade 3 and 4, iris-related pathologies, zonular dialysis, cataract, retinal hemorrhages, commotio retinae, zone of injury, and AR of >180°. Multivariate analysis was performed using Epi Info 7 software for factors having a P value of <0.2 after bivariate analysis. Odds ratios (ORs) were also calculated for both analyses. A P of <0.05 was considered to be a statistically significant value. Patients diagnosed as having traumatic glaucoma/OHT were initially given a trial of AGMs. Prostaglandin analogs were avoided in the trauma setting as they can worsen inflammation. Injection mannitol was given to patients who presented with an IOP of >40 mmHg. Other AGMs were also prescribed singly or in combination, depending upon the clinical profile: tablet dorzolamide (250 mg, 8–30 mg/day divided four times/day), timolol eyedrop (0.5%, twice a day), brimonidine eyedrop (0.2%, thrice a day), and dorzolamide eyedrop (2%, thrice a day). Often, such patients were prescribed topical steroids (prednisolone acetate 1% eyedrop, frequency varied according to the severity of anterior chamber [AC] inflammation) and cycloplegics (cyclopentolate 0.5% eyedrop, twice a day). Eyes with an IOP of >30 mmHg for 7 days or >50 for 5 days indicated the need of invasive approach as such high levels are a threat to the sensitive nerve fibers and eventually to vision. In such cases, AC wash was carried out in the presence of hyphema or corneal endothelial blood staining. Uncontrolled IOP for 10–14 days despite maximum AGMs + AC wash along with disc changes was an indication for glaucoma surgery. Trabeculectomy with mitomycin C (TMMC; 0.4 mg/ml) was the choice of filtration surgery if needed. A simplified treatment algorithm has been presented in . Data analysis was done among all early glaucoma cases and they were followed till 6 months. Patients were categorized into two groups based on the need of TMMC to control IOP. Those who underwent TMMC within 6 months were included in group A and the remaining patients were included in group B. Patients who underwent AC hyphema wash alone were also included in group B. Data entry was done in the Microsoft Excel worksheet (Microsoft Corporation, Redmond, Washington, USA), version 14. Continuous variables like age, IOP, and so on were presented as mean ± standard deviation (SD) and range. Mann–Whitney U test was used to compare nonparametric continuous variables. On the other hand, categorical variables were presented in the form of frequency and percentages. Chi-square test and Fischer’s exact test were used to identify statistical significance depending upon the frequency. Extensive bivariate analysis was performed to find the predictors of early surgery, which included presenting best corrected visual acuity (BCVA) of <3/60, high IOP (>50 mmHg) at presentation, corneal diffuse microcystic edema, hyphema, severe AC inflammation of grade 3 and 4, iris-related pathologies, zonular dialysis, cataract, retinal hemorrhages, commotio retinae, zone of injury, and AR of >180°. Multivariate analysis was performed using Epi Info 7 software for factors having a P value of <0.2 after bivariate analysis. Odds ratios (ORs) were also calculated for both analyses. A P of <0.05 was considered to be a statistically significant value. summarizes the demographic profile of the patients. A total of 139 treatment-naïve patients were identified as having traumatic glaucoma with at least 6 months of follow-up during the study duration. Of these, 19 patients having previous OGI were excluded. Thirty-five patients having delayed-onset glaucoma were further excluded. Thus, 85 patients (46 in group A and 39 in group B) fulfilled the necessary criteria and were included in this study. Mean age of the patients was 11.65 ± 3.13 years in group A and 11.12 ± 3.41 years in group B, with a strong overall male predominance (9.6:1). Patient presented to the hospital after 8.5 ± 8.2 days of incurring injury. Mean BCVA at presentation was significantly better in group B (1.59 ± 0.96 logMAR) compared to group A (2.19 ± 0.14 logMAR, P = 0.0023). Most of the patients in both the groups suffered trauma due to wooden objects (41.2%). Other common objects causing injury in descending order were stone (21.2%), rubber ball (20%), and blast injury due to firecrackers (10.6%) . shows the preoperative parameters related to glaucoma which were evaluated. Mean IOP at presentation was very high (≈40 mmHg), albeit similar in both the groups. Disc cupping was worse in group A, which had a P value of 0.06 that was almost statistically significant. IOP rise posttrauma was noticed early after a mean duration of approximately 9 days. Median value of AR was slightly more in group B (270° compared to 225° in group A). However, AR > 180° was present in 22 patients (68.7%) in group A, which was more than in group B ( n = 16, 55.2%, P = 0.27). shows an extensive comparison of various parameters at presentation between groups. We found that vision of < 3/60 (OR: 3.02), diffuse corneal microcystic edema (OR: 3.11), and severe AC reaction (OR: 6.82) were significantly more in group A patients in bivariate analysis. Zone of injury, AR, the IOP level, hyphema, and many other factors were not found to be significantly different. In multivariate analysis, only corneal microcystic edema and severe AC reaction were found to be statistically significant. In other words, these were the predictive factors which pointed toward a possible need of trabeculectomy in near future. The Kaplan–Meier curve is shown in , considering no need of TMMC as survival. Overall survival rate was 45.8%. We also calculated the survival rates for significant predictive factors, which was 36.4% with low BCVA, 31.7% with corneal microcystic edema, and 29.6% with severe AC reaction. A total of nine (19.5%) patients had to undergo TMMC within 2 weeks of trauma only. Highest frequency of TMMC was noted from 2 to 4 weeks of trauma (22, 47.8%) . A total of 12 patients underwent AC hyphema wash. Seven of these further required TMMC to control IOP. The mean number of surgeries done in group A was 1.95 (range 1–6). The mean number of AGMs required after 6 months was 0.32 in group A and 0.79 in group B which was significantly higher in group B ( P = 0.003). Significant improvement in BCVA was noticed in both the groups. Still, it was much better in group B than in group A (0.52 vs. 0.96 logMAR). Significant postoperative surgical events will be described briefly here, as we have discussed them in detail in our previous study. Totally 17 glaucoma related re-interventions were required in 12 (26.1%) subjects. They included needling in eight patients with 5-fluorouracil, bleb revision in four, autologous serum infusion in bleb in two, and repeat TMMC in three. Our study aimed at noting the clinical features and decoding the predictive factors for the need of trabeculectomy surgery in children with early traumatic glaucoma. Severe AC reaction was the most common anterior segment finding, followed by AR. Poor visual acuity at presentation, corneal microcystic edema, and severe grades of AC reaction at presentation were found to be significantly associated with increased odds of requirement of trabeculectomy within 6 months of trauma. Salient features noted in previous pediatric traumatic glaucoma studies were high baseline IOP, higher incidence of early glaucoma and subsequently, higher rate of surgery to control IOP. Similarly, in our study, baseline IOP was almost 40 mmHg, and 31/46 (63%) TMMCs had to be performed within a month of trauma. Considering the high rate of surgery to treat elevated IOP in children, there is a need to identify early markers to predict the need of surgery. Such patients might require very close and frequent follow-ups. Unnecessary delay and hoping for spontaneous resolution of the condition may result in the child becoming blind. The present study was undertaken to answer these questions. Mean age of the patients was around 11 years in our study, with a very strong male predominance (9.6:1). Kalamkar and Mukherjee, in their study, also reported similar mean age (10.25 years) with a male (84.4%) predominance. Kaur et al . reported similar mean age of 10.8 years, and a male predominance of 85.5%. Many studies involving OGI or CGI in the pediatric age group have commonly reported very high male preponderance, and this was attributed to more playful nature of boys and their involvement in unsupervised dangerous outdoor activities. Wooden objects were most commonly implicated for trauma, which is not surprising, considering the predominant rural population around our hospital. IOP rise posttrauma was seen very early after a mean duration of 9 days, which is expected as our study involved early glaucoma specifically. Our previous study, which aimed at evaluating the results of TMMC after traumatic glaucoma, also noticed a median of 13 days for increased IOP. Kaur et al . noted a mean of 1.67 weeks (roughly 11 days) following which increased IOP was documented in children. Similarly, 77% of pediatric patients developed raised IOP within a month of trauma in the study by Kalamkar and Mukherjee. These findings are very different from those of adult studies, where the onset of glaucoma is delayed, ranging from 3 to 16.5 years. We noted a mean IOP of almost 40 mmHg at presentation. This was much higher than 29.8 mmHg noted by Kalamkar and Mukherjee. Also, more than half of our patients required TMMC. This is again much higher than the values reported by Kalamkar and Mukherjee (37%) and Kaur et al . (13.3%). There could be several reasons for these. Firstly, ours is a tertiary level hospital, so we often receive much more severe cases. Another fact is the strict 6 months follow-up inclusion criterion set in our study. Still, there was a stark contrast when correlating pediatric study results with those of adult studies. Adult studies did report acute elevation of IOP in patients after CGI, but they could be treated successfully with AGMs. Regarding clinical features, the most common findings in our study were severe AC reaction in 63.5% cases, followed by AR in 56.4% and hyphema in 50.6%. Kalamkar and Mukherjee noted cataract and hyphema in 48% cases; however, AR was noted in only 19% eyes. Kaur et al . found hyphema as the most common presenting sign (57% cases), while AR was present in only 16% cases. Thus, presence of AR was much more common in our study compared to other pediatric series. This confirms our theory that we received much more severe cases, which was responsible for the higher rate of TMMC in our study. Ozer et al. , in a similar study, stated that visual acuity < 20/200, optic atrophy, corneal injury, and penetrating trauma were significantly associated with increased need of glaucoma surgery. They reported findings similar to ours (AR 52%, hyphema 49%); still, only 17% required glaucoma surgery. Inflammation was seen in only 5% of eyes in their study, compared to 63% noted by us. Despite the similar nature of studies, we strongly feel that this result cannot be applied or compared to the findings in the pediatric population. Their study involved a mixed population (mean age of 30–31 years) and mixed trauma (both OGI and CGI). Glaucoma was diagnosed after 6 months in 72% of cases and only 12% of blunt trauma cases needed glaucoma surgery in their study. Such differences and inhomogeneous nature of population forced us to evaluate our pediatric population. Three predictive factors for the need of surgery were noted in our study in bivariate analysis: poor visual acuity at presentation (<3/60), corneal microcystic edema, and severe grades of AC reaction. However, in multivariate analysis, poor visual acuity was not found to be significant. In other words, it was a confounding factor, as the latter two factors obviously had a causal relationship with poor visual acuity. Surprisingly, both our groups had similar, high levels of IOP, still microcystic edema was more common in group A. Corneal endothelial pump plays a major role in transparency of cornea. If the balance between the tendency to imbibe and export fluid is disrupted, stromal fluid accumulates and leads to corneal edema. We hypothesize that, patients in group A (trabeculectomy) might have had higher IOP for longer duration compared to their counterparts in group B. This might have disrupted the endothelial barrier and resulted in corneal edema. An indirect clue to support this hypothesis was increased cupping values in group A despite similar IOP values. Severe AC reaction, on the other hand, might be an indicator of more severe injury and may serve as a proxy marker in acute setting. AR and hyphema were not associated with increased need of glaucoma surgery. AR is a known risk factor for late-onset glaucoma, which may explain its nonrelevance in the current study. One of the important limitations of the study was its retrospective nature. Variable follow-up, possibility of inconsistent details, and multiple examiners can lead to some potential errors in such retrospective analysis. There was an unequal distribution of patients in both the groups. If the analysis is done prospectively, many such errors can be prevented. However, smaller number of such cases have prevented us and other researchers from prospectively analyzing this entity. We tried to maintain homogeneity of data as much as possible by enforcing stringent inclusion and exclusion criteria. Our study also involved predominantly severe cases, which may not be the situation at all places. To conclude, our study revealed that pediatric glaucoma cases presented with very high levels of IOP, which did not respond optimally to AGMs. Severe AC reaction and microcystic edema were important predictive factors for the need of early glaucoma surgery after traumatic glaucoma. Maximum number of patients underwent trabeculectomy within a month of trauma itself. Visual prognosis was poor in patients who needed filtration surgery. Pediatric glaucoma cases need to be very closely observed after trauma to identify the glaucoma early and eventually to minimize the extent of irreversible damage. Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. The authors certify that they have obtained all appropriate patient consent forms. In the form the patient (s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. Nil. There are no conflicts of interest.
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A novel Y-shaper and chopper for small pupil management in cataract surgery: The initial experience
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526acfec-2f6b-4200-9e16-c1086581ea64
|
10229943
|
Ophthalmology[mh]
|
Description of the instrument The instrument is made of stainless steel; at one end is a straight rod, and the other end has a curved chopper with a tip dimension of 0.8 mm which allows for easy entry through the side ports. In scenarios wherein we have moderately dilated pupil (5 mm) with any amount of iris billowing or miosis, it allows for simultaneous stretching of the floppy iris, thus enabling the phaco probe to hold a nuclear fragment and emulsifying the nucleus, whereas the curved chopper allows for easy nucleus chopping and allowing us to manage the small pupil and nucleus emulsification without the use of any pupil-expanding devices. Clinical applications Intra-operative miosis because of FIS: Intra-operative FIS is classified upon the presence of floppy iris stroma, which billows and ripples responding to phaco fluidics with progressive intra-operative miosis, independent of the use of mydriatic agents, and the iris stroma’s tendency to prolapse through the incisions. The Y-shaped chopper allows stretching the floppy iris to the periphery and preventing iris plugging in the phaco probe during aspiration and concomitantly performing nuclear fragmentation without risking anterior capsular tears or PCR and thus prevents the prolapse of the iris through the side ports or main wound . Small Pupil management: The Y-shaped chopper design allows for both capsular and iris stretching to enable the phacoemulsification in eyes in small pupils (<5 mm), allowing easy sculpting, nuclear fragmentation, and chopping of the nucleus fragments into smaller pieces. The rotation of the nucleus and epinuclear plate can also be achieved with ease, thus allowing the surgeon to sail through the cataract surgery without the use of pupil-expanding devices which can cause sphincter damage and post-operative photophobia to the patient . The instrument is made of stainless steel; at one end is a straight rod, and the other end has a curved chopper with a tip dimension of 0.8 mm which allows for easy entry through the side ports. In scenarios wherein we have moderately dilated pupil (5 mm) with any amount of iris billowing or miosis, it allows for simultaneous stretching of the floppy iris, thus enabling the phaco probe to hold a nuclear fragment and emulsifying the nucleus, whereas the curved chopper allows for easy nucleus chopping and allowing us to manage the small pupil and nucleus emulsification without the use of any pupil-expanding devices. Intra-operative miosis because of FIS: Intra-operative FIS is classified upon the presence of floppy iris stroma, which billows and ripples responding to phaco fluidics with progressive intra-operative miosis, independent of the use of mydriatic agents, and the iris stroma’s tendency to prolapse through the incisions. The Y-shaped chopper allows stretching the floppy iris to the periphery and preventing iris plugging in the phaco probe during aspiration and concomitantly performing nuclear fragmentation without risking anterior capsular tears or PCR and thus prevents the prolapse of the iris through the side ports or main wound . Small Pupil management: The Y-shaped chopper design allows for both capsular and iris stretching to enable the phacoemulsification in eyes in small pupils (<5 mm), allowing easy sculpting, nuclear fragmentation, and chopping of the nucleus fragments into smaller pieces. The rotation of the nucleus and epinuclear plate can also be achieved with ease, thus allowing the surgeon to sail through the cataract surgery without the use of pupil-expanding devices which can cause sphincter damage and post-operative photophobia to the patient . Successful surgical outcomes have been achieved with both mechanical iris dilation and iris retention devices, and these devices add to overall surgical cost and generally require more time in the operating theater than a mechanical pupillary stretch. One of the most important inventions in the history of mechanical pupil expansion was introduction of the iris hooks. Since the very first reports, the technique gained wide popularity all over the world. The advantages of this technique include ease of manipulations and wide availability of the hooks manufactured in different sizes, materials, and designs. However, there are chances of iris sphincter tears and risk of bleeding. It is generally recommended not to extend the pupil over 5.0 mm in size to decrease the chances of iris tissue over-stretching and in turn producing irregular and atonic pupils post-operatively, which can lead to post-operative photophobia and Haloes . Pupil stretching till date has been performed with the help of two instruments (spatulas, Kuglen hook, or similar) introduced through paracentesis incisions located contralateral to each other. However, pupillary stretching maneuvers are more traumatic to the iris and also possibly to the corneal endothelium, but this does not appear to detract from the surgical outcomes. The major drawback is that intra-operatively, the iris can get pulled into the phaco probe and in the corneal wounds, which makes it difficult to perform nuclear fragmentation and simultaneously salvage the iris. This problem gets alleviated with our Y-shaper chopper which allows us to manage the FIS and perform nuclear emulsification. In our initial experience, we have not observed any loss of iris tone or permanent damage or any patient complaining of post-operative photophobia. Significant variations in the ocular and systemic co-morbidities require the whole spectrum of pharmacological and surgical strategies to be in the armamentarium of the modern cataract surgeon. The easiness of manipulations and the final results vary significantly with different devices. Iris hooks and Malyugin Ring are the current standard of care for intra-operative mechanical pupil expansion in patients not responding to the pharmacological protocols. Some of these methods are associated with bleeding, loss of iris sphincter function, and an abnormal pupil shape post-operatively. The author’s initial experience with the Y-shaper and chopper minimizes the risk of intra-operative complications, yet enabling surgeons to aim at similar surgical outcomes. Further studies are needed to compare the various available techniques and assess the learning curve associated with this instrument. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest. www.ijo.in
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Surgical skills empowerment of ophthalmology residents in India –
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ea6822bc-d89a-4c4e-b180-98bb7cebef22
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10229956
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Ophthalmology[mh]
|
“ Sometimes less is more and sometimes more is less .” The constant debate that persists is between the power of observation versus immersive hands-on experience. A generous mentor or a senior surgeon passing on the tricks of the trade selflessly to a resident is an asset, and the resident must observe every move of the mentor and pay attention to the minute details. In little details, lie precious lessons. At the same time, learning from one’s own experiences remains unparalleled. There must be an ideal balance between these two processes for maximum learning. Indian residency programs are highly oriented towards knowledge empowerment, with surgical training often taking a backseat. The new National Medical Commission (NMC) competency-based curriculum for a master’s degree in ophthalmology does list the essential surgical skills but stops short of prescribing minimum required numbers. The All India Ophthalmological Society Ophthalmology Residency Curriculum lays elaborate guidelines for surgical training but does not state the desirable numbers. However, in the United States and the United Kingdom, quantification of surgical training is given significant importance, and for a resident to be qualified, a minimum number of 86 and 350 cataract surgeries respectively, as the primary surgeon is required. Singh et al . believe that greater practical training, brings in a sense of better judgment and ability to handle complications. However, the ideal number of surgeries to help make a resident confident, skillful, comfortable with handling complications with efficiency and appropriate speed and optimal outcome remains variable based on an individual learning curve and the mode of training. A didactic module providing adequate background knowledge, curriculum-based wet lab exposure to develop microsurgical and tissue-handling skills, staged rubric-based supervised surgical training, regular video recording of partially supervised and unsupervised resident surgeries and posthoc discussion and analysis leading to constant improvement are believed to be the best approach for optimal training. A study conducted to assess the effect of the lockdown on ophthalmic training programs across India stated that out of the 716 trainees, 81% felt a negative impact on their training due to the pandemic with a 50% drop in their surgical exposure. The authors of this study conducted a second phase of the survey carried out in 2022 after the pandemic, which is being brought to you in this issue of the Indian Journal of Ophthalmology. In this survey, 740 residents participated across India. It found that only 40% were independently performing cataract surgery and 14% were comfortable with phacoemulsification. However, it is impressive to see that 97% of this sub-set were trained to perform manual small incision cataract surgery (MSICS), which enables them to provide optimal eye care with minimal infrastructure and investment. The study also highlights the non-availability of training aids to at least 47% of the respondents. In times like the pandemic where there was a striking dip in the number of surgeries being performed, the residents’ surgical skills wouldn’t be compromised if high-quality training aids were to be part of the training program. A survey conducted in the pre-COVID times by the Academic Research Committee of the All-India Ophthalmology Society also showed that observed and assisted cataract surgeries were more prevalent in ophthalmology residency programs in India, with a predominance of SICS training and low sub-specialty exposure. These figures highlight the dire need for a standardized ophthalmology residency curriculum in India with quantification of a minimum desirable number of surgical opportunities. The Accreditation Council for Graduate Medical Education (ACGME) guidelines can be made the basis for developing the ideal system customized for the Indian scenario. These guidelines should be ideally utilized symmetrically (irrespective of the formal curriculum that is followed), nationwide with a deep understanding of the requirements and expectations of an evolving ophthalmologist: The importance and need for simulation training should be well understood and adopted by all the training institutes. Formal curriculum-based wet lab training should be made available to the residents either as institution-based facilities or shared regional hubs Competency-based surgical training curriculum with Rubric-based supervised training and posthoc discussion, with minimum numbers pegged in. National Ophthalmology Residency Resource Centres with emphasis on video-assisted surgical teaching. Honest inter-departmental discussions about the progress of each resident and appropriate tailored training. Promoting regular outreach eye care activities in underserved areas enables the trainees to develop independent thinking, management skills, and surgical skills while providing much-needed eye care services to the patients. Embracing the Halstedian concept of “See one, do one, teach one” with the senior residents involved in training the third and second-year residents and they all training the first year resident Focus on surgical skills ranging from extracapsular cataract extraction, MSICS to phacoemulsification, to prepare the residents for diverse practice scenarios. Optimal ocular surface, cornea, glaucoma, retina, oculoplasty, and squint subspecialty surgical exposure is needed to be a good comprehensive ophthalmologist Global interactions and exchange programs to help provide a broader perspective. While maintaining optimal background knowledge, a precise surgical skill set, and a high level of competency, the residents must adopt the step-wise training approach. It is important to understand and respect each phase of training and the lessons one is supposed to learn and imbibe during the journey. The metamorphosis might seem slow and tedious; however, the residents must trust the process and their trainers duly. Eventually, the outcome is impacted by aptitude and attitude maintained throughout the journey and the ability to embrace the transformation. “ Only those who will risk going too far can possibly find out how far one can go .” – T. S. Elliot
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Online patient ratings in ophthalmology
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02db4279-6a81-4489-b78b-646f3308c6d1
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10229971
|
Ophthalmology[mh]
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Nil. There are no conflicts of interest.
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Diary of an ophthalmology resident: Recollection of lessons learned at Dr. R. P. Centre, AIIMS, New Delhi
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f6c10771-6501-4f34-992d-76aa15dbcd94
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10229972
|
Ophthalmology[mh]
|
Nil. There are no conflicts of interest.
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Commentary: The never-ending story of COVID-19: Accustoming to the new abnormal in glaucoma practice
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025f0360-ca3b-4a5e-adde-955fc86dd4fb
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10229982
|
Ophthalmology[mh]
|
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) was causing devastation among the people and creating an economic, social, medical, and psychological burden. With the high infection and mortality rate, a safe and effective mode of providing services was imperative. As rightly said, necessity is the mother of invention; the pandemic made it a must to bring out new innovations to prevent disruption of medical services. That was when we brought in several changes to our practice pattern to provide a safe zone for both the patients as well as the ophthalmic service providers. From the pattern of emergence and reemergence, it is evident that we are still not clear in terms of coronavirus. Every time the world was lulled into the recovery state, the virus evolved and made a comeback, bringing new chaos in its wake. So, it is important to stay vigilant and not let our guard down and, make some permanent changes in your practice pattern to accommodate this new normal. Triaging became the most important barricade against disease control. Keeping in mind that ventilation is the key to success, we converted the underground hospital car parking garage into a triage zone . This was the primary step toward providing safe healthcare. An alternative location was provided for the patients to park their vehicles. The triage area was used as an information dispensing center to use the waiting time to garner the patient’s level of understanding of the disease and to simultaneously increase their awareness via Google Forms. Then came the corona cubicle (coronicle), which comprised a holistic setup of all the ophthalmic gadgets required to provide a comprehensive eye evaluation for cataract, glaucoma, retina, cornea, uvea, and paediatric services [ and ]. This cubicle created a barrier between the doctor and patients, thus hindering the transmission of aerosols. Also, only a minimal area was utilized for patient examination, thus reducing extensive patient movement inside the hospital premises, indirectly contributing toward optimal surface sanitization. The cubicle had the dimensions of 16 ft in length, 16 ft in breadth, and 8 ft in height, with a total area of 256 sq ft. Social distancing of 3 ft distance was maintained between the ophthalmic machines, which were installed inside the cubicle. The efficacy of the cubicle was further increased by interconnecting the various devices using a local area network (LAN) and making it available to the ophthalmic personnel in their computers, thereby reducing the need to move between places. This method of practice not only created an effective workflow but also paved the way for a greener workplace system. A sustainable healthcare system is the way to the future. The surgical practice of ophthalmology has been leaving behind a heavy carbon footprint. It is necessary to analyze the impact this will have on society and try to relieve the environmental burden. The pandemic has given us an opportunity to minimalize the carbon footprint and come up with better alternatives to safeguard the future. Difficulties were faced for certain procedures, like bowl perimetry, which needed to be addressed. It posed as a nidus for infection due to difficult cleaning protocols and erroneous data acquisition due to face mask positioning. This problem was tackled by the use of an enclosed chamber virtual reality perimeter (Advanced Vision Analyzer, Elisar). Additionally, this test could be conducted in the triage area during the patient’s wait time, reducing the overall hospital visit time . Fundus evaluation was another setting that invariably brought about contact between the doctor and the patient requiring constant sterilization of the lenses (90 and 20 D), besides this the dilatation process increased the overall patient waiting time in the hospital. So, bringing in mandatory fundus photography for all patients, using a non-contact, non-mydriatic, and auto-capture fundus camera was a game changer. It was deemed to be a beneficial tool in this pandemic. These innovations withstood the entirety of the last 2 years and have also proven successful during the Delta and Omicron variants. Considering that we are facing the new emergence of COVID-19 BF.7 variant, we need to be ever-ready and implement some permanent changes in practice patterns.
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Consensual eye intra-ocular pressure rise following unilateral glaucoma surgery
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f8642421-75af-4cbf-938a-c79406beba5d
|
10230001
|
Ophthalmology[mh]
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Data of consecutive patients ≥18 years of age who underwent surgery (trabeculectomy and AGV implant surgery) for primary or secondary glaucoma between January 1, 2019 and December 31, 2021, were prospectively inputted. Trabeculectomy (Moorfields Safer Surgery System) was performed when there was disease progression on near maximal medical therapy, or advanced glaucomatous damage on at least two or three medications. AGV implantation was performed in patients with secondary glaucoma who had superior conjunctival scarring as per standardized technique. Glaucoma surgeries were performed by three experienced surgeons. Post-operatively, patients received Moxifloxacin 0.5% four times a day and Pred Forte 1% eight times a day for one week. The steroids were gradually tapered over a period of six weeks. Fellow eye glaucoma status and data on previous glaucoma (medical and surgical) treatments were entered. Primary angle-closure suspects were defined as individuals with 180° or more irido-trabecular contact, normal IOP, no evidence of peripheral anterior synechiae, and a healthy optic nerve. Monocular subjects, patients on systemic or topical steroids because of ocular or systemic conditions, subjects with significant corneal opacity, or pathologies affecting IOP measurements were excluded. The following data were collected from the index eye (eye that underwent glaucoma surgery): best-corrected visual acuity (BCVA), cup disk ratio (CDR), glaucoma type and severity, use of acetazolamide and AGM number, pre-operative IOP, IOP at day 1, week 1, month 1, and month 3. AGM were stopped in the index eye following surgery. Fellow eye data included BCVA, CDR, glaucomatous or not, glaucoma type and severity, pre-operative IOP, IOP at day 1, week 1, month 1 and month 3, and AGM numbers. Two IOP measurements were recorded by an experienced optometrist using a Goldmann applanation tonometer, and the average was taken. When the two IOPs varied by more than 2 mmHg, a third measurement was taken and the median of the 3 was taken as the final IOP value. The IOP measurements were separated by at least one hour. Glaucoma severity was graded based on Hodapp–Parrish–Anderson classification. The primary outcome measure was IOP change in the FE that needed additional intervention to control the IOP. Intervention to control IOP was defined as increase in AGM or performing glaucoma surgery to decrease the IOP in the fellow eye. Any IOP increase associated with documented structural changes in the optic disc and/or functional changes in the visual fields was considered grounds to initiate additional intervention to control IOP. Secondary outcomes included mean change in IOP at follow-up visits from baseline (pre-operative IOP) and identification of factors that influence the change in IOP. The institutional review board has approved the conduct of the study. The study was performed in accordance with the tenets of Declaration of Helsinki. Statistics: IBM SPSS statistic software Version: 28.0.0.0 (190) was used to perform the analysis. Wilcoxon signed rank test to compare the mean IOP at baseline and at each follow-up time point in the consensual eye was performed. Kaplan–Meier survival analysis was performed to evaluate for failures. Failure was defined as need for additional intervention either an increase in AGM or performing glaucoma surgery to decrease the IOP in the fellow eye. Cox proportional hazard model to look for predictors for IOP change in the fellow eye was performed. Patients who needed additional intervention to reduce IOP were excluded from analysis at subsequent follow-up visits as primary outcome had occurred. These patients were included in the analysis in the period in which they were not prescribed any further medications or surgery for IOP control. The study analyzed prospectively inputted data of 187 consecutive glaucoma patients who underwent either trabeculectomy or AGV implant surgery. The mean age was 62.5 years, and majority were men (107, 57%). Glaucoma surgery in the Index eye was performed for primary open angle glaucoma in 85 patients (45.5%, n-187) and primary angle closure glaucoma in 76 patients (40.6%, n-187). Trabeculectomy was performed in 164 patients (87.7%) and AGV implant surgery in 23 patients (12.3%). The fellow eye was glaucomatous in 157 (84%) patients. The most common fellow eye glaucoma type was primary open angle glaucoma (82, 52.2%). Of them, 22 have had previous trabeculectomy surgery. Pre-operative acetazolamide was administered in 43 patients (23%). The mean pre-operative IOP in the Index eye and fellow eye were 17.6 mm Hg and 14.48 mm Hg, respectively. Among all patients (trabeculectomy and AGV implant surgery), the mean IOP in the fellow eyes rose from 14.48 ± 3.4 (n-187) in the pre-operative period to 14.54 ± 4.9 (p- 0.694) on day 1, 15.8 ± 5.5 (p-0.005) at week 1, 15.62 ± 5.7 (p-0.007) at month 1 and 15.09 ± 5.0 (n-135, p-0.279) at month 3. In patients who underwent trabeculectomy (n-164), there was a significant rise in fellow eye IOP at week 1 (15.89 ± 5.6, p-0.014) and month 1 (15.65 ± 5.6, p-0.02) from a baseline mean IOP of 14.61 ± 3.5. Among patients with AGV shunt surgery (n-23), the mean fellow eye IOP was higher than baseline at all-time points, with maximum rise on day 1 (15.91 ± 6.3, baseline 13.78 ± 2.7; n- 23, p-0.066). Pre-operative acetazolamide (n-43) strongly predicted a statistically significant increase in fellow eye mean IOP at week 1 (18.28 ± 6.8, P -0.023) and month 1 (17.13 ± 6.2, p-0.047) when compared to the baseline IOP (15.67 ± 4.5). The greatest rise in fellow eye IOP was noted when the IOP was < 5 mm Hg at month 1 in Index eye (n-11). The IOP rose from baseline of 13 ± 2.6 to 16.50 ± 3.4 (p-0.042) at week 1 and 15.5 ± 2.9 (p-0.072) at month 1 in the fellow eye with a maximum mean increase of nearly 3.5 mm Hg (p-0.042) at week 1. At one-month follow-up, there was a statistically significant increase in the mean number of AGM used in the fellow eyes. AGM was introduced in the index eye in only one patient in the follow-up period [ and ]. In the overall group, IOP increase requiring either increase in AGM or surgical intervention to control IOP in fellow eye was determined with the Kaplan–Meier survival estimates. 18, 8, and 33 patients needed additional intervention to control the IOP in the fellow eye in the 1–7, 7–30, and 30–90-day window periods . Nearly 15% of patients needed additional intervention to control the IOP in the fellow eye in the first 30 days and 33% in the first 90 days after glaucoma surgery in the index eye. A total of 27 patients (14.4%) needed glaucoma surgery to control IOP. In the trabeculectomy alone group, nearly 35% of patients needed either an increase in AGM or surgical intervention to control IOP in the fellow eye in the first 90 days (17% in the first 30 days). Of them, 26 patients (15.9%, n-164) needed surgical management (25: trabeculectomy, 1: AGV implantation). Compare this with 18% of patients who underwent shunt surgery needing an additional intervention to control FE IOP in the first 90 days. Of them, one patient underwent trabeculectomy in the FE for control of IOP. This patient had severe secondary open-angle glaucoma. Though there was trend toward significance, the difference noted between the two surgical groups was not statistically significant (p-0.097) . Further, fellow eye glaucoma status had no bearing on if additional intervention to control IOP in the fellow eye is required (p-0.55). Index eye pre-operative IOP, AGM numbers, pre-operative acetazolamide, glaucoma severity, fellow eye glaucoma status and severity, and AGM numbers did not predict need for additional intervention to control IOP in the Cox proportional hazard model. Intra-ocular pressure changes in the fellow eye following unilateral glaucoma surgery have been reported to occur in previous literature; however, it is unclear if the IOP increases or decreases in the post-operative period. In the current study, when all patients were considered (trabeculectomy and shunt surgery), we noted an elevation in mean IOP in the fellow eyes at all follow-up visits. There was a statistically significant increase in the mean IOP at week 1 and month 1 follow-up. To understand if this IOP increase was clinically significant, a more meaningful approach was to look for need for additional intervention to control IOP with time. A third of patients (33%) needed intervention to control IOP in the first 90 days, and 14.4% (27 patients) of patients eventually needed surgical intervention to reduce FE IOP. When we looked for differences between the surgical groups (trabeculectomy versus shunt surgery), we did not find a significant difference in the rates at which additional intervention was required to control IOP. A probable reason for lack of difference is less number of patients in the shunt surgery group. However, both in the trabeculectomy and the shunt surgery group, mean IOP remained elevated at all follow-up visits. In patients who underwent trabeculectomy, a statistically significant increase in mean IOP was noted at week 1 and month 1, and in patients with shunt surgery a significant increase in mean IOP was noted at day 1 post-operative period. Nearly twice the number of patients who had glaucoma or suspected angle closure needed intervention to control IOP as compared to when the fellow eye was normal; however, this difference was not statistically significant. Persistent hypotony in the index eye predicted the maximum rise in mean IOP with a mean increase of nearly 3.5 mm Hg in the fellow eye at week 1. Acetazolamide pre-operatively resulted a significant increase in IOP at week 1 and month 1. Similar to our study, Meshksar et al . (2021) noted a statistically significant mean IOP elevation in the FE up to 6 months after glaucoma surgery. The greatest increase in IOP was noted at 1 week following surgery (mean increase of 3.16 mm Hg). Thereafter, though there was a progressive drop, the mean IOP remained above the baseline at 6 months. Tube shunt surgery, glaucoma following cataract surgery in the FE, drop in IOP to ≤5 mm Hg in the index eye on first post-operative day, and pre-operative acetazolamide use were strongly associated with significant IOP elevation in the FE. Glaucoma medications were added to 18 fellow eyes and seven fellow eyes on maximal medical therapy needed surgical intervention due to increase in IOP. Further, Kaushik et al . (2016) noted a significant increase in FE IOP compared with baseline at all-time points (P < 0.001), with a maximum rise at 6 weeks post-operatively (4.8 mm Hg). By the sixth post-operative month, 24 patients required surgery or needed an increase in medications in the fellow eye for IOP control (n-71). Akin to our observation, Yarangümeli et al . (2003) noted elevated mean IOP in the FE compared to the baseline in 33% (n-107) in the 3 months following glaucoma surgery. IOP was elevated by 30% or more in 14% of eyes, and greater than five rise in IOP was noted in 12% of the eyes. One of the probable explanation could have been withdrawal of the anti-glaucoma medications from index eye following surgery. However, this theory has been debated as patients with glaucoma in the contralateral eye continued with the anti-glaucoma medications, and there was no correlation between the number of anti-glaucoma medications and the rise in IOP. Other potential explanation for the rise in IOP would be the sequelae of underperfusion of the scleral meshwork due to preferential flow through the sclerostomy site. Underperfusion results in meshwork densification, activation of endothelial cells and increase in extracellular matrix both in the ipsilateral and contralateral eye offering resistance to aqueous outflow and in turn contributing to the rise in consensual eye IOP. Further, it has been theorized that the scleral spur consists of special cells that play an important role in the afferent pathway of the reflex that controls bilateral ciliary body and meshwork contractile tone. In an early aqueous dynamics study, Diestelhorst et al . (1991) noticed a statistically significant increase in average aqueous flow from baseline at day 5 post-trabeculectomy in the consensual eye. They hypothesized that following filtration surgery, a reflex increase in aqueous flow occurs in the treated eye to maintain anterior chamber stability. In addition, this ocular-CNS reflex appears to affect aqueous flow dynamics bilaterally and hence explains the increase in aqueous flow in the fellow eye following unilateral trabeculectomy. Despite an increase in aqueous flow, the IOP increase in the consensual eye was not statistically significant. The authors attributed the lack in IOP increase to a compensatory increase aqueous outflow through the trabecular meshwork. To the contrary, Vysniauskienee at al. (2005) noted drop in IOP in the FE at day 1 and 1 month following glaucoma surgery causing a > 45% drop in IOP in the Index eye (n-24). The drop was significant when the FE was treated with ocular hypotensive medications. However, the FE IOP drop was marginal when no AGM was used. Similarly, Radcliffe et al . (2010) through the Collaborative Initial Glaucoma Treatment Study noted a modest drop in IOP in the contralateral eye at 3-month to 24-month follow-up. The drop in IOP was statistically significant only at 12 months. None of the index and fellow eye received topical hypotensive medications at baseline or follow up. In addition, there was strong but marginal correlation between the magnitude of drop in IOP in the Index eye and the fellow eye at 3 months. The drawback, however, was that they did not look at FE IOP in the first few months after glaucoma surgery. Why IOP decreases following glaucoma surgery is unclear and is yet to be elucidated. Previously, authors have explored if the topical medications played a role and if the reflex response can be abated by blocking the reflex arc. Gibbens (1988) noted that in the presence of ocular hypertension, a drop in IOP in the FE occurred when the index eye (IE) was treated with either timolol or pilocarpine. The plausible explanation was drug effect on fellow eye by the systemically absorbed drug. However, this was refuted by Smith et al . (1979), as they observed a drop in FE IOP despite no significant drug levels in plasma. The drop following topical administration was similar to that following Intravenous administration. Henceforth, an ocular-central nervous system reflex (O-CNS) was considered as probable cause for the consensual ophthalmotonic reaction. To further support the above hypothesis, the FE response was abated when retro-bulbar cocaine was administered in the treated eye (Leplat, 1924) and following cervical sympathetic blockade (Akagi et al. ,1955). To the contrary, Prijot and Stone (1956) observed abatement of the reflex response in atropinized rabbits, and they proposed a parasympathetic efferent pathway. Lastly, Perkins (1957) found that sectioning of the fifth cranial nerve eliminated the reflex response and concluded that fifth nerve may be the efferent limb of this reflex. Our study with a large sample (n-187) is an important addition to the sparse literature from this demographic population. An important observation was elevated mean IOP at all follow-up visits with significant increase at week 1 and month 1. We performed Kaplan–Meier survival estimates to assess the need for further intervention to control IOP in the post-operative period. The need for intervention to control IOP in fellow eye in the 30- to 90-day period was similar in the to the first 30 days. A similar trend was noted in both surgical groups (trabeculectomy and shunt surgery) and in patients with preexisting glaucoma (or suspected angle closure) in the fellow eye. Further, 14.4% surgical intervention rates are comparable to rates reported in literature ranging from 5% to 33.8%. Pre-operative acetazolamide administration and more interestingly persistent hypotony at one month in the index eye at 1 month seem to predispose to greater magnitude increase in IOP in the contralateral eye. Interestingly, persistent hypotony predicted the maximum mean increase in IOP. This study informs clinician on the need for monitoring IOP in fellow eye and also about the high-risk group. Limitation of the study includes a small number of patients in the shunt surgery group. This could have precluded eliciting a more robust increase in IOP in the fellow eye in this group. Further, patients were not controlled for the number of the AGM used in the fellow eye. This may have prevented the understanding of the de novo IOP changes in the absence of AGMs. Nevertheless, the study informs the need for stringent IOP monitoring of the fellow eye in the post-operative period. The current study raises the possibility of a real risk of IOP increase in the fellow eye following ipsilateral glaucoma surgery. Nearly a third of patients needed an intervention to control IOP in the overall group. Similar rates of intervention to control IOP were noted in the 30- to 90-day period and 0- to 30-day period. Nearly a sixth of the patients required surgical intervention to control IOP. This study paves the way to consider the need for a randomized controlled trial aimed at evaluating FE IOP changes early after surgery. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest. Nil. There are no conflicts of interest.
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Mumbai metropolitan region eye care facility survey: Mumbai Eye Disease Study (MEDiS) report #1
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5b782507-bc19-473e-a7fc-6cd2e4591dfd
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10230003
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Internal Medicine[mh]
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The study was approved by Sigma Institutional Review Board (New Delhi; 10084/IRB/21-22), and followed the tenets of the Declaration of Helsinki for human-related study. We adopted a mixed-methods research of quantitative and qualitative analyses. It included primary (data generation) and secondary (data collation and synthesis) research. The primary research was conducted across MMR to determine the demand and supply estimation and the health-care finances. We divided the MMR into five zones – North (Andheri, Borivali, Malad, and Virar), Central (Bandra, Dadar, Dharavi, and Wadala), South (Colaba, Fort, Malabar Hills, and Marine Lines), East (CBD Belapur, Navi Mumbai, Panvel, and Vashi), and Mumbai suburbs (Bhandup, Dombivali, Kalyan, Mulund, and Thane) . We chose Wadala area as it is situated in the center of MMR and on the central railway line of Mumbai local railways; also, Wadala is easily accessible. Using ESRI-ArcGIS software (Redlands, CA, USA), we mapped 473 critical facilities to assess eye care delivery in these five zones. The selected facilities included a mix of public and private eye hospitals, medical schools, multispecialty hospitals with eye departments, and large and small eye clinics. To analyze the eye care demand, we used the area within a 20-min drive distance from Wadala railway station (central zone in our definition). The primary research included an interview with the patients, eye care providers, and key opinion leaders in diverse fields of health care, including ophthalmology. Only permanent residents of MMR were included in the patient group. At the facility level, interviews were conducted only with the hospital administrators and senior physicians. Given that these interviews were conducted during the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic (April–October 2020), all necessary health-related protocols were followed, such as wearing proper personal protective equipment and frequent use of hand sanitizers. All direct physical contacts were avoided. The questionnaire of primary interview of various groups of people was framed after the data from the health-care facilities and insurance companies were collected and analyzed. The responses were analyzed separately. We selected four different groups for primary interview. These were (1) students (currently pursuing medicine [undergraduates], ophthalmology residency, and optometry); (2) practicing ophthalmologists providing eye care services in the region; (3) key opinion leaders or top professionals and experts in eye care; and (4) adults who could need eye care in the near future. The people were selected from each of the five regions. Only permanent residents of MMR with the government-issued photo ID (voter or Aadhaar card; Aadhaar card is the proof of residence in India) were included in the survey. We defined permanent residents as those who have lived in Mumbai for at least 1 year and were not planning to move out of the city for another year. At the facility level, interviews were conducted with the hospital administrators and senior physicians. Other hospital staff was excluded. The data were collected after obtaining a written consent. It was kept confidential, and the participants were explained that the anonymized data could be used for research and possible publications. The secondary research included analyzing data from the city- and state-based professional ophthalmology societies, data available in the public domain, and the health insurance providers. Data were collected from the hospital ( n = 75) websites to identify the service mix, qualification of key doctors, type of accreditation, empanelment with insurance providers, so also provision and practice of medical tourism. Data on the clinicians ( n = 63) was gathered to identify the clinician engagement models used by these hospitals, average charges for consultation services, and qualification/years of experience. Using the collected data, inter-/intra-hospital comparison was drawn, and 20 key eye care hospitals/clinics were identified for benchmarking. These hospitals/clinics were also examined using the Ayushman Bharat PM-JAY and MJP-JAY (Pradhan Mantri Jan Arogya Yojana, national and Mahatma Jyotirao Phule Jan Arogya Yojana, state public insurance, respectively) data to assess the insurance penetration and identify key packages in ophthalmology in-patient volume, patient profiles (age and sex), and insured beneficiary base. Data was also collected from key third-party assurance (TPA) providers ( n = 23) empaneled with major eye hospitals, eye clinics, and multispecialty hospitals in the region. To measure the demand, we divided the target population ( n = 513) into three income groups by annual income – low, middle, and high, which are less than INR 300,000, between INR 300,000 and INR 1.8 million, and above INR 1.8 million, respectively. Two important questions we asked were (1) Do you know the importance of eye examination? and (2) Do you regularly practice annual eye examination? Additional information on the patient feedback was also collected from various health-care apps such as Practo, Google review, and Just dial. Interviews All interviews were conducted by field investigators. It consisted of 11 people (eight men, three women) selected locally with a minimum educational qualification of graduation in any discipline. They received training for 2 days to identify key respondents, conduct interviews, and record the correct answers. Virtual interviews were conducted with key opinion leaders or top professionals and experts in eye care because of coronavirus disease 2019 (COVID-19) pandemic (second wave). The data collection was done utilizing a mixed method of quantitative and qualitative techniques. We used KoBo survey tool ( http://www.kobotoolbox.org ). KoBo Collect survey application, an open source tool, was installed on android mobile phones given to the field investigators for conducting primary data collection. KoBo is a controlled application protected by password and provides collective user-wise data. The categorized and open-ended questions used in the survey are show in Supplementary . Sample size We assumed that 5.1 million people, one-quarter of the MMR population, could possibly need eye care any time in their life. This number was used to calculate the sample size for interviewing the Mumbai residents. Using a random sampling methodology, and population proportion needs, we used the following formula: S = [Z2* p* (1 − p)/E2]/1+([Z2 *p* (1 − p)/E2 − 1]/N), where S: sample size Z: Z-score (which corresponds to the 95% confidence level) p: population proportion (assumed to be 50%) E: margin of error (assumed as 5%) N: population size (5,100,000- possible number of people needing eye care in MMR) S = [(1.96) 2 *0.5*(1 − 0.5)/(0.05) 2]/1+([(1.96) 2 *0.5*(1 − 0.5)/(0.05) 2 − 1]/5,100,000) S = 384.13 (~approx. 384) Incorporating a nonresponse rate of 20%: final sample size = 384.13/0.8 = 480.16 (~approx. 480) across defined eye service categories in proportion to their population needs. Minimum sample size required to draw statistically significant conclusion was 480 residents, though in an attempt to increase the precision, we have included a larger sample of 513 residents.
All interviews were conducted by field investigators. It consisted of 11 people (eight men, three women) selected locally with a minimum educational qualification of graduation in any discipline. They received training for 2 days to identify key respondents, conduct interviews, and record the correct answers. Virtual interviews were conducted with key opinion leaders or top professionals and experts in eye care because of coronavirus disease 2019 (COVID-19) pandemic (second wave). The data collection was done utilizing a mixed method of quantitative and qualitative techniques. We used KoBo survey tool ( http://www.kobotoolbox.org ). KoBo Collect survey application, an open source tool, was installed on android mobile phones given to the field investigators for conducting primary data collection. KoBo is a controlled application protected by password and provides collective user-wise data. The categorized and open-ended questions used in the survey are show in Supplementary .
We assumed that 5.1 million people, one-quarter of the MMR population, could possibly need eye care any time in their life. This number was used to calculate the sample size for interviewing the Mumbai residents. Using a random sampling methodology, and population proportion needs, we used the following formula: S = [Z2* p* (1 − p)/E2]/1+([Z2 *p* (1 − p)/E2 − 1]/N), where S: sample size Z: Z-score (which corresponds to the 95% confidence level) p: population proportion (assumed to be 50%) E: margin of error (assumed as 5%) N: population size (5,100,000- possible number of people needing eye care in MMR) S = [(1.96) 2 *0.5*(1 − 0.5)/(0.05) 2]/1+([(1.96) 2 *0.5*(1 − 0.5)/(0.05) 2 − 1]/5,100,000) S = 384.13 (~approx. 384) Incorporating a nonresponse rate of 20%: final sample size = 384.13/0.8 = 480.16 (~approx. 480) across defined eye service categories in proportion to their population needs. Minimum sample size required to draw statistically significant conclusion was 480 residents, though in an attempt to increase the precision, we have included a larger sample of 513 residents.
We chose the Wadala area as our point of reference because of its relatively central location, large suburbs with economically middle-class habitats, proximity to a large urban slum (Dharavi), and dense habitation (40,400 population per km 2 ). By annual income (2015 standards), using our division of economic class, we categorized 43%, 31%, and 26% people into low-, middle- and high-income groups, respectively. The investigators ensured that all date were available from all health-care facilities ( n = 473) and residents ( n = 513). We mapped 473 key facilities as follows: 56 eye hospitals, 246 multispecialty eye hospitals with an ophthalmology department, and 171 eye clinics. The majority of these facilities were on the western railway line. National Accreditation Board for Hospitals (NABH) accreditation : One-fifth (92/473 = 19.5%) of these eye care facilities had NABH accreditation- 68.5% ( n = 63/92) had full accreditation and 31.5% ( n = 29/92) had entry-level accreditation. Ophthalmic education : Some of these facilities offered ophthalmic and para-ophthalmic courses. These included a bachelor’s degree ( n = 56) and diploma ( n = 38) in optometry, a postgraduate degree in ophthalmology (DNB, n = 19 of 473), and a fellowship (32 of 473) in ophthalmology subspecialty and optometry. Demand and availability of eye care services: The demand for cataract, glaucoma, and vitreoretinal services was high in the MMR region, and so was the availability of these services, usually within a 5 km radius of the reference location. But the provision for uvea, pediatric ophthalmology, and ocular oncology was low . We benchmarked 20 eye care services; 14 (70%) of these were stand-alone large eye care facilities (eye hospitals) and six (30%) were the ophthalmology services in a large multispecialty hospital (eye department). Either location provided a range of eye care services and included both out- and in-patient facilities. On an average, the outpatient department (OPD) had approximately 80–90 patients per day in eye specialty hospitals and 65–75 patients per day in multispecialty facilities. Annual eye surgery was in the range of 12,228–29,460. Practice pattern . Most of the time, the engagement of hospitals with clinicians is primarily part-time and visiting consultant mode. Clinicians with more than 15 years of experience tend to establish their private practice while continuing to serve as visiting consultants or specialists with other hospitals. Half of the interviewed clinicians worked part-time at eye care facilities, and the other half had a full-time engagement with one facility. Typically, small and medium-sized providers hired less-experienced clinicians and large and multispecialty providers engaged more experienced physicians. OPD services in most facilities were in two shifts – morning, beginning at 10 AM (ending at 2 PM), and evening, beginning at 4 PM (ending at 9 PM). In most places, there is no regular service between 2 PM and 4 PM on weekdays and none on Sundays. Emergency and inpatient department (IPD) services are available 24 × 7. The ophthalmology residents and fellows-in-training conduct outreach programs for their respective hospitals. Cost of care : Most people pay all medical bills out of pocket or by private insurance . In the MMR region, the senior clinicians do not provide free consultations for outpatients or surgery. Nominal services are available for people seeking free service. However, free-of-cost surgeries are available in government facilities under the national- or state-level blindness control programs, but patients pay for all additional medicines and all high-cost consumables. Willingness to pay : All respondents were price sensitive. The average outpatient consultation was INR 857 (INR 500, INR 800, and INR 1200 for the low-, middle- and high-income groups, respectively). The willingness to pay for surgery was procedure dependent. It varied from INR 5000 to INR 150,000 per surgical procedure. We categorized them into three grades for convenience and easy understanding; the weighted average was INR 30,000–INR 70,000. The willingness to pay for the surgical procedure by grades is shown in . Ophthalmic medicine: The willingness to pay for medicine was based on the economic group: 58% of people were comfortable with spending up to INR 1000/- (predominantly lower-economic group), 39% of the respondents were comfortable up to INR 2000/- (predominantly middle-income group), and 3% of the respondents were willing to pay above INR 2000/- (only higher-income group). The weighted average for ophthalmic medicine was INR 1225. Spectacles : More than 60% of respondents were inclined to buy better-quality spectacles, ranging from INR 1000/- to INR 2000/-. The lower-income group preferred a modest variety, costing less than INR 1000/-, and were price sensitive. Again, with an increase in the annual income, the tendency was to purchase more expensive and superior-quality spectacles. In this segment, too, the average cost echoed with the competitor analysis, that is, INR 1365 . In our analysis, all 473 eye hospitals, eye clinics, and multispecialty hospitals were empaneled with various TPAs – 60% ( n = 284) of them with less than five TPAs, 34% ( n = 161) hospitals with 6–10 TPAs, and 6% ( n = 28) hospitals with more than 10 TPAs. Government insurance scheme such as AB PM-JAY offers additional incentives based on the accreditation levels of the health-care facility. Cataract surgery is not a part of the state health insurance scheme MJP-JAY in Maharashtra; hence, the beneficiaries were unable to take the facilities of the public health insurance scheme. Secondary research was undertaken using the data available on the National Programme for Control of Blindness &Visual Impairment (NPCB VI) website (2018–2019). In the financial year 2021–2022, a reimbursement claim was made for less than 1% (3199 of approximately 400,000) eye surgeries performed in the MMR. Demand estimation The health-seeking behavior improved with the increase in the income level. The people got their eyes examined when required –65% in the low-income group and 87% and 89% in the middle- and higher-income groups, respectively. But the routine annual eye examination was low in the lower- and middle-income groups (50% and 48%, respectively); it was much better at 85% in the high-income group. The preference for public and private eye care facilities was 65% versus 35%, respectively, in the low-income group, 56% versus 44%, respectively, in the middle-income group, and 13% versus 75%, respectively, in the high-income group; additionally, 12% of the high-income group preferred to seek eye care treatment outside of the state of Maharashtra . The cost of care was higher in private facilities. In addition to the medical cost, travel distance to the eye care facility was also an important determinant. Nearly all participants across income groups preferred not to travel beyond 5 km for eye care; it was 16%, 23%, and 20% in low-, middle-, and high-income groups, respectively. Other influencing factors were the familiarity with the treating physician (such as the physician had earlier treated one of the family members) and referral by the family physician.
The health-seeking behavior improved with the increase in the income level. The people got their eyes examined when required –65% in the low-income group and 87% and 89% in the middle- and higher-income groups, respectively. But the routine annual eye examination was low in the lower- and middle-income groups (50% and 48%, respectively); it was much better at 85% in the high-income group. The preference for public and private eye care facilities was 65% versus 35%, respectively, in the low-income group, 56% versus 44%, respectively, in the middle-income group, and 13% versus 75%, respectively, in the high-income group; additionally, 12% of the high-income group preferred to seek eye care treatment outside of the state of Maharashtra . The cost of care was higher in private facilities. In addition to the medical cost, travel distance to the eye care facility was also an important determinant. Nearly all participants across income groups preferred not to travel beyond 5 km for eye care; it was 16%, 23%, and 20% in low-, middle-, and high-income groups, respectively. Other influencing factors were the familiarity with the treating physician (such as the physician had earlier treated one of the family members) and referral by the family physician.
Mumbai city in India has many unique characters. It is the economic corridor of India with 33% of India’s income tax collections, 20% of all central excise tax collections, and 40% of India’s foreign trade. It is crowded with high population density , and over 35% of people live in urban slums. The per capita income is three times higher than the rest of the country, though 43% of people are either poor or live below the poverty line. The city also is home to 17% of migrant workers. It is cosmopolitan, with half of the people living in the city coming from other states of India. The current survey is the first systematically designed analysis of the eye care facilities in any metro in India (Medline search). The survey of the MMR collected data from a large number of eye care facilities and gathered information from a large adult population and diverse group of professionals. It showed several eye care practice patterns, possibly unique to the city. MMR has a large spread of eye care practices and facilities. The city HReH is good and boasts 80 ophthalmologists per million, which is better than the Indian average of 13.0/million. Despite this advantage, the study identified two significant disparities in eye care: (1) deficient spread of all specialties other than cataract and glaucoma care and (2) insecure health financing where there is a high proportion of out-of-pocket spending. The city also has a large population of “floating” specialist consultant ophthalmologists who visit several eye care facilities for a few fixed hours every day or on predetermined days. The advantage is that it brings the specialists closer to the people’s residence; but the disadvantage is that it neither provides the specialists enough time to spend with the patients nor provides enough space to resolve technically difficult medical problems. The genesis of this pattern of care partly owes to Mumbai’s traffic conditions. In a survey conducted of 403 cities in 53 countries, Mumbai was found to be the world’s most congested city by traffic; the TomTom N.V (Amsterdam, the Netherlands) reported a 53% congestion level in 2021. This means that the travel time by road is 53% longer than the baseline uncongested conditions; for example, a 30-min trip driven in a free-flow condition will take 16 min longer when the congestion level is at 53%. We also believe that the combination of the traffic situation and the consultant practice in Mumbai city could be behind the origin of three other phenomena in the city: one, low volume of voluntary eye care in the middle of the day (from 2 to 4 PM); two, people like to avail services within 5 km of their residence ; and three, nonexistence of large eye care institutes with equal emphasis on eye health research and public health similar to those available in a few states in South India. Provision and proximity of health facilities are the first steps to good eye care. But, by itself, it does not necessarily improve regular health-seeking behaviors. In a study conducted in Hyderabad (South India) city, nearly half of people did not voluntarily seek eye care despite decreased vision, and in a third of the people, routine eye care was not one of the health-care practices. A Singapore study has shown that annual eye examination ranged from 22.4% to 40.4% in three ethnic groups (Chinese, Malaya, Indian) and refractive service was better than other specific eye care services. Our study did not analyze specific services; instead, it looked for the proportion of people visiting the eye care facilities in the event of decreased vision and the practice of annual eye examination . It was over 65% in the former and 48% in the latter. The utilization of eye care was less in the lower-income group, and the practice of annual eye examination was less in the lower- and middle-income groups than in the higher economic group. The poor utilization of eye care services even in cities is related to “felt need” and “health literacy.” Mobile health service is one of the modalities to improving eye care uptake. A quality-assured mobile eye service is known to enhance eye care uptake across many economically less-privileged regions of the world. Also, one could improve eye care services using advanced technology. But these measures do not necessarily improve the health-seeking behavior of people. Often, the cost of care is reduced with insurance cover. Our study showed that 60%–83% of people paid for their eye care expenses , 57% of people opted for low-premium eye surgery , and 37% of people opted for less-expensive spectacles . While expensive eye surgery may not always parallel better outcomes, superior-quality devices, such as a toric or multifocal intraocular lens and spectacles lens with protective coatings, are also more expensive. Shifting from out-of-pocket spending to prepayment through insurance or pooled funds is likely to ease the financial burden on people. The public insurance, state (MJP-JAY) and national (AB PM-JAY), offers financial support, but not enough for the most common eye surgery, the cataract surgery. It was surprising that reimbursement claim was made for less than 1% (3199 of approximately 400,000) eye surgeries performed in the financial year 2021–2022 (April 2021–March 2022), even accounting that the number of surgeries was comparatively less due to SARS-CoV-2 infection (Maharashtra achieved 67.4% of target cataract surgery in 2020–2021, and the state cataract surgical rate at 4178/million population is 94.7% of the national average at 4410/million population). Limitations and strengths Limitations: The current study was conducted during the SARS-CoV-2 infection time, and hence, we could not reach more number of people. Also, many people would have been too depressed during this pandemic to respond more intelligently. We also suspect many needing eye care may have neglected to seek timely eye care and have gotten into this habit. Also, we did not collect ocular disorder-specific data. Strengths: This is the first survey in the MMR, looking at several aspects of eye care from the perspective of people, eye care providers, and health finance. The study included data from public and private eye care providers and interviewed people in various sections of society – prospective patients, eye care professionals, and key opinion leaders in health care outside of ophthalmology.
Limitations: The current study was conducted during the SARS-CoV-2 infection time, and hence, we could not reach more number of people. Also, many people would have been too depressed during this pandemic to respond more intelligently. We also suspect many needing eye care may have neglected to seek timely eye care and have gotten into this habit. Also, we did not collect ocular disorder-specific data. Strengths: This is the first survey in the MMR, looking at several aspects of eye care from the perspective of people, eye care providers, and health finance. The study included data from public and private eye care providers and interviewed people in various sections of society – prospective patients, eye care professionals, and key opinion leaders in health care outside of ophthalmology.
The data suggest that the eye health system in the MMR needs different strategic planning. With a literacy rate of 89.2% in Mumbai, higher than the national average of 74.04%, and with a higher per capita income, Mumbai region could consider the following: (1) improve affordable and accessible eye care for ophthalmic disorders not only confined to cataract and glaucoma; (2) incorporate strong public health surveillance system in ophthalmology; (3) research into the application of newer technologies to provide less-expensive home care for the elderly and minimize their hospital visits; and (4) collect and analyze big data to address city-specific eye health issues. Financial support and sponsorship Shantilal Shanghvi Foundation, Mumbai (2020). Conflicts of interest There are no conflicts of interest.
Shantilal Shanghvi Foundation, Mumbai (2020).
There are no conflicts of interest.
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Gasdermins assemble; recent developments in bacteriology and pharmacology
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dda34bb6-04ff-4c0d-9628-806a4ec7608e
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10230072
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Microbiology[mh]
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The discovery of pyroptosis is inherently linked to bacterial immunology. Early studies of macrophages lysing in a fiery demise after exposure to the lethal toxin of anthrax were, at the time, unappreciated observations of pyroptosis ( , ). The interaction between the Shigella flexneri protein ipaB and caspase-1 in macrophages led to the first designations of pyroptosis; typically characterised by release of pro-inflammatory cytokines through pores consisting of N-terminal oligomers of one of the gasdermin protein family, followed by inflammatory cell death ( , ). The history of pyroptosis and the historical context of bacterial-gasdermin interactions have been extensively covered elsewhere ( – ) and so here we will instead focus on bringing together the very recent discoveries in the context of bacteriology and gasdermins, before discussing the pharmacological landscape and associated challenges. Briefly, the gasdermin family consists of six proteins in human (GSDMA, GSDMB, GSDMC, GSDMD, GSDME and GSDMF/PJVK) and ten proteins in mouse (GSDMA1-3, GSDMC1-4, GSDMD, GSDME, PJVK) ( ). All except GSDMF have been shown to exhibit pyroptotic functionality via the N-terminal pore-forming domain, which exhibits lipid preferences depending on the family member ( ). Since the discovery of GSDMD downstream of caspase-11 (4/5 in human) by two independent groups ( , ), an explosion of mechanisms that drive gasdermin activation and inhibition have been uncovered. The first characterisation of GSDMD activation and induction of pyroptosis was through cytosolic LPS-induced caspase-11 cleavage of GSDMD at Asp275 and release of the C-terminal domain ( ). This leads to the oligomerisation of consequently uninhibited N-terminal fragments that traverse to and insert in the plasma membrane and form a pore in the cell, resulting in cytokine release and eventual cell death through lysis ( , ). It was recently discovered that gasdermins are required for the release of IL-1 family cytokines (IL-1α, IL-1β and IL-18), but an additional protein NINJ1 is critical for the plasma membrane rupture (PMR) and HMGB1 release characteristic of inflammasome activation ( ). Through mouse studies, it has been shown that both cytokine release as well as cell death is important for anti-bacterial host defence to pathogens such as Citrobacter rodentium ( , ). This all occurs rapidly and without the usual organisation of apoptosis, leading to cellular contents flooding, and consequently activating, the local environment to flag immune cells to the location of danger ( – ). Since these seminal discoveries, the functions, regulation, and consequences of gasdermin activation and/or inhibition has expanded considerably. Just focussing on GSDMD, there is a now a well-established contribution from inputs other than inflammatory caspases. Apoptotic caspases such as caspase-3 and caspase-8 have emerged as inflammasome-independent contributors to gasdermin activation ( ). Caspase independent contributions to gasdermin function also exist in the form of cathepsins ( , ), granzymes ( , ) and elastase, neutrophil expressed (ELANE) ( , ). These different drivers have both distinct and overlapping biological consequences dependent on the cellular context and/or sensed DAMP/pathogen, including switches between pyroptosis to apoptosis and necroptosis. There are emerging examples of virulence factors interfering with gasdermin function including targeting GSDMB and GSDMD for proteasomal degradation ( – ). Additionally, subcellular organelle-gasdermin interactions exist including in the mitochondria ( – ), endoplasmic reticulum ( ), autophagosome ( ) and the nucleus ( ) suggesting that subcellular localization patterns may regulate biological functions. Moreover, non-pore forming functions have emerged including full length GSDMB regulating proliferation, migration and adhesion in the context of the gut ( ), full length GSDMD mediating the homeostasis of intestinal epithelium and control of IL-1β containing extracellular vesicles in an inflammasome, activation-independent function ( ), as well as mucus secretion and barrier integrity ( ), reviewed in ( ). This review will bring together canonical pyroptotic functions of the gasdermin family with these alternative activation options, non-pyroptotic elements and subcellular interactions within the framework and context of bacterial infection and immunity.
As the pyroptosis pioneer, GSDMD is by far the best characterised gasdermin of the family. One major advantage to studying GSDMD is in the simplicity of driving inflammasome activation with clear assay-amenable cellular responses. Inflammasomes were defined in 2002 ( ) and have well established in vitro and in vivo assays with clear end points, that we now appreciate as, at least partially, GSDMD-mediated. It is entirely possible, like in the case for GSDMB ( ), we are already using stimulation conditions of lesser-understood gasdermin activation but are currently unaware of the gasdermin link. Now the field has elucidated some activation conditions for the pore forming behaviours of the other gasdermins, these authors wait with anticipation for the next wave of studies with the lesser understood gasdermins. Given the exciting roles emerging in bacteriology and disease, we suspect gasdermins have a lot more to teach us including GSDMD. GSDMD’s pyroptotic potential was uncovered by two complementary studies in 2015, one using an in vivo forward genetic screen ( ) and the other an in vitro CRISPR-Cas9 screen in mouse bone marrow derived macrophages (BMDM) ( ). In both cases, GSDMD emerged as a key player in the lipopolysaccharide (LPS)-induced non-canonical inflammasome model deployed by both studies. Since, the family (except GSDMF) were shown to possess N-terminal pore-forming and C-terminal autoinhibitory functions ( ), and GSDMD specifically has been shown to be activated by other caspases, in both activation and inhibition, as well as by other proteases ( – , ). It should be noted that GSDMB can engage lipid binding in vitro , even in the presence of the C-terminal domain, indicating a possible divergent role associated with lipid interactions for full length GSDMB ( ). Like the first in vitro screen, most of the GSDMD work to date has focussed on its function in macrophages, considered to be most relevant for pyroptotic function. In addition to cellular pyroptosis, GSDMD also possess direct bactericidal activity ( ) as well as functions independent from pyroptosis. Here, we will cover those most relevant to bacterial infection contexts, with a particular focus on those that would benefit from modulation by pharmacological intervention highlighting the potential in GSDM-based therapies.
One area of particular interest is the interplay between autophagy and pyroptosis, and how GSDMD is involved to carefully balance host response to pathogenicity of invader, and how invaders inhibit these pathways to shift the balance in their favour ( ). Although covered excellently by Harvest and Miao ( ), who also provide an interesting hypothesis to legitimise the relationship between autophagy and pyroptosis, it is worth briefly covering some of the key findings in the context of this review. As covered above, GSDMD connects direct pathogen sensing to pyroptotic response. This occurs due to the exposure of bacterial LPS by IFNγ-induced guanylate-binding proteins (GBP) to create a docking site and activation platform on the bacteria for caspase-4, leading to GSDMD cleavage and activation, thereby protecting the host from pathogen replication ( ). Although incredibly effective, pyroptosis is also potentially dangerous to the host and, as such, evolution has had to consider the relevance of pyroptosis to the invading pathogen, the benefit/risk ratio in the tissue niche and the ability of virulence factors to limit weapon potency. S. flexneri , a commonly used model system as covered above, is incredibly pathogenic requiring very low numbers of invading bacteria to colonise and cause shigellosis, primarily a diarrheal condition with high prevalence in young children ( ). S. flexneri uses its Type 3 Secretion system (T3SS) and subsequent injection of virulence factors into cells to invade and infect the host. In the context of pyroptosis, S. flexneri uses Osc3C to inhibit caspase-4/11, avoiding the LPS-detection from the non-canonical inflammasome ( – ). To do this, S. flexneri deploys a covalent modification of caspase-4 and 11 via ADP-riboxination of Arg314 and Arg310, respectively, inhibiting caspase autoprocessing and pyroptotic response. In doing so, S. flexneri has exposed itself to the T3SS-detection system of NAIP/NLRC4 but S. flexneri is one step ahead, through Ipa7.8H-mediated degradation of GSDMD (and GSDMB) directly, rendering NAIP-NLRC4 without pyroptotic bite ( , , ). These virulence factors likely explain the high pathogenicity of S. flexneri whereas in the case of Burkholderia thailandensis , another Gram-negative bacteria possessing a Shigella-like T3SS, humans and mice are extraordinarily resistant to infection. This may be due to the activities of caspase-4/11 and GSDMD, with neutrophilic pyroptosis as the main tool to remove B. thailandensis infection ( , ). Interestingly, a closely related pathogen Burkholderia cepacia lacks the highly pathogenic Shigella-like T3SS but can still invade the cytosol and activate caspase-11 ( ). The cytosolic presence of B. cepacia also activates autophagy in a caspase-11/GSDMD dependent manner, leads to GSDMD-mediated mitochondrial dysfunction and enhances reactive oxygen species (ROS)-induced autophagy, and ultimately restricting the growth of the bacterium without driving pyroptosis ( – ). The balance between pyroptosis and autophagy seems quite contrasting, yet Harvest and Miao present an excellent hypothesis suggesting that autophagy directly deals with intracellular bacteria without sacrificing the host, whereas pyroptosis deals with the infection by recruitment of local phagocytes and immune components ( ). Through an intricate balance of GSDMD-mediated autophagy activation coupled with self-capture of caspase-11 and subsequent limiting of GSDMD-driven pyroptosis, the cell can carefully remove the pathogen without committing explosive suicide, with any remaining pores rescued by the repair machinery ( ). The cell can use this rheostat to respond accordingly to the level of pathogenicity in the invader. For example, B. thailandensis possess BopA, an inhibitor of autophagy. Therefore, the cell cannot sequester the activated caspase-11 via autophagy and so, in the absence of inhibitory GSDMD virulence factors, the cell commits to pyroptosis and clears the pathogen at the sacrifice of the host cell. In an opposite way, Escherichia coli outer membrane vesicles possess autophagy inhibitors so the macrophage engages the non-canonical inflammasome instead ( ). At the extreme end of pathogenicity, S. flexneri is able to inhibit caspase-11, GSDMD and autophagy, meaning the cell has limited options and thus explains the lack of innate immunity against S. flexneri . We highly recommend a detailed insight into the processes and proposed mechanisms in the focussed review for a deeper dive into the autophagy-pyroptosis axis in control of bacterial infection ( ). Bacteria deploy a wide range of pyroptosis-interfering virulence factors in an attempt to subvert innate immunity through disrupting gasdermin-mediated response. Mycobacterium tuberculosis deploys PtpB, a phospholipid phosphatase capable of dephosphorylating phosphatidylinositol-4-monophosphate and phosphatidylinositol-(4,5)-bisphosphate and as such disrupting GSDMD pore formation in the membrane ( ). This relies on PtpB to hijack ubiquitin to activate the dephosphorylation function. Mutating either the phosphatase function or the ubiquitin binding site of PtpB renders M. tuberculosis vulnerable to attack. In another attempt to subvert immunity Leptospira interrogans , the bacteria responsible for leptospirosis, possesses an atypical LPS to avoid detection by caspase-4/11 ( ). It is so different in fact that it can actively antagonise caspase-4/11 from detecting LPS from other organisms, such as E. coli ( ). Consistent with this mechanism, the authors found that IL-1R plays little role in regulating L. interrogans growth in vivo , highlighting another strategy to subvert the innate immune pathways relevant to avoiding GSDMD-mediated pyroptosis. As the field uncovers more about the detailed molecular mechanisms controlling the intricate rheostat which balances protective GSDMD-mediated responses as described above without full blown unrestrained pyroptosis, we gain the puzzle pieces that will enable the design of small molecules to selectively modulate this process and boost host defences against pathogens which aim to subvert them.
Evidence for internal rheostats to control the commitment to full pyroptosis depending on pathogenicity and cellular state is building. Recent observations in this context around metabolic control of pyroptosis, and specifically the contribution of mitochondrial ROS, are of interest in the context of bacterial infections. The Ragulator-Rag complex is known to regulate mTORC1 activation through trafficking to the lysosome and links amino acid sensing to the downstream signalling of mTOR, a major cell signalling node ( ). Two groups used CRISPR-Cas9 in vitro screening approaches to implicate Ragulator-Rag to GSDMD function and control of pyroptosis ( , ). Evavold et al. used engineered BMDMs in a forward genome wide screen to find regulators of GSDMD oligomerisation in an inducible N-terminal mediated pore formation model ( ), whereas Zheng et al. compared LPS electroporation versus LPS + TAK1 inhibitor to replicate Yersinia pestis infection, the pathogen that causes plague, in a genome wide screen ( ). In both cases subunits of the Rag-Ragulator complex were identified as screen hits. Together the studies highlight the requirement for Rag-Ragulator in GSDMD oligomerisation and pyroptotic response to Yersinia , through caspase-8. In addition, Evavold et al. found that addition of electron transport chain inhibitors enhanced GSDMD oligomerisation in a Ragulator-Rag independent fashion, and addition of N-acetyl cysteine (NAC) blocked such response, implicating mitochondrial dysfunction and specifically mitochondrial ROS. The lack of requirement for Ragulator-Rag in this model suggests the role of Ragulator-Rag might be to drive mitochondrial dysfunction and ROS production to enhance GSDMD oligomerisation and consequently pyroptosis, as has been previously observed ( ). Building on this, a recent observation elucidated cysteine 192 of murine GSDMD (cysteine 191 in human GSDMD) to be critical in sensing mitochondrial ROS to promote oligomerisation and goes some way to explain the inhibitory effect of C192-targeting disulfiram ( , ). In Zheng et al., there is also evidence of a direct engagement between Rag-Ragulator, FADD, RIPK1 and caspase-8, and suggestion that mTORC1 is not necessary for pyroptosis ( ). More evidence is available to strengthen the connection between metabolic homeostasis and gasdermin function. In the case of LRRK2 G2019S gain of function mutations, macrophages exhibit considerable mitochondrial dysfunction, mediated by GSDMD-mitochondria binding driving a switch from pyroptosis to necroptosis, mediated by RIPK1/RIPK3/MLKL ( ). What isn’t clear is why GSDMD preferentially selects mitochondrial membranes in this scenario, but it is known that LRRK mutations drive changes in the mitochondrial structure, dynamics and ER-tethering as well as lipid dysfunction, which may increase the possibility of GSDMD-mitochondrial binding over the plasma membrane ( – ). Relevant to this review though is the consequence on infection. Particularly interesting is that this mechanism is evolutionary conserved as the authors show both LRRK2 G2019S expressing flies infected with Pseudomonas entomophila and LRRK2 G2019S mice infected with Mycobacterium tuberculosis exhibit hyperinflammation, higher bacterial burden and enhanced immunopathology ( ). This mitochondrial ROS-GSDMD axis is also seen to be important in surface expression of the synovial T cell receptor-γδ ligand on CD14 + CD16 + monocytes, in conjunction with toll-like receptor (TLR) stimulation, mitochondrial metabolism and inflammasome activation in a redox sensitive manner ( ). This is important in regulating the response of γδ T cells in infection and autoimmunity, where overactive GSDMD may contribute to overexpression of surface γδ ligand, driving aberrant adaptive immune response, and targeting GSDMD at the mitochondrial ROS-GSDMD axis in inflammatory monocytes may provide a useful approach. Indeed, targeting GSDMD in any of the above scenarios may provide an alternative approach to targeting metabolism or mitochondrial dysfunction which have their challenges as pharmacological platforms. The opposite is also true whereby modulating autophagy, metabolism or redox balance is an opportunity to control GSDMD function, and ultimately pyroptotic response. The authors await studies that further detail the molecular processes and biophysical properties that control GSDMD function to selectively modulate these processes with knowledge-based drug design.
As above, examples of host cells dialling back pyroptosis to limit cellular damage whilst ensuring a robust immune response are clear. These observations highlight the need for cells, particularly at barrier sites, to flag the invasion to neighbouring immune cells without compromising barrier integrity and exacerbating any pathology. Although not bacterial, Entamoeba histolytic implicates a similar inflammasome-gasdermin response in the gut upon infection ( ). Most typically E. histolytica infects the gut of a host while the host remains asymptomatic. However, in some individuals the pathogen can break through the gut and drive inflammation. Due to its size, E. histolytica is detected by macrophages through a Gal-lectin surface adhesin, which activates the NLRP3 response and recruits the immune system via caspase-4 and GSDMD, potentiated by ROS and K + efflux ( ). In addition, E. histolytica infected macrophages downregulate NINJ1, a key cell lysis regulatory protein, coupled with processing of GSDMD and IL-1β via caspase-1 and caspase-4 ( , ). This culminates in a hyperactivated macrophage, with active GSDMD pores and cytokine release, but no cell lysis. It is not clear what mechanisms control NINJ1 regulation in this context but understanding this may provide opportunities to control damaging NINJ1-mediate cell lysis driven pathology associated with over-inflammation such as sepsis. Controlling GSDMD contribution to pyroptosis is important in the case of Streptococcal infections . Streptococci are a Gram-positive, typically diplococci bacterial genus that exist mostly symbiotically with hosts, except a number of strains that are major causes of infections ( ). Pathogenic Streptococci cause mild to severe disease ranging from strep throat to meningitis and pneumonia and are a major socioeconomic burden globally. The emergence of antimicrobial resistant strains has accelerated the need for new therapies in this space to tackle associated pathology, such as sepsis. Although GSDMD has been linked to protecting against some Streptococci infections ( ), failure to control infection and the subsequent inflammation, upon which gasdermins play a critical role, can lead to sepsis and death. GSDMD has been linked to accelerating sepsis by providing a conduit for passive release of both SQSTM1, a regulator of innate immunity, and F3, a blood coagulation initiator downstream of LPS or STING activation, respectively ( , ). In both cases, pharmacological interference of the mediating pathways was beneficial, highlighting the potential for GSDMD as a legitimate target in this therapeutic space. There are also examples of evolution-directed mechanisms to limit gasdermin function in Streptococci infection. As will be covered in more detail later, recent seminal studies have uncovered a protective function of keratinocyte GSDMA in response to Group A Streptococcal protease SpeB ( , ) (see GSDMA section below). As for GSDMD and GSDME, the cytokine IL-6 has been proposed to have a protective role in lung pneumococcal infections ( ). In this case, IL-6 exhibited an inhibitory effect on GSDMD and GSDME mediated pyroptosis in lung macrophages, preventing cell death and lung injury, through a post-translational modification somewhere along the cascade. It is not clear from this study what the specific mechanism of IL-6 is, nor whether it is acting on GSDMD/E specifically, but the finding suggests a mechanism exists by which host cytokine balance can control pyroptotic flux to manage inflammation and tissue damage. Appreciating this in the context of disease and cytokine dysregulation would be insightful, and defining the specific cytokine-induced modifications could provide a novel approach to control GSDMD/E activity. In another case of evolved protection against overactive permeable membrane-cell death, Nozaki and Maltez et al. elucidated a role for caspase-7 in eliciting membrane repair in the face of both perforin and gasdermin pore formation ( ). Traditionally, caspase-7 was seen to be an ineffective secondary to caspase-3, with no independent function except an intriguing ability to be activated by caspase-1 ( ). In this study, the authors noticed correlation between cleaved caspase-7 and the ability of intestinal epithelial cells to extrude upon infection with Salmonella in vivo . Without caspase-7, these intestinal cells could not extrude and instead were dysfunctional in morphology and function, and in Casp7 -/- organoids GSDMD pore formation occurred much faster than wildtype. Mechanistically, the acid sphingomyelinase (ASM)-driven endocytosis membrane repair process is regulated by caspase-7, driving ceramide production and therefore membrane repair, limiting GSDMD driven cell lysis. Fascinatingly, it was lysosomal GSDMD pores that allows conduit of caspase-7 to ASM, which then generates ceramide and provides a membrane repair process against the forming GSDMD pores. The authors propose caspase-7 evolved to protect against perforin pores, and they prove this in CTL and NK-driven clearance of Listeria infections, as well as Salmonella and Chromobacterium violaceum in IECs and hepatocytes, respectively. However, caspase-7 can also protect against GSDMD pores using this same mechanism. It would be interesting to see whether that mechanism exists across other gasdermins as well. This mechanism provides a method to actively kill invading bacteria, whilst maintaining structural integrity, and therefore limiting inflammation to the invaded cell and not the wider tissue. Thus, a potential application in the context of disease would be realized by acutely enhancing caspase-7 activity in IEC during infection.
Neutrophils are a key player in the fight against invading pathogens. One of the key weapons they possess is the ability to deploy neutrophil extracellular traps (NETs), via a process called NETosis ( ). Particularly effective against large pathogens, NETs are chromatin based structures that act to trap their prey before killing and, although particularly effective, if dysregulated can contribute to immunopathology ( ). Both human and murine neutrophils possess elevated levels of GSDMD and GSDME, and two complementary papers originally highlighted GSDMD in the generation of NETs ( , , ). Chen et al. show that cytosolic LPS or invasion by Salmonella or Citrobacter rodentium activates GSDMD-mediated NETosis via caspase-4/11 non-canonical inflammasome signalling ( ), whereas Sollberger and colleagues used a small molecule screen to discover LDC7559, a pyrazolo-oxazepine type compound which inhibits PMA-induced NETosis, mediated by neutrophil elastase ( ). Beyond this, there is also evolutionary evidence that GSDME drives pyroptosis-mediated NET formation in zebrafish, and that GSDMD-mediated NET formation is negatively associated with outcome in severe COVID-19 ( , ). These findings may implicate a role for GSDMD in the formation and propagation of NETs as an immune strategy within the conditions evaluated. However, very recent observations have called into question the role of GSDMD in NET formation, or more accurately the involvement of GSDMD at distinct stages and contexts of NET formation and NETosis ( – ). Firstly, Chauhan et al. show that GSDMD is indispensable for NETosis induced by PMA, but is important in canonical inflammasome activation of neutrophil pyroptosis ( ). This is contrary to observations seen by Sollberger et al. ( ). However, since it was discovered, LDC7559 is now known to bind and activate phosphofructokinase, liver (PFKL) and not GSDMD ( ). In doing so, LDC7559 actually blocks NADPH oxidase-driven ROS formation and suppresses phagocytic function, including NETosis in a GSDMD-independent fashion ( ). Going even further, a recent study from Stojkov et al. claims that NET formation is completely independent of GSDMD and pyroptosis ( ). In this study, Stojkov and colleagues test C5a and LPS, both known NET inducers in primed neutrophils, as well as transfected LPS as a non-canonical inflammasome activator and LPS followed by nigericin as an established canonical inflammasome activator. In no scenario did Gsdmd -/- neutrophils form less NETs than wildtype equivalents, asking questions about GSDMD involvement in neutrophil NET formation entirely ( ). An interesting observation in neutrophils was that optimal production of IL-1β requires GSDMD, surprisingly in the absence of plasma-membrane pore formation and subsequent pyroptosis. This data suggests that whilst NETosis may not require GSDMD, the associated inflammatory conditions are mediated by GSDMD in neutrophils. The role of GSDMD during infection is likely cell type and pathogen specific, highlighted well in Pseudomonas aeruginosa lung infection models ( ). In this case, LPS from E. coli , Klebsiella pneumoniae and P. aeruginosa was evaluated for NET formation in Clec5a -/- , Tlr2 -/- and Tlr4 -/- neutrophils. NET formation mediated by P. aeruginosa LPS was abrogated only when CLEC5a was removed, yet LPS from E. coli and K. pneumoniae still had effect. Taking this further the authors were able to show a GSDMD-dependent release of cytokine in alveolar macrophages, yet GSDMD was dispensable in neutrophils for caspase-1 driven NET formation. However, it is not clear whether GSDME could compensate in this context. Additionally, P. aeruginosa triggered caspase-1 in both human and mouse neutrophils, which was limited by bacterial exotoxins, in a flagellin-mediated manner ( ). Here, NLRC4 was activated in the neutrophils, activating caspase-1 and GSDMD, triggering calcium and peptidyl arginine deiminase 4 (PADI4)-mediated histone citrullination, trafficking of neutrophil DNA into the cytoplasm, but without committing to NET formation. Overall, there is contrasting data in the context of neutrophil NET formation and GSDMD involvement and more clarity is needed to advise on targeting GSDMD in neutrophil-mediated pathology. Caution should be taken around the nomenclature used to define NET formation and whether NETosis has been induced with or without cell death taking place ( ). In addition, understanding the effect of NET inducers and their global effect on the neutrophil is important.
GSDMA is primarily expressed in the upper gastrointestinal tract and the skin ( ). Associations of GSDMA with asthma and respiratory pathology largely dominated the early literature on this gasdermin ( – ). Early work to assess the function of GSDMA focused on mitochondrial homeostasis ( ). The N-terminal functional domain can regulate mitochondrial homeostasis, specifically via mitochondrial oxidative stress and Hsp90/Trap1/Tom70 axis, culminating in mitochondrial permeability transition pore (mPTP) opening and mitochondrial collapse. This response is alleviated by the presence of the autoinhibitory GSDMA C-terminal domain, or pharmacological intervention at the points of ROS, mPTP activation or mitochondrial translocation, and an independent study showed GSDMA has a preference for mitochondrial membranes over plasma membranes ( ). Using overexpressed N-terminal GSDMD and GSDMA, there was a large differential between the two with regards to pyroptotic response, measured by propidium iodide and lactate dehydrogenase release assays. To address the caveats that come with overexpression of N-terminal fractions, a chimeric GSDMA-GSDMD protein was generated to enable cleavage by GSDMD activators caspase-1 or 4/5 to release an N-terminal GSDMA fragment. Relative to GSDMD, activated GSDMA N-terminal fragments exhibited delayed cell death, despite being cleaved at similar kinetic rates. There was also a clear abundance of N-terminal GSDMA in the mitochondrial fraction as tracked using a NEON-tag and microscopy, as well as functional data showing mitochondrial dysfunction upon N-GSDMA deposition. These observations are of course intriguing and shed light on the divergence of different gasdermin family members in driving cellular/subcellular consequence, even in a somewhat artificial, but informative, system. Specifically, an endogenous activator of GSDMA has yet to be discovered and so the intracellular activation of GSDMA and the context in which it occurs remains to be elucidated. Another consideration is the bacterial-origin of the mitochondria and what this means for direct GSDMA-bacterial interactions. Understanding a key activator of GSDMA endogenous to cells would be important in both the field of mitochondrial biology and immunology. Despite the endogenous activator of GSDMA remaining elusive, there has been progress in elucidating exogenous activators of GSDMA. Very recently, two seminal papers have linked GSDMA activation to an exogenous factor, SpeB from Streptococcus ( , ). In keratinocytes, which express elevated levels of GSDMA, the presence of SpeB, a Group A Streptococcus cysteine protease virulence factor, can drive GSDMA mediated pyroptosis. The two studies came to this conclusion from different perspectives yet concluded on the same observation. The LaRock et al. study assessed the cleavage ability of SpeB with all GSDM family members, based on the hypothesis that SpeB can cleave IL-1β, and observed cleaved flag-tagged GSDMA, GSDMC and GSDMD in HEK293 cells ( ). Given the skin-site expression of GSDMA in relation to Streptococcus infection, this group decided to focus on GSDMA and observed GSDMA can defend against Streptococcus mediated skin infections in mice through SpeB-driven GSDMA-mediated keratinocyte pyroptosis. Revisiting GSDMC in this context may also be of future interest. Alternatively, Deng et al. focussed on the fact SpeB drives epithelial pyroptosis and sought to understand the key mediators of the model ( ). A genome wide CRISPR screen in A431 keratinocytes was performed to further understand the role of GSDMA. A431 cells were electroporated with SpeB, cells enriched in GSDMA sgRNAs were resistance to SpeB-dependent pyroptosis. This was confirmed with follow up studies showing the cleavage site at Gln246 and a preference for acidic lipid membranes ( ). Both studies then highlight the sensitivity of GSDMA null mouse models to Group A Streptococcus infections. LaRock et al. used a Gsdma1 -/- / Gsdma2 -/- / Gsdma3 -/- model whereas Deng et al. focussed on Gsdma1 -/- only due to the fact only Gsdma1 and 3 are expressed in the skin, and Gsdma3 does not possess the Gln246 cleavage site. Neither study addresses the mitochondrial observations (discussed above), but this would be of interest in the future, especially given the delayed pyroptotic response compared to GSDMD activators, and relationship between mitochondrial function and immunity. Overall, early observations of genetic links to disease and tendencies to engage mitochondrial membranes give GSDMA a unique and divergent element relative to GSDMD. Exploiting the unique tendencies such as enhanced mitochondrial membrane engagement may allow preferential targeting of gasdermins to trigger protective host defense. The direct cleavage by an exogenous bacterial protease has shed some light onto the potential anti-bacterial function of GSDMA. Whether an endogenous activator can also drive GSDMA functionality with regards to its N-terminal pore forming domain remains to be seen. However, given the genetic link to disease and divergent functions compared to GSDMD, this would be of considerable interest and a possible therapeutic target in those associated pathologies.
GSDMB is hypothesized to have arisen from a gene duplication event in the 17q12-21 loci, at the evolutionary level of the synapsid clade with resulting presence of GSDMB in humans and bovine species but not mouse, rat or other non-mammals ( ). Before the pore-forming function of gasdermins was discovered, and similar to GSDMA, GSDMB was genetically linked to asthma, ulcerative colitis, Crohn’s disease, and rheumatoid arthritis ( , – ). Interestingly, the role of GSDMB in respiratory pathology may also come from an unknown regulatory role via arachidonate 5-lipoxygenase (5-LO) and transforming growth factor-β which isn’t related to inducing inflammation or pyroptosis ( ), or from the alteration in GSDMB structure caused by disease-associated SNPs ( ). A recent study focussing on the inflammatory bowel disease (IBD) role of GSDMB showed that GSDMB is able to regulate epithelial cell proliferation and migration, enhancing the repair process independent of pyroptosis ( ). Mechanistically this is linked to a pyroptosis-independent function mediated by GSDMB-dependent enhanced adhesion through focal adhesion kinase (FAK) phosphorylation of platelet-derived growth factor subunit A (PDGFA). This in turn drives vinculin focal adhesion, which is disrupted in disease associated GSDMB SNPs. Outside of inflammatory diseases there is a well-established literature base for the role of GSDMB expression changes and in promoting invasion and metastatic potential in cancer ( – ). GSDMB shares the typical gasdermin family domains of a pore-forming domain, linker region and autoinhibitory C-terminal section, and can partake in N-terminal driven pyroptosis ( ). Uniquely GSDMB prefers to bind phosphoinositides and sulfatides unlike other gasdermin family members ( ). There is evidence for endogenous activators of GSDMB, namely caspase-1, 3, 6 and 7 ( ), but more recently exogenous activators and interactors relevant to bacterial infection and immunity have emerged. Granzyme A originating from cytotoxic lymphocytes is able to cleave GSDMB at Lys244 ( ). Specifically, GSDMB expression in a HEK293 system was the only gasdermin able to induce NK-mediated cell killing. Mechanistically, the perforin-granzyme mechanism is critical for the induction of cell death in this context, specifically Granzyme A. Low or undetectable levels of GSDMB can be upregulated by IFNγ in a number of cell lines, making them sensitive to pyroptosis ( ). Critically, Granzyme A was able to cleave GSDMB in vivo with enhanced tumour clearance, highlighting the likely reason many cancers downregulate GSDMB in an oncogenic fashion ( , ). Considering the role of GSDMB in bacterial infection, recent studies provide evidence that bacteria have evolved mechanisms to shut down this Granzyme A driven mechanism of pyroptosis for immune evasion ( , ). Much like the tumour example described above, Granzyme A-mediated killing of infected cells is an effective mechanism to remove host cells harbouring pathogens, and it is likely GSDMB plays a key role in driving pyroptosis and/or has direct bactericidal activity to achieve this. Interestingly, S. flexneri has developed a T3SS-type virulence factor termed IpaH7.8, which has E3 ligase capabilities ( , ). IpaH7.8 can actively hijack the host ubiquitin system to ubiquitinate GSDMB marking GSDMB for degradation via the proteosome, which also happens to GSDMD ( ). This renders the Granzyme A mediated clearance process ineffective, promoting bacterial survival. GSDMB was shown to directly attack the bacteria itself, due to its preference for alternative lipids, and avoid pyroptosis of the whole cell, thereby protecting the epithelial barrier. GSDMD is also affected by IpaH7.8 mediated tagging ( , ). This is intriguing given that S. flexneri had been shown to induce caspase-1 mediated pyroptosis in macrophages previously ( ). This is explained by the fact the binding affinity of Ipa7.8H is lower for GSDMD than for GSDMB. If GSDMD is engineered based on structural interaction studies to resemble GSDMB, this restores the high binding affinity ( ). It is likely that cell-specific contexts play an important role in the consequence of Shigella and Ipa7.8H effectiveness, for example whilst S. flexneri causes caspase-1-mediated pyroptosis in a macrophage, in epithelial systems Ipa7.8H was shown to target GSDMD and tag it for degradation ( ). Gasdermins are not the only targets of Ipa7.8H and altogether these mechanisms are deployed by Shigella to avoid cell lysis and loss of the replicating niche and promote bacterial survival. Expansion of these findings to other bacterial strains with E3 ligase-like virulence factors and other gasdermins at infection sites may provide wider insight into the battle between bug and host at the gasdermin level with clinical interest and provide insight to enable drug design to either prevent or promote gasdermin degradation depending on the pathological context.
GSDMC was first uncovered in the early 2000s in metastatic melanoma and referred to as MLZE ( ) and then recognised as a gasdermin and renamed in 2007 as GSDMC ( ). GSDMC has a leucine zipper region in the C-terminal domain, which is not found in other gasdermins, suggesting a DNA binding function. Other functions of GSDMC have been proposed, including evidence that gene manipulation of GSDMC limited or exacerbated cell proliferation in colorectal cancer cell lines upon siRNA or overexpression, respectively ( ). With regards to activating cascades and consequences, there is now a body of evidence suggesting that TNF-activated caspase-8 can cleave GSDMC and switch the cell from apoptosis to pyroptosis. This occurred in the context of hypoxia under the driver of PD-L1, which upregulated GSDMC expression, with the TNF generated from tumour associated macrophages ( ). Intriguingly however, there are mixed observations on the role of GSDMC in tumour biology. In some cases, enhanced expression of GSDMC promotes tumour aggression and growth, suggesting the pyroptotic function of GSDMC is not a critical determinant of its ability to influence tumour homeostasis. To further complicate the role of GSDMC in tumour biology, α-ketoglutarate (KG) has been shown to drive caspase-8 mediated activation of GSDMC and subsequent pyroptosis in cancer cell lines and mouse models. Here, GSDMC expression was correlated to a reduction in tumour growth and volume, suggesting α-KG treatment shifts the GSDM from a pro-tumoral role to an anti-tumour pyroptotic function ( ). In terms of the gasdermins, GSDMC is the least well understood in the context of bacterial pathogenesis. There are some data suggesting GSDMC may play a role in skin immunity. GSDMC has been shown to play a role in MMP-1 expression in skin keratinocytes in the context of UV light exposure ( ). While MMP and ECM proteins can be exploited by pathogens to promote invasion and infection (as reviewed in ( )) it is not clear if pathogens can exploit GSDMC to modulate MMP-1 expression in the skin. There is also evidence that the intravenous delivery of an immunotherapy based on Listeria monocytogenes encapsulated in red blood cell membrane was able to drive GSDMC-mediated pyroptosis in the tumour, successfully dampening the suppressive environment of the tumour ( ). Although further work needs to be done to understand the key drivers of these processes, this is an interesting concept of exploiting bacteria-gasdermin interactions in tumour therapy, as has been covered elsewhere ( ). Outside of bacteriology, there are studies concerning the role of GSDMC in response to helminth infection, which suggests a role for GSDMC in gut immunity. Here, IL-4 and IL-13 induced typical type 2 responses as expected, but also induced the upregulation of GSDMC in gut organoid models ( ). Moving to an in vivo Nippostrongylus infection model, there is enhanced lytic cell death and evidence of a p30 band associated with activated GSDMC in enterocytes. In a similar vein, Zhao et al. show that downstream of helminth infection the release of IL-33, an important alarmin for inducing type 2 responses, was GSDMC dependent in gut epithelium, and was induced by the O-linked N-acetylglucosamine modification of STAT6 ( ). Together this evidence puts GSDMC central to anti-helminth immune response and controlling Type 2 immune response. Furthering our understanding of GSDMC’s role in gut inflammation, both infection or autoimmunity driven may provide novel approaches to gut pathology and insight enabling strategies to target GSDMC in disease ( , ).
DFNA5, or now more commonly known as GSDME, is another pore forming family member of the gasdermin family, but with marked divergence in expression patterns ( ). Unlike its cousins, GSDME is expressed in the heart, brain, placenta, and kidney. Evolutionary speaking GSDME is well conserved and is more aligned to its non-pyroptotic partner PJVK and has homologs in coral and teleost species, where it has a clear anti-bactericidal activity ( – ). Before the emergence of the pyroptotic function of gasdermins, GSDME was linked to a hearing loss disorder associated with a mutation that results in a lack of the C-terminal domain ( , ). There are also relationships between GSDME, p53 and cancer progression, as well as methylation of the GSDME gene that influences metastasis in breast cancer and p53 stabilisation in colorectal cancer ( – ). More recent evidence has also implicated GSDME in photoreceptor degeneration, IBD, RA and virus-induced pathology ( – ). Activation of GSDME is mediated by caspase-3 during apoptosis, and in response to chemotherapeutic agents, virus or death receptor driven apoptotic signalling ( , ). Intriguingly, the rheostat for cell committal to apoptosis or caspase-3 mediated-pyroptosis seems to be determined by the cellular expression of GSDME. Depending on the level of GSDME the cell will either commit to apoptosis driven by activation of caspases as per usual, or if given the opportunity with an abundance of substrate, caspase-3 will drive GSDME-mediated pyroptosis, or as traditionally known secondary necrosis. In GSDMD null cell systems, caspase-4/5 driven non-canonical inflammasome signals are completely abrogated; however, in canonical inflammasome signalling, GSDME can act as a back-up in the absence of GSDMD, largely through the recruitment of caspase-8 (and subsequent activation of caspase-3) by ASC, or activation of caspase-1 by caspase-3 ( ). This balance does seem to rely somewhat on the expression level of GSDME, and likely other contributing factors such as caspases and NINJ1, as to whether there is a built-in redundancy to the system ( , , ). In the context of bacterial pathology, there are examples of GSDME influencing bacterial invasion and survival. As above, this depends on the expression profile of the infected cell, and it would be interesting to understand the balance of GSDMD versus GSDME expression patterns relative to likelihood of encountering a pathogen. Interestingly, there is some evidence that GSDME function is dependent on expression level that controls sublytic cytokine release versus full lytic death response ( ). There is a dependence on the caspase-1/3/8 driven activation of GSDME in the context of NLRP3 activation or, in Salmonella infection, through NLRC4 ( ). In another example, the Gram-negative bacterium Brucella presents lipoproteins that initiate unique cascades, of which only one terminated in GSDME activation ( ). The two major lipoproteins, L16 and L19, both induce typical pro-inflammatory cytokine release but only L19 can induce IL-18 secretion. Upon further investigation L16 could only drive pro-IL-18 production, rendered so by an upregulation of caspase-3/8. Whilst L19 could cause phosphorylation of X-linked inhibitor of apoptosis (XIAP), thereby inhibiting caspase-3 function, L16 could not and instead activated caspase-3, GSDME formation and pyroptosis of the Thp1 cells used in this model ( ). In addition to immune cells, GSDME also plays a role in colonic epithelial cells in response to Campylobacter jejuni , the bacteria responsible for the majority of foodborne gastroenteritis globally ( ). One of the key virulence factors involved in C. jejuni infection is cytolethal descending toxin (CDT), which is also present in several other bacteria strains including E. coli and Helicobacter pylori ( ). CDT can drive a number of cell-specific responses including caspase-9/3 activation in colonic epithelial cells, which subsequently activates GSDME to drive cell death, with dependency on oxidative stress. This observation was independent to caspase-8 and caspase-1 and, given the role of caspase-9 and ROS as shown by addition of NAC in abrogating the effect, it would be interesting to understand the role of GSDME in release of cytochrome c from the mitochondria in this setting, to activate the apoptasome and caspase-9. GSDME is also involved in periodontitis in response to Fusobacterium nucleatum , a common oral bacterium ( ). In this case, F. nucleatum was able to skew macrophages to a proinflammatory state, accompanied by elevated levels of ZBP1 and GSDME-mediated pyroptosis, which was reduced by shutting down ZBP1. Interestingly, understanding the key regulatory mechanisms of GSDME and associated cellular death is important in deciphering host-pathogen relationships in common inflammatory conditions ( ) as well as in providing the relevant information to enable therapeutic targeting of GSDME in these conditions. Albeit not bacteria, a recent study highlighting GSDME in combating Candida infection is worth noting for the involvement of a subset of Th17 cells ( ). In this case, these Th17 cells can utilise NLRP3-caspase-8-caspase-3 to drive IL-1α production and secretion, which is dependent on GSDME. GSDME transcript is upregulated in response to Th17 stimuli (TGF-β and IL-1β) but not Th12 (Th1) or Th4 (Th2), and GSDME possesses binding sites for Th17-associated RORα and BATF, whereas GSDMD was not influenced by any T cell cytokines. Much like the above examples, Th17 cells do not progress to full pyroptosis and instead sit at a sublytic level driving GSDME-mediated release of IL-1α. Critically however, this function of Th17s is fundamental for their ability to protect against fungal pathogens, namely Candida , yet Staphylococcus nor polyclonal TCR activation drove GSDME-mediated IL-1α production. Whether this is an intentional restriction to this clone, or a caveat of the system used is unclear, but it will be interesting to see whether GSDME-mediated release of IL-1α and other cytokines in additional model systems plays an important physiological role via similar cell types. Th17 cells have a well appreciated pathogenic role in many autoimmune diseases so understanding this further will also be important for defining the role of GSDME in Th17-driven pathology.
PJVK is the only gasdermin family member lacking a N-terminal domain and so cannot induce N-terminal pyroptosis, at least in the systems tested ( ). There are links again to hearing disorders in humans and equivalent phenotypes in Gsdmf -/- mice ( – ), but the relevance of PJVK to cell death and immunity is unclear. Saying this, there is considerable similarity between PJVK and GSDME, albeit without the pore forming ability. Despite the similarity, one major difference in PJVK compared to other gasdermins is the presence of a small C-germinal zinc-finger domain with no known role or function. Given the divergence of GSDMF from its relatives it will be interesting to see whether this gene retains any similar functions to other gasdermins away from pyroptosis, or instead has taken on a new function of its own.
The development of tool molecules is required early in target validation to probe the function of a protein and provide insights into its mechanistic and phenotypic roles in biology and disease phenotypes. Tool molecules are generally potent, selective small molecules that demonstrate cellular target engagement and have a clearly defined mechanism of action (MoA) ( – ). To ensure all these criteria are met, tool compounds must be quality controlled and thoroughly profiled. If they are not, observed phenotypes cannot be confidently linked to the biological target, leading to spurious conclusions. Our knowledge of the GSDM family of proteins and their roles in various pathological pathways, whilst expanding, is hampered by an absence of high-quality tools. Efforts into the discovery of tool compounds for the GSDM protein family have primarily focussed around pyroptotic executor protein GSDMD, due to the reasons explained above in assay development and clear cellular endpoints. Going forward, utilising the more recent observations in other gasdermin family members may provide new avenues of molecule discovery. The inhibition of GSDMD, alike the other members of the GSDM family, could occur via numerous mechanisms. However, it was Liu et al. that discovered the key role C191 plays in N-terminal (NT) GSDMD oligomerisation and subsequent ability to form pores in cell membranes ( ). While investigating NT-oligomerisation, an immunoblot study was carried and NT-GSDMD was placed under reducing and non-reducing conditions. The monomeric and oligomeric forms were observed under non-reducing conditions and only the monomeric form under reducing conditions. This suggested that NT-oligomerisation involved disulphide cross-linking. Following these results, individual mutagenesis of all cysteine residues to alanine on murine GSDMD revealed that C39A and C192A impaired GSDMD oligomerisation. These residues correspond to human C38 and C191. This work uncovered a characteristic of GSDMD that could be leveraged to provide a viable strategy to target inhibition of GSDMD pore formation. Furthermore, amongst the GSDM family members this characteristic has only been reported for GSDMD, providing an opportunity to selectively engage one GSDM protein while sparing other family members. This is important in the context of findings from Huang et al. that implicate GSDMA in proper epidermal differentiation and cornification in skin barrier maintenance ( ). As a result of research by Liu et al. numerous tools were subsequently reported to prevent GSDMD pore formation via the covalent modification of C191 ( , , ). Necrosulfonamide (NSA) ( ) was the first of these ( ). NSA had previously been identified as a MLKL inhibitor, a necroptosis mediator protein ( ). The compound acted by modifying C86 of human MLKL, preventing MLKL polymer formation and subsequent necrotic cell death. Rathkey et al. hypothesised that NSA may act in a comparable manner with GSDMD ( ). NSA was shown to prevent LPS-induced pyroptosis in vitro , and LPS-induced sepsis in vivo . NSA did not impair cleavage of the full-length (FL)-GSDMD to the NT, instead, a western blot study showed that NSA impaired NT-oligomerisation. GSDMD inhibition was believed to be via direct binding to C191, as reported through precipitation experiments between NSA and C191A-mutated GSDMD. While these experiments indicated that NSA prevented pyroptosis via covalent modification of GSDMD, useful mass-spectrometry (MS) based experiments would have installed further confidence and confirmed the proposed MoA. Furthermore, later studies revealed that NSA inhibited the activation of inflammasome NLRP3, caspase-1, and IL-1β, key components of the pyroptotic pathway ( ). Concurrently, Hu et al. identified GSDMD inhibitors disulfiram and Bay 11-7082 ( ) in a small-molecule high-throughput screening (HTS) liposome leakage assay ( , ). Disulfiram was the most potent compound identified and 22-fold more potent than Bay 11-7082 with an IC 50 of 0.30 ± 0.01 µM in comparison to 6.88 ± 0.10 µM. NSA had also been screened in the liposome leakage assay and was the least potent of the three covalent binders with an IC 50 of 9.28 µM ± 0.70 µM. To identify the site of covalent modification, disulfiram and Bay 11-7082 were incubated with FL-GSDMD. Following trypsin digest and MS analysis, C191 was identified as the major site of covalent modification. Both compounds were confirmed to inhibit pyroptosis in cellular studies where LPS-primed cells were treated with the compounds and cell death was observed by CyTox98 assay ( , ). However, disulfiram and Bay-11-7082 have been shown to also inhibit inflammatory caspases through non-specific binding to their catalytic cysteine residues ( , ). Furthermore, Bay 11-7082 is a widely reported NLRP3 inflammasome inhibitor ( ). More recently, while investigating the effect of Kreb’s cycle intermediates on pyroptosis, Humphries et al. reported a new covalent GSDMD inhibitor, dimethyl fumarate (DMF) ( ) ( ). LPS-induced pyroptosis was recovered upon treatment with DMF in vitro , this was also observed in vivo whereby treatment of DMF protected mice from LPS-induced shock. The small molecule, which succinates GSDMD cysteine residues, was shown to inhibit cleavage of FL-GSDMD, thereby preventing pore-formation, a notably different MoA to disulfiram and NSA. Incubation of DMF with purified FL-GSDMD, followed by trypsin digest, revealed that DMF covalently modified five cysteine residues, C56, C191, C268, C309, C467, where C191 was the major modified residue. It is worth noting that the covalent modification of C191 had not previously been linked to the inhibition of FL-GSDMD cleavage, solely the prevention of NT oligomerisation. It is therefore possible that the MoA observed is the product of covalent modification of one of the remaining cysteine residues, such as C268, a residue located in the flexible linker region of FL-GSDMD in proximity to the site of caspase cleavage. Nonetheless it would be interesting to investigate whether DMF also impaired the oligomerisation of NT-GSDMD, utilising the non-reducing western blot conditions described by Rathkey and Hu et al. for disulfiram and NSA ( , ). It is also worth highlighting that the multiple covalent binding events observed is indicative of non-specific binding to GSDMD, with covalent modification being driven by intrinsic reactivity, and as such, promiscuity is to be expected. DMF was also included in an ABPP-proteomics experiment carried out by the Cravatt Lab ( ). DMF did not significantly enrich any GSDMD peptides, demonstrating limited cellular engagement with GSDMD. The in vitro and in vivo profile described by Humphries et al. is therefore likely driven by interactions with several additional proteins in the pyroptotic cascade. Finally, as observed with the aforementioned covalent tools, LDC7559 ( ) was also initially reported to prevent NETosis by reversibly inhibiting GSDMD, as covered above ( ). This was disproven in various cellular and recombinant protein assays by Amara et al., whereby LDC7759 was unable to recover LPS induced pyroptosis, in addition to failing to prevent GSDMD pore formation in a liposome leakage assay ( ). Numerous covalent and reversible tools for GSDMD have been reported in the literature. Whilst initial investigation into these tools appeared promising, characterisation was insufficient. Further studies reported a lack of selectivity to GSDMD, with numerous off-targets within the pyroptotic pathway described. Furthermore, the exact manner in which GSDMD is inhibited was poorly understood. As previously described, both factors are key for instilling confidence in a tool and any observed phenotypic changes. As such, higher quality tools for GSDMD investigation, and the wider GSDM family, are needed. The findings highlighted above noting C191 as a key residue in regulating GSDMD-mediated pyroptosis provide important groundwork in directing the discovery of more selective GSDMD tool inhibitors using reactive compound libraries. Moving forward, compounds that are found to engage a GSDM protein must therefore be fully characterised to ensure the in vivo profile aligns with the in vitro and recombinant profile. Western-blot studies have proven useful to investigate potential MoA, with antibodies available for all GSDM proteins. The GSDMD liposomal leakage assay reported by Hu et al. should be transferable to the broader GSDM proteins and is another powerful tool to assess the efficacy and potency of GSDM inhibitors ( ). Where covalent tools are investigated, mass-spectrometry workflows should be utilised to characterise covalent binding to the GSDM protein of interest, and the broader proteome using chemoproteomics in a cellular setting. It is worth noting that tool optimisation and discovery is limited by the absence of available crystallography for these pore-forming proteins. Indeed, due to the disordered linker region, entirely resolved crystal structures have thus far not been obtained. GSDMD C-terminal and a partial FL-GSDMD is currently accessible, however the linker region which contains the key C191 residue is absent. As such, the discovery and optimisation of tool compounds will need to be ligand-driven and through screening by HTS and fragment screening until further structures are defined. We await additional molecular and biophysical findings as discussed throughout this review that will allow novel more specific strategies to target the various GSDM in host defence and immune-mediated diseases.
Since 2015 the gasdermin protein family have been front and centre for innate immunological research. As the field expanded beyond the discovery of GSDMD in pyroptosis, we have witnessed new influencers of GSDMD biology, alternative roles for gasdermins beyond that of pore formation, how GSDMA, GSDMB, GSDMC and GSDME are modulated and, most importantly, how gasdermins help protect us against pathogen invasion, and how pathogens have fought back via evolution. Furthering our insights into the pathogen-gasdermin relationship will be crucial to appreciating where gasdermin-based therapies could be efficacious. In the context of bacterial pathogens which evade detection by blocking inflammasomes, transient enhancement of specific protective GSDMD-mediated functions such as appropriate mitochondrial targeting may be beneficial. In contrast, immune-mediated diseases which are augmented by GSDMD activity such as sepsis and Familial Mediterranean Fever (FMF) may benefit from blockade of GSDMD-mediated pore formation. Interestingly, recent work from Jorch et al. demonstrated GSDMD-dependent secretion of alarmins driving autoinflammation in FMF which further highlights the need for GSDMD inhibitors ( ). As gasdermins gather more traction and undoubtedly become even more integral to several immunological processes, the need for pharmacological tools as well as inhibitors for clinical use will become critical. We look forward to developments in this area as researchers overcome hurdles associated with gasdermin biology and assess how effective modulating gasdermin biology is in multiple indications. While we have journeyed far in expanding our understanding and appreciation of GSDMs in host defence and disease, the endgame is not yet near.
CG, MW-D, AB and LB collectively researched and wrote this article. All authors contributed to the article and approved the submitted version.
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Initial characteristics and course of disease in patients with suspected COVID-19 managed in general practice: a prospective, multicentre cohort study
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b766caf8-324c-4564-a4ef-dc3aa877a7ee
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10230337
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Family Medicine[mh]
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The first wave of COVID-19 in France prompted a lockdown from mid-March to mid-May 2020. General practitioners (GPs) were in the front line ; they referred severe cases to hospital and managed less severe cases. Early on in the epidemic, researchers sought to describe the demographic and clinical characteristics of patients with COVID-19 and their course of disease. However, these studies were fully or partly conducted in hospital. The most frequently reported initial signs were fever, cough and dyspnoea. Anosmia and ageusia were also prevalent, and their concomitant presence was quite specific for a SARS-CoV-2 infection. At the time when our study data were collected, some researchers had highlighted ‘long COVID-19’ as an entity with some or all the following symptoms 3–12 months after disease onset : persistent asthenia, headache, dyspnoea, sleep difficulties, anxiety or depression, and anosmia. The significance of these symptoms is subject to debate, particularly since the literature data were somewhat contradictory; however, some researchers have suggested that these symptoms are correlated with the severity of the initial disease and the number of initial symptoms. Most of these studies of ‘long COVID-19’ estimated the frequency of persistent symptoms or adverse outcomes in hospital cohorts of patients with a confirmed diagnosis of COVID-19 but lacked a control group. Hence, these studies were not representative of patients in primary care—even though most COVID-19 cases are diagnosed by GPs. Therefore, the objectives of the present study were to (1) describe and compare the initial clinical characteristics of a cohort of patients with suspected COVID-19 managed by GPs and whose COVID-19 status (‘confirmed’, ‘no-COVID’ and ‘uncertain’ cases) was determined by the GP after he/she had received the laboratory test results; (2) determine whether persistent symptoms at 3 months were more frequent among confirmed cases than among no-COVID cases; and (3) identify factors predictive of persistent symptoms and adverse outcomes among confirmed cases.
Patient and public involvement All patients received an information sheet and gave their verbal consent to participation. They were not involved in the study design, conduct or reporting or the plans for dissemination. Study design This prospective, multicentre cohort study was conducted in four counties in the Paris region: Val-de-Marne, Seine-et-Marne, Essonne and Seine-Saint-Denis. Forty-four GPs were recruited from multiprofessional primary care practices affiliated with the Faculty of Health at Université Paris-Est Créteil (Créteil, France), because some of the GPs tutored the university’s medical students. The GPs’ characteristics are summarised in . 10.1136/bmjopen-2022-068424.supp1 Supplementary data Population During the first wave’s lockdown period, we prospectively included all consecutive adult patients who consulted one of the participating GPs for a suspected COVID-19 infection. The exclusion criteria were age under 18, no suspicion of COVID-19 and residence in an institution. The first patient was included on 6 March 2020, and the last was included on 12 May 2020. Patients were followed up for 3 months, and study data were extracted on 22 October 2020. Data sources The patients’ data were extracted from the GPs’ electronic medical records. The clinical criteria for a diagnosis of COVID-19 were left to the GP’s discretion. Patients were followed up as usual by their GP, and all consultations with healthcare professionals and/or hospital visits were registered. Three months after inclusion, the GP phoned or visited patients to collect data on persistent symptoms or recovery. For confirmed cases, they also looked for COVID-19-related hospital admissions, referrals to an emergency department, admissions to an intensive care unit and deaths. These data were completed with information from hospital discharge reports, if available. COVID-19 status The GPs prescribed SARS-CoV-2 serology and/or reverse transcription PCR (RT-PCR) tests and/or a CT scan of the chest, in line with the French national guidelines. During the first wave of COVID-19 (mid-March to mid-May 2020), RT-PCR and serology tests were not widely available. An RT-PCR test was recommended for patients with severity criteria and/or with comorbidities, and for healthcare professionals. The French national guidelines recommended a CT scan if the patient had trouble breathing, in order to assess the extent of any lung damage and to have a reference examination. Serology tests became available from May 2020 and were prescribed a posteriori to (1) patients with compatible symptoms and who had not had an RT-PCR test and (2) patients with a negative RT-PCR test. The patient’s COVID-19 status was ultimately classified by the GP as ‘confirmed COVID’, ‘no-COVID’ or ‘uncertain COVID’ after he/she had received the laboratory test results. Confirmed COVID status was defined as a positive RT-PCR and/or serology test, and/or a chest CT result suggestive of COVID-19. ‘No-COVID’ status was defined as both a negative RT-PCR test and a negative serology test, a negative RT-PCR test in the absence of a positive serology test or a positive chest CT, or a negative serology test in the absence of a positive RT-PCR test or a positive chest CT. ‘Uncertain COVID’ status was defined as the presence of suggestive symptoms and the absence of both RT-PCR and serology test and chest CT results. Outcomes We considered the two following outcomes: the persistence of symptoms 3 months after study inclusion (as assessed by the GP), and (for confirmed cases only) adverse outcomes defined by a composite criterion that included COVID-19-related hospital admissions, referral to an emergency department, intensive care unit admissions and deaths. The relationship with COVID-19 was determined from hospital records. The GP identified and recorded the patient’s persistent symptoms (if any), according to his/her usual clinical practice. We asked the GPs three questions: ‘ Do you consider that the patient has been cured? ’, ‘ If not, which symptoms persisted? ’ and ‘ Do you attribute those symptoms to the initial disease? ’. Persistent symptoms (if any) were not rated on a scale or using a questionnaire. Potential factors predictive of 3-month persistent symptoms and adverse outcomes Among confirmed cases, the following variables collected at the initial consultation were considered as potentially predictive factors for persistent symptoms and adverse outcomes: demographic characteristics (age, sex, being a caregiver), smoking, obesity, comorbidities, initial COVID-19 symptoms, the number of symptoms, systemic symptoms (ie, fever, headache, asthenia and skin symptoms), ear-nose-throat symptoms and data from an initial clinical examination. 10.1136/bmjopen-2022-068424.supp2 Supplementary data Statistical analysis Qualitative variables were described as the number (percentage), and quantitative variables were described as the median (IQR) or tertile values, as appropriate. Univariate analyses used the χ 2 test, the Fisher’s test or the Kruskal-Wallis test, as appropriate. Given the hierarchical nature of the data (level 1: the patient; level 2: the GP), we used multilevel logistic models to estimate univariate and multivariate ORs and their 95% CIs. The distribution of the patient initial characteristics was compared across the three groups (confirmed, no-COVID and uncertain). When the p value was ≤0.15, we used age-adjusted multilevel logistic models to perform post hoc pairwise comparisons for confirmed cases versus no-COVID cases on one hand, and between confirmed cases and uncertain cases on the other. Next, we compared the prevalence of persistent symptoms in the confirmed versus no-COVID groups. To assess predictive factors for 3-month persistent symptoms and adverse outcomes among the COVID-confirmed cases, we compared the groups with versus without persistent symptoms and with versus without adverse outcomes in univariate analyses. Factors with p<0.15 in the univariable analysis were considered for inclusion in multivariable multilevel logistic analyses after the assessment of confounders and interactions in bivariate models. As ‘older age’and ‘at least one comorbidity’ were highly correlated, we built the following composite variable: ‘age>70 and/or at least one comorbidity’. Lastly, in a sensitivity analysis, patients with both anosmia and ageusia but no test results were moved from the ‘uncertain COVID’ group to the ‘confirmed COVID’ group, and similar analyses were performed. All tests were two sided, and the threshold for statistical significance was set to p≤0.05. We used the false discovery rate method for post hoc analyses. All analyses were performed with Stata software (V.14.2, StataCorp, College Station, Texas, USA).
All patients received an information sheet and gave their verbal consent to participation. They were not involved in the study design, conduct or reporting or the plans for dissemination.
This prospective, multicentre cohort study was conducted in four counties in the Paris region: Val-de-Marne, Seine-et-Marne, Essonne and Seine-Saint-Denis. Forty-four GPs were recruited from multiprofessional primary care practices affiliated with the Faculty of Health at Université Paris-Est Créteil (Créteil, France), because some of the GPs tutored the university’s medical students. The GPs’ characteristics are summarised in . 10.1136/bmjopen-2022-068424.supp1 Supplementary data
During the first wave’s lockdown period, we prospectively included all consecutive adult patients who consulted one of the participating GPs for a suspected COVID-19 infection. The exclusion criteria were age under 18, no suspicion of COVID-19 and residence in an institution. The first patient was included on 6 March 2020, and the last was included on 12 May 2020. Patients were followed up for 3 months, and study data were extracted on 22 October 2020.
The patients’ data were extracted from the GPs’ electronic medical records. The clinical criteria for a diagnosis of COVID-19 were left to the GP’s discretion. Patients were followed up as usual by their GP, and all consultations with healthcare professionals and/or hospital visits were registered. Three months after inclusion, the GP phoned or visited patients to collect data on persistent symptoms or recovery. For confirmed cases, they also looked for COVID-19-related hospital admissions, referrals to an emergency department, admissions to an intensive care unit and deaths. These data were completed with information from hospital discharge reports, if available.
The GPs prescribed SARS-CoV-2 serology and/or reverse transcription PCR (RT-PCR) tests and/or a CT scan of the chest, in line with the French national guidelines. During the first wave of COVID-19 (mid-March to mid-May 2020), RT-PCR and serology tests were not widely available. An RT-PCR test was recommended for patients with severity criteria and/or with comorbidities, and for healthcare professionals. The French national guidelines recommended a CT scan if the patient had trouble breathing, in order to assess the extent of any lung damage and to have a reference examination. Serology tests became available from May 2020 and were prescribed a posteriori to (1) patients with compatible symptoms and who had not had an RT-PCR test and (2) patients with a negative RT-PCR test. The patient’s COVID-19 status was ultimately classified by the GP as ‘confirmed COVID’, ‘no-COVID’ or ‘uncertain COVID’ after he/she had received the laboratory test results. Confirmed COVID status was defined as a positive RT-PCR and/or serology test, and/or a chest CT result suggestive of COVID-19. ‘No-COVID’ status was defined as both a negative RT-PCR test and a negative serology test, a negative RT-PCR test in the absence of a positive serology test or a positive chest CT, or a negative serology test in the absence of a positive RT-PCR test or a positive chest CT. ‘Uncertain COVID’ status was defined as the presence of suggestive symptoms and the absence of both RT-PCR and serology test and chest CT results.
We considered the two following outcomes: the persistence of symptoms 3 months after study inclusion (as assessed by the GP), and (for confirmed cases only) adverse outcomes defined by a composite criterion that included COVID-19-related hospital admissions, referral to an emergency department, intensive care unit admissions and deaths. The relationship with COVID-19 was determined from hospital records. The GP identified and recorded the patient’s persistent symptoms (if any), according to his/her usual clinical practice. We asked the GPs three questions: ‘ Do you consider that the patient has been cured? ’, ‘ If not, which symptoms persisted? ’ and ‘ Do you attribute those symptoms to the initial disease? ’. Persistent symptoms (if any) were not rated on a scale or using a questionnaire.
Among confirmed cases, the following variables collected at the initial consultation were considered as potentially predictive factors for persistent symptoms and adverse outcomes: demographic characteristics (age, sex, being a caregiver), smoking, obesity, comorbidities, initial COVID-19 symptoms, the number of symptoms, systemic symptoms (ie, fever, headache, asthenia and skin symptoms), ear-nose-throat symptoms and data from an initial clinical examination. 10.1136/bmjopen-2022-068424.supp2 Supplementary data
Qualitative variables were described as the number (percentage), and quantitative variables were described as the median (IQR) or tertile values, as appropriate. Univariate analyses used the χ 2 test, the Fisher’s test or the Kruskal-Wallis test, as appropriate. Given the hierarchical nature of the data (level 1: the patient; level 2: the GP), we used multilevel logistic models to estimate univariate and multivariate ORs and their 95% CIs. The distribution of the patient initial characteristics was compared across the three groups (confirmed, no-COVID and uncertain). When the p value was ≤0.15, we used age-adjusted multilevel logistic models to perform post hoc pairwise comparisons for confirmed cases versus no-COVID cases on one hand, and between confirmed cases and uncertain cases on the other. Next, we compared the prevalence of persistent symptoms in the confirmed versus no-COVID groups. To assess predictive factors for 3-month persistent symptoms and adverse outcomes among the COVID-confirmed cases, we compared the groups with versus without persistent symptoms and with versus without adverse outcomes in univariate analyses. Factors with p<0.15 in the univariable analysis were considered for inclusion in multivariable multilevel logistic analyses after the assessment of confounders and interactions in bivariate models. As ‘older age’and ‘at least one comorbidity’ were highly correlated, we built the following composite variable: ‘age>70 and/or at least one comorbidity’. Lastly, in a sensitivity analysis, patients with both anosmia and ageusia but no test results were moved from the ‘uncertain COVID’ group to the ‘confirmed COVID’ group, and similar analyses were performed. All tests were two sided, and the threshold for statistical significance was set to p≤0.05. We used the false discovery rate method for post hoc analyses. All analyses were performed with Stata software (V.14.2, StataCorp, College Station, Texas, USA).
Study population During the study period, 521 patients were included. Of these, 516 were analysed: 166 (32.2%) were classified as ‘confirmed COVID’, 180 (34.9%) were classified as ‘no-COVID’ and 170 (32.9%) were classified as ‘uncertain COVID’ . The characteristics of the groups’ test results and disease classifications are summarised in . Characteristics of the population and intergroup comparisons In the overall population, median (IQR) age was 43 years (33–56), 62.2% were female, 12.5% were caregivers and 40.7% had at least one comorbidity . The three groups differed significantly with regard to the following initial characteristics: age, being a caregiver, having been in contact with a positive case, having at least one comorbidity, fever or feeling feverish, having muscle ache, chest pain, dyspnoea, a sore throat, anosmia, ageusia, diarrhoea and the number of systemic symptoms. Relative to the no-COVID group, confirmed cases were significantly older and were more likely to be caregivers, to have been in contact with a confirmed case of COVID-19 and to have had anosmia or ageusia. A non-significant trend towards an association with a higher number of systemic symptoms was also observed. In contrast, chest pain and sore throat were less frequent in the ‘confirmed case’ group. Relative to the uncertain COVID group, confirmed cases were significantly older and were more likely to be caregiver, to have been in contact with a confirmed case of COVID-19, to have had fever or feeling feverish, muscle ache, anosmia, ageusia, diarrhoea and more than two systemic symptoms. In contrast, they were less likely to be male. Three-month persistent symptoms in the ‘confirmed COVID’ and ‘no-COVID’ groups Overall, the percentage of 3-month persistent symptoms was higher in the confirmed COVID group than in the no-COVID group, although the difference was not statistically significant (p=0.090) . The confirmed COVID group was more likely to have persistent anosmia (OR=8.51; 95% CI 1.03–70.43; p=0047). Similar results were found in the sensitivity analysis . Predictive factors for 3-month persistent symptoms and adverse outcomes in confirmed COVID cases In a univariate analysis, the factors associated with 3-month persistent symptoms were fever or feeling feverish and anosmia . In a multivariate analysis, fever and anosmia were independently associated with 3-month persistent symptoms. Similar results were found in the sensitivity analysis (OR fever =8.49; 95% CI 1.34–53.83; p=0023 and OR anosmia =4.24; 95% CI 0.99–18.23; p=0052). Among the confirmed cases, we observed 16 (9.8%) COVID-19-related hospital admissions, 3 (1.8%) admissions to an intensive care unit, 13 (37.1%) referrals to an emergency department and no death. In a univariate analysis, patients with 3-month adverse outcomes were older, and more likely to have at least one comorbidity (hypertension, dyslipidaemia, diabetes and cardiovascular disease), fever or feeling feverish and a higher number of systemic symptoms . A trend was observed for abnormalities in a lung clinical examination. In a multivariate analysis, the composite variable ‘age>70 and/or at least one comorbidity’, abnormalities in a lung clinical examination and two or more systemic symptoms were independently associated with 3-month adverse outcomes . Similar results were found in the sensitivity analysis (OR fever =6.72; 95% CI 1.24–36.54; p=0027, OR ≥2 systemic symptoms =44.52; 95% CI 2.67–741.89; p=0008 and OR abnormalities in a lung examination =17.58; 95% CI 1.80–171.63; p=0.047).
During the study period, 521 patients were included. Of these, 516 were analysed: 166 (32.2%) were classified as ‘confirmed COVID’, 180 (34.9%) were classified as ‘no-COVID’ and 170 (32.9%) were classified as ‘uncertain COVID’ . The characteristics of the groups’ test results and disease classifications are summarised in .
In the overall population, median (IQR) age was 43 years (33–56), 62.2% were female, 12.5% were caregivers and 40.7% had at least one comorbidity . The three groups differed significantly with regard to the following initial characteristics: age, being a caregiver, having been in contact with a positive case, having at least one comorbidity, fever or feeling feverish, having muscle ache, chest pain, dyspnoea, a sore throat, anosmia, ageusia, diarrhoea and the number of systemic symptoms. Relative to the no-COVID group, confirmed cases were significantly older and were more likely to be caregivers, to have been in contact with a confirmed case of COVID-19 and to have had anosmia or ageusia. A non-significant trend towards an association with a higher number of systemic symptoms was also observed. In contrast, chest pain and sore throat were less frequent in the ‘confirmed case’ group. Relative to the uncertain COVID group, confirmed cases were significantly older and were more likely to be caregiver, to have been in contact with a confirmed case of COVID-19, to have had fever or feeling feverish, muscle ache, anosmia, ageusia, diarrhoea and more than two systemic symptoms. In contrast, they were less likely to be male.
Overall, the percentage of 3-month persistent symptoms was higher in the confirmed COVID group than in the no-COVID group, although the difference was not statistically significant (p=0.090) . The confirmed COVID group was more likely to have persistent anosmia (OR=8.51; 95% CI 1.03–70.43; p=0047). Similar results were found in the sensitivity analysis .
In a univariate analysis, the factors associated with 3-month persistent symptoms were fever or feeling feverish and anosmia . In a multivariate analysis, fever and anosmia were independently associated with 3-month persistent symptoms. Similar results were found in the sensitivity analysis (OR fever =8.49; 95% CI 1.34–53.83; p=0023 and OR anosmia =4.24; 95% CI 0.99–18.23; p=0052). Among the confirmed cases, we observed 16 (9.8%) COVID-19-related hospital admissions, 3 (1.8%) admissions to an intensive care unit, 13 (37.1%) referrals to an emergency department and no death. In a univariate analysis, patients with 3-month adverse outcomes were older, and more likely to have at least one comorbidity (hypertension, dyslipidaemia, diabetes and cardiovascular disease), fever or feeling feverish and a higher number of systemic symptoms . A trend was observed for abnormalities in a lung clinical examination. In a multivariate analysis, the composite variable ‘age>70 and/or at least one comorbidity’, abnormalities in a lung clinical examination and two or more systemic symptoms were independently associated with 3-month adverse outcomes . Similar results were found in the sensitivity analysis (OR fever =6.72; 95% CI 1.24–36.54; p=0027, OR ≥2 systemic symptoms =44.52; 95% CI 2.67–741.89; p=0008 and OR abnormalities in a lung examination =17.58; 95% CI 1.80–171.63; p=0.047).
Principal findings We included 516 patients managed by GPs for suspected COVID-19 during the first wave of the disease in France: 32.2% were classified as ‘confirmed COVID’ cases, 34.9% were classified as ‘no-COVID’ cases and 32.9% were classified as ‘uncertain COVID’ cases. The clinical course was mainly benign, although the hospital admission rate (with no death) was 9.8% in the ‘confirmed COVID’ group. In the latter group, the variable ‘age>70 and/or at least one comorbidity’, abnormalities in a lung examination and two or more systemic symptoms were independently associated with 3-month hospital admission and referral to an emergency department. Moreover, ‘confirmed COVID’ patients tended to have more persistent symptoms at 3 months—mainly anosmia and ‘other persistent symptoms’. Fever or feeling feverish and anosmia were independently associated with the persistence of symptoms. Strengths and weaknesses of the study This is one of the few studies to have included solely patients consulting in general practice; most longitudinal studies of patients with COVID-19 assessed hospital-based or mixed cohorts. Moreover, our assessment of a prospective multicentre cohort recruited at different primary care health centres means that our results can be more readily extrapolated to the local or regional level. Another study strength was our comparison of ‘confirmed COVID’, ‘no-COVID’ and ‘uncertain COVID’ groups; this provided a more accurate comparison of the initial and subsequent signs and symptoms of COVID-19. The ‘no-COVID’ group was particularly relevant for comparing the prevalence of persistent symptoms because it probably comprised patients with other viral diseases. However, our study had some limitations. Selection bias might have been present because the RT-PCR test was only initially recommended for patients with severity criteria and/or with comorbidities and for healthcare professionals. This may explain some of the demographic characteristics of confirmed cases. However, this bias was limited by the prescription of serology tests a posteriori to patients with compatible symptoms and who had not had an RT-PCR test and to patients with a negative RT-PCR test. We did not include under-18 patients and institutionalised patients. The study was limited to the greater Paris region and so might not be representative of the French population as a whole. Moreover, the groups’ size might have led to a lack of statistical power. Given the small number of patients with persistent symptoms, the corresponding results should be interpreted with caution (especially the ORs with very broad CIs). The methods for determining the presence or absence of persistent symptoms were left to the GP’s discretion; the use of particular questionnaires or scales was not imposed on them. This lack of standardisation might have influenced the estimated prevalence of persistent symptoms. However, this unconstrained type of assessment was similar to that used in the GPs’ routine clinical medical practice. Lastly, COVID-19-related hospital admissions were recorded; it would have been useful to collect data on the symptom burden associated with all-cause hospital admissions. Comparison with other studies The demographic characteristics of our patients with COVID-19 consulting in general practice were similar to those in the literature, particularly with regard to the mean age (43 in our study and in Yordanov et al ’s study ), the proportion of caregivers and the most prevalent comorbidities (hypertension and diabetes). Several studies of ambulatory patients have shown that systemic symptoms (including asthenia, fever, cough, myalgia and headaches) were frequent. Anosmia and ageusia were also frequent and appeared later in the course of disease. Some experts consider that the anosmia-ageusia combination is specific for COVID-19. Digestive tract symptoms were less frequent. Our patients also varied with regard to the signs in the GPs’ clinical examination (including abnormalities in a lung examination), as found in systematic reviews. In line with our results, most studies of outpatients have found that the course of the disease is benign and that hospital admission is not required. As found in the present research, literature data have shown that a higher frequency of negative outcomes (hospital admission and death) is associated with older age and with comorbidities like cardiovascular disease and diabetes. In contrast to another study, we did not find an association with male sex. However, no other studies have found that more than two systemic symptoms at the initial GP visit and abnormalities in a lung examination are predictive of an adverse outcome. These present findings and the literature data highlight the need for a clinical consultation with the GP. It has been widely reported that patients can experience persistent symptoms more than 4 weeks after an episode of COVID-19. Here, we observed a non-significant trend towards a greater prevalence of persistent symptoms at 3 months in the ‘confirmed COVID’ group (15.7%) versus the no-COVID group (9.6%). This finding is in line with the results of a UK study in which 13.7% of outpatients had symptoms that persisted for at least 12 weeks. However, the association remained significant in our ‘confirmed COVID’ group for anosmia and ‘other symptoms’ (ie, deep vein thrombosis, alopecia, palpitations, feeling feverish and memory impairments), as also reported elsewhere. A recent large cohort study suggested that self-reported infection was positively associated with persistent physical symptoms, whereas a positive serology test result for SARS-CoV-2 was positively associated only with persistent anosmia. Furthermore, it appears that one of the factors determining the presence of persistent symptoms in our patients with COVID-19 was the presence of fever during the initial GP visit. This association with fever has only previously been found in one study of elderly people but not in other studies. In our study, a comparison at 3 months showed that some persistent symptoms (asthenia, cough, chest pain and dyspnoea) were not significantly more frequent in the ‘confirmed COVID’ group—suggesting they were not specific for ‘long COVID-19’. Asthenia and dyspnoea were the two most common persistent symptoms in hospitalised and non-hospitalised patients. However, we observed asthenia and dyspnoea, respectively, in only around 4% and 3% of our ‘confirmed COVID’ patients, and with much the same frequency as in no-COVID patients (4.5% and 4.5%, respectively). Outpatient studies with a control group found the presence of persistent symptoms up to 10 and 12 months after mild COVID-19, with miscellaneous symptoms: asthenia, headaches, smell and taste disorders, dyspnoea, memory disorders, insomnia and difficulty concentrating. The French health authorities also included neurological, cardiothoracic and sensory disorders in the list of persistent symptoms. The results of these ‘long COVID-19’ studies are relatively disparate and appear to show that this entity is non-specific because of the multisymptomatic, fluctuating nature of the clinical manifestations. Implications for clinicians and policymakers It is important to provide GPs with primary care-specific data that enable them to optimise patient management. GPs have an essential role in combating the pandemic and diagnose most patients with COVID-19. Identifying prognostic factors and examining patients for clinical abnormalities could help detect patients at risk, set up follow-up procedures and anticipate possible worsening. These strategies might be needed in France, with a view to enabling primary care to withstand future health emergencies and pandemics, as has been mentioned in Australia, New Zealand, Canada, the Netherlands, the UK and the USA. The trend towards more frequent persistent symptoms in patients with COVID-19 (more specifically anosmia and ‘other symptoms’) suggests that follow-up by the GP should take account of the disease’s impact on quality of life, overall health and life context via a patient-centred approach. Unanswered questions and future research Our findings (notably concerning persistent symptoms) need to be confirmed in the longer term and in other patient populations (eg, institutionalised people, children and adolescents). Our study was partly based on electronic medical records and showed that primary care can provide important public health data. This work could be expanded with patient surveys and GP interviews, so as to combine real-time data on patients’ symptoms and adverse outcomes with patient responses to public health messaging and information on the GPs’ adaptive coping mechanisms.
We included 516 patients managed by GPs for suspected COVID-19 during the first wave of the disease in France: 32.2% were classified as ‘confirmed COVID’ cases, 34.9% were classified as ‘no-COVID’ cases and 32.9% were classified as ‘uncertain COVID’ cases. The clinical course was mainly benign, although the hospital admission rate (with no death) was 9.8% in the ‘confirmed COVID’ group. In the latter group, the variable ‘age>70 and/or at least one comorbidity’, abnormalities in a lung examination and two or more systemic symptoms were independently associated with 3-month hospital admission and referral to an emergency department. Moreover, ‘confirmed COVID’ patients tended to have more persistent symptoms at 3 months—mainly anosmia and ‘other persistent symptoms’. Fever or feeling feverish and anosmia were independently associated with the persistence of symptoms.
This is one of the few studies to have included solely patients consulting in general practice; most longitudinal studies of patients with COVID-19 assessed hospital-based or mixed cohorts. Moreover, our assessment of a prospective multicentre cohort recruited at different primary care health centres means that our results can be more readily extrapolated to the local or regional level. Another study strength was our comparison of ‘confirmed COVID’, ‘no-COVID’ and ‘uncertain COVID’ groups; this provided a more accurate comparison of the initial and subsequent signs and symptoms of COVID-19. The ‘no-COVID’ group was particularly relevant for comparing the prevalence of persistent symptoms because it probably comprised patients with other viral diseases. However, our study had some limitations. Selection bias might have been present because the RT-PCR test was only initially recommended for patients with severity criteria and/or with comorbidities and for healthcare professionals. This may explain some of the demographic characteristics of confirmed cases. However, this bias was limited by the prescription of serology tests a posteriori to patients with compatible symptoms and who had not had an RT-PCR test and to patients with a negative RT-PCR test. We did not include under-18 patients and institutionalised patients. The study was limited to the greater Paris region and so might not be representative of the French population as a whole. Moreover, the groups’ size might have led to a lack of statistical power. Given the small number of patients with persistent symptoms, the corresponding results should be interpreted with caution (especially the ORs with very broad CIs). The methods for determining the presence or absence of persistent symptoms were left to the GP’s discretion; the use of particular questionnaires or scales was not imposed on them. This lack of standardisation might have influenced the estimated prevalence of persistent symptoms. However, this unconstrained type of assessment was similar to that used in the GPs’ routine clinical medical practice. Lastly, COVID-19-related hospital admissions were recorded; it would have been useful to collect data on the symptom burden associated with all-cause hospital admissions.
The demographic characteristics of our patients with COVID-19 consulting in general practice were similar to those in the literature, particularly with regard to the mean age (43 in our study and in Yordanov et al ’s study ), the proportion of caregivers and the most prevalent comorbidities (hypertension and diabetes). Several studies of ambulatory patients have shown that systemic symptoms (including asthenia, fever, cough, myalgia and headaches) were frequent. Anosmia and ageusia were also frequent and appeared later in the course of disease. Some experts consider that the anosmia-ageusia combination is specific for COVID-19. Digestive tract symptoms were less frequent. Our patients also varied with regard to the signs in the GPs’ clinical examination (including abnormalities in a lung examination), as found in systematic reviews. In line with our results, most studies of outpatients have found that the course of the disease is benign and that hospital admission is not required. As found in the present research, literature data have shown that a higher frequency of negative outcomes (hospital admission and death) is associated with older age and with comorbidities like cardiovascular disease and diabetes. In contrast to another study, we did not find an association with male sex. However, no other studies have found that more than two systemic symptoms at the initial GP visit and abnormalities in a lung examination are predictive of an adverse outcome. These present findings and the literature data highlight the need for a clinical consultation with the GP. It has been widely reported that patients can experience persistent symptoms more than 4 weeks after an episode of COVID-19. Here, we observed a non-significant trend towards a greater prevalence of persistent symptoms at 3 months in the ‘confirmed COVID’ group (15.7%) versus the no-COVID group (9.6%). This finding is in line with the results of a UK study in which 13.7% of outpatients had symptoms that persisted for at least 12 weeks. However, the association remained significant in our ‘confirmed COVID’ group for anosmia and ‘other symptoms’ (ie, deep vein thrombosis, alopecia, palpitations, feeling feverish and memory impairments), as also reported elsewhere. A recent large cohort study suggested that self-reported infection was positively associated with persistent physical symptoms, whereas a positive serology test result for SARS-CoV-2 was positively associated only with persistent anosmia. Furthermore, it appears that one of the factors determining the presence of persistent symptoms in our patients with COVID-19 was the presence of fever during the initial GP visit. This association with fever has only previously been found in one study of elderly people but not in other studies. In our study, a comparison at 3 months showed that some persistent symptoms (asthenia, cough, chest pain and dyspnoea) were not significantly more frequent in the ‘confirmed COVID’ group—suggesting they were not specific for ‘long COVID-19’. Asthenia and dyspnoea were the two most common persistent symptoms in hospitalised and non-hospitalised patients. However, we observed asthenia and dyspnoea, respectively, in only around 4% and 3% of our ‘confirmed COVID’ patients, and with much the same frequency as in no-COVID patients (4.5% and 4.5%, respectively). Outpatient studies with a control group found the presence of persistent symptoms up to 10 and 12 months after mild COVID-19, with miscellaneous symptoms: asthenia, headaches, smell and taste disorders, dyspnoea, memory disorders, insomnia and difficulty concentrating. The French health authorities also included neurological, cardiothoracic and sensory disorders in the list of persistent symptoms. The results of these ‘long COVID-19’ studies are relatively disparate and appear to show that this entity is non-specific because of the multisymptomatic, fluctuating nature of the clinical manifestations.
It is important to provide GPs with primary care-specific data that enable them to optimise patient management. GPs have an essential role in combating the pandemic and diagnose most patients with COVID-19. Identifying prognostic factors and examining patients for clinical abnormalities could help detect patients at risk, set up follow-up procedures and anticipate possible worsening. These strategies might be needed in France, with a view to enabling primary care to withstand future health emergencies and pandemics, as has been mentioned in Australia, New Zealand, Canada, the Netherlands, the UK and the USA. The trend towards more frequent persistent symptoms in patients with COVID-19 (more specifically anosmia and ‘other symptoms’) suggests that follow-up by the GP should take account of the disease’s impact on quality of life, overall health and life context via a patient-centred approach.
Our findings (notably concerning persistent symptoms) need to be confirmed in the longer term and in other patient populations (eg, institutionalised people, children and adolescents). Our study was partly based on electronic medical records and showed that primary care can provide important public health data. This work could be expanded with patient surveys and GP interviews, so as to combine real-time data on patients’ symptoms and adverse outcomes with patient responses to public health messaging and information on the GPs’ adaptive coping mechanisms.
Cases of COVID-19 seen in primary care have an essentially benign course. However, age >70 and/or at least one comorbidity, abnormalities in a lung examination and a higher number of systemic symptoms were associated with hospital admission and referral to an emergency department. Our results reinforce the need for a face-to-face medical consultation by the GP to identify patients at risk of severe disease. Almost one in six patients with COVID-19 had persistent symptoms at 3 months—emphasising the need for an overall patient-centred approach. This frequency of persistent symptoms tended to be higher in patients with COVID-19 than in no-COVID cases. Anosmia and a group of rarer symptoms were more prevalent in the ‘confirmed COVID’ group. Asthenia, chest pain, cough and dyspnoea were also present in the other groups and might not be specific for a possible ‘long COVID-19’. Our findings in primary care need to be confirmed in prospective studies with a longer follow-up period.
Reviewer comments Author's
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Digital health, digital medicine, and digital therapeutics in cardiology: current evidence and future perspective in Japan
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bbd8707f-bb76-449e-b21a-5cc50275341e
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10230462
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Internal Medicine[mh]
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Almost 10 years ago, Japan set out the Action Plan of Growth Strategy that declared the initiatives of digitalization for medicine, nursing care, and healthcare to achieve the world’s most advanced medical care. This action promoted a major push for digital health and digital medicine in Japan. Specific plans included the following: (1) construction of a digital infrastructure for medicine, nursing care, and healthcare; (2) utilization of the digital infrastructure; (3) advanced digitalization of on-site operations; and (4) system establishment for utilizing medical and personal information. These initiatives formed the foundation of the Japanese national strategy and have been continuously refined, resulting in the current environment of digital health and digital medicine . In this article, we define digital health-related terminologies. First, “digital health” is a comprehensive concept of the utilization of information and communication technology (ICT) for all medical, nursing care, or healthcare support. ICT includes digital technologies such as medical big data of genomic and electronic health information, artificial intelligence (AI), or extended reality (XR) . This term often implies the use of the latest and state-of-the-art digital technology to solve various problems in healthcare fields as the objective. Digital technology seems well suited in the following fields: patient treatment; health promotion including primary prevention; the conduct and support of clinical research including decentralized clinical trials; medical education; observation and evaluation of the patients’ clinical course; and public health monitoring for the general population or specific disease cohorts. Moreover, “digital medicine” refers to digital health related to medical care and broadly supporting medicine practice.” In digital medicine, the use of digital technology for disease treatment is referred to as “digital therapeutics (DTx)” (Fig. ) . For years, various studies and clinical applications other than digital technologies have been conducted to solve healthcare issues. However, digital technology has become one of the powerful tools to solve healthcare-related problems in any medical field and has strongly assisted the evolution of digital health, along with the recent leap in ICT development, miniaturization and technical advantages of mobile devices, easy access to vast and organized data and ample computational resources, and establishment of 5th (5 G) or higher-generation mobile communication systems that allow for high-capacity, low-latency, and multiple connections. Typical digital technologies include online medical services, AI and machine learning (AI/ML), Web 3.0 (web3) and blockchain technology, XR including metaverse, electronic health records (EHR) and personal health records (PHR), and mobile health (mHealth). Telehealth and telemedicine As for online medical service, the Ministry of Health, Labour, and Welfare (MHLW) in Japan has provided “the guidance for the appropriate implementation of telemedicine.” In this guidance, telehealth refers to health promotion and medically related activities using ICT equipment . It includes not only telemedicine but also online medical advice, remote healthcare communication, and real-time online consultation between physicians, similar in concept to digital health. In particular, telemedicine is strictly defined as real-time examination, diagnosis, explanation of laboratory results, and treatment between the physician and the patient, using remote communication tools installed on mobile devices or computers . Of note, regardless of whether it is an insurance-covered medical treatment or not, telemedicine based on this guidance is required. Owing to the recent coronavirus disease 2019 (COVID-19) pandemic, telemedicine has rapidly and mandatorily gained recognition in Japan. After the state of emergency was declared in April 2020, the MHLW has permitted the use of telemedicine from the first online consultation . Since then, the proportion of hospitals or clinics that could provide telemedicine increased to 15.2% in April 2021 . The revision for medical service fee in 2022 that raised several telemedicine fees (but still less than the face-to-face outpatient fees) may help accelerate the widespread use of telemedicine . However, the number of conducting telemedicine in Japan remains considerably lower than that in European and North American countries . Thus, the advantages and challenges of telemedicine should be reconsidered to further promote its use. AI/ML AI has no single definition. The Japanese Society of Artificial Intelligence defined AI as something aimed to perform advanced inference accurately on a large amount of knowledge data . However, the concept of AI is highly diversified and still under discussion. Thus, when using this term, we need to pay attention to what kind of specific AI technology is referred to Currently, rule-based and ML are frequently used AI technology in medical sciences. In addition, the development of computer resources and easy access to medical big data enable us to utilize ML and its subfield, that is, deep learning, for clinical applications. One of the primary approaches for medical AI implementation today would be to leverage ML, including deep learning or reinforcement learning, as a tool to obtain the target output . In other words, medical AI aims to maximize the performance of the output as “prediction,” “classification,” or “generation” of diseases or data that are currently required in medicine, and numerous efforts are being made to implement it in society. Recently, a large language model of Generative Pre-trained Transformer 3 (GPT-3) with supervised fine-tuning and reinforcement learning from human feedback as InstructGPT and its dialogue-optimized conversation web-console (ChatGPT) attracts huge attention worldwide. Surprisingly, the ChatGPT has already been scored at or near the passing threshold on the United States Medical Licensing Exam . The products applying these natural language processing models have the potential to rapidly penetrate in every aspect of medical fields soon. Web3.0/Metaverse/Blockchain Web3.0 is a next-generation Internet environment utilizing blockchain technology, and the metaverse supports part of this environment. Blockchain technology is a type of database that processes and records transactions using cryptography, directly connecting terminals on information communication networks . It has excellent tamper resistance for efficient monitoring and data management in clinical trials . The term “metaverse” refers to a virtual space where anyone can communicate similar to the real world and engage in economic activities involving money as both fiat currency and cryptocurrency . Metaverse uses cross reality (XR), including virtual reality (VR) as its utilization technology, and XR is becoming noteworthy in the medical field. XR VR is a technology that creates a virtual environment through a computer, stimulating the human senses and making such an environment perceived as “reality.” Currently, VR controls the visual and auditory senses, and it was often defined as an environment where the external “real” world is completely shut off by a full immersive head-mounted display (HMD). Similar concepts include Augmented Reality (AR) and Mixed Reality (MR), which mainly refer to real-time overlaying (for AR) or merging (for MR) of the environment and objects onto the actual reality we perceived using a see-through HMD or smartphones . Clearly distinguishing them is difficult; thus, a comprehensive concept of XR (cross reality or extended reality) emerged. In the medical field, XR has already been used for medical equipment-level surgical support system , medical education , and XR-based rehabilitation system . EHR/PHR EHR is a collection of electronic medical records stored in an electronic chart originally intended to use only in each hospital or clinic but made shareable and accessible in a specific region or nationwide . EHR contains sensitive personal information; thus, it has been managed mainly by medical institutions. Conversely, the PHR refers to securely usable online medical, health, care and well-being information collected and managed by the person who is being described in the record In PHR, health-related information can be shared and aggregated at the individual level. Thus, even if people visited multiple clinics, PHR can manage not only their medical records but also their lifelong data obtained by wearable devices during daily life. mHealth The term “mHealth” generally refers to digital health using mobile devices. Currently, the most used mobile devices are smartphones and wearable devices. With the advancement of “smart” devices, mobile devices can now measure and estimate not only steps or pulse rates but also electrocardiograms, skin temperature, blood oxygen levels, stress levels, blood pressure, or plasma glucose levels . The wearable devices can also be linked with smartphones to allow viewing, verifying, and processing of biometric data in detail and sharing of data with healthcare providers as needed. In mHealth, DTx is attracting attention as a novel third option for disease treatment; it is one of the three core treatment pillars, namely, medical, surgical, and digital therapies. I especially focus on DTx in the following chapters.
As for online medical service, the Ministry of Health, Labour, and Welfare (MHLW) in Japan has provided “the guidance for the appropriate implementation of telemedicine.” In this guidance, telehealth refers to health promotion and medically related activities using ICT equipment . It includes not only telemedicine but also online medical advice, remote healthcare communication, and real-time online consultation between physicians, similar in concept to digital health. In particular, telemedicine is strictly defined as real-time examination, diagnosis, explanation of laboratory results, and treatment between the physician and the patient, using remote communication tools installed on mobile devices or computers . Of note, regardless of whether it is an insurance-covered medical treatment or not, telemedicine based on this guidance is required. Owing to the recent coronavirus disease 2019 (COVID-19) pandemic, telemedicine has rapidly and mandatorily gained recognition in Japan. After the state of emergency was declared in April 2020, the MHLW has permitted the use of telemedicine from the first online consultation . Since then, the proportion of hospitals or clinics that could provide telemedicine increased to 15.2% in April 2021 . The revision for medical service fee in 2022 that raised several telemedicine fees (but still less than the face-to-face outpatient fees) may help accelerate the widespread use of telemedicine . However, the number of conducting telemedicine in Japan remains considerably lower than that in European and North American countries . Thus, the advantages and challenges of telemedicine should be reconsidered to further promote its use.
AI has no single definition. The Japanese Society of Artificial Intelligence defined AI as something aimed to perform advanced inference accurately on a large amount of knowledge data . However, the concept of AI is highly diversified and still under discussion. Thus, when using this term, we need to pay attention to what kind of specific AI technology is referred to Currently, rule-based and ML are frequently used AI technology in medical sciences. In addition, the development of computer resources and easy access to medical big data enable us to utilize ML and its subfield, that is, deep learning, for clinical applications. One of the primary approaches for medical AI implementation today would be to leverage ML, including deep learning or reinforcement learning, as a tool to obtain the target output . In other words, medical AI aims to maximize the performance of the output as “prediction,” “classification,” or “generation” of diseases or data that are currently required in medicine, and numerous efforts are being made to implement it in society. Recently, a large language model of Generative Pre-trained Transformer 3 (GPT-3) with supervised fine-tuning and reinforcement learning from human feedback as InstructGPT and its dialogue-optimized conversation web-console (ChatGPT) attracts huge attention worldwide. Surprisingly, the ChatGPT has already been scored at or near the passing threshold on the United States Medical Licensing Exam . The products applying these natural language processing models have the potential to rapidly penetrate in every aspect of medical fields soon.
Web3.0 is a next-generation Internet environment utilizing blockchain technology, and the metaverse supports part of this environment. Blockchain technology is a type of database that processes and records transactions using cryptography, directly connecting terminals on information communication networks . It has excellent tamper resistance for efficient monitoring and data management in clinical trials . The term “metaverse” refers to a virtual space where anyone can communicate similar to the real world and engage in economic activities involving money as both fiat currency and cryptocurrency . Metaverse uses cross reality (XR), including virtual reality (VR) as its utilization technology, and XR is becoming noteworthy in the medical field.
VR is a technology that creates a virtual environment through a computer, stimulating the human senses and making such an environment perceived as “reality.” Currently, VR controls the visual and auditory senses, and it was often defined as an environment where the external “real” world is completely shut off by a full immersive head-mounted display (HMD). Similar concepts include Augmented Reality (AR) and Mixed Reality (MR), which mainly refer to real-time overlaying (for AR) or merging (for MR) of the environment and objects onto the actual reality we perceived using a see-through HMD or smartphones . Clearly distinguishing them is difficult; thus, a comprehensive concept of XR (cross reality or extended reality) emerged. In the medical field, XR has already been used for medical equipment-level surgical support system , medical education , and XR-based rehabilitation system .
EHR is a collection of electronic medical records stored in an electronic chart originally intended to use only in each hospital or clinic but made shareable and accessible in a specific region or nationwide . EHR contains sensitive personal information; thus, it has been managed mainly by medical institutions. Conversely, the PHR refers to securely usable online medical, health, care and well-being information collected and managed by the person who is being described in the record In PHR, health-related information can be shared and aggregated at the individual level. Thus, even if people visited multiple clinics, PHR can manage not only their medical records but also their lifelong data obtained by wearable devices during daily life.
The term “mHealth” generally refers to digital health using mobile devices. Currently, the most used mobile devices are smartphones and wearable devices. With the advancement of “smart” devices, mobile devices can now measure and estimate not only steps or pulse rates but also electrocardiograms, skin temperature, blood oxygen levels, stress levels, blood pressure, or plasma glucose levels . The wearable devices can also be linked with smartphones to allow viewing, verifying, and processing of biometric data in detail and sharing of data with healthcare providers as needed. In mHealth, DTx is attracting attention as a novel third option for disease treatment; it is one of the three core treatment pillars, namely, medical, surgical, and digital therapies. I especially focus on DTx in the following chapters.
DTx is a novel therapeutic option that provides treatment for illnesses through software applications (apps) delivered via digital devices , and is expanding its scope to disease prevention and management. Currently, smartphones and VR HMDs are prevalently used for this purpose. This concept of DTx was introduced in Japan with the revision of the Act on Securing Quality, Efficacy, and Safety of Products Including Pharmaceuticals and Medical Devices, which demonstrated that software programs (i.e., Software as a Medical Device [SaMD]), including standalone apps themselves, could be certified as medical devices (Fig. ) . Currently, the software app that provides DTx is called “therapeutic apps.” The term “prescription digital therapeutics” is also used, considering that physicians or healthcare professionals “prescribe” the therapeutic app to the patients, let them install it on their digital device, and provide the intended treatment . DTx not only aims to treat illnesses but also provides a direct digital intervention to patients that have a scientifically proven treatment effect, and it has been approved by regulatory agencies. In addition, compared with medical care provided in hospitals or clinics, DTx can provide seamless treatment interventions through mobile digital devices even in patients’ daily lives.
Presently, the US and Germany are particularly leading the digital health-related policies, regulations, and their development status. In the US, regulation of mobile medical applications (MMAs), including DTx, was first issued by the Food and Drug Administration (FDA) in 2013, and was updated in 2015, 2019, and 2022 . Similar to traditional medical devices, MMAs that have a significant impact on patients or medical decision-making requires appropriate regulation processes, including clinical trials. However, the conventional medical device approval process does not automatically adopt the rapidly evolving digital technology used in software or MMA development. Therefore, in July 2017, the FDA launched the Digital Health Innovation Action Plan, which includes the Software Pre-Cert Pilot Program, a system used for faster and safer review and approval processes of digital health products, including MMAs . This innovative system assesses the development capabilities and safety of the development manufacturers rather than each individual medical device software, allowing the companies to bring their FDA-cleared software to market faster and more efficiently. Although the pilot program was completed in September 2022 , the FDA continues to develop policies with the Digital Health Center of Excellence, a digital health resource center, to improve regulatory processes related to medical device software, enabling digital health stakeholders to advance all aspects of healthcare through high-quality digital health innovation . Germany is releasing more DTx medical device software to the market than the US. Germany installs the same public health insurance system as Japan, and the implemented policies provide important hints on how to generalize DTx and medical device software. In November 2019, Germany launched the Digital Healthcare Act ( Digitale-Versorgung-Gesetz or DVG) , which explains the review and approval process of low-risk medical devices essentially based on digital technologies such as Digital Health Applications ( Digitale Gesundheitsanwendungen or DiGA) . Same as FDA, the German Federal Institute for Drug and Medical Devices ( Bundesinstitut für Arzneimittel und Medizinprodukte or BFArM) assesses DiGA according to the following requirements: safety, functionality, quality, data protection, data security, and positive effects on care under the DVG. However, the DVG’s striking point is that even the DiGA and its manufacturer satisfy all requirements excluding the “positive effects on care,” the DiGA can still provisionally be registered in the BFArM directory . Therefore, even if the manufacturer has not submitted the DiGA’s clinical efficacy validation data through regulation processes, such as clinical trials, the DiGA can still be registered and tentatively reimbursed by health insurance as long as the app’s safety, functionality, quality, data protection, and data security are satisfactory. The provisional reimbursement period is limited to 12 months (or can be extended to 24 months in a specific situation) until the clinical efficacy evaluation is confirmed. However, during this period, the manufacturer can conduct the DiGA’s pivotal trials, or real-world data can be collected while distributing the app in the market with health insurance coverage . As of February 2023, 48 DiGAs have been registered in the BFArM directory. Of these apps, 16 (33%) have reached permanent reimbursement, 5 (approximately 10%) were removed from the list, and 27 (56%) are in the provisional reimbursement period and are closely monitored if they can demonstrate sufficient clinical efficacy to obtain permanent reimbursement .
DTx in Japan has been led mainly by several start-ups since 2014 when the Act on Securing Quality, Efficacy, and Safety of Products Including Pharmaceuticals and Medical Devices were revised. Same in the US or Germany, Japan’s medical device software showing a therapeutic effect for diseases needs to be regulated by the MHLW. In 2020, to further promote early implementation of novel SaMD products, including DTx apps in Japan, the MHLW launched the Digital Transformation Action Strategies in Healthcare (DASH) for SaMD . This strategy included the following: (1) seeking promising technologies; (2) arranging and disclosing the concept of a review process specialized in SaMD; (3) centralizing the SaMD consultation service; (4) establishing a SaMD-compatible rapid, efficient, and flexible review system; and (5) reinforcing the review system for early SaMD implementation. Such regulatory efforts to implement SaMD have improved the related guidance and guidelines , leading to a better environment to develop medical device software in Japan. As of February 2023, two types of DTx (for nicotine addiction [CureApp SC TM ] and for hypertension [CureApp HT TM ]) have been approved and reimbursed by the MHLW in Japan. Additionally, DTx for insomnia (SUSMED Med CBT-i) has newly been cleared by MHLW . The following section focuses on the two former DTx apps relating to cardiovascular medicine. DTx system for nicotine dependence The CureApp SC TM DTx for nicotine dependence is a therapeutics system that aims to provide intervention and support for psychological dependence to quit smoking in addition to the 12-week standard smoking cessation program in Japan . This DTx system consists of a smartphone therapeutic app, a Bluetooth-paired mobile checker device for exhaled carbon monoxide (CO), and a web-based personal computer software for physicians . It provides individually tailored behavioral therapy and quit-smoking guidance content through a therapeutic app, thereby intensifying the treatment for psychological dependence on smoking. Moreover, an equipped mobile CO breath analyzer allows patients to measure their expiratory CO levels daily and view their cessation progress through a smartphone app or web-based software for physicians. A multicenter randomized controlled trial assessed the usefulness of the DTx for nicotine dependence . A total of 584 patients diagnosed with nicotine dependence were allocated to either of the following groups: intervention group (using the DTx system for nicotine dependence in addition to a standard smoking cessation program) and control group (using a sham app in addition to a standard smoking cessation program). The primary outcome of the continuous abstinence rate from weeks 9 to 24 was significantly higher in the DTx intervention group than in the control group (63.9% vs. 50.5%; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.24–2.42, P = 0.001), and this DTx add-on effect continued at least up to 52 weeks. Hence, the DTx system for nicotine dependence significantly improved the continuous abstinence rate when added to a standard smoking cessation program. Based on these results, the CureApp SC TM DTx system was approved and reimbursed by the MHLW in Japan in 2020 as the first DTx in Asia. SaMD DTx app for hypertension The CureApp HT TM DTx for hypertension is a SaMD therapeutic app that aims to provide continuous treatment for high blood pressure, not only during intermittent clinic visits but also in their daily life. This app was developed to efficiently support and maximize the blood pressure-lowering effect of lifestyle modification , which is recommended for all patients with high blood pressure by the hypertension management guidelines . Although many physicians think that hypertension treatment links directly to pharmacological therapy, nonpharmacological therapy has also demonstrated robust blood pressure-lowering effects. Nonpharmacological therapy includes a low-salt diet, weight reduction, regular exercise, moderate alcohol consumption, good sleep, stress management . With this background in the algorithm, the DTx app for hypertension aims to educate, practice, and habituate each nonpharmacological therapy for patients with hypertension through the app during daily life outside hospitals or clinics. The app first provides knowledge and techniques to the users for the six non-pharmacological therapy for hypertension (Step 1: input and education). Next, with the app’s support, the users implement specific lifestyle modifications related to the nonpharmacological therapy based on the knowledge and techniques obtained in Step 1 (Step 2: app-supported experiences). Finally, the users independently set, implement, and evaluate their own goals and achievements of lifestyle modification and truly habituate the target nonpharmacological therapy in their daily life (Step 3: self-planning and evaluation) . The efficacy of DTx for hypertension was tested in the HERB-DH1 pivotal clinical trial . The trial enrolled 390 patients aged 65 years or younger who had essential hypertension (grade I or II) but were not taking antihypertensive agents; they were then allocated to either of the DTx intervention group (received the DTx app for hypertension and lifestyle modification guidance according to the guidelines) or the control group (only received lifestyle modification education according to the guidelines) . The primary endpoint of the change in 24-hour systolic blood pressure by ambulatory blood pressure monitoring from baseline (week 0) to week 12 was −4.9 and −2.3 mmHg mmHg in the DTx intervention and control groups, respectively. Hence, the DTx app intervention group had a significantly greater reduction in blood pressure than the control group (mean difference, −2.4 mmHg; 95% CI, −4.5 to −0.3; P = 0.024). Additionally, the reduction of morning home systolic blood pressure from baseline to week 12 was greater in the DTx intervention group than in the control group (−10.6 mmHg vs. −6.2 mmHg; mean difference, −4.3 mmHg; 95% CI, −6.7 to −1.9; P < 0.001). Furthermore, these blood pressure reduction effects persisted at week 24 at least. In conclusion, the DTx for hypertension in addition to the guideline-based hypertension management was effective in patients aged 65 years or younger who had essential hypertension without antihypertensive agents. On top of that, we conducted a cost-effectiveness analysis of the DTx for hypertension by using the background characteristics and effect data of both intervention and control groups in the HERB-DH1 trial . In this analysis, we examined the medical economic effects of using the therapeutic app of DTx for hypertension with a time horizon. The differences in medical costs and quality-adjusted life years (QALY) between the DTx intervention group and the control group were 110 717 yen (higher in the DTx intervention group) and 0.092 (longer in the app intervention group). Therefore, the incremental cost-effectiveness ratio (ICER) was calculated to be 1 199 880 yen/QALY . This ICER value was lower than the “willingness-to-pay” threshold of 5 million yen/QALY, which is one of the acceptable medical costs for each increase in 1 QALY. Thus, prescribing the DTx app might be cost-effective through life. Considering these series of evidence, the CureApp HT TM DTx for hypertension was cleared and was reimbursed by the MLHW in Japan as the world’s first hypertension therapeutic app in 2022.
The CureApp SC TM DTx for nicotine dependence is a therapeutics system that aims to provide intervention and support for psychological dependence to quit smoking in addition to the 12-week standard smoking cessation program in Japan . This DTx system consists of a smartphone therapeutic app, a Bluetooth-paired mobile checker device for exhaled carbon monoxide (CO), and a web-based personal computer software for physicians . It provides individually tailored behavioral therapy and quit-smoking guidance content through a therapeutic app, thereby intensifying the treatment for psychological dependence on smoking. Moreover, an equipped mobile CO breath analyzer allows patients to measure their expiratory CO levels daily and view their cessation progress through a smartphone app or web-based software for physicians. A multicenter randomized controlled trial assessed the usefulness of the DTx for nicotine dependence . A total of 584 patients diagnosed with nicotine dependence were allocated to either of the following groups: intervention group (using the DTx system for nicotine dependence in addition to a standard smoking cessation program) and control group (using a sham app in addition to a standard smoking cessation program). The primary outcome of the continuous abstinence rate from weeks 9 to 24 was significantly higher in the DTx intervention group than in the control group (63.9% vs. 50.5%; odds ratio [OR], 1.73; 95% confidence interval [CI], 1.24–2.42, P = 0.001), and this DTx add-on effect continued at least up to 52 weeks. Hence, the DTx system for nicotine dependence significantly improved the continuous abstinence rate when added to a standard smoking cessation program. Based on these results, the CureApp SC TM DTx system was approved and reimbursed by the MHLW in Japan in 2020 as the first DTx in Asia.
The CureApp HT TM DTx for hypertension is a SaMD therapeutic app that aims to provide continuous treatment for high blood pressure, not only during intermittent clinic visits but also in their daily life. This app was developed to efficiently support and maximize the blood pressure-lowering effect of lifestyle modification , which is recommended for all patients with high blood pressure by the hypertension management guidelines . Although many physicians think that hypertension treatment links directly to pharmacological therapy, nonpharmacological therapy has also demonstrated robust blood pressure-lowering effects. Nonpharmacological therapy includes a low-salt diet, weight reduction, regular exercise, moderate alcohol consumption, good sleep, stress management . With this background in the algorithm, the DTx app for hypertension aims to educate, practice, and habituate each nonpharmacological therapy for patients with hypertension through the app during daily life outside hospitals or clinics. The app first provides knowledge and techniques to the users for the six non-pharmacological therapy for hypertension (Step 1: input and education). Next, with the app’s support, the users implement specific lifestyle modifications related to the nonpharmacological therapy based on the knowledge and techniques obtained in Step 1 (Step 2: app-supported experiences). Finally, the users independently set, implement, and evaluate their own goals and achievements of lifestyle modification and truly habituate the target nonpharmacological therapy in their daily life (Step 3: self-planning and evaluation) . The efficacy of DTx for hypertension was tested in the HERB-DH1 pivotal clinical trial . The trial enrolled 390 patients aged 65 years or younger who had essential hypertension (grade I or II) but were not taking antihypertensive agents; they were then allocated to either of the DTx intervention group (received the DTx app for hypertension and lifestyle modification guidance according to the guidelines) or the control group (only received lifestyle modification education according to the guidelines) . The primary endpoint of the change in 24-hour systolic blood pressure by ambulatory blood pressure monitoring from baseline (week 0) to week 12 was −4.9 and −2.3 mmHg mmHg in the DTx intervention and control groups, respectively. Hence, the DTx app intervention group had a significantly greater reduction in blood pressure than the control group (mean difference, −2.4 mmHg; 95% CI, −4.5 to −0.3; P = 0.024). Additionally, the reduction of morning home systolic blood pressure from baseline to week 12 was greater in the DTx intervention group than in the control group (−10.6 mmHg vs. −6.2 mmHg; mean difference, −4.3 mmHg; 95% CI, −6.7 to −1.9; P < 0.001). Furthermore, these blood pressure reduction effects persisted at week 24 at least. In conclusion, the DTx for hypertension in addition to the guideline-based hypertension management was effective in patients aged 65 years or younger who had essential hypertension without antihypertensive agents. On top of that, we conducted a cost-effectiveness analysis of the DTx for hypertension by using the background characteristics and effect data of both intervention and control groups in the HERB-DH1 trial . In this analysis, we examined the medical economic effects of using the therapeutic app of DTx for hypertension with a time horizon. The differences in medical costs and quality-adjusted life years (QALY) between the DTx intervention group and the control group were 110 717 yen (higher in the DTx intervention group) and 0.092 (longer in the app intervention group). Therefore, the incremental cost-effectiveness ratio (ICER) was calculated to be 1 199 880 yen/QALY . This ICER value was lower than the “willingness-to-pay” threshold of 5 million yen/QALY, which is one of the acceptable medical costs for each increase in 1 QALY. Thus, prescribing the DTx app might be cost-effective through life. Considering these series of evidence, the CureApp HT TM DTx for hypertension was cleared and was reimbursed by the MLHW in Japan as the world’s first hypertension therapeutic app in 2022.
This review introduces the latest and various digital health technologies with specific terminologies along with the DTx in cardiovascular medicine. Although only three DTx apps have been approved by MHLW in Japan, several manufacturers, including DTx start-ups and pharmaceutical companies, continuously develop DTx and conduct clinical research to obtain regulatory approval. The number of DTx development pipelines in Japan surpasses more than 30, which continues to increase every year (Table ). The movement to promote DTx in cardiovascular medicine, which applies various digital technologies to patients with cardiovascular diseases and considers the technologies’ safety, efficacy, and cost-effectiveness, will accelerate not only through basic experiments and clinical studies but also through social implementation.
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Interactions between Australian cancer physicians and the pharmaceutical industry: a qualitative study
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51094e13-8d8a-45e7-9a76-5cb92fb918fd
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10230892
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Internal Medicine[mh]
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Relationships between the pharmaceutical industry and physicians are widespread globally. These relationships inherently create conflicting priorities; physicians may perceive interactions with industry as a way to learn about new drugs, with an aim to provide the best possible treatment for their patients, while the commercial imperative of industry representatives is to sell their products. In this study, we define cancer physicians as medical oncologists and clinical haematologists. For industry, the motivation to interact with cancer physicians is high. Anticancer drugs are more lucrative to industry than any other therapeutic group, and this is an area of rapid drug development, with both the numbers and market share of cancer drugs increasing as a proportion of total pharmaceutical revenue. Physicians’ relationships with industry are important to understand, as industry financing can lead to both poorer prescribing practices and bias in research. In cancer care, this is of utmost concern: cancer is the second leading cause of death in the USA and contributed 18% of the burden of disease in Australia in 2018. Additionally, for many newer available cancer treatments, there is no evidence of survival benefits as compared with existing options. Industry-led trials are at the forefront of clinical cancer research and although not all new drugs have therapeutic advantages, a number of new cancer treatments that have been developed within the last couple of decades have been genuine breakthroughs. In the context of the preponderance of industry-funded studies, some form of working relationship with industry, such as a role as an investigator in industry-funded trials, is therefore inevitable for most cancer physicians. Previous research suggested that Australian cancer physicians interact with industry frequently, and that the majority had at some point received non-research payments from industry. The motivations behind these interactions, however, are poorly understood. For physicians other than cancer specialists, these relationships were last analysed in Australia in 2006 in a qualitative interview study. Physicians’ views varied on the potential risks and benefits of interactions with industry. They largely saw themselves as competent to manage these relationships and relied on their own moral compasses, with large individual variation in the types of interactions deemed to be acceptable. A 2014 Japanese interview study found that physicians’ attitudes tended to change over time as their careers progressed and they gained more experience of interactions with sales representatives, but this did not necessarily flow into altered behaviour. To our knowledge, no previous study has specifically explored the relationships between cancer physicians and the pharmaceutical industry. It is therefore unknown to what extent cancer physicians choose to maintain contact with industry, including both financial interactions, such as receiving gifts and payments, and non-financial interactions, such as meeting regularly with sales representatives. Nor is it known how they perceive these relationships and why they maintain them. The aim of this study was to understand how and why Australian cancer physicians interact with industry.
Design and participants We performed a qualitative analysis of in-depth, semistructured interviews with practising Australian consultant cancer physicians, reported in line with Consolidated Criteria for Reporting Qualitative Research guidelines . Development of the interview guide was based on topics raised in responses to a previous survey of Australian cancer physicians. Survey respondents were also invited to leave their details if they wished to be contacted to participate in a later interview study. Of 116 survey respondents, 39 agreed to be contacted. Following exclusion of trainees and coworkers of the lead researcher, 37 potential participants were identified, all of whom were invited to participate. 10.1136/bmjopen-2022-065719.supp1 Supplementary data Potential participants were emailed once and provided with a Participant Information Statement. Of the 37 people contacted, 18 agreed to an interview, 2 asked to be recontacted but did not respond to further queries, 1 declined upfront and 16 did not respond. Those who responded were asked to complete a consent form prior to arranging an interview using the Zoom platform on the University of Sydney secure server. Zoom has previously been considered a useful and effective platform to perform qualitative interviews. Sixteen interviews were ultimately completed after contact was lost with two potential participants, resulting in an ultimate response rate of 43% of those contacted. Interview process As noted above, interview questions were developed based on responses to an earlier survey and were further revised in discussions among all the researchers. These were intended as a guide to encourage flowing conversation around issues, rather than be prescriptive . 10.1136/bmjopen-2022-065719.supp2 Supplementary data A single researcher (AMJP) carried out all interviews and also had sole access to the recruitment list. Interviews were recorded with both video and sound, then transcribed verbatim and de-identified by AMJP, prior to distribution back to the interviewees for confirmation. Original recordings were then destroyed. Participant involvement Aside from confirming the content of the interview transcriptions, participants were not involved in the design or analysis of the study. Patient and public involvement Neither patients nor the general public were involved in the design, conduct or analysis of the study. Analysis Deductive codes, which had emerged from the results of the prior survey, were used initially, as well as codes that were based on two previous studies on this topic . Inductive codes were also developed based on the interviewee responses. Two researchers with postgraduate training in qualitative analysis but different clinical backgrounds (AMJP and EJM) initially independently coded two interviews to ensure inter-reviewer reliability and allow for reflexivity, with differences resolved through discussion, after which coding was performed exclusively by AMJP. Codes were formed into themes using Braun and Clarke’s six-step process: (1) data familiarisation, (2) code generation, (3) theme searching, (4) theme reviewing, (5) theme naming and definition and (6) report production. Analysis of themes was based on grounded theory. Data were managed using NVivo V.1.6.1 (QSR International, Melbourne, Australia). 10.1136/bmjopen-2022-065719.supp3 Supplementary data Reflexivity Three authors are practising medical oncologists in Australia (AMJP, PF and DJK), and four are researchers (BM, RM, LAB and EJM) with a background in research integrity and industry influence on health and healthcare. All the oncologist authors have contact with the pharmaceutical industry through drug access programmes and clinical trials. Two of these authors do not meet with sales representatives or attend sponsored educational events in person (AMJP and PF), while the third has received speaker fees from a company within the last year (DJK). None of the researcher authors (BM, RM, LAB and EJM) have any financial ties with industry. Prior to commencing the interview, each participant was informed of the varied levels of industry interactions of the researchers, with the overall neutrality of the team emphasised. Participants were encouraged to be open and honest, with the intention of the research being to understand their experiences, rather than hold preconceived judgements.
We performed a qualitative analysis of in-depth, semistructured interviews with practising Australian consultant cancer physicians, reported in line with Consolidated Criteria for Reporting Qualitative Research guidelines . Development of the interview guide was based on topics raised in responses to a previous survey of Australian cancer physicians. Survey respondents were also invited to leave their details if they wished to be contacted to participate in a later interview study. Of 116 survey respondents, 39 agreed to be contacted. Following exclusion of trainees and coworkers of the lead researcher, 37 potential participants were identified, all of whom were invited to participate. 10.1136/bmjopen-2022-065719.supp1 Supplementary data Potential participants were emailed once and provided with a Participant Information Statement. Of the 37 people contacted, 18 agreed to an interview, 2 asked to be recontacted but did not respond to further queries, 1 declined upfront and 16 did not respond. Those who responded were asked to complete a consent form prior to arranging an interview using the Zoom platform on the University of Sydney secure server. Zoom has previously been considered a useful and effective platform to perform qualitative interviews. Sixteen interviews were ultimately completed after contact was lost with two potential participants, resulting in an ultimate response rate of 43% of those contacted.
As noted above, interview questions were developed based on responses to an earlier survey and were further revised in discussions among all the researchers. These were intended as a guide to encourage flowing conversation around issues, rather than be prescriptive . 10.1136/bmjopen-2022-065719.supp2 Supplementary data A single researcher (AMJP) carried out all interviews and also had sole access to the recruitment list. Interviews were recorded with both video and sound, then transcribed verbatim and de-identified by AMJP, prior to distribution back to the interviewees for confirmation. Original recordings were then destroyed.
Aside from confirming the content of the interview transcriptions, participants were not involved in the design or analysis of the study.
Neither patients nor the general public were involved in the design, conduct or analysis of the study.
Deductive codes, which had emerged from the results of the prior survey, were used initially, as well as codes that were based on two previous studies on this topic . Inductive codes were also developed based on the interviewee responses. Two researchers with postgraduate training in qualitative analysis but different clinical backgrounds (AMJP and EJM) initially independently coded two interviews to ensure inter-reviewer reliability and allow for reflexivity, with differences resolved through discussion, after which coding was performed exclusively by AMJP. Codes were formed into themes using Braun and Clarke’s six-step process: (1) data familiarisation, (2) code generation, (3) theme searching, (4) theme reviewing, (5) theme naming and definition and (6) report production. Analysis of themes was based on grounded theory. Data were managed using NVivo V.1.6.1 (QSR International, Melbourne, Australia). 10.1136/bmjopen-2022-065719.supp3 Supplementary data
Three authors are practising medical oncologists in Australia (AMJP, PF and DJK), and four are researchers (BM, RM, LAB and EJM) with a background in research integrity and industry influence on health and healthcare. All the oncologist authors have contact with the pharmaceutical industry through drug access programmes and clinical trials. Two of these authors do not meet with sales representatives or attend sponsored educational events in person (AMJP and PF), while the third has received speaker fees from a company within the last year (DJK). None of the researcher authors (BM, RM, LAB and EJM) have any financial ties with industry. Prior to commencing the interview, each participant was informed of the varied levels of industry interactions of the researchers, with the overall neutrality of the team emphasised. Participants were encouraged to be open and honest, with the intention of the research being to understand their experiences, rather than hold preconceived judgements.
Sixteen interviews were completed between November 2021 and March 2022. Participant characteristics are described in . The median interview duration was 39.5 min (range 29–53 min). After coding each transcript, we developed six key themes that fell into two categories. An overarching theme ( desire for a ‘middle road’ ) then emerged from these two categories. shows the relationships between each theme. Some codes contributed to more than one theme within a category, and the two categories led to the overarching theme. Illustrative quotes attributable to each theme are shown in , , with the themes discussed in detail below. Box 1 Representative quotes for Overarching theme: Desire for a ‘middle road’ ‘I think we will work with them very closely indefinitely, and I think for each individual practitioner they need to navigate what that looks for them and where their moral compass is happy in the transactional part of the relationship.’ (P10) ‘I think both extremes of opinion [in] this matter, which [are], ‘I never talk to pharma, I never have any interactions with pharma reps’, and on the other spectrum, ‘there’s no problem, la la la, I’m an independent thinker.’ I think both those approaches are completely wrong. And there is actually a happy medium, and it’s about how do we navigate that particular pathway?’ (P13) ‘No one can cure cancer or disease alone without the corporate or industrial world. We need to learn how to engage in an open, honest and mature way, rather than this, really, this dichotomy of good and bad.’ (P15) Category I: views and experiences of interactions Transactional nature of relationships Access programmes, clinical trials and advisory boards Participants generally identified the transactional nature of all relationships with industry, with all beneficial relationships interpreted as having significant caveats. This was considered most pertinent in the context of access programmes, with some participants only maintaining contacts with industry for this purpose: ‘…that relationship [with industry] for the odd patient where you want to try and get access to drugs that you can’t get otherwise’ (participant (P)14). In Australia, cancer drugs found to be acceptably cost-effective are publicly funded under the Pharmaceutical Benefits Scheme, with patients required to pay a co-payment. Access programmes may allow unfunded medicines to be prescribed at either no cost to patients or at a discount on the retail price, with patients being required to contribute a co-payment. While interviewees saw these access programmes as generally beneficial for patients, required co-payments were often considered exorbitant and put cancer physicians in an awkward position with their patients. One noted that companies ‘weren’t generous’ (P7). The opaque nature of these programmes was also seen by some as a way of rewarding preferred clinicians who had provided benefits to the companies, such that these clinicians would learn about the existence of programmes prior to anybody else. Participants also discussed the benefits of these programmes to industry, underscoring their transactional nature. It was clear to most that companies used these programmes to gain useful data and create advocates who could then support the companies’ cases for funding of their drugs under Australia’s Pharmaceutical Benefits Scheme. This was similarly true for trial involvement or membership of advisory boards within companies, where participants felt there was likely to be just as much benefit to the company as clinicians or patients. One participant noted, for example, that while advisory board membership allowed them to ‘interact with people who I greatly respect within my field’, they concurrently ‘from the point of view of a pharmaceutical company, are probably incredibly potent marketing tools’ (P13). Education Interactions that are often proffered as beneficial for clinicians were interpreted with some scepticism. The educational role of industry, for example, both in the context of formal meetings and more broadly within medicine, was frequently considered ‘overstated’ (P6). It was seen as just as likely to benefit companies as clinicians and, accordingly, be potentially detrimental to the latter. Several participants expressed reluctance to attend sponsored education due to inherent biases such as the selection of speakers by sponsors, noting: … that’s how they censor speakers, basically. They pick people who they know will have a positive viewpoint. (P13) Sales representatives Interviewees’ scepticism often extended to the role of sales representatives, with some participants noting specific uncomfortable instances when it had become clear that the relationship was transactional, despite representatives ‘trying to be your bestie’ (P14). Others noted pragmatically that: …[sales representatives] are very pleasant people to interact with, but they are running a business. (P5) There were exceptions to this, with some other clinicians expressing enjoyment at the social aspect of meeting sales representatives, only acknowledging the likely transactional nature of these interactions as a secondary concern. For example: I really enjoy meeting them at third-party events and having social conversations with them. And I like that part of the relationship. (P10) Research dependence and associated risk There was a universal acknowledgement by participants of the role industry plays in funding research, though this was connected to an acknowledgement of the risk of bias from industry funding. Most participants identified, either directly or indirectly, that modern cancer research is dependent on the pharmaceutical industry, and no participant was able to see an alternative model for sustainable research funding. The risk of this dependence was clear, with participants noting instances where, for example: …the editorial for a large phase 3 trial is actually written by somebody who sits on the advisory board of the funding body. It’s impossible to say in that scenario that there’s not a level of bias. (P10) While participants were able to identify impressive drugs that could only have been developed with industry funding, some noted that further funding would be skewed towards ‘preferred centres’ (P15) and researchers (both in Australia and abroad), based on the strength of relationships with industry, noting: No one is publishing in NEJM [ New England Journal of Medicine ] with their little investigator-initiated study, right? So, all of those intangible advantages come from building relationships with them. (P15) Participants did not discuss why clinicians may need to produce high-impact publications, such as to maintain academic positions. They also did not explicitly discuss the motivation to seek industry assistance to obtain these high-impact publications. However, participants did report concerns that this funding model meant there were fewer trials focusing on patient care, such as dose reduction studies, studies using older drugs in new contexts or quality-of-life studies. When discussing the ‘profit motive’ (P16) of industry, one participant noted that these clinical questions would remain unresearched, stating that: …that’s the problem [with dependence on industry for research funding], is the gaps and holes, and the other questions that are nothing to do with therapeutics. (P16) Ethical challenge of industry payments Among all participants, the acceptance of non-research payments from industry was considered ‘just a norm that lots of people [do] ’ (P2). Even for those who refused payments, there was some reluctance to condemn colleagues for doing so for situations that were deemed broadly beneficial to patients, such as membership of advisory boards to guide clinical trial development. In these contexts, participants felt that ‘mostly I see people do it in very good faith’ (P8), even if in doing so, ‘the work [they] do by its nature must be biased in emphasis’ (P8). Conversely, most did not see it as reasonable for industry to be funding travel expenses to attend conferences or taking ‘a small bunch of doctors out to a very fancy very expensive restaurant’ (P1). Even those who had previously accepted these payments were sometimes critical. Indeed, those who had received payments that they felt were not commensurate with the amount of work put in described these with some contrition, such as: I haven’t accepted any for six or seven years, and part of that is some minor discomfort around [the ethics of accepting payments]. (P6) It was not clear in these discussions how an appropriate level of compensation should be determined, nor to whom it should be considered appropriate, be they clinicians or patients. Several participants also described a focus of industry largesse on clinicians deemed ‘key opinion leaders’: They pick the opinion leaders. They pick the people that are then going to go and influence the other people. (P12) The nature of disclosure of these payments was discussed by participants with an additional level of nuance. Many felt that the Australian public register of industry payments, administered by the industry trade association Medicines Australia, acted as disincentive for receiving payments (being ‘not a good look’ (P14)), but did not provide sufficient information to be of use. For example, several participants reported passing on their payments to their institution if the remunerated activity occurred during work hours. This was not considered in company reports recorded in the register; some felt that ‘there’s a big difference’ (P11) as this was seen as a way of mitigating any ethical dilemma and, further, providing financial benefit to their institution. Attitudes vary based on forms of interactions Approaches to interactions reflected the participants’ interpretation of each interaction’s value. The most frequent forms of interactions discussed were clinical trials, access programmes and meetings with sales representatives. The findings of the three prior themes—on the transactional nature of relationships, research dependence and ethical risks—informed the development of this theme. Clinical trials Universally, participants expressed confidence in participating in clinical trials run by the pharmaceutical industry. This reflected the willingness of industry to be involved in research, as: …negotiating for more funding with a pharma company is easier because they have more money… (P1) This was generally seen as a core aspect of participants’ jobs. Industry’s role in research was highly valued, and accessing trials was seen as ‘extremely important’ (P13), discussed further below. This was reflected by the confidence clinicians showed in approaching discussions around research. Access programmes Similarly, participants frequently expressed confidence in their interactions around compassionate drug access programmes, with most stating that they: …from time to time, will directly approach representatives of the pharmaceutical companies requesting for compassionate access to their drugs. (P2) While some expressed scepticism about both the putative compassionate nature of these programmes and their opacity, as discussed in our first theme ‘ Transactional nature of relationships ’, no participants felt that access programmes were inappropriate, and most demonstrated confidence in using them, although with understandable difficulties around discussing the co-payments with patients. Sales representatives Conversely, there were varying levels of comfort around interactions with sales representatives. Some felt confident in their ability to interact with sales representatives, while others would ‘usually avoid’ (P15) these relationships, or ‘just say no’ (P7) to meeting with them. Regardless, almost all participants questioned the value of sales representatives, with only a minority sensing any value in their role, and some finding the approach of representatives in building profiles about clinicians ‘really weird’ (P6) or invasive. One participant noted that: They collect information about who’s affiliated with whom, who’s married to whom, and then you get these kinds of comments that come out years down the track, and I found that was, I was peeved by that, actually. (P14) Because of this, most went out of their way to evade sales representatives at least some of the time, noting that they ‘don’t find [them] very valuable’ (P16) or that they ‘barely have time for those meetings’ (P10). The only situations in which sales representatives were seen as beneficial related to discrete periods of solo practice, in which information might be brought to the attention of a clinician that made them think, for example, ‘I didn’t actually know that I could [prescribe a given drug] for X cancer’ (P3). However, this was still met with ethical concern, noting ‘that’s another discomfort that I have with [sales representatives], knowing that I could potentially be impacting my practice [negatively],’ (P3) leading that participant to state they’ve ‘weaned off them’ (P3). Category II: management of interactions Lack of perceived useful guidance Participants were asked to discuss the role of both ethical guidelines and senior colleagues in managing interactions with industry. Most participants felt that ethical guidelines had little if any value. Even among those who felt they were useful, none had read any guidelines. This was despite the existence of ethical guidelines around industry interactions produced by the Australian Medical Association, the Royal Australasian College of Physicians and the industry association, Medicines Australia. Some participants felt that the guidelines were patronising or written as ‘rules to treat us like Kindergarten kids’ (P11), while others noted that in the absence of regulation, guidelines were likely to remain inconsequential. Most participants acknowledged that senior colleagues influenced more junior practitioners. Despite some participants describing positive experiences in this regard, others felt that this could be detrimental, as: …juniors probably have a better understanding about the ethical issues … than the senior colleagues. (P1) Additional education was also not consistently seen as a solution, though several participants felt there should be more discussions about industry interactions during training. Reduced interactions due to the COVID-19 pandemic This study was conducted during the COVID-19 pandemic. Many participants discussed the pandemic’s effects on these interactions during the interviews. Primarily, participants described COVID-19, and the associated travel restrictions, lockdowns and cancellation of in-person conferences, as providing a welcome buffer between them and industry. It inherently reduced the ability of sales representatives to visit cancer physicians, given: …that they’re finding it a lot more difficult to sort of reach people at this moment. (P2) Several participants expressed this as a relief, particularly among those who tended to avoid these interactions at a baseline. The COVID-19 pandemic was perceived as a ‘natural experiment’ to exclude sales representatives from cancer physicians’ practices, with no participant reporting this as detrimental to either them or their patients. Participants did not discuss the role of online scientific meetings during the COVID-19 pandemic, nor how the shift to online meetings may have affected industry’s influence on content and ability to interact with attendees. Overarching theme: desire for a ‘middle road’ Desire for a ‘middle road’ An overarching theme that emerged was the desire for a ‘middle road’ in managing these interactions, with all participants describing a nuance in their industry relationships. While participants were able to identify risks or potentials for harm arising from industry interactions, several described the presence of industry in cancer care as a ‘necessary evil’ (P1, P4, P13, P14). This referred in particular to industry’s fundamental role in drug development and distribution. In general, participants were able to identify some relationships that were unnecessary or unacceptable, such as meeting with sales representatives or receiving excessive remuneration for services, as well as others that were seen as necessary, such as participation in clinical trials and access programmes. The concept of a ‘middle road’ appeared to be an ethically acceptable navigation of the myriad interactions possible. Several participants proposed alternatives to the current industry funding structure that may reduce the risk of bias among recipients. A common proposition was a mixed pool of funding that could be administered and distributed independently of industry. In addition, participants expressed a desire to obtain the benefits of industry interactions, such as drug access programmes, without needing to develop uncomfortable relationships, such as with sales representatives.
Transactional nature of relationships Access programmes, clinical trials and advisory boards Participants generally identified the transactional nature of all relationships with industry, with all beneficial relationships interpreted as having significant caveats. This was considered most pertinent in the context of access programmes, with some participants only maintaining contacts with industry for this purpose: ‘…that relationship [with industry] for the odd patient where you want to try and get access to drugs that you can’t get otherwise’ (participant (P)14). In Australia, cancer drugs found to be acceptably cost-effective are publicly funded under the Pharmaceutical Benefits Scheme, with patients required to pay a co-payment. Access programmes may allow unfunded medicines to be prescribed at either no cost to patients or at a discount on the retail price, with patients being required to contribute a co-payment. While interviewees saw these access programmes as generally beneficial for patients, required co-payments were often considered exorbitant and put cancer physicians in an awkward position with their patients. One noted that companies ‘weren’t generous’ (P7). The opaque nature of these programmes was also seen by some as a way of rewarding preferred clinicians who had provided benefits to the companies, such that these clinicians would learn about the existence of programmes prior to anybody else. Participants also discussed the benefits of these programmes to industry, underscoring their transactional nature. It was clear to most that companies used these programmes to gain useful data and create advocates who could then support the companies’ cases for funding of their drugs under Australia’s Pharmaceutical Benefits Scheme. This was similarly true for trial involvement or membership of advisory boards within companies, where participants felt there was likely to be just as much benefit to the company as clinicians or patients. One participant noted, for example, that while advisory board membership allowed them to ‘interact with people who I greatly respect within my field’, they concurrently ‘from the point of view of a pharmaceutical company, are probably incredibly potent marketing tools’ (P13). Education Interactions that are often proffered as beneficial for clinicians were interpreted with some scepticism. The educational role of industry, for example, both in the context of formal meetings and more broadly within medicine, was frequently considered ‘overstated’ (P6). It was seen as just as likely to benefit companies as clinicians and, accordingly, be potentially detrimental to the latter. Several participants expressed reluctance to attend sponsored education due to inherent biases such as the selection of speakers by sponsors, noting: … that’s how they censor speakers, basically. They pick people who they know will have a positive viewpoint. (P13) Sales representatives Interviewees’ scepticism often extended to the role of sales representatives, with some participants noting specific uncomfortable instances when it had become clear that the relationship was transactional, despite representatives ‘trying to be your bestie’ (P14). Others noted pragmatically that: …[sales representatives] are very pleasant people to interact with, but they are running a business. (P5) There were exceptions to this, with some other clinicians expressing enjoyment at the social aspect of meeting sales representatives, only acknowledging the likely transactional nature of these interactions as a secondary concern. For example: I really enjoy meeting them at third-party events and having social conversations with them. And I like that part of the relationship. (P10) Research dependence and associated risk There was a universal acknowledgement by participants of the role industry plays in funding research, though this was connected to an acknowledgement of the risk of bias from industry funding. Most participants identified, either directly or indirectly, that modern cancer research is dependent on the pharmaceutical industry, and no participant was able to see an alternative model for sustainable research funding. The risk of this dependence was clear, with participants noting instances where, for example: …the editorial for a large phase 3 trial is actually written by somebody who sits on the advisory board of the funding body. It’s impossible to say in that scenario that there’s not a level of bias. (P10) While participants were able to identify impressive drugs that could only have been developed with industry funding, some noted that further funding would be skewed towards ‘preferred centres’ (P15) and researchers (both in Australia and abroad), based on the strength of relationships with industry, noting: No one is publishing in NEJM [ New England Journal of Medicine ] with their little investigator-initiated study, right? So, all of those intangible advantages come from building relationships with them. (P15) Participants did not discuss why clinicians may need to produce high-impact publications, such as to maintain academic positions. They also did not explicitly discuss the motivation to seek industry assistance to obtain these high-impact publications. However, participants did report concerns that this funding model meant there were fewer trials focusing on patient care, such as dose reduction studies, studies using older drugs in new contexts or quality-of-life studies. When discussing the ‘profit motive’ (P16) of industry, one participant noted that these clinical questions would remain unresearched, stating that: …that’s the problem [with dependence on industry for research funding], is the gaps and holes, and the other questions that are nothing to do with therapeutics. (P16) Ethical challenge of industry payments Among all participants, the acceptance of non-research payments from industry was considered ‘just a norm that lots of people [do] ’ (P2). Even for those who refused payments, there was some reluctance to condemn colleagues for doing so for situations that were deemed broadly beneficial to patients, such as membership of advisory boards to guide clinical trial development. In these contexts, participants felt that ‘mostly I see people do it in very good faith’ (P8), even if in doing so, ‘the work [they] do by its nature must be biased in emphasis’ (P8). Conversely, most did not see it as reasonable for industry to be funding travel expenses to attend conferences or taking ‘a small bunch of doctors out to a very fancy very expensive restaurant’ (P1). Even those who had previously accepted these payments were sometimes critical. Indeed, those who had received payments that they felt were not commensurate with the amount of work put in described these with some contrition, such as: I haven’t accepted any for six or seven years, and part of that is some minor discomfort around [the ethics of accepting payments]. (P6) It was not clear in these discussions how an appropriate level of compensation should be determined, nor to whom it should be considered appropriate, be they clinicians or patients. Several participants also described a focus of industry largesse on clinicians deemed ‘key opinion leaders’: They pick the opinion leaders. They pick the people that are then going to go and influence the other people. (P12) The nature of disclosure of these payments was discussed by participants with an additional level of nuance. Many felt that the Australian public register of industry payments, administered by the industry trade association Medicines Australia, acted as disincentive for receiving payments (being ‘not a good look’ (P14)), but did not provide sufficient information to be of use. For example, several participants reported passing on their payments to their institution if the remunerated activity occurred during work hours. This was not considered in company reports recorded in the register; some felt that ‘there’s a big difference’ (P11) as this was seen as a way of mitigating any ethical dilemma and, further, providing financial benefit to their institution. Attitudes vary based on forms of interactions Approaches to interactions reflected the participants’ interpretation of each interaction’s value. The most frequent forms of interactions discussed were clinical trials, access programmes and meetings with sales representatives. The findings of the three prior themes—on the transactional nature of relationships, research dependence and ethical risks—informed the development of this theme. Clinical trials Universally, participants expressed confidence in participating in clinical trials run by the pharmaceutical industry. This reflected the willingness of industry to be involved in research, as: …negotiating for more funding with a pharma company is easier because they have more money… (P1) This was generally seen as a core aspect of participants’ jobs. Industry’s role in research was highly valued, and accessing trials was seen as ‘extremely important’ (P13), discussed further below. This was reflected by the confidence clinicians showed in approaching discussions around research. Access programmes Similarly, participants frequently expressed confidence in their interactions around compassionate drug access programmes, with most stating that they: …from time to time, will directly approach representatives of the pharmaceutical companies requesting for compassionate access to their drugs. (P2) While some expressed scepticism about both the putative compassionate nature of these programmes and their opacity, as discussed in our first theme ‘ Transactional nature of relationships ’, no participants felt that access programmes were inappropriate, and most demonstrated confidence in using them, although with understandable difficulties around discussing the co-payments with patients. Sales representatives Conversely, there were varying levels of comfort around interactions with sales representatives. Some felt confident in their ability to interact with sales representatives, while others would ‘usually avoid’ (P15) these relationships, or ‘just say no’ (P7) to meeting with them. Regardless, almost all participants questioned the value of sales representatives, with only a minority sensing any value in their role, and some finding the approach of representatives in building profiles about clinicians ‘really weird’ (P6) or invasive. One participant noted that: They collect information about who’s affiliated with whom, who’s married to whom, and then you get these kinds of comments that come out years down the track, and I found that was, I was peeved by that, actually. (P14) Because of this, most went out of their way to evade sales representatives at least some of the time, noting that they ‘don’t find [them] very valuable’ (P16) or that they ‘barely have time for those meetings’ (P10). The only situations in which sales representatives were seen as beneficial related to discrete periods of solo practice, in which information might be brought to the attention of a clinician that made them think, for example, ‘I didn’t actually know that I could [prescribe a given drug] for X cancer’ (P3). However, this was still met with ethical concern, noting ‘that’s another discomfort that I have with [sales representatives], knowing that I could potentially be impacting my practice [negatively],’ (P3) leading that participant to state they’ve ‘weaned off them’ (P3).
Access programmes, clinical trials and advisory boards Participants generally identified the transactional nature of all relationships with industry, with all beneficial relationships interpreted as having significant caveats. This was considered most pertinent in the context of access programmes, with some participants only maintaining contacts with industry for this purpose: ‘…that relationship [with industry] for the odd patient where you want to try and get access to drugs that you can’t get otherwise’ (participant (P)14). In Australia, cancer drugs found to be acceptably cost-effective are publicly funded under the Pharmaceutical Benefits Scheme, with patients required to pay a co-payment. Access programmes may allow unfunded medicines to be prescribed at either no cost to patients or at a discount on the retail price, with patients being required to contribute a co-payment. While interviewees saw these access programmes as generally beneficial for patients, required co-payments were often considered exorbitant and put cancer physicians in an awkward position with their patients. One noted that companies ‘weren’t generous’ (P7). The opaque nature of these programmes was also seen by some as a way of rewarding preferred clinicians who had provided benefits to the companies, such that these clinicians would learn about the existence of programmes prior to anybody else. Participants also discussed the benefits of these programmes to industry, underscoring their transactional nature. It was clear to most that companies used these programmes to gain useful data and create advocates who could then support the companies’ cases for funding of their drugs under Australia’s Pharmaceutical Benefits Scheme. This was similarly true for trial involvement or membership of advisory boards within companies, where participants felt there was likely to be just as much benefit to the company as clinicians or patients. One participant noted, for example, that while advisory board membership allowed them to ‘interact with people who I greatly respect within my field’, they concurrently ‘from the point of view of a pharmaceutical company, are probably incredibly potent marketing tools’ (P13). Education Interactions that are often proffered as beneficial for clinicians were interpreted with some scepticism. The educational role of industry, for example, both in the context of formal meetings and more broadly within medicine, was frequently considered ‘overstated’ (P6). It was seen as just as likely to benefit companies as clinicians and, accordingly, be potentially detrimental to the latter. Several participants expressed reluctance to attend sponsored education due to inherent biases such as the selection of speakers by sponsors, noting: … that’s how they censor speakers, basically. They pick people who they know will have a positive viewpoint. (P13) Sales representatives Interviewees’ scepticism often extended to the role of sales representatives, with some participants noting specific uncomfortable instances when it had become clear that the relationship was transactional, despite representatives ‘trying to be your bestie’ (P14). Others noted pragmatically that: …[sales representatives] are very pleasant people to interact with, but they are running a business. (P5) There were exceptions to this, with some other clinicians expressing enjoyment at the social aspect of meeting sales representatives, only acknowledging the likely transactional nature of these interactions as a secondary concern. For example: I really enjoy meeting them at third-party events and having social conversations with them. And I like that part of the relationship. (P10)
Participants generally identified the transactional nature of all relationships with industry, with all beneficial relationships interpreted as having significant caveats. This was considered most pertinent in the context of access programmes, with some participants only maintaining contacts with industry for this purpose: ‘…that relationship [with industry] for the odd patient where you want to try and get access to drugs that you can’t get otherwise’ (participant (P)14). In Australia, cancer drugs found to be acceptably cost-effective are publicly funded under the Pharmaceutical Benefits Scheme, with patients required to pay a co-payment. Access programmes may allow unfunded medicines to be prescribed at either no cost to patients or at a discount on the retail price, with patients being required to contribute a co-payment. While interviewees saw these access programmes as generally beneficial for patients, required co-payments were often considered exorbitant and put cancer physicians in an awkward position with their patients. One noted that companies ‘weren’t generous’ (P7). The opaque nature of these programmes was also seen by some as a way of rewarding preferred clinicians who had provided benefits to the companies, such that these clinicians would learn about the existence of programmes prior to anybody else. Participants also discussed the benefits of these programmes to industry, underscoring their transactional nature. It was clear to most that companies used these programmes to gain useful data and create advocates who could then support the companies’ cases for funding of their drugs under Australia’s Pharmaceutical Benefits Scheme. This was similarly true for trial involvement or membership of advisory boards within companies, where participants felt there was likely to be just as much benefit to the company as clinicians or patients. One participant noted, for example, that while advisory board membership allowed them to ‘interact with people who I greatly respect within my field’, they concurrently ‘from the point of view of a pharmaceutical company, are probably incredibly potent marketing tools’ (P13).
Interactions that are often proffered as beneficial for clinicians were interpreted with some scepticism. The educational role of industry, for example, both in the context of formal meetings and more broadly within medicine, was frequently considered ‘overstated’ (P6). It was seen as just as likely to benefit companies as clinicians and, accordingly, be potentially detrimental to the latter. Several participants expressed reluctance to attend sponsored education due to inherent biases such as the selection of speakers by sponsors, noting: … that’s how they censor speakers, basically. They pick people who they know will have a positive viewpoint. (P13)
Interviewees’ scepticism often extended to the role of sales representatives, with some participants noting specific uncomfortable instances when it had become clear that the relationship was transactional, despite representatives ‘trying to be your bestie’ (P14). Others noted pragmatically that: …[sales representatives] are very pleasant people to interact with, but they are running a business. (P5) There were exceptions to this, with some other clinicians expressing enjoyment at the social aspect of meeting sales representatives, only acknowledging the likely transactional nature of these interactions as a secondary concern. For example: I really enjoy meeting them at third-party events and having social conversations with them. And I like that part of the relationship. (P10)
There was a universal acknowledgement by participants of the role industry plays in funding research, though this was connected to an acknowledgement of the risk of bias from industry funding. Most participants identified, either directly or indirectly, that modern cancer research is dependent on the pharmaceutical industry, and no participant was able to see an alternative model for sustainable research funding. The risk of this dependence was clear, with participants noting instances where, for example: …the editorial for a large phase 3 trial is actually written by somebody who sits on the advisory board of the funding body. It’s impossible to say in that scenario that there’s not a level of bias. (P10) While participants were able to identify impressive drugs that could only have been developed with industry funding, some noted that further funding would be skewed towards ‘preferred centres’ (P15) and researchers (both in Australia and abroad), based on the strength of relationships with industry, noting: No one is publishing in NEJM [ New England Journal of Medicine ] with their little investigator-initiated study, right? So, all of those intangible advantages come from building relationships with them. (P15) Participants did not discuss why clinicians may need to produce high-impact publications, such as to maintain academic positions. They also did not explicitly discuss the motivation to seek industry assistance to obtain these high-impact publications. However, participants did report concerns that this funding model meant there were fewer trials focusing on patient care, such as dose reduction studies, studies using older drugs in new contexts or quality-of-life studies. When discussing the ‘profit motive’ (P16) of industry, one participant noted that these clinical questions would remain unresearched, stating that: …that’s the problem [with dependence on industry for research funding], is the gaps and holes, and the other questions that are nothing to do with therapeutics. (P16)
Among all participants, the acceptance of non-research payments from industry was considered ‘just a norm that lots of people [do] ’ (P2). Even for those who refused payments, there was some reluctance to condemn colleagues for doing so for situations that were deemed broadly beneficial to patients, such as membership of advisory boards to guide clinical trial development. In these contexts, participants felt that ‘mostly I see people do it in very good faith’ (P8), even if in doing so, ‘the work [they] do by its nature must be biased in emphasis’ (P8). Conversely, most did not see it as reasonable for industry to be funding travel expenses to attend conferences or taking ‘a small bunch of doctors out to a very fancy very expensive restaurant’ (P1). Even those who had previously accepted these payments were sometimes critical. Indeed, those who had received payments that they felt were not commensurate with the amount of work put in described these with some contrition, such as: I haven’t accepted any for six or seven years, and part of that is some minor discomfort around [the ethics of accepting payments]. (P6) It was not clear in these discussions how an appropriate level of compensation should be determined, nor to whom it should be considered appropriate, be they clinicians or patients. Several participants also described a focus of industry largesse on clinicians deemed ‘key opinion leaders’: They pick the opinion leaders. They pick the people that are then going to go and influence the other people. (P12) The nature of disclosure of these payments was discussed by participants with an additional level of nuance. Many felt that the Australian public register of industry payments, administered by the industry trade association Medicines Australia, acted as disincentive for receiving payments (being ‘not a good look’ (P14)), but did not provide sufficient information to be of use. For example, several participants reported passing on their payments to their institution if the remunerated activity occurred during work hours. This was not considered in company reports recorded in the register; some felt that ‘there’s a big difference’ (P11) as this was seen as a way of mitigating any ethical dilemma and, further, providing financial benefit to their institution.
Approaches to interactions reflected the participants’ interpretation of each interaction’s value. The most frequent forms of interactions discussed were clinical trials, access programmes and meetings with sales representatives. The findings of the three prior themes—on the transactional nature of relationships, research dependence and ethical risks—informed the development of this theme. Clinical trials Universally, participants expressed confidence in participating in clinical trials run by the pharmaceutical industry. This reflected the willingness of industry to be involved in research, as: …negotiating for more funding with a pharma company is easier because they have more money… (P1) This was generally seen as a core aspect of participants’ jobs. Industry’s role in research was highly valued, and accessing trials was seen as ‘extremely important’ (P13), discussed further below. This was reflected by the confidence clinicians showed in approaching discussions around research. Access programmes Similarly, participants frequently expressed confidence in their interactions around compassionate drug access programmes, with most stating that they: …from time to time, will directly approach representatives of the pharmaceutical companies requesting for compassionate access to their drugs. (P2) While some expressed scepticism about both the putative compassionate nature of these programmes and their opacity, as discussed in our first theme ‘ Transactional nature of relationships ’, no participants felt that access programmes were inappropriate, and most demonstrated confidence in using them, although with understandable difficulties around discussing the co-payments with patients. Sales representatives Conversely, there were varying levels of comfort around interactions with sales representatives. Some felt confident in their ability to interact with sales representatives, while others would ‘usually avoid’ (P15) these relationships, or ‘just say no’ (P7) to meeting with them. Regardless, almost all participants questioned the value of sales representatives, with only a minority sensing any value in their role, and some finding the approach of representatives in building profiles about clinicians ‘really weird’ (P6) or invasive. One participant noted that: They collect information about who’s affiliated with whom, who’s married to whom, and then you get these kinds of comments that come out years down the track, and I found that was, I was peeved by that, actually. (P14) Because of this, most went out of their way to evade sales representatives at least some of the time, noting that they ‘don’t find [them] very valuable’ (P16) or that they ‘barely have time for those meetings’ (P10). The only situations in which sales representatives were seen as beneficial related to discrete periods of solo practice, in which information might be brought to the attention of a clinician that made them think, for example, ‘I didn’t actually know that I could [prescribe a given drug] for X cancer’ (P3). However, this was still met with ethical concern, noting ‘that’s another discomfort that I have with [sales representatives], knowing that I could potentially be impacting my practice [negatively],’ (P3) leading that participant to state they’ve ‘weaned off them’ (P3).
Universally, participants expressed confidence in participating in clinical trials run by the pharmaceutical industry. This reflected the willingness of industry to be involved in research, as: …negotiating for more funding with a pharma company is easier because they have more money… (P1) This was generally seen as a core aspect of participants’ jobs. Industry’s role in research was highly valued, and accessing trials was seen as ‘extremely important’ (P13), discussed further below. This was reflected by the confidence clinicians showed in approaching discussions around research.
Similarly, participants frequently expressed confidence in their interactions around compassionate drug access programmes, with most stating that they: …from time to time, will directly approach representatives of the pharmaceutical companies requesting for compassionate access to their drugs. (P2) While some expressed scepticism about both the putative compassionate nature of these programmes and their opacity, as discussed in our first theme ‘ Transactional nature of relationships ’, no participants felt that access programmes were inappropriate, and most demonstrated confidence in using them, although with understandable difficulties around discussing the co-payments with patients.
Conversely, there were varying levels of comfort around interactions with sales representatives. Some felt confident in their ability to interact with sales representatives, while others would ‘usually avoid’ (P15) these relationships, or ‘just say no’ (P7) to meeting with them. Regardless, almost all participants questioned the value of sales representatives, with only a minority sensing any value in their role, and some finding the approach of representatives in building profiles about clinicians ‘really weird’ (P6) or invasive. One participant noted that: They collect information about who’s affiliated with whom, who’s married to whom, and then you get these kinds of comments that come out years down the track, and I found that was, I was peeved by that, actually. (P14) Because of this, most went out of their way to evade sales representatives at least some of the time, noting that they ‘don’t find [them] very valuable’ (P16) or that they ‘barely have time for those meetings’ (P10). The only situations in which sales representatives were seen as beneficial related to discrete periods of solo practice, in which information might be brought to the attention of a clinician that made them think, for example, ‘I didn’t actually know that I could [prescribe a given drug] for X cancer’ (P3). However, this was still met with ethical concern, noting ‘that’s another discomfort that I have with [sales representatives], knowing that I could potentially be impacting my practice [negatively],’ (P3) leading that participant to state they’ve ‘weaned off them’ (P3).
Lack of perceived useful guidance Participants were asked to discuss the role of both ethical guidelines and senior colleagues in managing interactions with industry. Most participants felt that ethical guidelines had little if any value. Even among those who felt they were useful, none had read any guidelines. This was despite the existence of ethical guidelines around industry interactions produced by the Australian Medical Association, the Royal Australasian College of Physicians and the industry association, Medicines Australia. Some participants felt that the guidelines were patronising or written as ‘rules to treat us like Kindergarten kids’ (P11), while others noted that in the absence of regulation, guidelines were likely to remain inconsequential. Most participants acknowledged that senior colleagues influenced more junior practitioners. Despite some participants describing positive experiences in this regard, others felt that this could be detrimental, as: …juniors probably have a better understanding about the ethical issues … than the senior colleagues. (P1) Additional education was also not consistently seen as a solution, though several participants felt there should be more discussions about industry interactions during training. Reduced interactions due to the COVID-19 pandemic This study was conducted during the COVID-19 pandemic. Many participants discussed the pandemic’s effects on these interactions during the interviews. Primarily, participants described COVID-19, and the associated travel restrictions, lockdowns and cancellation of in-person conferences, as providing a welcome buffer between them and industry. It inherently reduced the ability of sales representatives to visit cancer physicians, given: …that they’re finding it a lot more difficult to sort of reach people at this moment. (P2) Several participants expressed this as a relief, particularly among those who tended to avoid these interactions at a baseline. The COVID-19 pandemic was perceived as a ‘natural experiment’ to exclude sales representatives from cancer physicians’ practices, with no participant reporting this as detrimental to either them or their patients. Participants did not discuss the role of online scientific meetings during the COVID-19 pandemic, nor how the shift to online meetings may have affected industry’s influence on content and ability to interact with attendees.
Participants were asked to discuss the role of both ethical guidelines and senior colleagues in managing interactions with industry. Most participants felt that ethical guidelines had little if any value. Even among those who felt they were useful, none had read any guidelines. This was despite the existence of ethical guidelines around industry interactions produced by the Australian Medical Association, the Royal Australasian College of Physicians and the industry association, Medicines Australia. Some participants felt that the guidelines were patronising or written as ‘rules to treat us like Kindergarten kids’ (P11), while others noted that in the absence of regulation, guidelines were likely to remain inconsequential. Most participants acknowledged that senior colleagues influenced more junior practitioners. Despite some participants describing positive experiences in this regard, others felt that this could be detrimental, as: …juniors probably have a better understanding about the ethical issues … than the senior colleagues. (P1) Additional education was also not consistently seen as a solution, though several participants felt there should be more discussions about industry interactions during training.
This study was conducted during the COVID-19 pandemic. Many participants discussed the pandemic’s effects on these interactions during the interviews. Primarily, participants described COVID-19, and the associated travel restrictions, lockdowns and cancellation of in-person conferences, as providing a welcome buffer between them and industry. It inherently reduced the ability of sales representatives to visit cancer physicians, given: …that they’re finding it a lot more difficult to sort of reach people at this moment. (P2) Several participants expressed this as a relief, particularly among those who tended to avoid these interactions at a baseline. The COVID-19 pandemic was perceived as a ‘natural experiment’ to exclude sales representatives from cancer physicians’ practices, with no participant reporting this as detrimental to either them or their patients. Participants did not discuss the role of online scientific meetings during the COVID-19 pandemic, nor how the shift to online meetings may have affected industry’s influence on content and ability to interact with attendees.
Desire for a ‘middle road’ An overarching theme that emerged was the desire for a ‘middle road’ in managing these interactions, with all participants describing a nuance in their industry relationships. While participants were able to identify risks or potentials for harm arising from industry interactions, several described the presence of industry in cancer care as a ‘necessary evil’ (P1, P4, P13, P14). This referred in particular to industry’s fundamental role in drug development and distribution. In general, participants were able to identify some relationships that were unnecessary or unacceptable, such as meeting with sales representatives or receiving excessive remuneration for services, as well as others that were seen as necessary, such as participation in clinical trials and access programmes. The concept of a ‘middle road’ appeared to be an ethically acceptable navigation of the myriad interactions possible. Several participants proposed alternatives to the current industry funding structure that may reduce the risk of bias among recipients. A common proposition was a mixed pool of funding that could be administered and distributed independently of industry. In addition, participants expressed a desire to obtain the benefits of industry interactions, such as drug access programmes, without needing to develop uncomfortable relationships, such as with sales representatives.
An overarching theme that emerged was the desire for a ‘middle road’ in managing these interactions, with all participants describing a nuance in their industry relationships. While participants were able to identify risks or potentials for harm arising from industry interactions, several described the presence of industry in cancer care as a ‘necessary evil’ (P1, P4, P13, P14). This referred in particular to industry’s fundamental role in drug development and distribution. In general, participants were able to identify some relationships that were unnecessary or unacceptable, such as meeting with sales representatives or receiving excessive remuneration for services, as well as others that were seen as necessary, such as participation in clinical trials and access programmes. The concept of a ‘middle road’ appeared to be an ethically acceptable navigation of the myriad interactions possible. Several participants proposed alternatives to the current industry funding structure that may reduce the risk of bias among recipients. A common proposition was a mixed pool of funding that could be administered and distributed independently of industry. In addition, participants expressed a desire to obtain the benefits of industry interactions, such as drug access programmes, without needing to develop uncomfortable relationships, such as with sales representatives.
Key findings Although interactions between Australian cancer physicians and the pharmaceutical industry occur frequently and are in many ways seen as integral to clinical practice, there remained a feeling of unease about specific types of interactions. Some interactions, such as involvement in industry-sponsored research and using access programmes to obtain new drugs for patients, were seen positively. Conversely, interactions where the benefit to the company was much clearer than any benefit to either the involved practitioner or their patients were seen negatively. This distinction was best observed in participants’ views on sales representatives and payments from industry. Most participants interpreted both meeting sales representatives and receiving payments as reasonable if there was a recognisable benefit for patients and, for payments, if the amount received was commensurate with the work involved. When the focus of an industry representative was purely sales, or when payments to a clinician were seen as clearly excessive, participants expressed discomfort or, in some circumstances, overt disapproval. The exact reason for this discomfort was not made clear by participants. No interviewee explicitly articulated whether such meetings provoked an ethical contest per se, but this seems plausible based on their approaches to other forms of interaction. Some of our themes broadly reflect previous research in this area for other populations. An interview study of Australian patient groups’ interactions with industry highlighted the transactional nature of these relationships. Similarly, a study of Irish general practitioners found frequent discomfort around sales interactions. In addition, the framing of interactions that benefit patients as altruistic and therefore without ethical contest is consistent with focus groups with primary care physicians in the USA, France and Canada. However, unlike other groups, cancer physicians in our study may feel they need to maintain relationships with industry to conduct clinical trials or access unfunded drugs while concurrently feeling discomfort about other interactions. The predominance of oncology as a therapeutic area for both drug access programmes and clinical trials has been established previously. Participants frequently dismissed ethical guidelines as a strategy to navigate these interactions. At times, these guidelines were met with open hostility and seen as out of touch with the system in which cancer physicians work. This attitude did not necessarily reflect familiarity with the guidelines’ provisions as no participant discussed having read any guidelines; most assumed that their contents would be unacceptably restrictive. There was therefore no consideration that ethical guidelines could be either inadequate or even flawed in endorsing problematic relationships with industry. Several participants discussed how the COVID-19 pandemic has forced distance from industry, eliminating, although transiently, many of the interactions that were seen as challenging. This was expressed with relief, suggesting that the pandemic has provided a real-world experience of separation from industry. Notably, this dissolution of non-essential contact was not seen as detrimental to patients and, instead, was felt to be a positive experience for cancer physicians. It follows, then, that there was a clear feeling of discontent with the current state of industry relationships. Among the minority of participants with particularly positive experiences, there was minimal understanding of the marketing intent of sales representatives. Yet, even for those who felt confident about their interactions, there were issues identified that could be improved upon or that made these clinicians feel uncomfortable or disparaged by industry. We found, in this way, that some interactions with cancer physicians occur reluctantly and with discomfort on the part of the clinician, and changes could occur to improve these while maintaining benefits for patients. The attitudes expressed in this study were broadly similar to our previous survey of Australian cancer physicians, but in the setting of a qualitative interview, participants were able to expand on their motivations and responses. For example, while we previously determined that most Australian cancer physicians would accept industry payments or funding at some point, we have now shown that the perceived appropriateness of these payments varies based on the perceived benefit to patients or the extent of work being remunerated. Further, while we previously showed frequent interactions between industry and Australian cancer physicians, this study assessed the reasons for and responses to these relationships. We now have a deeper understanding of interactions between cancer physicians and industry, noting circumstances, such as the presence of sales representatives, which are perceived as having little if any value in practice, and others, such as involvement in clinical trials, which are considered beneficial for patients. A previous study by Doran et al classified Australian internal medicine physicians into avoiders, confident engagers and ambivalent engagers when interacting with the pharmaceutical industry. Similarly, a study by Larkin et al on Irish general practitioners described reluctant meeters, anti-meeters and eager meeters. By focusing specifically on cancer physicians, we found that these categories were not applicable, primarily due to the large number of different interactions that cancer physicians report having with industry. Our study created new categories, reflective of the unique challenges faced by cancer physicians in navigating industry interactions. Strengths and limitations To our knowledge, this is the first qualitative study of cancer physicians looking specifically at their relationships with industry. As cancer drugs provide more profit to industry than any other therapeutic group, and increasingly so with an expanding range of medicines, it is vital to understand the interactions of this group of prescribing clinicians. The results of our study may therefore be useful in shaping policies to manage these interactions effectively. As with all qualitative studies in this area, there is a risk that the extent of relationships with industry will be incompletely disclosed due to social desirability bias, particularly with the interviewer being a colleague. However, interviewees may conversely have been more open with a fellow cancer physician, which seems likely based on the breadth of disclosures discussed, so this may be seen as a strength of our study. Using only one author for the majority of coding and thematic development may also bias our results. Another limitation is that our sampling was restricted by the limited pool of those who offered to be contacted in a previous study. This also does not appear to have affected our results in a negative way, as a broad range of opinions and experiences were discussed, to the point that saturation was met. Meaning of results As discussed, the overarching theme within these interviews was a desire among cancer physicians for a ‘middle road’ in navigating interactions, indicative of some level of internal contest, as has been suggested in previous studies. While participants frequently expressed feelings of discontent, they also frequently expressed the need to maintain some connection with industry. Despite this, it was clear that clinicians felt uncomfortable with the current state of these relationships, even when expressing confidence in their own ability to manage interactions. Two issues particularly reflected this. First, several participants described passing payments from industry on to their institutions, with the implication being that they were therefore absolved of influence. In doing so, however, these clinicians ignored the less tangible aspects associated with payments from industry, such as the formation of an ego-boosting relationship with industry and the professional status gain that may exist from redistributing payments to one’s institution. They did not consider the risk of influence from building these relationships over time, regardless of the setting or direct financial benefit. Second, when discussing the potential influence industry interactions may have on prescribing, some participants deferred to the protective role of the Pharmaceutical Benefits Scheme, which independently determines public funding of medicines in Australia. However, this may be seen as a form of diffused responsibility or bystander effect, where individuals may assume responsibility is attributed to others. In doing so, clinicians discounted the increasing competition within classes of drugs available on the Pharmaceutical Benefits Scheme, the ethical issues of partially funded access programmes and the possibility of skewed research findings that may negatively affect patient care. It is conceivable that such a diffusion arose from an avoidance of internal contest. The best way to manage interactions remained unclear, but the total disregard for ethical guidelines was notable, suggesting a need for regulation of these relationships. This study has provided another example of the limits of self-regulation, supporting previous research suggesting regulatory solutions are likely to be more effective at limiting the influence of pharmaceutical and other corporations on clinical practice than voluntary processes. One of our key study findings was participants’ lack of attention and contempt for ethical guidelines. Given these attitudes, it seems unlikely that strengthened ethical guidelines or further education on their use would alter behaviour. Assessment of the effectiveness of similar types of interventions in other physicians is limited. Implications for further research This study has identified several areas that could be altered to improve relationships between industry and cancer physicians. This may include the removal of sales representatives as a presence in clinicians’ lives, or the creation of a centralised database of drug access programmes independent of industry control. Medicines Australia is preparing to launch a database of access programmes in Australia, though it remains to be seen how comprehensive this will be or whether the link between individual companies and clinicians will be broken, given that this programme remains industry controlled. As discussed, the COVID-19 pandemic provided a real-world experiment of reduced contact with industry, and future research should concentrate on the purposeful introduction of similar policies to assess how physicians respond. Based on our analysis, it seems likely these changes would be welcomed by clinicians, although perhaps not by industry.
Although interactions between Australian cancer physicians and the pharmaceutical industry occur frequently and are in many ways seen as integral to clinical practice, there remained a feeling of unease about specific types of interactions. Some interactions, such as involvement in industry-sponsored research and using access programmes to obtain new drugs for patients, were seen positively. Conversely, interactions where the benefit to the company was much clearer than any benefit to either the involved practitioner or their patients were seen negatively. This distinction was best observed in participants’ views on sales representatives and payments from industry. Most participants interpreted both meeting sales representatives and receiving payments as reasonable if there was a recognisable benefit for patients and, for payments, if the amount received was commensurate with the work involved. When the focus of an industry representative was purely sales, or when payments to a clinician were seen as clearly excessive, participants expressed discomfort or, in some circumstances, overt disapproval. The exact reason for this discomfort was not made clear by participants. No interviewee explicitly articulated whether such meetings provoked an ethical contest per se, but this seems plausible based on their approaches to other forms of interaction. Some of our themes broadly reflect previous research in this area for other populations. An interview study of Australian patient groups’ interactions with industry highlighted the transactional nature of these relationships. Similarly, a study of Irish general practitioners found frequent discomfort around sales interactions. In addition, the framing of interactions that benefit patients as altruistic and therefore without ethical contest is consistent with focus groups with primary care physicians in the USA, France and Canada. However, unlike other groups, cancer physicians in our study may feel they need to maintain relationships with industry to conduct clinical trials or access unfunded drugs while concurrently feeling discomfort about other interactions. The predominance of oncology as a therapeutic area for both drug access programmes and clinical trials has been established previously. Participants frequently dismissed ethical guidelines as a strategy to navigate these interactions. At times, these guidelines were met with open hostility and seen as out of touch with the system in which cancer physicians work. This attitude did not necessarily reflect familiarity with the guidelines’ provisions as no participant discussed having read any guidelines; most assumed that their contents would be unacceptably restrictive. There was therefore no consideration that ethical guidelines could be either inadequate or even flawed in endorsing problematic relationships with industry. Several participants discussed how the COVID-19 pandemic has forced distance from industry, eliminating, although transiently, many of the interactions that were seen as challenging. This was expressed with relief, suggesting that the pandemic has provided a real-world experience of separation from industry. Notably, this dissolution of non-essential contact was not seen as detrimental to patients and, instead, was felt to be a positive experience for cancer physicians. It follows, then, that there was a clear feeling of discontent with the current state of industry relationships. Among the minority of participants with particularly positive experiences, there was minimal understanding of the marketing intent of sales representatives. Yet, even for those who felt confident about their interactions, there were issues identified that could be improved upon or that made these clinicians feel uncomfortable or disparaged by industry. We found, in this way, that some interactions with cancer physicians occur reluctantly and with discomfort on the part of the clinician, and changes could occur to improve these while maintaining benefits for patients. The attitudes expressed in this study were broadly similar to our previous survey of Australian cancer physicians, but in the setting of a qualitative interview, participants were able to expand on their motivations and responses. For example, while we previously determined that most Australian cancer physicians would accept industry payments or funding at some point, we have now shown that the perceived appropriateness of these payments varies based on the perceived benefit to patients or the extent of work being remunerated. Further, while we previously showed frequent interactions between industry and Australian cancer physicians, this study assessed the reasons for and responses to these relationships. We now have a deeper understanding of interactions between cancer physicians and industry, noting circumstances, such as the presence of sales representatives, which are perceived as having little if any value in practice, and others, such as involvement in clinical trials, which are considered beneficial for patients. A previous study by Doran et al classified Australian internal medicine physicians into avoiders, confident engagers and ambivalent engagers when interacting with the pharmaceutical industry. Similarly, a study by Larkin et al on Irish general practitioners described reluctant meeters, anti-meeters and eager meeters. By focusing specifically on cancer physicians, we found that these categories were not applicable, primarily due to the large number of different interactions that cancer physicians report having with industry. Our study created new categories, reflective of the unique challenges faced by cancer physicians in navigating industry interactions.
To our knowledge, this is the first qualitative study of cancer physicians looking specifically at their relationships with industry. As cancer drugs provide more profit to industry than any other therapeutic group, and increasingly so with an expanding range of medicines, it is vital to understand the interactions of this group of prescribing clinicians. The results of our study may therefore be useful in shaping policies to manage these interactions effectively. As with all qualitative studies in this area, there is a risk that the extent of relationships with industry will be incompletely disclosed due to social desirability bias, particularly with the interviewer being a colleague. However, interviewees may conversely have been more open with a fellow cancer physician, which seems likely based on the breadth of disclosures discussed, so this may be seen as a strength of our study. Using only one author for the majority of coding and thematic development may also bias our results. Another limitation is that our sampling was restricted by the limited pool of those who offered to be contacted in a previous study. This also does not appear to have affected our results in a negative way, as a broad range of opinions and experiences were discussed, to the point that saturation was met.
As discussed, the overarching theme within these interviews was a desire among cancer physicians for a ‘middle road’ in navigating interactions, indicative of some level of internal contest, as has been suggested in previous studies. While participants frequently expressed feelings of discontent, they also frequently expressed the need to maintain some connection with industry. Despite this, it was clear that clinicians felt uncomfortable with the current state of these relationships, even when expressing confidence in their own ability to manage interactions. Two issues particularly reflected this. First, several participants described passing payments from industry on to their institutions, with the implication being that they were therefore absolved of influence. In doing so, however, these clinicians ignored the less tangible aspects associated with payments from industry, such as the formation of an ego-boosting relationship with industry and the professional status gain that may exist from redistributing payments to one’s institution. They did not consider the risk of influence from building these relationships over time, regardless of the setting or direct financial benefit. Second, when discussing the potential influence industry interactions may have on prescribing, some participants deferred to the protective role of the Pharmaceutical Benefits Scheme, which independently determines public funding of medicines in Australia. However, this may be seen as a form of diffused responsibility or bystander effect, where individuals may assume responsibility is attributed to others. In doing so, clinicians discounted the increasing competition within classes of drugs available on the Pharmaceutical Benefits Scheme, the ethical issues of partially funded access programmes and the possibility of skewed research findings that may negatively affect patient care. It is conceivable that such a diffusion arose from an avoidance of internal contest. The best way to manage interactions remained unclear, but the total disregard for ethical guidelines was notable, suggesting a need for regulation of these relationships. This study has provided another example of the limits of self-regulation, supporting previous research suggesting regulatory solutions are likely to be more effective at limiting the influence of pharmaceutical and other corporations on clinical practice than voluntary processes. One of our key study findings was participants’ lack of attention and contempt for ethical guidelines. Given these attitudes, it seems unlikely that strengthened ethical guidelines or further education on their use would alter behaviour. Assessment of the effectiveness of similar types of interventions in other physicians is limited.
This study has identified several areas that could be altered to improve relationships between industry and cancer physicians. This may include the removal of sales representatives as a presence in clinicians’ lives, or the creation of a centralised database of drug access programmes independent of industry control. Medicines Australia is preparing to launch a database of access programmes in Australia, though it remains to be seen how comprehensive this will be or whether the link between individual companies and clinicians will be broken, given that this programme remains industry controlled. As discussed, the COVID-19 pandemic provided a real-world experiment of reduced contact with industry, and future research should concentrate on the purposeful introduction of similar policies to assess how physicians respond. Based on our analysis, it seems likely these changes would be welcomed by clinicians, although perhaps not by industry.
Australian cancer physicians have numerous interactions with industry, with attitudes towards these interactions usually reflecting their perceived benefits for patients, but frequent discomfort expressed with some forms of interactions. Most clinicians could identify interactions they felt were problematic, and there was a general desire for a ‘middle road’ approach to managing these. More research is needed to determine the acceptability of potential management strategies to clinicians.
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Educational readiness among health professionals in rheumatology: low awareness of EULAR offerings and unfamiliarity with the course content as major barriers—results of a EULAR-funded European survey
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Health professionals in rheumatology (HPR) are a heterogeneous group of professionals with different roles, responsibilities and scopes of practice. Their training differs depending on the level of professional qualification and varying health systems across countries. Educational needs of HPR differ considerably across countries and professions. While health professionals in rheumatology (HPR) in Europe have a solid knowledge about the existence of the European Alliance of Associations for Rheumatology (EULAR), they have little awareness about its educational offerings: approximately 90% do not know the ‘Teach the Teacher Course’, up to 80% are not aware of travel bursaries. English as the primary language in educational offerings of EULAR is a significant obstacle for more than 50% of HPR whose mother tongue is not English. Even among HPR who, ‘felt comfortable taking a course in English’, would prefer educational offerings in the national language (52%). Higher educated HPR and HPR with more extended work experience are more likely to attend the annual EULAR congress, whereas HPR with less work experience are more likely to take up EULAR School of Rheumatology offerings. Better-educated HPRs provide better care. Increasing awareness and use of EULAR educational offerings can increase up-to-date knowledge of HPR, promote research and collaboration and, ultimately, contribute to better patient outcomes. Active involvement of national HPR organisations is needed to ensure the leading role of EULAR in educational opportunities in rheumatology. Millions of people worldwide lack access to high-quality healthcare because of shortages, the uneven geographical distribution of service provision and not sufficiently trained health professionals. Higher education of health professionals contributes to better patient outcomes; a study even demonstrated that a higher number of nurses with a bachelor’s degree (compared with a diploma or an associate degree) reduced hospital mortality rates. To ensure that health professionals continue their education and training over a more extended period of their working life and, thus, contribute to a high quality of healthcare, many countries decided to make continuing education mandatory. As a result, health professionals are increasingly required to participate in continuing education after completing their basic training. Health professionals in rheumatology (HPR) play a critical role in the care of people with rheumatic and musculoskeletal diseases (RMDs). HPR are from different professions and include nurses, occupational therapists, physical therapists, social workers, psychologists, pharmacists and others. The EULAR definition of HPR does not include registered medical practitioners. Basic training varies across countries, and harmonised postgraduate education could guarantee that patients with similar diseases receive similar quality of care in different countries. Vliet Vlieland et al conducted an educational needs survey in 2015, being the first inventory of the educational needs of HPR in Europe. However, after the EULAR School of Rheumatology (ESOR) was launched in 2017, the educational needs of HPR were not reassessed, and EULAR’s new educational offerings for HPR have not yet been evaluated. In addition, changes in the legislation in some countries, such as the mandatory registration (and reregistrations) of HPR in Austria, Croatia, Cyprus, Ireland, Serbia, the UK and others and the need for accreditation of postgraduate courses in some countries, may also have led to changes in educational requirements. Compared to 2015, national offerings may have also changed over time. Moreover, the COVID-19 pandemic has significantly changed some of the didactic preferences of postgraduate education. For example, it has made online access to high-level specialists, unbound by time and space, more widely accepted. This study aimed to (1) determine current HPR educational readiness, needs and preferences, (2) identify barriers to taking part in postgraduate education and (3) ask for feedback on the current offerings and activities of ESOR for HPR. Design and participants The Educational Subcommittee of the EULAR Committee of HPR, in collaboration with the EULAR Committee of Education and Training and the Paediatric Rheumatology European Society, developed an online survey. The questionnaire was adapted from the 2015 version by Vliet Vlieland et al and extended to include questions asking for feedback on current courses. The following main topics were covered in the survey: (1) characteristics of the respondents, current educational needs in terms of clinical practice and theoretical knowledge, RMDs that should be addressed in the courses, wishes and expectations regarding the organisation of EULAR/ESOR courses, (2) barriers for participation in the courses, familiarity and awareness of the educational offerings from EULAR/ESOR, and (3) feedback on the current educational offerings of EULAR/ESOR. The online survey was distributed using a free software programme ( www.soscisurvey.com ). It was tested in advance by the Educational Sub-Committee of the EULAR Committee of HPR, and the feedback was used to adapt the questionnaire. We intended to distribute the questionnaire in as many European countries as possible. For this reason, we translated the questionnaire into 24 languages (Czech, Croatian, Danish, Dutch, English, Estonian, Finnish, French, German, Greek, Hungarian, Italian, Norwegian, Polish, Portuguese, Romanian, Russian, Serbian, Slovak, Slovenian, Spanish, Swedish, Turkish and Ukrainian). With these languages, we could cover the 25 national member organisations of EULAR and, at the same time, the 20 most populous European countries. Each translation was peer debriefed by at least two native-speaking HPR. The English version of the questionnaire is found in the supplement . The entire questionnaire, with all translations and response options, can be requested by the corresponding author. 10.1136/rmdopen-2023-003120.supp1 Supplementary data The questionnaire was distributed at the end of June 2021 via networks of the national professional organisations, the EULAR HPR Newsletter, participants in previous EULAR HPR activities, national liaison persons, national HPR associations, universities and other educational institutions with a request to forward it to all health professionals in the field of rheumatology. A similar invitation/reminder to participate in the questionnaire was sent in September 2021. Reporting of the results followed the ‘Checklist for Reporting Results of Internet E-Surveys’ guideline. Data analysis The responses of all participants were recorded anonymously. Absolute frequencies and percentages were given for categorical data. Ordinal variables were reported with median and IQR, metric variables with mean and SD as well as median and IQR, both for the whole study population and subgroups (HPR in adult care (HPR-A) and HPR in paediatric care (HPR-P)). As a final step, we conducted subgroup analyses for the three most prominent professional groups (nurses, physiotherapists, occupational therapists) and the North, South, East and Central European regions. We used natural language processing and the Latent Dirichlet Allocation (LDA) to analyse the questionnaire’s free text fields (feedback on current EULAR/ESOR offerings). LDA is a technique used for unsupervised generative probabilistic topic modelling. It aims to extract the meanings of a predefined number of topics. Each topic is characterised by high-frequency words and words that are best suited to distinguish it from other topics. To perform LDA, we created a semantic space by downloading and translating all the answers into English. Then, we took the following steps: stemming the words, removing stop words, casting the text into lowercase only and removing punctuations, names and personal references. We then explored the frequency of words and correlations between the co-occurrence of words and extracted five topics based on LDA. The number of topics was determined by examining the heatmaps and the coherence of the words in the topics. Heatmaps are a popular graphical method for visualising high-dimensional data. In a heatmap, tables of data are coded as coloured cells. The matrix’s rows and columns are arranged, so patterns become highlighted. The dendrogram reflects the inter-relationships of the topics and shows similarities between responses. Regression analysis We used multiple logistic regression models to determine higher postgraduate educational readiness factors. We operationalised educational readiness by means of four variables: first, in terms of knowledge (Have you ever heard of EULAR/ESOR?) and second, in terms of action taken (Have you ever attended the annual EULAR Congress/ESOR offerings?). The independent variables were selected from participants’ personal data (age, gender, occupation, work with children/adults, the highest level of education completed, work experience with people with RMD and time spent working in clinical patient care/research/organisational roles). All variables were tested for multicollinearity prior to the regression analysis. All analyses were conducted using R ( http://www.r-project.org ). The Educational Subcommittee of the EULAR Committee of HPR, in collaboration with the EULAR Committee of Education and Training and the Paediatric Rheumatology European Society, developed an online survey. The questionnaire was adapted from the 2015 version by Vliet Vlieland et al and extended to include questions asking for feedback on current courses. The following main topics were covered in the survey: (1) characteristics of the respondents, current educational needs in terms of clinical practice and theoretical knowledge, RMDs that should be addressed in the courses, wishes and expectations regarding the organisation of EULAR/ESOR courses, (2) barriers for participation in the courses, familiarity and awareness of the educational offerings from EULAR/ESOR, and (3) feedback on the current educational offerings of EULAR/ESOR. The online survey was distributed using a free software programme ( www.soscisurvey.com ). It was tested in advance by the Educational Sub-Committee of the EULAR Committee of HPR, and the feedback was used to adapt the questionnaire. We intended to distribute the questionnaire in as many European countries as possible. For this reason, we translated the questionnaire into 24 languages (Czech, Croatian, Danish, Dutch, English, Estonian, Finnish, French, German, Greek, Hungarian, Italian, Norwegian, Polish, Portuguese, Romanian, Russian, Serbian, Slovak, Slovenian, Spanish, Swedish, Turkish and Ukrainian). With these languages, we could cover the 25 national member organisations of EULAR and, at the same time, the 20 most populous European countries. Each translation was peer debriefed by at least two native-speaking HPR. The English version of the questionnaire is found in the supplement . The entire questionnaire, with all translations and response options, can be requested by the corresponding author. 10.1136/rmdopen-2023-003120.supp1 Supplementary data The questionnaire was distributed at the end of June 2021 via networks of the national professional organisations, the EULAR HPR Newsletter, participants in previous EULAR HPR activities, national liaison persons, national HPR associations, universities and other educational institutions with a request to forward it to all health professionals in the field of rheumatology. A similar invitation/reminder to participate in the questionnaire was sent in September 2021. Reporting of the results followed the ‘Checklist for Reporting Results of Internet E-Surveys’ guideline. The responses of all participants were recorded anonymously. Absolute frequencies and percentages were given for categorical data. Ordinal variables were reported with median and IQR, metric variables with mean and SD as well as median and IQR, both for the whole study population and subgroups (HPR in adult care (HPR-A) and HPR in paediatric care (HPR-P)). As a final step, we conducted subgroup analyses for the three most prominent professional groups (nurses, physiotherapists, occupational therapists) and the North, South, East and Central European regions. We used natural language processing and the Latent Dirichlet Allocation (LDA) to analyse the questionnaire’s free text fields (feedback on current EULAR/ESOR offerings). LDA is a technique used for unsupervised generative probabilistic topic modelling. It aims to extract the meanings of a predefined number of topics. Each topic is characterised by high-frequency words and words that are best suited to distinguish it from other topics. To perform LDA, we created a semantic space by downloading and translating all the answers into English. Then, we took the following steps: stemming the words, removing stop words, casting the text into lowercase only and removing punctuations, names and personal references. We then explored the frequency of words and correlations between the co-occurrence of words and extracted five topics based on LDA. The number of topics was determined by examining the heatmaps and the coherence of the words in the topics. Heatmaps are a popular graphical method for visualising high-dimensional data. In a heatmap, tables of data are coded as coloured cells. The matrix’s rows and columns are arranged, so patterns become highlighted. The dendrogram reflects the inter-relationships of the topics and shows similarities between responses. We used multiple logistic regression models to determine higher postgraduate educational readiness factors. We operationalised educational readiness by means of four variables: first, in terms of knowledge (Have you ever heard of EULAR/ESOR?) and second, in terms of action taken (Have you ever attended the annual EULAR Congress/ESOR offerings?). The independent variables were selected from participants’ personal data (age, gender, occupation, work with children/adults, the highest level of education completed, work experience with people with RMD and time spent working in clinical patient care/research/organisational roles). All variables were tested for multicollinearity prior to the regression analysis. All analyses were conducted using R ( http://www.r-project.org ). Response rate In total, the questionnaire was viewed 3589 times, and 998 times it was started to be filled in. Of these, 667 HPR (66.8%) from 34 European countries completed the questionnaire ( and ). Participant characteristics The respondents were mainly women (n=762; 76.4%), and the mean age was 40.53 (±11.5) years. More than 50% of the participants had a bachelor’s degree or similar, 24.9% (n=245) had a master’s degree and 9.1% (n=90) had a PhD . Physiotherapists (n=350; 34.8%), nurses (n=248; 25.0%) and occupational therapists (n=189; 18.8%) were the most frequent professions who responded to the questionnaire. Of all responses, n=913 (91.5%) came from HPR working with adults, and n=85 (8.5%) from HPR in children’s/youth’s care. Educational readiness In terms of knowledge, familiarity with EULAR or ESOR was mainly associated with being a nurse, being older (in years), having more experience in the field of rheumatology, having a formally higher level of education and being more involved in research. We observed a similar pattern in participation at the annual EULAR congress. Nurses and HPR with higher levels of education and increased research activities were also associated with taking part in the congress. This result contrasts with attendance in ESOR courses. In particular, people with less work experience seem to feel more addressed by the offer than more experienced people (OR 0.91, CI 95% 0.81 to 0.99) . 10.1136/rmdopen-2023-003120.supp3 Supplementary data Educational needs: differences between health professionals in adult and paediatric care The highest-rated educational need in terms of clinical practice was professional development for paediatric HPR (3.91; on a scale of 1 to 5) and prevention for HPR in adult care (3.70). ‘Clinical characteristics’ were rated as the most important theoretical knowledge for both HPR-A and HPR-P (3.62 and 4.00, respectively). Practice organisation and management were rated lowest by HPR-A (3.08), and training in diagnostic assessments by HPR-P (2.78). According to the ratings provided by HPR-A and HPR-P, the RMDs that need to be covered in the courses received all high scores, ranging from 3.76 to 4.49 . Course organisation: live courses and prerecorded (online) lectures were preferred Live courses taking place face-to-face (n=463; 38.6%) and prerecorded online lectures accessible without time constraints (n=338; 28.2%) were preferred over other modalities. Almost half of the participants (n=377; 44.0%) considered a course duration of 1–2 days optimal, and two-thirds of HPR preferred the organisation of the courses by EULAR compared with national organisations. However, following the responses of the HPR, offerings should be available in national languages rather than in English . Even among HPR who said they ‘felt comfortable taking a course in English’ (n=419), more than half would still prefer courses in the national language (n=219; 52.3%). Barriers: lack of awareness of educational offerings and content Lack of awareness of the educational offerings (3.30; ±1.49) and lack of knowledge of the content of the offerings (3.51; ±1.50) were mentioned as the most important barriers to non-participation in EULAR/ESOR educational offerings (on a scale of 1–5). Likewise, the participants perceived the costs of ESOR courses (3.29; ±1.36) and the annual congress (3.46; ±1.34) as too high. Participants reported that despite their interest in EULAR’s educational offerings, they did not receive support in the form of time resources during work hours from their employers to attend the courses (3.32; ±1.39). The technical requirements for participation in an online course, such as the availability of computers, laptops or tablets, and an internet connection, were not considered obstacles to participation in the courses or classes . Feedback on the current offerings of ESOR for HPR Although almost two-thirds of HPR-A and half of HPR-P were familiar with EULAR, they reported little awareness or use of EULAR’s offerings for HPR. Three-quarters of HPR only had limited or no knowledge of travel bursaries, and 85% (HPR-A), respectively, 97% (HPR-P), were not aware of the Teach the Teacher Course. Sixty to 80% have never visited the EULAR/ESOR communication platforms, and 73.9% (HPR-A), respectively 90.7% (HPR-P) had never participated in the annual EULAR congress . Using LDA, we generated two topics from positive feedback and three from negative feedback ( and ). The comprehensive and up-to-date course content, along with the flexibility to attend classes without any time restrictions, were highly valued, resulting in overall positive responses ( online courses offer time flexibility and the possibility to complete the course at your own pace ). Barriers to participation in training were: uncertainty if there would be a profession-specific gain from participating in interdisciplinary courses ( The online course should have a clear benefit for the respective professional groups, even if it is a multidisciplinary course ), language limitations during the classes ( English is challenging in classes, especially when you want to ask questions ) and problems with the English language in the final exams ( English is challenging, especially in the exams ). 10.1136/rmdopen-2023-003120.supp2 Supplementary data Differences in educational preferences but not the delivery method As expected, our subgroup analysis revealed differences in educational preferences related to course content across professions and countries. However, we found surprisingly limited variation in the preferences for the delivery method of educational offerings. The most common selection for live/on-site courses lasting 1–2 days and being conducted in the national language remained consistent across professions and countries. The only tendency found (not significant, p=0.056) was that Eastern countries favoured discipline-specific content (37.1% to 34.3% multidisciplinary) compared with the other countries. Nurses were more likely to attend the EULAR Congress (36.3%) than other professional groups (PT 16.9%, OT 12.6%; OR 2.35 CI 95% 0.98 to 5.87, p=0.05). Detailed results of the subgroup analysis are found in appendices E-I and J-O. In total, the questionnaire was viewed 3589 times, and 998 times it was started to be filled in. Of these, 667 HPR (66.8%) from 34 European countries completed the questionnaire ( and ). The respondents were mainly women (n=762; 76.4%), and the mean age was 40.53 (±11.5) years. More than 50% of the participants had a bachelor’s degree or similar, 24.9% (n=245) had a master’s degree and 9.1% (n=90) had a PhD . Physiotherapists (n=350; 34.8%), nurses (n=248; 25.0%) and occupational therapists (n=189; 18.8%) were the most frequent professions who responded to the questionnaire. Of all responses, n=913 (91.5%) came from HPR working with adults, and n=85 (8.5%) from HPR in children’s/youth’s care. In terms of knowledge, familiarity with EULAR or ESOR was mainly associated with being a nurse, being older (in years), having more experience in the field of rheumatology, having a formally higher level of education and being more involved in research. We observed a similar pattern in participation at the annual EULAR congress. Nurses and HPR with higher levels of education and increased research activities were also associated with taking part in the congress. This result contrasts with attendance in ESOR courses. In particular, people with less work experience seem to feel more addressed by the offer than more experienced people (OR 0.91, CI 95% 0.81 to 0.99) . 10.1136/rmdopen-2023-003120.supp3 Supplementary data The highest-rated educational need in terms of clinical practice was professional development for paediatric HPR (3.91; on a scale of 1 to 5) and prevention for HPR in adult care (3.70). ‘Clinical characteristics’ were rated as the most important theoretical knowledge for both HPR-A and HPR-P (3.62 and 4.00, respectively). Practice organisation and management were rated lowest by HPR-A (3.08), and training in diagnostic assessments by HPR-P (2.78). According to the ratings provided by HPR-A and HPR-P, the RMDs that need to be covered in the courses received all high scores, ranging from 3.76 to 4.49 . Live courses taking place face-to-face (n=463; 38.6%) and prerecorded online lectures accessible without time constraints (n=338; 28.2%) were preferred over other modalities. Almost half of the participants (n=377; 44.0%) considered a course duration of 1–2 days optimal, and two-thirds of HPR preferred the organisation of the courses by EULAR compared with national organisations. However, following the responses of the HPR, offerings should be available in national languages rather than in English . Even among HPR who said they ‘felt comfortable taking a course in English’ (n=419), more than half would still prefer courses in the national language (n=219; 52.3%). Lack of awareness of the educational offerings (3.30; ±1.49) and lack of knowledge of the content of the offerings (3.51; ±1.50) were mentioned as the most important barriers to non-participation in EULAR/ESOR educational offerings (on a scale of 1–5). Likewise, the participants perceived the costs of ESOR courses (3.29; ±1.36) and the annual congress (3.46; ±1.34) as too high. Participants reported that despite their interest in EULAR’s educational offerings, they did not receive support in the form of time resources during work hours from their employers to attend the courses (3.32; ±1.39). The technical requirements for participation in an online course, such as the availability of computers, laptops or tablets, and an internet connection, were not considered obstacles to participation in the courses or classes . Although almost two-thirds of HPR-A and half of HPR-P were familiar with EULAR, they reported little awareness or use of EULAR’s offerings for HPR. Three-quarters of HPR only had limited or no knowledge of travel bursaries, and 85% (HPR-A), respectively, 97% (HPR-P), were not aware of the Teach the Teacher Course. Sixty to 80% have never visited the EULAR/ESOR communication platforms, and 73.9% (HPR-A), respectively 90.7% (HPR-P) had never participated in the annual EULAR congress . Using LDA, we generated two topics from positive feedback and three from negative feedback ( and ). The comprehensive and up-to-date course content, along with the flexibility to attend classes without any time restrictions, were highly valued, resulting in overall positive responses ( online courses offer time flexibility and the possibility to complete the course at your own pace ). Barriers to participation in training were: uncertainty if there would be a profession-specific gain from participating in interdisciplinary courses ( The online course should have a clear benefit for the respective professional groups, even if it is a multidisciplinary course ), language limitations during the classes ( English is challenging in classes, especially when you want to ask questions ) and problems with the English language in the final exams ( English is challenging, especially in the exams ). 10.1136/rmdopen-2023-003120.supp2 Supplementary data As expected, our subgroup analysis revealed differences in educational preferences related to course content across professions and countries. However, we found surprisingly limited variation in the preferences for the delivery method of educational offerings. The most common selection for live/on-site courses lasting 1–2 days and being conducted in the national language remained consistent across professions and countries. The only tendency found (not significant, p=0.056) was that Eastern countries favoured discipline-specific content (37.1% to 34.3% multidisciplinary) compared with the other countries. Nurses were more likely to attend the EULAR Congress (36.3%) than other professional groups (PT 16.9%, OT 12.6%; OR 2.35 CI 95% 0.98 to 5.87, p=0.05). Detailed results of the subgroup analysis are found in appendices E-I and J-O. This study aimed to identify the educational needs of HPR in paediatric and adult care. For the first time, we anonymously asked for feedback on the current educational offerings of EULAR/ESOR in 34 countries and described current barriers to the attendance of EULAR/ESOR offerings. It is already widely recognised that the successful implementation of postgraduate HPR education is of great importance to ‘increase the quantity, quality and relevance of health professionals, and in so doing strengthen the country health systems and improve population health outcomes’. Our study’s two most important findings were that (1) EULAR only succeeds in reaching a minority of HPRs in Europe and (2) that services do not appeal equally to the broad range and different educational levels of HPRs. Our study found that higher age, more professional experience, being a nurse, and a higher level of education contributed significantly to the knowledge of EULAR and ESOR and attendance at the annual EULAR congress. In terms of higher education level, we were able to show, for example, that having a PhD increases the likelihood of knowing about ESOR (OR 3.70) and whether one has ever attended the EULAR congress (OR 7.50). Accordingly, the EULAR congress is (more) attractive to older and formally more educated HPR. However, we observed a contrasting picture with the ESOR offerings. The courses were more likely to be attended by HPRs with less experience in the field of rheumatology (OR 0.91). Several strategies can be derived from our findings on how to achieve a better use of educational offerings among HPR in Europe. Our findings suggest that several strategies can be implemented to enhance the utilisation of educational offerings among HPR in Europe. For instance, educational providers could target HPR at earlier stages of their career or those with less formal education by reviewing their existing offerings to identify which groups of HPR are being addressed. This would enable providers to develop tailored educational programmes that meet the specific needs of these groups, thereby increasing their engagement and participation. To address the varying educational qualification levels of HPR in Europe, educational courses could be developed at different levels to lower the access barrier for early career HPR while also providing more formally educated and later career HPR with opportunities to continue their education at a higher level. This could be achieved by involving national organisations in disseminating educational offerings through their networks in national languages. Strengthening these organisations would increase the accessibility of educational opportunities to HPR in different regions of Europe. To minimise costs for EULAR while enhancing the utilisation of its course offerings in national countries, a ‘franchise’ model could be considered in addition to the teach-the-teacher courses. Under this model, EULAR would act as a ‘franchisor’ and permit national organisations to use its brand and course offerings. In exchange, national organisations would agree to pay a franchise fee to EULAR. Another important finding of our survey was that English as an educational and examination language is a major barrier for HPR, whose native language is not English. Between 40% and 55% of all respondents indicated they did not feel comfortable taking a course in English. This problem was particularly cited when HPR were under time pressure and had to ask questions or take an exam in English. Even among HPR who reported having sufficient English competence to feel comfortable taking a course in English, more than half would still prefer their national language. HPR from northern countries (Estonia, Denmark, Finland, Norway, Sweden) were more likely to consider themselves advanced or fluent in English than HPR from other countries; however, the majority of them (58.8%) still preferred their national language. Therefore, one feasible approach to increase attendance in EULAR courses could be to translate the content (and exams) or offer courses with subtitles in the national language. English as a language was also identified in the first Education Needs Survey as an important barrier to using EULAR offerings. Apparently, this has not changed in the last 7 years. One lesson learnt from the survey is that EULAR messages/evidence may need to be translated into other languages to facilitate their implementation in clinical care across Europe. A strength of our study was that we also analysed the free text entries (positive and negative feedback to the ESOR classes) with a natural language processing tool. We applied an unsupervised generative probabilistic method specifically designed for short text inputs, such as those typically found in questionnaires. The results of our project go beyond the interests of EULAR and ESOR. Although the findings are initially based on EULAR’s existing educational programme, they can also be useful for national education providers, for example, with regards to preferences in course content or delivery methods. We are aware that our study has certain limitations. Although we translated the questionnaire into 24 languages, we could not exceed the overall response rate of the 2015 survey. Our study had limited representation from northern and eastern European countries. One reason may have been the timing during the (summer) vacations and/or the COVID-19 pandemic, which limited overall opportunities for training and education and may have changed the priorities for HPR. The results of our survey show that the proportions of some professions were different across the countries. In Portugal and Denmark, for example, more nurses participated compared with other countries, such as Austria or Italy. Such differences could limit the generalisability of our results. In addition, we had some missing data because it was not made mandatory to answer all questions in the survey. We decided not to make the responses mandatory because we wanted to give HPR as much freedom as possible in responding to the questions. However, the missing data could, of course, distort the results. Due to the study design and the partly unequally distributed responses in terms of country and profession, the sample’s representativeness is not given, and the results should be generalised only with caution. HPR is often involved in supporting people with RMD in managing their disease. Their knowledge directly impacts people’s well-being and is critical to utilising other health resources. Therefore, ongoing education and training for the various healthcare professions are essential to ensure optimal care. Improving awareness of educational offers among national organisations and the challenge of reducing costs and language barriers are points of attention to promote future dissemination. EULAR and other international postgraduate training providers could use a ‘franchise’ model of their offerings tailored to local contexts. 10.1136/rmdopen-2023-003120.supp4 Supplementary data 10.1136/rmdopen-2023-003120.supp5 Supplementary data 10.1136/rmdopen-2023-003120.supp6 Supplementary data
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Occurrence of adverse events and change in disease activity after initiation of etanercept in paediatric patients with juvenile psoriatic arthritis in the CARRA Registry
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3f9691ea-9f82-40b2-a6f0-961ff71bf7e0
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10230983
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Internal Medicine[mh]
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Juvenile psoriatic arthritis (JPsA) is one of seven categories of juvenile idiopathic arthritis (JIA) and constitutes approximately 5% of JIA. Etanercept is commonly used to treat JIA, including JPsA; however, information on the safety and effectiveness of etanercept in treatment of patients with JPsA in real-world clinical practice is limited. Here, we evaluated etanercept’s safety and effectiveness in JPsA using data from the Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry, which contains data from over 10 000 children with JIA.
Results from analysis of data in the CARRA Registry showed that etanercept treatment in JPsA was effective at 6 months of follow-up and remained effective at 12 months, with low rates of adverse events of special interest (including malignancy and uveitis) and serious adverse events. The analysis also showed that etanercept dosing for JPsA was consistent with the product label dose for JIA of 0.8 mg/kg weekly, with a maximum of 50 mg/week.
These results inform treatment choice for JPsA, supporting the use of etanercept as a safe and effective treatment option in this disease setting, including in children under 12 years old, an age group for whom there is no drug approval.
Juvenile psoriatic arthritis (JPsA) is one of seven categories of juvenile idiopathic arthritis (JIA) and constitutes approximately 5% of JIA. According to the International League of Associations for Rheumatology (ILAR), JPsA is classified by chronic arthritis before age 16 years, which is associated with either psoriasis or at least two of the following: dactylitis, nail pitting, onycholysis or psoriasis in a first-degree relative. JPsA has a bimodal distribution based on age at onset. JPsA occurring in children 1–4 years of age is categorised as early onset and typically manifests as peripheral arthritis involving a few joints, whereas JPsA occurring in children over 4 years of age is categorised as older onset and more commonly has features of adult psoriatic arthritis, including spondyloarthritis with increased risk of axial joint involvement and enthesitis. Dactylitis and uveitis are common clinical features of both early-onset and older-onset JPsA. Prior to the approval of secukinumab for treatment of JPsA in December 2021, etanercept was the only biologic treatment approved for JPsA (in children over 12 years old) in the European Union ; however, etanercept is not yet approved for JPsA in the USA. The low incidence of JPsA makes systematic collection of data on treatment outcomes challenging. As such, information on the safety and effectiveness of etanercept in treatment of patients with JPsA in real-world clinical practice is limited. The Childhood Arthritis and Rheumatology Research Alliance (CARRA) Registry is comprised of paediatric rheumatology research centres dedicated to advancing the field of paediatric rheumatology and contains data from over 10 000 children with JIA. In this study, we analysed data from the CARRA Registry to evaluate the safety and effectiveness of etanercept in JPsA.
Data source, study design and patient population The CARRA Registry was started in 2015. The general design and rationale of the registry and the characteristics of patients enrolled have been previously described in detail. Briefly, the CARRA Registry is an international observational registry of paediatric patients with rheumatic diseases, including JIA. At the inception of the registry, there was selective enrolment of paediatric patients who were most likely to be treated with biologics. The registry includes retrospective data collected at the time of enrolment and prospective observational data collected approximately every 6 months at patient visits in the context of routine clinical care and ideally at the time of initiation of any new JIA medication. Data collected include physician-assigned ILAR category, detailed medication logs, clinical features, laboratory data, imaging results and adverse events of special interest (AESIs), including serious adverse events (SAEs). The CARRA Registry also collects the clinical Juvenile Arthritis Disease Activity Score with an active joint count up to 10 (clinical Juvenile Arthritis Disease Activity Score 10-joint (cJADAS-10)), patient-reported outcomes, Childhood Health Assessment Questionnaire, the patient/parent global assessment and pain intensity. The present analysis used CARRA Registry data from approximately 70 clinical sites in the USA and Canada beginning 30 June 2015 until the data cut-off date for this analysis of 2 August 2021. Data were analysed for patients aged ≥2–<18 years at etanercept initiation who had a JPsA diagnosis as determined by a rheumatologist and had ever used etanercept. Patients were excluded from the analysis if they had an overlap >31 days of other biologics use after the start of etanercept and follow-up (etanercept start date for incident use or registry enrolment date for ongoing etanercept use) and/or a history of other rheumatic diseases. The safety cohort included patients who had received etanercept at any time after enrolment in the registry; the effectiveness cohort included patients with a registry visit within 14 days from etanercept initiation (ie, treatment inception cohort). Patients in the effectiveness cohort were etanercept naïve. Baseline period data were collected at the start of follow-up; consequently, different lengths of time constituted the baseline period for patients depending on the time elapsed between disease onset and enrolment (for ongoing etanercept users) or etanercept initiation. Start dates for study follow-up time at risk were based on receipt of etanercept during participation in the CARRA Registry. Follow-up was censored at date of death, registry discontinuation date, latest data collection date or specific censoring dates for each cohort. For the assessment of non-malignancy safety events, study follow-up started at initiation of incident use of etanercept or at registry enrolment for patients with ongoing use of etanercept. Follow-up was censored 91 days after starting another biologic disease-modifying antirheumatic drug (DMARD) or 91 days after discontinuing etanercept unless restarted within those 91 days. For the assessment of malignancy safety events, study follow-up started at initiation of incident use of etanercept or at registry enrolment for patients with ongoing use of etanercept. Follow-up was not censored because of discontinuation of etanercept or initiation of other biologic therapy. For the effectiveness cohort, study follow-up started at initiation of etanercept therapy and continued during ongoing uninterrupted etanercept use until outcomes were assessed. Follow-up was censored 91 days after starting another biologic DMARD or 91 days after discontinuing etanercept unless restarted within those 91 days. Study outcomes Baseline characteristics including patient demographics, duration of disease and concomitant use of non-biologic therapy were assessed at the registry visit closest to start of follow-up for etanercept receipt for the safety and effectiveness cohorts. For the effectiveness cohort only, baseline characteristics including disease activity/severity measures were assessed at the registry visit ±14 days from start of etanercept. Safety was assessed by calculating the rates of 31 prespecified AESIs (see for the complete list) and SAEs in the safety cohort. Effectiveness was assessed by determining changes in the American College of Rheumatology (ACR)-Pediatric Response (ACR-Pedi Response: 30/50/70/90/100), cJADAS-10 and ACR provisional inactive disease criteria (see for definition of outcomes) assessed at the 6-month and 12-month study follow-up visits in the effectiveness cohort. The 6-month follow-up included patients who were ≤9 months post-etanercept initiation at time of data cut-off or who had completed a registry visit 3–9 months post-etanercept start; if more than one visit was completed in the period 3–9 months post-etanercept initiation window, the visit closest to 183 days post-etanercept initiation was selected. The 12-month follow-up included patients who were ≤15 months post-etanercept initiation at time of data cut-off or who had a completed registry visit 9–15 months post-etanercept start; if more than one visit was completed in the 9–15 months post-etanercept initiation window, the visit closest to 365 days post-etanercept initiation was selected. The starting dose of etanercept expressed as weekly mg/kg was assessed for the effectiveness cohort only, based on recorded patient weight (at the visit closest to etanercept initiation) and administered dose of etanercept. 10.1136/rmdopen-2022-002943.supp1 Supplementary data Statistical analysis Summary statistics are presented using means, SDs, medians and IQRs (quartile 1 (Q1), quartile 3 (Q3)) for continuous variables and proportions for categorical variables with 95% CIs. AESI and SAE rates are presented as counts per 100 person-years of study follow-up at risk with 95% CIs. ACR-Pedi, cJADAS-10 and ACR provisional clinical inactive disease responses at 6-month and 12-month follow-up visits were assessed in three ways: (1) restricted to patients with ongoing etanercept use and outcome data available at respective 6-month and 12-month intervals from etanercept start (ie, complete case analysis); (2) the outcome from the last visit with ongoing etanercept use was carried forward for patients with missing outcome data or who discontinued etanercept before outcome determination (ie, last observation carried forward (LOCF)), as a sensitivity analysis; and (3) non-response was assumed for patients with missing outcome data or who discontinued etanercept before outcome ascertainment (ie, non-responder imputation (NRI)), as another sensitivity analysis.
The CARRA Registry was started in 2015. The general design and rationale of the registry and the characteristics of patients enrolled have been previously described in detail. Briefly, the CARRA Registry is an international observational registry of paediatric patients with rheumatic diseases, including JIA. At the inception of the registry, there was selective enrolment of paediatric patients who were most likely to be treated with biologics. The registry includes retrospective data collected at the time of enrolment and prospective observational data collected approximately every 6 months at patient visits in the context of routine clinical care and ideally at the time of initiation of any new JIA medication. Data collected include physician-assigned ILAR category, detailed medication logs, clinical features, laboratory data, imaging results and adverse events of special interest (AESIs), including serious adverse events (SAEs). The CARRA Registry also collects the clinical Juvenile Arthritis Disease Activity Score with an active joint count up to 10 (clinical Juvenile Arthritis Disease Activity Score 10-joint (cJADAS-10)), patient-reported outcomes, Childhood Health Assessment Questionnaire, the patient/parent global assessment and pain intensity. The present analysis used CARRA Registry data from approximately 70 clinical sites in the USA and Canada beginning 30 June 2015 until the data cut-off date for this analysis of 2 August 2021. Data were analysed for patients aged ≥2–<18 years at etanercept initiation who had a JPsA diagnosis as determined by a rheumatologist and had ever used etanercept. Patients were excluded from the analysis if they had an overlap >31 days of other biologics use after the start of etanercept and follow-up (etanercept start date for incident use or registry enrolment date for ongoing etanercept use) and/or a history of other rheumatic diseases. The safety cohort included patients who had received etanercept at any time after enrolment in the registry; the effectiveness cohort included patients with a registry visit within 14 days from etanercept initiation (ie, treatment inception cohort). Patients in the effectiveness cohort were etanercept naïve. Baseline period data were collected at the start of follow-up; consequently, different lengths of time constituted the baseline period for patients depending on the time elapsed between disease onset and enrolment (for ongoing etanercept users) or etanercept initiation. Start dates for study follow-up time at risk were based on receipt of etanercept during participation in the CARRA Registry. Follow-up was censored at date of death, registry discontinuation date, latest data collection date or specific censoring dates for each cohort. For the assessment of non-malignancy safety events, study follow-up started at initiation of incident use of etanercept or at registry enrolment for patients with ongoing use of etanercept. Follow-up was censored 91 days after starting another biologic disease-modifying antirheumatic drug (DMARD) or 91 days after discontinuing etanercept unless restarted within those 91 days. For the assessment of malignancy safety events, study follow-up started at initiation of incident use of etanercept or at registry enrolment for patients with ongoing use of etanercept. Follow-up was not censored because of discontinuation of etanercept or initiation of other biologic therapy. For the effectiveness cohort, study follow-up started at initiation of etanercept therapy and continued during ongoing uninterrupted etanercept use until outcomes were assessed. Follow-up was censored 91 days after starting another biologic DMARD or 91 days after discontinuing etanercept unless restarted within those 91 days.
Baseline characteristics including patient demographics, duration of disease and concomitant use of non-biologic therapy were assessed at the registry visit closest to start of follow-up for etanercept receipt for the safety and effectiveness cohorts. For the effectiveness cohort only, baseline characteristics including disease activity/severity measures were assessed at the registry visit ±14 days from start of etanercept. Safety was assessed by calculating the rates of 31 prespecified AESIs (see for the complete list) and SAEs in the safety cohort. Effectiveness was assessed by determining changes in the American College of Rheumatology (ACR)-Pediatric Response (ACR-Pedi Response: 30/50/70/90/100), cJADAS-10 and ACR provisional inactive disease criteria (see for definition of outcomes) assessed at the 6-month and 12-month study follow-up visits in the effectiveness cohort. The 6-month follow-up included patients who were ≤9 months post-etanercept initiation at time of data cut-off or who had completed a registry visit 3–9 months post-etanercept start; if more than one visit was completed in the period 3–9 months post-etanercept initiation window, the visit closest to 183 days post-etanercept initiation was selected. The 12-month follow-up included patients who were ≤15 months post-etanercept initiation at time of data cut-off or who had a completed registry visit 9–15 months post-etanercept start; if more than one visit was completed in the 9–15 months post-etanercept initiation window, the visit closest to 365 days post-etanercept initiation was selected. The starting dose of etanercept expressed as weekly mg/kg was assessed for the effectiveness cohort only, based on recorded patient weight (at the visit closest to etanercept initiation) and administered dose of etanercept. 10.1136/rmdopen-2022-002943.supp1 Supplementary data
Summary statistics are presented using means, SDs, medians and IQRs (quartile 1 (Q1), quartile 3 (Q3)) for continuous variables and proportions for categorical variables with 95% CIs. AESI and SAE rates are presented as counts per 100 person-years of study follow-up at risk with 95% CIs. ACR-Pedi, cJADAS-10 and ACR provisional clinical inactive disease responses at 6-month and 12-month follow-up visits were assessed in three ways: (1) restricted to patients with ongoing etanercept use and outcome data available at respective 6-month and 12-month intervals from etanercept start (ie, complete case analysis); (2) the outcome from the last visit with ongoing etanercept use was carried forward for patients with missing outcome data or who discontinued etanercept before outcome determination (ie, last observation carried forward (LOCF)), as a sensitivity analysis; and (3) non-response was assumed for patients with missing outcome data or who discontinued etanercept before outcome ascertainment (ie, non-responder imputation (NRI)), as another sensitivity analysis.
Baseline patient demographics and disease activity Overall, 3155 paediatric patients with JIA in the CARRA Registry had received etanercept and were screened for eligibility for this analysis. Of the 3155 patients, 226 patients had JPsA and of these, 191 met criteria for the safety cohort. The 191 patients in the safety cohort were predominantly white (80.6%) and female (66.0%) . At the start of follow-up for this analysis, median age in the safety cohort was 12.0 years and median disease duration was 2.4 years. Fifty-six per cent of the patients were taking etanercept at the time they enrolled in the CARRA Registry with a median of 14.9 months (mean of 29.5 months) of etanercept use before registry enrolment. Median age of etanercept initiation was 10.0 years. More than half (59.2%) of the patients had concomitant use of a non-biologic DMARD at the start of follow-up, with most (56.0% of all patients; 94.7% of concomitant non-biologic DMARD users) receiving methotrexate. Of the 226 patients with JPsA and etanercept exposure, 43 met the criteria for the effectiveness cohort. Patients in the effectiveness cohort had similar demographics to those in the safety cohort . The start of follow-up time for the effectiveness cohort was defined as when etanercept was initiated, so this cohort had a shorter disease duration at start of follow-up (median 0.4 years) compared with the safety cohort (median 2.4 years). Median physician global assessment of disease activity was 3.5, median patient/parent global assessment of overall well-being was 3.0, 27.9% of patients had active psoriasis skin lesions reported and median cJADAS-10 was 10.0. No patients had active uveitis at etanercept initiation. Rates of AESIs and SAEs with etanercept in the safety cohort The 191 patients in the safety cohort had a low incidence of AESIs, SAEs and malignancy . The incidence of AESIs (excluding malignancy) was based on the three cases of uveitis (non-serious) reported during the observed use of etanercept, with an incidence rate of new-onset uveitis of 0.55 (95% CI: 0.18, 1.69) per 100 person-years. All three new-onset uveitis events were consistent with JIA-associated uveitis and responded clinically to treatment with topical glucocorticoid eye drops. The three patients with uveitis (patients 1, 2 and 3) had been diagnosed with JIA at ages 2.4, 3.4 and 9.4 years, respectively, and were diagnosed with uveitis at ages 4.3, 5.7 and 10.0 years, respectively. Patient 1 had been taking etanercept only at the time of uveitis onset; patients 2 and 3 had been treated with etanercept and methotrexate. One SAE of new-onset neuropathy was reported, for an overall rate of 0.18 (95% CI: 0.03, 1.29) per 100 person-years during observed use of etanercept. The new-onset neuropathy occurred approximately 4 months after initiation of etanercept. The patient was concurrently treated with methotrexate. The patient who experienced the event had tingling sensation of the foot that was classified as medically significant by the investigator, indicating the potential to escalate to another serious outcome if not treated. The event ultimately resolved and was not considered suggestive of demyelination. Etanercept use was continued following the event. In the specific assessment for malignancy following etanercept use, one AESI of malignancy was reported representing an overall rate of 0.13 (95% CI: 0.02, 0.90) per 100 person-years during 789.2 person-years of follow-up including observed time after discontinuation of etanercept. The patient who experienced the malignancy presented with an abdominal mass that was diagnosed as a liver sarcoma by biopsy specimen approximately 30 months after initiation of etanercept. The patient had a history of prior treatment with adalimumab and methotrexate and was taking etanercept and methotrexate at the time of the malignancy diagnosis. ACR-Pedi, cJADAS-10 and ACR provisional clinical inactive disease responses with etanercept in the effectiveness cohort Of the 43 patients in the effectiveness cohort, 32 had evaluable data for the reported outcomes and uninterrupted etanercept use at the 6-month follow-up . For the 15 patients evaluable for the ACR-Pedi Response at the 6-month follow-up, 80.0% (12 of 15) of the patients showed an ACR30 response and 46.7% (7 of 15) of the patients showed an ACR90 response ( and ). Only five patients were evaluable for the ACR-Pedi Response at the 12-month follow-up, with 80.0% (4 of 5) of the patients showing an ACR30 response and 20.0% (1 of 5) of the patients showing an ACR90 response. For the 25 patients evaluable for cJADAS-10 at the 6-month follow-up, 36.0% (9 of 25) of the patients had cJADAS-10 ≤1.0 ( and ). Only 13 patients were evaluable for the cJADAS-10 at the 12-month follow-up and 53.8% (7 of 13) of these patients had cJADAS-10 ≤ 1.1. Median (Q1, Q3) change from baseline in cJADAS-10 was –2.8 (–6.0, –1.0) at the 6-month follow-up and –5.5 (–7.0, –2.8) at the 12-month follow-up . The ACR provisional criteria for inactive disease were met by 51.9% (14 of 27) of patients at the 6-month follow-up and 43.8% (7 of 16) of patients at the 12-month follow-up ( and ). Additionally, 55.8% (24 of 43) of patients were concurrently treated with methotrexate at the 6-month follow-up and 39.5% (17 of 43) were taking methotrexate at the 12-month follow-up. In sensitivity analyses, etanercept effectiveness was determined using LOCF and NRI. By LOCF, the observed effectiveness of etanercept was attenuated; nevertheless, approximately 30% of patients met the ACR provisional criteria for clinical inactive disease at the 6-month follow-up (13 of 41 patients) and 12-month follow-up (10 of 34 patients) ( and ). By NRI, which is the most conservative statistical approach, etanercept effectiveness was further attenuated, with the proportions of patients meeting the ACR provisional criteria for clinical inactive disease at the 6-month and 12-month follow-up of 34.1% (14 of 41 patients) and 20.6% (7 of 34 patients), respectively ( and ). Etanercept dosing in the effectiveness cohort Initial etanercept dosing was determined in the effectiveness cohort . Thirty-seven patients weighed less than 62.5 kg and the median dose was the etanercept-labelled dose for JIA of 0.8 mg/kg. At least 65% of patients received doses between 0.7 and 0.9 mg/kg. Among six patients weighing greater than 62.5 kg, five (83.3%) received the labelled dose of 50 mg weekly and one (16.7%) received less than 50 mg weekly. No patients received more than the labelled dose of 50 mg weekly.
Overall, 3155 paediatric patients with JIA in the CARRA Registry had received etanercept and were screened for eligibility for this analysis. Of the 3155 patients, 226 patients had JPsA and of these, 191 met criteria for the safety cohort. The 191 patients in the safety cohort were predominantly white (80.6%) and female (66.0%) . At the start of follow-up for this analysis, median age in the safety cohort was 12.0 years and median disease duration was 2.4 years. Fifty-six per cent of the patients were taking etanercept at the time they enrolled in the CARRA Registry with a median of 14.9 months (mean of 29.5 months) of etanercept use before registry enrolment. Median age of etanercept initiation was 10.0 years. More than half (59.2%) of the patients had concomitant use of a non-biologic DMARD at the start of follow-up, with most (56.0% of all patients; 94.7% of concomitant non-biologic DMARD users) receiving methotrexate. Of the 226 patients with JPsA and etanercept exposure, 43 met the criteria for the effectiveness cohort. Patients in the effectiveness cohort had similar demographics to those in the safety cohort . The start of follow-up time for the effectiveness cohort was defined as when etanercept was initiated, so this cohort had a shorter disease duration at start of follow-up (median 0.4 years) compared with the safety cohort (median 2.4 years). Median physician global assessment of disease activity was 3.5, median patient/parent global assessment of overall well-being was 3.0, 27.9% of patients had active psoriasis skin lesions reported and median cJADAS-10 was 10.0. No patients had active uveitis at etanercept initiation.
The 191 patients in the safety cohort had a low incidence of AESIs, SAEs and malignancy . The incidence of AESIs (excluding malignancy) was based on the three cases of uveitis (non-serious) reported during the observed use of etanercept, with an incidence rate of new-onset uveitis of 0.55 (95% CI: 0.18, 1.69) per 100 person-years. All three new-onset uveitis events were consistent with JIA-associated uveitis and responded clinically to treatment with topical glucocorticoid eye drops. The three patients with uveitis (patients 1, 2 and 3) had been diagnosed with JIA at ages 2.4, 3.4 and 9.4 years, respectively, and were diagnosed with uveitis at ages 4.3, 5.7 and 10.0 years, respectively. Patient 1 had been taking etanercept only at the time of uveitis onset; patients 2 and 3 had been treated with etanercept and methotrexate. One SAE of new-onset neuropathy was reported, for an overall rate of 0.18 (95% CI: 0.03, 1.29) per 100 person-years during observed use of etanercept. The new-onset neuropathy occurred approximately 4 months after initiation of etanercept. The patient was concurrently treated with methotrexate. The patient who experienced the event had tingling sensation of the foot that was classified as medically significant by the investigator, indicating the potential to escalate to another serious outcome if not treated. The event ultimately resolved and was not considered suggestive of demyelination. Etanercept use was continued following the event. In the specific assessment for malignancy following etanercept use, one AESI of malignancy was reported representing an overall rate of 0.13 (95% CI: 0.02, 0.90) per 100 person-years during 789.2 person-years of follow-up including observed time after discontinuation of etanercept. The patient who experienced the malignancy presented with an abdominal mass that was diagnosed as a liver sarcoma by biopsy specimen approximately 30 months after initiation of etanercept. The patient had a history of prior treatment with adalimumab and methotrexate and was taking etanercept and methotrexate at the time of the malignancy diagnosis.
Of the 43 patients in the effectiveness cohort, 32 had evaluable data for the reported outcomes and uninterrupted etanercept use at the 6-month follow-up . For the 15 patients evaluable for the ACR-Pedi Response at the 6-month follow-up, 80.0% (12 of 15) of the patients showed an ACR30 response and 46.7% (7 of 15) of the patients showed an ACR90 response ( and ). Only five patients were evaluable for the ACR-Pedi Response at the 12-month follow-up, with 80.0% (4 of 5) of the patients showing an ACR30 response and 20.0% (1 of 5) of the patients showing an ACR90 response. For the 25 patients evaluable for cJADAS-10 at the 6-month follow-up, 36.0% (9 of 25) of the patients had cJADAS-10 ≤1.0 ( and ). Only 13 patients were evaluable for the cJADAS-10 at the 12-month follow-up and 53.8% (7 of 13) of these patients had cJADAS-10 ≤ 1.1. Median (Q1, Q3) change from baseline in cJADAS-10 was –2.8 (–6.0, –1.0) at the 6-month follow-up and –5.5 (–7.0, –2.8) at the 12-month follow-up . The ACR provisional criteria for inactive disease were met by 51.9% (14 of 27) of patients at the 6-month follow-up and 43.8% (7 of 16) of patients at the 12-month follow-up ( and ). Additionally, 55.8% (24 of 43) of patients were concurrently treated with methotrexate at the 6-month follow-up and 39.5% (17 of 43) were taking methotrexate at the 12-month follow-up. In sensitivity analyses, etanercept effectiveness was determined using LOCF and NRI. By LOCF, the observed effectiveness of etanercept was attenuated; nevertheless, approximately 30% of patients met the ACR provisional criteria for clinical inactive disease at the 6-month follow-up (13 of 41 patients) and 12-month follow-up (10 of 34 patients) ( and ). By NRI, which is the most conservative statistical approach, etanercept effectiveness was further attenuated, with the proportions of patients meeting the ACR provisional criteria for clinical inactive disease at the 6-month and 12-month follow-up of 34.1% (14 of 41 patients) and 20.6% (7 of 34 patients), respectively ( and ).
Initial etanercept dosing was determined in the effectiveness cohort . Thirty-seven patients weighed less than 62.5 kg and the median dose was the etanercept-labelled dose for JIA of 0.8 mg/kg. At least 65% of patients received doses between 0.7 and 0.9 mg/kg. Among six patients weighing greater than 62.5 kg, five (83.3%) received the labelled dose of 50 mg weekly and one (16.7%) received less than 50 mg weekly. No patients received more than the labelled dose of 50 mg weekly.
Results from our analysis of data from patients with JPsA enrolled in the CARRA Registry showed that patients receiving etanercept had a low incidence of prespecified AESIs (three incidences of new-onset uveitis and one incidence of malignancy) and SAEs (one incidence of new-onset neuropathy). Etanercept was effective in JPsA treatment as assessed by the ACR-Pedi, cJADAS-10 and ACR provisional clinical inactive disease responses, and maintained effectiveness over 12 months. Additionally, etanercept dosing was consistent with the product label dose for JIA of 0.8 mg/kg weekly, with a maximum of 50 mg/week. When we consider the safety of biologic therapies, including etanercept, we can categorise safety events in three categories including serious infection, neoplasia and secondary autoimmunity, of which the first two are of highest concern in paediatrics. It is important to note that this study did not identify any events of serious infection. One patient developed malignancy. The observed overall incidence rate for the one AESI of malignancy of 0.18 (95% CI: 0.03, 1.29) per 100 person-years is within the range of previously published studies. In the German Biologics in Pediatrics Rheumatology Registry (BiKeR) Study, a malignancy rate of 0.05 (95% CI: 0.02, 0.2) per 100 person-years was reported in paediatric patients with JIA treated with etanercept (three cases of malignancy) and a rate of 0.05 (95% CI: 0.01, 0.2) was reported in paediatric patients with JIA who were biologic-naïve (two cases of malignancy). In a large claims database study in the USA, a malignancy rate of 0.05 per 100 person-years was reported for patients with JIA treated with tumour necrosis factor inhibitors, and a rate of 0.03 per 100 person-years was reported in paediatric patients with JIA who were not treated with tumour necrosis factor inhibitors. Because this is a small study, it is challenging to make conclusions about malignancy risk with only one malignancy event. This highlights the importance of long-term safety monitoring in large databases such as the CARRA Registry. Other than uveitis, which is a known complication of JIA, no patients developed a secondary autoimmune disease. The observed overall incidence rate for the three AESIs of new-onset uveitis of 0.55 (95% CI: 0.18, 1.69) per 100 person-years was substantially lower than that observed in other JIA cohorts, most likely because our analysis focused on a subpopulation with JPsA and did not include the population with oligoarthritis JIA that has a higher risk of uveitis. One patient developed neuropathy, but this was not thought to be a demyelinating disease. The observed SAE rate for the one incidence of new-onset neuropathy of 0.18 (95% CI: 0.03, 1.29) per 100 person-years in our analysis is substantially lower than SAE rates of 3.8 (95% CI: 3.3, 4.3) per 100 person-years reported in patients with JIA treated with etanercept and 1.4 (95% CI: 1.1, 1.8) per 100 person-years in patients with JIA who were biologic-naïve as reported in the BiKeR Study. This is also lower than the SAE rate of 3.3 per 100 person-years reported in a long-term (6-year) follow-up of the open-label clinical trial of etanercept for the treatment of JPsA. Of note, in our analysis, 62% of patients in the safety cohort had initiated etanercept, a mean of more than 2 years before enrolment in the study, and ongoing or recurrent users of etanercept would be expected to have fewer SAEs than new initiators. However, the nature of SAE reporting in the CARRA Registry may also contribute to the lower than anticipated observed event rate (ie, the clinical sites may not be aware of the occurrence of all SAEs). Etanercept administered at the labelled dose for JIA of 0.8 mg/kg weekly, with a maximum of 50 mg/week, appeared to be effective for the treatment of JPsA, although the interpretation of results was limited by missing clinical measures. Of the 43 patients in the effectiveness cohort, 15 patients were evaluable for the ACR-Pedi Response at the 6-month follow-up and 5 at the 12-month follow-up. Overall, etanercept treatment showed effectiveness by ACR30/50/70/90/100 response criteria, cJADAS-10, and ACR provisional clinical inactive disease criteria. However, in the most conservative statistical approach with missing data treated as treatment failure (NRI), the observed effectiveness of etanercept was substantially attenuated. Etanercept effectiveness observed in our analysis is consistent with that reported in earlier studies. In our analysis, the proportions of patients with ACR30/50/70 were 80.0%, 66.7% and 66.7%, respectively, among the 15 patients with complete data and uninterrupted etanercept use at the 6-month follow-up. Similar overall results were reported in the open-label clinical trial of etanercept for the treatment of JPsA that included 29 patients, and showed approximate proportions of patients with ACR30/50/70 of 90%, 90% and 60%, respectively. The BiKeR Study in JIA also showed similar results, with reported ACR30/50/70 response rates of 82%, 79% and 71%, respectively, after 9 years of treatment with etanercept. Our analysis has a number of limitations. First, the baseline assessment was up to 14 days after initiation of etanercept, which for some patients may have underestimated disease activity at etanercept onset. Additionally, the assessment of effectiveness was limited by the availability of clinical data in this observational registry study. For much of the effectiveness data, our sample sizes were quite small, as small as five patients in some cases, and thus we cannot be certain that our effectiveness findings are representative of the population with JPsA as a whole. To assess the ACR-Pedi Response criteria, clinical data at the time of treatment initiation are required. Among 72 patients who initiated etanercept after CARRA Registry enrolment, 26 (36.1%) did not have all of the required baseline clinical data for inclusion in the effectiveness assessment. Among patients included in the effectiveness assessment, there were patients with missing follow-up visits or patients with missing clinical assessments for the visits that occurred. Traditional clinical trial single imputation methods (ie, LOCF) were limited because the 6-month follow-up was typically the first data collection point following etanercept initiation. Inflammatory markers were not always assessed during clinic visits, which limited the utility of the ACR-Pedi Response criteria. Due to missing data for the parent global assessment of overall well-being, the ACR provisional criteria for clinical inactive disease was calculable for a greater proportion of patients compared with the ACR-Pedi Response criteria and cJADAS-10. It is possible that not all safety events were reported to the CARRA Registry. We did not assess rate or reason for discontinuation of etanercept in patients with JPsA because it was beyond the scope of this study, but it is likely that the rates and reasons for discontinuation are similar to those seen in a previous study of all etanercept use in the CARRA Registry. Sites report safety events as they become aware of their occurrence, which may result in incomplete or delayed identification of events, especially those that occur remotely from the rheumatology care centre’s institution. Given that the CARRA Registry remains active and open for data collection, there is also the potential that additional safety events will be identified and reported after publication of this study. Further, our analysis did not evaluate whether there is a difference in etanercept effectiveness in axial and non-axial peripheral JPsA. Finally, no comparator groups of patients who received treatments other than etanercept were included in our analysis. Results from our analysis of data in the CARRA Registry showed that etanercept treatment in JPsA was effective over 12 months, with low rates of AESIs and SAEs; however, further research is needed to evaluate whether there is a difference in etanercept effectiveness in axial and non-axial peripheral JPsA, and whether the effectiveness is sustained in the longer term. No signals were observed to suggest that etanercept is less effective or safe in JPsA than JIA in general.
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DETECT: DEveloping and testing a model to identify preventive vision loss among older paTients in gEneral praCTice – protocol for a complex intervention in Denmark
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afbeba35-f080-4a11-a287-66836055a4b1
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10230986
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Family Medicine[mh]
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It is estimated that 2.2 billion people have impaired vision, and of these, at least 1 billion people have a vision loss that could have been prevented or reduced by earlier detection or by access to treatment. The most common causes of moderate to severe visual impairment are uncorrected refractive errors, unoperated cataract, age-related macular degeneration (AMD), glaucoma and diabetic retinopathy. Also in affluent welfare states such as Denmark—constituting the setting of this study—visual impairment is a problem. The incidence of visual impairment increases with age, and due to the sociodemographical trajectory of an increasing elderly population, the prevalence of patients with visual impairment will increase. Timely access to healthcare has a major influence on the progression of eye conditions. The consequences of vision loss significantly affect the person’s quality of life, dependence and increases the risk of recurrent falls and fractures, which is a significant threat to mobility in old age. A YouGov poll showed that sight was by far the sensory function, people fear losing the most. Vision loss can result in worsened mental health, cognition and social functioning. Disease progression can be complicated by other chronic conditions and can complicate management of multimorbidity due to decreased self-care, ability to visit clinics and adherence to medication. Finally, vision loss can increase the risks of placement in nursing homes. Thus, visual impairment and loss has a great impact on the individual, their relatives, society in general and on the healthcare system. General practice and visual impairment General practitioners (GPs) handle preventive healthcare, diagnostics, treatment and care of chronic conditions as well as coordinate services from various healthcare professionals. In the Global North, the GPs handle the majority of all medical matters. A survey set in English general practice from 1998 concluded that eye problems, including undiagnosed glaucoma and AMD, were quite frequent among elderly patients consulting their GP. One study found that patients were more likely to have their eyes checked if their GP suggests it. In addition, an increased focus on eye health in at-risk populations in general practice is suggested to be more effective for early detection than broader screening programmes. However, a recent UK-based survey of GPs indicated that although up to 5% of the primary consultations were eye related, GPs ability to identify red flags was low. The literature points to a gap where, even though patients are in contact with their GP concerning symptoms related to their vision, an unidentified number of patients may suffer from unrecognised visual impairment that is not detected in general practice. Collaboration across healthcare professions Since vision problems are rarely detected in general practice, the need for research into how patients and health professionals collaborate to identify and manage visual impairment becomes a relevant matter. This is in line with a recent Cochrane review, which concludes that future research should look at optimised primary care-based vision screening interventions. Patients with visual impairment often have contacts with many different healthcare professionals. Therefore, it is important to incorporate collaboration across health professions and sectors in a GP intervention aimed at improving identification of patients with visual impairment. Optometrists constitute an occupational group who may be the first line of contact for some patients who experience visual changes. Optometrists will in most cases operate independently without a formal collaboration with other health professionals such as GPs. Given the optometrists contact with the patients and level of equipment for measuring vision and evaluating the eye, they pose a potentially important resource when collaboration across healthcare professions is rethought and are therefore important to include in the study. Their commercial agenda may influence their work and this will be evaluated in the collaboration. Aim and objectives In this study, we aim to develop a health intervention in a Danish general practice setting to improve the detection and care of visual impairment. The patient target group is middle-aged and older adults and their relatives, with GP’s constituting the primary professional target group. We define visual impairment broadly to be the patient experience of symptoms related to vision and findings identified by health professionals—such as reduced vision field—not yet experienced by the patient, but a serious threat to patient vision. Visual impairment is in this respect not connected to specific diagnoses, but as previously stated, we assume frequent eye diseases such as glaucoma and AMD will be well represented. The overall aim of DETECT is thus to: Develop an intervention in general practice aimed at identifying visual impairment among elderly patients with chronic conditions. Test the feasibility of the intervention model in general practice with a focus on ensuring improved patient support and education.
General practitioners (GPs) handle preventive healthcare, diagnostics, treatment and care of chronic conditions as well as coordinate services from various healthcare professionals. In the Global North, the GPs handle the majority of all medical matters. A survey set in English general practice from 1998 concluded that eye problems, including undiagnosed glaucoma and AMD, were quite frequent among elderly patients consulting their GP. One study found that patients were more likely to have their eyes checked if their GP suggests it. In addition, an increased focus on eye health in at-risk populations in general practice is suggested to be more effective for early detection than broader screening programmes. However, a recent UK-based survey of GPs indicated that although up to 5% of the primary consultations were eye related, GPs ability to identify red flags was low. The literature points to a gap where, even though patients are in contact with their GP concerning symptoms related to their vision, an unidentified number of patients may suffer from unrecognised visual impairment that is not detected in general practice.
Since vision problems are rarely detected in general practice, the need for research into how patients and health professionals collaborate to identify and manage visual impairment becomes a relevant matter. This is in line with a recent Cochrane review, which concludes that future research should look at optimised primary care-based vision screening interventions. Patients with visual impairment often have contacts with many different healthcare professionals. Therefore, it is important to incorporate collaboration across health professions and sectors in a GP intervention aimed at improving identification of patients with visual impairment. Optometrists constitute an occupational group who may be the first line of contact for some patients who experience visual changes. Optometrists will in most cases operate independently without a formal collaboration with other health professionals such as GPs. Given the optometrists contact with the patients and level of equipment for measuring vision and evaluating the eye, they pose a potentially important resource when collaboration across healthcare professions is rethought and are therefore important to include in the study. Their commercial agenda may influence their work and this will be evaluated in the collaboration.
In this study, we aim to develop a health intervention in a Danish general practice setting to improve the detection and care of visual impairment. The patient target group is middle-aged and older adults and their relatives, with GP’s constituting the primary professional target group. We define visual impairment broadly to be the patient experience of symptoms related to vision and findings identified by health professionals—such as reduced vision field—not yet experienced by the patient, but a serious threat to patient vision. Visual impairment is in this respect not connected to specific diagnoses, but as previously stated, we assume frequent eye diseases such as glaucoma and AMD will be well represented. The overall aim of DETECT is thus to: Develop an intervention in general practice aimed at identifying visual impairment among elderly patients with chronic conditions. Test the feasibility of the intervention model in general practice with a focus on ensuring improved patient support and education.
The study will be conducted in Denmark and is thus inscribed in a Scandinavian health system with universal access to healthcare. The general health status in Denmark is relatively high, and as far as vision is concerned, the incidence of legal blindness has decreased along with improved treatment options. The average life expectancy has increased over the last 70 years, which is positive, but it also entails a rise in age-related sight-threatening eye diseases, such as glaucoma and AMD. Despite the decreasing incidence of legal blindness due to AMD, many patients are diagnosed late with irreversible vision loss. It seems relevant to diagnose eye diseases earlier and optimise the coordination of care. It is difficult to provide an exact number of people in Denmark who live with visual impairment. A national survey of health, quality of life and morbidity from 2007 shows that 3.8% of the population over 60 reported difficulties in reading a newspaper text, while the Danish Eye Association estimate that 50.000 people in Denmark above 60 years are blind or visually impaired (total population 60+: 1.554.542 ). In Denmark, the GP is the patient’s primary entry point to the healthcare system, and the GP treats 90% of all medical cases. All Danes are assigned to a default general practice, and as many as 80% consult their GP at least annually, with an increased frequency among patients aged 50 or older. People with chronic conditions are offered an annual health check at their GP. A Danish survey from 2019 showed that 82.4% of men and 86.7% of women had their vision measured at their GP within the last 3 years. However, these figures are from 2010 to 2017. In 2017, regulations regarding driving licenses were changed, resulting in vision being measured only every 15 years. This may result in a lower frequency of vision acuity measurements at the GP today, but from the 2017 figures we can assume that the practice of performing vision measurements during the annual consultation for older adults is a well-known procedure. Ophthalmologists can diagnose, treat and carry out the necessary checks of, for example, glaucoma and atrophic AMD in the primary sector. If indicated, patients are referred to secondary care in the hospitals’ eye departments. Examples of referral indications may be neovascular AMD, proliferative diabetic retinopathy and medically uncontrollable glaucoma. At the hospitals, ophthalmologists work publicly funded and university hospital clinics have an obligation to do research within the field. Consultations with the GP or ophthalmologist are tax financed and without an out-of-pocket fee to patients. Hospital-based eye clinics are also free of charge, but the patient must be referred by a primary sector ophthalmologist for treatment. In cases of acute vision loss or pain, patients can be seen directly in the emergency room and referred from there to the on-call ophthalmologist in the hospital eye department. Anyone can book an appointment with the ophthalmologist in the primary sector without a referral, but due to a low number of ophthalmologists compared with the increasing demand, it is often difficult to book a consultation within a reasonable time frame and geographical distance. On the other hand, the GP must be available to all patients inscribed in his/her practice and have an in-depth knowledge of the patient’s general health and condition. The GPs, therefore, seem to be in an ideal situation to identify visual impairment and coordinate the management. It is estimated that around 2000 optometrists operate in Denmark (total population 5.8 million) and opticians shops can be found in most smaller cities, making it accessible even in rural areas to visit an optician shop. In most optician shops, it is free of charge to have vision tested. Many optometrists offer intraocular pressure measurements and fundus photographs as additional procedures for a fee. Optometrists are thus a professional stakeholder that we find interesting to explore further. Study phases We apply the Medical Research Council guidance on developing, testing and evaluating complex interventions. To operationalise the framework for complex interventions, we apply a temporal structure from the tradition of human-centred design to divide the project cycle into three main phases: (I) identify the problem, (II) develop the model and (III) test the feasibility of the model. Phase I+II focus on intervention development applying qualitative methods and phase III aims to first pilot test the intervention for feasibility and following implement it broader in a cohort study to measure effect. The intervention explores what patients, carers and professionals perceive as pivotal to improve regarding detection, navigating care and health services and support possibilities for people living with visual impairment. We are explicitly reflective on, how process and product are interwoven and to a very high extent dependent on the context it unfolds within. Following a structured and well-documented design process, we identify the changes made and insights produced (see ). Phase I: identify—identifying key issues to address in the health intervention The aim of phase I is to explore the problem we are addressing. We will perform a literature search on detection of eye diseases in general practice and conduct background interviews with a broad selection of relevant stakeholders to help us map the current practice in detecting and diagnosing eye conditions across sectors in the healthcare system (see ). An essential element in human-centred design processes is to create understanding and empathy for the end-user —in our case people living with visual impairment and their relatives. We aim to incorporate a wide spectrum of eye diagnoses and develop a model to identify and diagnose visual impairment that works from the onset of the patient’s early symptoms, such as stumbling over doorsteps, difficulties in reading, distorted vision or difficulties related to the transition from dark to light spaces and vice versa. The intervention aim of increased patient support also includes support of relatives, who carry a large part of the disease burden and may experience stress and depression due to their loved one’s visual impairment. Patient and public involvement in phase I Patients, relatives and professionals will be involved by providing their perspectives in various stages of the study and codesign core elements of the model constituting the intervention. The patient engagement process is informed by a thorough report produced by the Danish Center for Social Science Research on older adults with visual impairment and vision loss. The report underlines the need for increased knowledge on how visual impairment affects patients’ every day life in all aspects. The experiences, needs, preferences, and values of patients and relatives will thus be explored. See for an overview of the involvement of participants across the three project phases. In phase I, we perform semistructured interviews with patients and relatives, preferably in their home to gain an insight in their experiences in the context in which they occur. Here, we focus on the patient journey from the time patients experience symptoms leading to a diagnosis and handling life afterwards with a diagnosis. After the interview, patients and relatives are encouraged to contact the researcher if they would like her/him to participate in, for example, a visit to the ophthalmologist or if they have further input or concerns at a later stage. We will furthermore perform focus group interviews with older adults to investigate (1) the expectations to vision in old age and (2) which health professionals’ older adults identify as relevant when they experience vision changes. The focus group interviews supplement the interviews with patient and relative with a view to gain insight into the social norms and prominent attitudes towards visual impairment among older adults. The participants in the focus group interviews are asked to complete the validated Visual Function Questionnaire-25 to provide more individual knowledge about the participants own perception of their vision function. Through participant observations, we will generate knowledge on the everyday working environment and challenges that health professionals, private optometrists and communal workers navigate in concerning people with visual impairment. Phase II: develop—developing the intervention model In phase II, we operationalise the insights from phase I. This will be done through three consecutive content-developing workshops using the creative method of graphic facilitation. Graphic facilitation is well suited when elaborating on ideas and problem-solving processes because it allows for a transparent process open to multiple agendas. The method can be relevant for redistributing power and expertise in a codesign activity and the physical product of the three content workshops will act as design principles for the intervention. CTS facilitates the workshops and we invite a graphic facilitator to analogue draw and write inputs from the participants on a wall-to-wall paper during the workshops. The graphical recordings from the three workshops constitute a collective overview of core elements to include in the intervention model (see description of specifics on the workshops below and for details on participants). Choosing a visual method to engage participants could seem an unusual choice in a project focusing on visual impairment and vision loss. However, the participating patients are not blind. They live with a visual impairment, which poses a range of consequences and constraints in their every day life, but it does not prevent them from being able to participate in a graphical facilitated workshop. If needed, relevant aids will be provided—in example to enlarge the graphical recordings on a tablet Flow of the three workshops: The graphic facilitator and project researchers develop a template for the workshops. Participants for the workshops are recruited among participants in phase I. Workshop 1 In this workshop, the graphic facilitator engages patients and relatives to formulate a graphical recording on what is lacking in the identification, diagnosis and patient support concerning visual impairment. Workshop 2 The focus for this workshop is to include perspectives from relevant health professionals in the design phase. The health professionals are asked to identify possibilities and barriers of an intervention concerning vision impairment in general practice, including a discussion on key eye examinations and measures that would be feasible in a general practice setting as well as to identify the most relevant eye diseases. Workshop 3 Aims to synthesise the knowledge produced in the previous two workshops by formulating the specific activities, concrete consultation type and identify final intervention effect measurements. This also includes choosing the relevant guidelines for screening and diagnosing to apply in the intervention. Participants are GP’s and project researchers. Other stakeholders will be invited if relevant based on the insights from workshop 1+2. Phase III: feasibility—feasibility test in general practice Part 1: pilot test The intervention model will be first be validated and adjusted accordingly by patient representatives and ophthalmologists. The model will then be tested in a general practice setting to establish face validity and adjust according to the experiences. The model must be clinically relevant and feasible for implementation in a clinical practice. Data production continues in this phase through observational studies and interviews with GPs to disseminate the experiences with the model in general practice and whether the model can be part of improved collaboration between health professionals. Part 2: cohort study Based on findings from the pilot test, we expand the intervention by including 10–15 GP practices in the Capital and Zeeland Region, Denmark to participate in testing the GP’s possibilities and barriers to detect visual impairment and vision loss. According to previous literature, we need to include 1500–2000 patients in general practice aged 65 or older to identify 150–200 with visual impairment. The practices will receive the developed intervention model and recruit in up to 18 months. Patients 65+ who consult their GP as part of an annual consultation for a chronic condition will be informed about the study in the waiting room and asked to complete a questionnaire based on the validated Visual functioning Questionnaire-25. The questionnaire measures the dimensions of self-reported vision-targeted health status that are most important to individuals who have chronic eye diseases. A dedicated staff or GP will examine the vision according to the guidelines formulated in the model. If visual impairment is detected, the patient will undergo further examinations assessed by an ophthalmologist. The specifics of the examinations in general practice and at an ophthalmologist cannot be reported until the phases I+II have been completed, since these are to be developed in the codesign process. At present, we assume an ordinary vision test, visual fields and contrast vision as well as a function test could be included in the GP setting. We assume that measurement of the visual acuity, tonometry, macular and parapapillary optical coherence tomography (OCT) scans and fundus photographs could be part of the extended examinations at an ophthalmologist. An important outcome of phase II is thus detailed information on effect measurements, chosen guidelines and possibly a narrowed focus on specific eye diseases to address in the intervention. The results from the cohort study will function as a reference standard and allow us to study the prevalence of visual impairments as well as eye diseases and study predictor’s for visual impairment as well. Due to the Danish registers, it is possible to follow the cohort for a long period. This follow-up study is not part of the present project, but will be planned later. Analysis The analytical process will be carried out iteratively, which covers analytical steps taken between each of the three project phases to ensure appropriate adjustments in the design of the intervention model. All formal interviews from the study will be audio recorded and transcribed following a project guideline to ensure uniformity. The three phases will generate observations and informal talks, which will be documented through field notes and pictures. Details on the analytical steps are illustrated in .
We apply the Medical Research Council guidance on developing, testing and evaluating complex interventions. To operationalise the framework for complex interventions, we apply a temporal structure from the tradition of human-centred design to divide the project cycle into three main phases: (I) identify the problem, (II) develop the model and (III) test the feasibility of the model. Phase I+II focus on intervention development applying qualitative methods and phase III aims to first pilot test the intervention for feasibility and following implement it broader in a cohort study to measure effect. The intervention explores what patients, carers and professionals perceive as pivotal to improve regarding detection, navigating care and health services and support possibilities for people living with visual impairment. We are explicitly reflective on, how process and product are interwoven and to a very high extent dependent on the context it unfolds within. Following a structured and well-documented design process, we identify the changes made and insights produced (see ).
The aim of phase I is to explore the problem we are addressing. We will perform a literature search on detection of eye diseases in general practice and conduct background interviews with a broad selection of relevant stakeholders to help us map the current practice in detecting and diagnosing eye conditions across sectors in the healthcare system (see ). An essential element in human-centred design processes is to create understanding and empathy for the end-user —in our case people living with visual impairment and their relatives. We aim to incorporate a wide spectrum of eye diagnoses and develop a model to identify and diagnose visual impairment that works from the onset of the patient’s early symptoms, such as stumbling over doorsteps, difficulties in reading, distorted vision or difficulties related to the transition from dark to light spaces and vice versa. The intervention aim of increased patient support also includes support of relatives, who carry a large part of the disease burden and may experience stress and depression due to their loved one’s visual impairment.
Patients, relatives and professionals will be involved by providing their perspectives in various stages of the study and codesign core elements of the model constituting the intervention. The patient engagement process is informed by a thorough report produced by the Danish Center for Social Science Research on older adults with visual impairment and vision loss. The report underlines the need for increased knowledge on how visual impairment affects patients’ every day life in all aspects. The experiences, needs, preferences, and values of patients and relatives will thus be explored. See for an overview of the involvement of participants across the three project phases. In phase I, we perform semistructured interviews with patients and relatives, preferably in their home to gain an insight in their experiences in the context in which they occur. Here, we focus on the patient journey from the time patients experience symptoms leading to a diagnosis and handling life afterwards with a diagnosis. After the interview, patients and relatives are encouraged to contact the researcher if they would like her/him to participate in, for example, a visit to the ophthalmologist or if they have further input or concerns at a later stage. We will furthermore perform focus group interviews with older adults to investigate (1) the expectations to vision in old age and (2) which health professionals’ older adults identify as relevant when they experience vision changes. The focus group interviews supplement the interviews with patient and relative with a view to gain insight into the social norms and prominent attitudes towards visual impairment among older adults. The participants in the focus group interviews are asked to complete the validated Visual Function Questionnaire-25 to provide more individual knowledge about the participants own perception of their vision function. Through participant observations, we will generate knowledge on the everyday working environment and challenges that health professionals, private optometrists and communal workers navigate in concerning people with visual impairment.
In phase II, we operationalise the insights from phase I. This will be done through three consecutive content-developing workshops using the creative method of graphic facilitation. Graphic facilitation is well suited when elaborating on ideas and problem-solving processes because it allows for a transparent process open to multiple agendas. The method can be relevant for redistributing power and expertise in a codesign activity and the physical product of the three content workshops will act as design principles for the intervention. CTS facilitates the workshops and we invite a graphic facilitator to analogue draw and write inputs from the participants on a wall-to-wall paper during the workshops. The graphical recordings from the three workshops constitute a collective overview of core elements to include in the intervention model (see description of specifics on the workshops below and for details on participants). Choosing a visual method to engage participants could seem an unusual choice in a project focusing on visual impairment and vision loss. However, the participating patients are not blind. They live with a visual impairment, which poses a range of consequences and constraints in their every day life, but it does not prevent them from being able to participate in a graphical facilitated workshop. If needed, relevant aids will be provided—in example to enlarge the graphical recordings on a tablet Flow of the three workshops: The graphic facilitator and project researchers develop a template for the workshops. Participants for the workshops are recruited among participants in phase I. Workshop 1 In this workshop, the graphic facilitator engages patients and relatives to formulate a graphical recording on what is lacking in the identification, diagnosis and patient support concerning visual impairment. Workshop 2 The focus for this workshop is to include perspectives from relevant health professionals in the design phase. The health professionals are asked to identify possibilities and barriers of an intervention concerning vision impairment in general practice, including a discussion on key eye examinations and measures that would be feasible in a general practice setting as well as to identify the most relevant eye diseases. Workshop 3 Aims to synthesise the knowledge produced in the previous two workshops by formulating the specific activities, concrete consultation type and identify final intervention effect measurements. This also includes choosing the relevant guidelines for screening and diagnosing to apply in the intervention. Participants are GP’s and project researchers. Other stakeholders will be invited if relevant based on the insights from workshop 1+2.
In this workshop, the graphic facilitator engages patients and relatives to formulate a graphical recording on what is lacking in the identification, diagnosis and patient support concerning visual impairment.
The focus for this workshop is to include perspectives from relevant health professionals in the design phase. The health professionals are asked to identify possibilities and barriers of an intervention concerning vision impairment in general practice, including a discussion on key eye examinations and measures that would be feasible in a general practice setting as well as to identify the most relevant eye diseases.
Aims to synthesise the knowledge produced in the previous two workshops by formulating the specific activities, concrete consultation type and identify final intervention effect measurements. This also includes choosing the relevant guidelines for screening and diagnosing to apply in the intervention. Participants are GP’s and project researchers. Other stakeholders will be invited if relevant based on the insights from workshop 1+2.
Part 1: pilot test The intervention model will be first be validated and adjusted accordingly by patient representatives and ophthalmologists. The model will then be tested in a general practice setting to establish face validity and adjust according to the experiences. The model must be clinically relevant and feasible for implementation in a clinical practice. Data production continues in this phase through observational studies and interviews with GPs to disseminate the experiences with the model in general practice and whether the model can be part of improved collaboration between health professionals. Part 2: cohort study Based on findings from the pilot test, we expand the intervention by including 10–15 GP practices in the Capital and Zeeland Region, Denmark to participate in testing the GP’s possibilities and barriers to detect visual impairment and vision loss. According to previous literature, we need to include 1500–2000 patients in general practice aged 65 or older to identify 150–200 with visual impairment. The practices will receive the developed intervention model and recruit in up to 18 months. Patients 65+ who consult their GP as part of an annual consultation for a chronic condition will be informed about the study in the waiting room and asked to complete a questionnaire based on the validated Visual functioning Questionnaire-25. The questionnaire measures the dimensions of self-reported vision-targeted health status that are most important to individuals who have chronic eye diseases. A dedicated staff or GP will examine the vision according to the guidelines formulated in the model. If visual impairment is detected, the patient will undergo further examinations assessed by an ophthalmologist. The specifics of the examinations in general practice and at an ophthalmologist cannot be reported until the phases I+II have been completed, since these are to be developed in the codesign process. At present, we assume an ordinary vision test, visual fields and contrast vision as well as a function test could be included in the GP setting. We assume that measurement of the visual acuity, tonometry, macular and parapapillary optical coherence tomography (OCT) scans and fundus photographs could be part of the extended examinations at an ophthalmologist. An important outcome of phase II is thus detailed information on effect measurements, chosen guidelines and possibly a narrowed focus on specific eye diseases to address in the intervention. The results from the cohort study will function as a reference standard and allow us to study the prevalence of visual impairments as well as eye diseases and study predictor’s for visual impairment as well. Due to the Danish registers, it is possible to follow the cohort for a long period. This follow-up study is not part of the present project, but will be planned later.
The intervention model will be first be validated and adjusted accordingly by patient representatives and ophthalmologists. The model will then be tested in a general practice setting to establish face validity and adjust according to the experiences. The model must be clinically relevant and feasible for implementation in a clinical practice. Data production continues in this phase through observational studies and interviews with GPs to disseminate the experiences with the model in general practice and whether the model can be part of improved collaboration between health professionals.
Based on findings from the pilot test, we expand the intervention by including 10–15 GP practices in the Capital and Zeeland Region, Denmark to participate in testing the GP’s possibilities and barriers to detect visual impairment and vision loss. According to previous literature, we need to include 1500–2000 patients in general practice aged 65 or older to identify 150–200 with visual impairment. The practices will receive the developed intervention model and recruit in up to 18 months. Patients 65+ who consult their GP as part of an annual consultation for a chronic condition will be informed about the study in the waiting room and asked to complete a questionnaire based on the validated Visual functioning Questionnaire-25. The questionnaire measures the dimensions of self-reported vision-targeted health status that are most important to individuals who have chronic eye diseases. A dedicated staff or GP will examine the vision according to the guidelines formulated in the model. If visual impairment is detected, the patient will undergo further examinations assessed by an ophthalmologist. The specifics of the examinations in general practice and at an ophthalmologist cannot be reported until the phases I+II have been completed, since these are to be developed in the codesign process. At present, we assume an ordinary vision test, visual fields and contrast vision as well as a function test could be included in the GP setting. We assume that measurement of the visual acuity, tonometry, macular and parapapillary optical coherence tomography (OCT) scans and fundus photographs could be part of the extended examinations at an ophthalmologist. An important outcome of phase II is thus detailed information on effect measurements, chosen guidelines and possibly a narrowed focus on specific eye diseases to address in the intervention. The results from the cohort study will function as a reference standard and allow us to study the prevalence of visual impairments as well as eye diseases and study predictor’s for visual impairment as well. Due to the Danish registers, it is possible to follow the cohort for a long period. This follow-up study is not part of the present project, but will be planned later.
The analytical process will be carried out iteratively, which covers analytical steps taken between each of the three project phases to ensure appropriate adjustments in the design of the intervention model. All formal interviews from the study will be audio recorded and transcribed following a project guideline to ensure uniformity. The three phases will generate observations and informal talks, which will be documented through field notes and pictures. Details on the analytical steps are illustrated in .
Ethical issues will be a consideration at all levels of the study both when involving patients in the participatory design and during the cohort study. The study is registered in the records of research projects containing personal data at University of Copenhagen (J.nr: 514-0701/22-3000). It will be conducted according to the ethical standards of the Declaration of Helsinki and general data protection regulations (GDPR). Ethical approval was waived by the Danish National Ethical Research Committee because no biomaterial is included in the study. In data production, all participants will be asked to read and sign a consent form regarding their specific participation and kind of information, including how we will handle the information provided to us as, well as information on how to withdraw consent at a later stage. In the qualitative data production, we will produce photographical and graphical material, which requires further ethical reflections in terms of anonymisation. The cohort study involves a risk of overdiagnostic practice due to the tests and screening involved. Any potential harms, overdiagnosis, labelling effect and consequences of receiving the intervention will be scrutinised during the study. Age-related visual impairment diagnoses including glaucoma and AMD meet the requirements for screening formulated by WHO. During the analysis, both benefits and harms of the intervention will be investigated and presented as results. Results will be published in peer-reviewed journals, preferably open-access. Patients, relatives and health professionals are invited in as coauthors where relevant. We will present our results in relevant fora nationally and internationally (conferences, annual meetings, etc). In addition, we will organise a symposium directed at stakeholders from health and social care sector and employers. The participants from the three content-developing workshops in phase II will be invited to participate in the symposia and share their experiences of being part of the research process. For communication to lay persons, we will produce a podcast on sensory loss in old age focused on vision and participate in the yearly Danish democracy and community festival ‘Folkemødet’, which has a specific focus on communicating public health science. The proposed study is relevant for ensuring kind and empathic care with time to guide and comfort patients. This requires knowledge about how the patient experiences visual impairment as well as identification of the current challenges in the health services provided, which we aim to improve following the DETECT intervention. Specifically relevant in this study is the focus on general practice in relation to visual impairment, which is currently an understudied area. Implications Collectively, the output of intervention will help us understand, how to support and treat patients with impaired vision and to define an expedient role for general practice. In this respect, adding knowledge on the GP perspective will strengthen the feasibility of the intervention. The development of a codesigned intervention can have an important impact on the delivered quality in the diagnosis and management of patients with visual impairment in primary care.
Collectively, the output of intervention will help us understand, how to support and treat patients with impaired vision and to define an expedient role for general practice. In this respect, adding knowledge on the GP perspective will strengthen the feasibility of the intervention. The development of a codesigned intervention can have an important impact on the delivered quality in the diagnosis and management of patients with visual impairment in primary care.
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Demographic trends of patients undergoing ophthalmic surgery in Ontario, Canada: a population-based study
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Ophthalmology[mh]
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Ophthalmic surgery case volumes and wait times have been rapidly growing in recent years in Ontario, Canada as well as many other regions in North America. Wait times are important surrogate measures of the quality of health system delivery. However, there is currently limited research investigating the demographic variations in patients on the ophthalmic surgery wait list. This study found that women have consistently longer wait times compared with men across all ophthalmic procedure types, priority levels and geographic regions. This study also shows that the average age at the time of ophthalmic surgery has been steadily rising over the last decade, although at a slower rate compared with the general Ontario population. Women were found to be slightly older than men, on average. The results of this study indicate the presence of systematic sex-based differences that could be affecting the timeliness of ophthalmic surgery for women. This work provides the first step towards reaching health equity in ophthalmic surgery, and further research should be conducted to measure the potential magnitude of this effect on outcomes, in addition to other possible inequities that could be faced by women and other groups. Ophthalmic surgeries are among the most cost-effective interventions, and with the increasing demand, there is significant pressure being placed on the healthcare system. Case volumes and wait times for ophthalmic surgeries have been growing by more than 50% over the last decade in Ontario. Wait times are important and increasingly popular metrics of healthcare delivery that have important implications for both individual patients and health systems as a whole. Long waits for surgeries may burden patients with significant emotional distress and physical harm from prolonging untreated conditions, which may also result in worse outcomes. Furthermore, there are broad socioeconomic consequences of inappropriately long wait times, such as absenteeism, disability, reduced productivity and requiring assistance from others to perform basic activities. This universal healthcare coverage in Canada, which is managed at the provincial level, has been shown to have persisting inequities in health delivery for both patients and providers. To date, there is limited research and data investigating demographic variations in these patient populations. As the first step to achieving healthcare equity, it is necessary to first detect disparities and measure their magnitude as well as understand their causes. Herein, we present the demographic trends of ophthalmic case volumes and wait-times in Ontario, Canada. This was a population-based retrospective cohort study based on the Ontario Health Wait Times Information System (WTIS) database from January 2010 to December 2021. This study adhered to the Reporting of studies Conducted using Observational Routinely collected Data guidelines. Ethics approval for the conduction of this study was obtained from University of Toronto Research Ethics Board (RIS protocol number: 41582). Data source The Ontario Health Insurance Plan is the publicly-funded healthcare service in Ontario, Canada for residents who meet eligibility criteria. In order to track and address the issues with healthcare access in a centralised system, the WTIS was fully deployed in 2008. The WTIS collects and stores wait time information from non-emergent surgeries performed in publicly funded hospitals in Ontario, Canada. Each case is assigned a priority level by the surgeon according to WTIS guidelines, ranging from level 1 emergent cases (not included in this database) to high priority (level 2, severe symptoms which are likely getting worse), medium priority (level 3, some pain or other symptoms which do not dramatically impact the quality of life) and low priority (level 4, the patient’s condition may be worsening and medical management may be failing to help). The WTIS defines wait time as the number of days from the surgeon and patient’s joint decision to operate to the time of surgery. Wait list queues with only 1–5 cases per month are coded as low volume in the WTIS database and were excluded from this study. Annual wait time data are captured for the following non-emergent ophthalmic subspecialty surgeries: cataract, cornea, glaucoma, oculoplastic, vitreoretinal and adult strabismus surgery. This data set is further stratified into geographical regions, referred to as Local Health Integration Networks (effective April 2021, the 14 local health integration networks (LHINs) were consolidated into 5 regions), which constitute the subdivision of healthcare delivery across Ontario. The original geographical regions (LHINs) encompassed the following: Erie St. Clair, South West, Waterloo Wellington, Hamilton Niagara Haldimand Brant, Central West, Mississauga Halton, Toronto Central, Central, Central East, South East, Champlain, North Simcoe Muskoka, North East, and North West. Study objectives We aimed to summarise demographic shifts over the past decade while stratifying by geographic region, priority level and procedure type. We also sought to examine whether there were any notable differences in patient demographics across strata, with particular focus on sex-based differences in wait-times. Statistical analysis Cohort characteristics were stratified by ophthalmic procedure type, priority level, geographic region, year, age and sex (self-identified gender is not collected) and reported descriptively. To assess whether there was a monotonic upward or downward trend in the data over time, the Mann-Kendall test was chosen as it does not require that the data be linear or normally distributed. Other methods such as simple regression would not have been appropriate given that the underlying assumptions required (eg, normal distribution) are not met in this data set. Wait time rates of change were calculated using the Theil-Sen estimator along with corresponding 95% CIs. This estimator, also referred to as Sen’s Slope, is a well-established, non-parametric method for measuring linear trends while remaining robust to values that do not fit a linear trend and insensitive to outliers. It fits the data by efficiently computing the median slope of all lines through all pairs of points. An independent-samples t-test was used to analyse the aggregate data for continuous outcomes (mean with SD and sample number volumes) for the two groups. The χ 2 test was performed for comparison of categorical variables. All analyses were conducted using R V.3.5.0 (R Foundation for Statistical Computing, Vienna, Austria). Since multiple statistical tests are performed, a conservative significance level of p<0.001 was chosen a priori to reduce the probability of type I errors. Patients and the public were not involved in the design, conduct, reporting or dissemination of this study. The Ontario Health Insurance Plan is the publicly-funded healthcare service in Ontario, Canada for residents who meet eligibility criteria. In order to track and address the issues with healthcare access in a centralised system, the WTIS was fully deployed in 2008. The WTIS collects and stores wait time information from non-emergent surgeries performed in publicly funded hospitals in Ontario, Canada. Each case is assigned a priority level by the surgeon according to WTIS guidelines, ranging from level 1 emergent cases (not included in this database) to high priority (level 2, severe symptoms which are likely getting worse), medium priority (level 3, some pain or other symptoms which do not dramatically impact the quality of life) and low priority (level 4, the patient’s condition may be worsening and medical management may be failing to help). The WTIS defines wait time as the number of days from the surgeon and patient’s joint decision to operate to the time of surgery. Wait list queues with only 1–5 cases per month are coded as low volume in the WTIS database and were excluded from this study. Annual wait time data are captured for the following non-emergent ophthalmic subspecialty surgeries: cataract, cornea, glaucoma, oculoplastic, vitreoretinal and adult strabismus surgery. This data set is further stratified into geographical regions, referred to as Local Health Integration Networks (effective April 2021, the 14 local health integration networks (LHINs) were consolidated into 5 regions), which constitute the subdivision of healthcare delivery across Ontario. The original geographical regions (LHINs) encompassed the following: Erie St. Clair, South West, Waterloo Wellington, Hamilton Niagara Haldimand Brant, Central West, Mississauga Halton, Toronto Central, Central, Central East, South East, Champlain, North Simcoe Muskoka, North East, and North West. We aimed to summarise demographic shifts over the past decade while stratifying by geographic region, priority level and procedure type. We also sought to examine whether there were any notable differences in patient demographics across strata, with particular focus on sex-based differences in wait-times. Cohort characteristics were stratified by ophthalmic procedure type, priority level, geographic region, year, age and sex (self-identified gender is not collected) and reported descriptively. To assess whether there was a monotonic upward or downward trend in the data over time, the Mann-Kendall test was chosen as it does not require that the data be linear or normally distributed. Other methods such as simple regression would not have been appropriate given that the underlying assumptions required (eg, normal distribution) are not met in this data set. Wait time rates of change were calculated using the Theil-Sen estimator along with corresponding 95% CIs. This estimator, also referred to as Sen’s Slope, is a well-established, non-parametric method for measuring linear trends while remaining robust to values that do not fit a linear trend and insensitive to outliers. It fits the data by efficiently computing the median slope of all lines through all pairs of points. An independent-samples t-test was used to analyse the aggregate data for continuous outcomes (mean with SD and sample number volumes) for the two groups. The χ 2 test was performed for comparison of categorical variables. All analyses were conducted using R V.3.5.0 (R Foundation for Statistical Computing, Vienna, Austria). Since multiple statistical tests are performed, a conservative significance level of p<0.001 was chosen a priori to reduce the probability of type I errors. Patients and the public were not involved in the design, conduct, reporting or dissemination of this study. From 2010–2021, there were on average 1.49 adults/year/100 000 scheduled for non-emergency ophthalmic surgery in Ontario, Canada. The mean wait time over the study period for all procedure types and priority levels was 82 days (SD 85). The mean age was 71 years (SD 11) and 44% of all patients were men. Surgical volumes Over the last decade, an average of 83 783 women/year (SD 7297) underwent ophthalmic surgery annually in Ontario compared with 65 555 (SD 5266) men/year. Women underwent more eye surgeries than men across all ophthalmic specialties, with the exception of retinal procedures (3119 women/year vs 3819 men/year). The greatest difference in volume occurred with cataracts, where 76 177 women/year (SD 6791) had cataract surgery compared with 57 763 men/year (SD 4837). summarises the sex-stratified volume of surgery for each procedure and year. There were significantly higher proportions of men compared with women with high priority (level 2) eye surgery on the wait list. Specifically, 1739 men (SD 573, 2.7% of all men on the wait list) and 1482 women (SD 605, 1.8% of all women on the wait list) received high priority (level 2) surgery annually, 7594 men (SD 1869, 11.6% of all men on the wait list) and 8769 women (SD 2434, 10.5% of all women on the wait list) received medium priority (level 3) surgery annually and 56 222 men (SD 6141, 85.8% of all men on the wait list) compared with 73 532 women (SD 7861, 87.8% of all women on the wait list) received low priority (level 4) surgery annually (χ 2 p<0.001). Cataract surgery was the only procedure that still had a higher volume of women than men (82 more individual cases annually) for high priority (level 2) operations. Patient sex trends Men had consistently shorter wait times compared with women across all surgeries, with an average of 4.9 days shorter wait in the province over the last decade (p<0.001) . Oculoplastic (6.8 days longer wait for females, p=0.013) and corneal (5.1 days longer wait for women, p=0.38) surgeries had the greatest difference in wait times between men and women from 2010 to 2021, while glaucoma (0.6 days longer wait for women, p=0.77) and cataract surgery (3.6 days longer wait for women, p<0.001)) had the smallest difference . When stratifying by priority level, women continued to have longer wait times . Across all ophthalmic subspecialties in Ontario, women waited 6.1 days longer than men for high priority surgery (p<0.001), 7.2 days longer for medium priority surgery (p<0.001) and 3.7 days longer for low priority surgery (p<0.001). When stratified by geography over all priority levels and procedure types, again women waited longer than men in every region . The wait time difference between men and females was greatest in the Toronto Central, North West and North Simcoe Muskoka LHINs, where women waited 9.3 (p<0.001), 7.2 (p=0.034) and 6.7 (p=0.01) days longer than men, respectively. The North East, Central and Hamilton Niagara Haldimand Brant LHINs had the smallest difference with women waiting 2.0 (p=0.27), 2.4 (p=0.01) and 3.1 (p=0.01) days longer than men, respectively. 10.1136/bmjophth-2023-001253.supp3 Supplementary data 10.1136/bmjophth-2023-001253.supp1 Supplementary data Patient age trends Across Ontario, average age at the time of eye surgery has been increasing (+0.02 years/year; 95% CI (0.00 to 0.05)) over the past decade for all ophthalmic specialties . Cornea, glaucoma, strabismus and oculoplastic procedures all had statistically significant increases in age over time . Cornea procedures had the greatest increase in mean age in Ontario over the past decade (+0.62 years/year; 95% CI (0.49 to 0.71)). Glaucoma (+0.28 years/year; 95% CI (0.23 to 0.37)), strabismus (+0.24 years/year; 95% CI (0.10 to 0.40)) and oculoplastic surgery (+0.24 years/year; 95% CI (0.11 to 0.20)) all had similar increases in age. These patterns were consistent across different levels of surgical priority as well. For these four specialties, when reviewing the change in age across different geographies, there were no LHINs with statistically significant increases in age for oculoplastic surgery, while both adult strabismus and glaucoma surgery had significant increases in age in the Toronto Central region. Cornea procedures had significant increases in age in each of the following LHINs: Central West, Champlain, Erie St. Clair, Hamilton Niagara Haldimand Brant, South West, Toronto Central and Waterloo Wellington. 10.1136/bmjophth-2023-001253.supp2 Supplementary data Combined age and sex trends Overall, Ontario women on the wait list for eye surgery were 0.6 years older than men from 2010 to 2021 (p<0.001). The only ophthalmic specialty where women were younger (by 1.1 years on average) was oculoplastic surgery (p=0.08). The greatest differences were in corneal (women were 4.0 years older on average, p<0.001) and glaucoma surgery (women were 3.6 years older on average, p<0.001). The difference in age between men and women over the last decade has been decreasing slightly for most procedures . However, it increased with increasing levels of surgical priority, with women being 2.6 years older than men for high priority surgery (p<0.001) and 0.4 years older for low priority surgery (p<0.001). Over the last decade, an average of 83 783 women/year (SD 7297) underwent ophthalmic surgery annually in Ontario compared with 65 555 (SD 5266) men/year. Women underwent more eye surgeries than men across all ophthalmic specialties, with the exception of retinal procedures (3119 women/year vs 3819 men/year). The greatest difference in volume occurred with cataracts, where 76 177 women/year (SD 6791) had cataract surgery compared with 57 763 men/year (SD 4837). summarises the sex-stratified volume of surgery for each procedure and year. There were significantly higher proportions of men compared with women with high priority (level 2) eye surgery on the wait list. Specifically, 1739 men (SD 573, 2.7% of all men on the wait list) and 1482 women (SD 605, 1.8% of all women on the wait list) received high priority (level 2) surgery annually, 7594 men (SD 1869, 11.6% of all men on the wait list) and 8769 women (SD 2434, 10.5% of all women on the wait list) received medium priority (level 3) surgery annually and 56 222 men (SD 6141, 85.8% of all men on the wait list) compared with 73 532 women (SD 7861, 87.8% of all women on the wait list) received low priority (level 4) surgery annually (χ 2 p<0.001). Cataract surgery was the only procedure that still had a higher volume of women than men (82 more individual cases annually) for high priority (level 2) operations. Men had consistently shorter wait times compared with women across all surgeries, with an average of 4.9 days shorter wait in the province over the last decade (p<0.001) . Oculoplastic (6.8 days longer wait for females, p=0.013) and corneal (5.1 days longer wait for women, p=0.38) surgeries had the greatest difference in wait times between men and women from 2010 to 2021, while glaucoma (0.6 days longer wait for women, p=0.77) and cataract surgery (3.6 days longer wait for women, p<0.001)) had the smallest difference . When stratifying by priority level, women continued to have longer wait times . Across all ophthalmic subspecialties in Ontario, women waited 6.1 days longer than men for high priority surgery (p<0.001), 7.2 days longer for medium priority surgery (p<0.001) and 3.7 days longer for low priority surgery (p<0.001). When stratified by geography over all priority levels and procedure types, again women waited longer than men in every region . The wait time difference between men and females was greatest in the Toronto Central, North West and North Simcoe Muskoka LHINs, where women waited 9.3 (p<0.001), 7.2 (p=0.034) and 6.7 (p=0.01) days longer than men, respectively. The North East, Central and Hamilton Niagara Haldimand Brant LHINs had the smallest difference with women waiting 2.0 (p=0.27), 2.4 (p=0.01) and 3.1 (p=0.01) days longer than men, respectively. 10.1136/bmjophth-2023-001253.supp3 Supplementary data 10.1136/bmjophth-2023-001253.supp1 Supplementary data Across Ontario, average age at the time of eye surgery has been increasing (+0.02 years/year; 95% CI (0.00 to 0.05)) over the past decade for all ophthalmic specialties . Cornea, glaucoma, strabismus and oculoplastic procedures all had statistically significant increases in age over time . Cornea procedures had the greatest increase in mean age in Ontario over the past decade (+0.62 years/year; 95% CI (0.49 to 0.71)). Glaucoma (+0.28 years/year; 95% CI (0.23 to 0.37)), strabismus (+0.24 years/year; 95% CI (0.10 to 0.40)) and oculoplastic surgery (+0.24 years/year; 95% CI (0.11 to 0.20)) all had similar increases in age. These patterns were consistent across different levels of surgical priority as well. For these four specialties, when reviewing the change in age across different geographies, there were no LHINs with statistically significant increases in age for oculoplastic surgery, while both adult strabismus and glaucoma surgery had significant increases in age in the Toronto Central region. Cornea procedures had significant increases in age in each of the following LHINs: Central West, Champlain, Erie St. Clair, Hamilton Niagara Haldimand Brant, South West, Toronto Central and Waterloo Wellington. 10.1136/bmjophth-2023-001253.supp2 Supplementary data Overall, Ontario women on the wait list for eye surgery were 0.6 years older than men from 2010 to 2021 (p<0.001). The only ophthalmic specialty where women were younger (by 1.1 years on average) was oculoplastic surgery (p=0.08). The greatest differences were in corneal (women were 4.0 years older on average, p<0.001) and glaucoma surgery (women were 3.6 years older on average, p<0.001). The difference in age between men and women over the last decade has been decreasing slightly for most procedures . However, it increased with increasing levels of surgical priority, with women being 2.6 years older than men for high priority surgery (p<0.001) and 0.4 years older for low priority surgery (p<0.001). This retrospective population-based study highlighted the demographic trends of ophthalmic surgical wait times from 2010 to 2021 in Ontario, Canada. Consistent with previous reports and epidemiological data, more women undergo ophthalmic surgery (56%). While men represent a higher proportion of high-priority cases in our data set, it is important to note that women experienced longer wait times across all priority levels when compared with their male counterparts within the same priority category. The proportion of women undergoing ophthalmic surgery is slightly higher than the general Ontario population, which has 50% women overall and 54% women in the 65-or-older age group. Our study also shows that the average age at the time of ophthalmic surgery has been rising slowly over the last decade, and women are slightly older than men. The most striking finding of this study is that there are sex-based differences in surgical wait times, where men have shorter wait times compared with women across ophthalmic subspecialties, geographic regions and levels of priority. Although the difference in wait time might seem modest in terms of absolute difference when compared with the mean wait times, it is important to consider the cumulative impact of these differences on patients and the healthcare system as a whole. It is important to note that our study included data from the COVID-19 pandemic period (2020–2021), which has had a significant impact on healthcare systems and patient care. The disparities in wait times between sexes observed in our study existed prior to the pandemic, and recent work highlights that these disparities have been gradually worsening with time, especially during the pandemic. In fact, the COVID-19 pandemic exacerbated pre-existing wait time disparities between sexes, with women waiting 4.1 days longer than men overall to receive surgery in 2010–2019 compared with waiting 8.8 days longer in 2020–2021 (117% increase) in Ontario, Canada. This suggests that the pandemic has further intensified the systemic sex-based biases that may be affecting the care of women. Overall, men waited 5 days less for surgery than women in our study, with oculoplastic and corneal procedures having the greatest difference. Women experienced longer wait times across all priority levels and geographic regions, with medium-to-high priority levels and Toronto Central, North West and North Simcoe Muskoka LHINs having the greatest disparities from 2010 to 2021. Previous studies have also investigated sex-based differences in surgical wait times with varying results depending on the medical specialty, region and data availability. A study based in the USA found that women had a 2.95-day delay in receiving retinal detachment repair surgery compared with men along with 34% reduced odds of receiving surgery. Similarly, a study based in Japan found that men had 83% higher odds of early (within 1 week) retinal detachment surgery compared with women. In our study, women waited 4.6 days longer than men on average for retinal surgeries. Aligned with our study findings, an analysis of data from the Swedish National Cataract Register found longer waiting times for women that persisted across priority levels. Women waited 6 days longer than men, and this difference increased proportionally with increasing overall waiting times. Another study in Sweden found that longer waiting times were associated with good visual acuity, older age, low income, low level of education and being women. They found that even when adjusting for factors unrelated to wait time in addition to month of operation and surgical centre, women persistently waited 3.7 days longer than men for cataract surgery. When intersecting multiple inequalities together, such as a low-income female patient with no education, the authors found that wait times additively increased. Similarly, in our study, women waited 3.6 days longer than men for cataract surgery and this difference persisted across all priority levels and geographic regions. Beyond ophthalmology, the differences in wait time between men and women are more variable. For example, a longitudinal analysis of bariatric surgery wait times in Ontario found that men had significantly increased odds of longer wait times, with an effect size of 34 additional days compared with women. Conversely, another Ontario-based study that used the full WTIS database found that women waited 3.1 days longer than men across all surgical specialties. Other studies outside of Ontario have also demonstrated significant sex-based differences in wait times, such as in female Medicare beneficiaries waiting 13% longer to undergo pancreatectomy, female trauma patients experiencing longer delays in trauma care or women with shoulder injury waiting 18 days longer than men to receive surgery. Some reports have also shown differences in specialist referral wait times, such as one in Southwestern Ontario that found female patients waiting 4 days longer than men to see a specialist. The wait time differences discovered in this study may be suggestive of systemic sex-based biases. These biases may be affecting the care of women or other groups in other meaningful ways that are not currently measured. Previous studies have suggested that a complex interplay of sociopolitical and cultural factors, such as taking on the role of a caregiver and postponing appointments for the sake of other family members, has contributed to women having longer wait times. While this may be true and contribute to the wait time disparity along with other patient factors, it will be equally as important to further investigate the practice patterns and surgical referrals of ophthalmologists or other physicians in Ontario and beyond. It should also be noted that currently less than a third of all Canadian and Ontario ophthalmologists are women, and despite this being the largest proportion of women in Canadian history, ophthalmology is still severely lagging behind most other medical specialties. The average patient age at the time of eye surgery has been gradually increasing at a rate of +0.02 years/year over the past decade across all ophthalmic specialties, though this is at a much lower rate compared with the general Ontario population over the same time period (+0.18 years/year; 95% CI (0.15 to 0.20)). However, patient groups in the cornea, glaucoma, strabismus and oculoplastic subspecialties have been outpacing general population growth over the last 10 years, and have been doing so primarily in the Toronto Central region. This may be due to a variety of reasons, including greater accessibility to resources and availability of doctors and social supports, improvements in practice standards and enhanced screening which results in elderly patients having higher chances of receiving eye surgery. We also found that Ontario women on the wait list for eye surgery were slightly older than men across all ophthalmic subspecialties with the exception to oculoplastic surgery, and that this difference increased with increasing priority level. The general female population in Ontario is noted to be 1.8 years older than men on average. As such, the difference in our study is likely attributable to the fact that women represent the majority of patients receiving ophthalmic surgery and are older than men due to their higher life expectancy. Each of the 14 LHINs of Ontario had longer surgical wait times for women compared with men. The Toronto Central LHIN, which has the highest ratio of ophthalmologists per 100 000 in Ontario (8.87), also had the greatest overall difference in wait times at 9.3 days. This pattern did not persist for most other LHINs. For example, the North Simcoe Muskoka region had the third highest wait-time disparity at 6.7 days, yet it has one of the lowest ratios of ophthalmologists per 100 000 at 2.05. Limitations and future directions The authors would like to acknowledge limitations to this study. First, this was a retrospective study using aggregated data limited to the province of Ontario. Due to the limitations of our retrospective study design and the aggregate data available in the WTIS database, we were unable to adjust for patient-level and hospital-level covariates in our analysis. We also did not have data on the time from primary care referral to diagnostic work-up, nor did we have data on the time between surgical consultation and the day of operation. Furthermore, the WTIS database only collects patient sex, not gender or other self-reported identities. Thus, the findings of this study may not represent the true wait-time differences between self-identifying men and women, and they also do not incorporate potential disparities that may be experienced by non-binary individuals. Additionally, there may be some variability with respect to patient priority-level assignments in the WTIS database (eg, one high priority case being worse than another, or a high priority case which should have been classified as medium priority) that may have affected results. Finally, as we only have access to aggregated data, we cannot verify or account for any potential repeated measures. However, it is important to note that the waiting times reported in this study are not self-reported by patients, but rather are collected automatically through administrative information technology systems, which minimises the likelihood of reporting errors or biases. Future studies may explore the associations in the observed trends with important patient and provider characteristics. The authors would like to acknowledge limitations to this study. First, this was a retrospective study using aggregated data limited to the province of Ontario. Due to the limitations of our retrospective study design and the aggregate data available in the WTIS database, we were unable to adjust for patient-level and hospital-level covariates in our analysis. We also did not have data on the time from primary care referral to diagnostic work-up, nor did we have data on the time between surgical consultation and the day of operation. Furthermore, the WTIS database only collects patient sex, not gender or other self-reported identities. Thus, the findings of this study may not represent the true wait-time differences between self-identifying men and women, and they also do not incorporate potential disparities that may be experienced by non-binary individuals. Additionally, there may be some variability with respect to patient priority-level assignments in the WTIS database (eg, one high priority case being worse than another, or a high priority case which should have been classified as medium priority) that may have affected results. Finally, as we only have access to aggregated data, we cannot verify or account for any potential repeated measures. However, it is important to note that the waiting times reported in this study are not self-reported by patients, but rather are collected automatically through administrative information technology systems, which minimises the likelihood of reporting errors or biases. Future studies may explore the associations in the observed trends with important patient and provider characteristics. This population-based retrospective cohort study summarised the demographic trends of Ontario patients undergoing ophthalmic surgery from 2010 to 2021. Our findings suggest that women have consistently longer wait times than men across all ophthalmic procedure types, priority levels and geographic regions. Further research should be conducted to investigate the institutional-level and patient-level covariates that may be contributing to the disparities discovered in this study, in addition to other possible inequities as well as the impact they may have on health outcomes.
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Development of quality indicators for the diagnosis and treatment of urinary tract infections in general practice: a RAND appropriateness method
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3bcd1833-f266-42be-9c5d-f7740ea4f1a3
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10231022
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Family Medicine[mh]
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To improve antibiotic use in general practice, it is necessary to focus on both the diagnostic process and the prescribing patterns.
A total of 24 quality indicators for the management of patients with suspected urinary tract infections have been developed.
This set of indicators may be used to identify potential quality problems as a basis for reflection and opportunities for improvements.
Antibiotic use is associated with the emergence of resistant bacteria, which is considered to be a major threat to human health worldwide. It is estimated that around 700 000 people die annually due to an infection that no longer can be successfully treated with antibiotics. The overall antibiotic use in Denmark is low compared with many other European countries, although higher than in some of the other Nordic countries. In Denmark, antibiotic consumption has decreased over the last decade, however, a large use of antibiotics, including a relatively high use of quinolones, has recently been found in the elderly population. About 80% of antibiotics are prescribed in general practice with urinary tract infections (UTIs) being one of the most common indications for antibiotic prescribing, particularly for women and elderly patients. In order to improve the quality of antibiotic use for UTIs in general practice, it is necessary to identify any quality problems. Use of quality indicators (QIs) is a structured method allowing thorough detection of potential quality problems. QIs are defined as ‘specific and measureable elements of practice that can be used to assess the quality of care’, as they provide a quantitative measure of quality. Indicators are often constructed as a proportion ; defined with a numerator (number of patients receiving a specific investigation or treatment) and a denominator (number of patients included in the quality assessment). Quality is multidimensional and the development and interpretation of an indicator is not always straightforward. It is important to keep in mind that QIs do not provide definitive answers but indicate problems or good quality. QIs for the management of patients with suspected UTI have previously been developed. Some of these indicators are developed for use in outpatient care ; others in hospitals and some are adapted to a specific group of patients. However, no QIs for outpatient care encompass the diagnostic process even though it is well known that a rational decision to prescribe antibiotics is based on a proper diagnosis. Despite a clear demand, no QIs comprising both the diagnostic process and the decision to prescribe antibiotics for UTI, have so far been developed for use in general practice. The aim of this project was to develop a set of QIs for the diagnosis and antibiotic treatment of patients aged ≥18 years with suspected UTI in general practice.
A Research and Development (RAND)/University of California Los Angeles (UCLA) appropriateness method was used for the development of the indicators. This method was established in the 1980s by the RAND health organisation in corporation with UCLA. It is a consensus method described as the only thoroughly tested systematic method combining evidence with expert opinion. The RAND/UCLA appropriateness method is widely used for the development of QIs in healthcare systems, and comprises four steps: Development of preliminary QIs based on scientific evidence and guidelines. First assessment of proposed QIs by a panel of experts using an emailed fact sheet (round 1). Consensus meeting in which the panellists discuss the indicators. Second assessment of QIs during the meeting (round 2). Preparatory phase A national research team comprising four researchers/physicians (including three of the authors) was established. The assignment of the research team was to generate a set of proposed QIs as well as to prepare and lead the consensus process. The team identified all evidence-based Danish guidelines for the management of patients with suspected UTI in general practice. Only guidelines available online were included. Several main themes for quality measurement were identified from the guidelines. Whenever guidelines presented conflicting recommendations the most recent recommendation was used. The recommendations were operationalised as preliminary QIs with accompanying standards. Further evidence-based literature was sought whenever needed to complete the development of each of the preliminary QIs. This set of indicators was divided into three quality domains focusing on either; the diagnostic process (eg, the assessment of specific symptoms, signs and test results), the treatment decision (eg, to prescribe or withhold antibiotics) or the choice of antibiotics prescribed (eg, pivmecillinam, sulfamethizole, nitrofurantoin, trimethoprim or ciprofloxacin). The indicators comprised both lower and upper UTI for patients aged ≥18 years. Some QIs concerned only specific patient groups, such as catheter users and pregnant women. Each proposed QI was developed with an accompanying standard (acceptable range) to encompass the optimal performance addressed by that indicator. Standards were based on most recent national guidelines. Low quality of the care provided is indicated if a performance falls outside the standard range. Furthermore, a registration chart to collect data for quality investigation was developed to match the proposed indicators. 10.1136/bmjoq-2022-002156.supp1 Supplementary data Patients or the public were not involved in the design, conduct, reporting or dissemination plans of this research. The expert panel A broad national nine-person panel representing all five regions of healthcare administration of Denmark was established. The panel comprised seven general practitioners (GPs), one microbiologist and one infectious disease specialist. Seven women and two men participated (please see the Acknowledgement section for further details about the panel members). To be deemed as an expert, the person needed to have profound knowledge and experience within the management of UTI, general practice and/or quality assessment. The experts were purposively sampled to ensure participants from diverse geographical areas and different professional positions. Round 1 A fact sheet was prepared for each proposed QI including the definition of the QI, standard and domain, relevant patient population and evidence with reference to relevant literature. Fact sheets with the 27 proposed QIs were distributed to all experts via email. The experts were asked to rate each indicator on a 9-point Likert scale ranging from 1 (completely disagree) to 9 (completely agree). Each indicator had to be rated for the relevance of measuring the quality of a health professional’s management of patients with suspected UTI in general practice. The experts were provided with links to relevant guidelines and encouraged to use this evidence-based information when assessing the indicators. The experts were asked to rate each indicator with the accompanying standard as an integral unit. However, if the experts disagreed with the standard they were asked to explain the disagreement and rate the QI separately. Round 2 All nine experts attended the 2.5 hours online meeting held in November 2020, 7 days after the first round of rating was completed. One expert (a microbiologist) was unable to attend the entire meeting, but completed the process through postmeeting rating. Each expert was provided with feedback on their own ratings by means of a bar chart showing the distribution of ratings from the first round, with the expert’s own rating marked in the figure. Only QIs that did not reach consensus during the first round were discussed at the online meeting. Each of these indicators was discussed and experts were encouraged to propose new QIs or rephrase the already existing ones. The discussion was facilitated by a moderator (LTS) from the research team and evidence-based literature was cited whenever appropriate. Finally, the experts were asked to rate these indicators again at the meeting. Analysis For each QI, medians of the Likert scores were calculated and the indicators were classified into three levels of appropriateness based on the recommendation from the RAND team : (1) appropriate (accepted and not further assessed) was defined as a panel median of 7–9 with agreement; (2) uncertain (and included in the next assessment round) was defined as a panel median of 4–6 or any median without agreement and (3) inappropriate (excluded and not further assessed) was defined as a panel median of 1–3 with agreement. Agreement was defined as: no more than one expert rated the indicator outside the three-point region (1–3, 4–6 and 7–9) containing the median. Those indicators classified as appropriate from either the first or the second round were included in the final set of indicators.
A national research team comprising four researchers/physicians (including three of the authors) was established. The assignment of the research team was to generate a set of proposed QIs as well as to prepare and lead the consensus process. The team identified all evidence-based Danish guidelines for the management of patients with suspected UTI in general practice. Only guidelines available online were included. Several main themes for quality measurement were identified from the guidelines. Whenever guidelines presented conflicting recommendations the most recent recommendation was used. The recommendations were operationalised as preliminary QIs with accompanying standards. Further evidence-based literature was sought whenever needed to complete the development of each of the preliminary QIs. This set of indicators was divided into three quality domains focusing on either; the diagnostic process (eg, the assessment of specific symptoms, signs and test results), the treatment decision (eg, to prescribe or withhold antibiotics) or the choice of antibiotics prescribed (eg, pivmecillinam, sulfamethizole, nitrofurantoin, trimethoprim or ciprofloxacin). The indicators comprised both lower and upper UTI for patients aged ≥18 years. Some QIs concerned only specific patient groups, such as catheter users and pregnant women. Each proposed QI was developed with an accompanying standard (acceptable range) to encompass the optimal performance addressed by that indicator. Standards were based on most recent national guidelines. Low quality of the care provided is indicated if a performance falls outside the standard range. Furthermore, a registration chart to collect data for quality investigation was developed to match the proposed indicators. 10.1136/bmjoq-2022-002156.supp1 Supplementary data Patients or the public were not involved in the design, conduct, reporting or dissemination plans of this research.
A broad national nine-person panel representing all five regions of healthcare administration of Denmark was established. The panel comprised seven general practitioners (GPs), one microbiologist and one infectious disease specialist. Seven women and two men participated (please see the Acknowledgement section for further details about the panel members). To be deemed as an expert, the person needed to have profound knowledge and experience within the management of UTI, general practice and/or quality assessment. The experts were purposively sampled to ensure participants from diverse geographical areas and different professional positions.
A fact sheet was prepared for each proposed QI including the definition of the QI, standard and domain, relevant patient population and evidence with reference to relevant literature. Fact sheets with the 27 proposed QIs were distributed to all experts via email. The experts were asked to rate each indicator on a 9-point Likert scale ranging from 1 (completely disagree) to 9 (completely agree). Each indicator had to be rated for the relevance of measuring the quality of a health professional’s management of patients with suspected UTI in general practice. The experts were provided with links to relevant guidelines and encouraged to use this evidence-based information when assessing the indicators. The experts were asked to rate each indicator with the accompanying standard as an integral unit. However, if the experts disagreed with the standard they were asked to explain the disagreement and rate the QI separately.
All nine experts attended the 2.5 hours online meeting held in November 2020, 7 days after the first round of rating was completed. One expert (a microbiologist) was unable to attend the entire meeting, but completed the process through postmeeting rating. Each expert was provided with feedback on their own ratings by means of a bar chart showing the distribution of ratings from the first round, with the expert’s own rating marked in the figure. Only QIs that did not reach consensus during the first round were discussed at the online meeting. Each of these indicators was discussed and experts were encouraged to propose new QIs or rephrase the already existing ones. The discussion was facilitated by a moderator (LTS) from the research team and evidence-based literature was cited whenever appropriate. Finally, the experts were asked to rate these indicators again at the meeting.
For each QI, medians of the Likert scores were calculated and the indicators were classified into three levels of appropriateness based on the recommendation from the RAND team : (1) appropriate (accepted and not further assessed) was defined as a panel median of 7–9 with agreement; (2) uncertain (and included in the next assessment round) was defined as a panel median of 4–6 or any median without agreement and (3) inappropriate (excluded and not further assessed) was defined as a panel median of 1–3 with agreement. Agreement was defined as: no more than one expert rated the indicator outside the three-point region (1–3, 4–6 and 7–9) containing the median. Those indicators classified as appropriate from either the first or the second round were included in the final set of indicators.
A total of 16 QIs reached consensus after the first round of ratings. The remaining 11 indicators were discussed and reassessed at the following online meeting. Two QIs (QI9 and QI16, ) were rephrased at the meeting and one additional indicator (QI17) was proposed by the panel of experts. Following consensus of appropriateness, the final set comprised 24 QIs for the management of patients with suspected UTI in general practice. Consensus of appropriateness was attained for all of the 11 proposed QIs focusing on the diagnostic process . For example, the experts agreed on the relevance of measuring the performance of urine culture and susceptibility testing for patients diagnosed with complicated lower UTI (QI4). The importance of assessing the use of urine culture and susceptibility testing was likewise agreed on when referring to patients with pyelonephritis and specific patient groups (pregnant women or catheter users) who might have a UTI (QI6, QI10 and QI11). The panel of experts also agreed on the relevance of measuring the appearance of UTI symptoms and high probability of bacteriuria (a positive urinary dipstick and/or a positive microscopy) when patients were diagnosed with UTI (QI2). Consensus of appropriateness was attained for six of the eight proposed QIs focusing on the treatment decision . The experts agreed on the relevance of measuring antibiotic treatment for catheter users with suspected UTI (QI16), and concurrently proposed an additional indicator measuring change of catheter for catheter users with symptoms of UTI (QI17). Consensus of appropriateness was attained for six of the eight QIs focusing on the choice of antibiotics prescribed . The experts agreed on the relevance of measuring the use of pivmecillinam as first choice antibiotic for treatment of patients with suspected lower or upper UTI and for pregnant women with a possible UTI (QI19, QI21 and QI24). The panel also agreed on the relevance of measuring the use of ciprofloxacin for patients with either lower or upper UTI, and for catheter users with suspected UTI (QI20, QI22 and QI23). Three standards were modified during the consensus process. For example, the proposed standard for the indicator concerning patients with suspected lower UTI treated with ciprofloxacin was set to 0%–10%, but changed to 0%–5% according to the expert’s recommendation and consensus (QI20).
Main findings A panel of Danish experts agreed on a total of 24 QIs for the management of patients with suspected UTI in general practice. All QIs focusing on the diagnostic process achieved consensus of appropriateness, while the experts agreed on three quarters of the proposed QIs concerning either the treatment decision or the choice of antibiotics. Strengths and limitations Previous studies have demonstrated the need for validated QIs, developed by an appropriate methodology, to be able to assess the diagnostic process and the treatment decision for patients with suspected UTI in general practice. This study adhered to a widely recognised, systematic method combining evidence with expert opinion ensuring a transparent and scientific process for the development of indicators. During the consensus process, the experts evaluated the face validity of the proposed QIs, that is, if the indicators reflected the quality issue that was intended to be measured. Moreover, the QIs were based on guidelines and scientific evidence, which are considered to provide QIs with content validity. Thus, validation of each QI was an inherent part of the development process. The feasibility of the QIs has been evaluated by application of the indicators to relevant data. Previous studies have demonstrated that adherence to guidelines on antibiotic use is protective against treatment failure and mortality. This set of QIs is based on the most updated evidence-based national guidelines for UTI. All indicators only concerned day 1, that is, the day of the first contact to general practice. Consequently, no indicators were designed to include, for example, the result of a urine culture. We chose this design because most antibiotics for UTI are prescribed at day 1. Information about patient’s symptoms and signs and results of point-of-care tests are likewise available at day 1. Accordingly, we believe that insight into the care provided at day 1 is sufficient to generate useful and comprehensive knowledge about the quality of care for patients with suspected UTI in general practice. One expert attended only 1 hour of the consensus meeting. However, this expert was provided with comprehensive information from the last part of the meeting before asked to rate the indictors for the second time. Comparisons with other studies QIs for the management of patients with suspected UTI have previously been developed. However, to our knowledge, the indicators from this study are the first set of indicators developed for use in general practice, comprising both the diagnostic process and the decision to prescribe antibiotics for UTI. The indicator (QIB) that evaluated if patients with high probability of uncomplicated lower UTI (≥1 symptom of lower UTI and a positive urinary dipstick and/or a positive microscopy) were treated with antibiotics, did not obtain consensus. It is well documented that antibiotic treatment is superior to non-antibiotic treatment regimens in terms of achieving bacteriological cure and symptomatic relief. However, the results on the risk of complications such as pyelonephritis are debated. Some studies have documented an increased risk of pyelonephritis when ibuprofen was prescribed instead of antibiotics. Meanwhile previous studies have found very few cases of complications and no increased risk of pyelonephritis when treatment with pivmecillinam was compared with placebo. The European Surveillance of Antimicrobial Consumption Network managed to develop a similar indicator: percentage of female patients older than 18 years with cystitis/other urinary infection prescribed antibiotics for systemic use, standard range 80%–100%, although the indicator did not reach consensus for all dimensions during the consensus process. The result of the urinary dipstick was included in several of the indicators proposed. Diagnostic accuracy for UTI improves considerably when symptoms and signs are combined with the result of the dipsticks test. Dipstick results are also included in a model to predict antibiotic prescriptions for UTI. Several studies have explored the link between the non-specific symptom confusion and UTI, however, the association is not clearly documented. Interestingly, two of the QIs in our set of proposed indicators were rephrased (QI9 and QI16). The rephrasing of both indicators involved a deletion of the proposed specific symptom: emerging confusion in catheter users with suspected UTI. The rephrased indicators ended up with the inclusion of only four specific symptoms for catheter users with suspected UTI: (1) fever, (2) shivering, (3) flank pain and (4) systemically unwell. Examination of a urine sample is not recommended in patients without UTI symptoms and guidelines recommend no antibiotic treatment for asymptomatic bacteriuria. Nonetheless, it is very likely that, in every day general practice it may happen anyway. The experts agreed on the indicator concerning urinalysis in patients with no UTI symptoms (QI1). Contrarily, the indicator that evaluated if patients without UTI symptoms but with a high probability of bacteriuria (positive urinary dipstick and/or a positive microscopy) were treated with antibiotics, did not reach consensus (QIA). Some of the experts did not agree on QIA because it included two not recommended actions: (1) to examine urine in patients without UTI symptoms and (2) to treat patients without UTI symptoms with antibiotics. Perspectives A set of 24 QIs for the diagnosis and treatment of patients with suspected UTI in general practice has been developed. Studies have shown that GPs in general have a positive attitude towards the use of QIs. This set of indicators may be used to strengthen GPs’ focus on their management of patients with suspected UTI and identify potential quality problems. The indicators are not only applicable to Danish general practice but may be applied to for example general practices in the Nordic countries with antibiotic use, resistance pattern and practice setting similar to Danish conditions. In Denmark, such as many other countries, general practices do not have detailed systematic data registration systems. Therefore, application of indicators to measure and improve patient care is currently a time-consuming activity, hampering a systematic application of these indicators. Since 2018, Danish GPs have been joined in ‘quality clusters’ for quality discussion and support. This set of QIs can ideally be applied to data and used as a basis for reflection and discussion of opportunities for improvements in these ‘quality clusters’. Furthermore, the indicators can advantageously be used for an intervention programme aiming at improving the quality of the diagnostic approach and antibiotic use for patients with suspected UTI.
A panel of Danish experts agreed on a total of 24 QIs for the management of patients with suspected UTI in general practice. All QIs focusing on the diagnostic process achieved consensus of appropriateness, while the experts agreed on three quarters of the proposed QIs concerning either the treatment decision or the choice of antibiotics.
Previous studies have demonstrated the need for validated QIs, developed by an appropriate methodology, to be able to assess the diagnostic process and the treatment decision for patients with suspected UTI in general practice. This study adhered to a widely recognised, systematic method combining evidence with expert opinion ensuring a transparent and scientific process for the development of indicators. During the consensus process, the experts evaluated the face validity of the proposed QIs, that is, if the indicators reflected the quality issue that was intended to be measured. Moreover, the QIs were based on guidelines and scientific evidence, which are considered to provide QIs with content validity. Thus, validation of each QI was an inherent part of the development process. The feasibility of the QIs has been evaluated by application of the indicators to relevant data. Previous studies have demonstrated that adherence to guidelines on antibiotic use is protective against treatment failure and mortality. This set of QIs is based on the most updated evidence-based national guidelines for UTI. All indicators only concerned day 1, that is, the day of the first contact to general practice. Consequently, no indicators were designed to include, for example, the result of a urine culture. We chose this design because most antibiotics for UTI are prescribed at day 1. Information about patient’s symptoms and signs and results of point-of-care tests are likewise available at day 1. Accordingly, we believe that insight into the care provided at day 1 is sufficient to generate useful and comprehensive knowledge about the quality of care for patients with suspected UTI in general practice. One expert attended only 1 hour of the consensus meeting. However, this expert was provided with comprehensive information from the last part of the meeting before asked to rate the indictors for the second time.
QIs for the management of patients with suspected UTI have previously been developed. However, to our knowledge, the indicators from this study are the first set of indicators developed for use in general practice, comprising both the diagnostic process and the decision to prescribe antibiotics for UTI. The indicator (QIB) that evaluated if patients with high probability of uncomplicated lower UTI (≥1 symptom of lower UTI and a positive urinary dipstick and/or a positive microscopy) were treated with antibiotics, did not obtain consensus. It is well documented that antibiotic treatment is superior to non-antibiotic treatment regimens in terms of achieving bacteriological cure and symptomatic relief. However, the results on the risk of complications such as pyelonephritis are debated. Some studies have documented an increased risk of pyelonephritis when ibuprofen was prescribed instead of antibiotics. Meanwhile previous studies have found very few cases of complications and no increased risk of pyelonephritis when treatment with pivmecillinam was compared with placebo. The European Surveillance of Antimicrobial Consumption Network managed to develop a similar indicator: percentage of female patients older than 18 years with cystitis/other urinary infection prescribed antibiotics for systemic use, standard range 80%–100%, although the indicator did not reach consensus for all dimensions during the consensus process. The result of the urinary dipstick was included in several of the indicators proposed. Diagnostic accuracy for UTI improves considerably when symptoms and signs are combined with the result of the dipsticks test. Dipstick results are also included in a model to predict antibiotic prescriptions for UTI. Several studies have explored the link between the non-specific symptom confusion and UTI, however, the association is not clearly documented. Interestingly, two of the QIs in our set of proposed indicators were rephrased (QI9 and QI16). The rephrasing of both indicators involved a deletion of the proposed specific symptom: emerging confusion in catheter users with suspected UTI. The rephrased indicators ended up with the inclusion of only four specific symptoms for catheter users with suspected UTI: (1) fever, (2) shivering, (3) flank pain and (4) systemically unwell. Examination of a urine sample is not recommended in patients without UTI symptoms and guidelines recommend no antibiotic treatment for asymptomatic bacteriuria. Nonetheless, it is very likely that, in every day general practice it may happen anyway. The experts agreed on the indicator concerning urinalysis in patients with no UTI symptoms (QI1). Contrarily, the indicator that evaluated if patients without UTI symptoms but with a high probability of bacteriuria (positive urinary dipstick and/or a positive microscopy) were treated with antibiotics, did not reach consensus (QIA). Some of the experts did not agree on QIA because it included two not recommended actions: (1) to examine urine in patients without UTI symptoms and (2) to treat patients without UTI symptoms with antibiotics.
A set of 24 QIs for the diagnosis and treatment of patients with suspected UTI in general practice has been developed. Studies have shown that GPs in general have a positive attitude towards the use of QIs. This set of indicators may be used to strengthen GPs’ focus on their management of patients with suspected UTI and identify potential quality problems. The indicators are not only applicable to Danish general practice but may be applied to for example general practices in the Nordic countries with antibiotic use, resistance pattern and practice setting similar to Danish conditions. In Denmark, such as many other countries, general practices do not have detailed systematic data registration systems. Therefore, application of indicators to measure and improve patient care is currently a time-consuming activity, hampering a systematic application of these indicators. Since 2018, Danish GPs have been joined in ‘quality clusters’ for quality discussion and support. This set of QIs can ideally be applied to data and used as a basis for reflection and discussion of opportunities for improvements in these ‘quality clusters’. Furthermore, the indicators can advantageously be used for an intervention programme aiming at improving the quality of the diagnostic approach and antibiotic use for patients with suspected UTI.
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Variation in Temperature Dependences across Europe Reveals the Climate Sensitivity of Soil Microbial Decomposers
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c1fa94e2-d695-432f-93ce-46042968a93e
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10231190
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Microbiology[mh]
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Temperature is a dominant controller of biological process rates at all levels of biological organization ( ). Thus, changes in thermal regimes will influence how C is processed through all ecosystems. Microorganisms dominate the metabolic activity underpinning decomposition and therefore constitute a major determinant of C fluxes in the biosphere. To accurately predict how warming will alter C dynamics we need to understand how microorganisms depend on temperature. Warmer temperatures will accelerate microbial respiration within the studied environment’s temperature range ( , ). Consequently, it is generally expected that warming will stimulate losses of C into the atmosphere ( , ) representing positive feedback to climate warming, accelerating change. Despite considerable scientific attention in recent decades, there are multiple gaps in our understanding of microbial responses to warming. For instance, by simultaneously determining microbial temperature dependences of both primary production and decomposition in running water ecosystems, it was shown that warming induced a shift toward more heterotrophy ( , ), while other studies found similar responses of primary production and decomposition, resulting in no net change in metabolic balance ( ). In soils, long-term warming experiments have shown that the initial increase in soil respiration diminishes with time, gradually recovering to ambient values ( ). It has been suggested that this can be due to substrate depletion ( , , ), changes due to plant feedbacks ( ), shifts in microbial community composition ( ), or microbial thermal adaptation ( ). The existence of microbial thermal adaptation is of critical importance since it can modulate the positive feedback between climate warming and ecosystem CO 2 release. In addition, it is unknown if microbial communities will share a temperature dependence for respiration (immediate C loss from soil) and for synthesis of microbial biomass (potential microbial-C input to soil) ( ) and how these will respond to warming. This balance has implications for the fate of C as stored microbially derived C or atmospheric CO 2 release ( ). Although the thermal adaptation of microbial respiration and microbial community growth has been studied along climate gradients ( , , ), in laboratory experiments ( ), and in field warming experiments ( , , ), how and to what extent it will affect predictions of the microbial feedback to climate warming remains uncertain on a global scale ( ). It is anticipated that the environmental temperature will determine the temperature sensitivity of the microbial community by selecting for a community with trait distributions adapted to facilitate rates of growth and respiration at the prevailing temperature regime ( , ). Yet the strength by which trait distributions are shaped by the thermal regime, i.e., how much microbial temperature relationships shift per degree Celsius warmer temperature, has yet to be generalized. At low temperatures, growth and activity rates are low, starting from zero at the apparent temperature minimum ( T min ). When temperatures increase above T min , rates also increase until a maximal rate at the optimum temperature ( T opt ). At temperatures exceeding T opt , rates will again decrease until zero is reached at an apparent maximum temperature ( T max ), altogether making up the temperature relationship for a community. The square root (or “Ratkowsky”) model is a simple model that can represent these microbial temperature relationships ( , ). This model enables the estimation of useful indices that can be used to compare microbial community temperature trait distributions, including the lower temperature limit for activity (minimum temperature [ T min ]), the temperature for optimal rates (the optimum temperature [ T opt ]), and the upper temperature limit for activity (the maximum temperature [ T max ]). Any of these can be used to screen for changes or differences in temperature relationships, where higher values denote communities with warm-adapted traits, and lower values denote cold-adapted traits. The T min can be determined with high accuracy, which is why it has been used as an effective index for temperature relationships in comparative assessments in very different ecosystems ( , , ), making it possible to compare the level of the temperature adaptation for trait distributions for microbial growth and respiration ( , ). In this study, we set out to define the variance of temperature relationships of both microbial growth and soil respiration at a continental scale to determine if and how microbial communities’ distributions of temperature traits are adapted to the environmental temperature regime. Since latitudinal gradients have stable and large temperature differences, the assembly of microbial communities should have had sufficient time for ecological and evolutionary processes to act, allowing us to distinguish if soil microbial communities’ temperature relationships are matched to the local climate. To this end, we investigated a latitudinal gradient across Europe with 72 sites that spanned through a comprehensive gradient of mean annual temperatures (MAT) from −3.1 to 18.3°C ( ). The surveyed soils were intentionally selected to include wide ranges of soil organic matter (SOM), pH, and land uses in all climates. We resolved if thermal trait distributions that define the temperature relationships for bacterial growth, fungal growth, and respiration were adapted to the climate of the site. We did this by determining the temperature relationships for growth rates of bacteria and fungi, along with soil respiration rate in different sites. We estimated the indices for the temperature relationships for microbial growth and respiration, including T min , which were used for a comparative assessment of the variation of microbial temperature relationships across Europe, where higher values denoted communities with warm-adapted trait distributions ( , ). Additionally, we determined the consequences of the temperature sensitivity ( Q 10 ) for microbial growth and respiration resulting from these differences. The indices for the temperature relationships for each process were then related to environmental temperatures (MAT), allowing us to quantify the strength of the dependence of microbial temperature relationships on the environmental temperature regime. To interlink the microbial community composition to the differences in microbial temperature traits, we sequenced bacterial (16S) and fungal (internal transcribed spacer [ITS]) amplicons from the different sites to assess the α- and β-diversity variance across the latitudinal gradient.
We characterized the temperature relationships for bacterial growth, fungal growth, and respiration for all sites (see Table S1 in the supplemental material). Temperature relationships varied systematically with MAT, where higher MAT resulted in microbial communities with warm-adapted trait distributions for growth and respiration ( and ). The modeled temperature relationships for microbial growth rate, started at a minimum temperature for growth ( T min ) ( and ) between −14 and −5°C for bacteria and from −11 to −4°C for fungi ( ). From T min , microbial growth continually increased with higher temperature until a maximal value at the optimum temperature ( T opt ) ( and ) of 30 to 35°C for bacterial growth and of 30 to 43°C for fungal growth ( ). At temperatures above T opt , microbial growth rates decreased until they reached zero, thus defining the maximal temperature ( T max ) of the modeled temperature relationships ( and ) for growth, which varied between 43 and 51°C for bacteria and 40 to 52°C for fungi ( ). Bacteria and fungi showed differences in T opt and T max, with fungi having wider ranges ( and ). The temperature relationships for respiration started at T min values between −8 and −2°C ( ); rates increased with higher temperatures throughout the studied temperature interval without dropping during the brief assays ( ). In general, sites with higher historical MAT had warm-adapted temperature trait distributions for bacterial growth and respiration (bacterial F 1,69 = 22.1, P < 0.001; respiration F 1,70 = 22.3, P < 0.001, where F -test statistic and the numbers are degrees of freedom), while for fungal growth there were only tendencies ( F 1,70 = 3.9, P = 0.052) ( ; Table S3). We observed a similar pattern for the Q 10(5-15°C) values (bacterial F 1,69 = 16.9, P < 0.001; fungal F 1,70 = 4.2, P = 0.04; respiration F 1,70 = 24.4, P < 0.001), which increased in sites with higher MAT ( ). Our results suggest that an increase in MAT of 1°C will result in warm-shifted microbial temperature relationships equivalent to an increase of T min of 0.20°C for bacteria, 0.07°C for fungi, and 0.10°C for respiration. For the temperature sensitivity ( Q 10 ), an increase in MAT of 1°C will result in an increase of Q 10 of 0.03 units for bacteria, 0.02 units for fungi, and 0.03 units for respiration. Our survey was conducted during the summer and winter of 2019 and 2020. To control for the explanation that the determined microbial temperature relationships reflected the seasonal temperature at the time of sampling, we evaluated the effect of season and MAT through an analysis of covariance (ANCOVA). We found that only fungal T min had a significant effect of season ( F 1,68 = 5.3, P = 0.03), due to lower values in the summer sampling compared to the winter (Fig. S1B). However, we found no significant effect of the interactions between season and MAT in any of the temperature relationship indices. In addition, we selected a subset of 10 soils from nearby sites that were sampled both in winter and summer (Table S2). A paired t test revealed that the soils sampled in both summer and winter have indistinguishable temperature relationships for bacteria ( T min t 3 = −1.2, P = 0.33; T opt t 3 = −1.5, P = 0.23; T max t 3 = −1.1, P = 0.36, where t is the t -test statistic and the number is the degrees of freedom), fungi ( T min t 4 = −1.6; P = 0.18; T opt t 4 = −1.1; P = 0.34; T max t 4 = 1.4; P = 0.25), and respiration ( T min t 4 = −1.0, P = 0.39), respectively. Finally, we found no indication that the annual amplitude in environmental temperature variation could explain microbial adaptation to a stronger variation, since there was no significant relationship of the difference between summer and winter temperatures and the width of the temperature relationships ( T max − T min ) for bacteria or fungi (Fig. S2). To determine what environmental conditions had given rise to the variation in microbial temperature relationships, the indices for temperature relationships ( T min , T opt , T max ) were regressed against both mean annual summer and mean annual winter temperatures (Table S3). These comparisons indicated steeper responses to summer temperatures than to winter temperatures (Table S3). Other environmental variables were also regressed against the indices for temperature relationships (Table S4). We found that pH and SOM were positively related with some of the indices for microbial growth and respiration. Fungal T min was more strongly linked to soil pH ( R 2 = 0.09) than to MAT, while fungal T opt was better explained by pH ( R 2 = 0.26) and SOM ( R 2 = 0.25) (Table S4). The multiple regression analysis (Table S5) showed that MAT was the only significant explanatory variable for the T min s of bacterial growth ( p MAT ( p -value for MAT variable) < 0.001; model F 4,66 = 5.5, R 2 = 0.25, P = 0.001) and respiration ( p MAT = 0.001; model F 4,67 = 6.2, R 2 = 0.27, P < 0.001), while there was no significant predictor for fungal growth T min . For bacterial T opt , both SOM and MAT were significant predictors ( p SOM ( p -value for SOM variable) = 0.02; p MAT = 0.02; model F 4,66 = 5.3, R 2 = 0.24, P = 0.001), while we did not find any significant predictors for fungal growth T opt or bacterial and fungal growth T max s (Table S5). Finally, we found no correlation between C availability estimated as the rate of microbial C use per SOM and MAT (Pearson’s correlation [corr] = 0.18, P = 0.16; Fig. S3). To investigate if the microbial community’s taxonomic composition was linked to differences in thermal trait distributions of the microbial community, we regressed the bacterial and fungal growth T min s against bacterial and fungal α-diversity (Table S6). The bacterial growth T min was positively related to the bacterial α-diversity ( F 1,69 = 10.6, R 2 = 0.13, P = 0.002; ), while no patterns could be discerned for fungal growth T min ( F 1,70 = 1.6, R 2 = 0.02, P = 0.21; ). MAT and soil pH were also positively related to bacterial α-diversity, while SOM was negatively related (Table S6). Moreover, soil pH was also positively related to fungal α-diversity (Table S6). Furthermore, the envfit function showed that the bacterial community composition was significantly correlated with the bacterial growth T min ( R 2 = 0.17, P = 0.002; vector in ). Similarly, the fungal community composition was also significantly correlated with the variation in fungal growth T min ( R 2 = 0.24, P = 0.001; vector in ). We identified 1,501 bacterial amplicon sequence variants (ASVs) that correlated significantly with high T min values and 2,171 ASVs that correlated with low T min values (Supplemental File 1; Table S7). For fungi, we found 270 ASVs that correlated significantly with high T min values and 425 ASVs that correlated with low T min values (Appendix 1; Table S7). Several additional environmental factors were also significantly correlated with the microbial community composition (Table S8). The results indicated that pH, SOM, MAT, and latitude were highly correlated with both the bacterial and the fungal community composition (vectors in , ).
Previous large-scale surveys attempting to link microbial thermal adaptation to climatic differences have found differences between sites, but most of them have been unable to demonstrate a systematic link between the temperature relationships of microbial communities with differences in environmental temperatures ( , , ). For instance, temperature sensitivities of acidic boreal forest floors have been found to diverge from both arctic (cooler) and tropical (warmer) environments ( ), and while biomass-specific respiration rates with excess substrate have been found to decrease in warmer environments, temperature sensitivities ( Q 10 s) were independent of environmental MAT in a global dryland survey ( ) and in a geographic survey across the United States ( ). Only a recent large-scale study in China was able to demonstrate the link between temperature relationships for respiration and MAT ( ). In contrast, in our study, we demonstrated that soil microbial temperature relationships for both growth and respiration varied systematically with environmental temperatures at a continental scale, where higher MATs were associated with microbial communities with warm-adapted trait distributions for both growth and respiration. This variance in temperature relationships covering a wide MAT range from −3.1 to 18.3°C across Europe also quantified the strength with which the historical temperature regime had formed the microbial trait distributions for temperature. Using the obtained relationships in a space-for-time substitution, our results suggest that an increase in MAT of 1°C will result in warm-shifted microbial temperature trait distributions equivalent to an increase in T min of 0.20°C (and Q 10(5-15°C) of 0.03 units) for bacterial growth, 0.07°C (and Q 10(5-15°C) increase of 0.02 units) for fungal growth, and 0.10°C (and Q 10(5-15°C) increase of.03 units) for respiration. This provides evidence for microbial communities with temperature traits adapted to the local environment for microbial growth and respiration and is the first assessment over a continental scale. Previous studies of altitudinal and regional climatic gradients and field warming experiments have estimated that T min for bacterial growth and respiration increases by 0.2 to 0.8°C per 1°C increase in MAT ( , , , , , ). This closely corresponds with the estimates in this study. Similarly, previous assessments of temperature gradients in Antarctic soils ( ) and altitudinal gradients in tropical soils ( ) found that Q 10 increased by 0.03 and 0.05 units per 1°C temperature increase, respectively. Therefore, we can extend and refine the previous observations made across temperature gradients at smaller spatial scales to a continental-scale temperature gradient. The temperature relationships for bacterial growth had a stronger response to MAT than those for respiration or fungal growth. Consequently, our results suggest that the adaptation of bacterial temperature trait distributions will be more responsive to warming than those for respiration or fungal growth. It has been suggested that fungi are generally more resistant than bacteria to environmental changes such as changes in pH ( ), and moisture ( ), presumably due to their physiological plasticity. We also note that the method used to estimate fungal growth rates in intact soil samples ex situ included free-living fungi and excluded any contribution by symbiotic fungi that relied on, e.g., an intact plant host C supply ( ). While the few attempts to estimate temperature relationships for mycorrhizal fungi suggest that they match those of saprotrophic fungi from the same environment ( ), it is possible that the close physiological dependence that mycorrhizal fungi have on plants would differentiate them ( ). In the case of respiration, it is a broad microbial function contributed to by both bacterial and fungal communities, which can increase as a response to both physiological stress and enhanced conditions for microbial growth ( ), ambiguities that may weaken the link to climate temperatures compared to those for microbial growth. Soil sampling was conducted during summer and winter; however, the determined microbial temperature relationships did not reflect the seasonal temperature at the time of sampling, since sites sampled in both winter and summer had indistinguishable temperature relationships (as revealed by the paired t test and Fig. S1). Our results therefore suggest that the continental patterns for microbial temperature trait adaptation were stable across seasonal changes in temperature. Additionally, the indices for microbial temperature trait distributions determined had steeper dependences on summer temperatures than winter temperatures, consistent with previous suggestions that warm periods dominate the environmental control of microbial temperature relationships. That is, periods during which temperatures reach values close to or exceeding the microbial T opt are more important for determining the soil microbial temperature traits ( , , ). Further, some sites had larger seasonal variation in temperature. This could have selected for wider temperature relationships in seasonally variable sites. To test for such a link, we calculated the difference between summer and winter temperatures and the width of the temperature relationships ( T max − T min ) for bacteria and fungi. However, the width of temperature relationships was not systematically affected by the seasonal variation in temperature in the studied survey (Fig. S2). Other environmental variables such as pH and SOM were also correlated with the temperature relationships. Previous studies have shown that temperature and precipitation are important factors that control soil pH ( , ). Temperature mainly affects the rock weathering rate, while precipitation affects material flow. Additionally, along with climate, vegetation and its productivity will also play an important role in the regulation of soil pH ( ). In the surveyed soils, intentionally selected to include wide ranges of SOM, pH, and land uses in all climates, environmental temperatures explained a dominant proportion of the microbial temperature trait variation, having stronger relationships with microbial temperature trait indices than other variables. This was also confirmed by a multiple regression analysis, which showed that climate temperature was the strongest factor shaping the variation in microbial temperature trait distributions for bacterial growth and respiration. However, this was not true for the temperature traits of fungal growth, since the measured variables did not significantly explain the variation in the fungal temperature relationship indices. This suggested that the variation in fungal temperature relationships was more strongly shaped by environmental variables not measured in this study, such as plant productivity, changes in plant community composition, or plant diversity ( ). Moreover, the influence of substrate availability on temperature relationships is a topic that has received a lot of attention ( ). It has widely been assumed that substrate independence is needed to isolate the temperature relationship from the influence of variable substrate availability ( , , ). However, a recent study found that the temperature sensitivity was independent of the difference in chemical recalcitrance or C quality ( ). In our study, using the rate of microbial C use per SOM as a proxy for C quality, we found no correlation between MAT and C availability in the different sites (Fig. S3). This showed that the observed differences in microbial thermal trait distributions and temperature sensitivities were not explained by differences in C quality along the gradient. Variance in the microbial community composition was linked to differences in microbial temperature trait distributions. These links suggest that the long-term adaptation of microbial temperature traits is likely to have arisen from differences in microbial community composition ( , ). It has been argued that in response to changes in temperature in the short-term, microbes can respond with physiological adjustment and acclimate to new temperatures ( , ). However, in the long-term, species that thrive at the new temperature will outcompete others, more likely resulting in species turnover to yield higher relative abundances and activities of warm-adapted species, translating to differences in the microbial community composition ( , , ). Several additional environmental factors were also correlated with the microbial β-diversity. The envfit test indicated that pH, SOM, and MAT were highly correlated with the bacterial and fungal community composition. Since wide ranges of soil properties and land uses were included in all climates, it was expected that taxonomic breadth would be extensive in each climate. This is consistent with earlier findings revealing that pH is a major driver for differences in bacterial communities ( ), while the structure of fungal communities also correlated with other drivers, possibly via plant community differences ( , ). It has also been shown that MAT can be a significant predictor of the β-diversity of bacterial and fungal communities ( , ), which indicates that MAT can have an important role in structuring the microbial community composition. However, the sensitivity to detect MAT-associated differences in taxonomic composition is likely to be higher when ranges of other environmental factors powerfully regulating taxonomic composition are smaller, yielding a higher signal-to-noise ratio. Given the high level of microbial β-diversity in soil samples, with taxonomic overlap often being <1% in pairwise comparisons of samples along steep environmental gradients ( , , ), it is likely that the list of taxa with warm-adapted temperature traits would be nonoverlapping at pH 4 and 8. Such patterns would compromise the ability of ordination techniques to capture the link between temperature traits and microbial taxa ( ). This could be addressed by using a stratified sampling where study sites across a wide climate gradient are selected to have smaller ranges of pH and SOM. We also tried to identify warm- and cold-adapted taxa by investigating which ASVs’ relative abundance was correlated with high T min values for warm-adapted taxa and with low T min values for cold-adapted taxa. We found several bacterial ASVs that correlated significantly with high T min values and low T min values. Using the database Microbe Atlas ( ), we found some indications that cold-adapted ASVs have been found mainly in samples classified as forest and tundra soils, while warm-adapted ASVs have been mainly found in samples classified as farm, field, agricultural, and desert soils (Table S7). For fungi, we used the database GlobalFungi ( ) and found some indications that warm-adapted ASVs were more frequently sampled in sites with a MAT higher than 4°C, while cold-adapted ASVs were more frequently found in sites with a MAT lower than 8°C (Table S7). However, these findings only reveal an initial qualitative exploration. To be able to identify warm- and cold-adapted ASVs, observations from different manipulative experiments and natural temperature gradients should be combined to identify “common denominators” or “bioindicator taxa” that respond similarly to changes in temperature ( ). Understanding how ecosystem C balances are related to environmental temperatures and to warming is crucial for generating robust predictions from coupled climate-C models. Currently, soil C models that form the basis of Earth system models (ESMs) (e.g., Roth C, Century, Daycent, Candy) used to advise the Intergovernmental Panel on Climate Change (IPCC), assume a single, global temperature dependence for all microbial processes that is universal for all climates ( ). Here, we show that the temperature trait distributions for microbial growth and respiration are adapted to their environment and are thus climate specific. We also quantified the strength of this dependence. These differences in temperature trait distributions resulted in associated differences in temperature sensitivity ( Q 10 ) for microbial growth and respiration, where communities with warm-shifted temperature trait distributions in warmer environments had higher temperature sensitivities. This validated earlier reports from separate ecosystems ( , ), refined the estimate of the strength of the dependence, and extended them to a continental scale. Therefore, the use of a static temperature rate modifier currently used in ESMs ( ) will not correctly represent the variation in microbial temperature relationships across the globe or in response to climate change. Thus, soil C models need to incorporate the climate adaptation of microbial temperature traits into soil C climate feedback predictions. Moreover, we show that the temperature trait distributions for bacterial growth, fungal growth, and respiration rate are differentially adapted to temperature, requiring further revision of current soil C models, by delineating these different microbial processes. The partitioning of microbial-used C into growth, which potentially can be stored, and respiration, which is immediately lost to the atmosphere, has been shown to be a fundamentally decisive parameter that will define the long-term balance of C dynamics ( , ). We showed that temperature relationships for microbial growth will be more responsive to changes in temperature than respiration and that bacterial decomposers will respond more strongly than fungi will, which may affect the persistence of microbial necromass in soil due to their contrasting life history strategies ( , ). Several studies have found that fungi can accumulate more necromass in both agricultural and forest soils ( ) and that bacterial residues have a faster turnover rate than fungal residues ( , ), suggesting that fungi contribute more to soil C stabilization. Asymmetrical responses of the temperature relationships for microbial growth and respiration to warmer temperatures have also been observed in aquatic ecosystems, where respiration was found to be more responsive ( , ). The outcome of the asymmetrical sensitivities to warmer temperatures of microbial temperature traits for growth and respiration, and between the growth of different microbial groups, will affect the atmosphere-land C balance and thus the land-ecosystem feedback to climate warming.
Soil sampling and characterization. Soils were sampled across a European gradient in 72 sites that differed in their climate and soil characteristics and land use. The surveyed soils were intentionally selected to include wide ranges of SOM, pH, and land uses in all climates. Coordinates of the different sites were used to obtain mean annual temperature (MAT) and mean annual precipitation (MAP) from the Lund University Data Guru ( https://dataguru.lu.se/ ). For MAT, monthly mean historical temperatures of 10 years (2009 to 2018) were obtained from the WFDEI data set ( ). For MAP, monthly mean historical precipitation levels of 10 years (2002 to 2011) were obtained from the CRUTS version 3.20 data set ( ). A map to illustrate the temperature range and the sampling site locations was created in R version 4.0.3 ( ) with the raster package ( ) using historical climatic data ( ). Composite soil samples from each site were taken by sampling several soil pits with a spade from the upper 5 cm until reaching ca. 300 g of soil. Soils were collected between June 2019 and June 2020 in different seasons (Table S1). All samples were stored at 4°C. Soils were sieved (<4 mm), moisture adjusted to 50% water holding capacity (WHC), and subsequently left in the dark at room temperature (20°C) for 1 week before being processed. Soil pH and electrical conductivity (EC) were measured in a 1:5 (wt/vol) soil/water extraction (5 g soil plus 25 mL H 2 O) using electrodes (combined pH electrodes, Radiometer Analytical, France and 4520 conductivity, Jenway, England, respectively). Soil organic matter (SOM) was measured using a loss-on-ignition procedure ( ). The maximum amount of water that soils could hold after gravity loss was measured to assess the WHC of the soils ( ). Information about sampling location, collection date, environmental data, and soil characteristics can be found in Table S1. Microbial temperature dependences. Subsamples of the soils were transferred to different vials. Each vial was exposed to 1 of 10 different temperatures from 0°C to 45°C in 5°C intervals in water baths. Respiration, bacterial growth, and fungal growth were measured simultaneously for the different soils and temperatures in independent vials ( n = 10 for each soil and each process; see below and File S2). The exposure to different temperatures during the incubation step was always kept to a duration corresponding to approximately 2 h at 20 to 25°C for bacterial growth, adapting the time of the incubation period for the other temperatures (i.e., 1 h at 30°C, 4 h at 15°C, etc., to ensure a similar level of C use in all treatments; see File S2). Similarly, incubations for fungal growth and respiration corresponded to ca. 4 h and 18 h, respectively, at 20 to 25°C and adapted for other temperatures (i.e., 2 h and 6 h at 30°C, 8 h and 30 h at 15°C, etc.; see File S2). Within these time periods, no change in growth rates or respiration due to altered conditions occurred ( ), with the exception of the direct temperature effect on rates. Microbial growth and respiration. Bacterial growth was measured with 3 H-leucine incorporation ( ; see File S2). The amount of leucine incorporated into extracted bacteria (pmol leucine incorporated g −1 soil h −1 ) was used as a proxy for bacterial growth. Fungal growth was determined using 14 C-acetate in the fungus-specific lipid ergosterol ( , ; see File S2). The amount of acetate incorporated into extracted ergosterol (pmol acetate incorporated g −1 soil h −1 ) was used as a proxy for fungal growth. Soil respiration was measured as CO 2 production using a gas chromatograph equipped with a methanizer and a flame ionization detector (YL6500 GC, YL Instruments, South Korea). Microbial community. DNA was extracted from 250 mg of freeze-dried soil using Power Soil Pro kits (MoBio, Carlsbad, USA) following the manufacturer’s instructions. DNA concentration was determined using a NanoDrop spectrophotometer system (Thermo Scientific, Wilmington, NC, USA), and DNA extracts were sent to BGI Tech Solutions (Hong Kong, China) for amplicon sequencing according to their standard protocol (see File S2). For bacterial communities, the V3 to V4 region of the 16S gene was amplified using the primers 341F (5′- CCTAYGGGRBGCASCAG -3′) and 806R (5′- GGACTACNNGGGTATCTAAT -3′) ( ). For fungal communities the ITS1 to ITS2 region was amplified using the primers ITS1 (5′- CTTGGTCATTTAGAGGAAGTAA -3′) ( ) and ITS2 (5′- GCTGCGTTCTTCATCGATGC -3′) ( ). All sequence data were processed using DADA2 and the DADA2 ITS Pipeline Workflow version 1.8 ( ) to determine the amplicon sequence variants (ASVs). Data analyses. Temperature dependences of microbial growth and respiration were modeled using the Ratkowsky model ( ) according to . (1) R 1/2 = a ( T − T min ) × ( 1 − e b ( T − T max ) ) where R is the rate of leucine incorporation, acetate incorporation, or respiration, a and b are slope parameters, T is the screening temperature (°C), T min is the minimum temperature (the first x axis intercept), and T max is the maximum temperature (the second x axis intercept). At temperatures below optimum (0 to 25°C) and for respiration rate, the simplified was used. (2) R 1/2 = a ( T − T min ) where parameters are the same as in . was first used to estimate T min and the slope a , which could be used as constants in for the entire temperature interval. T max was estimated with . With the derivative of , the optimum temperature for growth ( T opt ) was estimated. Values were then normalized to the maximum rate (rate at T opt ). The temperature coefficient ( Q 10 ) was estimated as an index for the temperature sensitivity, according to . (3) Q 10 = [ a ( ( T + 10 ) − T min ) ] 2 ∕ [ a ( T − T min ) ] 2 where Q 10 indicates how the rate ( R ) changes with a difference of 10°C. Parameters are as defined in and , and the temperature interval 5 to 15°C was used. To understand the relationship between the temperature regime and the temperature relationships and temperature sensitivity calculated, we regressed the indices for temperature relationships ( T min , T opt , T max ) and the temperature sensitivity ( Q 10 ) against soil MAT. Additionally, the temperature relationship indices were regressed with the mean temperature of the warmest month (summer temperature) and the mean temperature of the coldest month (winter temperature). The difference between summer and winter temperatures was also regressed against the width of the temperature relationships ( T max − T min ) (see File S2 for details). To control for the seasonal temperature at the time of sampling, we studied the effect of the season on the regression between T min , T opt , T max , and MAT through an ANCOVA analysis taking MAT as the predictor variable, T min , T opt , or T max , as the response variable, and sampling season as a covariate. We also used a t test to identify if sampling season had a significant effect on the temperature relationships of a subset of 10 soils (Table S2) that were sampled in both winter and summer. Additionally, the temperature relationship indices were regressed against the other environmental and soil properties measured (MAP, pH, and SOM). To evaluate if C availability and temperature were interrelated in our survey, we estimated C availability as the rate of microbial C use per SOM and correlated it with MAT (see File S2 for details). Finally, to confirm the predictors of the temperature relationship indices, we used a multiple regression model. Using the lm function, we modeled the temperature relationship indices as a function of the different environmental factors and soil properties according to the regression . (4) y = a + b pH *pH + b SOM *SOM + b MAT *MAT + b MAP *MAP where y is one of the temperature relationship indices ( T min , T opt , or T max ) as a dependent variable, a is the intercept, and b pH , b SOM , b MAT , and b MAP are the regression coefficients of pH, SOM, MAT, and MAP, respectively. The data were checked for multicollinearity using the variance inflation factors before analyses. With the ASVs obtained from the microbial community analyses, we calculated diversity metrics for bacteria and fungi. We calculated the Shannon index (α-diversity) and richness for bacterial and fungal communities in each site before filtering and transformation to even sampling depth. Moreover, in the β-diversity analysis, ASVs were filtered by keeping only ASVs with at least 5 counts, and samples were then transformed to even sampling depth with the function transform_sample_counts from the phyloseq package ( ). We calculated the Bray-Curtis dissimilarity (β-diversity) matrix and visualized the differences in the bacterial and fungal communities across sites with a nonmetric multidimensional scaling (NMDS) ordination. To interlink microbial diversity with temperature trait distributions, we regressed the α-diversity and richness against bacterial and fungal T min values. Additionally, environmental parameters and soil properties were also regressed to bacterial and fungal α-diversity and richness. Further, bacterial or fungal growth T min values, environmental parameters, and soil properties were fitted onto the ordination space (Bray-Curtis NMDS) to assess correlations between these variables and the bacterial and fungal community composition using the function envfit from the vegan package ( ). The significance of fitted vectors was assessed using permutation of the variables ( ). Finally, we correlated the ASVs’ relative abundance with T min values to identify warm- and cold-adapted taxa (see File S2 for details). All statistical analyses were done in R version 4.0.3 ( ). Data availability. The sequencing data obtained have been deposited in European Nucleotide Archive (ENA) with the primary accession number PRJEB45259 . The raw data for bacterial growth, fungal growth, and respiration rate have been deposited in figshare under doi 10.6084/m9.figshare.21967388.
Soils were sampled across a European gradient in 72 sites that differed in their climate and soil characteristics and land use. The surveyed soils were intentionally selected to include wide ranges of SOM, pH, and land uses in all climates. Coordinates of the different sites were used to obtain mean annual temperature (MAT) and mean annual precipitation (MAP) from the Lund University Data Guru ( https://dataguru.lu.se/ ). For MAT, monthly mean historical temperatures of 10 years (2009 to 2018) were obtained from the WFDEI data set ( ). For MAP, monthly mean historical precipitation levels of 10 years (2002 to 2011) were obtained from the CRUTS version 3.20 data set ( ). A map to illustrate the temperature range and the sampling site locations was created in R version 4.0.3 ( ) with the raster package ( ) using historical climatic data ( ). Composite soil samples from each site were taken by sampling several soil pits with a spade from the upper 5 cm until reaching ca. 300 g of soil. Soils were collected between June 2019 and June 2020 in different seasons (Table S1). All samples were stored at 4°C. Soils were sieved (<4 mm), moisture adjusted to 50% water holding capacity (WHC), and subsequently left in the dark at room temperature (20°C) for 1 week before being processed. Soil pH and electrical conductivity (EC) were measured in a 1:5 (wt/vol) soil/water extraction (5 g soil plus 25 mL H 2 O) using electrodes (combined pH electrodes, Radiometer Analytical, France and 4520 conductivity, Jenway, England, respectively). Soil organic matter (SOM) was measured using a loss-on-ignition procedure ( ). The maximum amount of water that soils could hold after gravity loss was measured to assess the WHC of the soils ( ). Information about sampling location, collection date, environmental data, and soil characteristics can be found in Table S1.
Subsamples of the soils were transferred to different vials. Each vial was exposed to 1 of 10 different temperatures from 0°C to 45°C in 5°C intervals in water baths. Respiration, bacterial growth, and fungal growth were measured simultaneously for the different soils and temperatures in independent vials ( n = 10 for each soil and each process; see below and File S2). The exposure to different temperatures during the incubation step was always kept to a duration corresponding to approximately 2 h at 20 to 25°C for bacterial growth, adapting the time of the incubation period for the other temperatures (i.e., 1 h at 30°C, 4 h at 15°C, etc., to ensure a similar level of C use in all treatments; see File S2). Similarly, incubations for fungal growth and respiration corresponded to ca. 4 h and 18 h, respectively, at 20 to 25°C and adapted for other temperatures (i.e., 2 h and 6 h at 30°C, 8 h and 30 h at 15°C, etc.; see File S2). Within these time periods, no change in growth rates or respiration due to altered conditions occurred ( ), with the exception of the direct temperature effect on rates.
Bacterial growth was measured with 3 H-leucine incorporation ( ; see File S2). The amount of leucine incorporated into extracted bacteria (pmol leucine incorporated g −1 soil h −1 ) was used as a proxy for bacterial growth. Fungal growth was determined using 14 C-acetate in the fungus-specific lipid ergosterol ( , ; see File S2). The amount of acetate incorporated into extracted ergosterol (pmol acetate incorporated g −1 soil h −1 ) was used as a proxy for fungal growth. Soil respiration was measured as CO 2 production using a gas chromatograph equipped with a methanizer and a flame ionization detector (YL6500 GC, YL Instruments, South Korea).
DNA was extracted from 250 mg of freeze-dried soil using Power Soil Pro kits (MoBio, Carlsbad, USA) following the manufacturer’s instructions. DNA concentration was determined using a NanoDrop spectrophotometer system (Thermo Scientific, Wilmington, NC, USA), and DNA extracts were sent to BGI Tech Solutions (Hong Kong, China) for amplicon sequencing according to their standard protocol (see File S2). For bacterial communities, the V3 to V4 region of the 16S gene was amplified using the primers 341F (5′- CCTAYGGGRBGCASCAG -3′) and 806R (5′- GGACTACNNGGGTATCTAAT -3′) ( ). For fungal communities the ITS1 to ITS2 region was amplified using the primers ITS1 (5′- CTTGGTCATTTAGAGGAAGTAA -3′) ( ) and ITS2 (5′- GCTGCGTTCTTCATCGATGC -3′) ( ). All sequence data were processed using DADA2 and the DADA2 ITS Pipeline Workflow version 1.8 ( ) to determine the amplicon sequence variants (ASVs).
Temperature dependences of microbial growth and respiration were modeled using the Ratkowsky model ( ) according to . (1) R 1/2 = a ( T − T min ) × ( 1 − e b ( T − T max ) ) where R is the rate of leucine incorporation, acetate incorporation, or respiration, a and b are slope parameters, T is the screening temperature (°C), T min is the minimum temperature (the first x axis intercept), and T max is the maximum temperature (the second x axis intercept). At temperatures below optimum (0 to 25°C) and for respiration rate, the simplified was used. (2) R 1/2 = a ( T − T min ) where parameters are the same as in . was first used to estimate T min and the slope a , which could be used as constants in for the entire temperature interval. T max was estimated with . With the derivative of , the optimum temperature for growth ( T opt ) was estimated. Values were then normalized to the maximum rate (rate at T opt ). The temperature coefficient ( Q 10 ) was estimated as an index for the temperature sensitivity, according to . (3) Q 10 = [ a ( ( T + 10 ) − T min ) ] 2 ∕ [ a ( T − T min ) ] 2 where Q 10 indicates how the rate ( R ) changes with a difference of 10°C. Parameters are as defined in and , and the temperature interval 5 to 15°C was used. To understand the relationship between the temperature regime and the temperature relationships and temperature sensitivity calculated, we regressed the indices for temperature relationships ( T min , T opt , T max ) and the temperature sensitivity ( Q 10 ) against soil MAT. Additionally, the temperature relationship indices were regressed with the mean temperature of the warmest month (summer temperature) and the mean temperature of the coldest month (winter temperature). The difference between summer and winter temperatures was also regressed against the width of the temperature relationships ( T max − T min ) (see File S2 for details). To control for the seasonal temperature at the time of sampling, we studied the effect of the season on the regression between T min , T opt , T max , and MAT through an ANCOVA analysis taking MAT as the predictor variable, T min , T opt , or T max , as the response variable, and sampling season as a covariate. We also used a t test to identify if sampling season had a significant effect on the temperature relationships of a subset of 10 soils (Table S2) that were sampled in both winter and summer. Additionally, the temperature relationship indices were regressed against the other environmental and soil properties measured (MAP, pH, and SOM). To evaluate if C availability and temperature were interrelated in our survey, we estimated C availability as the rate of microbial C use per SOM and correlated it with MAT (see File S2 for details). Finally, to confirm the predictors of the temperature relationship indices, we used a multiple regression model. Using the lm function, we modeled the temperature relationship indices as a function of the different environmental factors and soil properties according to the regression . (4) y = a + b pH *pH + b SOM *SOM + b MAT *MAT + b MAP *MAP where y is one of the temperature relationship indices ( T min , T opt , or T max ) as a dependent variable, a is the intercept, and b pH , b SOM , b MAT , and b MAP are the regression coefficients of pH, SOM, MAT, and MAP, respectively. The data were checked for multicollinearity using the variance inflation factors before analyses. With the ASVs obtained from the microbial community analyses, we calculated diversity metrics for bacteria and fungi. We calculated the Shannon index (α-diversity) and richness for bacterial and fungal communities in each site before filtering and transformation to even sampling depth. Moreover, in the β-diversity analysis, ASVs were filtered by keeping only ASVs with at least 5 counts, and samples were then transformed to even sampling depth with the function transform_sample_counts from the phyloseq package ( ). We calculated the Bray-Curtis dissimilarity (β-diversity) matrix and visualized the differences in the bacterial and fungal communities across sites with a nonmetric multidimensional scaling (NMDS) ordination. To interlink microbial diversity with temperature trait distributions, we regressed the α-diversity and richness against bacterial and fungal T min values. Additionally, environmental parameters and soil properties were also regressed to bacterial and fungal α-diversity and richness. Further, bacterial or fungal growth T min values, environmental parameters, and soil properties were fitted onto the ordination space (Bray-Curtis NMDS) to assess correlations between these variables and the bacterial and fungal community composition using the function envfit from the vegan package ( ). The significance of fitted vectors was assessed using permutation of the variables ( ). Finally, we correlated the ASVs’ relative abundance with T min values to identify warm- and cold-adapted taxa (see File S2 for details). All statistical analyses were done in R version 4.0.3 ( ).
The sequencing data obtained have been deposited in European Nucleotide Archive (ENA) with the primary accession number PRJEB45259 . The raw data for bacterial growth, fungal growth, and respiration rate have been deposited in figshare under doi 10.6084/m9.figshare.21967388.
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Statement in Support of: “Virology under the Microscope—a Call for Rational Discourse”
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e80f6887-3c42-4fd6-9e42-bcb07ca31955
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10231235
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Microbiology[mh]
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We, members of the Australasian Virology Society, agree with and support the statement entitled “Virology under the Microscope—a Call for Rational Discourse” ( ). Like virologists everywhere, we have worked with scientist and clinician colleagues worldwide to develop knowledge, tests, and interventions which collectively have reduced the number of deaths due to COVID-19 and curtailed its economic impact. Such work adds to the extraordinary achievements resulting from virology research that have delivered vaccines and/or antivirals against a long list of diseases and global scourges, including AIDS, smallpox, and polio ( ). We believe the question of the origin of SARS-CoV-2 should be approached with an open mind and in consideration of the best scientific evidence available. We concur with the view that the zoonosis hypothesis has the strongest supporting evidence ( ), and this is a scenario that has been observed repeatedly in the past ( ), including in Australia ( ). Recent data strongly support the zoonosis hypothesis ( ). We share the concern that emotive and fear-based dialogues in this area add to public confusion and can lead to ill-informed condemnation of virology research. We believe the current narrative used by some parties—that gain-of-function research is synonymous with high-risk or nefarious activity—fails to appreciate, first, the true scientific value of this legitimate approach to experimental design and, second, the strength and effectiveness of current regulations. There is an extensive history of gain-of-function research safely and effectively contributing to the development of vaccines and antivirals ( ). A recent review of gain-of-function studies conducted by the Australian Government defined gain-of-function research as “a change to the genome of any biological entity—a living organism such as an animal, insect, plant, virus, bacterium, or fungus—through any process so that it acquires a new or enhanced function”. The review concluded that oversight of gain-of-function research in Australia is comprehensive and robust ( ). We do not believe virology research needs additional legislative controls. As in the United States, regulations in our region applying to virology research are strong, effective, and provide powerful oversight of manipulations of viruses by researchers. We support the call to legislators to resist fear-based campaigns that might lead to unnecessary and counter-productive restrictions being placed on virology research and may limit progress toward new antiviral drugs and vaccines. We echo the call for policy makers, virologists, and biosafety experts to work together to ensure that research is conducted safely, with the common goal of reducing the burden of disease caused by viruses.
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Leafy Green Farm-to-Customer Process Model Predicts Product Testing Is Most Effective at Detecting Contamination When Conducted Early in the System before Effective Interventions
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7933ac98-3465-43f3-bcaf-07436e5c055b
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10231246
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Microbiology[mh]
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Leafy greens are an increasingly important commodity in the United States ( ). Unfortunately, foodborne disease outbreaks associated with the consumption of leafy greens have increased ( , ). In the United States, from 2018 to 2020, 3 foodborne outbreaks have been linked to spinach and lettuce contaminated with Escherichia coli O157:H7 ( ). These repeated outbreaks have caused concern among producers, packers, buyers, and consumers, causing them to manage risk through multiple pathways, including food safety interventions and test-and-reject (sampling) plans. Now as part of the Shiga toxin-producing E. coli (STEC) action plan, the FDA has expressed the importance of prevention methods, especially as new research has identified seasonal effects linked to previous E. coli outbreaks ( , ). Contamination of produce can occur at multiple stages of the farm-to-facility process ( ). In the preharvest stage, produce may be contaminated through contaminated soil, manure, wildlife intrusion, dust particles, water used for irrigation, and other field activities ( ). In the harvest and postharvest stages, contamination may occur through direct human contact, cross-contamination from previously contaminated equipment, or other activities ( , ). Food safety interventions must be considered to manage the residual risk and provide consumers with relatively safe food ( ). To mitigate risk, pre- and postharvest interventions are required as part of regulations and standards ( , ). Preharvest food safety interventions include controlling adjacent hazards by limiting proximity to animal-rearing operations and urban areas, reducing animal activity through physical barriers, improving worker and equipment hygiene, proper aging controls for manure and soil amendments, and adequate control of water source and irrigation systems. Postharvest interventions include proper handling and temperature controls, product inspection at receiving, and incorporation of a chlorinated wash after cutting ( , ). The literature has identified process control verification testing (frequent samples over time) as the most desirable testing to be conducted for many foods, as it demonstrates that a system’s preventive controls are working as intended and minimizes the cost of testing programs ( ). However, more traditional lot testing programs such as “hold and release” testing for pathogens at the preharvest or finished product stage are often required by importers and buyers. An example of this is the Canadian Food Inspection Agency (CFIA) requiring romaine lettuce imported from the United States to be sampled and tested for the absence of E. coli O157:H7 ( ). Another example may be to comply with product specifications set by buyers, which may require the product to be sampled and tested at different stages to meet acceptance criteria ( ). However, the detection of pathogens by product sampling is challenging when prevalence and or levels are low, as in the case of leafy greens ( , ). The power of sampling is dependent on the total mass collected, the number of grabs, and contamination patterns ( , ). Validated simulations have shown that many preharvest sampling plans do not reliably detect low-level or low-prevalence contamination ( , ). Processing typically reduces levels due to the application of interventions, such as washing, spray washing, and preharvest holding time, but the impact of those on sampling power has not been thoroughly studied in leafy greens. Duffy and Schaffner evaluated the general effect of interventions, analyzing microbial sampling pre- and postprocessing for a mock liquid product. The study showed that sampling at the raw material stage was more useful than sampling at the finished product stage due to lower contamination levels postprocessing ( ). The leafy green industry would greatly benefit from a study that evaluates sampling’s ability to detect a high-level contamination event at multiple process stages (preharvest to finished product packing). This study aims to assess the effect of product food safety sampling at different stages of the farm-to-customer process on the total adulterant cells (TACs) reaching the endpoint of the farm-to-customer system in the context of systems with different food safety interventions. Three contamination spreads were modeled to cover the wide range of uncertainty around this parameter: (i) random uniform (widespread 100% cluster), (ii) a large cluster (10% coverage), and (iii) a small cluster (1% coverage). The literature shows that the spread of preharvest contamination is uncertain, ranging from widespread contamination to small clusters of contamination depending on the contamination source ( , ). Seven systems were modeled to represent different levels of adherence to food safety practices. (i) An all-intervention system (preharvest holding, precooling, prewash, wash, and processing line sanitation), (ii) a no-intervention system (a system without those five interventions), and (iii) five other in-between systems where each one of the five food safety interventions fails. The model implements sampling at seven process stages from preharvest to customer. The combinations of all these treatments resulted in 147 combinations ( ). As a measure of food safety management, these combinations will be compared to determine the effect of sampling at different stages and under different systems on the endpoint TACs.
Individual interventions reduced contamination levels throughout the system, and combined effective interventions achieved a greater reduction. The main objective of this model was to determine the effect of sampling at different stages of the farm-to-customer system in the context of the system with other intervention strategies. To achieve that, we first studied the effect of five interventions: (i) preharvest holding, (ii) precooling at receiving, (iii) prewash, (iv) chlorinated wash, and (v) processing line sanitation. Contamination in the system, total adulterant cells (TACs), and progression were tracked for the all-intervention system, the no-intervention system, and the systems with single interventions ( ). When all interventions were applied to the system, the total reduction of adulterant cells at the endpoint compared to no interventions averaged 3.43 log TACs (95% confidence interval [CI], 3.33 to 3.56). The two most effective interventions were the chlorinated wash and the prewash, which reduced the final TACs in the system by 1.32 log TACs (95% CI, 1.22 to 1.45) and 1.27 log TACs (95% CI, 1.18 to 1.38), respectively. The third most effective intervention was the implementation of holding time at preharvest, providing an average 0.80 log TACs (95% CI, 0.73 to 0.90) reduction. Interventions that had minor effects were conducting sanitation of the processing lines and precooling, providing an average reduction of 0.06 log TACs (95% CI, 0.02 to 0.15) and 0.01 log TACs (95% CI, −0.10 to 0.11), respectively. The poor efficacy of sanitation was due to the transfer coefficients between produce and surfaces being low, as well as contamination during processing being relatively low; therefore, contamination did not accumulate on the processing surfaces. The power of sampling plans depends on the contamination levels at each sampling point. Sampling plan power describes the ability of a given sampling plan to detect contamination at a given system stage under the sampling conditions stated in . Sampling plan power refers to the number of iterations where the sampling plan was able to detect contamination over the total number of iterations ( n = 10,000). Higher power means the sampling plan is more likely to detect contamination at a given system stage. The power to detect contamination of the 7 sampling plans ( ) at the 7 sampling stages and 3 contamination scenarios (147 total combinations) is summarized in (see also Table S2 in the supplemental material). First, sampling plan power was lower for those sampling plans that occurred later in the system; this was due to contamination at sampling points being lower as the system progressed, due to natural die-off through the system or reduction due to effective interventions. Second, except for preharvest sampling 4 days before harvest (PHS 4D), effective interventions reduced the power of sampling plans; this was because the application of effective interventions resulted in lower TAC contamination at each sampling point. Third, the degree of clustering matters; highly clustered contamination (e.g., 1% cluster) is harder to detect (lower power) earlier in the system. The most effective sampling plan across all systems was the preharvest sampling for 4 days (PHS 4D), with power ranging between 14.1% and 29.6%. Opposite to all the other sampling plans, the power of the PHS 4D sampling plan increased when an intervention was in place, in this case, the preharvest holding intervention. This intervention limited the contamination window to 2 to 8 days before harvest, compared to the 0 to 8 days when it was not in place. With a narrower contamination window, the probability of a contamination event occurring before PHS 4D was higher, as well as contamination at the sampling point being higher, and hence the higher sampling plan power. The disadvantage of this sampling plan was that contamination could still happen after sampling occurred, since sampling occurred 4 days before harvest, causing the plan to potentially miss a contamination event between PHS 4D and harvest. Contamination levels at PHS 4D for the systems that applied holding as an intervention were, on average, between 17,431 and 17,447 TACs, whereas for the systems that did not apply holding as an intervention (no-intervention and no holding), the contamination levels were lower, 12,983 (585 to 69,661, 90% variability interval) and 13,024 (587 to 70,032, 90% variability interval) TACs, respectively. Demonstrating that for this step, if the holding intervention were to fail, sampling would have reduced power for informing a contamination event. Preharvest sampling 4 h (PHS 4H), preharvest sampling intense (PHS Int), and harvest sampling (HS) were sampling plans that were solely affected by the preharvest holding intervention. These three sampling plans showed lower sampling plan power when the holding intervention was in place, meaning that the all-intervention, no-washing, no-prewash, no-precooling, and no-sanitation systems had lower sampling plan power than the no-intervention and no-holding systems at these three sampling stages. TAC levels at each sampling point were lower when holding was in place, on average, between 401 and 503 TACs for PHS 4H, PHS Int, and HS. When holding was not in place (no-intervention and no-holding systems), the contamination levels were meaningfully higher, between 2,600 to 3,360 TACs for PHS 4H, PHS Int, and HS, demonstrating that (i) sampling plan power depends on the contamination levels at the sampling point and (ii) effective interventions reduce contamination, therefore, making the remaining contamination harder to detect. (iii) If the holding intervention were to fail, sampling would provide more information than when this intervention is working as intended. Receiving sampling (RS), in addition to being affected by holding, was also affected by the precooling intervention. When holding or precooling interventions were not in place, TACs at RS were, on average, between 484 to 490 TACs. TACs were lower compared to when there was no precooling (492 TACs), when there was no holding (3,170 TACs), or when no interventions were in place (3,205 TACs). This demonstrates that if the precooling system were to fail, sampling would not provide much additional information about the failure of this intervention. In contrast, if the holding intervention were to fail, sampling would have a higher chance of detecting the remaining contamination. Finished product sampling (FPS) and customer sampling (CS) could be affected by all five interventions. These two sampling plans followed the same pattern as the other sampling plans. When effective interventions failed, contamination levels were higher at the sampling point, making it easier for sampling plans to detect contamination events. When the most effective intervention, washing, was not implemented in the system, TACs were, on average, 13 to 27 at FPS and CS. When holding was not in place, TACs, on average, were 4 to 9. When all interventions were in place, TACs were, on average, 1 for both FPS and CS. These results show that even when effective interventions fail, contamination levels remained relatively low because of other interventions. When no interventions were applied, endpoint TACs were, on average, between 1,550 and 3,200 at FPS and CS. shows that sampling plan power remained close to 0% for all systems when one intervention failed. This means the small differences in contamination when one intervention fails have small effects on sampling plan power. Sampling might still be able to detect a complete system failure, as seen by the higher power in the no-intervention system. The efficacy of sampling plans is dependent on interventions and system stage. The endpoint TACs were additionally used to compare two patterns: (i) the relative efficacy in endpoint TACs between sampling plans for each of the 7 systems and (ii) the log 10 difference and the relative efficacy of each system (losing an intervention) on endpoint TACs compared to the all-intervention system ( ). These two patterns allowed us to compare the relative effect that product sampling and removing one intervention at a time had on the endpoint TACs. The same relative efficacy achieved by incorporating each sampling plan was quantified for each of the 147 combinations. Table S3 shows the relative efficacies. A visualization of the 147 combinations is shown in . The main pattern suggests that sampling plans had a lower effect on reducing endpoint TACs when multiple effective interventions or all interventions were in place, except for PHS 4D. PHS 4D had the highest relative effect when the all-intervention system and those systems that included the holding intervention were in place, for reasons explained above. For PHS 4D, the relative efficacy in endpoint TACs ranged between 5% and 25% for systems that included the holding intervention. For those systems that did not include the holding intervention (no intervention and no holding), relative efficacy was lower, ranging between 3% to 8%. This result predicts that PHS 4D alone had at most an 8% relative reduction in the endpoint TACs, compared to the higher relative efficacy achieved (5% to 25%) when it was paired with the holding intervention. The assessment predicted that the remaining sampling plans had a lower relative effect on reducing endpoint TACs when multiple effective interventions or all interventions were in place for all other sampling plans. The most effective sampling plan across all systems was preharvest sampling intense (PHS Int). PHS Int showed a relative efficacy in endpoint TACs of between 68% and 78% for systems that did not apply the holding intervention, while for those systems affected by the holding intervention, the relative efficacy in endpoint TACs was lower, between 24% and 37%, demonstrating that sampling provides greater relative reduction to the endpoint TACs when no effective interventions or no interventions are in place compared to when effective interventions such as holding are in place. A similar pattern can be observed for other sampling plans later in the system. Finished product sampling (FPS) and customer sampling (CS) may be affected by all 5 interventions. When no interventions were in place, the relative efficacy in endpoint TACs ranged between 47% and 54% for FPS and 32% to 38% for CS. When all interventions were in place, the relative efficacy in endpoint TACs was between 0% and 1%, predicting that sampling at these later stages provided no effect when the optimal system was in place. Similarly, for the other systems, when one intervention at a time failed, FPS and CS did not provide much value; the relative efficacies ranged between 0% and 3%, predicting that for these effective systems, it is most beneficial to conduct sampling earlier in the system, potentially detecting high contamination loads, rather than after multiple reduction steps occur. The second pattern was to quantify the effect on relative efficacy that removing one intervention at a time had compared to having all and no interventions. A summary of the results can be found in . The results indicate that even if one intervention fails, the endpoint TACs will still be reduced by at least 99% by the remaining effective interventions. When all interventions are in place, the relative reduction to endpoint TACs increases to 99.2%. While some sampling plans were able to have a relatively meaningful effect on the endpoint TACs, the relative reduction achieved by sampling does not compare to the relative reduction of endpoint TACs achieved by implementing effective interventions, 99.96%. Even when effective interventions such as washing, prewashing, and holding were removed, the relative efficacy of the systems remained high, 99.27%, 99.31%, and 99.75%, respectively. Nevertheless, in all these scenarios, adulterant cells still reached the endpoint of the system, showing that even with effective interventions, the risk is not fully mitigated under these outbreak-like conditions; even the best-simulated system has residual risk. Factor sensitivity shows that sampling before processing interventions leads to greater adulterant reductions than sampling after processing interventions. Factor sensitivity (FS) analysis ( ) assessed the effect of sampling plans across each of the seven what-if systems. The FS provides information on which sampling plans would most efficiently reduce endpoint TACs if a specific intervention were to fail, as well as on how removing interventions decreases the overall efficacy of the optimal all-intervention system. The factor sensitivity analysis reiterates and shows very similar results to what was previously shown in and . The all-intervention system will have an efficacy reduction when the most effective interventions are removed from the system; this is the distance between the black lines and the red line in . In the FS analysis, removing washing, prewashing, and holding alone decreased the all-intervention system’s efficacy by 1.30, 1.24, and 0.81 logs, respectively. Removing interventions with lower efficacy, such as precooling and sanitation, reduced all intervention efficacy by 0.02, and 0.06 logs. When all interventions were removed from the system the effectiveness was reduced by 3.42 logs. The most effective sampling plan across all systems was the preharvest intense system (PHS Int), with added reductions between 0.13 and 0.66 log endpoint TACs. This is represented by the distance between the black line and the end of the bar in . For the no-intervention system, the second and third most effective sampling plans were the finished product sampling (FPS) and receiving sampling (RS) systems, with added reduction of 0.26 log and 0.17, respectively. For the all-intervention system, the second and third most effective plans had limited effects, the preharvest sampling 4 days (PHS 4D) and receiving sampling (RS), had an added log reduction of 0.053 and 0.054, respectively. For the no-sanitation, no-precooling, no-wash, and no-prewash systems, the second and third most effective sampling stages were the preharvest sampling 4 days (PHS 4D) followed by preharvest sampling 4 h (PHS 4H), with reductions of between 0.084 to 0.11 log and 0.054 to 0.092 log, respectively. For the no-holding system, the second and third most effective sampling plans were receiving sampling (RS), and harvest sampling (HS), with reductions of 0.34 and 0.30, respectively. In addition, for all the systems except for the no-intervention system, the least effective sampling plans were finished product sampling (FPS) and customer sampling (CS), with added reductions ranging between 0 and 0.037, and 0 and 0.031 log, respectively. Beyond assessing major system interventions and sampling plans with an FS, a partial rank correlation coefficient (PRCC) sensitivity analysis ( ) was performed to obtain a complete profile of endpoint TACs of all randomized input variables. The inputs with the largest absolute PRCCs, indicating the most sensitivity in the time between the contamination event and harvest (−0.58), were chlorinated wash on (−0.29), prewash on (−0.21), and transportation time to consumer (−0.06). The PRCC also showed sampling plans to have the smallest PRCC values, ranging between −0.005 and 0.021. These sensitivity analyses together suggest that the incorporation of effective interventions, such as preharvest holding or process washing, meaningfully reduce endpoint TACs. These also show that when effective interventions are in place, sample testing and rejection before processing result in larger reductions. If no interventions are in place, sample testing and rejection of the finished product lead to larger reductions.
The main objective of this model was to determine the effect of sampling at different stages of the farm-to-customer system in the context of the system with other intervention strategies. To achieve that, we first studied the effect of five interventions: (i) preharvest holding, (ii) precooling at receiving, (iii) prewash, (iv) chlorinated wash, and (v) processing line sanitation. Contamination in the system, total adulterant cells (TACs), and progression were tracked for the all-intervention system, the no-intervention system, and the systems with single interventions ( ). When all interventions were applied to the system, the total reduction of adulterant cells at the endpoint compared to no interventions averaged 3.43 log TACs (95% confidence interval [CI], 3.33 to 3.56). The two most effective interventions were the chlorinated wash and the prewash, which reduced the final TACs in the system by 1.32 log TACs (95% CI, 1.22 to 1.45) and 1.27 log TACs (95% CI, 1.18 to 1.38), respectively. The third most effective intervention was the implementation of holding time at preharvest, providing an average 0.80 log TACs (95% CI, 0.73 to 0.90) reduction. Interventions that had minor effects were conducting sanitation of the processing lines and precooling, providing an average reduction of 0.06 log TACs (95% CI, 0.02 to 0.15) and 0.01 log TACs (95% CI, −0.10 to 0.11), respectively. The poor efficacy of sanitation was due to the transfer coefficients between produce and surfaces being low, as well as contamination during processing being relatively low; therefore, contamination did not accumulate on the processing surfaces.
Sampling plan power describes the ability of a given sampling plan to detect contamination at a given system stage under the sampling conditions stated in . Sampling plan power refers to the number of iterations where the sampling plan was able to detect contamination over the total number of iterations ( n = 10,000). Higher power means the sampling plan is more likely to detect contamination at a given system stage. The power to detect contamination of the 7 sampling plans ( ) at the 7 sampling stages and 3 contamination scenarios (147 total combinations) is summarized in (see also Table S2 in the supplemental material). First, sampling plan power was lower for those sampling plans that occurred later in the system; this was due to contamination at sampling points being lower as the system progressed, due to natural die-off through the system or reduction due to effective interventions. Second, except for preharvest sampling 4 days before harvest (PHS 4D), effective interventions reduced the power of sampling plans; this was because the application of effective interventions resulted in lower TAC contamination at each sampling point. Third, the degree of clustering matters; highly clustered contamination (e.g., 1% cluster) is harder to detect (lower power) earlier in the system. The most effective sampling plan across all systems was the preharvest sampling for 4 days (PHS 4D), with power ranging between 14.1% and 29.6%. Opposite to all the other sampling plans, the power of the PHS 4D sampling plan increased when an intervention was in place, in this case, the preharvest holding intervention. This intervention limited the contamination window to 2 to 8 days before harvest, compared to the 0 to 8 days when it was not in place. With a narrower contamination window, the probability of a contamination event occurring before PHS 4D was higher, as well as contamination at the sampling point being higher, and hence the higher sampling plan power. The disadvantage of this sampling plan was that contamination could still happen after sampling occurred, since sampling occurred 4 days before harvest, causing the plan to potentially miss a contamination event between PHS 4D and harvest. Contamination levels at PHS 4D for the systems that applied holding as an intervention were, on average, between 17,431 and 17,447 TACs, whereas for the systems that did not apply holding as an intervention (no-intervention and no holding), the contamination levels were lower, 12,983 (585 to 69,661, 90% variability interval) and 13,024 (587 to 70,032, 90% variability interval) TACs, respectively. Demonstrating that for this step, if the holding intervention were to fail, sampling would have reduced power for informing a contamination event. Preharvest sampling 4 h (PHS 4H), preharvest sampling intense (PHS Int), and harvest sampling (HS) were sampling plans that were solely affected by the preharvest holding intervention. These three sampling plans showed lower sampling plan power when the holding intervention was in place, meaning that the all-intervention, no-washing, no-prewash, no-precooling, and no-sanitation systems had lower sampling plan power than the no-intervention and no-holding systems at these three sampling stages. TAC levels at each sampling point were lower when holding was in place, on average, between 401 and 503 TACs for PHS 4H, PHS Int, and HS. When holding was not in place (no-intervention and no-holding systems), the contamination levels were meaningfully higher, between 2,600 to 3,360 TACs for PHS 4H, PHS Int, and HS, demonstrating that (i) sampling plan power depends on the contamination levels at the sampling point and (ii) effective interventions reduce contamination, therefore, making the remaining contamination harder to detect. (iii) If the holding intervention were to fail, sampling would provide more information than when this intervention is working as intended. Receiving sampling (RS), in addition to being affected by holding, was also affected by the precooling intervention. When holding or precooling interventions were not in place, TACs at RS were, on average, between 484 to 490 TACs. TACs were lower compared to when there was no precooling (492 TACs), when there was no holding (3,170 TACs), or when no interventions were in place (3,205 TACs). This demonstrates that if the precooling system were to fail, sampling would not provide much additional information about the failure of this intervention. In contrast, if the holding intervention were to fail, sampling would have a higher chance of detecting the remaining contamination. Finished product sampling (FPS) and customer sampling (CS) could be affected by all five interventions. These two sampling plans followed the same pattern as the other sampling plans. When effective interventions failed, contamination levels were higher at the sampling point, making it easier for sampling plans to detect contamination events. When the most effective intervention, washing, was not implemented in the system, TACs were, on average, 13 to 27 at FPS and CS. When holding was not in place, TACs, on average, were 4 to 9. When all interventions were in place, TACs were, on average, 1 for both FPS and CS. These results show that even when effective interventions fail, contamination levels remained relatively low because of other interventions. When no interventions were applied, endpoint TACs were, on average, between 1,550 and 3,200 at FPS and CS. shows that sampling plan power remained close to 0% for all systems when one intervention failed. This means the small differences in contamination when one intervention fails have small effects on sampling plan power. Sampling might still be able to detect a complete system failure, as seen by the higher power in the no-intervention system.
The endpoint TACs were additionally used to compare two patterns: (i) the relative efficacy in endpoint TACs between sampling plans for each of the 7 systems and (ii) the log 10 difference and the relative efficacy of each system (losing an intervention) on endpoint TACs compared to the all-intervention system ( ). These two patterns allowed us to compare the relative effect that product sampling and removing one intervention at a time had on the endpoint TACs. The same relative efficacy achieved by incorporating each sampling plan was quantified for each of the 147 combinations. Table S3 shows the relative efficacies. A visualization of the 147 combinations is shown in . The main pattern suggests that sampling plans had a lower effect on reducing endpoint TACs when multiple effective interventions or all interventions were in place, except for PHS 4D. PHS 4D had the highest relative effect when the all-intervention system and those systems that included the holding intervention were in place, for reasons explained above. For PHS 4D, the relative efficacy in endpoint TACs ranged between 5% and 25% for systems that included the holding intervention. For those systems that did not include the holding intervention (no intervention and no holding), relative efficacy was lower, ranging between 3% to 8%. This result predicts that PHS 4D alone had at most an 8% relative reduction in the endpoint TACs, compared to the higher relative efficacy achieved (5% to 25%) when it was paired with the holding intervention. The assessment predicted that the remaining sampling plans had a lower relative effect on reducing endpoint TACs when multiple effective interventions or all interventions were in place for all other sampling plans. The most effective sampling plan across all systems was preharvest sampling intense (PHS Int). PHS Int showed a relative efficacy in endpoint TACs of between 68% and 78% for systems that did not apply the holding intervention, while for those systems affected by the holding intervention, the relative efficacy in endpoint TACs was lower, between 24% and 37%, demonstrating that sampling provides greater relative reduction to the endpoint TACs when no effective interventions or no interventions are in place compared to when effective interventions such as holding are in place. A similar pattern can be observed for other sampling plans later in the system. Finished product sampling (FPS) and customer sampling (CS) may be affected by all 5 interventions. When no interventions were in place, the relative efficacy in endpoint TACs ranged between 47% and 54% for FPS and 32% to 38% for CS. When all interventions were in place, the relative efficacy in endpoint TACs was between 0% and 1%, predicting that sampling at these later stages provided no effect when the optimal system was in place. Similarly, for the other systems, when one intervention at a time failed, FPS and CS did not provide much value; the relative efficacies ranged between 0% and 3%, predicting that for these effective systems, it is most beneficial to conduct sampling earlier in the system, potentially detecting high contamination loads, rather than after multiple reduction steps occur. The second pattern was to quantify the effect on relative efficacy that removing one intervention at a time had compared to having all and no interventions. A summary of the results can be found in . The results indicate that even if one intervention fails, the endpoint TACs will still be reduced by at least 99% by the remaining effective interventions. When all interventions are in place, the relative reduction to endpoint TACs increases to 99.2%. While some sampling plans were able to have a relatively meaningful effect on the endpoint TACs, the relative reduction achieved by sampling does not compare to the relative reduction of endpoint TACs achieved by implementing effective interventions, 99.96%. Even when effective interventions such as washing, prewashing, and holding were removed, the relative efficacy of the systems remained high, 99.27%, 99.31%, and 99.75%, respectively. Nevertheless, in all these scenarios, adulterant cells still reached the endpoint of the system, showing that even with effective interventions, the risk is not fully mitigated under these outbreak-like conditions; even the best-simulated system has residual risk.
Factor sensitivity (FS) analysis ( ) assessed the effect of sampling plans across each of the seven what-if systems. The FS provides information on which sampling plans would most efficiently reduce endpoint TACs if a specific intervention were to fail, as well as on how removing interventions decreases the overall efficacy of the optimal all-intervention system. The factor sensitivity analysis reiterates and shows very similar results to what was previously shown in and . The all-intervention system will have an efficacy reduction when the most effective interventions are removed from the system; this is the distance between the black lines and the red line in . In the FS analysis, removing washing, prewashing, and holding alone decreased the all-intervention system’s efficacy by 1.30, 1.24, and 0.81 logs, respectively. Removing interventions with lower efficacy, such as precooling and sanitation, reduced all intervention efficacy by 0.02, and 0.06 logs. When all interventions were removed from the system the effectiveness was reduced by 3.42 logs. The most effective sampling plan across all systems was the preharvest intense system (PHS Int), with added reductions between 0.13 and 0.66 log endpoint TACs. This is represented by the distance between the black line and the end of the bar in . For the no-intervention system, the second and third most effective sampling plans were the finished product sampling (FPS) and receiving sampling (RS) systems, with added reduction of 0.26 log and 0.17, respectively. For the all-intervention system, the second and third most effective plans had limited effects, the preharvest sampling 4 days (PHS 4D) and receiving sampling (RS), had an added log reduction of 0.053 and 0.054, respectively. For the no-sanitation, no-precooling, no-wash, and no-prewash systems, the second and third most effective sampling stages were the preharvest sampling 4 days (PHS 4D) followed by preharvest sampling 4 h (PHS 4H), with reductions of between 0.084 to 0.11 log and 0.054 to 0.092 log, respectively. For the no-holding system, the second and third most effective sampling plans were receiving sampling (RS), and harvest sampling (HS), with reductions of 0.34 and 0.30, respectively. In addition, for all the systems except for the no-intervention system, the least effective sampling plans were finished product sampling (FPS) and customer sampling (CS), with added reductions ranging between 0 and 0.037, and 0 and 0.031 log, respectively. Beyond assessing major system interventions and sampling plans with an FS, a partial rank correlation coefficient (PRCC) sensitivity analysis ( ) was performed to obtain a complete profile of endpoint TACs of all randomized input variables. The inputs with the largest absolute PRCCs, indicating the most sensitivity in the time between the contamination event and harvest (−0.58), were chlorinated wash on (−0.29), prewash on (−0.21), and transportation time to consumer (−0.06). The PRCC also showed sampling plans to have the smallest PRCC values, ranging between −0.005 and 0.021. These sensitivity analyses together suggest that the incorporation of effective interventions, such as preharvest holding or process washing, meaningfully reduce endpoint TACs. These also show that when effective interventions are in place, sample testing and rejection before processing result in larger reductions. If no interventions are in place, sample testing and rejection of the finished product lead to larger reductions.
Process interventions reduce contamination that reaches the system endpoint. The analysis predicted an overall impact of food safety interventions of around 3.5 log, with individual interventions achieving 0.8- to 1.3-log reductions, a range consistent with other models in the literature that have tracked microbial loads through produce systems ( ). Washing of leafy greens has been predicted to be a food safety intervention, as also predicted in this model. Pang et al. demonstrated that a chlorinated wash would result in a 12.7-fold reduction of illness (from 2,160 to 170 illness per year in the US) caused by leafy greens compared to the baseline system in their quantitative microbial risk assessment (QMRA) ( ). In a different study, Danyluk and Schaffner indicated that washing may not only provide a reduction in contamination but may also reduce the degree of cross-contamination that occurs during the washing process, providing a significant reduction of the E. coli O157:H7 levels on leafy greens ( ). Mokhtari et al. showed that compliance and an effective preliminary spray wash are an effective intervention to reduce contamination levels in the finished product ( ). A holding time between final irrigation and harvest is another critical strategy to control the bacterial load on harvested produce. A QMRA conducted by Ottoson et al. showed that holding time is extremely important for illness reduction. In that study, the baseline model, 0 days of holding time, resulted in 436 illnesses per 10,000 servings. In contrast, the system with 2 days of holding time reduced the illness prevalence to 52 illnesses per 10,000 servings, a significant reduction ( ). These studies highlight the importance of implementing systems such as good agricultural and manufacturing practices (GAPs and GMPs) to control contamination as recommended by the FDA ( ). Contamination levels at the sampling point and sampling plan characteristics determine sampling plan power. The power of sampling plans depends on the adulterant cells at the sampling point. Overall samples taken earlier in the system (such as preharvest, harvest, and receiving) were more powerful than those taken after effective processing interventions (such as at finished product sampling and customer sampling). Statistical analysis ( ) supports that sampling plan power is higher when contamination levels are higher. Simulation analysis ( ) specifically shows that as the contamination level decreases through effective food safety interventions, the power of the sampling plan decreases. The power of sampling also depends on the sampling plan characteristics (composite sample mass and the number of individual grabs taken). In our scenario analysis, preharvest sampling intense (PHS Int) had a 4-fold increase in the composite sample mass and number of grabs taken compared to harvest sampling (HS), at the same sampling point. Preharvest sampling intense outperformed harvest sampling by 2.5- to 4.0-fold. Therefore, our study agrees with other studies that show that sampling plans with higher sample mass and more sampling grabs perform better in detecting contamination ( , , ). The sampling plans analyzed in this study would mostly fail to detect a preharvest contamination event with contamination levels of 1 CFU/lb. Other studies have also concluded that typical sampling strategies would fail to detect in-field contamination specifically at low levels and prevalence ( , , ). The total sample mass and the total number of grabs would need to increase to improve the performance of the sampling plans ( ). However, increases are constrained by the feasibility of labor and laboratory analysis costs. Our study adds another suggestion to increase power. Sampling before effective process interventions (preharvest, harvest, or receiving) will typically result in a higher power due to higher contamination levels at the sampling point. The predicted power of sampling for a test-and-reject program does not fully describe the impact of that sampling program. Sampling plan power provides information on how well the sampling plan performs for a given sampling stage. However, that metric does not directly addresses how many contaminants reach the customer when sampling is used for the testing and rejection of lots. A sampling plan might have higher power because it detects contamination when the contamination levels are high (preharvest), making detection more likely. But effective food safety interventions later in the system might otherwise reduce that contamination, lowering the relative immediate food safety impact of sampling, where many of the cells detected early would already be managed and reduced, though such detection would still be valuable in the long term for understanding what causes contamination and informing continuous improvement. For example, the most powerful preharvest sampling in our model (PHS 4D) had similar effects on reducing the contamination that reached the customer compared to other preharvest sampling plans. The sensitivity analysis showed that the most important factor in reducing preharvest contamination levels is the preharvest die-off. Belias et al. ( ) showed that contamination at preharvest declined at a variable rate with a mean and standard deviation of −0.77 and 0.21 log(adulterant cells per day), respectively. If the event happens 8 days before harvest, the microbial population might naturally be reduced to very low levels before harvest occurs, limiting the direct food safety impact of preharvest sampling. The model suggests that the most powerful samples would be those taken as close as possible to the contamination event, as shown by Duffy and Schaffner ( ). The value of testing. Buchanan and Schaffner describe different types of microbial testing within a food safety system ( ). In food systems, the most beneficial and desired type of testing is process control verification testing, as it may be able to detect when preventive measures may be out of control. In addition, the produce industry is often required to conduct hold-and-release testing to comply with import or buyer requirements as a preventive control, to prevent contaminated products from entering the market. While beneficial if contamination is detected, the effectiveness of this type of testing decreases substantially when the prevalence of contamination is low, as many samples would be needed to detect contamination. Our results show that effective food safety interventions reduce contamination more than the effect of sampling for rejecting contaminated lots. As mentioned above, test-and-reject sampling is powerful when contamination is high, tests identify an adulterant, and rejection effectively reduces the number of cells that would otherwise pass through the system. Here, we show that sampling is typically more powerful early in the system, but then subsequent interventions would otherwise reduce contamination if it were not detected, reducing the impact of testing for rejection. In addition, sampling at later stages after effective interventions take place has lower detection power because of the lower contamination prevalence and levels as an effect of effective interventions. Therefore, the value of the test-and-reject sampling could be in detecting critical, high-level contamination events that the system may not otherwise adequately control and preventing them from entering the system. As mentioned earlier, another use for sampling beyond test and reject sampling is microbiological testing used for verification, specifically, process control verification testing ( , ). A smaller number of samples is taken over time, facilitating analysis of the overall performance of a process. Here, testing is a tool to continuously improve food safety by maintaining or improving the performance of other controls; testing is not used as the primary control ( ). This second type of testing could provide value to a leafy green food safety system by identifying unknown or underappreciated pathways of contamination, novel hazards, or other potential risks. For example, pathogen testing environmental data and meteorological data could be used to predict environmental reservoirs of pathogens ( ). These types of analyses, particularly if done on data aggregated across producers to represent large portions of the industry, would allow producers and processors to improve their food safety controls to manage new risks across the whole industry. (i) Future steps. Our work represents a variety of contamination spreads, systems, and sampling plans—a total of 147 scenarios. Still, scenarios not addressed in this model are possible. Developing an application programming interface (API) or another user interphase would be beneficial for growers and packers to tune the model to their specific food safety systems and questions. While this model aims to represent a farm-to-customer system for leafy greens, there is still uncertainty about the contamination levels and clustering parameters. As data gaps are filled, the parameters and functions in this model can be updated to deal with this uncertainty, allowing for future analyses with less uncertainty. Future work could also use the model to provide effective sampling plans for a given food safety objective. This would help growers and producers set a testing standard and develop effective sampling plans to meet that standard. (ii) Conclusion. This model was built to assess the efficacy of test-and-reject sampling for farm-to-customer leafy green safety. For this model, the focus was on seven sampling strategies, for three contamination spreads, across seven systems. The results indicate that sampling is less impactful at reducing the endpoint adulterant cells when effective systems-based interventions are in place. The model showed that the sampling plans in this study have limited power to detect contamination levels such as the ones that caused a foodborne disease outbreak in 2018. The model showed that conducting sampling too early in the system may not be as beneficial as sampling closer to a contamination event. Sampling plans should focus on stages at which contamination may be high enough for detection. The stages are preharvest, harvest, and receiving. Other process interventions reduce contamination, resulting in finished product sampling and customer sampling having very low power. However, this study shows that finished product sampling could detect contamination if all interventions were to fail. This study suggests that interventions are effective at reducing incoming contamination. Producers and buyers should focus on implementing good food safety interventions as primary preventive controls. Once interventions are implemented, test-and-reject sampling plans can be used to detect incoming high-level contamination or unappreciated or otherwise uncontrolled sources of contamination.
The analysis predicted an overall impact of food safety interventions of around 3.5 log, with individual interventions achieving 0.8- to 1.3-log reductions, a range consistent with other models in the literature that have tracked microbial loads through produce systems ( ). Washing of leafy greens has been predicted to be a food safety intervention, as also predicted in this model. Pang et al. demonstrated that a chlorinated wash would result in a 12.7-fold reduction of illness (from 2,160 to 170 illness per year in the US) caused by leafy greens compared to the baseline system in their quantitative microbial risk assessment (QMRA) ( ). In a different study, Danyluk and Schaffner indicated that washing may not only provide a reduction in contamination but may also reduce the degree of cross-contamination that occurs during the washing process, providing a significant reduction of the E. coli O157:H7 levels on leafy greens ( ). Mokhtari et al. showed that compliance and an effective preliminary spray wash are an effective intervention to reduce contamination levels in the finished product ( ). A holding time between final irrigation and harvest is another critical strategy to control the bacterial load on harvested produce. A QMRA conducted by Ottoson et al. showed that holding time is extremely important for illness reduction. In that study, the baseline model, 0 days of holding time, resulted in 436 illnesses per 10,000 servings. In contrast, the system with 2 days of holding time reduced the illness prevalence to 52 illnesses per 10,000 servings, a significant reduction ( ). These studies highlight the importance of implementing systems such as good agricultural and manufacturing practices (GAPs and GMPs) to control contamination as recommended by the FDA ( ).
The power of sampling plans depends on the adulterant cells at the sampling point. Overall samples taken earlier in the system (such as preharvest, harvest, and receiving) were more powerful than those taken after effective processing interventions (such as at finished product sampling and customer sampling). Statistical analysis ( ) supports that sampling plan power is higher when contamination levels are higher. Simulation analysis ( ) specifically shows that as the contamination level decreases through effective food safety interventions, the power of the sampling plan decreases. The power of sampling also depends on the sampling plan characteristics (composite sample mass and the number of individual grabs taken). In our scenario analysis, preharvest sampling intense (PHS Int) had a 4-fold increase in the composite sample mass and number of grabs taken compared to harvest sampling (HS), at the same sampling point. Preharvest sampling intense outperformed harvest sampling by 2.5- to 4.0-fold. Therefore, our study agrees with other studies that show that sampling plans with higher sample mass and more sampling grabs perform better in detecting contamination ( , , ). The sampling plans analyzed in this study would mostly fail to detect a preharvest contamination event with contamination levels of 1 CFU/lb. Other studies have also concluded that typical sampling strategies would fail to detect in-field contamination specifically at low levels and prevalence ( , , ). The total sample mass and the total number of grabs would need to increase to improve the performance of the sampling plans ( ). However, increases are constrained by the feasibility of labor and laboratory analysis costs. Our study adds another suggestion to increase power. Sampling before effective process interventions (preharvest, harvest, or receiving) will typically result in a higher power due to higher contamination levels at the sampling point.
Sampling plan power provides information on how well the sampling plan performs for a given sampling stage. However, that metric does not directly addresses how many contaminants reach the customer when sampling is used for the testing and rejection of lots. A sampling plan might have higher power because it detects contamination when the contamination levels are high (preharvest), making detection more likely. But effective food safety interventions later in the system might otherwise reduce that contamination, lowering the relative immediate food safety impact of sampling, where many of the cells detected early would already be managed and reduced, though such detection would still be valuable in the long term for understanding what causes contamination and informing continuous improvement. For example, the most powerful preharvest sampling in our model (PHS 4D) had similar effects on reducing the contamination that reached the customer compared to other preharvest sampling plans. The sensitivity analysis showed that the most important factor in reducing preharvest contamination levels is the preharvest die-off. Belias et al. ( ) showed that contamination at preharvest declined at a variable rate with a mean and standard deviation of −0.77 and 0.21 log(adulterant cells per day), respectively. If the event happens 8 days before harvest, the microbial population might naturally be reduced to very low levels before harvest occurs, limiting the direct food safety impact of preharvest sampling. The model suggests that the most powerful samples would be those taken as close as possible to the contamination event, as shown by Duffy and Schaffner ( ).
Buchanan and Schaffner describe different types of microbial testing within a food safety system ( ). In food systems, the most beneficial and desired type of testing is process control verification testing, as it may be able to detect when preventive measures may be out of control. In addition, the produce industry is often required to conduct hold-and-release testing to comply with import or buyer requirements as a preventive control, to prevent contaminated products from entering the market. While beneficial if contamination is detected, the effectiveness of this type of testing decreases substantially when the prevalence of contamination is low, as many samples would be needed to detect contamination. Our results show that effective food safety interventions reduce contamination more than the effect of sampling for rejecting contaminated lots. As mentioned above, test-and-reject sampling is powerful when contamination is high, tests identify an adulterant, and rejection effectively reduces the number of cells that would otherwise pass through the system. Here, we show that sampling is typically more powerful early in the system, but then subsequent interventions would otherwise reduce contamination if it were not detected, reducing the impact of testing for rejection. In addition, sampling at later stages after effective interventions take place has lower detection power because of the lower contamination prevalence and levels as an effect of effective interventions. Therefore, the value of the test-and-reject sampling could be in detecting critical, high-level contamination events that the system may not otherwise adequately control and preventing them from entering the system. As mentioned earlier, another use for sampling beyond test and reject sampling is microbiological testing used for verification, specifically, process control verification testing ( , ). A smaller number of samples is taken over time, facilitating analysis of the overall performance of a process. Here, testing is a tool to continuously improve food safety by maintaining or improving the performance of other controls; testing is not used as the primary control ( ). This second type of testing could provide value to a leafy green food safety system by identifying unknown or underappreciated pathways of contamination, novel hazards, or other potential risks. For example, pathogen testing environmental data and meteorological data could be used to predict environmental reservoirs of pathogens ( ). These types of analyses, particularly if done on data aggregated across producers to represent large portions of the industry, would allow producers and processors to improve their food safety controls to manage new risks across the whole industry. (i) Future steps. Our work represents a variety of contamination spreads, systems, and sampling plans—a total of 147 scenarios. Still, scenarios not addressed in this model are possible. Developing an application programming interface (API) or another user interphase would be beneficial for growers and packers to tune the model to their specific food safety systems and questions. While this model aims to represent a farm-to-customer system for leafy greens, there is still uncertainty about the contamination levels and clustering parameters. As data gaps are filled, the parameters and functions in this model can be updated to deal with this uncertainty, allowing for future analyses with less uncertainty. Future work could also use the model to provide effective sampling plans for a given food safety objective. This would help growers and producers set a testing standard and develop effective sampling plans to meet that standard. (ii) Conclusion. This model was built to assess the efficacy of test-and-reject sampling for farm-to-customer leafy green safety. For this model, the focus was on seven sampling strategies, for three contamination spreads, across seven systems. The results indicate that sampling is less impactful at reducing the endpoint adulterant cells when effective systems-based interventions are in place. The model showed that the sampling plans in this study have limited power to detect contamination levels such as the ones that caused a foodborne disease outbreak in 2018. The model showed that conducting sampling too early in the system may not be as beneficial as sampling closer to a contamination event. Sampling plans should focus on stages at which contamination may be high enough for detection. The stages are preharvest, harvest, and receiving. Other process interventions reduce contamination, resulting in finished product sampling and customer sampling having very low power. However, this study shows that finished product sampling could detect contamination if all interventions were to fail. This study suggests that interventions are effective at reducing incoming contamination. Producers and buyers should focus on implementing good food safety interventions as primary preventive controls. Once interventions are implemented, test-and-reject sampling plans can be used to detect incoming high-level contamination or unappreciated or otherwise uncontrolled sources of contamination.
Model overview. The model was developed using Python version 3.9.12 ( ). It simulated a variety of contamination spreads, food safety interventions, and sampling plans involved in the harvesting, receiving, and processing of leafy greens. A modular process model approach was taken to represent the microbial dynamics of growth, inactivation, mixing, partitioning, removal, and cross-contamination. Figure S1 describes the modules and microbial dynamics, and Table S1 describes the parameters for the model and product flow. Product flow. The initial mass was 100,000 lb of romaine lettuce, chosen to represent a mass reasonably harvested and processed in 1 day by a grower and packer ( ). The initial mass was split into 50-lb units to represent the mass as it moves through the system. These 50-lb units were aggregated, mixed, and partitioned as needed to represent processing units such as the production rate, pallet load, and finished product packages. Partitioning and mixing processes were modeled as described by Nauta ( ). Preharvest contamination. Contamination was introduced at preharvest in one of three different patterns ( ), representing the uncertainty around the actual spread of a contamination event. (i) Random uniform (widespread 100% cluster) contamination: this contamination spread covered the whole mass to be harvested and processed, representing an event such as a field irrigated with contaminated water ( , ) or rainfall that splashed contaminated soil onto the leafy green leaves, leading to the widespread contamination of the field ( ). (ii) Large-cluster contamination: 10% (10,000 lb) of the product was contaminated with an adulterant concentration of 10 CFU/lb. This larger cluster represented a contamination event due to run-off of cattle feces from adjacent farms, contamination from dust containing the adulterant, or other field activities that may have led to contamination in the form of a large cluster ( , ). (iii) Small cluster contamination: 1% (1,000 lb) of the total mass was highly contaminated (100 CFU/lb). A small-cluster contamination event represented contamination due to animal intrusion or fecal contamination from wild animals, such as bird droppings directly contaminating the leaves or fecal pellets deposited in the field that could be transferred to the leaves due to irrigation or rainfall splash ( , , ). The total adulterant cells in the process model were fixed at 100,000 cells to provide consistent hazard pressure for empirical scenarios. The 100,000 adulterant cells were distributed over the 100,000-lb mass in patterns dependent on the contamination scenario (random uniform: widespread 100% cluster, large 10% cluster, small 1% cluster). The overall contamination level of the mass was 1 adulterant cell/lb based on reverse engineering performed by the 2020 United Fresh workgroup of the 2018 E. coli O157:H7 romaine lettuce outbreak ( ), where the average contamination that likely led to the outbreak was determined to be 0.81 CFU/lb. The goal was to realistically represent a rare event that actual food safety sampling failed to detect, with contamination levels high enough to result in a foodborne disease outbreak. Sporadic background-level contamination was applied to the system at 636 adulterant cells distributed randomly and uniformly across the entire mass of the product; this sporadic contamination level was consistent with the simulated low background contamination in leafy green fields as assumed by Quintanilla Portillo et al. ( ). Sampling model. The calculation for the presence of STEC in an individual grab sample was calculated as in Jongenburger et al. ( ) following the low-level heterogeneous contamination assumption since it allowed for obtaining the probability of detection of each grab: (1) P detect = 1 − e − C ⋅ M GrabSample where P detect is the probability of detection, C is the concentration of the adulterant (adulterant cells/g) in the sampling unit (50-lb unit), and M GrabSample is the mass of the individual grab sample (g). Once P detect was obtained, the presence or absence of the adulterant in the given sample was calculated by checking if a random number between 0 and 1 drawn from a uniform distribution was less than P detect , meaning the adulterant is present in the grab sample and detected; otherwise, the adulterant is absent from the grab sample and not detected. At the end of each sampling process, if any of the grabs detected an adulterant cell, the product was rejected as part of an International Commission on Microbial Specification for Foods (ICMSF) 2-class attribute sampling plan ( ). Growth, survival, and die-off models. Growth and survival were modeled during the transportation and storage of the product. The primary growth model used for this study was a three-phase linear model as proposed by Buchanan et al. ( ). The specific growth rate was modeled with a square root model as proposed by Ratkowsky et al. ( ). The lag time was calculated as a function of temperature using the power law equation obtained from Mishra et al. ( ). It was adapted to represent partial lag consumption under dynamic temperature conditions. When the temperature was under 5°C a log-linear survival model for the adulterant was used as proposed by McKellar et al. ( ). The in-field die-off model was adapted from Belias et al. ( ), using parameters for E. coli in lettuce; the log-linear parameters were implemented into the model. The combined growth, survival, and die-off models, equations, and parameters are described in detail in File S1. Processing steps. Processing consisted of 6 processing modules: (i) preliminary spray wash, (ii) shredding, (iii) conveyor belt transportation, (iv) flume washing, (v) shaker table, and (vi) dewatering centrifuge as described by Pérez Rodríguez et al. ( ). Inactivation and cross-contamination were the primary microbial dynamics during processing. For the preliminary spray wash, the product underwent a microbial reduction as an effect of spray washing as described by Mokhtari et al. ( ). Microbial contaminants were washed off, and the spray wash water was free from contaminants. The reduction of the spray wash was modeled to be between 1.10 and 1.46 log CFU as described by Pahariya et al. ( ). Shredding, the conveyor belt, the shaker table, and the dewatering centrifuge represented product-to-surface cross-contamination steps. The approach discussed by Hoelzer et al. ( ) and Mokhtari and Van Doren ( ) was used to represent cross-contamination between two objects caused by a tactile even, where the total adulterant cells in a 50-lb unit of the total mass were cross-contaminated to the cells on the processing equipment. Specific equations for both the spray wash and cross-contamination are found in File S1. Washing. Flume tank washing was adapted from the model developed by Munther et al. ( ). Additional data and parameters were obtained from other studies to fully develop the washing system ( , , , ). The dynamic chlorine wash evaluated changes in free chlorine (FC) inside the flume tank as a factor of chemical oxygen demand (COD) ( ). The product wash rate was 100 lb/min, which determines the level of organic matter entering the flume wash. The developed system followed the dosing scheme shown by Luo et al. ( ), where sodium hypochlorite was added to the flume tank in 12-min intervals. These dosing periods allowed the FC levels to rise back to desired levels. The inactivation and cross-contamination achieved were simulated for the product entering the wash. Equations and parameters for washing dynamics are found in File S1. The code repository contains information on the washing step adaptation and validation. See the “Data Availability” section. Processing line sanitation. The sanitation of four processing equipment surfaces (conveyor belt, shredder, shaker table, and dewatering centrifuge) was simulated as an intervention strategy. The contamination in the processing line was updated for every processing step as every 50-lb portion of the product was processed. Sanitation was modeled as a function of three parameters to represent sanitation at the following steps: (i) sanitation compliance, (ii) sanitation frequency, and (iii) sanitation reduction, where the values for these three parameters are uncertain values randomized on every iteration as shown in Table S1, where sanitation compliance is described as the probability of sanitation occurring, sanitation frequency is the pounds of product that are processed before the processing line is sanitized, and sanitation reduction refers to the log reduction on adulterant cells achieved by sanitation. If the sanitation step occurred, then the new contamination of the processing surface was calculated by applying the corresponding reduction to the processing line as indicated by Mokhtari et al. ( ). Scenario analysis development. A scenario analysis was developed, as shown in . The scenario analysis consists of three different contamination spreads, seven systems, and seven sampling plans. The combination of the different factors yielded a total of 147 scenario combinations. Production scenarios and interventions. Five food safety interventions were adapted to represent interventions in a farm-to-customer system. The interventions were preharvest holding, precooling, prewash, chlorinated wash, and processing line sanitation. A detailed description of the interventions can be found in . The goal of modeling different systems was to represent the wide range of uncertainty regarding adherence to food safety practices in different operations. Seven systems were generated to address the wide range of uncertainty: an all-intervention system, where all five interventions are applied, a no-intervention system, where no food safety interventions are applied, and five in-between systems, where one of the five food safety interventions was removed at a time. The five additional systems are no holding, no washing, no prewash, no sanitation, and no precooling. This evaluates the benefit of conducting product sampling when a specific food safety intervention fails. Sampling scenarios. Seven separate sampling plans were simulated at different system stages. Preharvest 4 days (PHS 4D): sampling occurred 4 days before harvest. Preharvest 4 h (PHS 4H): sampling occurred 4 hours before harvest. Preharvest intense (PHS Int): sampling occurred immediately before harvest. Harvest sampling (HS): sampling occurred at harvest. Receiving sampling (RS): sampling occurred after temporary storage at the facility. Finished product sampling (FPS): sampling was performed as the shredded product was packed into 5-lb bags. Customer sampling (CS): simulated sampling occurred after transportation from a processing facility to a retail or food service customer. The sampling plans were designed to match a 1,500-g total composite sample mass and 60 total grabs ( ). These sampling plan characteristics were selected to observe guidelines from the International Commission on Microbial Specification for Foods (ICMSF) sampling plan stringency (cases), in this case, ICMSF’s case 15 for severe hazards for which growth may happen ( ). The sampling plans also adhered to recommendations from Western Growers’ (WG) Appendix C, which guides growers and processors on preharvest product testing as specified in the Leafy Greens Marketing Agreement (LGMA) approved guidelines ( ). These documents recommend that 60 total individual 25-g grabs be tested, resulting in a 1,500-g composite mass. All sampling adhered to the 60 grabs, 1,500-g composite recommendations, except for preharvest sampling intense (PHS Int). For PHS Int, the composite sample mass and the number of grabs were increased 4-fold to observe recommendations made by WG’s Appendix C under the intensified sampling recommendations. WG recommends that for intensified sampling, the maximum sampling area be 1 acre. Since our model simulated a total mass of 100,000 lb, this translated to approximately 4 acres of romaine lettuce harvested ( ). Therefore, a total of four 1,500-g 60-grab samples were taken under this scenario. Scenario analysis metrics. A total of 147 combinations were generated for analysis. A total of 10,000 iterations were conducted per combination. The number of iterations needed was computed for the all-intervention and no-intervention systems as a factor of (i) S, the standard deviation of the outputs (endpoint TACs), (ii) E, the desired margin of error (10% of the mean endpoint TACs), and (iii) Z α/2 = 1.96, the critical Z value of a normal distribution at a 95% confidence interval ( ). More information can be found in “Determination of Total Iterations” in File S1. The following metrics were used to assess the performance of interventions and sampling plans across the scenarios. Sampling power was a way to measure how well a sampling plan performed at detecting contamination. It is defined as the percentage of times that the sampling plan detected contamination out of the total ( n = 10,000) number of iterations: (2) Sampling power = Model iterations where sampling plan detected pathogen Total iterations × 100 The relative efficacy in reducing endpoint total adulterant cells (TACs) from reaching the system’s endpoint was used to quantify how well sampling plans performed in a specific system. The endpoint TAC relative efficacy between the 7 sampling plans was quantified across all 7 systems and compared to each system without sampling as a baseline: (3) Realtive efficacy = 1 − Endpoint ( TAC ) , what - if scenario Endpoint ( TAC ) , baseline scenario Factor sensitivity analysis (FS) was used to compare interventions and sampling plans. Factor sensitivity is the log reduction between endpoint TACs from each scenario and the system with no food safety interventions or sampling plans (no interventions). The greater the absolute FS, the greater the effect that a specific scenario or condition had on total consumer reduction of endpoint TACs ( , ): (4) FS =log Output (intervention or sampling plan) Output (baseline no - intervention or sampling plan) The partial rank correlation coefficient (PRCC) sensitivity analysis was done as described by Marino et al. ( ). The analysis of the outputs was performed in R version 3.6.1 with the function PRCC from the package “sensitivity.” The sensitivity analysis was performed in a system where interventions were turned on, the all-intervention system. The model’s sensitivity was used to determine the system’s most influential parameters for final adulterant cells. Data availability. The code for the process model and the analysis can be found on GitHub ( https://github.com/foodsafetylab/Farm-to-Consumer-LG-Sim ). Validation documents and a descriptive file with basic instructions on the model files are included in the repository.
The model was developed using Python version 3.9.12 ( ). It simulated a variety of contamination spreads, food safety interventions, and sampling plans involved in the harvesting, receiving, and processing of leafy greens. A modular process model approach was taken to represent the microbial dynamics of growth, inactivation, mixing, partitioning, removal, and cross-contamination. Figure S1 describes the modules and microbial dynamics, and Table S1 describes the parameters for the model and product flow.
The initial mass was 100,000 lb of romaine lettuce, chosen to represent a mass reasonably harvested and processed in 1 day by a grower and packer ( ). The initial mass was split into 50-lb units to represent the mass as it moves through the system. These 50-lb units were aggregated, mixed, and partitioned as needed to represent processing units such as the production rate, pallet load, and finished product packages. Partitioning and mixing processes were modeled as described by Nauta ( ).
Contamination was introduced at preharvest in one of three different patterns ( ), representing the uncertainty around the actual spread of a contamination event. (i) Random uniform (widespread 100% cluster) contamination: this contamination spread covered the whole mass to be harvested and processed, representing an event such as a field irrigated with contaminated water ( , ) or rainfall that splashed contaminated soil onto the leafy green leaves, leading to the widespread contamination of the field ( ). (ii) Large-cluster contamination: 10% (10,000 lb) of the product was contaminated with an adulterant concentration of 10 CFU/lb. This larger cluster represented a contamination event due to run-off of cattle feces from adjacent farms, contamination from dust containing the adulterant, or other field activities that may have led to contamination in the form of a large cluster ( , ). (iii) Small cluster contamination: 1% (1,000 lb) of the total mass was highly contaminated (100 CFU/lb). A small-cluster contamination event represented contamination due to animal intrusion or fecal contamination from wild animals, such as bird droppings directly contaminating the leaves or fecal pellets deposited in the field that could be transferred to the leaves due to irrigation or rainfall splash ( , , ). The total adulterant cells in the process model were fixed at 100,000 cells to provide consistent hazard pressure for empirical scenarios. The 100,000 adulterant cells were distributed over the 100,000-lb mass in patterns dependent on the contamination scenario (random uniform: widespread 100% cluster, large 10% cluster, small 1% cluster). The overall contamination level of the mass was 1 adulterant cell/lb based on reverse engineering performed by the 2020 United Fresh workgroup of the 2018 E. coli O157:H7 romaine lettuce outbreak ( ), where the average contamination that likely led to the outbreak was determined to be 0.81 CFU/lb. The goal was to realistically represent a rare event that actual food safety sampling failed to detect, with contamination levels high enough to result in a foodborne disease outbreak. Sporadic background-level contamination was applied to the system at 636 adulterant cells distributed randomly and uniformly across the entire mass of the product; this sporadic contamination level was consistent with the simulated low background contamination in leafy green fields as assumed by Quintanilla Portillo et al. ( ).
The calculation for the presence of STEC in an individual grab sample was calculated as in Jongenburger et al. ( ) following the low-level heterogeneous contamination assumption since it allowed for obtaining the probability of detection of each grab: (1) P detect = 1 − e − C ⋅ M GrabSample where P detect is the probability of detection, C is the concentration of the adulterant (adulterant cells/g) in the sampling unit (50-lb unit), and M GrabSample is the mass of the individual grab sample (g). Once P detect was obtained, the presence or absence of the adulterant in the given sample was calculated by checking if a random number between 0 and 1 drawn from a uniform distribution was less than P detect , meaning the adulterant is present in the grab sample and detected; otherwise, the adulterant is absent from the grab sample and not detected. At the end of each sampling process, if any of the grabs detected an adulterant cell, the product was rejected as part of an International Commission on Microbial Specification for Foods (ICMSF) 2-class attribute sampling plan ( ).
Growth and survival were modeled during the transportation and storage of the product. The primary growth model used for this study was a three-phase linear model as proposed by Buchanan et al. ( ). The specific growth rate was modeled with a square root model as proposed by Ratkowsky et al. ( ). The lag time was calculated as a function of temperature using the power law equation obtained from Mishra et al. ( ). It was adapted to represent partial lag consumption under dynamic temperature conditions. When the temperature was under 5°C a log-linear survival model for the adulterant was used as proposed by McKellar et al. ( ). The in-field die-off model was adapted from Belias et al. ( ), using parameters for E. coli in lettuce; the log-linear parameters were implemented into the model. The combined growth, survival, and die-off models, equations, and parameters are described in detail in File S1.
Processing consisted of 6 processing modules: (i) preliminary spray wash, (ii) shredding, (iii) conveyor belt transportation, (iv) flume washing, (v) shaker table, and (vi) dewatering centrifuge as described by Pérez Rodríguez et al. ( ). Inactivation and cross-contamination were the primary microbial dynamics during processing. For the preliminary spray wash, the product underwent a microbial reduction as an effect of spray washing as described by Mokhtari et al. ( ). Microbial contaminants were washed off, and the spray wash water was free from contaminants. The reduction of the spray wash was modeled to be between 1.10 and 1.46 log CFU as described by Pahariya et al. ( ). Shredding, the conveyor belt, the shaker table, and the dewatering centrifuge represented product-to-surface cross-contamination steps. The approach discussed by Hoelzer et al. ( ) and Mokhtari and Van Doren ( ) was used to represent cross-contamination between two objects caused by a tactile even, where the total adulterant cells in a 50-lb unit of the total mass were cross-contaminated to the cells on the processing equipment. Specific equations for both the spray wash and cross-contamination are found in File S1.
Flume tank washing was adapted from the model developed by Munther et al. ( ). Additional data and parameters were obtained from other studies to fully develop the washing system ( , , , ). The dynamic chlorine wash evaluated changes in free chlorine (FC) inside the flume tank as a factor of chemical oxygen demand (COD) ( ). The product wash rate was 100 lb/min, which determines the level of organic matter entering the flume wash. The developed system followed the dosing scheme shown by Luo et al. ( ), where sodium hypochlorite was added to the flume tank in 12-min intervals. These dosing periods allowed the FC levels to rise back to desired levels. The inactivation and cross-contamination achieved were simulated for the product entering the wash. Equations and parameters for washing dynamics are found in File S1. The code repository contains information on the washing step adaptation and validation. See the “Data Availability” section.
The sanitation of four processing equipment surfaces (conveyor belt, shredder, shaker table, and dewatering centrifuge) was simulated as an intervention strategy. The contamination in the processing line was updated for every processing step as every 50-lb portion of the product was processed. Sanitation was modeled as a function of three parameters to represent sanitation at the following steps: (i) sanitation compliance, (ii) sanitation frequency, and (iii) sanitation reduction, where the values for these three parameters are uncertain values randomized on every iteration as shown in Table S1, where sanitation compliance is described as the probability of sanitation occurring, sanitation frequency is the pounds of product that are processed before the processing line is sanitized, and sanitation reduction refers to the log reduction on adulterant cells achieved by sanitation. If the sanitation step occurred, then the new contamination of the processing surface was calculated by applying the corresponding reduction to the processing line as indicated by Mokhtari et al. ( ).
A scenario analysis was developed, as shown in . The scenario analysis consists of three different contamination spreads, seven systems, and seven sampling plans. The combination of the different factors yielded a total of 147 scenario combinations.
Five food safety interventions were adapted to represent interventions in a farm-to-customer system. The interventions were preharvest holding, precooling, prewash, chlorinated wash, and processing line sanitation. A detailed description of the interventions can be found in . The goal of modeling different systems was to represent the wide range of uncertainty regarding adherence to food safety practices in different operations. Seven systems were generated to address the wide range of uncertainty: an all-intervention system, where all five interventions are applied, a no-intervention system, where no food safety interventions are applied, and five in-between systems, where one of the five food safety interventions was removed at a time. The five additional systems are no holding, no washing, no prewash, no sanitation, and no precooling. This evaluates the benefit of conducting product sampling when a specific food safety intervention fails.
Seven separate sampling plans were simulated at different system stages. Preharvest 4 days (PHS 4D): sampling occurred 4 days before harvest. Preharvest 4 h (PHS 4H): sampling occurred 4 hours before harvest. Preharvest intense (PHS Int): sampling occurred immediately before harvest. Harvest sampling (HS): sampling occurred at harvest. Receiving sampling (RS): sampling occurred after temporary storage at the facility. Finished product sampling (FPS): sampling was performed as the shredded product was packed into 5-lb bags. Customer sampling (CS): simulated sampling occurred after transportation from a processing facility to a retail or food service customer. The sampling plans were designed to match a 1,500-g total composite sample mass and 60 total grabs ( ). These sampling plan characteristics were selected to observe guidelines from the International Commission on Microbial Specification for Foods (ICMSF) sampling plan stringency (cases), in this case, ICMSF’s case 15 for severe hazards for which growth may happen ( ). The sampling plans also adhered to recommendations from Western Growers’ (WG) Appendix C, which guides growers and processors on preharvest product testing as specified in the Leafy Greens Marketing Agreement (LGMA) approved guidelines ( ). These documents recommend that 60 total individual 25-g grabs be tested, resulting in a 1,500-g composite mass. All sampling adhered to the 60 grabs, 1,500-g composite recommendations, except for preharvest sampling intense (PHS Int). For PHS Int, the composite sample mass and the number of grabs were increased 4-fold to observe recommendations made by WG’s Appendix C under the intensified sampling recommendations. WG recommends that for intensified sampling, the maximum sampling area be 1 acre. Since our model simulated a total mass of 100,000 lb, this translated to approximately 4 acres of romaine lettuce harvested ( ). Therefore, a total of four 1,500-g 60-grab samples were taken under this scenario.
A total of 147 combinations were generated for analysis. A total of 10,000 iterations were conducted per combination. The number of iterations needed was computed for the all-intervention and no-intervention systems as a factor of (i) S, the standard deviation of the outputs (endpoint TACs), (ii) E, the desired margin of error (10% of the mean endpoint TACs), and (iii) Z α/2 = 1.96, the critical Z value of a normal distribution at a 95% confidence interval ( ). More information can be found in “Determination of Total Iterations” in File S1. The following metrics were used to assess the performance of interventions and sampling plans across the scenarios. Sampling power was a way to measure how well a sampling plan performed at detecting contamination. It is defined as the percentage of times that the sampling plan detected contamination out of the total ( n = 10,000) number of iterations: (2) Sampling power = Model iterations where sampling plan detected pathogen Total iterations × 100 The relative efficacy in reducing endpoint total adulterant cells (TACs) from reaching the system’s endpoint was used to quantify how well sampling plans performed in a specific system. The endpoint TAC relative efficacy between the 7 sampling plans was quantified across all 7 systems and compared to each system without sampling as a baseline: (3) Realtive efficacy = 1 − Endpoint ( TAC ) , what - if scenario Endpoint ( TAC ) , baseline scenario Factor sensitivity analysis (FS) was used to compare interventions and sampling plans. Factor sensitivity is the log reduction between endpoint TACs from each scenario and the system with no food safety interventions or sampling plans (no interventions). The greater the absolute FS, the greater the effect that a specific scenario or condition had on total consumer reduction of endpoint TACs ( , ): (4) FS =log Output (intervention or sampling plan) Output (baseline no - intervention or sampling plan) The partial rank correlation coefficient (PRCC) sensitivity analysis was done as described by Marino et al. ( ). The analysis of the outputs was performed in R version 3.6.1 with the function PRCC from the package “sensitivity.” The sensitivity analysis was performed in a system where interventions were turned on, the all-intervention system. The model’s sensitivity was used to determine the system’s most influential parameters for final adulterant cells.
The code for the process model and the analysis can be found on GitHub ( https://github.com/foodsafetylab/Farm-to-Consumer-LG-Sim ). Validation documents and a descriptive file with basic instructions on the model files are included in the repository.
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Greening Family Medicine clinic operations and clinical care,
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35ab99bb-2a14-4a67-accf-b19f9b880a34
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10231359
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Family Medicine[mh]
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The climate crisis is a healthcare crisis. Left unabated, worsening environmental health will increase major diseases including cardiovascular disease, respiratory disease, cancer, mental illness, and suffering imposed by extreme weather events. The healthcare sector plays a vital role in mitigating the deleterious impact of rising greenhouse gases. Many healthcare organizations are committing to green policies, setting targets to support environmentally sustainable healthcare. Family physicians, given their central role in community healthcare provision, are strategically placed to lead, support, and promote sustainable healthcare. However, guidance on how to do this is fragmented and busy family physicians, while supportive of this culture shift in theory, are often unclear how to implement change in their clinical practice. A collated evidence-based resource to guide practice change could empower family physicians to implement sustainable planetary health practices at a community level, with cumulative national and global impact. Healthcare contributes globally to greenhouse gas production, an estimated 4%–7% of total national emissions in developed countries. Eminent professional healthcare organizations have called for action, including the World Health Organisation, the American Medical Association, the Canadian Association of Physicians for the Environment, and the National Health System, United Kingdom. , , The specific role of primary care is widely advocated in supporting sustainable healthcare practices, endorsed by family medicine societies including WONCA (Global Family Doctors) and multiple associations from Canada, Australia, New Zealand, Scotland, and the United Kingdom. , , , Organizations recognize the central position of family physicians to support planetary health directly in clinical patient care and indirectly through operations, procedures, and processes in their clinics. In clinical care, e.g. family physicians can avoid prescribing medications which contribute to greenhouse gas generation and advise on lifestyles that support “co-benefits” for patient and planet such as plant-based diets. As small businesses, family physicians can green their clinical operations by adopting circular economic procurement practices and sourcing locally where possible. There is a willingness by family physicians to respond to these calls to action and integrate change by “greening” their practice through clinic operations or patient care. Several grassroots organizations have developed aids to assist community-based family doctors (general practitioners) in improving their clinic operations to reduce their environmental footprint and support changes in clinical care that prioritize the health of the patient while acknowledging and prioritizing the health of the planet. , The aids that have been developed range from solely educational materials and commentaries to very advanced, multilayered, intervention strategies with corresponding tools to support the strategies. To date, as far as we are aware, these aids have not been collated or curated. Family doctors, challenged by time and resources across most countries, need aids readily available. The purpose of this study was to identify and evaluate toolkits and aids on sustainable healthcare to act as a resource for family physicians interested in delivering evidence-based sustainable healthcare in their clinical practices.
Our study consisted of 2 steps: first, we conducted a scoping review , of published and grey literature to identify toolkits and aids, and secondly, we evaluated the toolkits identified. Scoping reviews support an exploration of the breadth and depth of the literature and identify knowledge gaps for a specific subject. The structure of the scoping review allowed for investigation of the breadth of the literature, to include aids, singular projects, tools, toolkits, or educational interventions in family medicine that could be considered for replication by a family doctor and their clinical team, to integrate clinic greening policies in their setting. This review follows the Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols—extension for Scoping Reviews. The protocol is available on the Open Science Framework, osf.io/xp4t3. Inclusion and exclusion criteria We established a priori criteria for inclusion and exclusion of published studies that address environmental mitigation through clinic operations and clinical care in family medicine . We defined “clinic operations” as processes involved in the actual running of a clinic, such as energy use, procurement of equipment, recycling and waste handling, and utility use. “Clinical care” referred to the interaction between doctor and patient, the assessment and management of health concerns. “Toolkits” were defined as collections of adaptable documents to inform and facilitate implementation of evidence-based clinical interventions, in contrast to articles describing a single aid, such as a checklist or single intervention. Empirical research studies (qualitative, quantitative, mixed methods), thesis, and abstracts and conference proceedings, published in English between 1990 and June 2022, were included. Commentaries or opinion pieces which did not include a tool were excluded. Some topics, which could support sustainable healthcare practices were excluded if articles lacked a direct link with environmental outcomes (antibiotic stewardship and deprescribing), and/or where the literature on the topic is vast and extrapolating data was beyond the scope of the research team (all). On this basis we excluded: antibiotic stewardship, deprescribing, green-prescribing (prescribing of nature-based health interventions), and advice on plant protein-based diets. We also excluded studies focussing on building energy analysis as that was beyond our qualifications as a team. Search strategy We searched 4 databases (PubMed, Embase, Scopus, and CINAHL) and 2 search engines (Google Scholar and Google). We completed a preliminary search on Google Scholar to identify search terms, worked with a medical librarian (Diane Lorenzetti) to develop a search string, which was then piloted and refined. The search strategies for each database and the search engine can be found in . Article tracking, review, and extraction were completed on Covidence (covidence.org). Duplicates were removed. Screening and full article review were completed by 2 or more independent reviewers (Sonja Wicklum, Kate Nuique, Jessica Zhang, and Colleen Nesbitt) with all disagreements concluded by a third reviewer after discussion. Manual searching on organizational websites about sustainable healthcare was also completed. Data extraction Data extraction was completed on Covidence by independent reviewers (Sonja Wicklum, Kate Nuique, Jessica Zhang, and Colleen Nesbitt). Data extraction included author, title, publication source and date, target audience of article or aid, presence of educational materials (toolkits only), aim and description of articles presenting a singular tool/aid/project, and methods and results for articles describing interventions. We were unable to locate 1 article, despite assistance from our institutional librarian. Toolkit evaluation All toolkits were evaluated separately. We acknowledge that assessment of article quality or risk of bias is not included in routine scoping reviews. In this circumstance, as our objective was to support family doctors integrating environmentally sustainable clinical care and operations, we deemed it important to provide basic evaluative elements for the reader. To the authors’ knowledge there is no formal critical appraisal tool for toolkits, therefore the evaluation was based on work by Yamada et al., a systematic review of the effectiveness of toolkits as knowledge translation strategies. As per Yamada et al., we applied the following appraisal criteria: (i) clear description of purpose; (ii) evidence-based elements; (iii) detailed implementation process; and (iv) rigorous evaluation plan combining outcome and process measures. In addition, we added a criterion to indicate if strategies to adapt to a community-based family practice setting were included.
We established a priori criteria for inclusion and exclusion of published studies that address environmental mitigation through clinic operations and clinical care in family medicine . We defined “clinic operations” as processes involved in the actual running of a clinic, such as energy use, procurement of equipment, recycling and waste handling, and utility use. “Clinical care” referred to the interaction between doctor and patient, the assessment and management of health concerns. “Toolkits” were defined as collections of adaptable documents to inform and facilitate implementation of evidence-based clinical interventions, in contrast to articles describing a single aid, such as a checklist or single intervention. Empirical research studies (qualitative, quantitative, mixed methods), thesis, and abstracts and conference proceedings, published in English between 1990 and June 2022, were included. Commentaries or opinion pieces which did not include a tool were excluded. Some topics, which could support sustainable healthcare practices were excluded if articles lacked a direct link with environmental outcomes (antibiotic stewardship and deprescribing), and/or where the literature on the topic is vast and extrapolating data was beyond the scope of the research team (all). On this basis we excluded: antibiotic stewardship, deprescribing, green-prescribing (prescribing of nature-based health interventions), and advice on plant protein-based diets. We also excluded studies focussing on building energy analysis as that was beyond our qualifications as a team.
We searched 4 databases (PubMed, Embase, Scopus, and CINAHL) and 2 search engines (Google Scholar and Google). We completed a preliminary search on Google Scholar to identify search terms, worked with a medical librarian (Diane Lorenzetti) to develop a search string, which was then piloted and refined. The search strategies for each database and the search engine can be found in . Article tracking, review, and extraction were completed on Covidence (covidence.org). Duplicates were removed. Screening and full article review were completed by 2 or more independent reviewers (Sonja Wicklum, Kate Nuique, Jessica Zhang, and Colleen Nesbitt) with all disagreements concluded by a third reviewer after discussion. Manual searching on organizational websites about sustainable healthcare was also completed.
Data extraction was completed on Covidence by independent reviewers (Sonja Wicklum, Kate Nuique, Jessica Zhang, and Colleen Nesbitt). Data extraction included author, title, publication source and date, target audience of article or aid, presence of educational materials (toolkits only), aim and description of articles presenting a singular tool/aid/project, and methods and results for articles describing interventions. We were unable to locate 1 article, despite assistance from our institutional librarian.
All toolkits were evaluated separately. We acknowledge that assessment of article quality or risk of bias is not included in routine scoping reviews. In this circumstance, as our objective was to support family doctors integrating environmentally sustainable clinical care and operations, we deemed it important to provide basic evaluative elements for the reader. To the authors’ knowledge there is no formal critical appraisal tool for toolkits, therefore the evaluation was based on work by Yamada et al., a systematic review of the effectiveness of toolkits as knowledge translation strategies. As per Yamada et al., we applied the following appraisal criteria: (i) clear description of purpose; (ii) evidence-based elements; (iii) detailed implementation process; and (iv) rigorous evaluation plan combining outcome and process measures. In addition, we added a criterion to indicate if strategies to adapt to a community-based family practice setting were included.
Our database search identified 17,751 articles after duplicates were removed. When assessing full texts for eligibility 4 articles were commentaries directing the reader to toolkits. These articles were not included in our review but the 4 toolkits they identified were. Complete screening of eligibility resulted in 20 relevant published articles that include an aid and 11 toolkits . Most of the articles and toolkits originate in the Western hemisphere and Europe: 7 from the United Kingdom, 8 from the United States, 4 from Canada, and 1 each from the Americas, Brazil, and Switzerland. Only 1 article came from another continent (Africa). Many articles and toolkits focus on clinical operations, covering a broad scope of topics, as overviewed in . The clinical care areas that were addressed included active transportation, , , appropriate prescribing, , social prescribing, , , mercury and lead as environmental contaminants, respiratory diseases, the value of connection of care providers to patients through community gardens, and medication disposal education. , , , , We start by describing the articles found in the scoping review and then present toolkits identified and their appraisal. Articles Of the 20 articles found, 14 describe a single aid and 6 describe an intervention . Articles describing a single aid Of the 14 articles, 7 focussed on clinic operations, 4 clinical care, and 3 addressed both areas. Most of articles reported simple checklists. , , , Three articles focussed on carbon footprint analyses. , , This included demonstrating the environmental value of e-learning by reducing participants’ use of physical transport, the potential benefit of local GP follow-up over more distant hospital-based care and the variability of the carbon footprint in differing GP offices. One book chapter was included given its relevance to rural practitioners based in small-sized, rural hospitals. It introduced an approach to management called “demand-side management,” which advocated responding to a clearly defined need e.g. closing the clinic when not needed, or building a single room only when required. Articles describing interventions to promote sustainable healthcare Six articles described interventions to promote sustainable healthcare , 4 of which described educational interventions. , , , Educational interventions ranged from a large scale massive open online course (MOOC) to smaller educational initiatives and exploratory studies. , The MOOC reached many individuals—midway through that project, the scope changed from practitioners to the public due to high demand. The MOOC included “action plan development” as part of the course, supporting implementation of change. Other interventions evaluated the impact of “action plans” of a primary care and health promotion partnership ; the benefit of a medical clinic establishing a community garden and exploring GP awareness and promotion of active transport, such as walking or biking to complete daily activities in place of motorized transport. No study evaluated the implementation of integrating sustainable healthcare into practice. Toolkits We identified 11 toolkits . Three toolkits, Practice Greenhealth, The Smart Hospital Toolkit, and The GHG + H 2 O Green Facility Toolkit are primarily hospital focussed but include information about small- and medium-sized facilities and were included as they may inform rural family practices that operate in clinics attached to small-sized healthcare facilities. The scope of material covered by toolkits ranged from several topic areas (Green Impact for Health and My Green Doctor) , to those which focussed on 1 specific area, such as The Greener Practice Asthma Toolkit and The Greener Respiratory Pathway. , All toolkits provided basic knowledge and explained terms commonly used in climate change, planetary health, or sustainable healthcare. Most tools included educational material for healthcare providers’ clinic staff, and 7 provided resources for patients . The focus of most toolkits was on clinical operations, spanning procurement of office supplies, energy use, and waste and recycling . Clinical care topics were less commonly addressed and included prescribing and medication disposal. Three toolkits focussed on medical education, one describes medical curriculum and training program opportunities, a second requires trainees, member doctors, and/or members of the team to complete and submit proof of courses/training, and 1 included QI projects that trainees could implement. Toolkit appraisal In relation to critical appraisal toolkits that include implementation and adaptation processes, and discussion around barriers are considered most effective. Two toolkits made suggestions for how to implement change. My Green Doctor recommended easing into change by adding 5 min to medical staff meetings until interest and skills increased sufficiently for greening projects to be undertaken. In contrast, Green Impact for Health presented a suite of topics, the clinic can choose a topic and identify the degree of change to target. However, participation is limited to doctors practicing in the United Kingdom (nonresidents can visit and utilize tools but cannot track or report). My Green Doctor, Green Impact for Health, Practice Greenhealth, and the Smart Hospitals Toolkit recommend monitoring and reporting on a number of items including energy, water, waste, and cost-savings, and provide tiered options that allow a clinic to adapt the approach best suited for their unique situation. Only the Green Impact for Health Toolkit has robust tracking, evaluating, and reporting information. To date, an estimated 37,700 kg of CO 2 has been saved through the combined efforts of participating practices. At the time of writing, My Green Doctor and Green Impact for Health have large numbers of practices enrolled, and Practice Greenhealth, which focusses on hospital-based changes but supports Community Health Centers, required login and we were unable to obtain detailed information beyond that listed in , , and . Overall, there was a lack of evaluation of toolkit efficacy in terms of benefits or outcomes. All toolkits are freely available but the functionality of some is limited by geographical location, or the inability to track progress. Few toolkits addressed barriers to implementing change. Only 5 toolkits , , , , are updated regularly.
Of the 20 articles found, 14 describe a single aid and 6 describe an intervention . Articles describing a single aid Of the 14 articles, 7 focussed on clinic operations, 4 clinical care, and 3 addressed both areas. Most of articles reported simple checklists. , , , Three articles focussed on carbon footprint analyses. , , This included demonstrating the environmental value of e-learning by reducing participants’ use of physical transport, the potential benefit of local GP follow-up over more distant hospital-based care and the variability of the carbon footprint in differing GP offices. One book chapter was included given its relevance to rural practitioners based in small-sized, rural hospitals. It introduced an approach to management called “demand-side management,” which advocated responding to a clearly defined need e.g. closing the clinic when not needed, or building a single room only when required. Articles describing interventions to promote sustainable healthcare Six articles described interventions to promote sustainable healthcare , 4 of which described educational interventions. , , , Educational interventions ranged from a large scale massive open online course (MOOC) to smaller educational initiatives and exploratory studies. , The MOOC reached many individuals—midway through that project, the scope changed from practitioners to the public due to high demand. The MOOC included “action plan development” as part of the course, supporting implementation of change. Other interventions evaluated the impact of “action plans” of a primary care and health promotion partnership ; the benefit of a medical clinic establishing a community garden and exploring GP awareness and promotion of active transport, such as walking or biking to complete daily activities in place of motorized transport. No study evaluated the implementation of integrating sustainable healthcare into practice.
Of the 14 articles, 7 focussed on clinic operations, 4 clinical care, and 3 addressed both areas. Most of articles reported simple checklists. , , , Three articles focussed on carbon footprint analyses. , , This included demonstrating the environmental value of e-learning by reducing participants’ use of physical transport, the potential benefit of local GP follow-up over more distant hospital-based care and the variability of the carbon footprint in differing GP offices. One book chapter was included given its relevance to rural practitioners based in small-sized, rural hospitals. It introduced an approach to management called “demand-side management,” which advocated responding to a clearly defined need e.g. closing the clinic when not needed, or building a single room only when required.
Six articles described interventions to promote sustainable healthcare , 4 of which described educational interventions. , , , Educational interventions ranged from a large scale massive open online course (MOOC) to smaller educational initiatives and exploratory studies. , The MOOC reached many individuals—midway through that project, the scope changed from practitioners to the public due to high demand. The MOOC included “action plan development” as part of the course, supporting implementation of change. Other interventions evaluated the impact of “action plans” of a primary care and health promotion partnership ; the benefit of a medical clinic establishing a community garden and exploring GP awareness and promotion of active transport, such as walking or biking to complete daily activities in place of motorized transport. No study evaluated the implementation of integrating sustainable healthcare into practice.
We identified 11 toolkits . Three toolkits, Practice Greenhealth, The Smart Hospital Toolkit, and The GHG + H 2 O Green Facility Toolkit are primarily hospital focussed but include information about small- and medium-sized facilities and were included as they may inform rural family practices that operate in clinics attached to small-sized healthcare facilities. The scope of material covered by toolkits ranged from several topic areas (Green Impact for Health and My Green Doctor) , to those which focussed on 1 specific area, such as The Greener Practice Asthma Toolkit and The Greener Respiratory Pathway. , All toolkits provided basic knowledge and explained terms commonly used in climate change, planetary health, or sustainable healthcare. Most tools included educational material for healthcare providers’ clinic staff, and 7 provided resources for patients . The focus of most toolkits was on clinical operations, spanning procurement of office supplies, energy use, and waste and recycling . Clinical care topics were less commonly addressed and included prescribing and medication disposal. Three toolkits focussed on medical education, one describes medical curriculum and training program opportunities, a second requires trainees, member doctors, and/or members of the team to complete and submit proof of courses/training, and 1 included QI projects that trainees could implement. Toolkit appraisal In relation to critical appraisal toolkits that include implementation and adaptation processes, and discussion around barriers are considered most effective. Two toolkits made suggestions for how to implement change. My Green Doctor recommended easing into change by adding 5 min to medical staff meetings until interest and skills increased sufficiently for greening projects to be undertaken. In contrast, Green Impact for Health presented a suite of topics, the clinic can choose a topic and identify the degree of change to target. However, participation is limited to doctors practicing in the United Kingdom (nonresidents can visit and utilize tools but cannot track or report). My Green Doctor, Green Impact for Health, Practice Greenhealth, and the Smart Hospitals Toolkit recommend monitoring and reporting on a number of items including energy, water, waste, and cost-savings, and provide tiered options that allow a clinic to adapt the approach best suited for their unique situation. Only the Green Impact for Health Toolkit has robust tracking, evaluating, and reporting information. To date, an estimated 37,700 kg of CO 2 has been saved through the combined efforts of participating practices. At the time of writing, My Green Doctor and Green Impact for Health have large numbers of practices enrolled, and Practice Greenhealth, which focusses on hospital-based changes but supports Community Health Centers, required login and we were unable to obtain detailed information beyond that listed in , , and . Overall, there was a lack of evaluation of toolkit efficacy in terms of benefits or outcomes. All toolkits are freely available but the functionality of some is limited by geographical location, or the inability to track progress. Few toolkits addressed barriers to implementing change. Only 5 toolkits , , , , are updated regularly.
In relation to critical appraisal toolkits that include implementation and adaptation processes, and discussion around barriers are considered most effective. Two toolkits made suggestions for how to implement change. My Green Doctor recommended easing into change by adding 5 min to medical staff meetings until interest and skills increased sufficiently for greening projects to be undertaken. In contrast, Green Impact for Health presented a suite of topics, the clinic can choose a topic and identify the degree of change to target. However, participation is limited to doctors practicing in the United Kingdom (nonresidents can visit and utilize tools but cannot track or report). My Green Doctor, Green Impact for Health, Practice Greenhealth, and the Smart Hospitals Toolkit recommend monitoring and reporting on a number of items including energy, water, waste, and cost-savings, and provide tiered options that allow a clinic to adapt the approach best suited for their unique situation. Only the Green Impact for Health Toolkit has robust tracking, evaluating, and reporting information. To date, an estimated 37,700 kg of CO 2 has been saved through the combined efforts of participating practices. At the time of writing, My Green Doctor and Green Impact for Health have large numbers of practices enrolled, and Practice Greenhealth, which focusses on hospital-based changes but supports Community Health Centers, required login and we were unable to obtain detailed information beyond that listed in , , and . Overall, there was a lack of evaluation of toolkit efficacy in terms of benefits or outcomes. All toolkits are freely available but the functionality of some is limited by geographical location, or the inability to track progress. Few toolkits addressed barriers to implementing change. Only 5 toolkits , , , , are updated regularly.
Family doctors experience climate anxiety, as do their patients, and many want to change their personal behaviours at home and work, and support their patients to do the same, but are unsure of their role in addressing the topic. , This scoping review was developed to support family doctors in greening their clinical care and operations, map the literature, evaluate the toolkits and aids, and identify gaps. The literature identified covered a wide range of topics, with more emphasis placed on clinical operations than direct patient care. Resources ranged from singular tools (typically checklists) to toolkits. The singular tools that were checklists, or singular projects covering 1 topic, generally lacked information about implementation and did not explore barriers. Though lacking in detail and breadth of addressing the issues, they may prove useful for clinics with limited staff interest or resources (time or financial), or they may serve as an introduction to the concepts of sustainable healthcare that does not seem overwhelming, but rather can feel like an encouraging first step. Toolkits also varied in their scope; some focussed on specific areas such as respiratory disease, while others (Green Impact for Health, My Green Doctor ) provided in-depth and comprehensive information across a range of healthcare and environmental sustainability topics. All the toolkits identified were free to use and accessible online, and their curation herein as a single resource we anticipate being beneficial for family physicians ready to transition to environmentally sustainable clinical care but without the time to identify resources to hand. Family physicians are well positioned to leverage trusting patient relationships to both educate and turn concern into action. However, our review also identified some shortcomings in the literature. Few of the articles or toolkits described provided guidance on how to implement change and address barriers to implementation. Another gap in the literature is around capacity for estimating carbon foot printing of various processes and interventions in healthcare. Carbon footprinting was introduced as an aid in several of the articles and toolkits, although we found no studies that looked at the influence of carbon footprinting on behaviour in the family practice setting, or how this intersects with larger governmental initiatives to reduce carbon footprints and meet international emission commitments. Identifying and quantifying carbon “hotspots” in healthcare and how they intersect with providing care in the context of cost effectiveness and social resources is complex, but necessary to understand the compromises that may need to be discussed to make our healthcare systems viable and sustainable. These studies support the notion that family doctors in community can have a significant impact on the carbon footprint of healthcare but have not yet been proven; this is a fruitful topic for future inquiry. Significantly, none of the resources (aids or toolkits) identified in our review have been evaluated for implementation or cost effectiveness, and we suggest integrating a robust evaluation process that includes environmental, social, and economic impacts (the “triple bottom line”) as balanced against outcomes for patients and populations to determine the sustainable value of implementation strategies. Limitations Despite our best attempts to develop a robust search strategy, it is possible that we may have overlooked or omitted articles or toolkits, and this challenge is compounded by the wide range of terms used in relation to planetary health. Also, although we searched the grey literature it is possible that we failed to identify resources. Our review was limited to the English language and consequently the resources we found predominantly originated in Western countries, and it is possible that additional resources in different languages exist, particularly given the geographical and local health system focus of several of the resources we found. To complement our search, we contacted leading authors in the field for further suggestions of resources we may not have found and to discuss our findings.
Despite our best attempts to develop a robust search strategy, it is possible that we may have overlooked or omitted articles or toolkits, and this challenge is compounded by the wide range of terms used in relation to planetary health. Also, although we searched the grey literature it is possible that we failed to identify resources. Our review was limited to the English language and consequently the resources we found predominantly originated in Western countries, and it is possible that additional resources in different languages exist, particularly given the geographical and local health system focus of several of the resources we found. To complement our search, we contacted leading authors in the field for further suggestions of resources we may not have found and to discuss our findings.
There have been several commentaries and “calls to action” to family doctors and primary care to make changes to their clinical operations and clinical care. , Our scoping review highlights the beginnings of educational initiatives and interventions, and the availability of toolkits to support family doctors, clinic managers, and staff to implement evidence-based changes in both their clinic operations and clinical care. Despite knowledge and best intentions, doctors struggle to change practice behaviour. , , Toolkits that support change are a step in the right direction. Though limited, excellent toolkits exist to support family doctors and their clinics to provide more environmentally conscious and sustainable healthcare.
cmad006_suppl_Supplementary_Table_A Click here for additional data file. cmad006_suppl_Supplementary_Checklist Click here for additional data file.
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Tackling climate change and health inequalities in primary care
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33b2e868-c32c-4e9d-be26-2003bad72294
|
10231381
|
Family Medicine[mh]
|
The Climate Emergency is now widely accepted as the biggest public health crisis facing humanity. Previous research has highlighted how social and health inequalities shape the health impacts of climate change in the United Kingdom, but there has been little attention to the role of general practice in deprived areas. Scotland’s Deep End GP practices are practices with the highest percentage of registered patients living in the most socioeconomically deprived postcodes—the majority (roughly 70%) of these practices are based in the city of Glasgow. Since 2009, the Scottish Deep End Project has been a collaboration between frontline GPs and academics working and advocating to improve the volume and quality of primary care in areas of socioeconomic disadvantage. In November 2021, world politicians convened in Glasgow for the 26th United Nations Climate Change Conference (COP26). In anticipation, a group of 10 GP colleagues from a variety of Deep End settings contributed to an online roundtable event to explore climate change in the context of health inequalities. Discussion centred on the various factors that influence Deep End patients and practices in particular, but also explored the urgent need for system-wide solutions to tackle this burden. The full report is available on the Deep End website ; what follows is a brief summary.
The discussion opened with a sense from many participants that GPs are “not experts in climate change” but that there was an urgent need for climate justice and action to future proof and protect the lives of our patients and the services we deliver. Participants reflected that while GPs may lack specific expertise on climate science, in the words of Henry Sigerist, GPs and their teams “ well know the factors that paralyse all their efforts . They are not only scientists but also responsible citizens, and if they did not raise their voices, who else should? ” The group recognized that when our patients are malnourished because of damaging food systems, or when patients struggle with weight, inactivity, and chronic respiratory and cardiovascular conditions because of unhealthy transport systems and environments, or when stressed patients use cigarettes, alcohol, drugs, and violence to cope with economic and social injustices, these are the drivers of climate change intersecting within the communities we serve. Participants expressed the hope that collective action could facilitate shifts to more sustainable approaches and models of care. It was recognized, however, that the most effective drivers of change are upstream and that we need to “ apply pressure at a structural and political level ” to move from “ an unequal, sickness economy ” of unsustainable growth to an economy guided by a compass where every individual enjoys just social foundations, yet humanity collectively operates within viable planetary boundaries. As we recover from a global pandemic, this shift in thinking will help facilitate a transition to a healthier, fairer, greener, safer future.
Despite the urgent need to act, participants recognized that meaningful progress can only be achieved with genuine inclusivity and collectivism, as a radical shift to policies with a climate action focus could attract negative views. Traditional societal markers of “success” have for generations included car ownership, overseas holidays, and numerous gifts for children—advantages that have been disproportionately enjoyed by the most affluent sections of society. One participant raised the point that globally “ the richest 10% produce half of lifestyle carbon emissions, while the poorest 50% produce only a tenth .” Shifting cultural norms and expectations will not be easy. In the health sector, there was agreement that improved knowledge sharing and shared decision tools could help staff and patients to engage in a patient-centred approach for greener, lower carbon health interventions. Primary care networks (GP clusters in Scotland) could build on the potential of community-oriented primary care teams to engage with schools, families, and communities to improve public health at a local level. Collaborative and inclusive decision-making with patients was felt to be pivotal to any successful local neighbourhood and system-wide changes. For individual practices and practitioners, participants proposed standing in solidarity with patients, amplifying their voices on issues that affect them, and ensuring that change is introduced with humility, sensitivity, and a sense of collective action and trust. Practical examples included improved insulation in housing to reduce fuel poverty, improved active travel options, improved access to green spaces, improved access to technology to facilitate access to health care, and other essential services.
At its core, sustainable general practice is based on preventative medicine. The principles of “realistic medicine,” outlined in the Chief Medical Officer for Scotland’s annual reports since 2016, include reducing health care-generated waste (e.g. carbon contributions), addressing unwarranted variation in practice and outcomes, and building a more personalized approach to care with a focus on shared decision-making. Participants supported the wider rollout of resources such as the RCGP Greener Impact for Health toolkit, which align with these principles. The shift in focus to sustainability and wellbeing at Scottish Government and Local Authority levels was welcomed. It was recognized that both in Scotland and UK-wide, there is higher air pollution in more deprived areas, despite lower levels of vehicle ownership. Participants agreed that joined up working with councils and local grassroots groups could improve the safety of pedestrianized and cycling networks surrounding GP practices, encouraging active travel amongst staff and patients. It was also recognized that increased advocacy of active travel, equitable access to green space, and nonpharmacological methods of improving health and wellbeing by primary care teams empowers patients toward better health, while reducing the collective climate footprint. UK appointment length differs from comparable high income health systems. Participants favoured the trend for 15-min GP appointments as a minimum standard, against the UK norm of 10 min, to enhance opportunities for social prescribing and work with embedded support workers, to prioritize social wellbeing, financial wellbeing, mental health and personalized, holistic care.
It was recognized that prescribing has the single largest carbon impact in general practice. As well as promoting nonpharmacological approaches to health and wellbeing, participants agreed on the need for more rational prescribing, and deprescribing where appropriate. In particular, it was noted that inhaler prescriptions alone account for approximately 13% of the carbon footprint of General Practice. Careful work to switch from carbon-intense Metered Dose Inhalers (MDIs) to Dry Powder Inhaler (DPI) alternatives is already being carried out in practices, but it was agreed that this work requires consistent support from health boards and widespread dissemination of helpful resources. , There was an accompanying call for action at formulary level for the carbon impact of medicines to be considered and for sustainable prescribing to be promoted via formularies and toolkits with the carbon cost of medications outlined, sharing examples of good practice. Similarly, the use of well-designed decision aids , could help both patients and clinicians to choose more sustainable medications across a range of disease areas, in line with “realistic medicine” principles.
The use of digital technology and telemonitoring by health care teams provides opportunities to reduce patient and staff travel and associated carbon footprint. However, participants cautioned that decision makers be mindful of unintended widening of health inequalities. Ongoing evaluation of the inequalities impact of remote consulting is needed. ,
Participants agreed that sustainability should be part of teaching and training at undergraduate and postgraduate levels. Climate change and health (and health inequalities) should be integrated in sustainable health care themes across curricula. Participants were supportive of initiatives such as the “Planetary Health Report Card,” which compares medical school teaching of planetary health, with recommendations for change at the institutional level. Other opportunities for Quality Improvement (QI) projects with a sustainability focus should be encouraged at both undergraduate (e.g. during “Student Selected Components” [SSCs]) and postgraduate levels, with improved mechanisms for sharing QI project ideas across practices and clusters/networks. Supported education time and posts for sustainability champions at different career stages (medical students, trainees, qualified GPs, those nearing retirement) would enable this.
It was widely recognized that NHS estates will need to be retrofitted and rebuilt, and waste streams will need optimization to effect change at local board level and nationwide. While a degree of independence in estate management exists amongst GP teams, participants highlighted a significant need for sustainability assessments to be rolled out consistently and collectively, and for skilled teams to examine building energy supplies, insulation and waste management streams as well as carry out detailed data collection. With current unmanageable workloads in primary care, it is unrealistic to expect GP teams to do this work out of goodwill. There is a need for dedicated funding to support sustainable general practice. At present, NHS Health Boards have sustainability governance groups with very little input from general practice. Initiatives such as NHS England’s Healthier Futures Action Fund and RCGP’s Net Zero Primary Care Development programme are welcomed, but further funding for Primary Care Sustainability Leads would help support early adopters and networks of good practice and allow GP input and governance for sustainable change.
The Scottish Deep End Group supports ongoing collaboration between key stakeholders to deliver more sustainable general practice. The linked issues of climate change and health inequalities require proportionate resourcing. Indeed, many existing Deep End projects—practice-attached Community Links Workers Programme and welfare advisors, Govan SHIP and the Pioneer Scheme —all aim to strengthen communities, address unmet patient needs, and free up GP time for more targeted interventions. These initiatives allow GP teams to build stronger ties with local third sector organizations and support community health and wellbeing through nonpharmacological means. Commitments made for NHS Scotland to be “net-zero” by 2040 will only be achieved if general practice is on board, but additional resource is required where needs are greatest, or health inequalities will inevitably widen.
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Antihypertensive Medications Use among Chronic Hemodialysis Patients Visiting the Outpatient Department of Nephrology of a Tertiary Care Centre: A Descriptive Cross-sectional Study
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2e1b97dc-1f10-4b1e-bcfa-f16885349a4f
|
10231535
|
Internal Medicine[mh]
|
Hypertension is one of the leading causes of mortality (>50%) in chronic hemodialysis patients. Antihypertensive medication like calcium channel blockers (CCB-amlodipine), alpha-blockers (prazosin), betablockers (atenolol, bisoprolol, carvedilol, metoprolol), angiotensin-converting enzyme inhibitors (ACEI), angiotensin receptor blockers (ARB), centrally acting alpha-2 agonists (clonidine), diuretics (torsemide), vasodilators, and direct renin inhibitors (aliskiren) are prescribed for the management of high blood pressure. , The practice pattern of anti-hypertensive medication may vary in chronic hemodialysis patients as it can impair the drug's pharmacokinetic properties. The objective of our study was to find out the prevalence of anti-hypertensive medication use among chronic hemodialysis patients visiting the outpatient Department of Nephrology of a tertiary care centre.
This was a descriptive cross-sectional study conducted on chronic hemodialysis patients visiting the Department of Nephrology at Nepal Medical College and Teaching Hospital (NMCTH), Kathmandu, Nepal. The study commenced on 2 April 2022 to 30 September 2022 for 6 months. Ethical approval was taken from the Institutional Review Committee (Reference number: 062-078/079). Patients who visited the Nephrology Department for hemodialysis and who were willing to participate voluntarily in the study were included. Drugs other than anti-hypertensive medication and follow-up patients were also excluded from this study. A convenience sampling method was used. The sample size was calculated by using the following formula: n = Z 2 × p × q e 2 = 1.96 2 × 0.50 × 0.50 0.10 2 = 97 Where, n = minimum required sample size Z = 1.96 at 90% Confidence Interval (CI) p = prevalence taken as 50% for maximum sample size calculation q = 1-p e = margin of error, 10% The calculated minimum required sample size was 97. However, 105 patients were included in the study. Data on the patient's details and anti-hypertensive medication profile were collected from the dialysis unit of the Department of Nephrology by the researcher by interviewing individually through the preformed self-constructed questionnaire. The predesigned questionnaire was used. Anti-hypertensive drugs used for the management of complications during dialysis were also reported. Data were entered and analyzed with IBM SPSS Statistics 16.0. Point estimate and 90% CI were calculated.
The prevalence of anti-hypertensive medication use was 102 (97.14%) (93.95-100, 90% CI) among patients undergoing chronic hemodialysis. There were a total of 60 (58.82%) males and 42 (41.17%) females. The patient's age group was ranging from 21 to 81 years with a mean age of 45.75±14.91 years. The three common antihypertensive medications prescribed for patients on chronic hemodialysis patients were amlodipine 79 (77.45%), torsemide 59 (57.84%), and prazosin 48 (47.05%) . During dialysis, intradialytic hypotension was reported in 4 (3.92%) while intradialytic hypertension was reported in 18 (17.64%) of the patients. Intradialytic hypertension was managed with centrally acting alpha-2 agonists (clonidine) and intradialytic hypotension was managed with crystalloid fluids like normal saline and IV dextrose.
This study found the prevalence of anti-hypertensive medications used in chronic hemodialysis patients was 97.14%. A study conducted in Kolkata reported that the drugs prescribed to the majority of hemodialysis patients were anti-hypertensive medicines (23.41%). Among the anti-hypertensive medicines, diuretics (9.29%), calcium channel blockers (5.92%), and alphablockers (2.91%) were commonly prescribed. In our study, we observed that calcium channel blocker (amlodipine- 77.5%) was frequently prescribed to the patients undergoing hemodialysis followed by diuretics (torsemide- 57.8%) and alpha-blockers (prazosin- 47.1%). Although in the study, diuretics were prescribed the most, other studies have reported calcium channel blockers as the most common drug to be prescribed. , A study reported that CCB has been associated with decreased mortality when compared with alpha-1 blockers, beta-blockers, ACEI, ARB, and nitrates. Calcium channel blockers are mainly eliminated by hepatic metabolism and can usually be administered in standard dosages in patients with severe renal impairment. However, some of the CCBs (nifedipine, verapamil, diltiazem) have active metabolites that may require renal clearance. CCBs have also been found to reduce proteinuria and slow the progression of renal insufficiency independently of their blood pressure lowering effect. , Similarly, a-blocker have also been found to have renoprotective action. In our study, we did not observe any ACE Inhibitors prescribed to our patients. Although ACE inhibitors and ARB slows the progression of renal failure; they were not prescribed for patients with ESRD as these medications can lead to hyperkalemia. The thiazide group of diuretics is less efficacious in a patient with advanced renal disease. The efficacy of furosemide also decreases in a patient with advanced CKD and therefore the dose needs to be increased with a subsequent increase in its adverse effect i.e., ototoxicity. However, torsemide was found to be effective and its pharmacokinetics parameter remains unchanged even in advanced kidney disease. Torsemide was also found to have renoprotective action. Potassium-sparing diuretics should be avoided in patients with eGFR less than 35-45 ml/min due to a marked increase in hyperkalemia. End-stage renal disease (ESRD) patients are susceptible to cardiac failure. Therefore, beta blockers are prescribed for hemodialysis patients. Beta blockers have reduced the risk of cardiovascular disease and favour cardioprotective action among hemodialysis patients. In our study, beta-blockers like metoprolol-25.5%, carvedilol-3.9%, atenolol-2% and bisoprolol-1% were prescribed to hemodialysis patients. Another study observed the use of the majority of beta blockers like carvedilol, metoprolol, atenolol, bisoprolol, and acebutolol in chronic hemodialysis patients. β-Blockers can reduce renal blood flow and adversely affect renal function in patients with severe renal impairment. The dose of β-blockers should be reduced especially if eliminated through the renal route, e.g. atenolol. Renally excreted β-blockers (especially atenolol) may accumulate in ESRD and cause profound bradycardia and subsequent hypotension and collapse. β-blockers like metoprolol are preferred. β-blockers like metoprolol and carvedilol are eliminated by the liver and do not require dose adjustment in renal failure while hydrophilic beta blockers like atenolol and bisoprolol need a dose adjustment. Although hemodialysis is a life-sustaining procedure for end-stage kidney disease (ESKD) patients, there is a tendency for blood pressure (BP) to change frequently during hemodialysis. Two of the complication that occurs during hemodialysis are intra-dialytic hypotension and intra-dialytic hypertension. Intradialytic hypertension as defined by the National Kidney Foundation's Kidney Disease Outcomes Quality Initiative [KDOQI] as a post-dialysis BP >130/80 mmHg). Patients with intradialytic hypertension are at high risk for renal failure and antihypertensive medications should be prescribed. Intradialytic hypertension occurs regularly in 10-15% of hemodialysis patients. In our study, intradialytic hypertension was reported in 18 (17.6%) of the patients. The use of the highly dialysable drug like metoprolol was associated with a high risk of intradialytic hypertension. In our study, metoprolol was used in 26 (25.49%) which could have led to a higher rate of intradialytic hypertension. In our study, intradialytic hypertension was managed with clonidine. Intradialytic hypertension can be prevented by the use of less dialyzable drugs like carvedilol. Clonidine has been found to be effective at reducing systolic blood pressure in hemodialysis patients. Only 5% of clonidine is removed by hemodialysis. Intradialytic hypotension occurs in 10-12% of the patients. An intradialytic systolic BP of less than 90mm Hg is associated with mortality. In our study, we observed intradialytic hypotension was observed in 4 (3.9%) of the patients and was managed with normal saline and IV dextrose. Intradialytic hypotension was more common in those on carvedilol (a poorly dialyzable P-blocker) compared with those on metoprolol (a highly dialysable P-blocker). In our study, the poorly dialysable drug carvedilol was used in 4 (3.92%) which coincides with the number of intradialytic hypotension cases. One of the limitations of this study was that the patient who had come for hemodialysis could not recall the names of the medications that were consumed. Such patients had to be followed up on the next visit to reconfirm the name of the medication from their record file. During the dialysis procedure, a few patients collapsed and were therefore excluded from our study.
The prevalence of antihypertensive medication was found to be higher than in other studies done in similar settings. Anti-hypertensive medications were prescribed for both hypertensive and non-hypertensive patients on chronic hemodialysis which could be due to renoprotection action. We did not observe the use of newer anti-hypertensive medications which could be due to a lack of efficacy and safety in hemodialysis patients. We also would like to suggest the use of an alternative drug to metoprolol as it could be associated with a higher rate of intradialytic hypertension.
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Pregnancy Induced Hypertensive Disorders among Patients Admitted to the Department of Obstetric and Gynecology in a Tertiary Care Centre: A Descriptive Cross-sectional Study
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7d429e81-ca87-4e9b-9469-d7b6f54ed695
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10231536
|
Gynaecology[mh]
|
Hypertensive disorder of pregnancy are among the leading cause of maternal and perinatal mortality in developing countries. Hypertension affects more than 5-8% of all pregnancies in the world. Maternal complications includes acute renal failure, hepatic failure, postpartum haemorrhage, disseminated intravascular coagulation, abruptio, and cerebrovascular accident. Foetal complications includes premature deliveries, intrauterine growth restriction, stillbirth, and neonatal deaths. , Although there are some tests for prediction or early detection of preeclampsia like uterine artery doppler and maternal serum markers, there is not enough evidence to suggest their use in clinical practice more so in poor resource clinical settings. - In Nepal, there are only few studies regarding this topic so this study helps us to improve our management protocol thereby reducing maternal and foetal morbidity and mortality. The aim of this study was to find out the prevalence of pregnancy induced hypertensive disorders among patients admitted to the Department of Obstetrics and Gynaecology in a tertiary care centre. This descriptive cross sectional study was done in the Department of Obstetrics and Gynaecology of a Patan Academy of Health Sciences (PAHS). The duration of study was one year from 30 July 2020 to 30 July 2021. Ethical approval for the study was taken from the Institutional Review Committee of the PAHS (Reference number: 2007211399). All pregnant women admitted in the maternity ward within the study period were included in the study. Patients with chronic hypertension (when hypertension begins before 20 weeks of gestation or exists before pregnancy), chronic hypertension with superimposed preeclampsia and those with pre-existing medical conditions like renal, vascular, systemic lupus erythematosus, thyrotoxicosis were excluded from the study. Convenience sampling method was used. The sample size was calculated using the formula: n = Z 2 × p × q e 2 = 1.96 2 × 0.50 × 0.50 0.02 2 = 1068 Where, n = minimum required sample size Z = 1.96 at 95% Confidence interval (CI) p = prevalence taken as 50% for maximum sample size calculation q = 1-p e = margin of error, 2% The minimum sample size obtained was 2401. Since convenience sampling was used, the sample size was quadrupled which was 4,272. However, 4303 patients were included in the study. According to the American College of Obstetrics and Gynaecology, pregnancy induced hypertension is divided into four groups: gestational hypertension where blood pressure is 140/90 mm Hg or more after 20 th week of gestation without proteinuria, preeclampsia is raised BP with proteinuria, chronic hypertension is that exists before pregnancy or begins in the first 20 weeks of gestation, preeclampsia superimposed on chronic hypertension (chronic hypertension with proteinuria) and eclampsia is preeclampsia with seizures. Data were collected from patient's files regarding maternal age, parity, gestational age, type of hypertensive disorder, type of delivery, maternal complications, and foetal birth weight, preterm, intrauterine foetal death, neonatal death, neonatal intensive care unit, and nursery admission were noted. Data were entered and analysed by using Microsoft Excel 2016. Point estimate and 95% Confidence Interval were calculated. Among 4,303 women, who delivered in our hospital during the study period, the prevalence of hypertensive disorder in pregnancy was 110 (2.55%) (2.08-3.03, 95% CI). In this study, 69 (62.72%) of the patients had preeclampsia, 34 (30.90%) had gestational hypertension and 7 (6.36%) had eclampsia. Among eclampsia, 5 (71.43%) patients had antepartum eclampsia and 2 (28.57%) had postpartum eclampsia . Out of 110 patients with hypertensive disorders of pregnancy 78 (70.90%) patients had caesarean section among which 60 (54.54%) were elective and 18 (16.36%) were emergency caesarean section. There were a total 32 (29.09%) vaginal deliveries. There were 2 (0.90%) patients who had acute renal failure, 1 (50%) of the patients had six cycles of haemodialysis. There were 4 (3.63%) postpartum haemorrhage in which 3 (75%) were managed medically and also received blood products and in 1 (25%) patient bilateral uterine artery ligation was done and condom tamponade was also inserted. There was 1 (0.90%) re-laparotomy following hemoperitoneum. A total of 9 (8.18%) patients were COVID-19 positive. There were 2 (1.81%) mortality, in which first died due to disseminated intravascular coagulation, acute renal failure and pulmonary oedema, second one died due to acute respiratory distress syndrome and pulmonary oedema. Both the patients also had COVID-19 pneumonia. Both died in the postpartum period and both were intubated. One expired on day 7 and the other on day 11 . There were 17 (15.45%) babies who were <1.5 kg. In complication, the majority of the cases were intrauterine growth restriction 49 (44.54%) and prematurity 40 (36.36%). There were 9 (8.18%) intrauterine foetal death . The mean±SD of age was 29.48±4.95 years varying from a minimum of 19 years to maximum of 35 years. There were 55 (50%) patients in either group; primigravida and multigravida group. The mean±SD of gestational age was 35.66±3.47 weeks (minimum= 20 weeks and maximum= 40 weeks) . Hypertensive disorders in pregnancy are considered a major problem affecting both maternal and foetal morbidity and mortality. The prevalence of hypertensive disorder in pregnancy differs worldwide. In Sweden it is 1.5%, 7.5% in Brazil and 6-8% in India. Out of total 4,303 deliveries, 110 deliveries were found to be complicated by hypertensive disorder in pregnancy hence the prevalence was 2.55% in our study. The extremes of age are well known risk factors for hypertension in pregnancy. In our study most of the participants were in the age group of 30 to 35 (48.18%) where as a previously studied showed, the participants were in the age group of 21 to 25 years, and 20 to 30. Another study found that the age above 30 years were associated with risk for preeclampsia. In our study number of primigravida and multigravida was equal 50% whereas in previously study showed 60.8% was primigravida and 39.2% multigravida. Primigravida has 15 times greater risk for developing preeclampsia as compared to multigravida. The most common maternal complications were HELLP, abruptio and oligohydramnios in our study. Similar study also showed HELLP syndrome as common complication followed by abruptio, acute renal failure and postpartum haemorrhage. Another study found a significant association between the occurrence of HELLP syndrome and maternal mortality and morbidity. In our study 5.4% patients had HELLP syndrome. In our study there were 2 (0.90%) maternal mortalities, in which first died due to disseminated intravascular coagulation, acute renal failure and pulmonary oedema, second one died due to acute respiratory distress syndrome and pulmonary oedema. Both the patients were also COVID-19 positive. Whereas as in a previous study also showed had two mortality which were due to intracranial haemorrhage. A study showed the majority of the patients had eclampsia 40.6% whereas in our study most of the patients presented with features of preeclampsia. There were only seven cases of eclampsia in our study. Similar study showed majority of patients had preeclampsia (59.6%). In another study showed gestational hypertension was most common (65.62%), followed by preeclampsia (28.12%) and eclampsia (6.25%). In our study there were 70.90% caesarean section out of which 54.54% were emergency and 18% were elective and 29.09% were vaginal delivery. Similar study showed out of 123 patients 105 (85.2%) had caesarean section, had vaginal delivery and 1 had vaginal birth after caesarean section. whereas in another study 63% had vaginal delivery and 37% had caesarean section. The risk of prematurity with PIH is approximately 25-30%. A study showed IUGR and prematurity as the commonest foetal complications which was similar to our study. In our study 36.36% of the pregnancies ended up in preterm deliveries, majority of which occurred due to maternal and foetal indication for termination of pregnancy. The definite treatment of preeclampsia is termination of pregnancy, which is done despite of risk of prematurity irrespective of the gestational age to avoid maternal complications and morbidity. Another study showed, out of 64 deliveries 18.75% babies required NICU admission for various causes with 1.56% IUFD and 1.56% neonatal death. 19 However our study showed 10% NICU admission, 8.18% IUFD and 4.54% neonatal death. There are some limitations in our study. Since preeclampsia is influenced by race, parity, ethnicity, environmental factors, socio-economic status, obesity, these parameters have not been included in our study so the association could not be explored. A multicentre based larger population study might have a different outcome compared to that of this study. The prevalence of hypertensive disorder among pregnancies was similar to the other studies done in similar settings. Emphasis should be on early registration, regular antenatal visits, identifying the high risk group, starting medication on time, timely referral to higher centres which can prevent and reduce severity and its associated complications. Awareness about hypertension should not just be spread from the hospital but should be from the community level as well. Women should be educated about regular blood pressure monitoring and to do routine urinary protein analysis at every antenatal visit.
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Colostrum Feeding among Newborns Visiting the Outpatient Department of Pediatrics of a Tertiary Care Centre: A Descriptive Cross-sectional Study
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706814a1-2947-40d1-8fbc-cb30d0679c3e
|
10231541
|
Pediatrics[mh]
|
Colostrum is the first milk that is very important for newborns in protecting against infections since it is rich in immunoglobin G. , Various bacterial, viral, fungal and protozoal infections of the neonates can be reduced by feeding colostrum. Optimal breastfeeding practices, reflected by early initiation and feeding of colostrum, avoidance of prelacteal feeds, and continued exclusivity or predominance of breastfeeding, are critical for assuring proper infant nutrition, growth and development. , Despite many global efforts being made, the practices of discarding colostrum and the early use of commercial milk formula are still prevalent. Lack of social support, poverty, disability, and perinatal complications could be some of the reasons for shorter breastfeeding duration and discarding of colostrum. There have different studies that show that children not feeding on colostrum are more at risk of developing infections, stunting underweight and wasting. The objective of this study was to find out the prevalence of colostrum feeding among infants visiting the Department of Pediatrics in a tertiary care centre. A descriptive cross-sectional study was conducted in the Outpatient Department of Pediatric of KIST Medical College and Teaching Hospital, Lalitpur, Nepal after receiving ethical approval from the Institutional Review Committee (Reference number: 2078/079/107). The duration of study was six month from 12 February 2022 to 12 August 2022. Infants less than 6 months of age were enrolled in the study. Mothers of twin/multiple infants were excluded from the study as there could be chances of inadequate breast milk production to fulfill the needs of two infants by a single mother. Apart from these mothers having chronic medical illnesses (active tuberculosis, leprosy, and AIDS) and conditions in which breastfeeding is contraindicated were also excluded. Infants with specific feeding problems (cleft lip or palate, congenital heart disease, severe illness during the neonatal period, or delayed developmental milestones) were also excluded. Convenience sampling was done. The sample size was calculated using the following formula: n = Z 2 × p × q e 2 = 1.96 2 × 0.835 × 0.165 0.04 2 = 331 Where, n = minimum required sample size Z = 1.96 at 95% Confidence Interval (CI) p = prevalence of colostrum feeding taken from the previous study, 83.5% q = 1-p e = margin of error, 4% The calculated minimum required sample size was 331. However, we enrolled 350 infants in our study. A pre-designed questionnaire was administered directly to the participants by the investigator via face-to-face interviews. Those infants that were fed with colostrum (thick yellowish breastmilk) were taken as our required criteria. Questionnaires were created in the Nepali language to optimize proper communication. Investigators read out questions written in the questionnaire and answers were marked by the investigator as per the reply of the participants. After completion of all the questions, all the responses given by the participant himself/herself were read out to the participants to ensure the recorded responses were correct. Data were entered and analyzed using IBM SPSS Statistics version 26.0. Point estimate and 95% Confidence Interval were calculated. Among 350 infants, 305 (87.14%) (83.63-90.65, 95% CI) were fed with colostrum. Out of them, 179 (58.69%) were male . Among mother who fed colostrum, 243 (79.67%) followed Hinduism religion, 42 (13.77%) followed Buddhism . A total of 180 (59.02%) were breastfed within 1 hour of delivery . Around 179 (58.69%) infants were breastfed 8 to 12 times in 24 hours while 7 (2.30%) of the infants were breastfed as per the demand of the infant . The birth weight of 262 (85.9%) infants was between 2500-4000 grams . This study revealed that 87.14% of infants were fed colostrum after birth which was slightly higher (83.5%) than a nation-wide study done in Nepal. In a similar study done in lowlands of Nepal, only 67% of infants were given colostrum. In our study 59.02% of infants were breastfed within one hour of delivery similar to the data from studies done in different parts of Nepal. , , But it is more as compared to some of the nation-based studies. , In a similar study done in an urban population of Western Nepal, Midwestern Nepal and Eastern Nepal it was 72.7% and 67.2% respectively, which is more as compared to our study. , Among our study population, the majority of infants were breastfed 8-12 times a day 179 (58.69%) while 76 (24.92%) of them were breastfed 4-8 times per day, 31 (10.16%) breastfed >12 times a day and only 12 (3.93%) breastfed 4 times per day. In one breastfeeding practice study among US mothers, the average frequency of breastfeeding was 8 times per day at 1 month of age which decreased with an increase in age to 3.5 times per day at 1 year of age. In another study conducted in Iran, the frequency of breastfeeding was observed to be higher than the developing countries. Similar studies conducted in a rural area of Bangladesh revealed the greater frequency of breastfeeding among housewives compared to working mothers. In our study, 37 (12.13%) of the infants weighed below 2500 grams at birth. According to a study done in Africa, neonates with low birth weight (<2.5 kg) were more likely to not begin breastfeeding on demand than full-weight neonates. With the introduction of commercial milk formula in this market-driven world, early cessation of breastfeeding and discarding colostrum is still prevalent. So, necessary efforts should be made in explaining to the mothers and families the importance of breastfeeding and colostrum. Studies have suggested that maternal education, working status, infant's birth order, birth weight, maturity, antenatal counselling, and mode of delivery influences the practice of breastfeeding. As we conducted a descriptive cross-sectional study in a single tertiary care centre, our result might not be generalized in other settings and is different as compared to other studies conducted in different demographic regions of Nepal. Also, inferential statistics like association, and correlation could not be established from this study due to the limitation of the study design. The prevalence of colostrum feeding among newborns was higher as compared to other studies done in similar settings. However, proper counselling should be given to mothers and families regarding the importance of colostrum and breastmilk.
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Hysteroscopy among Patients Attending the Outpatient Department of Gynaecology in a Tertiary Care Centre: A Descriptive Cross-sectional Study
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bcfe0da8-ebc6-4fa9-8eb3-3e9ab61ebf36
|
10231550
|
Gynaecology[mh]
|
Hysteroscopy is a diagnostic and operative procedure used worldwide for intrauterine pathology. Gradually over the years, it has become a tool of choice and it is replacing dilatation and curettage in treating conditions like abnormal uterine bleeding. , Hysteroscopy enables visualisation of the endometrial cavity and if possible treatment in the same setting avoiding the need for an invasive procedure. It helps in the complete visualization of endometrium revealing normal and abnormal intrauterine pathology. Even cases of missing Intrauterine Contraceptive devices (IUCD), synechiae removal and infertility can be tackled with ease. The objective of the study was to find out the prevalence of hysteroscopy among gynaecological patients attending the outpatient department of Obstetrics and Gynaecology in a tertiary care centre. A descriptive cross-sectional study was done for a duration of five years from 1 January 2016 to 1 January 2020 among gynaecological patients visiting the outpatient Department of Obstetrics and Gynaecology of Grande International Hospital (GIH), Dhapasi, Kathmandu, Nepal after taking ethical approval from the Institutional Review Committee of GIH (Registration number: 029/2021). All patients visiting outpatients Department of Gynaecology during study period were included in the study. Patients with irregular menstrual cycles and with continuous per vaginal bleeding were excluded. Convenience sampling was used. The sample size was calculated using the following formula: n = Z 2 × p × q e 2 = 1.96 2 × 0.5 × 0.5 0.06 2 = 267 Where, n = minimum required sample size Z = 1.96 at 95% of Confidence Interval (CI) p = prevalence taken as 50% for maximum sample size q= 1-p e = margin of error, 6% The calculated sample size was 267. However, a total of 319 patients were included. Each of the patients were given 400 mcg misoprostol per vaginally four hours prior to the procedure. The surgical instrument used was a rigid storz hysteroscope with 0° or 30° lenses. Cutting loop, coagulating electrode, biopsy forceps and scissors were commonly used. The media used was normal saline. All examinations were performed by a single gynaecologist with specialized training in gynaecological endoscopy experience of more than 15 years. The standard sequencing of visualization of the endometrial cavity was maintained. The order followed was a visualisation of the ectocervix, endocervical canal, walls of the uterus, uterine fundus, Ostia and finally the endometrial characteristics. Hysteroscopic findings were thus documented and video-recorded. As per need, diagnostic and therapeutic management was done. Obtained tissue was sent for histopathological examination. Demographic parameters of the patients like age, parity, abortion, menopause, symptoms, and diagnosis along with hysteroscopic and histopathological findings and procedures performed were retrieved from the electronic database maintained in the hospital record. Care was taken to maintain patient confidentiality. Data were analysed using IBM SPSS Statistics version 22.0. Point estimate and 95% CI were calculated. Among 319 gynaecological patients, hysteroscopy was done in 72 (22.57%) (17.98-27.16, 95% CI) patients. From them, 60 (83.33%) of those patients had endometrial tissue samples sent for histopathology. Among the remaining 12 cases, 5 (41.67%) patients had synechiae release, 3 (25%) had septum resections, 3 (25%) had IUCD removal and 1 (8.33%) had foreign body removal. A total of 33 (45.83%) patients were in the age group of 31-40 . Excessive vaginal bleeding during menstruation was seen in 28 (38.88%) as common presenting complaint followed by inability to conceive 26 (36.11%) . The commonest diagnosis at presentation was subfertility 16 (22.22%) followed by endometrial polyp 15 (20.83%) with both diagnoses together present in 5 (6.94%) . Total of 27 (37.50%) patients underwent polypectomy and 20 (27.78%) underwent endometrial sampling . The hysteroscopy findings of endometrial polyp 31 (43.05%) was confirmed in only 28 (46.67%) cases by histopathological examination . Hysteroscopy enables visualisation of the endometrial cavity and if possible treatment in the same setting. It is a minimally invasive procedure used worldwide. The American College of Obstetricians and Gynecologists and the American Association of Gynecologic Laparoscopists recommend the use of hysteroscopy for the diagnosis and treatment of intrauterine pathology. The recommendation of using 400 mcg per vaginal misoprostol 4 hours prior to the procedure was followed in this study. The present study showed the prevalence of hysteroscopy to be higher than in other studies. This could be because the present study was done in a centre where there were more infertile cases approaching and this centre was among the few in the country where hysteroscopy is performed. In the present study, the most common presenting complaint was excessive vaginal bleeding 38.88% followed by inability to conceive 36.11%. Similarly, in a study done abnormal uterine bleeding 32.5% was the presenting complaint. Endometrial polyp 43.05% was the commonest hysteroscopy finding in the present study similar to the study done in Libya was 53.6%. However, it was contrary to the study done in India where most patients presented with proliferative endometrium 34%. This could be because in their study they had included patients of 40 years and above with abnormal uterine bleeding only, however, we had patients with ages ranging from 20-65 years with various symptoms. There were five cases of uterine synechiae in which adhesiolysis was done after which three cases were able to conceive. A study done in Linkou that carried adhesiolysis in 85 females with Asherman's syndrome had also shown excellent results. Uterine septum resection was done in three cases with conception occurring within six months in two cases. The present study had 13 patients 43% of who conceived postsurgery mostly through in vitro fertilization. A study done in Belgium also showed a higher pregnancy rate of 63% after the removal of endometrial polyp prior to intrauterine insemination. Higher fertility rates thus usually occur in women seeking fertility treatment after hysteroscopic removal of an endometrial polyp, submucosal leiomyoma, uterine septum and intrauterine adhesions. Three cases of missing IUCD and one case of a foreign body were also successfully removed in the present study. No significant complications occurred except in two cases 2.77% of fluid overload was managed with diuretics. The complication rate was also 2% in a study done in Libya however, they were of different types of postoperative haemorrhage and perforation. This could be because of more number of cases in their study. Another reason could be the lack of consistency and experience of the surgeons doing hysteroscopy. Similar to the present study, a study done in Iran also showed complications of fluid overload in only a few cases. The point of confusion in the case of proliferative and secretory endometrium could be because of the history of intake of hormonal pills. In patients with such an intake of pills, the endometrium shows mixed characteristics. In the same endometrial cavity, there could be some areas of proliferative and some areas of secretory endometrium. Also, endometrial polyps and submucosal leiomyoma can present in the background of either secretory or proliferative endometrium leading to false results. The limitation of the study is thus its small sample size. Bigger sample populations with more named patterns on hysteroscopy are required as sometimes one histology may give more than one picture on hysteroscopic analysis. Also since it is a retrospective study, data might have been missed. The prevalence of hysteroscopy among gynaecological patients was higher than in the studies done in similar settings. Hysteroscopy enables visualisation of the endometrial cavity and if possible treatment in the same setting avoiding the need for an invasive procedure.
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