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Molecular pathology and synaptic loss in primary tauopathies: an | 67ce326f-76db-4f98-86b0-1cc35163ccd3 | 8967099 | Pathology[mh] | Synaptic loss is a feature of many neurodegenerative disorders. It is closely related to cognitive decline in symptomatic stages of disease, , but can begin long before symptom onset and neuronal loss. Synaptic loss and dysfunction may be an important mediator of decline even where atrophy is minimal or absent. , Conversely, synaptic connectivity may facilitate the spread of oligomeric misfolded proteins such as tau. The relationship between synaptic loss and the accumulation of misfolded proteins in primary tauopathies has yet to be determined in vivo. Preclinical models suggest early synaptotoxicity of oligomeric tau, leading to reduced synaptic plasticity and density. , In patients with mutations of microtubule-associated protein tau (MAPT), there are deficiencies in many synaptic pathways including GABA-mediated signalling and synaptic plasticity. The mechanisms of synapse loss following tau pathology include both direct and indirect pathways (reviewed by Spires-Jones and Hyman ); however, the severity of synaptic toxicity in the related tauopathy of Alzheimer’s disease appears to be dependent on the stage of disease in preclinical models and in patients post-mortem and in vivo . In animal models of Alzheimer’s disease, and at human post-mortem, there is differential expression of synaptic proteins in the early stages with increases in some proteins and reductions in others. , This may be an attempt to maintain cellular physiology in early disease, which fails as the disease progresses, leading to loss of synaptic function and synapse numbers in moderate and advanced disease. In clinical disorders, the in vivo pathologies of synaptic density and tau burden can be characterized by PET. Recent in vivo PET imaging in Alzheimer’s disease using 11 C-UCB-J as a marker of synaptic density and 18 F-AV-1451 or 18 F-MK-6240 PET as markers of tau pathology have shown decreased temporal lobe synaptic density with increasing pathological burden, but with individual variability depending on the severity of cortical pathology. However, the pathology of Alzheimer’s disease is multifaceted with amyloid and tau aggregation, vascular changes and neuroinflammation. In this study, we use progressive supranuclear palsy–Richardson’s syndrome (PSP) and corticobasal degeneration (CBD) as models of human tauopathy, with relevance to other tau-mediated neurodegenerative disorders, and examine the in vivo relationship between synaptic density and burden of molecular pathology. An advantage of studying PSP is the very high correlation between the clinical syndrome, and the specific 4R-tauopathy at autopsy. , The clinical phenotype of corticobasal syndrome (CBS), may be caused by CBD, but can also be mimicked by Alzheimer’s disease and less commonly by other forms of frontotemporal lobar degeneration. Here, we use the term CBD to refer to patients with CBS in whom Alzheimer’s disease is excluded by 11 C-PiB PET, whereby in the absence of amyloid pathology there is a high clinicopathological correlation with 4R-tauopathy at post-mortem. Both PSP and CBD demonstrate synaptic loss in vivo , and at post-mortem examination. , The distribution of tau pathology in both diseases is well characterized with cortical and subcortical involvement. , Animal models of tauopathy have illustrated the co-localization of tau aggregates at the synaptic bouton, associated with synaptic dysfunction and synaptic loss , but the tau–synapse association is yet to be determined in vivo . 18 F-AV-1451 signals are above normal in the cortex of patients with PSP and CBS/CBD, but there is relatively low affinity for 4R-tauopathy compared to Alzheimer’s disease, and off-target binding particularly in the basal ganglia. We therefore refer to 18 F-AV-1451 as a marker of ‘molecular pathology’, referring to the combination of tau and non-tau targets. illustrates our hypotheses. Previous studies suggest that the strength of connectivity within a region and between brain regions can promote the spread of tau pathology, in humans as in preclinical models. Therefore, we hypothesized that brain areas with higher synaptic density would develop more tau pathology (schematically represented by green arrows in ). We predicted that the spatial distribution of molecular pathology, as measured with the PET radioligand 18 F-AV-1451, would be correlated with synaptic density, as measured with the PET radioligand 11 C-UCB-J (which binds to the presynaptic vesicle glycoprotein SV2A that is ubiquitously expressed in brain synapses , ). Because pathology in a region may impair efferent projections, a corollary hypothesis is that tau accumulation in one region (source region) leads to diaschisis characterized by reduced synaptic density in the areas to which it connects (target regions). A second part of the model describes the consequence of the pathology, which is to reduce synaptic density (schematically represented by red arrows in ). The predicted result is a positive relationship between 18 F-AV-1451 binding and synaptic loss, negatively moderated by disease severity . Participant recruitment and study design Twenty-three patients with probable PSP–Richardson syndrome and 12 with probable CBS in whom Alzheimer’s disease was excluded with 11 C-PiB PET were recruited from a regional specialist National Health Service clinic at the Cambridge University Centre for Parkinson-plus. We refer to our amyloid-negative CBS cohort as having CBD but acknowledge other pathologies are possible. Nineteen healthy volunteers were recruited from the UK National Institute for Health Research Join Dementia Research register. Participants were screened using the inclusion/exclusion criteria set out in Holland et al . Eligible participants underwent clinical and cognitive assessments including the revised Addenbrooke’s Cognitive Examination (ACE-R), the Mini-Mental State Examination (MMSE), and the Institute of Cognitive Neurology (INECO) frontal screening; disease severity was measured with the PSP rating scale, and the Cortical Basal ganglia Functional Scale. Participants underwent 3 T MRI, 18 F-AV-1451 PET and 11 C-UCB-J PET. The research protocol was approved by the Cambridge Research Ethics Committee (reference 18/EE/0059) and the Administration of Radioactive Substances Advisory Committee. All participants provided written informed consent in accordance with the Declaration of Helsinki. PET data acquisition and kinetic analysis 11 C-UCB-J PET The procedure for 11 C-UCB-J synthesis, PET data acquisition, image reconstruction and kinetic analysis was the same as in Holland et al . In brief, dynamic PET data acquisition was performed on a GE SIGNA PET/MR (GE Healthcare) for 90 min immediately after injection, with attenuation correction using a multisubject atlas method and improvements to the MRI brain coil component. Emission image series were aligned using SPM12 ( www.fil.ion.ucl.ac.uk/spm/software/spm12/ ) and rigidly registered to the T 1 -weighted MRI acquired during PET data acquisition (repetition time = 3.6 ms, echo time = 9.2 ms, 192 sagittal slices, in plane resolution 0.55 × 0.55 mm, interpolated to 1.0 × 1.0 mm; slice thickness 1.0 mm). The Hammersmith atlas ( http://brain-development.org ) with modified posterior fossa regions was spatially normalized to the T 1 -weighted MRI of each participant using advanced normalization tools software. Regional time–activity curves were extracted following the application of geometric transfer matrix (GTM) partial volume correction (PVC ) to each dynamic PET image. Regions of interest were multiplied by a binary grey matter mask (>50% on the SPM12 grey matter probability map smoothed to PET spatial resolution), with the exception of the subcortical grey matter regions pallidum, substantia nigra, pons and medulla. To assess the impact of PVC, time–activity curves were also extracted from the same regions of interest without the application of GTM PVC (discussed below as ‘without partial volume correction’). To quantify SV2A density, 11 C-UCB-J non-displaceable binding potential (BP ND ) was determined using a basis function implementation of the simplified reference tissue model, with the reference tissue defined in the centrum semiovale. , 18 F-AV-1451 PET As for 11 C-UCB-J, PET data acquisition was performed on a GE SIGNA PET/MR for 90 min after 18 F-AV-1451 injection, with attenuation correction as described above for 11 C-UCB-J. Image processing was also as given above for 11 C-UCB-J, except that 18 F-AV-1451 BP ND was determined using a different basis function implementation of the simplified reference tissue model and the reference tissue was defined in inferior cerebellar grey matter using a 90% threshold on the grey matter probability map produced by SPM12 smoothed to PET resolution. 11 C-PiB PET Amyloid imaging using Pittsburgh Compound B ( 11 C-PiB) followed the protocol given in Holland et al . 11 C-PiB cortical standardized uptake value ratio (SUVR; 50–70 min post injection) was calculated using the whole cerebellum reference tissue as per the Centiloid Project methodology. A negative amyloid status was characterized by a cortical 11 C-PiB SUVR < 1.21 obtained by converting the Centiloid cut-off of 19 to SUVR using the Centiloid-to-SUVR transformation in Jack et al . Statistical analyses We compared demographic and clinical variables between the diagnostic groups using analysis of covariance (ANCOVA), and chi-square tests where appropriate. We used a linear mixed effects model to assess the overall relationship between 18 F-AV-1451 and 11 C-UCB-J BP ND , with age and scan interval as covariates. To adjust for normal levels of tracer uptake from off-target binding not present in the reference region (over and above the correction for non-specific binding in the reference region), we normalized the patient BP ND data against controls by subtracting the regional mean BP ND values in controls from the data of each patient, for each region, for each tracer. Furthermore, we removed regions with previously reported off-target binding of 18 F-AV-1451 (basal ganglia and substantia nigra ) The linear mixed effect model therefore included normalized 11 C-UCB-J as the dependent variable, normalized 18 F-AV-1451 as the independent variable, and age and scan interval as covariates of no interest. To investigate the effect of individual variability on the relationship between 11 C-UCB-J and 18 F-AV-1451 BP ND , we used a linear model with the slope of 11 C-UCB-J BP ND as a function of 18 F-AV-1451 BP ND for each individual (extracted from the previous linear mixed effect model) as the dependent variable, the PSP rating scale (a measure of disease severity) as the independent variable and age as a covariate of no interest. To explore the correlation between 11 C-UCB-J and 18 F-AV-1451 BP ND between regions, we calculated a correlation matrix between cortical 18 F-AV-1451 binding and synaptic density in cortical and subcortical regions. Analyses were performed with and without GTM partial volume correction, yielding similar results; we focus on partial volume-corrected BP ND to limit the potential effect of atrophy on our ligand cross-correlation, but present data without PVC in the . Statistical analyses were implemented in R (version 3.6.2). Data availability The data that support the findings of this study are available from the corresponding author, upon reasonable request for academic (non-commercial) purposes, subject to restrictions required to preserve participant confidentiality. Twenty-three patients with probable PSP–Richardson syndrome and 12 with probable CBS in whom Alzheimer’s disease was excluded with 11 C-PiB PET were recruited from a regional specialist National Health Service clinic at the Cambridge University Centre for Parkinson-plus. We refer to our amyloid-negative CBS cohort as having CBD but acknowledge other pathologies are possible. Nineteen healthy volunteers were recruited from the UK National Institute for Health Research Join Dementia Research register. Participants were screened using the inclusion/exclusion criteria set out in Holland et al . Eligible participants underwent clinical and cognitive assessments including the revised Addenbrooke’s Cognitive Examination (ACE-R), the Mini-Mental State Examination (MMSE), and the Institute of Cognitive Neurology (INECO) frontal screening; disease severity was measured with the PSP rating scale, and the Cortical Basal ganglia Functional Scale. Participants underwent 3 T MRI, 18 F-AV-1451 PET and 11 C-UCB-J PET. The research protocol was approved by the Cambridge Research Ethics Committee (reference 18/EE/0059) and the Administration of Radioactive Substances Advisory Committee. All participants provided written informed consent in accordance with the Declaration of Helsinki. 11 C-UCB-J PET The procedure for 11 C-UCB-J synthesis, PET data acquisition, image reconstruction and kinetic analysis was the same as in Holland et al . In brief, dynamic PET data acquisition was performed on a GE SIGNA PET/MR (GE Healthcare) for 90 min immediately after injection, with attenuation correction using a multisubject atlas method and improvements to the MRI brain coil component. Emission image series were aligned using SPM12 ( www.fil.ion.ucl.ac.uk/spm/software/spm12/ ) and rigidly registered to the T 1 -weighted MRI acquired during PET data acquisition (repetition time = 3.6 ms, echo time = 9.2 ms, 192 sagittal slices, in plane resolution 0.55 × 0.55 mm, interpolated to 1.0 × 1.0 mm; slice thickness 1.0 mm). The Hammersmith atlas ( http://brain-development.org ) with modified posterior fossa regions was spatially normalized to the T 1 -weighted MRI of each participant using advanced normalization tools software. Regional time–activity curves were extracted following the application of geometric transfer matrix (GTM) partial volume correction (PVC ) to each dynamic PET image. Regions of interest were multiplied by a binary grey matter mask (>50% on the SPM12 grey matter probability map smoothed to PET spatial resolution), with the exception of the subcortical grey matter regions pallidum, substantia nigra, pons and medulla. To assess the impact of PVC, time–activity curves were also extracted from the same regions of interest without the application of GTM PVC (discussed below as ‘without partial volume correction’). To quantify SV2A density, 11 C-UCB-J non-displaceable binding potential (BP ND ) was determined using a basis function implementation of the simplified reference tissue model, with the reference tissue defined in the centrum semiovale. , 18 F-AV-1451 PET As for 11 C-UCB-J, PET data acquisition was performed on a GE SIGNA PET/MR for 90 min after 18 F-AV-1451 injection, with attenuation correction as described above for 11 C-UCB-J. Image processing was also as given above for 11 C-UCB-J, except that 18 F-AV-1451 BP ND was determined using a different basis function implementation of the simplified reference tissue model and the reference tissue was defined in inferior cerebellar grey matter using a 90% threshold on the grey matter probability map produced by SPM12 smoothed to PET resolution. 11 C-PiB PET Amyloid imaging using Pittsburgh Compound B ( 11 C-PiB) followed the protocol given in Holland et al . 11 C-PiB cortical standardized uptake value ratio (SUVR; 50–70 min post injection) was calculated using the whole cerebellum reference tissue as per the Centiloid Project methodology. A negative amyloid status was characterized by a cortical 11 C-PiB SUVR < 1.21 obtained by converting the Centiloid cut-off of 19 to SUVR using the Centiloid-to-SUVR transformation in Jack et al . C-UCB-J PET The procedure for 11 C-UCB-J synthesis, PET data acquisition, image reconstruction and kinetic analysis was the same as in Holland et al . In brief, dynamic PET data acquisition was performed on a GE SIGNA PET/MR (GE Healthcare) for 90 min immediately after injection, with attenuation correction using a multisubject atlas method and improvements to the MRI brain coil component. Emission image series were aligned using SPM12 ( www.fil.ion.ucl.ac.uk/spm/software/spm12/ ) and rigidly registered to the T 1 -weighted MRI acquired during PET data acquisition (repetition time = 3.6 ms, echo time = 9.2 ms, 192 sagittal slices, in plane resolution 0.55 × 0.55 mm, interpolated to 1.0 × 1.0 mm; slice thickness 1.0 mm). The Hammersmith atlas ( http://brain-development.org ) with modified posterior fossa regions was spatially normalized to the T 1 -weighted MRI of each participant using advanced normalization tools software. Regional time–activity curves were extracted following the application of geometric transfer matrix (GTM) partial volume correction (PVC ) to each dynamic PET image. Regions of interest were multiplied by a binary grey matter mask (>50% on the SPM12 grey matter probability map smoothed to PET spatial resolution), with the exception of the subcortical grey matter regions pallidum, substantia nigra, pons and medulla. To assess the impact of PVC, time–activity curves were also extracted from the same regions of interest without the application of GTM PVC (discussed below as ‘without partial volume correction’). To quantify SV2A density, 11 C-UCB-J non-displaceable binding potential (BP ND ) was determined using a basis function implementation of the simplified reference tissue model, with the reference tissue defined in the centrum semiovale. , F-AV-1451 PET As for 11 C-UCB-J, PET data acquisition was performed on a GE SIGNA PET/MR for 90 min after 18 F-AV-1451 injection, with attenuation correction as described above for 11 C-UCB-J. Image processing was also as given above for 11 C-UCB-J, except that 18 F-AV-1451 BP ND was determined using a different basis function implementation of the simplified reference tissue model and the reference tissue was defined in inferior cerebellar grey matter using a 90% threshold on the grey matter probability map produced by SPM12 smoothed to PET resolution. C-PiB PET Amyloid imaging using Pittsburgh Compound B ( 11 C-PiB) followed the protocol given in Holland et al . 11 C-PiB cortical standardized uptake value ratio (SUVR; 50–70 min post injection) was calculated using the whole cerebellum reference tissue as per the Centiloid Project methodology. A negative amyloid status was characterized by a cortical 11 C-PiB SUVR < 1.21 obtained by converting the Centiloid cut-off of 19 to SUVR using the Centiloid-to-SUVR transformation in Jack et al . We compared demographic and clinical variables between the diagnostic groups using analysis of covariance (ANCOVA), and chi-square tests where appropriate. We used a linear mixed effects model to assess the overall relationship between 18 F-AV-1451 and 11 C-UCB-J BP ND , with age and scan interval as covariates. To adjust for normal levels of tracer uptake from off-target binding not present in the reference region (over and above the correction for non-specific binding in the reference region), we normalized the patient BP ND data against controls by subtracting the regional mean BP ND values in controls from the data of each patient, for each region, for each tracer. Furthermore, we removed regions with previously reported off-target binding of 18 F-AV-1451 (basal ganglia and substantia nigra ) The linear mixed effect model therefore included normalized 11 C-UCB-J as the dependent variable, normalized 18 F-AV-1451 as the independent variable, and age and scan interval as covariates of no interest. To investigate the effect of individual variability on the relationship between 11 C-UCB-J and 18 F-AV-1451 BP ND , we used a linear model with the slope of 11 C-UCB-J BP ND as a function of 18 F-AV-1451 BP ND for each individual (extracted from the previous linear mixed effect model) as the dependent variable, the PSP rating scale (a measure of disease severity) as the independent variable and age as a covariate of no interest. To explore the correlation between 11 C-UCB-J and 18 F-AV-1451 BP ND between regions, we calculated a correlation matrix between cortical 18 F-AV-1451 binding and synaptic density in cortical and subcortical regions. Analyses were performed with and without GTM partial volume correction, yielding similar results; we focus on partial volume-corrected BP ND to limit the potential effect of atrophy on our ligand cross-correlation, but present data without PVC in the . Statistical analyses were implemented in R (version 3.6.2). The data that support the findings of this study are available from the corresponding author, upon reasonable request for academic (non-commercial) purposes, subject to restrictions required to preserve participant confidentiality. Demographics The patients (PSP and CBD) and control groups were similar in age, sex, education and injected activity of 11 C-UCB-J and 18 F-AV-1451 . We observed typical cognitive profiles for people with PSP and CBD: impaired on verbal fluency, memory and visuospatial domains of the ACE-R, MMSE and INECO frontal screening tool. Relationship between 11 C-UCB-J BP ND and 18 F-AV-1451 BP ND Compared to controls, patients had significantly higher 18 F-AV1451 binding in the caudate nucleus, pallidum, putamen and substantia surviving correction for multiple comparison [ P < 0.05, false discovery rate (FDR) corrected; and ]. As previously reported in a smaller cohort, , patients had significantly lower 11 C-UCB-J binding across all cortical and subcortical areas compared to controls, which survived FDR correction ( and ). Summary statistics for regional 18 F-AV1451 and 11 C-UCB-J binding potentials in patients and controls are shown in , respectively. There was an overall positive relationship between normalized 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND across the patient cohort (β = 0.4, t = 3.6, P = 0.001; ). There was a significant region × 18 F-AV-1451 interaction ( P < 0.001) driven by subregions of the frontal, parietal and temporal cortices, as well as the hippocampus, subcallosal area and the thalamus, with all but the hippocampus surviving correction for multiple comparison. Age ( P = 0.9) and scan interval ( P = 0.5) did not have a significant effect on the overall model (note that brain regions with known off-target binding of 18 F-AV-1451 were removed before running this linear mixed model). The direction of the relationship between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND within each individual (i.e. the slope of each grey line in ) negatively correlated with disease severity (β = −0.02, t = −2.9, P = 0.007, R = − 0.41), independent of age (effect of age: β = 0.02, t = 2.6, P = 0.01; ). In other words, those patients with more severe disease displayed a less-positive relationship between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND . Of note, the positive correlation between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND across the patient cohort remains even if BP ND values are not normalized against the control data (β = 0.4, t = 4.0, P = 0.0001), as well as the negative relationship between disease severity and the slope of each individual in . Also note that similar findings were observed using BP ND derived from data without partial volume correction . The relationship between 18 F-AV-1451 and 11 C-UCB-J binding was also positive in an analogous linear mixed effect model in controls alone (β = 0.6, t = 4, P < 0.0001), with no main effect of age or scan interval, or 18 F-AV-1451 × Region interaction. The relationship between unadjusted 18 F-AV-1451 and 11 C-UCB-J binding in all three groups (controls, amyloid-negative CBS and PSP) is shown in . Cross-regional correlation between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND Synaptic density in a region is proposed to be affected by both local tau pathology and tau burden in connected regions from which it receives afferent projections. As a result, despite a positive correlation at a regional level, the synaptic density in any given region may be negatively affected by remote insult, with diaschisis between anatomically connected regions (illustrated schematically in ). As an exploratory analysis, we computed the asymmetric Pearson’s correlation matrix shown in , between normalized cortical 18 F-AV-1451 BP ND (horizontal axis of matrix) and normalized cortical and subcortical 11 C-UCB-J BP ND (vertical axis of matrix) in patients. We show that, overall, there are significant negative correlations between cortical (frontal, temporal, parietal, occipital) 18 F-AV-1451 BP ND and subcortical 11 C-UCB-J BP ND within the caudate nucleus and putamen (−0.52 < R < −0.37; P < 0.05; uncorrected for multiple comparisons). We observed a positive correlation between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND within the thalamus where strong local connections exist . We did not include subcortical 18 F-AV-1451 BP ND in the matrix in given the off-target binding in these regions, which undermines the interpretability of the signal. However, we include these regions as well as other subregions in the larger correlation matrix in for completeness. Similar findings are seen using BP ND from data without partial volume correction . The patients (PSP and CBD) and control groups were similar in age, sex, education and injected activity of 11 C-UCB-J and 18 F-AV-1451 . We observed typical cognitive profiles for people with PSP and CBD: impaired on verbal fluency, memory and visuospatial domains of the ACE-R, MMSE and INECO frontal screening tool. 11 C-UCB-J BP ND and 18 F-AV-1451 BP ND Compared to controls, patients had significantly higher 18 F-AV1451 binding in the caudate nucleus, pallidum, putamen and substantia surviving correction for multiple comparison [ P < 0.05, false discovery rate (FDR) corrected; and ]. As previously reported in a smaller cohort, , patients had significantly lower 11 C-UCB-J binding across all cortical and subcortical areas compared to controls, which survived FDR correction ( and ). Summary statistics for regional 18 F-AV1451 and 11 C-UCB-J binding potentials in patients and controls are shown in , respectively. There was an overall positive relationship between normalized 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND across the patient cohort (β = 0.4, t = 3.6, P = 0.001; ). There was a significant region × 18 F-AV-1451 interaction ( P < 0.001) driven by subregions of the frontal, parietal and temporal cortices, as well as the hippocampus, subcallosal area and the thalamus, with all but the hippocampus surviving correction for multiple comparison. Age ( P = 0.9) and scan interval ( P = 0.5) did not have a significant effect on the overall model (note that brain regions with known off-target binding of 18 F-AV-1451 were removed before running this linear mixed model). The direction of the relationship between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND within each individual (i.e. the slope of each grey line in ) negatively correlated with disease severity (β = −0.02, t = −2.9, P = 0.007, R = − 0.41), independent of age (effect of age: β = 0.02, t = 2.6, P = 0.01; ). In other words, those patients with more severe disease displayed a less-positive relationship between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND . Of note, the positive correlation between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND across the patient cohort remains even if BP ND values are not normalized against the control data (β = 0.4, t = 4.0, P = 0.0001), as well as the negative relationship between disease severity and the slope of each individual in . Also note that similar findings were observed using BP ND derived from data without partial volume correction . The relationship between 18 F-AV-1451 and 11 C-UCB-J binding was also positive in an analogous linear mixed effect model in controls alone (β = 0.6, t = 4, P < 0.0001), with no main effect of age or scan interval, or 18 F-AV-1451 × Region interaction. The relationship between unadjusted 18 F-AV-1451 and 11 C-UCB-J binding in all three groups (controls, amyloid-negative CBS and PSP) is shown in . 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND Synaptic density in a region is proposed to be affected by both local tau pathology and tau burden in connected regions from which it receives afferent projections. As a result, despite a positive correlation at a regional level, the synaptic density in any given region may be negatively affected by remote insult, with diaschisis between anatomically connected regions (illustrated schematically in ). As an exploratory analysis, we computed the asymmetric Pearson’s correlation matrix shown in , between normalized cortical 18 F-AV-1451 BP ND (horizontal axis of matrix) and normalized cortical and subcortical 11 C-UCB-J BP ND (vertical axis of matrix) in patients. We show that, overall, there are significant negative correlations between cortical (frontal, temporal, parietal, occipital) 18 F-AV-1451 BP ND and subcortical 11 C-UCB-J BP ND within the caudate nucleus and putamen (−0.52 < R < −0.37; P < 0.05; uncorrected for multiple comparisons). We observed a positive correlation between 18 F-AV-1451 BP ND and 11 C-UCB-J BP ND within the thalamus where strong local connections exist . We did not include subcortical 18 F-AV-1451 BP ND in the matrix in given the off-target binding in these regions, which undermines the interpretability of the signal. However, we include these regions as well as other subregions in the larger correlation matrix in for completeness. Similar findings are seen using BP ND from data without partial volume correction . We have identified an in vivo relationship between molecular pathology (estimated with 18 F-AV-1451 PET) and synaptic density (estimated with 11 C-UCB-J PET) in patients with the primary tauopathies of PSP and CBD (inferred in vivo from amyloid-negative CBS). There are three principal results: (i) regions with higher synaptic density have higher molecular pathology; (ii) within regions, synaptic density becomes less dependent on 18 F-AV-1451 binding as disease severity increases; and (iii) between regions, increased cortical 18 F-AV-1451 binding is associated with reduced subcortical synaptic density. We interpret these three findings in the context of synaptic connectivity-based susceptibility to tauopathy, the synaptotoxic effects of tauopathy and cortico-subcortical diaschisis, respectively. The above results are congruent with the model of tau-induced synaptic toxicity, acknowledging the caveat of off-target binding of 18 F-AV-1451. Our primary pathology of interest in the context of PSP and CBD is 4R-tau, but other tauopathies such as latent Alzheimer pathology in older adults and non-tau molecular pathologies may also contribute to 18 F-AV-1451 binding. The effect of hyperphosphorylated tau on synaptic function and density is complex. It involves both direct and indirect pathways of injury with changes in cellular physiology preceding the loss of neurons. Through direct pathways, pathological tau interferes with dendritic morphology, synaptic protein expression, the number of NMDA ( N -methyl- D- aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors on the presynaptic membrane, mitochondrial function, synaptic vesicle numbers and ultimately synaptic loss (for a review of animal studies illustrating various direct tau-induced synaptic abnormalities, see Jadhav et al . ). Tau also directly affects the axon cytoskeleton and trafficking, as well as the normal functioning of the soma. Indirectly, hyperphosphorylated tau adversely affects the functioning of the neuronal support network, including glia cells and astrocytes. These events are affected by the stage and severity of the disease process, and in relation to regional differences in connectivity which we discuss next (concepts schematically illustrated in ). We identified a positive relationship between the binding of 11 C-UCB-J and 18 F-AV-1451 such that areas of the brain with higher synaptic density had higher molecular pathology. This accords with preclinical and clinical models of tauopathy in which the strength of local network connectivity facilitates the transneuronal spread of tau pathology. , , However, the relationship between tau accumulation and synaptic density changes with disease progression, at least as inferred from the cross-sectional moderation by disease severity . With increasing scores on the PSP rating scale, synaptic density becomes less dependent on local accumulation of pathology. In other words, according to the model in areas with relatively low tau accumulation synaptic density is minimally affected, whereas in areas with higher tau accumulation there is reduction of synaptic density as the disease progresses, and this preferentially occurs in synapse-rich areas. As the disease progresses, other pathological processes may contribute to synaptic loss, such as inflammation, another predictor of prognosis and mediator of synaptic loss. Therefore, there is not a simple linear relationship between tau accumulation and synaptic density in moderate and advanced disease. This observation accords with human post-mortem and animal studies. In post-mortem studies of the tauopathy Alzheimer’s disease, there is a biphasic synaptic protein response during disease progression, with increases in synaptophysin/syntaxin/SNAP-25 in early Braak stages and synaptic loss observed only when the disease has progressed to the neocortex. In the P301L transgenic mouse model of PSP-like tauopathy, there is a differential loss of synapses, as well as synaptic proteins, depending on disease stage. These results have recently been replicated in vivo , where the relationship between synaptic density and tau burden in patients with Alzheimer’s disease is reported to be modulated by cortical tau load. Coomans et al . show that in patients with mild disease and low cortical tau burden, the relationship between tau and synaptic density is positive, whereas in those with increasing cortical tau load, this relationship changes direction; the relationship between the two tracers in controls is not reported. In our study, we also observe a positive relationship between 18 F-AV-1451 and 11 C-UCB-J binding potentials in controls , even though 18 F-AV-1451 binding is lower in controls. Disease-related 18 F-AV-1451 binding attributable to presence of PSP/CBD pathology is unlikely in the controls, as the prevalence of these conditions in the normal population is only 1/10 000. However, the presence of asymptomatic Alzheimer’s disease pathology in the normal older population is more likely. Rising from the age of 40, by the age of 85 two-thirds of cognitively normal individuals will show positive changes in the A/T/N classification for Alzheimer’s disease, whether by CSF, plasma or amyloid PET. , Some of the non-specific 18 F-AV-1451 signal, even in healthy controls, may therefore be attributable to latent/preclinical Alzheimer’s disease pathology. We control for this component of the signal by subtracting the mean regional control values from those of the patients. The positive correlation between 18 F-AV-1451 and 11 C-UCB-J binding potential in controls appears stronger compared to that seen in patients, as a group . One explanation for this observation is the heterogeneity in disease severity in PSP/CBD, given the interaction between 11 C-UCB-J, 18 F-AV-1451 and disease severity. This can be understood in terms of the model set out in . The patient group includes those with a strong positive correlation (at early stages of disease) and those with negligible correlation (as a consequence of more advanced disease). The net result for a group-wise test will be a reduction of the group correlation. This is not present in the control group, in whom the level of tau pathology is expected to be very much lower (even if present from Alzheimer type tau with high 18 F-AV-1451 affinity). To understand the biphasic relationship between molecular pathology and synaptic density, one must consider other key players in synaptotoxicity in tauopathies, such as neuroinflammation. Recent in vivo studies have confirmed the regional co-localization of inflammation and 18 F-AV-1451 binding in PSP, including in many cortical areas, in line with previous in vivo , and post-mortem reports of the tight interplay between neuroinflammation and tau accumulation in tauopathies. There is growing evidence that these two pathological processes affect synaptic function both independently and synergistically. The relationship between tauopathy and synaptic density is even more intriguing when considering the change in synaptic density in one region as a function of pathology in another. There are strong correlations between 11 C-UCB-J binding within the basal ganglia (in particular the caudate nucleus and putamen) and 18 F-AV-1451 binding in all major cortical areas. The reverse association, between subcortical 18 F-AV-1451 and cortical 11 C-UCB-J binding is also observed but is dismissed here as uninterpretable in view of subcortical off-target binding of 18 F-AV-1451. The significant negative correlation between cortical 18 F-AV-1451 binding and synaptic density in the basal ganglia could be a reflection of severe disease in the basal ganglia and accumulating pathology in the neocortex. In other words, synapses are severely affected in the basal ganglia as one of the earliest sites of pathology, with pathology spreading and accumulating in synapse-rich areas of the brain, for example the neocortex. A second possible explanation is that loss of descending cortico-striatal axons due to cortical pathology may cause diaschisis, affecting subcortical synaptic density even further. Previous analysis of diffusion tensor imaging in patients with PSP/CBD have revealed extensive white matter abnormalities (within the main association fibres) beyond the degree of cortical atrophy, , resulting in loss of cortical afferents onto subcortical structures. A third, although not mutually exclusive, potential explanation is the weakening of cortical–subcortical functional connectivity resulting from dysfunctional synapses rather than synaptic loss, although cortico-subcortical connectivity is inferred and was not directly measured in our study. Although at a regional level there is a positive correlation between 11 C-UCB-J and 18 F-AV-1451 BP ND , we are not directly measuring either synaptic function or the synaptotoxic tau oligomers. This caveat must be borne in mind when interpreting PET data. It is the preclinical models that have shown that oligomers of tau are toxic to synaptic function, even in the absence of tau polymers/fibrils. , By the time tau aggregates are established, oligomers of tau are expected cortically, and perhaps interfere with cortical function and the integrity of descending axons. There are other limitations to our study. First, the low affinity of 18 F-AV-1451 for PSP and CBD 4R tau. Even though the radioligand recapitulates the distribution of post-mortem neuropathology in PSP and CBD and binds PSP 4R tau, the affinity is very much lower than for 3R tau in Alzheimer’s disease. Second, there is well-established off-target binding of 18 F-AV-1451, particularly within subcortical structures where monoamine oxidase and neuromelanin are present. Off-target binding is most prominent in the basal ganglia and substantia nigra, which we excluded before running the linear mixed model and correlation matrix. We included these regions in the detailed descriptive correlation matrices in for completeness sake, noting the strong negative correlations between cortical 18 F-AV-1451 BP ND and subcortical 11 C-UCB-J BP ND . Furthermore, we normalized our patient data against those of controls to remove any additional normal levels of off-target binding, noting the caveat that the remaining signal in patients may still arise from tau and non-tau pathology; there is no evidence to suggest that 18 F-AV-1451 shares a common binding target with 11 C-UCB-J, which has a high specificity for SV2A in previous in vivo and in vitro validation studies. , Third, we note that in PET studies of neurodegeneration with atrophy, grey matter volume loss can affect the interpretation of PET signals. However, synaptic loss in PSP and CBD occurs even in areas of the brain without discernible atrophy on MRI. , Nonetheless, we used a stringent partial volume correction method (GTM) to minimize the effect of atrophy on our ligand cross-correlations. Of note, our data without partial volume correction yield similar results in all the main analyses . Fourth, although the sample size is small, it is adequately powered in view of the large effect sizes seen. However, more subtle relationships with phenotypic variants of PSP and CBS would require larger studies. Additionally, clinical diagnostic criteria for PSP–Richardson’s syndrome and amyloid-negative CBS (here called CBD) were used to select a clinical cohort with likely a 4R-tauopathy as the underlying pathological diagnosis. While both PSP–Richardson’s syndrome and amyloid-negative CBS are highly correlated with a 4R-tauopathy at post-mortem, both from our local brain bank and internationally, , , other pathologies are possible, and so are coexistent pathologies that may synergistically contribute to neurodegeneration. Neuropathological correlates, to test the correlations between phenotype and pathology, and between in vivo to post-mortem measures of synaptic density, as well as tau to synapse correlations would be useful but are not yet available for our cohort. Lastly, the cross-sectional design of this study limits the interpretation of the dynamic relationship between pathology and synaptic loss. Although we include patients at various stages of their illness, a longitudinal design is necessary to test the dynamic relationship we propose and the mediation of synaptic loss by progressive tauopathy. In conclusion, we demonstrate a widespread positive association between 18 F-AV-1451 and 11 C-UCB-J binding in patients with symptomatic PSP and amyloid-negative CBS. Individual variability in this association correlates with disease severity. The complex relationship between molecular pathology, including but not exclusive to tau, and synaptic density may explain changes in cognitive and motor physiology. We hope that these insights will inform the design of new clinical trials to arrest PSP and CBD. awab282_Supplementary_Data Click here for additional data file. |
Research Progress in Traditional Applications, Phytochemistry, Pharmacology, and Safety Evaluation of | 7e75f8da-01de-434b-8504-3403a3857817 | 10935076 | Pharmacology[mh] | Cynomorium is a genus containing two species, C. songaricum Rupr. and C. coccineum L., and is in the family Cynomoriaceae . These two types are mainly distributed in dry, rocky, or sandy soil areas, mainly appearing in the Northern Hemisphere. For centuries, folk medicine has been widely applied in countries such as Europe, North Africa, East Asia, and West Asia. Cynomorium songaricum Rupr. (CSR), a dried succulent stem of a rare endangered medicinal herb that belongs to the genus of Cynomorium L . , is mainly distributed in Xinjiang, Qinghai, Gansu, Ningxia, Inner Mongolia, Shaanxi and other northwestern regions in China . At present, CSR has been classified as vulnerable (VU) by the International Union for Conservation of Nature (IUCN), and as a Grade II protected plant by the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) . As a plant with homologous medicinal and edible properties, CSR is widely used both domestically and internationally. There are some common ways to consume CSR in North Africa, Europe, East Asia, and West Asia due to its nutritional and health value. For example, fresh CSR can be used for steaming rice, pancakes, etc., while dry CSR can be used for boiling soup, brewing wine, making health foods, etc. . The medicinal history of CSR can be traced back to the Yuan Dynasty, as recorded in the Ben Cao Yan Yi Bu Yi (Yuan Dynasty, A.D. 1347) which has the effects of tonifying kidney yang, benefiting essence and blood, and moistening the intestines and relieving constipation . The dried fleshy stem of CSR can tonify kidney Qi, enhance essence blood nourishment, and treat problems such as weakness and impotence . CSR whole grass can treat impotence caused by kidney deficiency, multiple dreams, spermatogenesis, waist and knee weakness, and other symptoms. However, it can also be used in the treatment of diarrhea, women’s leucorrhea, gum bleeding, and other diseases . CSR is rich in polyphenols and polysaccharides, both with biological activities such as scavenging free radicals in the body. As a result, CSR is also known as “the elixir of youth” because it prevents lipid oxidation, aging, cardiovascular disease, cancer, and radiation damage . In recent years, the chemical composition of CSR has been gradually revealed, and it has been reported that it contains flavonoids , triterpenoids , sugars and glycosides , steroids , organic acids and other components. Modern pharmacological studies have shown that the extract has the ability to promote cell regeneration and metabolism; enhance immune regulatory function ; has anti-cancer , anti-viral , and anti-aging properties ; and relieves fatigue . The present paper provides a comprehensive review of the botanical characteristics, traditional medicinal history, phytochemical composition, pharmacological research, and toxicological research progress of CSR. This systematic analysis aims to offer valuable insights into its clinical application and further research and development in the field of functional foods. Google Scholar ( http://scholar.google.com/ , accessed on 27 May 2023), PubMed ( http://www.ncbi.nlm.nih.gov/pubmed/ , accessed on 27 May 2023), Science Direct ( http://www.sciencedirect.com/ , accessed on 27 May 2023), Web of Science ( http://apps.webofknowledge.com/ , accessed on 27 May 2023) and China National Knowledge Infrastructure ( https://www.cnki.net/ , accessed on 27 May 2023) and other medical, ecological, and scientific databases were used to conduct an extensive literature search to classify the distribution of CSR and preliminary studies. Various keyword combinations such as “ Cynomorium songaricum Rupr.” and “traditional use”, “phytochemistry”, “pharmacology” and “isolated compounds” were used to collect scientific evidence on plant description, traditional use, phytochemical composition, and pharmacological properties of CSR. EndNote ( https://endnote.com/ , accessed on 27 May 2023) was used to collate the published literature. And we used the PubChem chemical database ( https://pubchem.ncbi.nlm.nih.gov/search/search.cgi/ , accessed on 27 May 2023), and other open access and redraw the chemical structure of CSR compounds. The structure of CSR compounds was plotted in Chem Draw 18.0 software. 3.1. Characteristics of Plants CSR is a perennial succulent parasitic herb with a reddish-brown coloration, predominantly submerged in sand and lacking chlorophyll . CSR possesses a few triangular scales in its middle and upper sections. Its inflorescence is terminal and clavate, adorned with scaly leaves, featuring bisexual flowers consisting of petals, stamens, and an ovary. Male flowers typically have four perianth pieces, while the pistil undergoes degradation. Female flowers exhibit a lower ovary with usually 5–6 perianth pieces enclosing one pendulous ovule at the apex, male flowers degenerate accordingly. The fruit resembles a nut . 3.2. Growth Environment and Regional Distribution CSR is a rare and endangered medicinal plant mainly distributed in northwest China, such as Inner Mongolia (Red), Gansu (Blue), Xinjiang (Orange), Qinghai (Purple), and Ningxia (Yellow) . Growing in temperate and subtropical regions, it can grow in areas with lower elevations or up to 2000 m above sea level. It likes warm and humid climates, has strong adaptability, and can grow in areas with full or partial sunlight. The minimum temperature is generally required to be no lower than −10 °C. It can withstand high temperatures even in hotter summers. It can adapt to different types of soil but it is best to avoid excessively humid or poor soil. Wild CSR is the main source, with a domestic accumulation of about 30,000 tons. Under normal circumstances, about 1500 tons are harvested annually. However, the existing wild CSR resources are only concentrated in the Hedong sandy land of Pingluo County and their distribution area has been decreasing year by year . There are five host plants, namely Nitraria sphaerocarpa , Nitraria sibirica , Nitraria tangutorum , Zygophyllum xanthoxylon , and Peganum multisectum . The lifecycle of CSR mainly includes several stages: seed germination, parasitic localization, parasitic attachment, growth cycle, and seed dispersal . Under natural conditions, CSR vegetative growth does not necessitate external light, and the entire growth process takes 4–5 years. However, through artificial cultivation, CSR can be harvested within 3–4 years . CSR seeds germinate under favorable conditions, producing a specialized “bud tube organ”. The terminal regions of this structure expand and adhere to the host plant’s root system, invading it. Once connected to the host plant’s vascular bundle, a parasitic relationship is established, leading to new gemmules . CSR is a perennial succulent parasitic herb with a reddish-brown coloration, predominantly submerged in sand and lacking chlorophyll . CSR possesses a few triangular scales in its middle and upper sections. Its inflorescence is terminal and clavate, adorned with scaly leaves, featuring bisexual flowers consisting of petals, stamens, and an ovary. Male flowers typically have four perianth pieces, while the pistil undergoes degradation. Female flowers exhibit a lower ovary with usually 5–6 perianth pieces enclosing one pendulous ovule at the apex, male flowers degenerate accordingly. The fruit resembles a nut . CSR is a rare and endangered medicinal plant mainly distributed in northwest China, such as Inner Mongolia (Red), Gansu (Blue), Xinjiang (Orange), Qinghai (Purple), and Ningxia (Yellow) . Growing in temperate and subtropical regions, it can grow in areas with lower elevations or up to 2000 m above sea level. It likes warm and humid climates, has strong adaptability, and can grow in areas with full or partial sunlight. The minimum temperature is generally required to be no lower than −10 °C. It can withstand high temperatures even in hotter summers. It can adapt to different types of soil but it is best to avoid excessively humid or poor soil. Wild CSR is the main source, with a domestic accumulation of about 30,000 tons. Under normal circumstances, about 1500 tons are harvested annually. However, the existing wild CSR resources are only concentrated in the Hedong sandy land of Pingluo County and their distribution area has been decreasing year by year . There are five host plants, namely Nitraria sphaerocarpa , Nitraria sibirica , Nitraria tangutorum , Zygophyllum xanthoxylon , and Peganum multisectum . The lifecycle of CSR mainly includes several stages: seed germination, parasitic localization, parasitic attachment, growth cycle, and seed dispersal . Under natural conditions, CSR vegetative growth does not necessitate external light, and the entire growth process takes 4–5 years. However, through artificial cultivation, CSR can be harvested within 3–4 years . CSR seeds germinate under favorable conditions, producing a specialized “bud tube organ”. The terminal regions of this structure expand and adhere to the host plant’s root system, invading it. Once connected to the host plant’s vascular bundle, a parasitic relationship is established, leading to new gemmules . The use of CSR in Asia has a long history, primarily for erectile dysfunction, premature ejaculation, and spermatogenesis enhancement. It was first recorded in Ben Cao Yan Yi Bu Yi (Yuan Dynasty, A.D. 1347). Later, the use of the plant was documented in other well-known medicinal works, including Ben Cao Meng Quan (Ming Dynasty, A.D. 1565), Ben Cao Gang Mu (Ming Dynasty, A.D. 1590), Ben Cao Qie Yao (Ming Dynasty, A.D. 1609), Ben Cao Bei Yao (Qing Dynasty, A.D. 1694). The traditional preparation of CSR primarily involves the formulation of pills, with notable examples being Huqian wan and Suoyang gujing wan. Its primary function lies in nourishing the kidneys and invigorating Yang, albeit with distinct effects . CSR is primarily utilized in clinical practice for the treatment of andrological and gynecological disorders. Its key therapeutic advantages include enhancing sexual function, regulating endocrine function, and promoting gastrointestinal health. Besides its significant medicinal value, it also finds application in culinary preparations such as steamed rice or pancakes when fresh. It is also incorporated into soups, wines, or health foods when dried. Prolonged consumption can improve immunity and prevent diabetes . Additionally, CSR can serve as a supplementary source of essential trace elements for the human body. CSR exhibits robust vitality and adaptability, thriving in arid deserts, rocky crevices, and harsh environments characterized by drought and wind erosion. It possesses significant ecological value in terms of enhancing environmental conditions, preserving soil stability, and maintaining ecological equilibrium . In recent years, a diverse range of potent chemical components have been isolated from various parts of the CSR plant. These components include flavonoids, triterpenoids, steroids, organic acids, sugars and glycosides, amino acids, and trace elements. Most of them have been extensively used in proprietary Chinese medicine and healthcare products. We classify the 98 compounds isolated and identified according to their types. The basic information and source areas of these compounds are summed up in , while their structures can be seen in , , , , , , and . 5.1. Flavonoids A class of natural compounds with a parent nucleus structure of 2-phenylchromogen (flavone) are called flavonoids, which are the main active ingredients in CSR. Their molecular basis as the antioxidant and anti-aging activities of CSR have been widely proven, and CSR flavonoids have also been found to be effective in antibacterial activity . Currently, a total of 27 types of flavonoids have been isolated from CSR sinensis. Phloridzin ( 1 ) , (−)-epicatechin ( 2 ), and naringenin ( 3 ) were obtained from the 70% acetone extract and chloroform extract of stems. Ethyl acetate extract was isolated from (−)-catechin ( 4 ) . Additionally, luteolin-7- O -glucoside ( 5 ) was isolated from the ethyl acetate fraction of the methanol extract derived from stems of CSR . Two anthocyanins, procyanidin B1 ( 6 ) and procyanidin B6 ( 7 ), were extracted from the aqueous extract of stems of CSR chinensis . The procyanidin B3 ( 8 ) was isolated and identified from the 70% acetone extract of fresh CSR stem through column chromatography. The identified compounds include catechin-(6′-8)-catechin ( 9 ), catechin-(6′-6)-catechin ( 10 ), epicatechin-(4 β -8)-epicatechin-(4 β -8)-catechin ( 11 ), epicatechin-(4 β -6)-epicatechin-(4 β -8)-catechin ( 12 ) and arecatannin A1 ( 13 ) . Furthermore, dehydrodiconiferyl alcohol-9- O - β -D-glu-copyranoside ( 14 ), 3′,4′,5,7-tetrahydroxy-flavanone-2(S)-3′- O - β -D-glucopyranoside ( 15 ), luteolin-4′- O - β -glucopyranoside ( 16 ), astragalin ( 17 ), quercetin-3- O -rutinoside ( 18 ), naringenin-7- O - β -D-glucopyranoside ( 19 ), naringenin-5- O - β -D-glucopyranoside ( 20 ) was isolated from the ethyl acetate fraction of 95% ethanol extract obtained from fresh stems of CSR and identified through NMR analysis . The compound naringenin-4′- O - β -pyranoglucose ( 21 ) was isolated from the n-butanol fraction of a 95% ethanol extract obtained from CSR whole grass . Two anthocyanin pigments were isolated from a 95% ethanol extract of CSR inflorescences. Cyanidin 3- O -glucoside ( 22 ) was identified as the predominant pigment, accounting for 92%, while cyanidin 3- O -rhamnosylglucoside ( 23 ) was identified as the minor component, comprising 8% . The compounds -catechin ( 24 ), isoquercetin ( 25 ), rutin ( 26 ), and (−)-epicatechin-3- O -gallate ( 27 ) were isolated from ethanol extract of CSR inflorescences . 5.2. Terpenoids Terpenoids, composed of isoprene polymers as the basic skeleton, exhibit diverse structures and functions in different plants. These compounds influence plant odor and flavor. Therefore, terpenoids have significant application value in fields such as natural drugs and spices. The studies have demonstrated that terpenoids are the secondary metabolites of CSR. Twelve terpenoids were isolated, which were as follows. Malonyl ursolic acid hemiester ( 28 ), ursolic acid ( 29 ), acetyl ursolic acid ( 30 ), oleanolic acid ( 31 ), betulinic acid ( 32 ) , and malonyl oleanolic acid hemiester ( 33 ) were isolated from dichloromethane extract of CSR stem. The glutaryl ursolic acid hemiester ( 34 ), oxalyl ursolic acid hemiester ( 35 ), succinyl ursolic acid hemiester ( 36 ), and ursolic acid methyl ester ( 37 ) were isolated from the ethyl acetate extract of CSR stem . Additionally, a diterpenoid compound 3 β , 28-dihydroxyoleana-11,13(18)-diene ( 38 ) was isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from the CSR stem . The isolation of maslinic acid ( 39 ) was achieved from the aqueous extract of CSR . The terpenoids discovered in CSR malonyl ursolic acid hemiester, ursolic acid, acetyl ursolic acid, and malonyl oleanolic acid hemiester are commonly occurring triterpenes that can also be found in other plant species . 5.3. Steroids The tetracyclic structure of cycloalkyl polyhydrophenanthrene is the parent nucleus of this class of compounds known as steroids. There are several substances found in fauna and flora, including cholesterol, steroid hormones (such as estrogen, androgen, and adrenal corticosteroids), and sterols. These substances play a crucial role in physiological functions with a wide range of biological functions. Ten steroid compounds were isolated from CSR was achieved. The compounds 5 α -Stigmast-9(11)-en-3 β -ol ( 40 ) and 5 α -Stigmast-9(11)-en-3 β -ol tetracosatrienoic acid ester ( 41 ) were isolated from ethyl acetate extract of stems of CSR . Daucosterol ( 42 ) and β -sitosterol ( 43 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained stem of CSR . The β -sitosteryl oleate ( 44 ), β -sitosteryl glucoside ( 45 ), and β -sitosteryl glucoside 6′- O -aliphatates ( 46 ) were isolated from the dichloromethane extract of CSR stem . Furthermore, β -sitosterol palmaitate ( 47 ) was isolated from the chloroform extract . The identification and analysis of campesterol ( 48 ) and γ -sitosterol ( 49 ) were conducted using Gas chromatography–mass spectrometry (GC-MS) in addition to other techniques . 5.4. Saccharides and Glycosides Saccharides are one kind of important bioactive compounds in CSR, which exhibit diverse biological and pharmacological activities. These two polysaccharides consist of galactose, glucose, arabinose, rhamnose, mannose, ribose, and uronic acid. Among these components, the latter two ingredients account for 10.7% and 10.5%, respectively . By report, a water-soluble heteropolysaccharide called CSPA, which is a heteropolysaccharide composed of arabinose (Ara), glucose (Glu), and galactose (Gal), was isolated from CSR. It has a molecular weight of 1.394 × 10 5 Da. The chemical structure consisted of the following units: “→3)- α -araf-(1→3)- α -d-glcp-(1→4)- α -d-GalpA6Me-(1→” . The polysaccharide extracted from CSR was fractionated into three components, namely CSG-F1, CSG-F2, and CSG-F3. CSG-F1 (yield of 21%) exhibited an average molecular weight of approximately 2.4 × 10 5 Da and primarily consisted of galactose, glucose, arabinose, and rhamnose. Similarly, the purified CSG-F2 (yield of 14%) displayed an average molecular weight of around 1.3 × 10 5 Da and contained galactose, glucose, arabinose, rhamnose, and ribose as its main constituents. Lastly, the purified CSG-F3 (yield of 37%) had an estimated average molecular weight of about 1.9 × 10 5 Da and glucose, arabinose, rhamnose, ribose, and mannose . The isolation of thirteen sugars and glycosides from CSR was achieved. Glucose ( 50 ) was isolated from the chloroform extract of CSR stem . Zingerone 4- O - β -D-glucopyranosid ( 51 ) was isolated from the dichloromethane extract of CSR stem. Three fructosides were isolated from the ethyl acetate extract. The structures of n -butyl- β -D-fructofuranoside ( 52 ) , n -butyl- α -D-fructofuranoside ( 53 ) , and n -butyl- β -D-fructopyranoside ( 54 ) were determined using spectroscopic methods. The compound piceid ( 55 ) was obtained from the ethyl acetate fraction of the methanol extract, while coniferin ( 56 ) and isoconiferin ( 57 ) were isolated from the n-butanol fraction. The isolation of adenosine ( 58 ) was achieved from the n-butanol fraction of the CSR methanol extract. The compounds (−)-isolariciresinol 4- O - β -D-glucopyranoside ( 59 ) and (7S,8R)-dehydrodiconiferyl alcohol 9′- β -glucopyranoside ( 60 ) were isolated from the aqueous extract . The compound nicoloside ( 61 ) was isolated from the aqueous fraction of the methanol extract obtained from CSR. Furthermore, songaricumone A ( 62 ) was isolated from the ethyl acetate fraction of 95% ethanol extract obtained from fresh stems of CSR and identified through NMR analysis . 5.5. Organic Acids and Organic Acid Ester Organic acids and esters can serve as carriers for drug delivery systems, improving the physical stability and solubility of drugs, regulating lipid metabolism, participating, and regulating various physiological processes as signaling molecules, and having anti-inflammatory and antibacterial effects. One of the important active ingredients of CSR is an acidic organic compound called organic acid. At present, 17 distinct types of organic acids and organic acid ester have been successfully isolated from CSR. Protocatechuic acid ( 63 ), gallic acid ( 64 ), n -butyric acid ( 65 ) , and 4-methoxycinnamic acid ( 66 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from stems of CSR. The compounds p -hydroxybenzoic acid ( 67 ) , methyl protocatechuicate ( 68 ) and p -hydroxybenzoic acid ( 69 ) , were isolated from the ethyl acetate fraction of the methanol extract. The compounds 3,4-dihydroxybenzoic acid ethyl ester ( 70 ) , 4-hydroxyphenethyl 2-(4-hydroxyphenyl) acetate ( 71 ) , and stearic acid α -monoglyceride ( 72 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from stems of CSR. The water parts are separated by succinic acid ( 73 ) . The compounds ferulic acid ( 74 ) were isolated from a 70% ethanol extract of stems of CSR. Additionally, gentisic acid ( 75 ), palmitic acid ( 76 ), and 3,4-dihydroxyphenethyl acetate ( 77 ) were obtained from a water extract . The vanillic acid ( 78 ) was extracted from an aqueous solution of 95% ethanol extract from whole grass, while the capilliplactone ( 79 ) was isolated from the ethyl acetate fraction. The structure was determined using spectroscopic techniques . 5.6. Phloroglucinol Adducts A type of compound formed by three hydroxyl groups (-OH) to replace the hydrogens at the 1,3,5 positions in benzene, is called phloroglucinol. It is a drug widely used in the medical field. Belonging to the class of phenobarbital drugs, it has pharmacological effects such as sedation, hypnosis, anticonvulsant, and antianxiety. It is considered an important drug that can be used to treat various diseases and symptoms. Six types of phloroglucinol compounds were isolated from CSR. The identification of three phloroglucinol compounds was achieved from the 70% acetone extract of fresh stems of CSR using LC-MS and HPLC retention time analysis. These compounds were identified as epicatechin-(4 β -2)-phloroglucinol ( 80 ), epicatechin-3- O -gallate-(4 β -2)-phloroglucinol ( 81 ) and catechin-(4 α -2)-phloroglucinol ( 82 ) . Two new compounds were recently isolated from a degraded mixture of cynomoriitannin and identified as cynomoriitannin-phloroglucinol A ( 83 ) and cynomoriitannin-phloroglucinol B ( 84 ) based on spectroscopic analyses . Phloroglucinol ( 85 ) was isolated from the stem’s aqueous extract of CSR . 5.7. Other Compounds The qualitative and quantitative analysis of amino acids revealed the presence of 20 common amino acids in CSR, which serve as essential nutritional elements for the human body . Furthermore, volatile components , trace elements , and tannins play a significant role in the pharmacological activity of CSR. Mannitol ( 86 ) was isolated from the aqueous extract . Protocatechualdehyde ( 87 ), chrysophanol ( 88 ), emodin ( 89 ), and physcion ( 90 ) were isolated from the 70% ethanol extract . The compounds (−)-lariciresinol ( 91 ) and 4-methylcatechol ( 92 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from the CSR stem. Additionally, the following compounds were also identified: 4 β -(L-cysteinyl)-catechin ( 93 ), 4 β -(L-cysteinyl)-epicatechin ( 94 ), 4 β -(L-cysteinyl)-epicatechin 3- O -gallate ( 95 ) three cysteine conjugates , 4 β -(L-acetylcysteinyl)-epicatechin ( 96 ), 4 β -(L-acetylcysteinyl)-epicatechin 3- O -gallate ( 97 ), 4 β -(L-acetylcysteinyl)-epiafzelechin ( 98 ) three acetylcysteine conjugates , and edible reagents from CSR were isolated and purified. The structures were elucidated via a combination of NMR and mass spectrometry techniques . A class of natural compounds with a parent nucleus structure of 2-phenylchromogen (flavone) are called flavonoids, which are the main active ingredients in CSR. Their molecular basis as the antioxidant and anti-aging activities of CSR have been widely proven, and CSR flavonoids have also been found to be effective in antibacterial activity . Currently, a total of 27 types of flavonoids have been isolated from CSR sinensis. Phloridzin ( 1 ) , (−)-epicatechin ( 2 ), and naringenin ( 3 ) were obtained from the 70% acetone extract and chloroform extract of stems. Ethyl acetate extract was isolated from (−)-catechin ( 4 ) . Additionally, luteolin-7- O -glucoside ( 5 ) was isolated from the ethyl acetate fraction of the methanol extract derived from stems of CSR . Two anthocyanins, procyanidin B1 ( 6 ) and procyanidin B6 ( 7 ), were extracted from the aqueous extract of stems of CSR chinensis . The procyanidin B3 ( 8 ) was isolated and identified from the 70% acetone extract of fresh CSR stem through column chromatography. The identified compounds include catechin-(6′-8)-catechin ( 9 ), catechin-(6′-6)-catechin ( 10 ), epicatechin-(4 β -8)-epicatechin-(4 β -8)-catechin ( 11 ), epicatechin-(4 β -6)-epicatechin-(4 β -8)-catechin ( 12 ) and arecatannin A1 ( 13 ) . Furthermore, dehydrodiconiferyl alcohol-9- O - β -D-glu-copyranoside ( 14 ), 3′,4′,5,7-tetrahydroxy-flavanone-2(S)-3′- O - β -D-glucopyranoside ( 15 ), luteolin-4′- O - β -glucopyranoside ( 16 ), astragalin ( 17 ), quercetin-3- O -rutinoside ( 18 ), naringenin-7- O - β -D-glucopyranoside ( 19 ), naringenin-5- O - β -D-glucopyranoside ( 20 ) was isolated from the ethyl acetate fraction of 95% ethanol extract obtained from fresh stems of CSR and identified through NMR analysis . The compound naringenin-4′- O - β -pyranoglucose ( 21 ) was isolated from the n-butanol fraction of a 95% ethanol extract obtained from CSR whole grass . Two anthocyanin pigments were isolated from a 95% ethanol extract of CSR inflorescences. Cyanidin 3- O -glucoside ( 22 ) was identified as the predominant pigment, accounting for 92%, while cyanidin 3- O -rhamnosylglucoside ( 23 ) was identified as the minor component, comprising 8% . The compounds -catechin ( 24 ), isoquercetin ( 25 ), rutin ( 26 ), and (−)-epicatechin-3- O -gallate ( 27 ) were isolated from ethanol extract of CSR inflorescences . Terpenoids, composed of isoprene polymers as the basic skeleton, exhibit diverse structures and functions in different plants. These compounds influence plant odor and flavor. Therefore, terpenoids have significant application value in fields such as natural drugs and spices. The studies have demonstrated that terpenoids are the secondary metabolites of CSR. Twelve terpenoids were isolated, which were as follows. Malonyl ursolic acid hemiester ( 28 ), ursolic acid ( 29 ), acetyl ursolic acid ( 30 ), oleanolic acid ( 31 ), betulinic acid ( 32 ) , and malonyl oleanolic acid hemiester ( 33 ) were isolated from dichloromethane extract of CSR stem. The glutaryl ursolic acid hemiester ( 34 ), oxalyl ursolic acid hemiester ( 35 ), succinyl ursolic acid hemiester ( 36 ), and ursolic acid methyl ester ( 37 ) were isolated from the ethyl acetate extract of CSR stem . Additionally, a diterpenoid compound 3 β , 28-dihydroxyoleana-11,13(18)-diene ( 38 ) was isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from the CSR stem . The isolation of maslinic acid ( 39 ) was achieved from the aqueous extract of CSR . The terpenoids discovered in CSR malonyl ursolic acid hemiester, ursolic acid, acetyl ursolic acid, and malonyl oleanolic acid hemiester are commonly occurring triterpenes that can also be found in other plant species . The tetracyclic structure of cycloalkyl polyhydrophenanthrene is the parent nucleus of this class of compounds known as steroids. There are several substances found in fauna and flora, including cholesterol, steroid hormones (such as estrogen, androgen, and adrenal corticosteroids), and sterols. These substances play a crucial role in physiological functions with a wide range of biological functions. Ten steroid compounds were isolated from CSR was achieved. The compounds 5 α -Stigmast-9(11)-en-3 β -ol ( 40 ) and 5 α -Stigmast-9(11)-en-3 β -ol tetracosatrienoic acid ester ( 41 ) were isolated from ethyl acetate extract of stems of CSR . Daucosterol ( 42 ) and β -sitosterol ( 43 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained stem of CSR . The β -sitosteryl oleate ( 44 ), β -sitosteryl glucoside ( 45 ), and β -sitosteryl glucoside 6′- O -aliphatates ( 46 ) were isolated from the dichloromethane extract of CSR stem . Furthermore, β -sitosterol palmaitate ( 47 ) was isolated from the chloroform extract . The identification and analysis of campesterol ( 48 ) and γ -sitosterol ( 49 ) were conducted using Gas chromatography–mass spectrometry (GC-MS) in addition to other techniques . Saccharides are one kind of important bioactive compounds in CSR, which exhibit diverse biological and pharmacological activities. These two polysaccharides consist of galactose, glucose, arabinose, rhamnose, mannose, ribose, and uronic acid. Among these components, the latter two ingredients account for 10.7% and 10.5%, respectively . By report, a water-soluble heteropolysaccharide called CSPA, which is a heteropolysaccharide composed of arabinose (Ara), glucose (Glu), and galactose (Gal), was isolated from CSR. It has a molecular weight of 1.394 × 10 5 Da. The chemical structure consisted of the following units: “→3)- α -araf-(1→3)- α -d-glcp-(1→4)- α -d-GalpA6Me-(1→” . The polysaccharide extracted from CSR was fractionated into three components, namely CSG-F1, CSG-F2, and CSG-F3. CSG-F1 (yield of 21%) exhibited an average molecular weight of approximately 2.4 × 10 5 Da and primarily consisted of galactose, glucose, arabinose, and rhamnose. Similarly, the purified CSG-F2 (yield of 14%) displayed an average molecular weight of around 1.3 × 10 5 Da and contained galactose, glucose, arabinose, rhamnose, and ribose as its main constituents. Lastly, the purified CSG-F3 (yield of 37%) had an estimated average molecular weight of about 1.9 × 10 5 Da and glucose, arabinose, rhamnose, ribose, and mannose . The isolation of thirteen sugars and glycosides from CSR was achieved. Glucose ( 50 ) was isolated from the chloroform extract of CSR stem . Zingerone 4- O - β -D-glucopyranosid ( 51 ) was isolated from the dichloromethane extract of CSR stem. Three fructosides were isolated from the ethyl acetate extract. The structures of n -butyl- β -D-fructofuranoside ( 52 ) , n -butyl- α -D-fructofuranoside ( 53 ) , and n -butyl- β -D-fructopyranoside ( 54 ) were determined using spectroscopic methods. The compound piceid ( 55 ) was obtained from the ethyl acetate fraction of the methanol extract, while coniferin ( 56 ) and isoconiferin ( 57 ) were isolated from the n-butanol fraction. The isolation of adenosine ( 58 ) was achieved from the n-butanol fraction of the CSR methanol extract. The compounds (−)-isolariciresinol 4- O - β -D-glucopyranoside ( 59 ) and (7S,8R)-dehydrodiconiferyl alcohol 9′- β -glucopyranoside ( 60 ) were isolated from the aqueous extract . The compound nicoloside ( 61 ) was isolated from the aqueous fraction of the methanol extract obtained from CSR. Furthermore, songaricumone A ( 62 ) was isolated from the ethyl acetate fraction of 95% ethanol extract obtained from fresh stems of CSR and identified through NMR analysis . Organic acids and esters can serve as carriers for drug delivery systems, improving the physical stability and solubility of drugs, regulating lipid metabolism, participating, and regulating various physiological processes as signaling molecules, and having anti-inflammatory and antibacterial effects. One of the important active ingredients of CSR is an acidic organic compound called organic acid. At present, 17 distinct types of organic acids and organic acid ester have been successfully isolated from CSR. Protocatechuic acid ( 63 ), gallic acid ( 64 ), n -butyric acid ( 65 ) , and 4-methoxycinnamic acid ( 66 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from stems of CSR. The compounds p -hydroxybenzoic acid ( 67 ) , methyl protocatechuicate ( 68 ) and p -hydroxybenzoic acid ( 69 ) , were isolated from the ethyl acetate fraction of the methanol extract. The compounds 3,4-dihydroxybenzoic acid ethyl ester ( 70 ) , 4-hydroxyphenethyl 2-(4-hydroxyphenyl) acetate ( 71 ) , and stearic acid α -monoglyceride ( 72 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from stems of CSR. The water parts are separated by succinic acid ( 73 ) . The compounds ferulic acid ( 74 ) were isolated from a 70% ethanol extract of stems of CSR. Additionally, gentisic acid ( 75 ), palmitic acid ( 76 ), and 3,4-dihydroxyphenethyl acetate ( 77 ) were obtained from a water extract . The vanillic acid ( 78 ) was extracted from an aqueous solution of 95% ethanol extract from whole grass, while the capilliplactone ( 79 ) was isolated from the ethyl acetate fraction. The structure was determined using spectroscopic techniques . A type of compound formed by three hydroxyl groups (-OH) to replace the hydrogens at the 1,3,5 positions in benzene, is called phloroglucinol. It is a drug widely used in the medical field. Belonging to the class of phenobarbital drugs, it has pharmacological effects such as sedation, hypnosis, anticonvulsant, and antianxiety. It is considered an important drug that can be used to treat various diseases and symptoms. Six types of phloroglucinol compounds were isolated from CSR. The identification of three phloroglucinol compounds was achieved from the 70% acetone extract of fresh stems of CSR using LC-MS and HPLC retention time analysis. These compounds were identified as epicatechin-(4 β -2)-phloroglucinol ( 80 ), epicatechin-3- O -gallate-(4 β -2)-phloroglucinol ( 81 ) and catechin-(4 α -2)-phloroglucinol ( 82 ) . Two new compounds were recently isolated from a degraded mixture of cynomoriitannin and identified as cynomoriitannin-phloroglucinol A ( 83 ) and cynomoriitannin-phloroglucinol B ( 84 ) based on spectroscopic analyses . Phloroglucinol ( 85 ) was isolated from the stem’s aqueous extract of CSR . The qualitative and quantitative analysis of amino acids revealed the presence of 20 common amino acids in CSR, which serve as essential nutritional elements for the human body . Furthermore, volatile components , trace elements , and tannins play a significant role in the pharmacological activity of CSR. Mannitol ( 86 ) was isolated from the aqueous extract . Protocatechualdehyde ( 87 ), chrysophanol ( 88 ), emodin ( 89 ), and physcion ( 90 ) were isolated from the 70% ethanol extract . The compounds (−)-lariciresinol ( 91 ) and 4-methylcatechol ( 92 ) were isolated from the ethyl acetate fraction of a 95% ethanol extract obtained from the CSR stem. Additionally, the following compounds were also identified: 4 β -(L-cysteinyl)-catechin ( 93 ), 4 β -(L-cysteinyl)-epicatechin ( 94 ), 4 β -(L-cysteinyl)-epicatechin 3- O -gallate ( 95 ) three cysteine conjugates , 4 β -(L-acetylcysteinyl)-epicatechin ( 96 ), 4 β -(L-acetylcysteinyl)-epicatechin 3- O -gallate ( 97 ), 4 β -(L-acetylcysteinyl)-epiafzelechin ( 98 ) three acetylcysteine conjugates , and edible reagents from CSR were isolated and purified. The structures were elucidated via a combination of NMR and mass spectrometry techniques . Scholars have combined traditional Chinese medicine with modern medicinal chemistry to explore the biological activity of chemical components in traditional Chinese medicine. Alcohol and water extracts exhibit significant pharmacological activities, including anti-oxidant and anti-tumor effects, among others ( and ). 6.1. Anti-Tumor Effects Inducing apoptosis serves as a method for preventing and treating tumors as it plays an essential role in tumor progression . Cancer stem cells are inhibited from proliferating and dying when exposed to CSR. It can be used to treat malignant tumors such as breast cancer, leukemia, colon cancer, and others. 6.1.1. Anti-Cancer There are four breast cancer cell lines inhibited by CSR extracts and its ethyl acetate extraction site, including MDA-MB-231 , MCF-7 , MB468 , and 4T1 . Furthermore, CSR extract induces Foxo3 expression in apoptosis and prevents the transition from G1 to S phases . It has been found that chloroform and ethyl acetate extraction sites from the CSR ethanol extract are capable of inhibiting the proliferation of the colon adenocarcinoma cell line Caco-2 . In cell research for cervical cancer treatment, Cynomrium songaricum polysaccharides (CSP) inhibit proliferative activity in HeLa cells . A further study showed that both methanol extract and anthocyanin 3- O -glucoside from CSR inhibited KBWT cell proliferation in a dose-dependent manner . By inhibiting telomerase reverse transcriptase (TERT) mRNA, CSP induced apoptosis in A549 cells . Methanol extract and aqueous extract from CSR inhibited the growth of B16 cells, which are used for studying skin cancer in humans . Further, CSR ethyl acetate extract inhibited both LNCaP and HepG2 cells, showing that it may have therapeutic effects on prostate cancer and liver cancer . Research has shown that the anticancer ingredients in CSR are concentrated in the ethyl acetate extraction site, and it may be related to activating and enhancing autophagy processes in cells to trigger. In autophagy and apoptotic cell death, mitochondrial-related proteins Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) and Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) play a critical role . 6.1.2. Leukemia CCRF-CEM and CCRF-SB cells were inhibited by methanol extract and anthocyanin 3- O -glucoside from CSR . Similarly, mitochondrial pathways modulate caspase-3 activity. Therefore, CSR ethanol extract causes apoptosis in leukemia cells by causing apoptosis in HL-60 cells . The above studies indicate that CSR has a certain inhibitory effect on two types of tumor cells, cancer, and leukemia. In cancer, it inhibits the growth of cancer cells by inducing the expression of Foxo3 and inhibiting telomerase reverse transcriptase mRNA to activate mitochondrial-related proteins BNIP3 and BNIP3L. Regulating caspase-3 activity through the mitochondrial pathway in leukemia induces cell apoptosis. In contrast, there is more research data on adenocarcinoma and less research on leukemia. However, it cannot be concluded with certainty that CSR has a better therapeutic effect on cancer than on leukemia. Therefore, more in-depth research is still needed . 6.2. Anti-Oxidation Function Different parts of CSR have different antioxidant activities when extracted from methanol. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals are best scavenged in the central part, hydroxyl radicals are best inhibited in the lower part, and superoxide anions are highly resisted in the upper part . Multiple solvent extracts of CSR exhibit antioxidant activity. A methanol extract of the CSR and an ethyl acetate extraction site strongly inhibit superoxide anions . DPPH radicals, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals, and hydroxyl radicals are also scavenged by the aqueous extract and ethyl acetate extraction site . Additionally, the aqueous extract was able to scavenge DPPH free radicals and inhibit superoxide anion formation . Different extracts exhibit significantly different antioxidant and radical scavenging properties. CSR aqueous extract scavenges DPPH free radicals and nitrates more effectively than ethanol extract. In contrast, ethanol extract inhibits xanthine oxidase (XO) and superoxide dismutase (SOD) more effectively than aqueous extract . To examine the categories of substances with superior antioxidant effects, compounds of the same type extracted from CSR were compared to their antioxidant activity. This was performed to clarify the strong antioxidant activity of CSR extract. Within a certain concentration range, CSP exhibits effective scavenging ability against superoxide anion radicals, DPPH radicals, and hydroxyl radicals . CSR flavonoids also scavenge DPPH and hydroxyl radicals . Crude polyphenols exhibited significantly higher antioxidant activity than crude polysaccharides when measured against DPPH radicals, ABTS free radicals, and crude polysaccharides in CSR . According to another study, microwave-extracted procyanidins exhibited superior scavenging activity against DPPH and hydroxyl free radicals . The main antioxidant component in the CSR ethyl acetate extraction site is catechin, which was isolated from protocatechuic acid, gallic acid, and catechins . The aqueous extract of CSR was separated into catechin, epicatechin, and olive saponin to determine the DPPH free radical scavenging capacity . In vivo experiments were used to verify the CSR extract’s significant antioxidant activity. CSR extracts (0.22 g/kg, 0.44 g/kg, 0.88 g/kg) can enhance the serum DPPH free radical scavenging ability of KM mice, and reduce oxidative damage caused by free radicals and lipid peroxides . Some in vivo and in vitro experiments have shown that the antioxidant components in CSR exert antioxidant effects by clearing free radicals, as well as inhibiting XO and SOD, etc. The antioxidant activity of CSR is one of its main functions, which can slow down the oxidative state of the body and fight against diseases . 6.3. Anti-Aging Effects Several studies have demonstrated the anti-aging effects of CSR through a variety of mechanisms. According to reports, adding CSR to the diet can extend the average and maximum lifespans of adult female flies. Ethanol extract of CSR suppresses age-related learning disabilities in elderly flies by reducing hydrogen peroxide levels and increasing antioxidants, extending their lifespan, improving mating readiness, increasing fertility, and inhibiting age-related learning disabilities . A transcriptome sequencing study found that CSR extract impacted wild-type Caenorhabditis elegans aging. The lifespan of Caenorhabditis elegans was extended and motor abilities were enhanced by ethyl acetate extract (0.4 mg/mL). Multiple pathways and genes collaborate to produce the effects of the ethyl acetate extract . Various research results have shown that extracts, CSP, and preparations from CSR can delay aging by inhibiting telomere length shortening , enhancing telomerase activity , improving immune function , inhibiting neuronal apoptosis , improving hippocampal CA1 neurons , enhancing antioxidant capacity . The aqueous extract of CSR can also improve the energy metabolism of liver mitochondria in aging model KM mice. It can also alleviate free radical damage to mitochondrial membrane structure and function and play a role in delaying aging . These findings not only reveal the potential of the polysaccharide and extract in combating aging but also lay the groundwork for future clinical research. It would be beneficial to further investigate the chemical composition of CSR and the mechanism underlying anti-aging as well as their safety and effectiveness to offer novel insights and possibilities for delaying the aging process in the future . 6.4. Anti-Fatigue and Anti-Hypoxia Activities The aqueous extract and ethanol extract of CSR are responsible for its anti-fatigue properties. By lowering the lactate index , inhibiting amino acid protein breakdown, and increasing glycogen reserves , they can improve energy metabolism. It also possesses the ability to increase the level of cyclic adenosine monophosphate (cAMP), reduce cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP) ratio , improve free radical metabolism . In addition, CSR flavonoids (CSF) reduce MAO activity and reactive oxygen species (ROS) levels by improving free radical metabolism . Oxygen deficiency can cause abnormal tissue metabolism, function, and morphology. The main cause of death is hypoxia of the brain and heart. CSR aqueous extract has positive atmospheric pressure anti-hypoxia and anti-acute cerebral ischemia and hypoxia effects , which increases blood hemoglobin content and enhances oxygen-carrying function . As well as reducing brain edema, it increases myocardial protein content . CSR exhibits remarkable anti-fatigue and anti-hypoxia properties. Research on anti-fatigue effects focuses on its active ingredients, such as water extract, ethanol extract, and flavonoids. Currently, hypoxic resistance studies are primarily focused on CSR water extract. Developing highly potent and pharmaceutically viable compounds from CSR for anti-fatigue and anti-hypoxia purposes will require further investigation . 6.5. Effects on Nervous System Ethyl acetate extract and methanol extract are both effective against A β 25–35 , hypoxanthine/xanthine oxidase (HPX/XO) , Xanthine dehydrogenase/xanthine oxidase (XDH/XO) induced SK-N-SH cells have protective effects. Among them, ethyl acetate extract is more effective against Amyloid β -Protein 25–35 (A β 25–35 ) and has an anti-Starosporin-induced injury effect . CSP and ethyl acetate extraction sites of CSR can protect PC12 cells against damage by H 2 O 2 and A β 25–35 . Ethyl acetate extract has cytotoxicity to Neuro2A cells (EC 50 = 116 mg/L) and increases the expression of synaptophysin through the mitogen-activated protein kinases (MAPK) pathway . In another study, the methanol extract of CSR inhibits A β 25–35 induced phosphorylation of dynamin-related protein 1 (Drp1) at Ser637 in HT22 cells and reduced the expression of Fission 1 Protein (Fis1) in H 2 O 2 induced model for the treatment of Alzheimer’s disease (AD) . Based on neuroprotective effects at the cellular level, scholars have further explored them through animal models. The ethyl acetate fraction of CSR improves the behavior of C57BL/6 male mice by reducing mitochondrial dynamics imbalance. It also downregulated the expression of the Drp1 protein and upregulated the expression of Optic Atrophy 1 (OPA1) and Mito Fusin 1 (MFN1) proteins . It also improves the spatial memory and learning ability of AD model mice by regulating fecal microbiota disorder . In the ovariectomized Sprague–Dawley (SD) rat model, it increased the expression of Growth-Associated Protein 43 (GAP-43) protein in the hippocampus , regulated the MAPK pathway, increased the expression of phosphorylation-cAMP response element-binding protein (p-CREB), and decreased the expression of p38, thereby promoting the survival and repair of hippocampal neurons. Other studies have shown that CSR ethyl acetate extract increases the expression levels of synaptic plasticity-related proteins Syn and postsynaptic density protein-95 (PSD-95) while upregulating the protein expression levels of phosphor-extracellular regulated protein kinases 1/2 (P-Erk1/2) and P-CREB in the MAPK/ERK1/2 signaling pathway . It increases the effect of Long-term Potential (LTP) in Morris water maze and neuroelectrophysiology, further improving cognitive dysfunction in chronic stress Institute of Cancer Research (ICR) mice after ovariectomy . The ethanol extract of CSR increased cAMP response element-binding protein /Brain-Derived Neurotrophic Factor (CREB/BDNF) expression in ovariectomized SD rats by inhibiting the p38MAPK/ERK pathway . It also reduced serum corticosterone levels, increased the expression of BDNF mRNA in this region, promoted the proliferation of mouse dentate gyrus cells and differentiation of neuroblasts, enhanced the potential for hippocampal plasticity in male C57BL/6J mice , and thus achieved neuroprotective effects on the nerves. The aqueous extract of CSR has a significant improvement effect on the learning and memory of scopolamine-induced KM male mice. Its mechanism may be related to reducing oxidative stress in brain tissue . In Wistar male rats, through upregulating the Brain-Derived Neurotrophic Factor/Tyrosine Kinase receptor B (BDNF/TrkB) signaling pathway, enhancing cognitive function, increasing acetylcholine (ACH) content in the central cholinergic system, inhibiting cell apoptosis, and enhancing synaptic plasticity, CSF improves the AD model induced by A β 1–42 . In addition, CSF inhibits oxidative stress and inflammatory reactions. It can also downregulate the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ROS, and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in the hippocampus, exerting neuroprotective effects . The main component of CSR, ursolic acid, at a concentration of 5–15 µM, can effectively protect SD rat hippocampal neurons from damage induced by kainic acid by regulating α -amino-3-hydroxy-5-methy1-4-isoxazole propionic acid (AMPA) receptors, protecting mitochondria, and reducing free radical generation . In summary, the neuroprotective active ingredients are mainly concentrated in the methanol, ethanol, and ethyl acetate extracts of CSR. However, the main components that play a key neuroprotective role are not yet clear because of the complex components in CSR extract. Therefore, future studies should explore compounds that play a major role in protecting the nervous system . 6.6. Effects on Reproductive System In geriatrics, benign prostatic hyperplasia (BPH) is a common genitourinary disorder characterized by prostate gland enlargement and urinary dysfunction . There is an inhibitory effect of ethanol extract of CSR (2.5 mg/mL) on testosterone 5 α -reductase . Moreover, it interferes with estrogen/androgen signals to inhibit prostate hyperplasia in Wistar rats and improves the disorder of prostate epithelial cells and abnormal proliferation of connective tissue in Wistar rats with BPH model, inhibiting Proliferating Cell Nuclear Antigen (PCNA), Androgen Receptor (AR), and estrogen receptor α (Er α ) Protein expression while promoting estrogen receptor β (ER β ) Protein expression while promoting ER β Protein expression . Meanwhile, Wistar male rat protein expression of prostate AR, ER α / β, and 3-oxo-5-alpha-steroid 4-dehydrogenase 1/2 (SRD5A1/2) were regulated to inhibit BPH . Additionally, CSR aqueous extract inhibits prostate hyperplasia, increases SOD and glutathione (GSH) activities, decreases malondialdehyde (MDA) content, and significantly reduces prostate wet weight and prostate index by improving testosterone propionate-induced oxidative stress levels . In vitro experiments, Luteolin, Gallic acid, Ferulic acid, Protocatechualdehyde from CSR suppressed BPH by downregulating the expression of AR and ER α in BPH-1 cells and upregulation ER β expression . CSRs containing luteolin, epicatechin, and epicatechin gallate all improve the contractility of Wistar male rats’ bladder detrusors . Infertility in men is complex and multifactorial, with idiopathic infertility accounting for approximately 30% of cases . It has been shown that CSR improves sexual hormone levels as a kidney tonifying traditional Chinese medicine . Under the intervention of CSR aqueous extract, serum testosterone and Follicle-stimulating Hormone (FSH) levels are reduced, and interstitial cell-stimulating hormone (ICSH) levels are increased to directly affect the spermatogenic effect of immature seminiferous tubules of Wistar rats . It can also promote the secretion of testosterone in SD rats and inhibit abnormal secretion of FSH and Luteinizing hormone (LH) by regulating gonadal hormone levels . Glial Cell Line-derived Neurotrophic Factor (GDNF) production in testes of SD rats and undifferentiated spermatogonia proliferation stimulates, increases testosterone levels, and improves sperm motility . Relieving sperm damage and serum testosterone levels are increased through the MAPK-3-mediated GDNF signaling pathway, thereby enhancing sperm motility . In addition, enhancing sperm production in Wistar rats and upregulating the expression pathway of GDNF in the testes to improve male fertility , enhancing sperm production in golden hamsters, and blocking the impact of short photoperiod on reproductive function by CSR aqueous extract. In summary, active compounds from CSR that inhibit BPH mainly exist in its ethanol extract, while the active ingredients that promote spermatogenesis are mainly concentrated in the aqueous extract, which has been proven to have good therapeutic effects in treating male infertility . 6.7. Anti-Virus Despite a significant increase in the number of approved antiviral drugs, these existing drugs are not always effective or well tolerated. It is becoming increasingly common for viruses to develop drug resistance. As of now, many polysaccharides have been approved as drugs as independent or major bioactive components . The methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of CSP on MT-4 cells, which showed that only sulfated polysaccharides (SCSP-M, SCSP-1, SCSP-2) are anti-HIV. Due to the interaction between sulfated polysaccharides and poly L-lysine, sulfated polysaccharides have antiviral properties . In addition to the CSP, the triterpenoids contained in CSR also have antiviral activity. Ursolic acid, half ursolic malonate, malonyl oleanolic acid hemiester , acetyl ursolic acid, and condensed tannin extracted from CSR all have the function of inhibiting human immunodeficiency virus (HIV) protease . Furthermore, triterpenoids in CSR also have inhibitory activity against hepatitis C virus (HCV) protease, with malonyl ursolic acid hemiester having the maximum inhibitory effect . The main components of CSR are polysaccharides and triterpenoids, which are potentially useful for developing antiviral drugs. Additionally, it is worth noting that the antiviral efficacy of CSR has predominantly been tested in vitro with limited reports on its in vivo effects. Consequently, the precise mechanism by which CSR is antiviral remains unclear. Future studies should explore this aspect further to uncover the antiviral mechanism of CSR and establish solid foundations for its application . 6.8. Anti-Diabetic Properties CSP as one of the pivotal active constituents in CSR, exhibits significant therapeutic effects on several diseases. Consequently, CSR is being considered a potential candidate for the development of novel anti-diabetic drugs . Oral administration of CSR water-soluble polysaccharide (CSPA) significantly reduced the blood glucose level, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, blood urea nitrogen, creatinine activity in streptozotocin (STZ) induced diabetes model rats, effectively increased the serum insulin level and liver glycogen content and promoted the recovery of pancreatic islet cells in the pancreas to near normal levels . CSP (300 mg/kg) can upregulate the expression of protein kinase B (AKT) and endothelial nitric oxide synthase (eNOS), and downregulate tumor necrosis factor α (TNF- α ) expression . It can also regulate phospholipid metabolism, including phosphatidylcholine, Lys phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to play a role in the treatment of diabetes . In addition to polysaccharides, the flavonoids and their amino acid derivatives contained in CSR can also exert hypoglycemic effects. Flavan-3-ol derivatives prepared from CSR and other reagents, including 3 cysteine conjugates and 3 acetylcysteine conjugates, were found to have significant effects on α-glucosidase, sucrase, and maltase have inhibitory effects . Furthermore, the flavane-3-ol oligomer and compound Pentamers (pentamer) in the stem have inhibitory effects on α-Glucosidase has inhibitory effects . The investigation of CSR’s anti-diabetic activity is limited to in vitro and in vivo experiments. It plays an anti-diabetes role by regulating blood sugar levels, improving insulin sensitivity, protecting islet cells, controlling the risk of complications, etc. While these studies have demonstrated some anti-diabetic effects of CSR, further clinical trials are necessary to confirm its efficacy and safety for human use . 6.9. Anti-Osteoporosis Effect A few studies have demonstrated the favorable anti-osteoporotic effects of CSR. After screening the methanol and water extracts of 60 natural medicinal herbs, it was found that the methanol extract of CSR has a stimulating effect on the proliferation ability of osteoblast UMR106 and an inhibitory activity on osteoclast formation . CSP (100 μg/mL) induces osteogenic differentiation in MC3T3-E1 cells by activating Phosphatidylinositide 3-kinases/AKT/glycogen synthase kinase-3 β / β -Catenin (PI3K/AKT/GSK3 β / β -Catenin) pathway and upregulates mRNA, PI3K, phos-pho-phosphatidylinositide 3-kinases (p⁃PI3K), AKT, phospho-protein kinase B (p⁃AKT), GSK3 β , phosphor-glycogen synthase kinase-3 β (p⁃GSK3 β ), β ⁃catenin protein expression . Ethanol extract of CSR can promote the differentiation of osteoblasts from MC3T3-E1 while inhibiting osteoblast apoptosis, upregulating the expression of Bax and caspase-3, and downregulating the expression of B-cell lymphoma-2 (Bcl-2) . CSR aqueous extract containing serum can promote the proliferation and differentiation of MC3T3-E1 osteoblasts, increase alkaline phosphatase (ALP) activity, and increase the number of calcified nodules . In in vitro experiments, CSP was administered to ovariectomized SD rats. The results express that CSP can increase the osteoclastogenesis inhibitory fac-tor/Receptor Activator for Nuclear Factor- κ B Ligand (OPG/RANKL) ratio, inhibit osteoclast activity by activating the OPG/Receptor Activator for Nuclear Factor- κ B (RANK)/RANKL signaling pathway, regulate osteocalcin levels to reduce bone turnover rate, restore the balance between bone formation and bone resorption, reduce bone loss, increase bone density, improve tibial biomechanical properties, reduce bone fragility and fracture risk, and promote osteoblast differentiation . The ethanol extract of CSR can accelerate bone formation, inhibit bone resorption, and alleviate oxidative stress. It can also increase ALP levels in ovariectomized SD rats and reduce the levels of bone resorption-related biomarkers tartrate-resistant acid phosphatase (TRAP), Cathepsin K, and DPD . At the same time, it can also mediate PI3K/AKT and Nuclear Factor- κ B (NF- κ B) through RANKL/RANK/ TNF receptor-associated factor 6 (TRAF6) pathway to play an anti-osteoporosis role . To summarize, the anti-osteoporotic effect of CSR is primarily attributed to its extract and polysaccharide constituents. By increasing bone density, slowing down the process of osteoporosis, enhancing the resistance to fractures, reducing the risk of fractures, improving blood circulation, and increasing the nutrient supply of bones, it plays its role. However, there are currently no mechanisms of action for specific components. Further investigations are still required to clarify the underlying anti-osteoporosis mechanisms associated with the active constituents of CSR . 6.10. Liver Protection Among its many functions, the liver plays a crucial role in immunity, metabolism, detoxification, and digestion. Fibrosis of the liver is an injury-repair response, which can be partially reversed. However, persistent damage can lead to chronic inflammation, which triggers the formation of liver fibers . In order to effectively treat patients with chronic liver disease, liver fibrosis must be halted or slowed down . Blood levels of glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) increase when the liver is damaged. In liver injury induced by Streptozocin (STZ) in Wistar rats, CSPA (200 mg/kg, 150 mg/kg) reduces levels of GOT and GPT . By increasing white blood cell (WBC) levels, hematocrit (HCT) levels, red blood cells (RBCs), mean corpuscular volumes (MCVs), and red blood cell distribution width (RDW) levels in the blood cells of SD male rats induced by carbon tetrachloride. CSR extract regulates the transforming growth factor β 1 (TGF- β 1) expression and increases levels of WBC, HCT, RBC, MCV, and RDW in the blood cells of SD male rats induced by carbon tetrachloride, to impact blood cell typing and alleviate symptoms of liver fibrosis . Furthermore, it can also reduce the liver’s exposure to the inflammatory factors TGF- β 1, TNF- α, and interleukin 1 (IL-1) stimulation, thereby reducing liver fibrosis . CSR aqueous extract (3.5 g/kg) alleviates the lipid peroxidation damage caused by free radicals attacking the liver cell membrane of male Wister rats and protects the liver tissue from normal physiological operation . The use of 60% ethanol extract from CSR has been found to reduce serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydro-genase (LDH), and laminin (LN) in KM male mice induced by carbon tetrachloride. Additionally, the extract of CSR reduced the content of Hyp and MDA in liver tissue, while increasing SOD and GSH. By increasing the body’s antioxidant level and scavenging free radicals, reducing collagen fiber production, and reducing extracellular matrix deposition, the extract of CSR protects the liver . HCY2 and ursolic acid isolated from the ethanol extract of CSR can enhance mitochondrial function and glutathione antioxidant status in liver tissue, inhibit plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and protect SD female rats from carbon tetrachloride damage . CSF enhances the activity of SOD and Glutathione peroxidase (GSH-Px) in formaldehyde-induced T6 cells , reduces the protein concentration and MDA content of H 2 O 2 -induced damage to T6 cells, increases the expression level of nitric oxide synthase (NOS) protein , and has a protective effect on oxidative damage to T6 cells. Along with the current increasing demand for hepatoprotective drugs, CSR has demonstrated promising potential in the field of drug development for liver protection. However, due to the intricate physiological functions of the liver, further investigations are required to elucidate more specific mechanisms underlying hepatoprotection. Additionally, it is also imperative to conduct comparative analyses of CSR constituents to assess their respective hepatoprotective abilities . 6.11. Other Pharmacological Effects 6.11.1. Intestinal Effects A specific effect of CSR is to promote intestinal peristalsis, facilitate bowel movement, and maintain intestinal moisture. CSP (14.28 mg/kg, 28.57 mg/kg, 57.14 mg/kg) counteracted atropine’s inhibitory effect on intestinal peristalsis in KM mice by modulating parasympathetic nervous system function, reducing phenol red residue, and increasing intestinal propulsion rates . Comparing the effects of aqueous extract with ethyl acetate, methanol, and the aqueous extraction site of CSR on intestinal defecation in KM mice, it was observed that the aqueous extract (3.9 g/kg) showed significant activity . As demonstrated by the aqueous extracts of CSR (0.01 g/mL, 0.015 g/mL, 0.02 g/mL), the CSR augments smooth muscle contraction frequency while attenuating smooth muscle contraction amplitude in New Zealand white rabbits, resulting in mild “intestinal moistening and purging” effects . 6.11.2. Mitigate Obesity Ursolic acid (UA), an active component of CSR, HCY2 significantly reduced both body weight gain and fat pad weight in ICR mice . Furthermore, the expression of mitochondrial uncoupling protein 3 in skeletal muscle can be increased by ursolic acid through the regulation of the Adenosine phosphate-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 (AMPK/PGC1) pathway, thereby potentially contributing to the treatment of obesity . 6.11.3. Renal Protective Effects With the aggravation of diabetes and the side effects of hypoglycemic drugs, kidney damage is gradually caused. Serum levels of blood urea nitrogen (BUN) and creatinine (Cr) are significantly increased, which is considered to be an important indicator of renal dysfunction. HCY2 (0.5 mg/kg, 1.0 mg/kg) and ursolic acid (0.35 mg/kg, 0.70 mg/kg), derived from the CSR, resulted in a reduction in BUN and Cr levels and provided protection against gentamicin-induced nephrotoxicity in female SD rats . CSPA (200 mg/kg, 150 mg/kg) in vivo significantly reduces the levels of BUN and Cr, thereby ameliorating renal dysfunction in streptozotocin-induced Wistar rats . The CSP concentrations (0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL) indirectly attenuated H 2 O 2 -induced apoptosis of VERO cells by suppressing caspase-3 activity in vitro, indicating the potential of CSR for the prevention and treatment of kidney-related diseases . In summary, CSR protects kidney function from further damage by improving renal blood circulation, anti-fibrotic, antioxidant, and anti-inflammatory effects. For acute kidney injury, renal protection can promote the repair and regeneration of kidney tissue, reduce oxidative damage and inflammatory reactions, and help alleviate the degree of kidney injury and restore kidney function. For chronic renal failure, renal protection can delay the progression of the disease and reduce the loss of renal function. 6.11.4. Immune System Modulation Studies have demonstrated that CSR exine levels effectively inhibit the autoimmune antibodies and enhance humoral immune function, thereby improving overall immune competence in the body. The 75% alcohol extract (0.1 g/kg, 0.2 g/kg, 0.4 g/kg) and aqueous extract (0.18 g/kg, 0.36 g/kg, 0.72 g/kg) of CSR significantly augmented the thymus index and spleen index in immunosuppressed KM mice while also enhancing phagocytic function within the immune system. They promoted hemolysin antibody production and increased serum levels, interferon- γ (IFN- γ ), and TNF- α secretion, thereby bolstering both humoral and cellular immunity responses; notably, aqueous extract to the ethanol extract . The aqueous extract of CSR part Ⅲ (300 mg/kg) demonstrated a protective effect on BALB/C mice immunosuppressed by cyclophosphamide (CTX). It enhanced the phagocytic capacity of macrophages towards foreign bodies and resulted in an elevation in serum, effectively improving the humoral immune function of mice . In addition to the immunomodulatory effects observed with CSR aqueous extract and ethanol extract, CSP exhibits significant immunomodulatory effects in vitro experiments. Specifically, CSP polysaccharide demonstrates remarkable potential as it promotes the proliferation and enhances the phagocytic activity of RAW264.7 macrophages at concentrations ranging from 25 to 400 μg/mL. Moreover, CSP also induces an increase in the secretion levels of IL-6, TNF-α, and NO . 6.11.5. Anti-Ulcer Effect In recent years, despite the efficacy of antiplatelet drugs such as aspirin and clopidogrel in managing arterial circulation disorders caused by excessive platelet aggregation, it is crucial to consider potential gastrointestinal complications like gastric bleeding and ulceration when administering these medications. CSR has also demonstrated positive outcomes in the restoration and optimization of digestive functionality. The following examples are provided. The administration of CSP (100 mg/kg, 200 mg/kg, 400 mg/kg) effectively inhibits the development of water immersion restraint stress-induced gastric ulcers and pyloric ligation-induced gastric ulcer index in Wister rats. It also enhances the microcirculation of the gastric mucosa and improves its defensive capabilities, thereby exerting an anti-ulcer effect . Additionally, CSR can stimulate the synthesis and release of endogenous prostaglandin E2 (PGE2) and epidermal growth factor (EGF), enhance mucosal blood defense and repair functions of gastric mucosa, suppress the inflammatory mediator platelet-activating factor (PAF), mitigate its damage to mucosa, and restore the balance and defense factors for achieving an anti-gastric ulcer effect . 6.11.6. Anti-Depressant Effect The therapeutic potential of CSF has garnered significant attention in research studies. The administration of CSF at doses of 0.2 g/kg, 0.1 g/kg, and 0.05 g/kg has mitigated perimenopausal depression in female SD rats by modulating the hypothalamic-pituitary-gonadal axis through an increase in E2 levels . Not singly but in pairs, CSF (400 mg/kg, 200 mg/kg, 100 mg/kg) also effectively demonstrates significant therapeutic efficacy in perimenopausal depression KM female mice, ameliorating the pathological alterations in the uterus, thymus, spleen, and hypothalamus . 6.11.7. Anti-Epileptic The maximum electroconvulsive seizure (MES) model is widely regarded as a robust experimental model for grand mal epilepsy. The clinical efficacy of drugs with potent anti-MES effects extends to grand mal seizures. Based on this, CSR aqueous extract (1 g/mL), which exhibits a potent anti-MES effect in KM mice, holds promising potential for the treatment of grand mal epilepsy . 6.11.8. Anti-Bacterial For good measure, the polyphenolic compounds and polymeric procyanidins present in CSR exhibit antibacterial properties. Cynomoriitannin (MIC = 64 μg/mL) demonstrates higher efficacy against methicillin-resistant staphylococcus aureus (MRSA) than other compounds separated from CSR . Inducing apoptosis serves as a method for preventing and treating tumors as it plays an essential role in tumor progression . Cancer stem cells are inhibited from proliferating and dying when exposed to CSR. It can be used to treat malignant tumors such as breast cancer, leukemia, colon cancer, and others. 6.1.1. Anti-Cancer There are four breast cancer cell lines inhibited by CSR extracts and its ethyl acetate extraction site, including MDA-MB-231 , MCF-7 , MB468 , and 4T1 . Furthermore, CSR extract induces Foxo3 expression in apoptosis and prevents the transition from G1 to S phases . It has been found that chloroform and ethyl acetate extraction sites from the CSR ethanol extract are capable of inhibiting the proliferation of the colon adenocarcinoma cell line Caco-2 . In cell research for cervical cancer treatment, Cynomrium songaricum polysaccharides (CSP) inhibit proliferative activity in HeLa cells . A further study showed that both methanol extract and anthocyanin 3- O -glucoside from CSR inhibited KBWT cell proliferation in a dose-dependent manner . By inhibiting telomerase reverse transcriptase (TERT) mRNA, CSP induced apoptosis in A549 cells . Methanol extract and aqueous extract from CSR inhibited the growth of B16 cells, which are used for studying skin cancer in humans . Further, CSR ethyl acetate extract inhibited both LNCaP and HepG2 cells, showing that it may have therapeutic effects on prostate cancer and liver cancer . Research has shown that the anticancer ingredients in CSR are concentrated in the ethyl acetate extraction site, and it may be related to activating and enhancing autophagy processes in cells to trigger. In autophagy and apoptotic cell death, mitochondrial-related proteins Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) and Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) play a critical role . 6.1.2. Leukemia CCRF-CEM and CCRF-SB cells were inhibited by methanol extract and anthocyanin 3- O -glucoside from CSR . Similarly, mitochondrial pathways modulate caspase-3 activity. Therefore, CSR ethanol extract causes apoptosis in leukemia cells by causing apoptosis in HL-60 cells . The above studies indicate that CSR has a certain inhibitory effect on two types of tumor cells, cancer, and leukemia. In cancer, it inhibits the growth of cancer cells by inducing the expression of Foxo3 and inhibiting telomerase reverse transcriptase mRNA to activate mitochondrial-related proteins BNIP3 and BNIP3L. Regulating caspase-3 activity through the mitochondrial pathway in leukemia induces cell apoptosis. In contrast, there is more research data on adenocarcinoma and less research on leukemia. However, it cannot be concluded with certainty that CSR has a better therapeutic effect on cancer than on leukemia. Therefore, more in-depth research is still needed . There are four breast cancer cell lines inhibited by CSR extracts and its ethyl acetate extraction site, including MDA-MB-231 , MCF-7 , MB468 , and 4T1 . Furthermore, CSR extract induces Foxo3 expression in apoptosis and prevents the transition from G1 to S phases . It has been found that chloroform and ethyl acetate extraction sites from the CSR ethanol extract are capable of inhibiting the proliferation of the colon adenocarcinoma cell line Caco-2 . In cell research for cervical cancer treatment, Cynomrium songaricum polysaccharides (CSP) inhibit proliferative activity in HeLa cells . A further study showed that both methanol extract and anthocyanin 3- O -glucoside from CSR inhibited KBWT cell proliferation in a dose-dependent manner . By inhibiting telomerase reverse transcriptase (TERT) mRNA, CSP induced apoptosis in A549 cells . Methanol extract and aqueous extract from CSR inhibited the growth of B16 cells, which are used for studying skin cancer in humans . Further, CSR ethyl acetate extract inhibited both LNCaP and HepG2 cells, showing that it may have therapeutic effects on prostate cancer and liver cancer . Research has shown that the anticancer ingredients in CSR are concentrated in the ethyl acetate extraction site, and it may be related to activating and enhancing autophagy processes in cells to trigger. In autophagy and apoptotic cell death, mitochondrial-related proteins Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) and Bcl-2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) play a critical role . CCRF-CEM and CCRF-SB cells were inhibited by methanol extract and anthocyanin 3- O -glucoside from CSR . Similarly, mitochondrial pathways modulate caspase-3 activity. Therefore, CSR ethanol extract causes apoptosis in leukemia cells by causing apoptosis in HL-60 cells . The above studies indicate that CSR has a certain inhibitory effect on two types of tumor cells, cancer, and leukemia. In cancer, it inhibits the growth of cancer cells by inducing the expression of Foxo3 and inhibiting telomerase reverse transcriptase mRNA to activate mitochondrial-related proteins BNIP3 and BNIP3L. Regulating caspase-3 activity through the mitochondrial pathway in leukemia induces cell apoptosis. In contrast, there is more research data on adenocarcinoma and less research on leukemia. However, it cannot be concluded with certainty that CSR has a better therapeutic effect on cancer than on leukemia. Therefore, more in-depth research is still needed . Different parts of CSR have different antioxidant activities when extracted from methanol. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radicals are best scavenged in the central part, hydroxyl radicals are best inhibited in the lower part, and superoxide anions are highly resisted in the upper part . Multiple solvent extracts of CSR exhibit antioxidant activity. A methanol extract of the CSR and an ethyl acetate extraction site strongly inhibit superoxide anions . DPPH radicals, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radicals, and hydroxyl radicals are also scavenged by the aqueous extract and ethyl acetate extraction site . Additionally, the aqueous extract was able to scavenge DPPH free radicals and inhibit superoxide anion formation . Different extracts exhibit significantly different antioxidant and radical scavenging properties. CSR aqueous extract scavenges DPPH free radicals and nitrates more effectively than ethanol extract. In contrast, ethanol extract inhibits xanthine oxidase (XO) and superoxide dismutase (SOD) more effectively than aqueous extract . To examine the categories of substances with superior antioxidant effects, compounds of the same type extracted from CSR were compared to their antioxidant activity. This was performed to clarify the strong antioxidant activity of CSR extract. Within a certain concentration range, CSP exhibits effective scavenging ability against superoxide anion radicals, DPPH radicals, and hydroxyl radicals . CSR flavonoids also scavenge DPPH and hydroxyl radicals . Crude polyphenols exhibited significantly higher antioxidant activity than crude polysaccharides when measured against DPPH radicals, ABTS free radicals, and crude polysaccharides in CSR . According to another study, microwave-extracted procyanidins exhibited superior scavenging activity against DPPH and hydroxyl free radicals . The main antioxidant component in the CSR ethyl acetate extraction site is catechin, which was isolated from protocatechuic acid, gallic acid, and catechins . The aqueous extract of CSR was separated into catechin, epicatechin, and olive saponin to determine the DPPH free radical scavenging capacity . In vivo experiments were used to verify the CSR extract’s significant antioxidant activity. CSR extracts (0.22 g/kg, 0.44 g/kg, 0.88 g/kg) can enhance the serum DPPH free radical scavenging ability of KM mice, and reduce oxidative damage caused by free radicals and lipid peroxides . Some in vivo and in vitro experiments have shown that the antioxidant components in CSR exert antioxidant effects by clearing free radicals, as well as inhibiting XO and SOD, etc. The antioxidant activity of CSR is one of its main functions, which can slow down the oxidative state of the body and fight against diseases . Several studies have demonstrated the anti-aging effects of CSR through a variety of mechanisms. According to reports, adding CSR to the diet can extend the average and maximum lifespans of adult female flies. Ethanol extract of CSR suppresses age-related learning disabilities in elderly flies by reducing hydrogen peroxide levels and increasing antioxidants, extending their lifespan, improving mating readiness, increasing fertility, and inhibiting age-related learning disabilities . A transcriptome sequencing study found that CSR extract impacted wild-type Caenorhabditis elegans aging. The lifespan of Caenorhabditis elegans was extended and motor abilities were enhanced by ethyl acetate extract (0.4 mg/mL). Multiple pathways and genes collaborate to produce the effects of the ethyl acetate extract . Various research results have shown that extracts, CSP, and preparations from CSR can delay aging by inhibiting telomere length shortening , enhancing telomerase activity , improving immune function , inhibiting neuronal apoptosis , improving hippocampal CA1 neurons , enhancing antioxidant capacity . The aqueous extract of CSR can also improve the energy metabolism of liver mitochondria in aging model KM mice. It can also alleviate free radical damage to mitochondrial membrane structure and function and play a role in delaying aging . These findings not only reveal the potential of the polysaccharide and extract in combating aging but also lay the groundwork for future clinical research. It would be beneficial to further investigate the chemical composition of CSR and the mechanism underlying anti-aging as well as their safety and effectiveness to offer novel insights and possibilities for delaying the aging process in the future . The aqueous extract and ethanol extract of CSR are responsible for its anti-fatigue properties. By lowering the lactate index , inhibiting amino acid protein breakdown, and increasing glycogen reserves , they can improve energy metabolism. It also possesses the ability to increase the level of cyclic adenosine monophosphate (cAMP), reduce cyclic adenosine monophosphate/cyclic guanosine monophosphate (cAMP/cGMP) ratio , improve free radical metabolism . In addition, CSR flavonoids (CSF) reduce MAO activity and reactive oxygen species (ROS) levels by improving free radical metabolism . Oxygen deficiency can cause abnormal tissue metabolism, function, and morphology. The main cause of death is hypoxia of the brain and heart. CSR aqueous extract has positive atmospheric pressure anti-hypoxia and anti-acute cerebral ischemia and hypoxia effects , which increases blood hemoglobin content and enhances oxygen-carrying function . As well as reducing brain edema, it increases myocardial protein content . CSR exhibits remarkable anti-fatigue and anti-hypoxia properties. Research on anti-fatigue effects focuses on its active ingredients, such as water extract, ethanol extract, and flavonoids. Currently, hypoxic resistance studies are primarily focused on CSR water extract. Developing highly potent and pharmaceutically viable compounds from CSR for anti-fatigue and anti-hypoxia purposes will require further investigation . Ethyl acetate extract and methanol extract are both effective against A β 25–35 , hypoxanthine/xanthine oxidase (HPX/XO) , Xanthine dehydrogenase/xanthine oxidase (XDH/XO) induced SK-N-SH cells have protective effects. Among them, ethyl acetate extract is more effective against Amyloid β -Protein 25–35 (A β 25–35 ) and has an anti-Starosporin-induced injury effect . CSP and ethyl acetate extraction sites of CSR can protect PC12 cells against damage by H 2 O 2 and A β 25–35 . Ethyl acetate extract has cytotoxicity to Neuro2A cells (EC 50 = 116 mg/L) and increases the expression of synaptophysin through the mitogen-activated protein kinases (MAPK) pathway . In another study, the methanol extract of CSR inhibits A β 25–35 induced phosphorylation of dynamin-related protein 1 (Drp1) at Ser637 in HT22 cells and reduced the expression of Fission 1 Protein (Fis1) in H 2 O 2 induced model for the treatment of Alzheimer’s disease (AD) . Based on neuroprotective effects at the cellular level, scholars have further explored them through animal models. The ethyl acetate fraction of CSR improves the behavior of C57BL/6 male mice by reducing mitochondrial dynamics imbalance. It also downregulated the expression of the Drp1 protein and upregulated the expression of Optic Atrophy 1 (OPA1) and Mito Fusin 1 (MFN1) proteins . It also improves the spatial memory and learning ability of AD model mice by regulating fecal microbiota disorder . In the ovariectomized Sprague–Dawley (SD) rat model, it increased the expression of Growth-Associated Protein 43 (GAP-43) protein in the hippocampus , regulated the MAPK pathway, increased the expression of phosphorylation-cAMP response element-binding protein (p-CREB), and decreased the expression of p38, thereby promoting the survival and repair of hippocampal neurons. Other studies have shown that CSR ethyl acetate extract increases the expression levels of synaptic plasticity-related proteins Syn and postsynaptic density protein-95 (PSD-95) while upregulating the protein expression levels of phosphor-extracellular regulated protein kinases 1/2 (P-Erk1/2) and P-CREB in the MAPK/ERK1/2 signaling pathway . It increases the effect of Long-term Potential (LTP) in Morris water maze and neuroelectrophysiology, further improving cognitive dysfunction in chronic stress Institute of Cancer Research (ICR) mice after ovariectomy . The ethanol extract of CSR increased cAMP response element-binding protein /Brain-Derived Neurotrophic Factor (CREB/BDNF) expression in ovariectomized SD rats by inhibiting the p38MAPK/ERK pathway . It also reduced serum corticosterone levels, increased the expression of BDNF mRNA in this region, promoted the proliferation of mouse dentate gyrus cells and differentiation of neuroblasts, enhanced the potential for hippocampal plasticity in male C57BL/6J mice , and thus achieved neuroprotective effects on the nerves. The aqueous extract of CSR has a significant improvement effect on the learning and memory of scopolamine-induced KM male mice. Its mechanism may be related to reducing oxidative stress in brain tissue . In Wistar male rats, through upregulating the Brain-Derived Neurotrophic Factor/Tyrosine Kinase receptor B (BDNF/TrkB) signaling pathway, enhancing cognitive function, increasing acetylcholine (ACH) content in the central cholinergic system, inhibiting cell apoptosis, and enhancing synaptic plasticity, CSF improves the AD model induced by A β 1–42 . In addition, CSF inhibits oxidative stress and inflammatory reactions. It can also downregulate the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ROS, and NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in the hippocampus, exerting neuroprotective effects . The main component of CSR, ursolic acid, at a concentration of 5–15 µM, can effectively protect SD rat hippocampal neurons from damage induced by kainic acid by regulating α -amino-3-hydroxy-5-methy1-4-isoxazole propionic acid (AMPA) receptors, protecting mitochondria, and reducing free radical generation . In summary, the neuroprotective active ingredients are mainly concentrated in the methanol, ethanol, and ethyl acetate extracts of CSR. However, the main components that play a key neuroprotective role are not yet clear because of the complex components in CSR extract. Therefore, future studies should explore compounds that play a major role in protecting the nervous system . In geriatrics, benign prostatic hyperplasia (BPH) is a common genitourinary disorder characterized by prostate gland enlargement and urinary dysfunction . There is an inhibitory effect of ethanol extract of CSR (2.5 mg/mL) on testosterone 5 α -reductase . Moreover, it interferes with estrogen/androgen signals to inhibit prostate hyperplasia in Wistar rats and improves the disorder of prostate epithelial cells and abnormal proliferation of connective tissue in Wistar rats with BPH model, inhibiting Proliferating Cell Nuclear Antigen (PCNA), Androgen Receptor (AR), and estrogen receptor α (Er α ) Protein expression while promoting estrogen receptor β (ER β ) Protein expression while promoting ER β Protein expression . Meanwhile, Wistar male rat protein expression of prostate AR, ER α / β, and 3-oxo-5-alpha-steroid 4-dehydrogenase 1/2 (SRD5A1/2) were regulated to inhibit BPH . Additionally, CSR aqueous extract inhibits prostate hyperplasia, increases SOD and glutathione (GSH) activities, decreases malondialdehyde (MDA) content, and significantly reduces prostate wet weight and prostate index by improving testosterone propionate-induced oxidative stress levels . In vitro experiments, Luteolin, Gallic acid, Ferulic acid, Protocatechualdehyde from CSR suppressed BPH by downregulating the expression of AR and ER α in BPH-1 cells and upregulation ER β expression . CSRs containing luteolin, epicatechin, and epicatechin gallate all improve the contractility of Wistar male rats’ bladder detrusors . Infertility in men is complex and multifactorial, with idiopathic infertility accounting for approximately 30% of cases . It has been shown that CSR improves sexual hormone levels as a kidney tonifying traditional Chinese medicine . Under the intervention of CSR aqueous extract, serum testosterone and Follicle-stimulating Hormone (FSH) levels are reduced, and interstitial cell-stimulating hormone (ICSH) levels are increased to directly affect the spermatogenic effect of immature seminiferous tubules of Wistar rats . It can also promote the secretion of testosterone in SD rats and inhibit abnormal secretion of FSH and Luteinizing hormone (LH) by regulating gonadal hormone levels . Glial Cell Line-derived Neurotrophic Factor (GDNF) production in testes of SD rats and undifferentiated spermatogonia proliferation stimulates, increases testosterone levels, and improves sperm motility . Relieving sperm damage and serum testosterone levels are increased through the MAPK-3-mediated GDNF signaling pathway, thereby enhancing sperm motility . In addition, enhancing sperm production in Wistar rats and upregulating the expression pathway of GDNF in the testes to improve male fertility , enhancing sperm production in golden hamsters, and blocking the impact of short photoperiod on reproductive function by CSR aqueous extract. In summary, active compounds from CSR that inhibit BPH mainly exist in its ethanol extract, while the active ingredients that promote spermatogenesis are mainly concentrated in the aqueous extract, which has been proven to have good therapeutic effects in treating male infertility . Despite a significant increase in the number of approved antiviral drugs, these existing drugs are not always effective or well tolerated. It is becoming increasingly common for viruses to develop drug resistance. As of now, many polysaccharides have been approved as drugs as independent or major bioactive components . The methyl thiazolyl tetrazolium (MTT) method was used to detect the toxicity of CSP on MT-4 cells, which showed that only sulfated polysaccharides (SCSP-M, SCSP-1, SCSP-2) are anti-HIV. Due to the interaction between sulfated polysaccharides and poly L-lysine, sulfated polysaccharides have antiviral properties . In addition to the CSP, the triterpenoids contained in CSR also have antiviral activity. Ursolic acid, half ursolic malonate, malonyl oleanolic acid hemiester , acetyl ursolic acid, and condensed tannin extracted from CSR all have the function of inhibiting human immunodeficiency virus (HIV) protease . Furthermore, triterpenoids in CSR also have inhibitory activity against hepatitis C virus (HCV) protease, with malonyl ursolic acid hemiester having the maximum inhibitory effect . The main components of CSR are polysaccharides and triterpenoids, which are potentially useful for developing antiviral drugs. Additionally, it is worth noting that the antiviral efficacy of CSR has predominantly been tested in vitro with limited reports on its in vivo effects. Consequently, the precise mechanism by which CSR is antiviral remains unclear. Future studies should explore this aspect further to uncover the antiviral mechanism of CSR and establish solid foundations for its application . CSP as one of the pivotal active constituents in CSR, exhibits significant therapeutic effects on several diseases. Consequently, CSR is being considered a potential candidate for the development of novel anti-diabetic drugs . Oral administration of CSR water-soluble polysaccharide (CSPA) significantly reduced the blood glucose level, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, blood urea nitrogen, creatinine activity in streptozotocin (STZ) induced diabetes model rats, effectively increased the serum insulin level and liver glycogen content and promoted the recovery of pancreatic islet cells in the pancreas to near normal levels . CSP (300 mg/kg) can upregulate the expression of protein kinase B (AKT) and endothelial nitric oxide synthase (eNOS), and downregulate tumor necrosis factor α (TNF- α ) expression . It can also regulate phospholipid metabolism, including phosphatidylcholine, Lys phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin to play a role in the treatment of diabetes . In addition to polysaccharides, the flavonoids and their amino acid derivatives contained in CSR can also exert hypoglycemic effects. Flavan-3-ol derivatives prepared from CSR and other reagents, including 3 cysteine conjugates and 3 acetylcysteine conjugates, were found to have significant effects on α-glucosidase, sucrase, and maltase have inhibitory effects . Furthermore, the flavane-3-ol oligomer and compound Pentamers (pentamer) in the stem have inhibitory effects on α-Glucosidase has inhibitory effects . The investigation of CSR’s anti-diabetic activity is limited to in vitro and in vivo experiments. It plays an anti-diabetes role by regulating blood sugar levels, improving insulin sensitivity, protecting islet cells, controlling the risk of complications, etc. While these studies have demonstrated some anti-diabetic effects of CSR, further clinical trials are necessary to confirm its efficacy and safety for human use . A few studies have demonstrated the favorable anti-osteoporotic effects of CSR. After screening the methanol and water extracts of 60 natural medicinal herbs, it was found that the methanol extract of CSR has a stimulating effect on the proliferation ability of osteoblast UMR106 and an inhibitory activity on osteoclast formation . CSP (100 μg/mL) induces osteogenic differentiation in MC3T3-E1 cells by activating Phosphatidylinositide 3-kinases/AKT/glycogen synthase kinase-3 β / β -Catenin (PI3K/AKT/GSK3 β / β -Catenin) pathway and upregulates mRNA, PI3K, phos-pho-phosphatidylinositide 3-kinases (p⁃PI3K), AKT, phospho-protein kinase B (p⁃AKT), GSK3 β , phosphor-glycogen synthase kinase-3 β (p⁃GSK3 β ), β ⁃catenin protein expression . Ethanol extract of CSR can promote the differentiation of osteoblasts from MC3T3-E1 while inhibiting osteoblast apoptosis, upregulating the expression of Bax and caspase-3, and downregulating the expression of B-cell lymphoma-2 (Bcl-2) . CSR aqueous extract containing serum can promote the proliferation and differentiation of MC3T3-E1 osteoblasts, increase alkaline phosphatase (ALP) activity, and increase the number of calcified nodules . In in vitro experiments, CSP was administered to ovariectomized SD rats. The results express that CSP can increase the osteoclastogenesis inhibitory fac-tor/Receptor Activator for Nuclear Factor- κ B Ligand (OPG/RANKL) ratio, inhibit osteoclast activity by activating the OPG/Receptor Activator for Nuclear Factor- κ B (RANK)/RANKL signaling pathway, regulate osteocalcin levels to reduce bone turnover rate, restore the balance between bone formation and bone resorption, reduce bone loss, increase bone density, improve tibial biomechanical properties, reduce bone fragility and fracture risk, and promote osteoblast differentiation . The ethanol extract of CSR can accelerate bone formation, inhibit bone resorption, and alleviate oxidative stress. It can also increase ALP levels in ovariectomized SD rats and reduce the levels of bone resorption-related biomarkers tartrate-resistant acid phosphatase (TRAP), Cathepsin K, and DPD . At the same time, it can also mediate PI3K/AKT and Nuclear Factor- κ B (NF- κ B) through RANKL/RANK/ TNF receptor-associated factor 6 (TRAF6) pathway to play an anti-osteoporosis role . To summarize, the anti-osteoporotic effect of CSR is primarily attributed to its extract and polysaccharide constituents. By increasing bone density, slowing down the process of osteoporosis, enhancing the resistance to fractures, reducing the risk of fractures, improving blood circulation, and increasing the nutrient supply of bones, it plays its role. However, there are currently no mechanisms of action for specific components. Further investigations are still required to clarify the underlying anti-osteoporosis mechanisms associated with the active constituents of CSR . Among its many functions, the liver plays a crucial role in immunity, metabolism, detoxification, and digestion. Fibrosis of the liver is an injury-repair response, which can be partially reversed. However, persistent damage can lead to chronic inflammation, which triggers the formation of liver fibers . In order to effectively treat patients with chronic liver disease, liver fibrosis must be halted or slowed down . Blood levels of glutamic oxalate transaminase (GOT) and glutamic pyruvate transaminase (GPT) increase when the liver is damaged. In liver injury induced by Streptozocin (STZ) in Wistar rats, CSPA (200 mg/kg, 150 mg/kg) reduces levels of GOT and GPT . By increasing white blood cell (WBC) levels, hematocrit (HCT) levels, red blood cells (RBCs), mean corpuscular volumes (MCVs), and red blood cell distribution width (RDW) levels in the blood cells of SD male rats induced by carbon tetrachloride. CSR extract regulates the transforming growth factor β 1 (TGF- β 1) expression and increases levels of WBC, HCT, RBC, MCV, and RDW in the blood cells of SD male rats induced by carbon tetrachloride, to impact blood cell typing and alleviate symptoms of liver fibrosis . Furthermore, it can also reduce the liver’s exposure to the inflammatory factors TGF- β 1, TNF- α, and interleukin 1 (IL-1) stimulation, thereby reducing liver fibrosis . CSR aqueous extract (3.5 g/kg) alleviates the lipid peroxidation damage caused by free radicals attacking the liver cell membrane of male Wister rats and protects the liver tissue from normal physiological operation . The use of 60% ethanol extract from CSR has been found to reduce serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydro-genase (LDH), and laminin (LN) in KM male mice induced by carbon tetrachloride. Additionally, the extract of CSR reduced the content of Hyp and MDA in liver tissue, while increasing SOD and GSH. By increasing the body’s antioxidant level and scavenging free radicals, reducing collagen fiber production, and reducing extracellular matrix deposition, the extract of CSR protects the liver . HCY2 and ursolic acid isolated from the ethanol extract of CSR can enhance mitochondrial function and glutathione antioxidant status in liver tissue, inhibit plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, and protect SD female rats from carbon tetrachloride damage . CSF enhances the activity of SOD and Glutathione peroxidase (GSH-Px) in formaldehyde-induced T6 cells , reduces the protein concentration and MDA content of H 2 O 2 -induced damage to T6 cells, increases the expression level of nitric oxide synthase (NOS) protein , and has a protective effect on oxidative damage to T6 cells. Along with the current increasing demand for hepatoprotective drugs, CSR has demonstrated promising potential in the field of drug development for liver protection. However, due to the intricate physiological functions of the liver, further investigations are required to elucidate more specific mechanisms underlying hepatoprotection. Additionally, it is also imperative to conduct comparative analyses of CSR constituents to assess their respective hepatoprotective abilities . 6.11.1. Intestinal Effects A specific effect of CSR is to promote intestinal peristalsis, facilitate bowel movement, and maintain intestinal moisture. CSP (14.28 mg/kg, 28.57 mg/kg, 57.14 mg/kg) counteracted atropine’s inhibitory effect on intestinal peristalsis in KM mice by modulating parasympathetic nervous system function, reducing phenol red residue, and increasing intestinal propulsion rates . Comparing the effects of aqueous extract with ethyl acetate, methanol, and the aqueous extraction site of CSR on intestinal defecation in KM mice, it was observed that the aqueous extract (3.9 g/kg) showed significant activity . As demonstrated by the aqueous extracts of CSR (0.01 g/mL, 0.015 g/mL, 0.02 g/mL), the CSR augments smooth muscle contraction frequency while attenuating smooth muscle contraction amplitude in New Zealand white rabbits, resulting in mild “intestinal moistening and purging” effects . 6.11.2. Mitigate Obesity Ursolic acid (UA), an active component of CSR, HCY2 significantly reduced both body weight gain and fat pad weight in ICR mice . Furthermore, the expression of mitochondrial uncoupling protein 3 in skeletal muscle can be increased by ursolic acid through the regulation of the Adenosine phosphate-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 (AMPK/PGC1) pathway, thereby potentially contributing to the treatment of obesity . 6.11.3. Renal Protective Effects With the aggravation of diabetes and the side effects of hypoglycemic drugs, kidney damage is gradually caused. Serum levels of blood urea nitrogen (BUN) and creatinine (Cr) are significantly increased, which is considered to be an important indicator of renal dysfunction. HCY2 (0.5 mg/kg, 1.0 mg/kg) and ursolic acid (0.35 mg/kg, 0.70 mg/kg), derived from the CSR, resulted in a reduction in BUN and Cr levels and provided protection against gentamicin-induced nephrotoxicity in female SD rats . CSPA (200 mg/kg, 150 mg/kg) in vivo significantly reduces the levels of BUN and Cr, thereby ameliorating renal dysfunction in streptozotocin-induced Wistar rats . The CSP concentrations (0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL) indirectly attenuated H 2 O 2 -induced apoptosis of VERO cells by suppressing caspase-3 activity in vitro, indicating the potential of CSR for the prevention and treatment of kidney-related diseases . In summary, CSR protects kidney function from further damage by improving renal blood circulation, anti-fibrotic, antioxidant, and anti-inflammatory effects. For acute kidney injury, renal protection can promote the repair and regeneration of kidney tissue, reduce oxidative damage and inflammatory reactions, and help alleviate the degree of kidney injury and restore kidney function. For chronic renal failure, renal protection can delay the progression of the disease and reduce the loss of renal function. 6.11.4. Immune System Modulation Studies have demonstrated that CSR exine levels effectively inhibit the autoimmune antibodies and enhance humoral immune function, thereby improving overall immune competence in the body. The 75% alcohol extract (0.1 g/kg, 0.2 g/kg, 0.4 g/kg) and aqueous extract (0.18 g/kg, 0.36 g/kg, 0.72 g/kg) of CSR significantly augmented the thymus index and spleen index in immunosuppressed KM mice while also enhancing phagocytic function within the immune system. They promoted hemolysin antibody production and increased serum levels, interferon- γ (IFN- γ ), and TNF- α secretion, thereby bolstering both humoral and cellular immunity responses; notably, aqueous extract to the ethanol extract . The aqueous extract of CSR part Ⅲ (300 mg/kg) demonstrated a protective effect on BALB/C mice immunosuppressed by cyclophosphamide (CTX). It enhanced the phagocytic capacity of macrophages towards foreign bodies and resulted in an elevation in serum, effectively improving the humoral immune function of mice . In addition to the immunomodulatory effects observed with CSR aqueous extract and ethanol extract, CSP exhibits significant immunomodulatory effects in vitro experiments. Specifically, CSP polysaccharide demonstrates remarkable potential as it promotes the proliferation and enhances the phagocytic activity of RAW264.7 macrophages at concentrations ranging from 25 to 400 μg/mL. Moreover, CSP also induces an increase in the secretion levels of IL-6, TNF-α, and NO . 6.11.5. Anti-Ulcer Effect In recent years, despite the efficacy of antiplatelet drugs such as aspirin and clopidogrel in managing arterial circulation disorders caused by excessive platelet aggregation, it is crucial to consider potential gastrointestinal complications like gastric bleeding and ulceration when administering these medications. CSR has also demonstrated positive outcomes in the restoration and optimization of digestive functionality. The following examples are provided. The administration of CSP (100 mg/kg, 200 mg/kg, 400 mg/kg) effectively inhibits the development of water immersion restraint stress-induced gastric ulcers and pyloric ligation-induced gastric ulcer index in Wister rats. It also enhances the microcirculation of the gastric mucosa and improves its defensive capabilities, thereby exerting an anti-ulcer effect . Additionally, CSR can stimulate the synthesis and release of endogenous prostaglandin E2 (PGE2) and epidermal growth factor (EGF), enhance mucosal blood defense and repair functions of gastric mucosa, suppress the inflammatory mediator platelet-activating factor (PAF), mitigate its damage to mucosa, and restore the balance and defense factors for achieving an anti-gastric ulcer effect . 6.11.6. Anti-Depressant Effect The therapeutic potential of CSF has garnered significant attention in research studies. The administration of CSF at doses of 0.2 g/kg, 0.1 g/kg, and 0.05 g/kg has mitigated perimenopausal depression in female SD rats by modulating the hypothalamic-pituitary-gonadal axis through an increase in E2 levels . Not singly but in pairs, CSF (400 mg/kg, 200 mg/kg, 100 mg/kg) also effectively demonstrates significant therapeutic efficacy in perimenopausal depression KM female mice, ameliorating the pathological alterations in the uterus, thymus, spleen, and hypothalamus . 6.11.7. Anti-Epileptic The maximum electroconvulsive seizure (MES) model is widely regarded as a robust experimental model for grand mal epilepsy. The clinical efficacy of drugs with potent anti-MES effects extends to grand mal seizures. Based on this, CSR aqueous extract (1 g/mL), which exhibits a potent anti-MES effect in KM mice, holds promising potential for the treatment of grand mal epilepsy . 6.11.8. Anti-Bacterial For good measure, the polyphenolic compounds and polymeric procyanidins present in CSR exhibit antibacterial properties. Cynomoriitannin (MIC = 64 μg/mL) demonstrates higher efficacy against methicillin-resistant staphylococcus aureus (MRSA) than other compounds separated from CSR . A specific effect of CSR is to promote intestinal peristalsis, facilitate bowel movement, and maintain intestinal moisture. CSP (14.28 mg/kg, 28.57 mg/kg, 57.14 mg/kg) counteracted atropine’s inhibitory effect on intestinal peristalsis in KM mice by modulating parasympathetic nervous system function, reducing phenol red residue, and increasing intestinal propulsion rates . Comparing the effects of aqueous extract with ethyl acetate, methanol, and the aqueous extraction site of CSR on intestinal defecation in KM mice, it was observed that the aqueous extract (3.9 g/kg) showed significant activity . As demonstrated by the aqueous extracts of CSR (0.01 g/mL, 0.015 g/mL, 0.02 g/mL), the CSR augments smooth muscle contraction frequency while attenuating smooth muscle contraction amplitude in New Zealand white rabbits, resulting in mild “intestinal moistening and purging” effects . Ursolic acid (UA), an active component of CSR, HCY2 significantly reduced both body weight gain and fat pad weight in ICR mice . Furthermore, the expression of mitochondrial uncoupling protein 3 in skeletal muscle can be increased by ursolic acid through the regulation of the Adenosine phosphate-activated protein kinase/peroxisome proliferator-activated receptor γ coactivator-1 (AMPK/PGC1) pathway, thereby potentially contributing to the treatment of obesity . With the aggravation of diabetes and the side effects of hypoglycemic drugs, kidney damage is gradually caused. Serum levels of blood urea nitrogen (BUN) and creatinine (Cr) are significantly increased, which is considered to be an important indicator of renal dysfunction. HCY2 (0.5 mg/kg, 1.0 mg/kg) and ursolic acid (0.35 mg/kg, 0.70 mg/kg), derived from the CSR, resulted in a reduction in BUN and Cr levels and provided protection against gentamicin-induced nephrotoxicity in female SD rats . CSPA (200 mg/kg, 150 mg/kg) in vivo significantly reduces the levels of BUN and Cr, thereby ameliorating renal dysfunction in streptozotocin-induced Wistar rats . The CSP concentrations (0.25 mg/mL, 0.5 mg/mL, 1.0 mg/mL) indirectly attenuated H 2 O 2 -induced apoptosis of VERO cells by suppressing caspase-3 activity in vitro, indicating the potential of CSR for the prevention and treatment of kidney-related diseases . In summary, CSR protects kidney function from further damage by improving renal blood circulation, anti-fibrotic, antioxidant, and anti-inflammatory effects. For acute kidney injury, renal protection can promote the repair and regeneration of kidney tissue, reduce oxidative damage and inflammatory reactions, and help alleviate the degree of kidney injury and restore kidney function. For chronic renal failure, renal protection can delay the progression of the disease and reduce the loss of renal function. Studies have demonstrated that CSR exine levels effectively inhibit the autoimmune antibodies and enhance humoral immune function, thereby improving overall immune competence in the body. The 75% alcohol extract (0.1 g/kg, 0.2 g/kg, 0.4 g/kg) and aqueous extract (0.18 g/kg, 0.36 g/kg, 0.72 g/kg) of CSR significantly augmented the thymus index and spleen index in immunosuppressed KM mice while also enhancing phagocytic function within the immune system. They promoted hemolysin antibody production and increased serum levels, interferon- γ (IFN- γ ), and TNF- α secretion, thereby bolstering both humoral and cellular immunity responses; notably, aqueous extract to the ethanol extract . The aqueous extract of CSR part Ⅲ (300 mg/kg) demonstrated a protective effect on BALB/C mice immunosuppressed by cyclophosphamide (CTX). It enhanced the phagocytic capacity of macrophages towards foreign bodies and resulted in an elevation in serum, effectively improving the humoral immune function of mice . In addition to the immunomodulatory effects observed with CSR aqueous extract and ethanol extract, CSP exhibits significant immunomodulatory effects in vitro experiments. Specifically, CSP polysaccharide demonstrates remarkable potential as it promotes the proliferation and enhances the phagocytic activity of RAW264.7 macrophages at concentrations ranging from 25 to 400 μg/mL. Moreover, CSP also induces an increase in the secretion levels of IL-6, TNF-α, and NO . In recent years, despite the efficacy of antiplatelet drugs such as aspirin and clopidogrel in managing arterial circulation disorders caused by excessive platelet aggregation, it is crucial to consider potential gastrointestinal complications like gastric bleeding and ulceration when administering these medications. CSR has also demonstrated positive outcomes in the restoration and optimization of digestive functionality. The following examples are provided. The administration of CSP (100 mg/kg, 200 mg/kg, 400 mg/kg) effectively inhibits the development of water immersion restraint stress-induced gastric ulcers and pyloric ligation-induced gastric ulcer index in Wister rats. It also enhances the microcirculation of the gastric mucosa and improves its defensive capabilities, thereby exerting an anti-ulcer effect . Additionally, CSR can stimulate the synthesis and release of endogenous prostaglandin E2 (PGE2) and epidermal growth factor (EGF), enhance mucosal blood defense and repair functions of gastric mucosa, suppress the inflammatory mediator platelet-activating factor (PAF), mitigate its damage to mucosa, and restore the balance and defense factors for achieving an anti-gastric ulcer effect . The therapeutic potential of CSF has garnered significant attention in research studies. The administration of CSF at doses of 0.2 g/kg, 0.1 g/kg, and 0.05 g/kg has mitigated perimenopausal depression in female SD rats by modulating the hypothalamic-pituitary-gonadal axis through an increase in E2 levels . Not singly but in pairs, CSF (400 mg/kg, 200 mg/kg, 100 mg/kg) also effectively demonstrates significant therapeutic efficacy in perimenopausal depression KM female mice, ameliorating the pathological alterations in the uterus, thymus, spleen, and hypothalamus . The maximum electroconvulsive seizure (MES) model is widely regarded as a robust experimental model for grand mal epilepsy. The clinical efficacy of drugs with potent anti-MES effects extends to grand mal seizures. Based on this, CSR aqueous extract (1 g/mL), which exhibits a potent anti-MES effect in KM mice, holds promising potential for the treatment of grand mal epilepsy . For good measure, the polyphenolic compounds and polymeric procyanidins present in CSR exhibit antibacterial properties. Cynomoriitannin (MIC = 64 μg/mL) demonstrates higher efficacy against methicillin-resistant staphylococcus aureus (MRSA) than other compounds separated from CSR . A growing awareness of food safety has led to a growing focus on CSR, which is a homology between medicine and food. The evaluation of a new potential drug’s safety is crucial not only in the concept of fitness and healthcare but also in its research and development. Several studies were conducted to evaluate the safety of CSR, including the contents of heavy metal ions detection and toxicity studies in vivo. 7.1. Heavy Metal Ions Detection The levels of Cu, Pb, Cd, Cr, As, and Hg in CSR have been determined by microwave digestion and high-resolution continuous light source atomic absorption spectrometry . The results indicated that the concentrations of these metals in CSR were far below the limitations of both the Green Industry Standard for Importing Medicinal Plants and Preparations as well as the national food safety standard named Maximum Levels of Contaminants in Foods (GB2762–2012). 7.2. Toxicity Studies In Vivo Currently, CSR aqueous extract has been confirmed to have no obvious toxicity by experiments on acute toxicity tests, teratogenicity tests, and subchronic toxicity tests. In acute toxicity experiments, the oral LD 50 of CSR aqueous extract exceeded 21.5 g/kg in all cases . The results of another study indicated that the maximum tolerable dose of CSR in KM mice is greater than 15 g/kg , also providing evidence of CSR aqueous extract nonobvious toxicity. Salmonella typhimurium reverse mutation test, bone marrow polychromatic red blood cell micronucleus test, and sperm aberration test in KM mice were adopted to further investigate the genetic toxicity of CSR aqueous extract. One of the studies indicated that CSR aqueous extract (7.5 g/kg, 3.75 g/kg, and 1.875 g/kg) could not induce tested strains (TA97, TA98, TA100, TA102) to form colonies, as well as did not cause any mutagenic effects against the somatic cells and germ cells . The results of the experiment are proved by another study conducted with CSR aqueous extract (2.25, 4.50, 9.00 g/kg), with the difference being the type of experimental strains (TA97, TA98, TA100, TA102, and TA1535) . As the third stage of food safety toxicological evaluation, a subchronic toxicity test was conducted for a ninety-day feeding trial. No significant toxicological findings were detected in hematological parameters or clinical and pathological examinations when feeding various concentrations of CSR aqueous extract (1.04 g/kg, 2.08 g/kg, 4.16 g/kg) to SD rats . Three Doses of CSR aqueous extract (2.83 g/kg, 5.66 g/kg, 8.49 g/kg) were fed to Wistar rats in the same year’s research. Fu. Et found that the medium (5.66 g/kg) and high (8.49 g/kg) dose groups exhibited significantly increased plasma prothrombin time (PT), as well as testicular organ coefficient and epididymal organ coefficient. The maximum no-observed-adverse-effect level (NOAEL) was determined to be 2.83 g/kg, while the minimum lowest-observed-adverse-effect level (LOAEL) was identified at 5.66 g/kg in this subchronic transoral toxicity study. However, according to clinical reports, a patient was diagnosed with acute renal function injury after taking a single Chinese medicine CSR 100–150 g aqueous extract for about 0.5 h, experiencing nausea and vomiting 4–6 times, non-jet like, with all vomit being gastric contents, without abdominal pain or diarrhea, headache, or fever. Therefore, it is still essential to exercise caution and avoid an overdose of CSR . Based on the current situation, most studies about the toxicity of CSR focus on its aqueous extract, and few on other extracts and extract components. In order to build a more comprehensive toxicity evaluation, subsequent studies are required to focus on CSR extracts from other solvents. Furthermore, further exploration of toxic compounds in CSR is also necessary. These will provide a more reliable scientific basis for the safe use of CSR. The levels of Cu, Pb, Cd, Cr, As, and Hg in CSR have been determined by microwave digestion and high-resolution continuous light source atomic absorption spectrometry . The results indicated that the concentrations of these metals in CSR were far below the limitations of both the Green Industry Standard for Importing Medicinal Plants and Preparations as well as the national food safety standard named Maximum Levels of Contaminants in Foods (GB2762–2012). Currently, CSR aqueous extract has been confirmed to have no obvious toxicity by experiments on acute toxicity tests, teratogenicity tests, and subchronic toxicity tests. In acute toxicity experiments, the oral LD 50 of CSR aqueous extract exceeded 21.5 g/kg in all cases . The results of another study indicated that the maximum tolerable dose of CSR in KM mice is greater than 15 g/kg , also providing evidence of CSR aqueous extract nonobvious toxicity. Salmonella typhimurium reverse mutation test, bone marrow polychromatic red blood cell micronucleus test, and sperm aberration test in KM mice were adopted to further investigate the genetic toxicity of CSR aqueous extract. One of the studies indicated that CSR aqueous extract (7.5 g/kg, 3.75 g/kg, and 1.875 g/kg) could not induce tested strains (TA97, TA98, TA100, TA102) to form colonies, as well as did not cause any mutagenic effects against the somatic cells and germ cells . The results of the experiment are proved by another study conducted with CSR aqueous extract (2.25, 4.50, 9.00 g/kg), with the difference being the type of experimental strains (TA97, TA98, TA100, TA102, and TA1535) . As the third stage of food safety toxicological evaluation, a subchronic toxicity test was conducted for a ninety-day feeding trial. No significant toxicological findings were detected in hematological parameters or clinical and pathological examinations when feeding various concentrations of CSR aqueous extract (1.04 g/kg, 2.08 g/kg, 4.16 g/kg) to SD rats . Three Doses of CSR aqueous extract (2.83 g/kg, 5.66 g/kg, 8.49 g/kg) were fed to Wistar rats in the same year’s research. Fu. Et found that the medium (5.66 g/kg) and high (8.49 g/kg) dose groups exhibited significantly increased plasma prothrombin time (PT), as well as testicular organ coefficient and epididymal organ coefficient. The maximum no-observed-adverse-effect level (NOAEL) was determined to be 2.83 g/kg, while the minimum lowest-observed-adverse-effect level (LOAEL) was identified at 5.66 g/kg in this subchronic transoral toxicity study. However, according to clinical reports, a patient was diagnosed with acute renal function injury after taking a single Chinese medicine CSR 100–150 g aqueous extract for about 0.5 h, experiencing nausea and vomiting 4–6 times, non-jet like, with all vomit being gastric contents, without abdominal pain or diarrhea, headache, or fever. Therefore, it is still essential to exercise caution and avoid an overdose of CSR . Based on the current situation, most studies about the toxicity of CSR focus on its aqueous extract, and few on other extracts and extract components. In order to build a more comprehensive toxicity evaluation, subsequent studies are required to focus on CSR extracts from other solvents. Furthermore, further exploration of toxic compounds in CSR is also necessary. These will provide a more reliable scientific basis for the safe use of CSR. In recent years, due to China’s aging population and growing demand for healthcare, CSR has emerged as a highly valuable Chinese herbal medicine. It is currently being investigated for its medicinal properties. A few studies have highlighted the beneficial effects of CSR on overall health. This has led to its incorporation into various compound preparations and health supplements, expanding its potential applications . This study summarizes various studies on Cynomorium songaricum Rupr. (CSR) from various aspects such as botany, ethnic pharmacology, phytochemistry, modern pharmacology, and toxicology. Compared with previous reviews describing the effects of CSR, the data on phytochemistry and pharmacology in this study are complete and more comprehensive, which helps to provide a data reference for professionals studying CSR. Traditional Chinese medicine CSR remains an outstanding kidney yang and tonifying remedy. Additionally, it nourishes the essence and blood, as well as moistening the intestines and bowel movements. Consequently, several traditional CSR prescriptions have been included in the Chinese Pharmacopoeia as modern clinical medications. Various active compounds such as flavonoids, terpenoids, and polysaccharides may be the molecular basis for CSR pharmacological activity. A portion of the 98 compounds isolated from CSR have pharmacological properties, including anti-tumor, antioxidant, neuroprotective, antiviral, and anti-diabetic properties, etc. However, most studies involving CSR mainly focus on simple validation of its effectiveness in extract administration. In vitro studies rarely involve in vivo mechanisms and there is a lack of modern pharmacological mechanism research on traditional Chinese medicine compound formulations. Looking back at the entire article, there are also some shortcomings in this study. The literature collection work is up to June 2023. Afterwards, more recently published CSR-related studies may not be included. Although CSR has become a well-known health medication in China, its popularity internationally is still insufficient. This may be related to its limited distribution in other countries. Therefore, it is inevitable that foreign research cited is relatively scarce. We hope that this study can attract more people to be interested in and involved in the study of CSR. Furthermore, we look forward to the future where traditional Chinese medicine of this kind will be included in foreign pharmacopoeias. Furthermore, in phytochemistry, there is a lack of research on the pharmacological activities of some compounds isolated from CSR, so their IC50 values were not included in this study. In a nutshell, the future direction of CSR should focus on further research into the pharmacology and toxicity of compounds. Further exploration of the pharmacological mechanisms underlying CSR needs to be conducted to address the current lack of research data, as well as the development of functional health products. |
Regional eye care survey: An essential prerequisite for an overall betterment of eye care services | 85b59a05-d3d9-4988-9028-7e2b41622280 | 10391444 | Ophthalmology[mh] | |
German radiation oncology’s next generation: a web-based survey of young biologists, medical physicists, and physicians—from problems to solutions | 6fcbb3ab-659a-4ca3-9462-573e550bbf56 | 11588816 | Internal Medicine[mh] | Radiation science is of the utmost significance not only due to its growing importance for clinical use, but also regarding safety issues related to environmental exposure to radiation, nuclear build outside of Germany, and decommissioning of existing power plants or possibly due to space travel . Because of the expected increase in cancer incidence (2.5% annually) due to an aging population and because of faster improvements and prevention measures in cardiovascular treatment, cancer is predicted to overtake cardiovascular disease as the leading cause of death. This further marks the growing importance of radiation in clinical cancer treatment and diagnostics . Surveys of the European Union show that 45% of all cancer patients are cured. Of these 45%, 22% undergo surgery alone, 12% radiotherapy alone, 6% a combination of surgery and radiotherapy, and 5% multimodal treatment combining surgery, radiotherapy, and chemotherapy . Moreover, radiotherapy is often implemented in palliative care to reduce symptomatic burden in advanced disease . Overall, these developments clearly demonstrate that ionizing radiation should not only be considered a risk factor for harmful effects in the public but also as a crucial factor for cancer treatment benefitting the public overall. In recent years, the field of radiation oncology has seen significant advances in therapeutic techniques, with new insights into radiation biology as well as advanced treatment planning algorithms, together contributing to improved quality of life and prolonged overall survival in patients with cancer . For application of advanced radiation therapy in the clinical context, various professional groups are needed, all of which make an important contribution to the overall treatment concept and success . Overall, technicians make up the biggest cohort of staff in radiation oncology and are of utmost importance; the following survey, however, focusses on the academic workforce. Here, physicians represent the largest cohort. The other academic professional groups include radiation biologists and medical physicists . All of these persons are of utmost importance not only for ensuring high-quality treatment and safety, but also to achieve the best possible clinical outcomes for patients and to pave the way to future scientific progress. The need for improved translational and multidisciplinary work has also been addressed by the “Vision 2030 for radiotherapy & radiation oncology in Germany” . To achieve this goal, the different specialties have different focus points: physicians usually have a primarily clinical focus on patient care. Medical physicists are more involved in technical treatment planning, accurate dosimetry, safe and effective delivery of radiation, and the implementation of new technologies . Biologists, on the other hand, usually focus on scientific questions, often work in laboratories outside of patient care, and are often challenged by the existing structures of the scientific world such as reduced and thus inadequate funding and/or limited career opportunities . Although 50% of cancer patients receive radiotherapy, the funding of radiation research appears to be rather low, and the opportunities may even be decreasing . In addition, it has been shown that 50% of funded research projects are carried out within a rather small number of research organizations, raising the question of how a more nationwide funding of research projects can be achieved . While working in radiation oncology can be highly motivating and fulfilling, this intersection of high-end technology and dedication to cancer patients also requires a highly trained and skilled workforce to maintain a high level of quality . While a number of surveys have reported high levels of satisfaction and a comfortable working environment in radiation oncology, other sources report an exponentially growing shortage of well-trained specialists, as driven by generational change and the retirement trend of the baby boomer generation . Leading radiation oncologists from teaching hospitals report problems with finding medical staff almost 50% of the time . Recent data suggest a potential shortage of about 18% for radiation oncologists in Europe overall . This means that the challenge of the future lies in inspiring and retaining young talents for the various professional specialties. Thus, factors that may discourage specialists from entering the field need to be identified and addressed by the radiation oncology community in order to maintain its independence from other departments and to remain a highly competitive medical specialty in its own right, without being incorporated into other specialties. One major challenge to acquiring sufficient numbers of young professionals is that each subspecialty has different requirements that have to be fulfilled to achieve job satisfaction. The aim of this study is therefore to examine the current situation of young professionals in all three subspecialties within the field of radiation oncology and to identify factors that may need to be addressed in order to provide a prosperous working and development environment for potential future professionals. Within the framework of the young DEGRO ( Deutsche Gesellschaft für Radioonkologie , German Society for Radiation Oncology) trial group and in close cooperation with the young DeGBS ( Deutsche Gesellschaft für Biologische Strahlenforschung , German Society for Biological Radiation Research) and the young Medical Physicists (MP; Deutsche Gesellschaft für Medizinphysik [DGMP], German Society for Medical Physics), a web-based questionnaire with one general and three occupation-specific questionnaires for physicians, biologists, and medical physicists working in radiation oncology and radiation research was developed. The aim of the survey was to identify the needs and wishes but also the level of satisfaction of the young workforce working in radiation oncology and radiation research. Survey An anonymous questionnaire was created using Survey Online® (Enuvo Inc., Switzerland). The survey consisted of a general part (13 questions) and 20–25 specialty-specific questions (physicians: 22 questions; biologists: 25 questions; medical physicists: 20 questions). 51 questions were mandatory (general: 9 questions; physicians: 14 questions; biologists: 17 questions; medical physicists: 11 questions), 29 questions were voluntary of which 17 were open-text questions (general: 4/3 voluntary/open-text questions; physicians: 8/4; biologists: 8/6; medical physicists: 9/4). The survey (for the full survey, see Supplementary Material 1) was designed with questions that allowed for single-choice or multiple-choice answers; some answers had an additional open-text field, and for the assessment of, e.g., workload or translational research opportunities, answers were ranked on a scale of 1 (very low) to 100 (very high) according to the participant’s subjective opinion. Free-text replies of participants were used to identify common problems and possible solutions. These insights from free-text replies were used to identify the most prominent questions, which are addressed in the Discussion. Prior to publishing the survey, a 3-day test phase was initiated, during which representatives of the subspecialties filled out the survey in order to identify potential shortfalls and problems. The survey was available for 6 weeks, from 22 June 2023 to 2 August 2023. Distribution Initial distribution of the questionnaires took place during the 29th Annual Meeting of the DEGRO in Kassel, Germany, on 22 June 2023. This was followed by distribution via the young DEGRO, young DeGBS, and young MP mailing lists, telephone calls, social media posts, and emails to various representatives from 42 (university) hospitals and 6 research institutes in Germany. Cohort and exclusion criteria As the survey was distributed at a conference, via social media, and via emails to various representatives, it is not known how many young academics were reached. In addition, 13% (Supplementary Table 1) of participants were aged 40 years or older, who are often not considered to be part of the young workforce. However, even those aged 40 years and older are expected to work for at least another 25 years. We thus chose to include this age group in the survey as, for example, having children or changing fields could result in a higher age while still holding a young professional position. Indeed, 27 of 34 participants from the 40 years and older age group also said that they were taking care of children or family members. Thus, all participants were included in the analyses provided they had completely filled out the survey. Statistics Statistical analysis was carried out using GraphPad Prism© (GraphPad Software, LLC, version 9.5.1, Boston, MA, USA). Descriptive statistics were used to visualize the answers given by the participants. For graphs showing the median distribution of the participants’ opinions, data are expressed as median + interquartile range (IQR; 75% percentile–25% percentile). An anonymous questionnaire was created using Survey Online® (Enuvo Inc., Switzerland). The survey consisted of a general part (13 questions) and 20–25 specialty-specific questions (physicians: 22 questions; biologists: 25 questions; medical physicists: 20 questions). 51 questions were mandatory (general: 9 questions; physicians: 14 questions; biologists: 17 questions; medical physicists: 11 questions), 29 questions were voluntary of which 17 were open-text questions (general: 4/3 voluntary/open-text questions; physicians: 8/4; biologists: 8/6; medical physicists: 9/4). The survey (for the full survey, see Supplementary Material 1) was designed with questions that allowed for single-choice or multiple-choice answers; some answers had an additional open-text field, and for the assessment of, e.g., workload or translational research opportunities, answers were ranked on a scale of 1 (very low) to 100 (very high) according to the participant’s subjective opinion. Free-text replies of participants were used to identify common problems and possible solutions. These insights from free-text replies were used to identify the most prominent questions, which are addressed in the Discussion. Prior to publishing the survey, a 3-day test phase was initiated, during which representatives of the subspecialties filled out the survey in order to identify potential shortfalls and problems. The survey was available for 6 weeks, from 22 June 2023 to 2 August 2023. Initial distribution of the questionnaires took place during the 29th Annual Meeting of the DEGRO in Kassel, Germany, on 22 June 2023. This was followed by distribution via the young DEGRO, young DeGBS, and young MP mailing lists, telephone calls, social media posts, and emails to various representatives from 42 (university) hospitals and 6 research institutes in Germany. As the survey was distributed at a conference, via social media, and via emails to various representatives, it is not known how many young academics were reached. In addition, 13% (Supplementary Table 1) of participants were aged 40 years or older, who are often not considered to be part of the young workforce. However, even those aged 40 years and older are expected to work for at least another 25 years. We thus chose to include this age group in the survey as, for example, having children or changing fields could result in a higher age while still holding a young professional position. Indeed, 27 of 34 participants from the 40 years and older age group also said that they were taking care of children or family members. Thus, all participants were included in the analyses provided they had completely filled out the survey. Statistical analysis was carried out using GraphPad Prism© (GraphPad Software, LLC, version 9.5.1, Boston, MA, USA). Descriptive statistics were used to visualize the answers given by the participants. For graphs showing the median distribution of the participants’ opinions, data are expressed as median + interquartile range (IQR; 75% percentile–25% percentile). Participants Figure gives an overview of the participants. While physicians in general are the largest group in the field of radiation oncology, there was also high participation of other subspecialties. Despite being the smallest group, biologists showed relatively high participation ( n = 59) compared to the larger groups of physicians ( n = 89) and (medical) physicists ( n = 70; Fig. ; Supplementary Table 1). In terms of age distribution, the cohort of biologists and physicists displayed a relatively even age distribution, while the participating physicians exhibited a peak between the ages of 26 and 35. While the gender distribution was balanced (51% female, 47% male), an examination of the subgroups showed a higher participation of females in biology (43 out of 59), whereas participants in the physicist (30 out of 70) and physician cohorts (37 out of 89) were predominantly male. An overview of participants’ characteristics can be found in Supplementary Table 1, Supplementary Material 2. Occupation-specific characteristics of participants While the majority of participating physicians were assistant physicians with a doctoral degree, the majority of participating biologists were PhD students with a bachelor’s or master’s degree (Fig. A.1 and A.2). Among the participating physicists, a large number of participants were certified medical physics experts ( Medizinphysikexperte , MPE) with a master’s or doctors’ degree or postdocs with a doctor’s degree and master’s students working on their master’s degree (Fig. A.3). Regarding the type of employment contract, the vast majority of biologists (74.6%) and physicians (76.4%) held temporary contracts, while the physicists were more likely to hold a permanent contract (44.3%; Fig. B). When asked about their involvement in teaching and supervision, the physician and biologist cohorts were more involved in teaching and supervision than the physicist group, where 38.6% replied that they were not involved in teaching at all (Fig. C). Biologists are the group most involved in supervising doctoral students (17%; Fig. C), while physicians and physicists are more involved in teaching (19.1% and 20%, respectively). All three groups are equally willing to increase their involvement. Level of satisfaction and future perspective in the younger workforce In our survey, university hospitals were found to be the most attractive employer (physicians 60.7%, biologists 56%, physicists 41.4%; Fig. A.1 to A.3). For physicians, private practices seem to be more attractive (23.6%) than employment at centers of maximal care (11.2%; Fig. A.1). Only around 5% of the physician participants envision a future outside of radiation oncology. While physicists, due to the nature of their profession, have the most opportunities per se, the numbers of physicists inclined to work in industry (25.7%; Fig. A.3) is equal to the number of biologists willing to work in industry (30.5%; Fig. A.2). While the vast majority of physicians (87.6%) and physicists (90%) confirmed their decision to enter the field of radiation oncology (Fig. B.1 and B.3), the biologist cohort has a large number of participants who are unsure of whether they would choose the same work field again (33.9%; Fig. B.2). The majority of physician participants rated the quality of their educational training as above average, with a median rating of 65 (IQR: 72–47) on a scale ranging from 1 (very low) to 100 (very high), as seen in Fig. C.1. In terms of subjective support in pursuing career goals, all cohorts report similar rates of lack of support (23.7 to 31.5%; Fig. D), with physicians taking the lead with a proportion of approximately 32% (Fig. D), while being the smallest group of participants not having spoken about the career goals with their employer (16.9%). Both physicists and biologists report a lack of communication about career goals in one third of cases. Approximately 50% of participants (45.7% to 51.7%) from all specialties report sufficient subjective support in achieving career goals. Concerns of all subspecialties about the future among the young workforce Three major concerns seem to be the leading causes limiting long-term career planning of physicians in radiotherapy: 1. economic pressure ( n = 48) as the biggest problem in pursuing a long-term career among all medical participants; 2. work–life balance ( n = 34); and 3. compatibility of career and family ( n = 32; Fig. A.1). For biologists, uncertain contract terms ( n = 15) seem to be the major concern when pursuing a career in radiation research (Fig. B). Further issues are very mediocre career options in the field (median: 51; IQR: 64–36; Fig. C) and a significant cohort even reports perceived worse career opportunities in radiation research than in other areas of biology (39%; Fig. D). For the participating physicists, the impeding factors seem to be a lot more varied, while the lack of work–life balance ( n = 22) and economic pressure ( n = 19) are also leading causes for not choosing a long-term career in radiation oncology (Fig. A.2). Subjective workload and weekly working hours For physicians and physicists working in daily patient care, the weekly workload appears to be very high (physicists: perceived median of 72.5 on a scale of 1 to 100; IQR: 85.5–50; Fig. A.3), with physicians having the highest subjective workload (median: 75; IQR: 92–5; Fig. A.1). The perceived workload for research is more evenly distributed (median 53 for physicians; IQR: 78–24 and 67 for physicists; IQR: 83.5–48; Fig. B.1 and B.3). Regarding the differences between full- and part-time employment, the survey results reveal that physicians are mostly employed full-time, while biologists have a higher percentage of employees working part-time (Fig. C.1 and C.2). In terms of the number of hours worked per week, physicians lead the field with a median of 48 h (IQR: 55–42; Fig. D.1), while biologists reported a median of 45 h (IQR: 50–40; Fig. D.2). There are no data for physicists, as physicists who participated in the original design of the survey had decided not to include these questions in the questionnaire. The distinction by contract type (temporary vs. permanent) shows no difference in subjective workload among physicians (median: 74 vs. 75; IQR: 95.5–56.3 vs. 91–51; Fig. A.1 on a scale of 1 to 100), while there seems to be a trend towards a greater subjective workload for staff on a permanent contract among biologists (median: 76 vs. 83; IQR: 90.3–61.8 vs. 94–55; Fig. A.2) and physicists (median: 60 vs. 80; IQR: 78–45 vs. 93–66; Fig. A.3). While the subjective workload in research does not differ for physicians (median: 52 vs. 53; IQR: 78–24.8 vs. 78–23.5; Fig. B.1), the subjective workload in research appears to be higher for employees with temporary contracts among physics (median: 73 vs. 62; IQR: 84–51 vs. 79.3–29.8; Fig. B.3). While full-time employment seems to be the rule for physicians, with very few exceptions, the biologist cohort shows a wider distribution of working time models and more employees working part-time (Fig. C.2), potentially due to the high numbers of participating doctoral students (Fig. ). This can also be seen from the fact that the median employment rate among temporarily employed biologists resembles 65% (IQR: 100–65), while the median employment percentage among those working on permanent contracts is 100% (IQR: 100–82.5; Fig. C.2). For the cohort of physicians, the median number of working hours per week seems to become smaller with a permanent contract (median 42 permanent contract vs. median 48 with a temporary contract; IQR: 50–40 vs. 56.5–42; Fig. D.1), while the opposite seems to be true for biologists (median 50 vs. 42.5; IQR: 52.5–38.5 vs. 50–40; Fig. D.2). Problems of subspecialties in day-to-day research work Concerning research time distribution, approximately 70% of physicians do not conduct research during their regular working hours, with 17% reporting that they conduct research during regular working hours most of the time. Only 5% reported doing research strictly during their working hours (Fig. A.1). This means that 50% of the participants invest less than 10 h per week in research while 30% manage to invest 10–15 h and only 10% are able to invest 15–20 h in research. Only 5% of physicians invest more than 20 h per week outside of their regular working hours (Fig. D). While only a small percentage (13.5%) of physicians report not having a research project, only 37% report receiving sufficient support for their research projects, while 50% report a lack of support for their research projects (Fig. C). On the other hand, physicists seem to be less involved in research projects (“I have no research project”: 21.4%; Fig. A.3), while more often receiving sufficient support for their research projects (55.7%; Fig. C). This is reflected by the fact that 53% of physicists carry out their research mostly during their regular working hours and 18% report that they only do research during their working hours (Fig. D). The cohort of biologists has 80% of participants doing research mostly during regular working hours, while 12% of the biologists carry out research strictly during regular hours only (Fig. A.2). Furthermore, 55.9% of biologists also reported that their tasks are not limited to the contractually agreed job description (data not shown). The perceived ability to carry out interdisciplinary and translational research at their institutions rated on a scale of 1 to 100 is lower for physicians (median: 63; IQR: 81–32; Fig. E) than for biologists (median: 78; IQR: 88–60; Fig. E) and physicists (median: 71; IQR: 84.8–34.5; Fig. E). In particular, among physicians and physicists, there appears to be a very broad distribution of perceived opportunities for translational research at their respective locations, thus lowering the overall median, whereas for biologists, there appears to be a greater presence of translational research. Participation and satisfaction with respective to professional societies Physicians show the highest rate of membership in a professional society (86.5%; Fig. a). Biologists have the highest number of participants in more than one society (13.6%; Fig. a). Physicists show the lowest number of participants in a professional society overall (44.3%; Fig. a) and feel inadequately represented by a society in 35% of cases (Fig. b). While the majority of physicians and biologists feel adequately represented (77% and 73.7%, respectively), around one quarter of participants still do not feel adequately represented by a society overall (Fig. b). Regarding the image of radiation research, physicians in particular have a positive perception of radiation research (positive: 42.7% and rather positive: 31.5%; Fig. c), while physicists (positive: 32.9% and rather positive: 51.4%; Fig. c) and biologists (positive: 25.4% and rather positive: 30.5%, Fig. c) are more reserved. Biologists are most likely to see the image of radiation research as neutral (32.2%) or even rather negative (11.9%). None of the participating professions indicated that radiation research is perceived negatively. Figure gives an overview of the participants. While physicians in general are the largest group in the field of radiation oncology, there was also high participation of other subspecialties. Despite being the smallest group, biologists showed relatively high participation ( n = 59) compared to the larger groups of physicians ( n = 89) and (medical) physicists ( n = 70; Fig. ; Supplementary Table 1). In terms of age distribution, the cohort of biologists and physicists displayed a relatively even age distribution, while the participating physicians exhibited a peak between the ages of 26 and 35. While the gender distribution was balanced (51% female, 47% male), an examination of the subgroups showed a higher participation of females in biology (43 out of 59), whereas participants in the physicist (30 out of 70) and physician cohorts (37 out of 89) were predominantly male. An overview of participants’ characteristics can be found in Supplementary Table 1, Supplementary Material 2. While the majority of participating physicians were assistant physicians with a doctoral degree, the majority of participating biologists were PhD students with a bachelor’s or master’s degree (Fig. A.1 and A.2). Among the participating physicists, a large number of participants were certified medical physics experts ( Medizinphysikexperte , MPE) with a master’s or doctors’ degree or postdocs with a doctor’s degree and master’s students working on their master’s degree (Fig. A.3). Regarding the type of employment contract, the vast majority of biologists (74.6%) and physicians (76.4%) held temporary contracts, while the physicists were more likely to hold a permanent contract (44.3%; Fig. B). When asked about their involvement in teaching and supervision, the physician and biologist cohorts were more involved in teaching and supervision than the physicist group, where 38.6% replied that they were not involved in teaching at all (Fig. C). Biologists are the group most involved in supervising doctoral students (17%; Fig. C), while physicians and physicists are more involved in teaching (19.1% and 20%, respectively). All three groups are equally willing to increase their involvement. In our survey, university hospitals were found to be the most attractive employer (physicians 60.7%, biologists 56%, physicists 41.4%; Fig. A.1 to A.3). For physicians, private practices seem to be more attractive (23.6%) than employment at centers of maximal care (11.2%; Fig. A.1). Only around 5% of the physician participants envision a future outside of radiation oncology. While physicists, due to the nature of their profession, have the most opportunities per se, the numbers of physicists inclined to work in industry (25.7%; Fig. A.3) is equal to the number of biologists willing to work in industry (30.5%; Fig. A.2). While the vast majority of physicians (87.6%) and physicists (90%) confirmed their decision to enter the field of radiation oncology (Fig. B.1 and B.3), the biologist cohort has a large number of participants who are unsure of whether they would choose the same work field again (33.9%; Fig. B.2). The majority of physician participants rated the quality of their educational training as above average, with a median rating of 65 (IQR: 72–47) on a scale ranging from 1 (very low) to 100 (very high), as seen in Fig. C.1. In terms of subjective support in pursuing career goals, all cohorts report similar rates of lack of support (23.7 to 31.5%; Fig. D), with physicians taking the lead with a proportion of approximately 32% (Fig. D), while being the smallest group of participants not having spoken about the career goals with their employer (16.9%). Both physicists and biologists report a lack of communication about career goals in one third of cases. Approximately 50% of participants (45.7% to 51.7%) from all specialties report sufficient subjective support in achieving career goals. Three major concerns seem to be the leading causes limiting long-term career planning of physicians in radiotherapy: 1. economic pressure ( n = 48) as the biggest problem in pursuing a long-term career among all medical participants; 2. work–life balance ( n = 34); and 3. compatibility of career and family ( n = 32; Fig. A.1). For biologists, uncertain contract terms ( n = 15) seem to be the major concern when pursuing a career in radiation research (Fig. B). Further issues are very mediocre career options in the field (median: 51; IQR: 64–36; Fig. C) and a significant cohort even reports perceived worse career opportunities in radiation research than in other areas of biology (39%; Fig. D). For the participating physicists, the impeding factors seem to be a lot more varied, while the lack of work–life balance ( n = 22) and economic pressure ( n = 19) are also leading causes for not choosing a long-term career in radiation oncology (Fig. A.2). For physicians and physicists working in daily patient care, the weekly workload appears to be very high (physicists: perceived median of 72.5 on a scale of 1 to 100; IQR: 85.5–50; Fig. A.3), with physicians having the highest subjective workload (median: 75; IQR: 92–5; Fig. A.1). The perceived workload for research is more evenly distributed (median 53 for physicians; IQR: 78–24 and 67 for physicists; IQR: 83.5–48; Fig. B.1 and B.3). Regarding the differences between full- and part-time employment, the survey results reveal that physicians are mostly employed full-time, while biologists have a higher percentage of employees working part-time (Fig. C.1 and C.2). In terms of the number of hours worked per week, physicians lead the field with a median of 48 h (IQR: 55–42; Fig. D.1), while biologists reported a median of 45 h (IQR: 50–40; Fig. D.2). There are no data for physicists, as physicists who participated in the original design of the survey had decided not to include these questions in the questionnaire. The distinction by contract type (temporary vs. permanent) shows no difference in subjective workload among physicians (median: 74 vs. 75; IQR: 95.5–56.3 vs. 91–51; Fig. A.1 on a scale of 1 to 100), while there seems to be a trend towards a greater subjective workload for staff on a permanent contract among biologists (median: 76 vs. 83; IQR: 90.3–61.8 vs. 94–55; Fig. A.2) and physicists (median: 60 vs. 80; IQR: 78–45 vs. 93–66; Fig. A.3). While the subjective workload in research does not differ for physicians (median: 52 vs. 53; IQR: 78–24.8 vs. 78–23.5; Fig. B.1), the subjective workload in research appears to be higher for employees with temporary contracts among physics (median: 73 vs. 62; IQR: 84–51 vs. 79.3–29.8; Fig. B.3). While full-time employment seems to be the rule for physicians, with very few exceptions, the biologist cohort shows a wider distribution of working time models and more employees working part-time (Fig. C.2), potentially due to the high numbers of participating doctoral students (Fig. ). This can also be seen from the fact that the median employment rate among temporarily employed biologists resembles 65% (IQR: 100–65), while the median employment percentage among those working on permanent contracts is 100% (IQR: 100–82.5; Fig. C.2). For the cohort of physicians, the median number of working hours per week seems to become smaller with a permanent contract (median 42 permanent contract vs. median 48 with a temporary contract; IQR: 50–40 vs. 56.5–42; Fig. D.1), while the opposite seems to be true for biologists (median 50 vs. 42.5; IQR: 52.5–38.5 vs. 50–40; Fig. D.2). Concerning research time distribution, approximately 70% of physicians do not conduct research during their regular working hours, with 17% reporting that they conduct research during regular working hours most of the time. Only 5% reported doing research strictly during their working hours (Fig. A.1). This means that 50% of the participants invest less than 10 h per week in research while 30% manage to invest 10–15 h and only 10% are able to invest 15–20 h in research. Only 5% of physicians invest more than 20 h per week outside of their regular working hours (Fig. D). While only a small percentage (13.5%) of physicians report not having a research project, only 37% report receiving sufficient support for their research projects, while 50% report a lack of support for their research projects (Fig. C). On the other hand, physicists seem to be less involved in research projects (“I have no research project”: 21.4%; Fig. A.3), while more often receiving sufficient support for their research projects (55.7%; Fig. C). This is reflected by the fact that 53% of physicists carry out their research mostly during their regular working hours and 18% report that they only do research during their working hours (Fig. D). The cohort of biologists has 80% of participants doing research mostly during regular working hours, while 12% of the biologists carry out research strictly during regular hours only (Fig. A.2). Furthermore, 55.9% of biologists also reported that their tasks are not limited to the contractually agreed job description (data not shown). The perceived ability to carry out interdisciplinary and translational research at their institutions rated on a scale of 1 to 100 is lower for physicians (median: 63; IQR: 81–32; Fig. E) than for biologists (median: 78; IQR: 88–60; Fig. E) and physicists (median: 71; IQR: 84.8–34.5; Fig. E). In particular, among physicians and physicists, there appears to be a very broad distribution of perceived opportunities for translational research at their respective locations, thus lowering the overall median, whereas for biologists, there appears to be a greater presence of translational research. Physicians show the highest rate of membership in a professional society (86.5%; Fig. a). Biologists have the highest number of participants in more than one society (13.6%; Fig. a). Physicists show the lowest number of participants in a professional society overall (44.3%; Fig. a) and feel inadequately represented by a society in 35% of cases (Fig. b). While the majority of physicians and biologists feel adequately represented (77% and 73.7%, respectively), around one quarter of participants still do not feel adequately represented by a society overall (Fig. b). Regarding the image of radiation research, physicians in particular have a positive perception of radiation research (positive: 42.7% and rather positive: 31.5%; Fig. c), while physicists (positive: 32.9% and rather positive: 51.4%; Fig. c) and biologists (positive: 25.4% and rather positive: 30.5%, Fig. c) are more reserved. Biologists are most likely to see the image of radiation research as neutral (32.2%) or even rather negative (11.9%). None of the participating professions indicated that radiation research is perceived negatively. Identifying the issues faced by the young workforce is an essential step towards improving the satisfaction of all subspecialties and improving the willingness to not only get involved but also to continue an active career in the field of radiation science. Despite physicians comprising the largest of the subspecialties according to the overall number of employees in Germany, it was especially biologists and physicists with a very high participation in the survey . The overall high involvement during a rather short period of 6 weeks in comparison to similar studies indicates that there is potential, ambition, and interest among the younger employees to identify problems and improve the field of radiation oncology and radiation research over the next decades. Our study revealed that each of the subspecialties has distinct problems (e.g., the contractual situation for biologists), while other factors are present cross-disciplinarily and thus need to be addressed by the societies as a whole. Subspecialties specific problems Biologists Radiation biology, despite not being present in all departments, makes a tremendous contribution to the field of radiation oncology and radiation protection by providing knowledge of dose fractionation, tumor hypoxia, tissue weighting factors, and radiation quality as well as regarding the optimized use of combination therapies. Radiation biologists have helped to define parameters needed for risk and exposure assessment, thus establishing dose limits and offering mechanistic explanations for outcomes observed in radiation epidemiology that provide the pivotal translation of laboratory data into human applications. Insights gained by radiation biologists are used in clinical work for radiation protection, diagnosis, risk assessment, and treatment. While our survey reports that most participants see only mediocre career options and about 40% even worse career opportunities than in other biological fields (Fig. ), one has to point out that the insights and knowledge gathered can be used not only in the public health domain but also for military purposes, space exploration, environmental stewardship, and national security, and also provide biological and biophysical information on radiation response to federal agencies setting exposure limits for workers and the general public. This can of course be used for creating job opportunities, and the gathered knowledge can be used as a transition into other scientific fields or jobs outside of the field of radiation oncology. In these cases, career seminars might be helpful in order to point out alternative career options for biologists working in radiation research. In this way, well-trained high-potential staff will not get lost to other disciplines or subjects but will find ways to remain in radiation research outside of the medical field. An uncontroversial fact is that securing research funding, which is often challenging to obtain, places a significant burden on employees, as it is an essential element for maintaining a position and providing the potential for career advancement, especially in biological research . The probability of funding, on the other hand, is strongly determined by strong mentorship, establishment of a collaborative network of scientific and clinical expertise within the host department, and scientific input from related departments both within the host department and from external institutions. Besides typical support with data analysis, biostatics, literature, and article writing, intensified help in preclinical animal studies with exceptionally hard and complex legal and ethical regulations should be considered . At the moment, the majority of postdocs are forced to cover their position using grant money, which basically makes them cheap, smart, and highly educated labor . Short-sighted hyper-competitive environments potentially even slow down scientific innovation and productivity and favor fabrication and publication of immature data, thereby wasting valuable resources. High expectations and success—despite the low probability of success in the whole research environment, ranging from grant applications to hiring decisions and career progression—strongly depend upon publications, which encourages scientific dishonesty . Improvement can be achieved by preparing trainees for different potential career paths, teaching training skills that can be used outside of the academic world, creating communication platforms, and giving postdocs more visibility and thus possibilities to be more proactive about their career. Another approach could be to optimize the postdoc salary with cost-of-living adjustments . Improvements need to be made towards individual grant application by postdocs and improving grant evaluation for younger researchers with lesser-known names and reputations, thus enabling high-potential scientists to focus on research instead of writing grants . The harsh working conditions are very well reflected by the low percentage of participants in our survey who would choose radiation biology again. In combination with the mediocre career options and even worse-perceived employment options overall compared to other biological research fields (Fig. ), attracting high-potential biologists to the field is becoming more challenging. However, as a high number of individuals in our survey are unsure about whether or not to remain in radiation research, it is evident that adequate measures can aid in keeping these individuals rooted in radiation research. (Medical) physicists Physicists represent the second largest cohort of employees involved in the field of radiation oncology. A regulatorily required number of (medical) physicists is of utmost importance for the safety of patients and the quality of radiation treatment. Important tasks carried out by physicists range from administrative and clinical services to informatics, equipment maintenance, and performance evaluations, as well as research, teaching, and training. The past has shown that implementation of new techniques like intensity-modulated radiotherapy (IMRT) leads to an increased demand for physicists . Despite being of such great importance, very few new academic positions are being created in research and education in health care physics and medical physics overall . Nevertheless, the profession will likely continue to further increase in importance, mostly due to the vast amount of new technology that will need to be implemented into current treatment protocols. In the present survey, physicists show the highest willingness to leave academia and transition into industry, while private practices are the employers least favored by physicists, which could be biased as participants of the survey are mostly employed at university hospitals. However, the fear of less challenging and versatile cases as well as repetitive daily tasks in peripheral institutions could also be a reason for this. As well-trained physicists can easily transition into other work fields, it is an essential task to better include these specialists in the society and keep these individuals in the field of radiation oncology in order to remain competitive in the years to come, as inadequate staffing will inevitably lead to loss of quality and safety in the long run. One possible way to achieve long-term commitment could be an increase in the permanent contracts offered by institutions. Physicists represent the subspecialty with the highest percentage of permanent contracts in the survey, which seems to reflect the particular need for long-term integration of well-trained personnel into workflows of the respective departments to ensure consistency and reliability, but also the legal requirements for the presence of MPEs. It remains unclear why a large percentage of (medical) physicists are not members of a society (Fig. ). It may be possible that bachelor’s/master’s students, due to their early career stage, and non-scientific MPs may not see any representation of interests in a scientific society. As there is a lack of a professional association for clinically working medical physics experts in Germany, the offer of (interdisciplinary) education and interaction for this group should eventually be further expanded within the existing scientific associations. At the same time, this may increase interest and active participation in the societies and strengthen interdisciplinary and translational cooperation between research and clinical settings. In general, the cohort of physicists seems to be rather satisfied with their career choice, reporting the highest percentage of participants who would choose the same specialty again and high satisfaction rates regarding the support offered by the employer (Fig. ). Only the subjective workload in science tends towards being higher compared to the physician cohort. Physicians Physicians are the largest cohort and also represent the subspecialty with the highest visibility due to the direct patient contact. In our survey, the most prominent fear among physicians is high economic pressure followed by a lack of work–life balance and compatibility of career and family (Fig. ). Regarding the general economic situation, lower financial reimbursement combined with higher costs for personnel, energy, and maintenance cause significant economic stress in the different departments. Profit targets set by the hospital administrations are a balancing act between the economic situation and a reasonable treatment for patients. In Germany, a large cohort of patients undergo treatment within private practices, which due to newer rules, regulations, and alterations in billing could possibly put those practices under significant economic pressure in the future, especially as insurance companies try to cut costs overall . Another significant point defining employee satisfaction is the subjective or perceived workload. The present survey shows that especially physicians have the highest subjective workload among all subspecialties (Figs. and ). Recent studies report that a continuously high workload can lead to depression and loss of workforce in the long term, resulting in a negative public perception of the field . As especially research work outside of regular working hours can contribute to the workload, protected research time needs to be implemented using specialized programs or research grants. Common problems in the younger workforce Work–life balance Another topic that needs to be addressed for all subspecialties in the future is the creation of a job environment with an adequate work–life balance, as this is one of the major concerns of the physician and physicist cohorts in our survey. Compatibility of career and family, especially with a higher number of female professionals (Fig. ), paired with significant changes in classical family roles, is also increasing in importance. The possibility of working part-time is an essential factor, facilitating better adaptation to private circumstances such as nursing a family member or taking time out for childcare. Another possible solution is implementing the concept of a 4-day working week that is currently discussed and even being tested in some clinics in Germany. While the biologist cohort shows a fair number of employees working part-time, this is rather unusual for the physician group, which shows the highest rate of full-time employment (Figs. and ). However, as doctoral candidates in biology or medical physics are employed part-time while usually working full-time, an in-depth analysis of these factors will need to be carried out in the future. Thus, this is in most cases only a pro-forma part-time contract, while PhD students actually work full-time. True part-time contracts might, however, increase the attractiveness of the field through improved work–life or work–family balance. The high percentage of full-time-employed participants in the physician group could possibly be explained by the need of finishing a residency program as the final step of their education. In the future, more liberal part-time models should be offered and will be inevitable as more and more female employees strive into the field. Working part-time in general needs to become more accepted by supervisors independent of gender to enable contemporary gender-equal divisions of labor and non-classical family roles. Working environment The most favored form of employment in this survey is academia among all subspecialties (Fig. ). One possible explanation could be that academia combines clinical, scientific, and teaching work, in contrast to non-academic clinics or private practice, and offers the chance to work in a cutting-edge and up-to-date environment that is usually less economically driven. University hospitals offer, besides a basic education, the possibility to acquire further scientific education, helping to implement new results into daily work as paradigm changes can occur very fast in modern medicine . However, the structure of universities overall will not make it possible for all participants to pursue a career in academia. Further considerations discouraging long-term academic careers include academic pressure, declining support for research, and increased bureaucracy , as also visible in the present study (Fig. ). Potential points of improvement A survey by Krug et al. about the young radiation oncology workforce showed that only 4% of participants reported a complete separation between clinical work and research and/or teaching activities. Among these, physicians reported the lowest number (9%) of protected time to carry out research or teaching in their regular working hours on a regular basis , suggesting that most of the time, teaching and research are carried out outside of regular working hours. This can potentially become problematic, as the quality of research and teaching but also of clinical work might suffer when tasks are mainly carried out outside of regular hours and during overtime . Research One possible point for improvement is the creation of easier options for entry into scientific work. While the volume and intensity of research will differ significantly between facilities, barriers impeding participation in research have to be clearly addressed. Obstacles that are frequently mentioned range from lack of time, limited access to statistical analysis through insufficient mentoring, and variable support from superiors, to great variability in research productivity at the department level, partially due to metafactors such as the size and composition of the department, advanced technology capability, limited access to funding, and the numbers of patients and degree of engagement with specialist medical research institutions . The present survey confirms that especially employees in the physician cohort lack protected research time (Fig. ). Combined with the fact that overtime in work covering the clinical routine is becoming the rule rather than the exception, this makes it virtually impossible for clinicians to engage in high-quality and pioneering research . Furthermore, according to our survey, physicians and physicists are less frequently involved in the supervision of doctoral students compared to biologists. This can be attributed to a higher level of clinical involvement and responsibilities; likewise, it is possible that these groups are more involved in the supervision of bachelor’s or master’s theses. As scientific work is time consuming, an approach to include mainly clinically working personnel in research, thus giving them an overview of current trends and developments outside of their daily work, could be to involve them in the reviewing process of scientific work after respective training. Other countries require medical candidates to complete mandatory scientific work before their final examination. In the case of clinical medical physicists in training, completion of a scientific project or an additional study before becoming eligible for certification could be obligatory—an approach that has been successfully implemented in the model medical study course in Cologne, Germany . Another recent improvement in this regard is the introduction of young scientist scholarships, enabling physicians to take protected research time as a clinician scientist , which could significantly improve the situation. This is especially important as fundamental research on combining radiation therapy with other treatment modalities like surgery and chemo-/immunotherapy will become more and more important in the future. Intensifying translational work cooperations will be an inevitable component of this. Teaching Likewise, lectures on physics and biology have to be implemented into standard residency programs to generate a basic understanding of the mode of operation of, e.g., immunotherapies, and to foster awareness of possible cooperations and interactions between subspecialties . Furthermore, the satisfaction of trainees with their occupational training is of utmost importance for choosing a certain career path. Participants from the medical field that were asked about their educational quality in our study, however, report a broad range of satisfaction, with a median of 65/100 (Fig. ). There is a significant number of participants that seem to be highly satisfied with their education versus 29% of participants reporting satisfaction rates of below 50. One option could be to hold written and oral examination on the topics of radiation biology and radiation physics early during residency, following the example of the radiation protection course. These results could also give more feedback to the teaching institutions from an educational standpoint . Cooperations between biologists and physicists also need to be intensified, as new radiation-generating devices show increasing complexity, requiring formal training in basic principles of radiation dosimetry and measurement for research. Our survey further reports that the involvement of physicians and biologists in teaching is higher than that of the subcohort of physicists (Fig. ). While medical physics most likely will continue to grow in importance due to increasing numbers of cancer cases as well as further scientific improvements leading to advanced technologies that increase the demand for well-trained professionals , the high number of participants not involved in either teaching or scientific work is problematic and needs to be improved in the future. Mentoring and networking One quarter of participants indicate insufficient support for their career goals, potentially causing them to leave the field in the long term. Among the participants, the cohorts of biologists and physicists seem to be a lot less prone to communicating their potential career goals to their employers (Fig. ), which could be interpreted as either being less career oriented or as being less informed regarding the possibilities in the field. Here, career courses could be an option to inform and train staff to better identify and communicate their goals. Another possibility might be a widespread implementation of mentoring programs that pair young professionals with those more experienced in the field who often have a more widespread network and can offer pivotal guidance in personal, clinical, and academic growth. Especially mentoring has been frequently mentioned during the current survey. The supervision by mentors could help to create a meaningful timeline for reaching certain milestones and complementary educational achievements such as a basic education in health care economics, as often required for higher positions , especially as mentors can give advice independently of the department’s own interests. Recently, the young scientist groups have reacted to this topic and offered a mentoring program for all three subspecialties involved in radiation oncology, highly supported by older members of the society in Germany. Future developments and generational change in radiation oncology Another point that needs to be discussed is how the clinical demand will change as a response to growth in the cancer incidence (approximately 2.5% per year), the impact of new technologies with possible acceleration of treatment-related workflows due to artificial intelligence, but also possible slowing of processes due to more adaptive and individualized treatment regimens . While future developments cannot be entirely foreseen, technical progress, which holds great potential for improvements in treatment overall, is not seen as a significant problem, neither by the physicist nor by the physician cohort (Fig. ). In contrast, new technologies can even lead to an increase in treatment time. Models and further predictions show that a 5% increase in overall workload could potentially lead to a significantly higher number of staff needed from all involved specialties . One example is the increase in demand for medical physicists due to the introduction of new technologies and an increased number of treatment machines . Combined with a general shortage of personnel due to aging of the existing workforce, which can only be compensated to a very limited basis, this could lead to a real decline in treatment capacity, safety, and overall quality. Limitations could be reduced through the extension of work contracts or consulting and by increasing the number of open clinical training positions for MPEs . While a lack of potential employers is present for a large variety of specialties, overall competition for motivated and well-trained employees due to suboptimal working conditions and payment level compared to some other European countries is inevitable. Radiation oncology will probably not be given more attention in undergraduate life science education, thus limiting its visibility to potential future employees at that early stage of their career path. Therefore, advertising the field by offering good and diversified training programs with a wide range of additional offers could serve as a letter of invitation, convincing potential candidates to take their decision in favor of radiation oncology in the end. In contrast to a lack of potential employers, the generational change benefits investor-managed hospital and practice chains primarily focusing on increasing margins rather than prioritizing improving patient treatment. Radiation oncology is underrepresented in oncological teaching in medical schools, which leads to underutilization and a lot of prejudices towards the field, while studies show improved knowledge with dedicated courses, especially favoring clinical exposure over dedicated lectures . The lack of knowledge potentially even impedes multidisciplinary oncological care. Furthermore, a recent study demonstrates that the introduction of radiation oncology into the preclinical part of medical education is feasible , an idea that is generally well perceived by students . This may aid in attracting more doctoral candidates and residents while improving overall knowledge in radiation oncology. Possibilities to teach radiation oncology outside of the existing curriculum include special lectures, seminars, and bedside training as well as the meaningful integration of e‑learning . Teaching in biology and medical physics is generally less frequent . The decision to choose a certain specialty is driven by different intrinsic (personal interest) and extrinsic (perceived prestige or potential income) as well as structural considerations like the influence of lifestyle, working hours, perceived stress from on-call duties, amount of time on-duty, and the possibility to work part-time. Although no one single factor can be seen as decisive, a beneficial combination of exactly these points could give the field of radiation oncology the edge, especially for students still undecided at the end of their studies . Awareness and basic knowledge in these fields, however, also needs to be established amongst general practitioners to provide patients with adequate advice and information . The role of societies Another promising step towards improvement is the creation of networks among younger professionals within the framework of existing societies. The creation of internal groups linking young radiation oncology scholars has been successfully implemented in other European countries . Structured collaboration between new and advanced professionals within the society can further address essential points such as the drafting of a good residential curriculum, including reliable long-term rotation plans covering all relevant aspects of clinical radiation oncology and naming individuals responsible for the supervision of residency training. Another possibility would be the creation of improved teaching including lecture series covering all the major relevant topics in oncology in a location-independent approach, ensuring that all residents can benefit from the knowledge of local specialists. Such a step was recently taken by the young DEGRO in Germany and has been met with a very positive response . Teaching, which is often not standardized throughout the different departments, has been further compromised by the COVID pandemic . The young DEGRO group has implemented online courses covering different topics in physics, biology, and clinical radiation oncology. The courses so far have been very well received, inviting new aspiring scientists and clinicians to present a talk on their research focus. A similar program is found in the DGMP and young MP, while the young DeGBS focuses on online seminars covering topics such as how to set up a lab or introduction of committees. Points that have to be further addressed include more time for self-study; increasing support by the DEGRO, DGMP, and DeGBS; participation in meetings; improved educational quality; and an institutional training schedule that focuses more on the guidelines drawn up by the German medical council . Another viable option could be the creation of a central research and teaching team organized by the DEGRO and young DEGRO in collaboration with the DeGBS and young DeGBS as well as the DGMP and young MP to assist in the setup of studies and offer help with valid statistical evaluation, research training, and the advancement of science overall . Another more decentralized possibility could be the creation of a core research team in each university clinic consisting of a clinical researcher, a biologist, and a physicist with clearly defined tasks and protected time for the generation and supervision of larger-scale studies as well as assisting in translational research approaches. More ambition in this field by DEGRO/young DEGRO could improve the number of members of all subspecialties in the society, as there will be an improved sense of personal benefit from joining the society (; Fig. ). In general, giving more power to young societies not only creates opportunities for getting involved but also allows for more uniform training, also including special techniques such as new imaging modalities (for example, PET-CT, MRI, special sequences) as well as insights into less mainstream areas of radiation oncology such as brachytherapy. Limitations As this is the first study combining a team of young DEGRO, young DeGBS, and young MP members, the present interrogation could not rely on a validated questionnaire but was composed of newly developed questions, thus leaving room for multiple interpretation possibilities that need to be further specified in the future. Nevertheless, this approach was chosen due to the nature of the survey at hand. We have included a wide number of occupations, workplaces, and research foci in this survey, as all of these professionals work in radiation oncology and radiation research and thus also have a wide range of specific occupational requirements. This approach thus allowed generation of a broader overview. Further studies will also have to include the specific needs of radiotherapy technologists, especially with regard to the declining numbers of this essential profession. Biologists Radiation biology, despite not being present in all departments, makes a tremendous contribution to the field of radiation oncology and radiation protection by providing knowledge of dose fractionation, tumor hypoxia, tissue weighting factors, and radiation quality as well as regarding the optimized use of combination therapies. Radiation biologists have helped to define parameters needed for risk and exposure assessment, thus establishing dose limits and offering mechanistic explanations for outcomes observed in radiation epidemiology that provide the pivotal translation of laboratory data into human applications. Insights gained by radiation biologists are used in clinical work for radiation protection, diagnosis, risk assessment, and treatment. While our survey reports that most participants see only mediocre career options and about 40% even worse career opportunities than in other biological fields (Fig. ), one has to point out that the insights and knowledge gathered can be used not only in the public health domain but also for military purposes, space exploration, environmental stewardship, and national security, and also provide biological and biophysical information on radiation response to federal agencies setting exposure limits for workers and the general public. This can of course be used for creating job opportunities, and the gathered knowledge can be used as a transition into other scientific fields or jobs outside of the field of radiation oncology. In these cases, career seminars might be helpful in order to point out alternative career options for biologists working in radiation research. In this way, well-trained high-potential staff will not get lost to other disciplines or subjects but will find ways to remain in radiation research outside of the medical field. An uncontroversial fact is that securing research funding, which is often challenging to obtain, places a significant burden on employees, as it is an essential element for maintaining a position and providing the potential for career advancement, especially in biological research . The probability of funding, on the other hand, is strongly determined by strong mentorship, establishment of a collaborative network of scientific and clinical expertise within the host department, and scientific input from related departments both within the host department and from external institutions. Besides typical support with data analysis, biostatics, literature, and article writing, intensified help in preclinical animal studies with exceptionally hard and complex legal and ethical regulations should be considered . At the moment, the majority of postdocs are forced to cover their position using grant money, which basically makes them cheap, smart, and highly educated labor . Short-sighted hyper-competitive environments potentially even slow down scientific innovation and productivity and favor fabrication and publication of immature data, thereby wasting valuable resources. High expectations and success—despite the low probability of success in the whole research environment, ranging from grant applications to hiring decisions and career progression—strongly depend upon publications, which encourages scientific dishonesty . Improvement can be achieved by preparing trainees for different potential career paths, teaching training skills that can be used outside of the academic world, creating communication platforms, and giving postdocs more visibility and thus possibilities to be more proactive about their career. Another approach could be to optimize the postdoc salary with cost-of-living adjustments . Improvements need to be made towards individual grant application by postdocs and improving grant evaluation for younger researchers with lesser-known names and reputations, thus enabling high-potential scientists to focus on research instead of writing grants . The harsh working conditions are very well reflected by the low percentage of participants in our survey who would choose radiation biology again. In combination with the mediocre career options and even worse-perceived employment options overall compared to other biological research fields (Fig. ), attracting high-potential biologists to the field is becoming more challenging. However, as a high number of individuals in our survey are unsure about whether or not to remain in radiation research, it is evident that adequate measures can aid in keeping these individuals rooted in radiation research. (Medical) physicists Physicists represent the second largest cohort of employees involved in the field of radiation oncology. A regulatorily required number of (medical) physicists is of utmost importance for the safety of patients and the quality of radiation treatment. Important tasks carried out by physicists range from administrative and clinical services to informatics, equipment maintenance, and performance evaluations, as well as research, teaching, and training. The past has shown that implementation of new techniques like intensity-modulated radiotherapy (IMRT) leads to an increased demand for physicists . Despite being of such great importance, very few new academic positions are being created in research and education in health care physics and medical physics overall . Nevertheless, the profession will likely continue to further increase in importance, mostly due to the vast amount of new technology that will need to be implemented into current treatment protocols. In the present survey, physicists show the highest willingness to leave academia and transition into industry, while private practices are the employers least favored by physicists, which could be biased as participants of the survey are mostly employed at university hospitals. However, the fear of less challenging and versatile cases as well as repetitive daily tasks in peripheral institutions could also be a reason for this. As well-trained physicists can easily transition into other work fields, it is an essential task to better include these specialists in the society and keep these individuals in the field of radiation oncology in order to remain competitive in the years to come, as inadequate staffing will inevitably lead to loss of quality and safety in the long run. One possible way to achieve long-term commitment could be an increase in the permanent contracts offered by institutions. Physicists represent the subspecialty with the highest percentage of permanent contracts in the survey, which seems to reflect the particular need for long-term integration of well-trained personnel into workflows of the respective departments to ensure consistency and reliability, but also the legal requirements for the presence of MPEs. It remains unclear why a large percentage of (medical) physicists are not members of a society (Fig. ). It may be possible that bachelor’s/master’s students, due to their early career stage, and non-scientific MPs may not see any representation of interests in a scientific society. As there is a lack of a professional association for clinically working medical physics experts in Germany, the offer of (interdisciplinary) education and interaction for this group should eventually be further expanded within the existing scientific associations. At the same time, this may increase interest and active participation in the societies and strengthen interdisciplinary and translational cooperation between research and clinical settings. In general, the cohort of physicists seems to be rather satisfied with their career choice, reporting the highest percentage of participants who would choose the same specialty again and high satisfaction rates regarding the support offered by the employer (Fig. ). Only the subjective workload in science tends towards being higher compared to the physician cohort. Physicians Physicians are the largest cohort and also represent the subspecialty with the highest visibility due to the direct patient contact. In our survey, the most prominent fear among physicians is high economic pressure followed by a lack of work–life balance and compatibility of career and family (Fig. ). Regarding the general economic situation, lower financial reimbursement combined with higher costs for personnel, energy, and maintenance cause significant economic stress in the different departments. Profit targets set by the hospital administrations are a balancing act between the economic situation and a reasonable treatment for patients. In Germany, a large cohort of patients undergo treatment within private practices, which due to newer rules, regulations, and alterations in billing could possibly put those practices under significant economic pressure in the future, especially as insurance companies try to cut costs overall . Another significant point defining employee satisfaction is the subjective or perceived workload. The present survey shows that especially physicians have the highest subjective workload among all subspecialties (Figs. and ). Recent studies report that a continuously high workload can lead to depression and loss of workforce in the long term, resulting in a negative public perception of the field . As especially research work outside of regular working hours can contribute to the workload, protected research time needs to be implemented using specialized programs or research grants. Radiation biology, despite not being present in all departments, makes a tremendous contribution to the field of radiation oncology and radiation protection by providing knowledge of dose fractionation, tumor hypoxia, tissue weighting factors, and radiation quality as well as regarding the optimized use of combination therapies. Radiation biologists have helped to define parameters needed for risk and exposure assessment, thus establishing dose limits and offering mechanistic explanations for outcomes observed in radiation epidemiology that provide the pivotal translation of laboratory data into human applications. Insights gained by radiation biologists are used in clinical work for radiation protection, diagnosis, risk assessment, and treatment. While our survey reports that most participants see only mediocre career options and about 40% even worse career opportunities than in other biological fields (Fig. ), one has to point out that the insights and knowledge gathered can be used not only in the public health domain but also for military purposes, space exploration, environmental stewardship, and national security, and also provide biological and biophysical information on radiation response to federal agencies setting exposure limits for workers and the general public. This can of course be used for creating job opportunities, and the gathered knowledge can be used as a transition into other scientific fields or jobs outside of the field of radiation oncology. In these cases, career seminars might be helpful in order to point out alternative career options for biologists working in radiation research. In this way, well-trained high-potential staff will not get lost to other disciplines or subjects but will find ways to remain in radiation research outside of the medical field. An uncontroversial fact is that securing research funding, which is often challenging to obtain, places a significant burden on employees, as it is an essential element for maintaining a position and providing the potential for career advancement, especially in biological research . The probability of funding, on the other hand, is strongly determined by strong mentorship, establishment of a collaborative network of scientific and clinical expertise within the host department, and scientific input from related departments both within the host department and from external institutions. Besides typical support with data analysis, biostatics, literature, and article writing, intensified help in preclinical animal studies with exceptionally hard and complex legal and ethical regulations should be considered . At the moment, the majority of postdocs are forced to cover their position using grant money, which basically makes them cheap, smart, and highly educated labor . Short-sighted hyper-competitive environments potentially even slow down scientific innovation and productivity and favor fabrication and publication of immature data, thereby wasting valuable resources. High expectations and success—despite the low probability of success in the whole research environment, ranging from grant applications to hiring decisions and career progression—strongly depend upon publications, which encourages scientific dishonesty . Improvement can be achieved by preparing trainees for different potential career paths, teaching training skills that can be used outside of the academic world, creating communication platforms, and giving postdocs more visibility and thus possibilities to be more proactive about their career. Another approach could be to optimize the postdoc salary with cost-of-living adjustments . Improvements need to be made towards individual grant application by postdocs and improving grant evaluation for younger researchers with lesser-known names and reputations, thus enabling high-potential scientists to focus on research instead of writing grants . The harsh working conditions are very well reflected by the low percentage of participants in our survey who would choose radiation biology again. In combination with the mediocre career options and even worse-perceived employment options overall compared to other biological research fields (Fig. ), attracting high-potential biologists to the field is becoming more challenging. However, as a high number of individuals in our survey are unsure about whether or not to remain in radiation research, it is evident that adequate measures can aid in keeping these individuals rooted in radiation research. Physicists represent the second largest cohort of employees involved in the field of radiation oncology. A regulatorily required number of (medical) physicists is of utmost importance for the safety of patients and the quality of radiation treatment. Important tasks carried out by physicists range from administrative and clinical services to informatics, equipment maintenance, and performance evaluations, as well as research, teaching, and training. The past has shown that implementation of new techniques like intensity-modulated radiotherapy (IMRT) leads to an increased demand for physicists . Despite being of such great importance, very few new academic positions are being created in research and education in health care physics and medical physics overall . Nevertheless, the profession will likely continue to further increase in importance, mostly due to the vast amount of new technology that will need to be implemented into current treatment protocols. In the present survey, physicists show the highest willingness to leave academia and transition into industry, while private practices are the employers least favored by physicists, which could be biased as participants of the survey are mostly employed at university hospitals. However, the fear of less challenging and versatile cases as well as repetitive daily tasks in peripheral institutions could also be a reason for this. As well-trained physicists can easily transition into other work fields, it is an essential task to better include these specialists in the society and keep these individuals in the field of radiation oncology in order to remain competitive in the years to come, as inadequate staffing will inevitably lead to loss of quality and safety in the long run. One possible way to achieve long-term commitment could be an increase in the permanent contracts offered by institutions. Physicists represent the subspecialty with the highest percentage of permanent contracts in the survey, which seems to reflect the particular need for long-term integration of well-trained personnel into workflows of the respective departments to ensure consistency and reliability, but also the legal requirements for the presence of MPEs. It remains unclear why a large percentage of (medical) physicists are not members of a society (Fig. ). It may be possible that bachelor’s/master’s students, due to their early career stage, and non-scientific MPs may not see any representation of interests in a scientific society. As there is a lack of a professional association for clinically working medical physics experts in Germany, the offer of (interdisciplinary) education and interaction for this group should eventually be further expanded within the existing scientific associations. At the same time, this may increase interest and active participation in the societies and strengthen interdisciplinary and translational cooperation between research and clinical settings. In general, the cohort of physicists seems to be rather satisfied with their career choice, reporting the highest percentage of participants who would choose the same specialty again and high satisfaction rates regarding the support offered by the employer (Fig. ). Only the subjective workload in science tends towards being higher compared to the physician cohort. Physicians are the largest cohort and also represent the subspecialty with the highest visibility due to the direct patient contact. In our survey, the most prominent fear among physicians is high economic pressure followed by a lack of work–life balance and compatibility of career and family (Fig. ). Regarding the general economic situation, lower financial reimbursement combined with higher costs for personnel, energy, and maintenance cause significant economic stress in the different departments. Profit targets set by the hospital administrations are a balancing act between the economic situation and a reasonable treatment for patients. In Germany, a large cohort of patients undergo treatment within private practices, which due to newer rules, regulations, and alterations in billing could possibly put those practices under significant economic pressure in the future, especially as insurance companies try to cut costs overall . Another significant point defining employee satisfaction is the subjective or perceived workload. The present survey shows that especially physicians have the highest subjective workload among all subspecialties (Figs. and ). Recent studies report that a continuously high workload can lead to depression and loss of workforce in the long term, resulting in a negative public perception of the field . As especially research work outside of regular working hours can contribute to the workload, protected research time needs to be implemented using specialized programs or research grants. Work–life balance Another topic that needs to be addressed for all subspecialties in the future is the creation of a job environment with an adequate work–life balance, as this is one of the major concerns of the physician and physicist cohorts in our survey. Compatibility of career and family, especially with a higher number of female professionals (Fig. ), paired with significant changes in classical family roles, is also increasing in importance. The possibility of working part-time is an essential factor, facilitating better adaptation to private circumstances such as nursing a family member or taking time out for childcare. Another possible solution is implementing the concept of a 4-day working week that is currently discussed and even being tested in some clinics in Germany. While the biologist cohort shows a fair number of employees working part-time, this is rather unusual for the physician group, which shows the highest rate of full-time employment (Figs. and ). However, as doctoral candidates in biology or medical physics are employed part-time while usually working full-time, an in-depth analysis of these factors will need to be carried out in the future. Thus, this is in most cases only a pro-forma part-time contract, while PhD students actually work full-time. True part-time contracts might, however, increase the attractiveness of the field through improved work–life or work–family balance. The high percentage of full-time-employed participants in the physician group could possibly be explained by the need of finishing a residency program as the final step of their education. In the future, more liberal part-time models should be offered and will be inevitable as more and more female employees strive into the field. Working part-time in general needs to become more accepted by supervisors independent of gender to enable contemporary gender-equal divisions of labor and non-classical family roles. Working environment The most favored form of employment in this survey is academia among all subspecialties (Fig. ). One possible explanation could be that academia combines clinical, scientific, and teaching work, in contrast to non-academic clinics or private practice, and offers the chance to work in a cutting-edge and up-to-date environment that is usually less economically driven. University hospitals offer, besides a basic education, the possibility to acquire further scientific education, helping to implement new results into daily work as paradigm changes can occur very fast in modern medicine . However, the structure of universities overall will not make it possible for all participants to pursue a career in academia. Further considerations discouraging long-term academic careers include academic pressure, declining support for research, and increased bureaucracy , as also visible in the present study (Fig. ). Another topic that needs to be addressed for all subspecialties in the future is the creation of a job environment with an adequate work–life balance, as this is one of the major concerns of the physician and physicist cohorts in our survey. Compatibility of career and family, especially with a higher number of female professionals (Fig. ), paired with significant changes in classical family roles, is also increasing in importance. The possibility of working part-time is an essential factor, facilitating better adaptation to private circumstances such as nursing a family member or taking time out for childcare. Another possible solution is implementing the concept of a 4-day working week that is currently discussed and even being tested in some clinics in Germany. While the biologist cohort shows a fair number of employees working part-time, this is rather unusual for the physician group, which shows the highest rate of full-time employment (Figs. and ). However, as doctoral candidates in biology or medical physics are employed part-time while usually working full-time, an in-depth analysis of these factors will need to be carried out in the future. Thus, this is in most cases only a pro-forma part-time contract, while PhD students actually work full-time. True part-time contracts might, however, increase the attractiveness of the field through improved work–life or work–family balance. The high percentage of full-time-employed participants in the physician group could possibly be explained by the need of finishing a residency program as the final step of their education. In the future, more liberal part-time models should be offered and will be inevitable as more and more female employees strive into the field. Working part-time in general needs to become more accepted by supervisors independent of gender to enable contemporary gender-equal divisions of labor and non-classical family roles. The most favored form of employment in this survey is academia among all subspecialties (Fig. ). One possible explanation could be that academia combines clinical, scientific, and teaching work, in contrast to non-academic clinics or private practice, and offers the chance to work in a cutting-edge and up-to-date environment that is usually less economically driven. University hospitals offer, besides a basic education, the possibility to acquire further scientific education, helping to implement new results into daily work as paradigm changes can occur very fast in modern medicine . However, the structure of universities overall will not make it possible for all participants to pursue a career in academia. Further considerations discouraging long-term academic careers include academic pressure, declining support for research, and increased bureaucracy , as also visible in the present study (Fig. ). A survey by Krug et al. about the young radiation oncology workforce showed that only 4% of participants reported a complete separation between clinical work and research and/or teaching activities. Among these, physicians reported the lowest number (9%) of protected time to carry out research or teaching in their regular working hours on a regular basis , suggesting that most of the time, teaching and research are carried out outside of regular working hours. This can potentially become problematic, as the quality of research and teaching but also of clinical work might suffer when tasks are mainly carried out outside of regular hours and during overtime . Research One possible point for improvement is the creation of easier options for entry into scientific work. While the volume and intensity of research will differ significantly between facilities, barriers impeding participation in research have to be clearly addressed. Obstacles that are frequently mentioned range from lack of time, limited access to statistical analysis through insufficient mentoring, and variable support from superiors, to great variability in research productivity at the department level, partially due to metafactors such as the size and composition of the department, advanced technology capability, limited access to funding, and the numbers of patients and degree of engagement with specialist medical research institutions . The present survey confirms that especially employees in the physician cohort lack protected research time (Fig. ). Combined with the fact that overtime in work covering the clinical routine is becoming the rule rather than the exception, this makes it virtually impossible for clinicians to engage in high-quality and pioneering research . Furthermore, according to our survey, physicians and physicists are less frequently involved in the supervision of doctoral students compared to biologists. This can be attributed to a higher level of clinical involvement and responsibilities; likewise, it is possible that these groups are more involved in the supervision of bachelor’s or master’s theses. As scientific work is time consuming, an approach to include mainly clinically working personnel in research, thus giving them an overview of current trends and developments outside of their daily work, could be to involve them in the reviewing process of scientific work after respective training. Other countries require medical candidates to complete mandatory scientific work before their final examination. In the case of clinical medical physicists in training, completion of a scientific project or an additional study before becoming eligible for certification could be obligatory—an approach that has been successfully implemented in the model medical study course in Cologne, Germany . Another recent improvement in this regard is the introduction of young scientist scholarships, enabling physicians to take protected research time as a clinician scientist , which could significantly improve the situation. This is especially important as fundamental research on combining radiation therapy with other treatment modalities like surgery and chemo-/immunotherapy will become more and more important in the future. Intensifying translational work cooperations will be an inevitable component of this. Teaching Likewise, lectures on physics and biology have to be implemented into standard residency programs to generate a basic understanding of the mode of operation of, e.g., immunotherapies, and to foster awareness of possible cooperations and interactions between subspecialties . Furthermore, the satisfaction of trainees with their occupational training is of utmost importance for choosing a certain career path. Participants from the medical field that were asked about their educational quality in our study, however, report a broad range of satisfaction, with a median of 65/100 (Fig. ). There is a significant number of participants that seem to be highly satisfied with their education versus 29% of participants reporting satisfaction rates of below 50. One option could be to hold written and oral examination on the topics of radiation biology and radiation physics early during residency, following the example of the radiation protection course. These results could also give more feedback to the teaching institutions from an educational standpoint . Cooperations between biologists and physicists also need to be intensified, as new radiation-generating devices show increasing complexity, requiring formal training in basic principles of radiation dosimetry and measurement for research. Our survey further reports that the involvement of physicians and biologists in teaching is higher than that of the subcohort of physicists (Fig. ). While medical physics most likely will continue to grow in importance due to increasing numbers of cancer cases as well as further scientific improvements leading to advanced technologies that increase the demand for well-trained professionals , the high number of participants not involved in either teaching or scientific work is problematic and needs to be improved in the future. Mentoring and networking One quarter of participants indicate insufficient support for their career goals, potentially causing them to leave the field in the long term. Among the participants, the cohorts of biologists and physicists seem to be a lot less prone to communicating their potential career goals to their employers (Fig. ), which could be interpreted as either being less career oriented or as being less informed regarding the possibilities in the field. Here, career courses could be an option to inform and train staff to better identify and communicate their goals. Another possibility might be a widespread implementation of mentoring programs that pair young professionals with those more experienced in the field who often have a more widespread network and can offer pivotal guidance in personal, clinical, and academic growth. Especially mentoring has been frequently mentioned during the current survey. The supervision by mentors could help to create a meaningful timeline for reaching certain milestones and complementary educational achievements such as a basic education in health care economics, as often required for higher positions , especially as mentors can give advice independently of the department’s own interests. Recently, the young scientist groups have reacted to this topic and offered a mentoring program for all three subspecialties involved in radiation oncology, highly supported by older members of the society in Germany. One possible point for improvement is the creation of easier options for entry into scientific work. While the volume and intensity of research will differ significantly between facilities, barriers impeding participation in research have to be clearly addressed. Obstacles that are frequently mentioned range from lack of time, limited access to statistical analysis through insufficient mentoring, and variable support from superiors, to great variability in research productivity at the department level, partially due to metafactors such as the size and composition of the department, advanced technology capability, limited access to funding, and the numbers of patients and degree of engagement with specialist medical research institutions . The present survey confirms that especially employees in the physician cohort lack protected research time (Fig. ). Combined with the fact that overtime in work covering the clinical routine is becoming the rule rather than the exception, this makes it virtually impossible for clinicians to engage in high-quality and pioneering research . Furthermore, according to our survey, physicians and physicists are less frequently involved in the supervision of doctoral students compared to biologists. This can be attributed to a higher level of clinical involvement and responsibilities; likewise, it is possible that these groups are more involved in the supervision of bachelor’s or master’s theses. As scientific work is time consuming, an approach to include mainly clinically working personnel in research, thus giving them an overview of current trends and developments outside of their daily work, could be to involve them in the reviewing process of scientific work after respective training. Other countries require medical candidates to complete mandatory scientific work before their final examination. In the case of clinical medical physicists in training, completion of a scientific project or an additional study before becoming eligible for certification could be obligatory—an approach that has been successfully implemented in the model medical study course in Cologne, Germany . Another recent improvement in this regard is the introduction of young scientist scholarships, enabling physicians to take protected research time as a clinician scientist , which could significantly improve the situation. This is especially important as fundamental research on combining radiation therapy with other treatment modalities like surgery and chemo-/immunotherapy will become more and more important in the future. Intensifying translational work cooperations will be an inevitable component of this. Likewise, lectures on physics and biology have to be implemented into standard residency programs to generate a basic understanding of the mode of operation of, e.g., immunotherapies, and to foster awareness of possible cooperations and interactions between subspecialties . Furthermore, the satisfaction of trainees with their occupational training is of utmost importance for choosing a certain career path. Participants from the medical field that were asked about their educational quality in our study, however, report a broad range of satisfaction, with a median of 65/100 (Fig. ). There is a significant number of participants that seem to be highly satisfied with their education versus 29% of participants reporting satisfaction rates of below 50. One option could be to hold written and oral examination on the topics of radiation biology and radiation physics early during residency, following the example of the radiation protection course. These results could also give more feedback to the teaching institutions from an educational standpoint . Cooperations between biologists and physicists also need to be intensified, as new radiation-generating devices show increasing complexity, requiring formal training in basic principles of radiation dosimetry and measurement for research. Our survey further reports that the involvement of physicians and biologists in teaching is higher than that of the subcohort of physicists (Fig. ). While medical physics most likely will continue to grow in importance due to increasing numbers of cancer cases as well as further scientific improvements leading to advanced technologies that increase the demand for well-trained professionals , the high number of participants not involved in either teaching or scientific work is problematic and needs to be improved in the future. One quarter of participants indicate insufficient support for their career goals, potentially causing them to leave the field in the long term. Among the participants, the cohorts of biologists and physicists seem to be a lot less prone to communicating their potential career goals to their employers (Fig. ), which could be interpreted as either being less career oriented or as being less informed regarding the possibilities in the field. Here, career courses could be an option to inform and train staff to better identify and communicate their goals. Another possibility might be a widespread implementation of mentoring programs that pair young professionals with those more experienced in the field who often have a more widespread network and can offer pivotal guidance in personal, clinical, and academic growth. Especially mentoring has been frequently mentioned during the current survey. The supervision by mentors could help to create a meaningful timeline for reaching certain milestones and complementary educational achievements such as a basic education in health care economics, as often required for higher positions , especially as mentors can give advice independently of the department’s own interests. Recently, the young scientist groups have reacted to this topic and offered a mentoring program for all three subspecialties involved in radiation oncology, highly supported by older members of the society in Germany. Another point that needs to be discussed is how the clinical demand will change as a response to growth in the cancer incidence (approximately 2.5% per year), the impact of new technologies with possible acceleration of treatment-related workflows due to artificial intelligence, but also possible slowing of processes due to more adaptive and individualized treatment regimens . While future developments cannot be entirely foreseen, technical progress, which holds great potential for improvements in treatment overall, is not seen as a significant problem, neither by the physicist nor by the physician cohort (Fig. ). In contrast, new technologies can even lead to an increase in treatment time. Models and further predictions show that a 5% increase in overall workload could potentially lead to a significantly higher number of staff needed from all involved specialties . One example is the increase in demand for medical physicists due to the introduction of new technologies and an increased number of treatment machines . Combined with a general shortage of personnel due to aging of the existing workforce, which can only be compensated to a very limited basis, this could lead to a real decline in treatment capacity, safety, and overall quality. Limitations could be reduced through the extension of work contracts or consulting and by increasing the number of open clinical training positions for MPEs . While a lack of potential employers is present for a large variety of specialties, overall competition for motivated and well-trained employees due to suboptimal working conditions and payment level compared to some other European countries is inevitable. Radiation oncology will probably not be given more attention in undergraduate life science education, thus limiting its visibility to potential future employees at that early stage of their career path. Therefore, advertising the field by offering good and diversified training programs with a wide range of additional offers could serve as a letter of invitation, convincing potential candidates to take their decision in favor of radiation oncology in the end. In contrast to a lack of potential employers, the generational change benefits investor-managed hospital and practice chains primarily focusing on increasing margins rather than prioritizing improving patient treatment. Radiation oncology is underrepresented in oncological teaching in medical schools, which leads to underutilization and a lot of prejudices towards the field, while studies show improved knowledge with dedicated courses, especially favoring clinical exposure over dedicated lectures . The lack of knowledge potentially even impedes multidisciplinary oncological care. Furthermore, a recent study demonstrates that the introduction of radiation oncology into the preclinical part of medical education is feasible , an idea that is generally well perceived by students . This may aid in attracting more doctoral candidates and residents while improving overall knowledge in radiation oncology. Possibilities to teach radiation oncology outside of the existing curriculum include special lectures, seminars, and bedside training as well as the meaningful integration of e‑learning . Teaching in biology and medical physics is generally less frequent . The decision to choose a certain specialty is driven by different intrinsic (personal interest) and extrinsic (perceived prestige or potential income) as well as structural considerations like the influence of lifestyle, working hours, perceived stress from on-call duties, amount of time on-duty, and the possibility to work part-time. Although no one single factor can be seen as decisive, a beneficial combination of exactly these points could give the field of radiation oncology the edge, especially for students still undecided at the end of their studies . Awareness and basic knowledge in these fields, however, also needs to be established amongst general practitioners to provide patients with adequate advice and information . Another promising step towards improvement is the creation of networks among younger professionals within the framework of existing societies. The creation of internal groups linking young radiation oncology scholars has been successfully implemented in other European countries . Structured collaboration between new and advanced professionals within the society can further address essential points such as the drafting of a good residential curriculum, including reliable long-term rotation plans covering all relevant aspects of clinical radiation oncology and naming individuals responsible for the supervision of residency training. Another possibility would be the creation of improved teaching including lecture series covering all the major relevant topics in oncology in a location-independent approach, ensuring that all residents can benefit from the knowledge of local specialists. Such a step was recently taken by the young DEGRO in Germany and has been met with a very positive response . Teaching, which is often not standardized throughout the different departments, has been further compromised by the COVID pandemic . The young DEGRO group has implemented online courses covering different topics in physics, biology, and clinical radiation oncology. The courses so far have been very well received, inviting new aspiring scientists and clinicians to present a talk on their research focus. A similar program is found in the DGMP and young MP, while the young DeGBS focuses on online seminars covering topics such as how to set up a lab or introduction of committees. Points that have to be further addressed include more time for self-study; increasing support by the DEGRO, DGMP, and DeGBS; participation in meetings; improved educational quality; and an institutional training schedule that focuses more on the guidelines drawn up by the German medical council . Another viable option could be the creation of a central research and teaching team organized by the DEGRO and young DEGRO in collaboration with the DeGBS and young DeGBS as well as the DGMP and young MP to assist in the setup of studies and offer help with valid statistical evaluation, research training, and the advancement of science overall . Another more decentralized possibility could be the creation of a core research team in each university clinic consisting of a clinical researcher, a biologist, and a physicist with clearly defined tasks and protected time for the generation and supervision of larger-scale studies as well as assisting in translational research approaches. More ambition in this field by DEGRO/young DEGRO could improve the number of members of all subspecialties in the society, as there will be an improved sense of personal benefit from joining the society (; Fig. ). In general, giving more power to young societies not only creates opportunities for getting involved but also allows for more uniform training, also including special techniques such as new imaging modalities (for example, PET-CT, MRI, special sequences) as well as insights into less mainstream areas of radiation oncology such as brachytherapy. As this is the first study combining a team of young DEGRO, young DeGBS, and young MP members, the present interrogation could not rely on a validated questionnaire but was composed of newly developed questions, thus leaving room for multiple interpretation possibilities that need to be further specified in the future. Nevertheless, this approach was chosen due to the nature of the survey at hand. We have included a wide number of occupations, workplaces, and research foci in this survey, as all of these professionals work in radiation oncology and radiation research and thus also have a wide range of specific occupational requirements. This approach thus allowed generation of a broader overview. Further studies will also have to include the specific needs of radiotherapy technologists, especially with regard to the declining numbers of this essential profession. The possibility of collaborating with different members of the radiation research community with diverse interests and expertise, the breadth of career opportunities, and the ability to adapt research to human needs are great strengths of the field . All factors that may discourage applicants from entering the field have to be identified and successively addressed to maintain a competitive field and independence from other departments. As pointed out by Professor Zietman, former president of the American Society for Radiation Oncology (ASTRO), there is a danger that, if radiation oncologists become simple guardians of a single therapeutic modality, as time goes by and techniques live on and improve, the specialty as a whole may not. This, however, requires a proactive attitude on the part of all the participants in the field of radiation oncology . Furthermore, it is the responsibility of researchers to engage a broader audience, illustrating the potential value they can provide to society. Supplementary Material 1 Complete online survey Supplementary Material 2 Table 1. Characteristics of survey participants ( n = 218). Absolute numbers are given in brackets. Numbers may not add up to 100% due to rounding error or missing values. Abbreviations: d diverse; f female; m male; n/a not applicable. |
Gesundheitskompetenz und Gesundheitsverhalten – Einblicke in ein sich ausdifferenzierendes Forschungs- und Handlungsfeld für Public Health | 7486a353-c5bf-4a2f-b8e0-18d5334ee4e1 | 11868217 | Health Literacy[mh] | Gesundheitskompetenz beschreibt den Umgang mit Gesundheitsinformationen als Grundlage von individuellen gesundheitsrelevanten Entscheidungen . Unabhängig davon, ob gesundheitsrelevante Entscheidungen bewusst oder impulsgesteuert getroffen werden, führen sie häufig zu konkreten Gesundheitsverhalten . Mit Gesundheitsverhalten sind konkrete Verhaltensweisen gemeint, welche die eigene Gesundheit positiv beeinflussen können (bspw. körperliche Aktivität, Ernährung, Schlafverhalten, Früherkennungsuntersuchungen oder Impfungen). Damit wird die Stärkung von Gesundheitskompetenz relevant für die Förderung des Gesundheitsverhaltens . Für die Förderung von Gesundheitskompetenz ist zu berücksichtigen, dass diese kontextabhängig ist und deswegen als ein „relationales Konzept“ bezeichnet wird : Auf individueller Ebene umfasst Gesundheitskompetenz Fähigkeiten, Motivation, Wissen, soziale Praktiken und Selbstwirksamkeit . Auf organisationaler und struktureller Ebene bezieht sich Gesundheitskompetenz auf komplexe situative Anforderungen, um gesundheitsrelevante Entscheidungen, z. B. für ein Gesundheitsverhalten, zu treffen . Es wird angenommen, dass sich Gesundheitskompetenz auf gesundheitsbezogene Einstellungen in der motivationalen und volitionalen Phase der Entscheidungsfindung und damit auf das Gesundheitsverhalten auswirkt . Dem breiten, mehrdimensionalen Konzept von Gesundheitskompetenz folgend, geht die Förderung von Gesundheitskompetenz im Hinblick auf ein gesundheitsförderliches Gesundheitsverhalten über ein enges Verständnis von Gesundheitsbildung und verhaltensorientierter Kommunikation hinaus und zielt auf kontextuelle, politische und soziale Einflussfaktoren . Mit Gesundheitskompetenz liegt ein Konstrukt vor, das die gesundheitliche Handlungs- und Kompetenzebene zum Umgang mit Wissen und Information, um gesundheitsrelevante Entscheidungen und Gesundheitsverhalten zu beeinflussen, adressiert. Somit setzt sich Gesundheitskompetenz klar von Gesundheitswissen und Gesundheitsverhalten ab und füllt gleichzeitig die Lücke aus, die auf dem Spektrum von Wissen zu Gesundheitsverhalten existiert. Somit ist Gesundheitskompetenz als Konstrukt zwischen diesen beiden einzuordnen. Die wissenschaftstheoretische Auseinandersetzung mit dem Konstrukt wie auch die Frage, ob Gesundheitskompetenz ein Mediator zwischen sozioökonomischen (z. B. Bildung) bzw. psychosozialen Faktoren (z. B. Selbstwirksamkeit) und gesundheitsbezogenen Faktoren wie dem Gesundheitsverhalten ist , haben erst begonnen. Der Begriff Gesundheitskompetenz bezieht sich in der Regel auf die „allgemeine Gesundheitskompetenz“. Allgemeine Gesundheitskompetenz beschreibt themen- und kontextübergreifende Fähigkeiten im Umgang mit Gesundheitsinformationen und wird auch als generische oder generelle Gesundheitskompetenz bezeichnet. Im Unterschied zur allgemeinen Gesundheitskompetenz beziehen sich spezifische Gesundheitskompetenzen auf bestimmte Handlungsfelder, zum Beispiel auf konkrete Gesundheitsverhalten wie Ernährung und Bewegung, bestimmte Erkrankungen oder digitale Gesundheitsinformationen . Neben dem Umgang mit Gesundheitsinformationen beinhalten die spezifischen Gesundheitskompetenzen teilweise weitere Kompetenzbereiche, die für das Verhalten wesentlich sind . Exemplarisch seien hier die Bewegungs- oder Steuerungskompetenz im Bereich der Förderung körperlicher Aktivität genannt . Die zunehmenden Erkenntnisse zu Gesundheitskompetenz und verschiedenen Gesundheitsverhalten sind Anlass, in diesem Beitrag einen Einblick in den aktuellen Forschungsstand hierzu zu geben. Neben Konzepten und Erkenntnissen zu allgemeiner Gesundheitskompetenz und Gesundheitsverhalten werden exemplarisch spezifische Gesundheitskompetenzen aus dem Bereich Ernährung und Bewegung vorgestellt. Den Abschluss bildet die Vorstellung des Konzepts „Behavioural and Cultural Insights“ und seiner Schnittmengen mit Gesundheitskompetenz im Hinblick auf die Förderung des Gesundheitsverhaltens. Gesundheitskompetenz und Gesundheitsverhalten im Erwachsenenalter Das Konzept der allgemeinen Gesundheitskompetenz hat sich in den letzten Jahrzehnten national und international in Forschung, Praxis und Politik etabliert und gilt als relevant für die Förderung des Gesundheitsverhaltens . Das Gesundheitsverhalten kann dabei im Kontext von Aktivitäten zur Gesundheitsförderung (z. B. als Settingmaßnahme) als auch zur Prävention (z. B. individueller Rauchausstieg) oder in der Krankenversorgung (z. B. Inanspruchnahme von Nachsorgeterminen) adressiert werden. Diese Gesundheitskompetenz beschreibt themen- und kontextübergreifende Fähigkeiten im Umgang mit Gesundheitsinformationen und eignet sich besonders, um auf Bevölkerungsebene Bedarfe zur Förderung der Gesundheitskompetenz zu identifizieren und ihre Verteilung und Entwicklung in der Bevölkerung zu beobachten, insbesondere im Hinblick auf vulnerable Gruppen . Gemessen wird die allgemeine Gesundheitskompetenz in der Regel in Form von (subjektiven) Selbsteinschätzungsinstrumenten wie dem Fragebogen des „Europäischen Health Literacy Survey“ (HLS-EU‑Q) oder des „Health Literacy Questionnaire“ (HLQ) . Bei diesen Instrumenten gibt die ausfüllende Person beispielsweise an, wie leicht bzw. schwierig es für sie ist, Informationen zu finden und zu verstehen (z. B. Informationen darüber, wie sie psychisch gesund bleiben kann). Das Messergebnis bildet die Passung zwischen den externen Anforderungen und den Fähigkeiten des Individuums ab [ , S. e29]. Dabei weisen die Selbsteinschätzungsinstrumente einen gewissen Interpretationsspielraum auf: Die Einschätzung einer Testperson, dass eine Aufgabe „schwierig“ sei, beruht zum einen darauf, dass die betreffende Person selbst nicht über die erforderlichen Kompetenzen verfügt, um die Aufgabe zu lösen. Zum anderen kann die Schwierigkeit der Aufgabe auch objektiv begründet sein, beispielsweise durch hohe Anforderungen oder komplexe Rahmenbedingungen. Auch sozial erwünschtes Antwortverhalten oder Selbstüberschätzung können die Aussagekraft der Ergebnisse einschränken. Leistungsbezogene (objektive bzw. performanzbasierte) Messinstrumente hingegen liefern objektivere Informationen durch die Testung auf Gesundheitswissen oder gesundheitsbezogene Lese- und Rechenfähigkeit. Beispiele dafür sind der „Newest Vital Sign Test“ (NVS), der „Rapid Estimate of Adult Literacy in Medicine“ (REALM) und der „Test of Functional Health Literacy in Adults“ (TOFHLA) . Diese Instrumente kommen in verschiedenen Studien zum Einsatz, um den Zusammenhang mit Gesundheitskompetenz und Gesundheitsverhalten zu erfassen, eignen sich aber nicht, um Gesundheitskompetenz mehrdimensional abzubilden, oder für den Einsatz in bevölkerungsweiten Studien. Daher sind Selbsteinschätzungsinstrumente trotz der oben genannten Limitationen für Bevölkerungsstudien praktikable und geeignete Messinstrumente. In Deutschland konnten Studien mit umfassenden Selbsteinschätzungsinstrumenten, insbesondere dem HLS-EU‑Q, in den letzten Jahren zeigen, dass die Hälfte oder etwas mehr als die Hälfte der Bevölkerung Schwierigkeiten hat, mit Gesundheitsinformationen umzugehen . Besonders betroffen sind Bevölkerungsgruppen mit niedrigem Sozialstatus, niedriger Bildung oder finanzieller Deprivation . Daten aus verschiedenen Ländern legen ebenso nahe, dass Individuen mit höherer Gesundheitskompetenz eine Tendenz zu einem gesünderen Lebensstil im Hinblick auf den Obst- und Gemüsekonsum und körperliche Aktivität aufweisen [ , S. 184–185]. Zwischen allgemeiner Gesundheitskompetenz und Gesundheitsverhalten zeigt sich in vielen Studien eine positive Korrelation . Zumeist werden der Umfang der körperlichen Aktivität, des Sporttreibens, Menge oder Häufigkeit des Obst- und Gemüsekonsums sowie Konsum bzw. Konsummenge von Tabak- und Alkohol untersucht. Hier zeigen sich studienspezifische Unterschiede: Je nach Studienpopulation, Messinstrument zur Gesundheitskompetenz bzw. dem Gesundheitsverhalten oder Analysemethoden sind in einigen Studien bzw. für bestimmte Teilpopulationen keine Assoziation zu beobachten . Im Hinblick auf die Absicht einer Verhaltensänderung, die oft das Ziel von Interventionen ist, hat sich Gesundheitskompetenz als moderierende Variable bei körperlicher Aktivität gezeigt . Im Hinblick auf das Gesundheitsverhalten fokussieren sich Forschung und Interventionspraxis häufig auf die individuelle Gesundheitskompetenz. Public-Health-Strategien wie der „Nationale Aktionsplan Gesundheitskompetenz“ oder die Strategie der Weltgesundheitsorganisation (WHO) zur Bekämpfung von nichtübertragbaren Erkrankungen legen den Schwerpunkt anders . Sie empfehlen den Fokus nicht nur auf die individuelle Ebene zu legen, sondern Gesundheitskompetenz als Teil von Verhältnisprävention zu verstehen, damit Normen und Kulturen sowie organisatorische und politische Strukturen verändert werden (soziale Praxis), die die Entwicklung von Gesundheitskompetenz in der Bevölkerung maßgeblich beeinflussen . Gesundheitskompetenz und Gesundheitsverhalten im Kindes- und Jugendalter Gesundheitskompetenz hat sich mittlerweile auch im Bereich der Kinder- und Jugendgesundheit als wichtiges Interventionsziel sowohl für die Förderung der körperlichen als auch der psychischen Gesundheit etabliert. Die WHO betont seit 10 Jahren die besondere Rolle der Schule für die Stärkung der Gesundheitskompetenz . In Deutschland werden solche Empfehlungen insbesondere durch den „Nationalen Aktionsplan Gesundheitskompetenz“ und die „Allianz für Gesundheitskompetenz in der Schule“ ausgesprochen . Konzeptionell orientieren sich Gesundheitskompetenzansätze für Kinder und Jugendliche an den Gesundheitskompetenzkonzeptionen für Erwachsene, die den Umgang mit Gesundheitsinformationen für gesundheitsbezogene Entscheidungen adressieren. Adaptierungen und Erweiterungen der Gesundheitskompetenzansätze für Kinder und Jugendliche zu spezifischen Gesundheitskompetenzen liegen insbesondere zu digitalen, psychischen und auf das Gesundheitsverhalten bezogenen Gesundheitskompetenzen vor . Basierend auf diesen Konzepten wurden in den letzten Jahren unterschiedliche Instrumente entwickelt , die ab dem Grundschulalter eingesetzt werden können . Diese reichen von (subjektiven) Selbsteinschätzungsinstrumenten bis zu leistungsbezogenen (objektiven bzw. performanzbasierten) Messverfahren, sowohl für allgemeine als auch spezifische Varianten von Gesundheitskompetenz. Derzeit finden Fragebogenverfahren, die teilweise oder ganz auf dem Fragebogen des „European Health Literacy Survey“ (HLS-EU‑Q) oder dem „Digital Health Literacy Instrument for Adolescents“ (DHLI) basieren bzw. verwandte Verfahren weite Verbreitung. Auch ethnografische Methoden werden zunehmend angewendet . Die Studien deuten auf einen Zusammenhang zwischen der Gesundheitskompetenz von Kindern und Jugendlichen und ihrem Gesundheitsverhalten hin . Eine Evidenzsynthese für das Europäische Schulnetzwerk kommt zu dem Schluss, dass eine höhere Gesundheitskompetenz häufiger mit gesundheitsförderlichen Verhaltensweisen einhergeht (z. B. in Bezug auf Ernährung, Sport und Bewegung, Substanzkonsum, Sexualverhalten, Ruhe und Schlafverhalten) . Dabei gibt es Unterschiede je nach Art des Gesundheitsverhaltens, insbesondere der Substanzkonsum scheint sich von Ernährung und Bewegung zu unterscheiden, wie beispielsweise die bundesweite Befragung zur „Gesundheitskompetenz von Jugendlichen“ (GeKoJu) zeigt: Gesundheitskompetenz ist mit dem täglichen Konsum von Obst und Gemüse assoziiert. Niedrigere Level in behavioralen, affektiven und konativen (absichtsorientierten) Dimensionen der Gesundheitskompetenz erhöhen die Chance, körperlich inaktiv zu sein und zu rauchen. Bei riskantem Alkoholkonsum spielen andere Faktoren (wie Alter oder familiärer Wohlstand) eine wichtigere Rolle als allgemeine Gesundheitskompetenz . In einer Studie zur Gesundheitskompetenz bei Jugendlichen an Schulen in Hessen sind mittlere und geringe digitale Gesundheitskompetenz mit weniger körperlicher Aktivität, nichttäglichem Obstkonsum und täglichem Konsum von zuckerhaltigen Getränken assoziiert . Die repräsentative Befragung von Grundschulkindern in der 4. Schulklasse zeigt , dass eine höhere Gesundheitskompetenz den stärksten Prädiktor für regelmäßiges Zähneputzen sowie Obst- und Gemüsekonsum darstellt . Soziale Ungleichheiten stellen auch im Kindes- und Jugendalter ein wiederkehrendes Phänomen dar, denn die Ausprägung von Gesundheitskompetenz folgt einem sozialen Gradienten , welcher sich auch bei der positiven Assoziation zwischen Gesundheitskompetenz und Gesundheitsverhalten im Jugendalter findet . Das Konzept der allgemeinen Gesundheitskompetenz hat sich in den letzten Jahrzehnten national und international in Forschung, Praxis und Politik etabliert und gilt als relevant für die Förderung des Gesundheitsverhaltens . Das Gesundheitsverhalten kann dabei im Kontext von Aktivitäten zur Gesundheitsförderung (z. B. als Settingmaßnahme) als auch zur Prävention (z. B. individueller Rauchausstieg) oder in der Krankenversorgung (z. B. Inanspruchnahme von Nachsorgeterminen) adressiert werden. Diese Gesundheitskompetenz beschreibt themen- und kontextübergreifende Fähigkeiten im Umgang mit Gesundheitsinformationen und eignet sich besonders, um auf Bevölkerungsebene Bedarfe zur Förderung der Gesundheitskompetenz zu identifizieren und ihre Verteilung und Entwicklung in der Bevölkerung zu beobachten, insbesondere im Hinblick auf vulnerable Gruppen . Gemessen wird die allgemeine Gesundheitskompetenz in der Regel in Form von (subjektiven) Selbsteinschätzungsinstrumenten wie dem Fragebogen des „Europäischen Health Literacy Survey“ (HLS-EU‑Q) oder des „Health Literacy Questionnaire“ (HLQ) . Bei diesen Instrumenten gibt die ausfüllende Person beispielsweise an, wie leicht bzw. schwierig es für sie ist, Informationen zu finden und zu verstehen (z. B. Informationen darüber, wie sie psychisch gesund bleiben kann). Das Messergebnis bildet die Passung zwischen den externen Anforderungen und den Fähigkeiten des Individuums ab [ , S. e29]. Dabei weisen die Selbsteinschätzungsinstrumente einen gewissen Interpretationsspielraum auf: Die Einschätzung einer Testperson, dass eine Aufgabe „schwierig“ sei, beruht zum einen darauf, dass die betreffende Person selbst nicht über die erforderlichen Kompetenzen verfügt, um die Aufgabe zu lösen. Zum anderen kann die Schwierigkeit der Aufgabe auch objektiv begründet sein, beispielsweise durch hohe Anforderungen oder komplexe Rahmenbedingungen. Auch sozial erwünschtes Antwortverhalten oder Selbstüberschätzung können die Aussagekraft der Ergebnisse einschränken. Leistungsbezogene (objektive bzw. performanzbasierte) Messinstrumente hingegen liefern objektivere Informationen durch die Testung auf Gesundheitswissen oder gesundheitsbezogene Lese- und Rechenfähigkeit. Beispiele dafür sind der „Newest Vital Sign Test“ (NVS), der „Rapid Estimate of Adult Literacy in Medicine“ (REALM) und der „Test of Functional Health Literacy in Adults“ (TOFHLA) . Diese Instrumente kommen in verschiedenen Studien zum Einsatz, um den Zusammenhang mit Gesundheitskompetenz und Gesundheitsverhalten zu erfassen, eignen sich aber nicht, um Gesundheitskompetenz mehrdimensional abzubilden, oder für den Einsatz in bevölkerungsweiten Studien. Daher sind Selbsteinschätzungsinstrumente trotz der oben genannten Limitationen für Bevölkerungsstudien praktikable und geeignete Messinstrumente. In Deutschland konnten Studien mit umfassenden Selbsteinschätzungsinstrumenten, insbesondere dem HLS-EU‑Q, in den letzten Jahren zeigen, dass die Hälfte oder etwas mehr als die Hälfte der Bevölkerung Schwierigkeiten hat, mit Gesundheitsinformationen umzugehen . Besonders betroffen sind Bevölkerungsgruppen mit niedrigem Sozialstatus, niedriger Bildung oder finanzieller Deprivation . Daten aus verschiedenen Ländern legen ebenso nahe, dass Individuen mit höherer Gesundheitskompetenz eine Tendenz zu einem gesünderen Lebensstil im Hinblick auf den Obst- und Gemüsekonsum und körperliche Aktivität aufweisen [ , S. 184–185]. Zwischen allgemeiner Gesundheitskompetenz und Gesundheitsverhalten zeigt sich in vielen Studien eine positive Korrelation . Zumeist werden der Umfang der körperlichen Aktivität, des Sporttreibens, Menge oder Häufigkeit des Obst- und Gemüsekonsums sowie Konsum bzw. Konsummenge von Tabak- und Alkohol untersucht. Hier zeigen sich studienspezifische Unterschiede: Je nach Studienpopulation, Messinstrument zur Gesundheitskompetenz bzw. dem Gesundheitsverhalten oder Analysemethoden sind in einigen Studien bzw. für bestimmte Teilpopulationen keine Assoziation zu beobachten . Im Hinblick auf die Absicht einer Verhaltensänderung, die oft das Ziel von Interventionen ist, hat sich Gesundheitskompetenz als moderierende Variable bei körperlicher Aktivität gezeigt . Im Hinblick auf das Gesundheitsverhalten fokussieren sich Forschung und Interventionspraxis häufig auf die individuelle Gesundheitskompetenz. Public-Health-Strategien wie der „Nationale Aktionsplan Gesundheitskompetenz“ oder die Strategie der Weltgesundheitsorganisation (WHO) zur Bekämpfung von nichtübertragbaren Erkrankungen legen den Schwerpunkt anders . Sie empfehlen den Fokus nicht nur auf die individuelle Ebene zu legen, sondern Gesundheitskompetenz als Teil von Verhältnisprävention zu verstehen, damit Normen und Kulturen sowie organisatorische und politische Strukturen verändert werden (soziale Praxis), die die Entwicklung von Gesundheitskompetenz in der Bevölkerung maßgeblich beeinflussen . Gesundheitskompetenz hat sich mittlerweile auch im Bereich der Kinder- und Jugendgesundheit als wichtiges Interventionsziel sowohl für die Förderung der körperlichen als auch der psychischen Gesundheit etabliert. Die WHO betont seit 10 Jahren die besondere Rolle der Schule für die Stärkung der Gesundheitskompetenz . In Deutschland werden solche Empfehlungen insbesondere durch den „Nationalen Aktionsplan Gesundheitskompetenz“ und die „Allianz für Gesundheitskompetenz in der Schule“ ausgesprochen . Konzeptionell orientieren sich Gesundheitskompetenzansätze für Kinder und Jugendliche an den Gesundheitskompetenzkonzeptionen für Erwachsene, die den Umgang mit Gesundheitsinformationen für gesundheitsbezogene Entscheidungen adressieren. Adaptierungen und Erweiterungen der Gesundheitskompetenzansätze für Kinder und Jugendliche zu spezifischen Gesundheitskompetenzen liegen insbesondere zu digitalen, psychischen und auf das Gesundheitsverhalten bezogenen Gesundheitskompetenzen vor . Basierend auf diesen Konzepten wurden in den letzten Jahren unterschiedliche Instrumente entwickelt , die ab dem Grundschulalter eingesetzt werden können . Diese reichen von (subjektiven) Selbsteinschätzungsinstrumenten bis zu leistungsbezogenen (objektiven bzw. performanzbasierten) Messverfahren, sowohl für allgemeine als auch spezifische Varianten von Gesundheitskompetenz. Derzeit finden Fragebogenverfahren, die teilweise oder ganz auf dem Fragebogen des „European Health Literacy Survey“ (HLS-EU‑Q) oder dem „Digital Health Literacy Instrument for Adolescents“ (DHLI) basieren bzw. verwandte Verfahren weite Verbreitung. Auch ethnografische Methoden werden zunehmend angewendet . Die Studien deuten auf einen Zusammenhang zwischen der Gesundheitskompetenz von Kindern und Jugendlichen und ihrem Gesundheitsverhalten hin . Eine Evidenzsynthese für das Europäische Schulnetzwerk kommt zu dem Schluss, dass eine höhere Gesundheitskompetenz häufiger mit gesundheitsförderlichen Verhaltensweisen einhergeht (z. B. in Bezug auf Ernährung, Sport und Bewegung, Substanzkonsum, Sexualverhalten, Ruhe und Schlafverhalten) . Dabei gibt es Unterschiede je nach Art des Gesundheitsverhaltens, insbesondere der Substanzkonsum scheint sich von Ernährung und Bewegung zu unterscheiden, wie beispielsweise die bundesweite Befragung zur „Gesundheitskompetenz von Jugendlichen“ (GeKoJu) zeigt: Gesundheitskompetenz ist mit dem täglichen Konsum von Obst und Gemüse assoziiert. Niedrigere Level in behavioralen, affektiven und konativen (absichtsorientierten) Dimensionen der Gesundheitskompetenz erhöhen die Chance, körperlich inaktiv zu sein und zu rauchen. Bei riskantem Alkoholkonsum spielen andere Faktoren (wie Alter oder familiärer Wohlstand) eine wichtigere Rolle als allgemeine Gesundheitskompetenz . In einer Studie zur Gesundheitskompetenz bei Jugendlichen an Schulen in Hessen sind mittlere und geringe digitale Gesundheitskompetenz mit weniger körperlicher Aktivität, nichttäglichem Obstkonsum und täglichem Konsum von zuckerhaltigen Getränken assoziiert . Die repräsentative Befragung von Grundschulkindern in der 4. Schulklasse zeigt , dass eine höhere Gesundheitskompetenz den stärksten Prädiktor für regelmäßiges Zähneputzen sowie Obst- und Gemüsekonsum darstellt . Soziale Ungleichheiten stellen auch im Kindes- und Jugendalter ein wiederkehrendes Phänomen dar, denn die Ausprägung von Gesundheitskompetenz folgt einem sozialen Gradienten , welcher sich auch bei der positiven Assoziation zwischen Gesundheitskompetenz und Gesundheitsverhalten im Jugendalter findet . Spezifische Gesundheitskompetenzen fokussieren auf Bedarfe hinsichtlich bestimmter Gesundheits- und Krankheitsthemen, um passgenaue Interventionen zu entwickeln . Konzepte spezifischer Gesundheitskompetenz sollen Anforderungen spezifischer gesundheitsrelevanter bewusster oder unbewusster Entscheidungssituationen oder Alltagsroutinen abbilden und ergänzen Erkenntnisse zu allgemeiner Gesundheitskompetenz. Für den Bereich des Gesundheitsverhaltens wurden je nach Verhaltensweise theoriebasiert eigene Dimensionen und Kompetenzbausteine für die spezifische Gesundheitskompetenz definiert, auch als Grundlage für die Entwicklung von spezifischen Messinstrumenten. Konzepte spezifischer Gesundheitskompetenzen gibt es in den Bereichen Ernährung und Bewegung, aber auch Rauchen, Alkoholkonsum, Schlafverhalten, Impfverhalten etc. . Im Folgenden werden exemplarisch 2 Bereiche spezifischer Gesundheitskompetenzen vorgestellt, für die in Forschung und Praxis ausdifferenzierte Konzepte, darauf aufbauende Erhebungsinstrumente und eine Interventionspraxis, zumindest in Modellprojekten, vorliegen: Ernährung und Bewegung. Food Literacy – Ernährungsbezogene Gesundheitskompetenz Für den Ernährungsbereich gibt es verschiedene Konzepte und Definitionen, die sich hinsichtlich der Handlungsebenen, Anwendungsbereiche und Domänen der Ernährungskompetenz und auch der Benennung der Gesundheitskompetenz unterscheiden . Funktionelle ernährungsbezogene Gesundheitskompetenz ( Nutrition Literacy ) bezieht sich auf das Verständnis von Informationen über Lebensmittel oder Ernährung . Sie umfasst vereinzelt zusätzlich interaktive und kritische Fähigkeiten . Food Literacy wird auch als Ernährungskompetenz bzw. ernährungsbezogene Gesundheitskompetenz bezeichnet. Sie geht über Wissen und Verstehen hinaus und beinhaltet praktische Fähigkeiten, z. B. Mahlzeitenplanung, Lebensmittelauswahl und -zubereitung , manchmal ergänzt um Kontextfaktoren (z. B. Verfügbarkeit gesunder Lebensmittel) oder psychologische Faktoren (z. B. motivationale, volitionale und behaviorale Aspekte). Food-Literacy-Konzepte für junge Menschen betonen „Food Systems“ („Ernährungssystem“: Gesamtheit der Lebensmittelversorgung und der gesellschaftlichen Ernährungsnormen) und soziale Gerechtigkeit bzw. soziale Aspekte zu Ernährung und Körperbild . Die Förderung der Ernährungskompetenz soll die Gesundheit auf Bevölkerungsebene verbessern und gesundheitliche Ungleichheit reduzieren . Damit ist Ernährungskompetenz relevant für Public Health, was die steigende Zahl an Publikationen zu diesem Konzept erklärt . Die Ausdifferenzierung von Ernährungskompetenz spiegelt sich in der Messung von Ernährungskompetenz wider . Die Nutrition Literacy erfassenden Instrumente sind performanzbasierte Messinstrumente, z. B. die validierten Instrumente „Nutrition Literacy Scale“ (NLS) und „Newest-Vital-Sign“(NVS)-Test (letzterer wird auch für die Erfassung allgemeiner funktionaler Gesundheitskompetenz eingesetzt). NLS und NVS-Test messen performanzbasiert die Fähigkeit, gedruckte Informationen zu verstehen. Beispielsweise sollen beim NVS-Test Befragte Nährwertangaben auf einer Eiscremepackung verstehen und 6 Fragen dazu beantworten, z. B. wie viele Kalorien sie beim Verzehr des gesamten Packungsinhalts zu sich nehmen würden. Von diesen 6 Fragen beantwortete knapp ein Drittel der Teilnehmenden in einer repräsentativen Befragungsstudie von Erwachsenen in Deutschland ( N = 1974) höchstens 3 Fragen richtig , ähnliche Ergebnisse zeigten sich in einer bundesweiten Befragung in Österreich . Das breitere Konzept Food Literacy wird mithilfe von multidimensionalen Selbsteinschätzungsinstrumenten erfasst , z. B. mit der validierten „Self-Perceived-Food-Literacy“(SPFL)-Skala. Sie umfasst 29 Fragen, aus deren Antworten ein Gesamtscore sowie 8 Subscores gebildet werden, die folgende Bereiche umfassen: „Selbst zubereiten“, „Gemeinsam essen“, „Gesund haushalten“, „Wahl der Vorräte“ „Widerstehen können“, „Smart snacken“, „Mahlzeiten planen“ und „Gesund vergleichen“ . Eine höhere Food Literacy ist mit einem häufigeren Obst‑, Gemüse- und Fischkonsum sowie mit mehr Selbstkontrolle und weniger Impulsivität assoziiert . Eine erste Studie für Deutschland fand eine höhere Ernährungskompetenz bei Frauen, steigendem Alter, höherem Bildungsabschluss und höherem Einkommen . Mahlzeitenplanung und Vergleich von Nährmittelangaben bewerteten die Befragten als am schwierigsten . Für Kinder und Jugendliche existieren ebenfalls unterschiedliche Instrumente, die Nutrition Literacy, Food Literacy oder beide Konstrukte messen, und für unterschiedliche Altersgruppen validiert wurden , z. B. „Nutrition Health Literacy Scale for Children“ (NHL‑C) [ , S. 27 ff.] und „Food Literacy Questionnaire for schoolchildren“ (FLQ-sc) . Es gibt Hinweise auf einen positiven Einfluss von verschiedenen Teilaspekten der Food Literacy auf die Nahrungsaufnahme und Ernährungsgewohnheiten von Jugendlichen . Die meisten Nutrition- und Food-Literacy-Instrumente für Kinder und Jugendliche werden allerdings als zu wenig umfassend bzw. als zu wenig lebensphasenspezifisch kritisiert oder erheben nur Teilaspekte von Food Literacy . Ernährungskompetenz liefert Ansatzpunkte auf individueller Ebene, um Wissen, Fähigkeiten und Ressourcen zu stärken, um sich in der Ernährungslandschaft zu orientieren, informierte Ernährungsentscheidungen zu treffen sowie nahrhafte Mahlzeiten herzustellen. Auf Bevölkerungsebene weist sie darauf hin, wo verhältnispräventive Public-Health-Strategien ansetzen sollten, z. B. Nährwertkennzeichnung (wie Nutri-Score) oder Gemeinschaftsverpflegung. Die vorhandenen bevölkerungsweiten Studien stellen dafür zentrale Befunde bereit (z. B. ). Physical Literacy – Bewegungsbezogene Gesundheitskompetenz Für den Bereich körperliche Aktivität und spezifische Gesundheitskompetenz ist eine konzeptionelle Vielfalt zu beobachten. Gemeinsam ist den Physical-Literacy- Konzepten das Verständnis von einer Kompetenz, welche Bewegung im gesamten Lebensverlauf fördern soll und im Kindes- und Jugendalter eine Bildungsaufgabe darstellt . Übergreifend werden neben motorischen Basiskompetenzen (funktionelle körperliche Ebene), Wissen bzw. Verständnis eines aktiven Lebensstils (kognitive Ebene), Motivation und Selbstbewusstsein (affektive Ebene) als zentrale Elemente von Physical Literacy gesehen . Ein weiteres Konzept wird als bewegungsbezogene Gesundheitskompetenz bezeichnet; es umfasst 3 Teilkompetenzen: Bewegungskompetenz, Steuerungskompetenz (Ausrichtung auf Wohlbefinden und Gesundheit) und Selbstregulationskompetenz (Motivation, Volition) . Neben personalen Komponenten werden in Konzepten der bewegungsbezogenen Gesundheitskompetenz auch Umgebungsfaktoren und soziale Teilhabe berücksichtigt und das Konzept um die Forderung nach „Physical Literate Societies“ ergänzt . Für die Erfassung von Physical Literacy liegen verschiedene Messinstrumente vor. Ein Mangel aller Messinstrumente besteht im Fehlen der sozialen Komponente, die der Physical Literacy inhärent ist . Als umfassende Messinstrumente für Physical Literacy werden auf Basis von systematischen Übersichtsarbeiten das „Canadian Assessment of Physical Literacy“ (CAPL) sowie der „Passport for Life“ für Kinder empfohlen (für Erwachsene gibt es keine klare Empfehlung für ein Instrument ). Das Tool CAPL erfragt für einen Physical Literacy Score verschiedene Aspekte aus den Domänen „Physical Competence“, „Daily Behaviour“, „Knowledge and Understanding“ und „Motivation and Confidence“ . Mit dem CAPL konnte in Studien ein positiver Zusammenhang zwischen Physical Literacy und körperlicher Aktivität bei Kindern von 8–12 Jahren gezeigt werden . Die meisten Instrumente decken nur eine oder 2 Ebenen von Physical Literacy ab, wobei körperliche und affektive Kompetenzen eher erfasst werden als kognitive Kompetenz . So erfasst ein Teil der Messinstrumente körperliche Kompetenzen, z. B. durch Aufgaben, die motorische Basisfertigkeiten, Balance, Kraft oder kardiovaskuläre Fitness erfordern, quantitativ mithilfe von Zeitmessung . Ein anderer Teil der Messinstrumente erfasst affektive und/oder kognitive Aspekte von Physical Literacy, z. B. die „Children’s Physical Activity Self-Efficacy Scale“ oder die „Children’s Self-Perception of Adequacy in and Predilection for Physical Activity Scale“ . Insgesamt gibt es bisher wenig Forschung zum Zusammenhang von Physical Literacy und körperlicher Aktivität bei Erwachsenen, vermutlich auch, da bisher nicht konsentiert ist, ob Bewegungsverhalten Teil des Physical-Literacy-Konzeptes selbst ist oder ein Outcome von Physical Literacy und damit eine außerhalb des Konstrukts liegende Variable darstellt [ , S. 1067]. Für Physical Literacy insgesamt sowie körperliche und motivationale, nicht aber kognitive Komponenten sind positive Zusammenhänge beschrieben . Im deutschsprachigen Raum wurden mithilfe validierter Lang- sowie Kurzinstrumente bei verschiedenen Bevölkerungsgruppen Zusammenhänge zwischen Komponenten bewegungsbezogener Gesundheitskompetenz und körperlicher Aktivität gezeigt . Physical-Literacy-Interventionen zeigen in Studien auf mehrere für körperliche Aktivität und Gesundheit relevante Endpunkte positive Effekte und verweisen damit auch auf Ansatzpunkte für verhältnispräventive Public-Health-Strategien. Für den Ernährungsbereich gibt es verschiedene Konzepte und Definitionen, die sich hinsichtlich der Handlungsebenen, Anwendungsbereiche und Domänen der Ernährungskompetenz und auch der Benennung der Gesundheitskompetenz unterscheiden . Funktionelle ernährungsbezogene Gesundheitskompetenz ( Nutrition Literacy ) bezieht sich auf das Verständnis von Informationen über Lebensmittel oder Ernährung . Sie umfasst vereinzelt zusätzlich interaktive und kritische Fähigkeiten . Food Literacy wird auch als Ernährungskompetenz bzw. ernährungsbezogene Gesundheitskompetenz bezeichnet. Sie geht über Wissen und Verstehen hinaus und beinhaltet praktische Fähigkeiten, z. B. Mahlzeitenplanung, Lebensmittelauswahl und -zubereitung , manchmal ergänzt um Kontextfaktoren (z. B. Verfügbarkeit gesunder Lebensmittel) oder psychologische Faktoren (z. B. motivationale, volitionale und behaviorale Aspekte). Food-Literacy-Konzepte für junge Menschen betonen „Food Systems“ („Ernährungssystem“: Gesamtheit der Lebensmittelversorgung und der gesellschaftlichen Ernährungsnormen) und soziale Gerechtigkeit bzw. soziale Aspekte zu Ernährung und Körperbild . Die Förderung der Ernährungskompetenz soll die Gesundheit auf Bevölkerungsebene verbessern und gesundheitliche Ungleichheit reduzieren . Damit ist Ernährungskompetenz relevant für Public Health, was die steigende Zahl an Publikationen zu diesem Konzept erklärt . Die Ausdifferenzierung von Ernährungskompetenz spiegelt sich in der Messung von Ernährungskompetenz wider . Die Nutrition Literacy erfassenden Instrumente sind performanzbasierte Messinstrumente, z. B. die validierten Instrumente „Nutrition Literacy Scale“ (NLS) und „Newest-Vital-Sign“(NVS)-Test (letzterer wird auch für die Erfassung allgemeiner funktionaler Gesundheitskompetenz eingesetzt). NLS und NVS-Test messen performanzbasiert die Fähigkeit, gedruckte Informationen zu verstehen. Beispielsweise sollen beim NVS-Test Befragte Nährwertangaben auf einer Eiscremepackung verstehen und 6 Fragen dazu beantworten, z. B. wie viele Kalorien sie beim Verzehr des gesamten Packungsinhalts zu sich nehmen würden. Von diesen 6 Fragen beantwortete knapp ein Drittel der Teilnehmenden in einer repräsentativen Befragungsstudie von Erwachsenen in Deutschland ( N = 1974) höchstens 3 Fragen richtig , ähnliche Ergebnisse zeigten sich in einer bundesweiten Befragung in Österreich . Das breitere Konzept Food Literacy wird mithilfe von multidimensionalen Selbsteinschätzungsinstrumenten erfasst , z. B. mit der validierten „Self-Perceived-Food-Literacy“(SPFL)-Skala. Sie umfasst 29 Fragen, aus deren Antworten ein Gesamtscore sowie 8 Subscores gebildet werden, die folgende Bereiche umfassen: „Selbst zubereiten“, „Gemeinsam essen“, „Gesund haushalten“, „Wahl der Vorräte“ „Widerstehen können“, „Smart snacken“, „Mahlzeiten planen“ und „Gesund vergleichen“ . Eine höhere Food Literacy ist mit einem häufigeren Obst‑, Gemüse- und Fischkonsum sowie mit mehr Selbstkontrolle und weniger Impulsivität assoziiert . Eine erste Studie für Deutschland fand eine höhere Ernährungskompetenz bei Frauen, steigendem Alter, höherem Bildungsabschluss und höherem Einkommen . Mahlzeitenplanung und Vergleich von Nährmittelangaben bewerteten die Befragten als am schwierigsten . Für Kinder und Jugendliche existieren ebenfalls unterschiedliche Instrumente, die Nutrition Literacy, Food Literacy oder beide Konstrukte messen, und für unterschiedliche Altersgruppen validiert wurden , z. B. „Nutrition Health Literacy Scale for Children“ (NHL‑C) [ , S. 27 ff.] und „Food Literacy Questionnaire for schoolchildren“ (FLQ-sc) . Es gibt Hinweise auf einen positiven Einfluss von verschiedenen Teilaspekten der Food Literacy auf die Nahrungsaufnahme und Ernährungsgewohnheiten von Jugendlichen . Die meisten Nutrition- und Food-Literacy-Instrumente für Kinder und Jugendliche werden allerdings als zu wenig umfassend bzw. als zu wenig lebensphasenspezifisch kritisiert oder erheben nur Teilaspekte von Food Literacy . Ernährungskompetenz liefert Ansatzpunkte auf individueller Ebene, um Wissen, Fähigkeiten und Ressourcen zu stärken, um sich in der Ernährungslandschaft zu orientieren, informierte Ernährungsentscheidungen zu treffen sowie nahrhafte Mahlzeiten herzustellen. Auf Bevölkerungsebene weist sie darauf hin, wo verhältnispräventive Public-Health-Strategien ansetzen sollten, z. B. Nährwertkennzeichnung (wie Nutri-Score) oder Gemeinschaftsverpflegung. Die vorhandenen bevölkerungsweiten Studien stellen dafür zentrale Befunde bereit (z. B. ). Für den Bereich körperliche Aktivität und spezifische Gesundheitskompetenz ist eine konzeptionelle Vielfalt zu beobachten. Gemeinsam ist den Physical-Literacy- Konzepten das Verständnis von einer Kompetenz, welche Bewegung im gesamten Lebensverlauf fördern soll und im Kindes- und Jugendalter eine Bildungsaufgabe darstellt . Übergreifend werden neben motorischen Basiskompetenzen (funktionelle körperliche Ebene), Wissen bzw. Verständnis eines aktiven Lebensstils (kognitive Ebene), Motivation und Selbstbewusstsein (affektive Ebene) als zentrale Elemente von Physical Literacy gesehen . Ein weiteres Konzept wird als bewegungsbezogene Gesundheitskompetenz bezeichnet; es umfasst 3 Teilkompetenzen: Bewegungskompetenz, Steuerungskompetenz (Ausrichtung auf Wohlbefinden und Gesundheit) und Selbstregulationskompetenz (Motivation, Volition) . Neben personalen Komponenten werden in Konzepten der bewegungsbezogenen Gesundheitskompetenz auch Umgebungsfaktoren und soziale Teilhabe berücksichtigt und das Konzept um die Forderung nach „Physical Literate Societies“ ergänzt . Für die Erfassung von Physical Literacy liegen verschiedene Messinstrumente vor. Ein Mangel aller Messinstrumente besteht im Fehlen der sozialen Komponente, die der Physical Literacy inhärent ist . Als umfassende Messinstrumente für Physical Literacy werden auf Basis von systematischen Übersichtsarbeiten das „Canadian Assessment of Physical Literacy“ (CAPL) sowie der „Passport for Life“ für Kinder empfohlen (für Erwachsene gibt es keine klare Empfehlung für ein Instrument ). Das Tool CAPL erfragt für einen Physical Literacy Score verschiedene Aspekte aus den Domänen „Physical Competence“, „Daily Behaviour“, „Knowledge and Understanding“ und „Motivation and Confidence“ . Mit dem CAPL konnte in Studien ein positiver Zusammenhang zwischen Physical Literacy und körperlicher Aktivität bei Kindern von 8–12 Jahren gezeigt werden . Die meisten Instrumente decken nur eine oder 2 Ebenen von Physical Literacy ab, wobei körperliche und affektive Kompetenzen eher erfasst werden als kognitive Kompetenz . So erfasst ein Teil der Messinstrumente körperliche Kompetenzen, z. B. durch Aufgaben, die motorische Basisfertigkeiten, Balance, Kraft oder kardiovaskuläre Fitness erfordern, quantitativ mithilfe von Zeitmessung . Ein anderer Teil der Messinstrumente erfasst affektive und/oder kognitive Aspekte von Physical Literacy, z. B. die „Children’s Physical Activity Self-Efficacy Scale“ oder die „Children’s Self-Perception of Adequacy in and Predilection for Physical Activity Scale“ . Insgesamt gibt es bisher wenig Forschung zum Zusammenhang von Physical Literacy und körperlicher Aktivität bei Erwachsenen, vermutlich auch, da bisher nicht konsentiert ist, ob Bewegungsverhalten Teil des Physical-Literacy-Konzeptes selbst ist oder ein Outcome von Physical Literacy und damit eine außerhalb des Konstrukts liegende Variable darstellt [ , S. 1067]. Für Physical Literacy insgesamt sowie körperliche und motivationale, nicht aber kognitive Komponenten sind positive Zusammenhänge beschrieben . Im deutschsprachigen Raum wurden mithilfe validierter Lang- sowie Kurzinstrumente bei verschiedenen Bevölkerungsgruppen Zusammenhänge zwischen Komponenten bewegungsbezogener Gesundheitskompetenz und körperlicher Aktivität gezeigt . Physical-Literacy-Interventionen zeigen in Studien auf mehrere für körperliche Aktivität und Gesundheit relevante Endpunkte positive Effekte und verweisen damit auch auf Ansatzpunkte für verhältnispräventive Public-Health-Strategien. Bei der Untersuchung der Frage, wie Gesundheitskompetenz und Gesundheitsverhalten zusammenhängen, kann auch der interdisziplinäre Ansatz der „Behavioural and Cultural Insights“ (BCI) herangezogen werden. BCI umfassen empirische Erkenntnisse, die erklären, wie Menschen in ihrem Alltag gesundheitsrelevante Entscheidungen treffen und welche Faktoren ihr Gesundheitsverhalten beeinflussen. Der BCI-Ansatz weist viele Ähnlichkeiten mit bestehenden Public-Health-Ansätzen auf, stellt allerdings das Verhalten als zentrale Zielgröße in den Mittelpunkt und zielt darauf ab, individuelle und strukturelle Barrieren und Einflussfaktoren zu identifizieren, um daraus präventive Maßnahmen abzuleiten . Der BCI-Ansatz bietet verschiedene Anknüpfungspunkte zur Gesundheitskompetenz, insbesondere im Hinblick auf die Analyse und Förderung des Gesundheitsverhaltens. Laut dem COM-B-Modell von Michie , das eine zentrale theoretische Grundlage des BCI-Ansatzes bildet, beeinflussen nicht nur Fähigkeiten (C = Capabilities) und Motivation (M = Motivation), sondern auch strukturelle und soziale Faktoren (O = Opportunity) das Verhalten (B = Behaviour) . Zum einen zeigen sich Parallelen zur Gesundheitskompetenz: So bedarf Gesundheitskompetenz nicht nur Gesundheitswissen und Fähigkeiten, sondern auch Anpassungen der Umwelt, z. B. durch organisationale Gesundheitskompetenz. So lenken beide Ansätze die Aufmerksamkeit darauf, dass Gesundheitsverhalten nicht nur von Wissen und Kompetenzen, sondern insbesondere von den sozialen Kontexten, dem Umfeld und den konkreten Lebenswelten geprägt wird. Zum anderen lässt sich Gesundheitskompetenz als eine Fähigkeit (Capability) im COM-B-Modell einordnen. Damit kann BCI dazu beitragen, den Zusammenhang zwischen Gesundheitskompetenz und Gesundheitsverhalten besser zu verstehen. Umfassende Definitionen von Gesundheitskompetenz schließen auch motivationale Aspekte ein, ein Bereich, der im COM-B-Modell einen eigenständigen Faktor (Motivation) darstellt. Diese Überschneidung sollte bei Analysen zum Gesundheitsverhalten berücksichtigt werden. Die Einbeziehung von BCI-Studien in die Gesundheitskompetenzforschung eröffnet neue Perspektiven, da für BCI auch unbewusste gesundheitsrelevante Entscheidungen untersucht werden. Der BCI-Ansatz adressiert die aus der Prävention und Gesundheitsförderung bekannte Beobachtung, dass Menschen trotz vorhandenen Wissens ihr Verhalten nicht ändern (der sogenannte Intention-Action-Gap) und hat so das Potenzial, strukturelle Barrieren zu identifizieren, an denen Gesundheitskompetenzförderung ansetzen kann. Der Ansatz „Behavioural and Cultural Insights“ wird in Public Health verstärkt verfolgt , andererseits kontrovers diskutiert. Das mag daran liegen, dass der Ansatz ursprünglich als „Behavioural Insights“ durch die Verhaltensökonomie geprägt wurde, mit einer engen Ausrichtung auf psychologische Verhaltens- und Entscheidungsmuster und im Hinblick auf sogenannte Nudging-Maßnahmen, die durch kleine Umgebungsänderungen oder Anreize erwünschte Verhaltensänderungen erleichtern . Die WHO hingegen betont, dass sich Verhaltenswissenschaften von einer individuellen, verhaltensbezogenen Perspektive deutlich erweitern müssen hin zu einem umfassenderen, systemischen Ansatz . Sie verdeutlicht diesen Wandel durch die Ergänzung von „Behavioural Insights“ um den Begriff „cultural,“ dadurch wird das soziokulturelle Umfeld adressiert . Damit wird deutlich, dass BCI wie auch Gesundheitskompetenzförderung nur dann erfolgreich soziale Chancengleichheit adressieren können, wenn sie im Sinne von „New Public Health“ Rahmen- und Lebensbedingungen verstehen und im Sinne von Verhältnisprävention positiv gestalten helfen, um das Gesundheitsverhalten in der Bevölkerung zu verbessern. Das Forschungs- und Handlungsfeld „Gesundheitskompetenz und Gesundheitsverhalten“ differenziert sich zunehmend aus. Studien zu allgemeiner Gesundheitskompetenz und Gesundheitsverhalten eignen sich in besonderem Maße für die vergleichende Beobachtung auf Bevölkerungsebene. Untersuchungen zu spezifischen Gesundheitskompetenzen hinsichtlich einzelner Gesundheitsverhalten liefern Informationen zu konkreten Ansatzpunkten für Interventionen. Die Erhebungsinstrumente ergänzen sich demnach. Allgemein zeigen die zumeist als Querschnittsstudien durchgeführten Studien eine positive Assoziation zwischen Gesundheitskompetenz und verschiedenen Gesundheitsverhalten. Dies gilt sowohl für die allgemeine Gesundheitskompetenz als auch für die hier näher untersuchten spezifischen Gesundheitskompetenzen zur körperlichen Aktivität und Ernährung. Eine höhere Gesundheitskompetenz geht häufiger mit einem verbesserten gesundheitsförderlichen Verhalten ein. In manchen Studien finden sich nicht bei allen untersuchten Gesundheitsverhaltensweisen diese Zusammenhänge und manchmal nur für bestimmte Gruppen. Das kann teilweise auf verschiedene Studiendesigns und Messinstrumente zurückzuführen sein, aber auch auf unterschiedliche Kontexte wie sozioökonomische Bedingungen und soziale Normen. Letztere verweisen auf den Bedarf, stets auch die Wechselwirkung von Verhalten (Mensch) und Verhältnissen (Struktur) zu untersuchen und im Rahmen von Interventionen zu berücksichtigen. Gesundheitskompetenz als Determinante von Gesundheitsverhalten ist in individuelle Lebenslagen und soziale Kontexte eingebettet. Der Ansatz „Behavioural and Cultural Insights“ kann für die Förderung von Gesundheitskompetenz im Hinblick auf verschiedene Gesundheitsverhalten wissenschaftliche Erkenntnisse liefern, zu persönlichen Barrieren und Förderfaktoren, die auf Lebensumstände und Rahmenbedingungen zurückgehen und die soziale Praxis berücksichtigen. BCI und Gesundheitskompetenz ergänzen sich und haben das Potenzial, effektivere und zielgerichtetere Strategien zur Verbesserung des Gesundheitsverhaltens zu entwickeln. |
Prognostic significance of CD8 and TCF1 double positive T cell subset in microsatellite unstable gastric cancer | 8a10193c-7696-45dd-881a-78c522d70368 | 11579470 | Anatomy[mh] | Gastric cancer (GC) is a highly heterogeneous disease and attempts have been made to categorize its molecular subtypes. One of the GC subtypes according to the classification proposed by the Cancer Genome Atlas and the Asian Cancer Research Group is microsatellite instability-high (MSI-H) or microsatellite-unstable GC. Recent studies have shown that the percentage of MSI-H GC in patients with GC differs (8–25%) depending on the racial, geographical, or tumor stage distribution of the cohorts . While the percentages of MSI-H GC are different, accumulating results have made it increasingly clear that MSI-H in GC is linked to a better prognosis – . Meta-analyses incorporating various studies compared the prognosis of MSI-H GC with MSI-low and microsatellite-stable GC, showing that MSI-H GC was associated with significantly improved survival , , . The previous studies regarding MSI-H GC showed that MSI-H GC has been associated with female sex, distal (middle to lower) location, earlier TNM stages, intestinal type of Lauren’s classification, older age, and fewer lymph node invasions , , , . The impact of chemotherapy on patients with MSI-H GC is still controversial, with results that they do not benefit from chemotherapy , , . Patients with MSI-H GC show high responses to immunotherapy such as PD-1 inhibitor , , but some MSI-H patients do not respond to the immunotherapy . Therefore, there is a need for further scrutinization of the tumor microenvironment of MSI-H GC. Microsatellites are short repetitive sequences that can be found throughout the whole human genome and MSI-H is regarded as a molecular fingerprint of a deficient DNA mismatch repair (dMMR) system . As a result, MSI-H GC is prone to hypermutation and an increased number of tumor-specific neoantigens that are recognized by T lymphocytes , . Because CD8 + T cells can recognize most tumor cells by the antigens they express, clinically detected cancers are a sign that the tumor cells have somehow evaded immune responses during their growth . Therefore, the tumor immune microenvironment is crucial for predicting tumor behavior. Within the tumor immune microenvironment, tumor-infiltrating lymphocytes (TILs) recognize abnormal tumor-specific neoantigens . Owing to excessive mutations and a higher quantity of tumor-specific neoantigens, MSI-H GCs typically exhibit high infiltration of TILs , . Several studies have reported a significant correlation between TILs and MSI or dMMR status in tumors , . TIL infiltration is a positive prognostic factor in GC. A meta-analysis of gastric cancer reported high levels of CD8+, CD3+, and CD4 + T cell infiltration as positive prognostic indicators . However, our previous study and another recent study demonstrated no significant association between CD8 + TIL infiltration and prognosis, particularly in MSI-H GC , . This lack of correlation may be attributed to the heterogeneity of T cell functions and exhausted T cells. Exhausted T cells are those whose response to tumors is compromised after continuous antigen stimulation , allowing a stalemate to occur between the pathogen and the host immune system, which can result in the coexistence of T cells and tumors . T cell factor 1 (T Cell Transcription Factor 1, TCF1) and CD103 (Alpha E Integrins) are two biomarkers reported to be related to T cell exhaustion. TCF1 is a transcription factor involved in the Wnt signaling pathway and is encoded by the TCF7 gene . TCF1 plays an important role in T cell differentiation and memory formation, with CD8+/TCF1 + TILs showing self-renewing abilities and the ability to differentiate into terminal effector T cells , . Additionally, TCF1 is essential for the generation of CD8 progenitor cells, the coordination of genes that regulate T cell differentiation, long-lasting CD8 responses, and also for regulating genes related to immune checkpoints that can cause CD8 T cell exhaustion . In epithelial tumors, CD103 binds to its ligand E-cadherin and stimulates and recruits activated CD8+/CD103 + TIL, while also promoting lymphocyte migration and activation . CD103 contributes to the recruitment of TILs and enhances their cytotoxicity, but is also frequently co-expressed with PD-1 and Tim3, which are known to be involved in T cell exhaustion . Concerning anti-PD-1 treatment, tumor-infiltrating CD8+/CD103 + T cells have been reported to be associated with significantly improved outcomes when anti-PD-1 immunotherapy is applied . In this study, we aimed to clarify the clinicopathological and prognostic significance of specific T-cell subsets in a large cohort of patients with MSI-H GC. We evaluated the densities of CD8+, TCF1+, and CD103 + cells in MSI-H GC cases using immunohistochemistry (IHC) and utilized the cell densities as criteria to categorize T cells into subsets. IHC was employed due to the long period of time required for collecting a large number of samples, since MSI-H GC accounts for around a tenth of total GC and sufficient time for follow-up was needed. Therefore, in our samples consisting of old FFPE blocks, other methods such as fluorescence-activated cell sorting and next-generation sequencing were unsuitable. We employed double-antibody and single-antibody IHC techniques to mark double-positive, CD8+/TCF1+, and CD8+/CD103 + cells, which allowed us to distinguish double-positive cells from single-positive cells. By analyzing the cell densities, prognosis, and survival outcomes of the cases included in this study, we examined and assessed the prognostic impact of each T-cell subgroup in a large cohort of patients with MSI-H GC. Clinicopathologic features of the patients Of the 382 patients included in the study, 230 (60.2%) were men and 152 (39.8%) were women. The median age was 69 years (range: 39–92 years). According to pT categories, 158 (41.4%), 66 (17.3%), 97 (25.4%), and 51 (16.0%) cases showed pT1, pT2, pT3, and pT4, respectively. Detailed clinicopathological features of the patients are shown in Table . Correlation between single IHC and double IHC In addition to single IHC for CD8, TCF1, and CD103, double IHC was performed to detect CD8+/TCF1 + cells and CD8+/CD103 + cells in 382 MSI-H GC cases (Fig. ). To confirm the concordance between the results obtained from single IHC and double IHC, the correlation between the detected densities of positive cells was calculated (Fig. a). The densities of CD8+, CD103+, and TCF1 + cells in single IHC images exhibited moderate to strong correlations (R 2 = 0.709, p < 0.001, and R 2 = 0.648, p < 0.001 for CD8; R 2 = 0.696, p < 0.001 for CD103; R 2 = 0.523, p < 0.001 for TCF1, respectively) with their corresponding counterparts in double IHC. Additionally, to investigate the association between cell densities, the correlation between CD8+, TCF1+, and CD103 + cell densities was analyzed (Fig. b). CD103 + cell densities were strongly correlated with CD8 + cell densities (R 2 = 0.810, p < 0.001 in single IHC and R 2 = 0.725, p < 0.001 in double IHC). However, TCF1 + cell densities were weakly correlated with CD8 + cell densities (R 2 = 0.295, p < 0.001 in single IHC and R 2 = 0.227, p < 0.001 in double IHC), possibly due to inclusion of TCF1-expressing tumor cells and CD8-negative lymphocytes in the TIL regions. TCF1 + cell densities also exhibited very weak correlations with CD103 + cell densities (R 2 = 0.124, p = 0.016 in single IHC; R 2 = 0.140, p = 0.006 in double IHC). When correlation analysis was restricted to CD8 + T cells, CD8+/CD103 + cell density was moderately correlated with CD8+/TCF1 + cell density (R 2 = 0.539, p < 0.001; Fig. c). Correlation between cell densities and clinicopathological characteristics The correlations between the aforementioned criteria defined by the ratio and clinicopathological characteristics are presented in Table . The high ratio of CD8+/TCF1 + to CD8 + cell density (CD8+/TCF1 + to CD8 + ratio) was significantly associated with less aggressive clinicopathological characteristics than the low CD8+/TCF1 + to CD8 + ratio, such as smaller tumor size ( p < 0.001), lower pT ( p < 0.001), pN ( p = 0.004), and pTNM ( p = 0.001), and was less likely to have lymphatic ( p = 0.031), vascular ( p = 0.037), or neural ( p = 0.001) invasion. However, the ratio of CD8+/CD103 + to CD8 + cell density (CD8+/CD103 + to CD8 + ratio) did not show any significant correlation with clinicopathological characteristics, although the density of CD8+/CD103 + cells was significantly correlated with histologic classifications by WHO ( p < 0.001) and Lauren ( p = 0.001), and lymphatic invasion ( p = 0.009). More detailed correlations between clinicopathologic features and single and double IHC results are presented in Supplementary Tables and . Survival analysis The median follow-up period for the survival analysis was 74.7 months (range, 0.1 to 96.0 months). To investigate the association between CD8+, TCF1+, and CD103 + cell densities and prognosis, survival analysis using the Kaplan-Meier survival curves was employed (Fig. ). Although there was a tendency for patients with high densities of CD8+, TCF1+, or CD103 + cells to exhibit better OS, these were not statistically significant ( p = 0.135, p = 0.065, and p = 0.272 for CD8, CD103, and TCF1, respectively). However, when the study population was dichotomized based on the density of CD8+/TCF1 + cells, patients with a high density of CD8+/TCF1 + cells exhibited significantly better prognosis ( p = 0.017) than those with a low density of CD8+/TCF1 + cells. The difference in prognosis between the two groups became more pronounced and significant ( p = 0.001) when the criterion employed to dichotomize the study population was the CD8+/TCF1 + to CD8 + ratio. The density of CD8+/CD103 + cells showed no discernible trend or significant association with prognosis regardless of the criteria used, including CD8+/CD103 + cell density ( p = 0.880) or CD8+/CD103 + to CD8 + ratio ( p = 0.828). For reference, Kaplan-Meier survival curves using the Q2 values as the threshold are shown in Supplementary Fig. . Within those curves, patients with high TCF1 + cell density ( p = 0.002) had significantly better OS while those with high CD8+/TCF1 + cell density only showed a tendency to have better OS ( p = 0.240). Multivariable Cox regression analyses were performed to assess whether each criterion was an independent prognostic predictor (Fig. and Supplementary Table ). To avoid interference when conducting multivariable analysis, only one feature was used when closely related clinicopathological features were present. pTNM stage was used to represent the stages and Lauren’s classification was used to represent the histologic types. CD8+/TCF1 + cell density ( p = 0.020) and the CD8+/TCF1 + to CD8 + ratio ( p = 0.002), as well as age ( p < 0.001), sex ( p = 0.008), tumor size ( p < 0.001), pTNM stage ( p < 0.001), Lauren’s classification ( p = 0.023), tumor border ( p < 0.001), lymphatic invasion ( p < 0.001), vascular invasion ( p = 0.001), and neural invasion ( p < 0.001) were all significantly associated with OS in univariable analysis. In multivariable Cox regression analysis, all the previously mentioned criteria shown to be significantly associated with OS in univariable analysis were included. As CD8+/TCF1 + cell density and the CD8+/TCF1 + to CD8 + ratio were both significantly associated with OS in univariable analysis, multivariable Cox regression analysis was performed twice to independently assess whether the two closely related criteria were significantly associated with OS. One analysis included the CD8+/TCF1 + T cell density, but not the CD8+/TCF1 + to CD8 + ratio and the other included the CD8+/TCF1 + to CD8 + ratio, but not the CD8+/TCF1 + cell density. The ratio of CD8+/TCF1 + cell density to total CD8 + cell density was an independent favorable prognostic factor for OS ( p = 0.028), whereas CD8+/TCF1 + cell density was not ( p = 0.146). In both multivariable analyses, age, sex, pTNM stage, tumor border, and neural invasion were other independent prognostic factors. Of the 382 patients included in the study, 230 (60.2%) were men and 152 (39.8%) were women. The median age was 69 years (range: 39–92 years). According to pT categories, 158 (41.4%), 66 (17.3%), 97 (25.4%), and 51 (16.0%) cases showed pT1, pT2, pT3, and pT4, respectively. Detailed clinicopathological features of the patients are shown in Table . In addition to single IHC for CD8, TCF1, and CD103, double IHC was performed to detect CD8+/TCF1 + cells and CD8+/CD103 + cells in 382 MSI-H GC cases (Fig. ). To confirm the concordance between the results obtained from single IHC and double IHC, the correlation between the detected densities of positive cells was calculated (Fig. a). The densities of CD8+, CD103+, and TCF1 + cells in single IHC images exhibited moderate to strong correlations (R 2 = 0.709, p < 0.001, and R 2 = 0.648, p < 0.001 for CD8; R 2 = 0.696, p < 0.001 for CD103; R 2 = 0.523, p < 0.001 for TCF1, respectively) with their corresponding counterparts in double IHC. Additionally, to investigate the association between cell densities, the correlation between CD8+, TCF1+, and CD103 + cell densities was analyzed (Fig. b). CD103 + cell densities were strongly correlated with CD8 + cell densities (R 2 = 0.810, p < 0.001 in single IHC and R 2 = 0.725, p < 0.001 in double IHC). However, TCF1 + cell densities were weakly correlated with CD8 + cell densities (R 2 = 0.295, p < 0.001 in single IHC and R 2 = 0.227, p < 0.001 in double IHC), possibly due to inclusion of TCF1-expressing tumor cells and CD8-negative lymphocytes in the TIL regions. TCF1 + cell densities also exhibited very weak correlations with CD103 + cell densities (R 2 = 0.124, p = 0.016 in single IHC; R 2 = 0.140, p = 0.006 in double IHC). When correlation analysis was restricted to CD8 + T cells, CD8+/CD103 + cell density was moderately correlated with CD8+/TCF1 + cell density (R 2 = 0.539, p < 0.001; Fig. c). The correlations between the aforementioned criteria defined by the ratio and clinicopathological characteristics are presented in Table . The high ratio of CD8+/TCF1 + to CD8 + cell density (CD8+/TCF1 + to CD8 + ratio) was significantly associated with less aggressive clinicopathological characteristics than the low CD8+/TCF1 + to CD8 + ratio, such as smaller tumor size ( p < 0.001), lower pT ( p < 0.001), pN ( p = 0.004), and pTNM ( p = 0.001), and was less likely to have lymphatic ( p = 0.031), vascular ( p = 0.037), or neural ( p = 0.001) invasion. However, the ratio of CD8+/CD103 + to CD8 + cell density (CD8+/CD103 + to CD8 + ratio) did not show any significant correlation with clinicopathological characteristics, although the density of CD8+/CD103 + cells was significantly correlated with histologic classifications by WHO ( p < 0.001) and Lauren ( p = 0.001), and lymphatic invasion ( p = 0.009). More detailed correlations between clinicopathologic features and single and double IHC results are presented in Supplementary Tables and . The median follow-up period for the survival analysis was 74.7 months (range, 0.1 to 96.0 months). To investigate the association between CD8+, TCF1+, and CD103 + cell densities and prognosis, survival analysis using the Kaplan-Meier survival curves was employed (Fig. ). Although there was a tendency for patients with high densities of CD8+, TCF1+, or CD103 + cells to exhibit better OS, these were not statistically significant ( p = 0.135, p = 0.065, and p = 0.272 for CD8, CD103, and TCF1, respectively). However, when the study population was dichotomized based on the density of CD8+/TCF1 + cells, patients with a high density of CD8+/TCF1 + cells exhibited significantly better prognosis ( p = 0.017) than those with a low density of CD8+/TCF1 + cells. The difference in prognosis between the two groups became more pronounced and significant ( p = 0.001) when the criterion employed to dichotomize the study population was the CD8+/TCF1 + to CD8 + ratio. The density of CD8+/CD103 + cells showed no discernible trend or significant association with prognosis regardless of the criteria used, including CD8+/CD103 + cell density ( p = 0.880) or CD8+/CD103 + to CD8 + ratio ( p = 0.828). For reference, Kaplan-Meier survival curves using the Q2 values as the threshold are shown in Supplementary Fig. . Within those curves, patients with high TCF1 + cell density ( p = 0.002) had significantly better OS while those with high CD8+/TCF1 + cell density only showed a tendency to have better OS ( p = 0.240). Multivariable Cox regression analyses were performed to assess whether each criterion was an independent prognostic predictor (Fig. and Supplementary Table ). To avoid interference when conducting multivariable analysis, only one feature was used when closely related clinicopathological features were present. pTNM stage was used to represent the stages and Lauren’s classification was used to represent the histologic types. CD8+/TCF1 + cell density ( p = 0.020) and the CD8+/TCF1 + to CD8 + ratio ( p = 0.002), as well as age ( p < 0.001), sex ( p = 0.008), tumor size ( p < 0.001), pTNM stage ( p < 0.001), Lauren’s classification ( p = 0.023), tumor border ( p < 0.001), lymphatic invasion ( p < 0.001), vascular invasion ( p = 0.001), and neural invasion ( p < 0.001) were all significantly associated with OS in univariable analysis. In multivariable Cox regression analysis, all the previously mentioned criteria shown to be significantly associated with OS in univariable analysis were included. As CD8+/TCF1 + cell density and the CD8+/TCF1 + to CD8 + ratio were both significantly associated with OS in univariable analysis, multivariable Cox regression analysis was performed twice to independently assess whether the two closely related criteria were significantly associated with OS. One analysis included the CD8+/TCF1 + T cell density, but not the CD8+/TCF1 + to CD8 + ratio and the other included the CD8+/TCF1 + to CD8 + ratio, but not the CD8+/TCF1 + cell density. The ratio of CD8+/TCF1 + cell density to total CD8 + cell density was an independent favorable prognostic factor for OS ( p = 0.028), whereas CD8+/TCF1 + cell density was not ( p = 0.146). In both multivariable analyses, age, sex, pTNM stage, tumor border, and neural invasion were other independent prognostic factors. The high density of CD8 + T cell infiltration has long been considered a good prognostic factor for conventional GC. However, recent studies , and the results of this study have shown that a high density of CD8 + T cell infiltration does not always indicate a better prognosis in MSI-H GC, which is characterized by a high density of TIL. Therefore, further investigation into the characteristics of TIL in MSI-H GC is needed. The lack of investigation may be attributed to the difficulty in amassing cases available for investigation, as MSI-H GC accounts for only 8–25% of all GCs . Multiplex IHC, which allows confirmation of T cell subsets, is expensive to apply to a large cohort . Therefore, we aggregated and analyzed a large cohort of 382 patients with MSI-H GC for the study. To the best of our knowledge, the present study investigated the prognostic value of T cell subsets using double IHC in the largest cohort of patients with MSI-H GC. TCF1 is the downstream transcription factor of the canonical Wnt signaling pathway and plays an important role in T cell development and maturation, including CD4 + and CD8 + T cells . TCF1 helps intensify the immune response against tumors, and CD8+/TCF1 + TILs can self-renew and differentiate into terminal effector T cells – . TCF1 can be also expressed on tumor cells; however, TCF1 expression on tumor cells is not necessarily associated with a better immune response , . A study on liver cancer stem cells identified that a long noncoding RNA, lncTCF7, which activates the TCF7 promoter by recruiting the SWI/SNF complex, activates the Wnt pathway via TCF7 , thereby promoting the renewal of liver cancer stem cells and tumor propagation . Other studies have suggested that lncTCF7 promotes tumor cell migration and invasion in colorectal cancer . As TCF1 can be expressed on cells other than CD8 + T cells, those other cells may hinder the investigation of CD8+/TCF1 + T cells. To differentiate CD8+/TCF1 + T cells from other TCF1 + cells, we used double IHC of TCF1 and CD8. Consequently, we deemed the single TCF1 + cell density unsuitable as a primary marker for our study, as our main interest was specifically in CD8 + T cells and as we cannot rule out the possibility that the association between the TCF1 + cells and prognosis may be interrupted due to cells other than CD8 + T cells. Therefore, we employed the Q3 values, which showed a better correlation between the CD8+/TCF1 + T cells and OS than the Q2 values, as the primary threshold for dichotomizing the study population. Our results showed that CD8+/TCF1 + cell density can be a positive prognostic factor, as in previous studies on lung adenocarcinoma , melanoma , and primary small cell carcinoma of the esophagus . During chronic infection, which is also an environment characterized by prolonged antigen exposure, TCF1 expression on CD8 T cells was shown to help sustain long-lasting T cell activity by enhancing the generation of Tim3-low CD8 progenitor cells, and to repress exhaustion of T cells by downregulating exhaustion-mediating pathways and expression of Tim3 and Blimp1 . Other studies showed that loss of TCF1-expressing CD8 T cells precludes the maintenance of a sizeable pool of effector T cells and viral clearance during recurrent infection , and that TCF1-expressing CD8 T cells are indispensable for the generation and maintenance of CXCR5+/CD8 + effector T cells during chronic infection . Given these findings and our result, CD8+/TCF1 + cells may be crucial in sustaining the activity of T cells in MSI-H GC, where they encounter continuous antigen stimulation. It is noteworthy that the CD8+/TCF1 + to CD8 + ratio predicts prognosis better than the absolute number of CD8+/TCF1 + or TCF1 + T cells. This result might be due to the abundance of TIL in MSI-H GC. A single subset of T cells may not have a significant impact on the tumor microenvironment if the subset is populous, but its proportion is small. Given these findings, our results imply that it is important to investigate subsets of T cells. Furthermore, our results suggest that the overall tendency of TIL to exhibit strong antitumor activity may be predicted by lymphocyte composition. The CD8+/TCF1 + to CD8 + ratio has also been reported as a significant prognostic predictor in primary small cell carcinoma of the esophagus . A similar method of stratifying CD8+/CD103 + cell density within the total CD8 + T cells was utilized in a previous study to assess intratumoral CD8+/CD103 + T cells in patients with GC, which reported that the prognostic value of CD8+/CD103 + T cells was significant for total GC . Therefore, we suggest further studies to discover and validate other subsets of T cells that serve as significant prognostic predictors in MSI-H GC, based on their proportion in the total T cell population. While TCF1 and CD103 are both known to be engaged in the activation of CD8 + T cells, a recent study reported that TCF1 binds to the ITGAE locus of the gene encoding CD103 and partly inhibits the TGF-β induced expression of CD103 . This is consistent with a study on pediatric gliomas that reported that CD103 + and TCF1 + T cells are distinct subsets of tissue-resident memory T cells and show distinctly different spatial location patterns . Another study on GC also confirmed that TCF1 was expressed with higher levels in CD8+/CD103- T cells than in CD8+/CD103 + T cells . The low correlation between TCF1 and CD103 density (R 2 = 0.124) shown in Fig. of our study corresponds to previous studies. Contrary to previous studies on GC, in the present study, which included MSI-H GC, neither CD103 + nor CD8+/CD103 + T cells showed prognostic significance. This may be attributed to the neoantigen-rich nature of MSI-H GC. CD8+/CD103 + T cells are characterized not only by their high cytotoxicity but also by the expression of co-inhibitory receptors , . Moreover, IFN-γ, the immune molecule secreted by TILs when encountered by tumor antigens and what CD8+/CD103 + T cells secrete more than do CD8+/CD103- T cells in GC , , is reported to upregulate PD-L1 expression on tumor cells in the adaptive immune response system , . Therefore, we can infer that CD8+/CD103 + T cells may secrete a large amount of IFN‐γ in the neoantigen-rich tumoral environment MSI-H GC, inducing the upregulation of PD-L1 on tumor cells which in turn hinders the activation of PD-1-expressing T cells. Considering the frequent co-expression of PD-1 and CD103, the activation of CD8+/CD103 + T cells may also be compromised by the upregulation of PD-L1 on tumor cells. This agrees with previous studies suggesting CD103 as a useful biomarker for anti-PD-1 therapy, showing that CD8+/CD103 + T cells restore their function in the presence of PD-1 blockade , , , and that CD103 expression is weakly but significantly correlated with PD-L1 expression . The main limitations of the present study are that the study was conducted retrospectively and that we performed single and double IHC using tissue microarray (TMA) method. In addition, double IHC for TCF1 and CD103, which would have better confirmed the correlation between TCF1 + and CD103 + cells, was not performed in the present study. Single and double IHC were performed using different sections of the same TMA block, which may have been one of the possible causes of some discrepant results between single and double IHC. In conclusion, our study revealed that the proportion of CD8+/TCF1 + cells in the total CD8 + T cell population could be used as an independent favorable prognostic indicator in MSI-H GC, but the CD8+/CD103 + T cell population did not show a significant correlation with prognosis in MSI-H GC. Our results also suggested that evaluating the proportion of each T cell subset might be helpful in further assessing the characteristics of TIL and their association with the prognosis of MSI-H GC. Clinical sample selection and tissue array construction A total of 411 samples were collected from patients diagnosed with MSI-H GC. Of these, 293 were obtained from patients at Seoul National University Bundang Hospital between January 2007 and December 2013. The remaining 118 samples were obtained from patients admitted to Seoul National University Hospital between 2012 and 2014. This study was approved by the Institutional Review Board of Seoul National University Hospital (reference: H-2010-179-1171) and Seoul National University Bundang Hospital (reference: B-2401-879-103), and was performed in accordance with the Declaration of Helsinki for biomedical research involving human subjects. Informed consent was obtained from all the patients enrolled in the present study. Surgically resected GC specimens from 411 patients were fixed in 10% formalin and embedded in paraffin. In formalin-fixed and paraffin-embedded (FFPE) blocks, core tissue biopsies from representative areas (2 mm in diameter) of the tumor were obtained and rearranged into new TMA blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, South Korea) . One representative tissue core was taken from each case, and the representative areas were selected by an experienced board-certified pathologist (HSL). Of these 411 patients, 29 were excluded during IHC due to loss of cores, low numbers of cancer cells (less than 100), or inapplicability of double IHC. Consequently, 382 patients with MSI-H GC were analyzed in this study. Microsatellite instability assessment The MSI status was assessed at five loci (BAT25, BAT26, D2S123, D5S346, and D17S250) according to the National Cancer Institute panel guidelines. DNA was extracted from macrodissected FFPE tumor tissues and their respective paired FFPE normal tissues. After polymerase chain reaction (PCR) amplification, the resulting products were analyzed using the ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). GC was categorized into three groups based on the analysis: MSI-H, if two or more loci were unstable; MSI-L, if only one locus was unstable; and MSS, if no unstable loci were detected. Single IHC IHC for the classification of T cell subsets was performed on serial 4-µm-thick TMA sections using a Benchmark XT automated slide staining system (Ventana Medical System, Inc., Tucson, AZ, USA) according to the manufacturer’s protocol. For staining, CD8 (pre-diluted, DAKO Agilent Technologies, Inc., Santa Clara, CA, USA), TCF1 (1:30, Cell Signaling Technology Inc., Danvers, MA, USA), and CD103 (1:500, Abcam, Cambridge, UK) antibodies were used. Double IHC To confirm the presence of CD8+/TCF1 + and CD8+/CD103 + cells, double IHC with two antibodies was performed using DoubleStain IHC Kit (Abcam) according to a modified manufacturer’s protocol. After deparaffinization and dehydration, the slides were treated with a blocking reagent (Vector Labs, Newark, CA, US), and subsequently, antigen retrieval was performed on the slides in Tris/EDTA buffer (1:100, pH 9.0, Vector Labs) in a microwave for 15 min. For dual staining, an anti-CD8 monoclonal antibody (mAb) (1:100, Abcam) was applied for 1 h in a humid chamber at room temperature. After primary incubation, a mixture of anti-CD8 mAb (1:50, Abcam) and anti-TCF1 mAb (1:50, Cell Signaling Technology Inc.), or anti-CD8 mAb (1:50, Abcam) and anti-CD103 mAb (1:200, Abcam) was applied for 2 h in a humidified chamber at room temperature. Subsequently, following the manufacturer’s protocol, the polymer binding and substrate processing steps were performed using the solutions in the kit. Finally, counterstaining was performed using hematoxylin (1:40, Abcam). IHC image analysis The IHC slides were scanned using an Aperio AT2 (Leica Biosystems, Wetzlar, Germany) at 40x magnification. Representative images of single and double IHC staining are shown in Fig. . Images of the slides were analyzed using QuPath version 0.4.2 . For single IHC images, the detection and identification of the cells were performed based on the sum of the optical density and average DAB staining intensity within the cell, respectively. Cells were classified as positive or negative based on a single-intensity threshold. For double IHC images, cells were detected based on the optical density sum. The cells were identified based on two staining intensity criteria (CD8 and TCF1 or CD8 and CD103): the average emerald green staining intensity within the cell for CD8, the average permanent red staining within the nucleus for TCF1, and the average permanent red staining within the cell for CD103. By applying the two criteria simultaneously to a single image, each cell was classified into four subgroups: CD8-/TCF1-, CD8+/TCF1-, CD8-/TCF1+, and CD8+/TCF1 + or CD8-/CD103-, CD8+/CD103-, CD8-/CD103+, and CD8+/CD103+. Throughout the image analyses, the quantitative analysis included immune cells located within the tumor tissues (TIL), and the calculated mean number of positive cells per mm 2 , including the mean number of double-positive (CD8+/TCF1 + or CD8+/CD103+) cells, was obtained for further statistical analyses. The study population was dichotomized using various densities of positive cells (mean number of positive cells per mm 2 ) obtained from IHC image analysis. The third quartile (Q3) values were used as thresholds to dichotomize the populations. The criteria for dichotomy included CD8 + cell density, TCF1 + cell density, and CD103 + cell density based on the analysis results of single IHC; CD8+/TCF1 + cell density, CD8+/CD103 + cell density, CD8+/TCF1 + to CD8 + ratio, and CD8+/CD103 + to CD8 + ratio from the analysis results of double IHC. Statistical analysis Statistical analyses and graph preparation were performed using R software (version 4.2.2, R Foundation for Statistical Computing, Vienna, Austria). Associations between immune cell density and clinicopathologic parameters were assessed using the Wilcoxon rank-sum test and chi-square test, as appropriate. Kaplan–Meier curves and log-rank tests were used to compare the OS. For both univariable and multivariable analyses, hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using the Cox proportional hazards model. In the survival analysis using Kaplan-Meier curves, univariable analysis, and multivariable analysis, 380 cases were analyzed because follow-up data for two cases were unavailable. Statistical significance was set at P < 0.05. A total of 411 samples were collected from patients diagnosed with MSI-H GC. Of these, 293 were obtained from patients at Seoul National University Bundang Hospital between January 2007 and December 2013. The remaining 118 samples were obtained from patients admitted to Seoul National University Hospital between 2012 and 2014. This study was approved by the Institutional Review Board of Seoul National University Hospital (reference: H-2010-179-1171) and Seoul National University Bundang Hospital (reference: B-2401-879-103), and was performed in accordance with the Declaration of Helsinki for biomedical research involving human subjects. Informed consent was obtained from all the patients enrolled in the present study. Surgically resected GC specimens from 411 patients were fixed in 10% formalin and embedded in paraffin. In formalin-fixed and paraffin-embedded (FFPE) blocks, core tissue biopsies from representative areas (2 mm in diameter) of the tumor were obtained and rearranged into new TMA blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, South Korea) . One representative tissue core was taken from each case, and the representative areas were selected by an experienced board-certified pathologist (HSL). Of these 411 patients, 29 were excluded during IHC due to loss of cores, low numbers of cancer cells (less than 100), or inapplicability of double IHC. Consequently, 382 patients with MSI-H GC were analyzed in this study. The MSI status was assessed at five loci (BAT25, BAT26, D2S123, D5S346, and D17S250) according to the National Cancer Institute panel guidelines. DNA was extracted from macrodissected FFPE tumor tissues and their respective paired FFPE normal tissues. After polymerase chain reaction (PCR) amplification, the resulting products were analyzed using the ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). GC was categorized into three groups based on the analysis: MSI-H, if two or more loci were unstable; MSI-L, if only one locus was unstable; and MSS, if no unstable loci were detected. IHC for the classification of T cell subsets was performed on serial 4-µm-thick TMA sections using a Benchmark XT automated slide staining system (Ventana Medical System, Inc., Tucson, AZ, USA) according to the manufacturer’s protocol. For staining, CD8 (pre-diluted, DAKO Agilent Technologies, Inc., Santa Clara, CA, USA), TCF1 (1:30, Cell Signaling Technology Inc., Danvers, MA, USA), and CD103 (1:500, Abcam, Cambridge, UK) antibodies were used. To confirm the presence of CD8+/TCF1 + and CD8+/CD103 + cells, double IHC with two antibodies was performed using DoubleStain IHC Kit (Abcam) according to a modified manufacturer’s protocol. After deparaffinization and dehydration, the slides were treated with a blocking reagent (Vector Labs, Newark, CA, US), and subsequently, antigen retrieval was performed on the slides in Tris/EDTA buffer (1:100, pH 9.0, Vector Labs) in a microwave for 15 min. For dual staining, an anti-CD8 monoclonal antibody (mAb) (1:100, Abcam) was applied for 1 h in a humid chamber at room temperature. After primary incubation, a mixture of anti-CD8 mAb (1:50, Abcam) and anti-TCF1 mAb (1:50, Cell Signaling Technology Inc.), or anti-CD8 mAb (1:50, Abcam) and anti-CD103 mAb (1:200, Abcam) was applied for 2 h in a humidified chamber at room temperature. Subsequently, following the manufacturer’s protocol, the polymer binding and substrate processing steps were performed using the solutions in the kit. Finally, counterstaining was performed using hematoxylin (1:40, Abcam). The IHC slides were scanned using an Aperio AT2 (Leica Biosystems, Wetzlar, Germany) at 40x magnification. Representative images of single and double IHC staining are shown in Fig. . Images of the slides were analyzed using QuPath version 0.4.2 . For single IHC images, the detection and identification of the cells were performed based on the sum of the optical density and average DAB staining intensity within the cell, respectively. Cells were classified as positive or negative based on a single-intensity threshold. For double IHC images, cells were detected based on the optical density sum. The cells were identified based on two staining intensity criteria (CD8 and TCF1 or CD8 and CD103): the average emerald green staining intensity within the cell for CD8, the average permanent red staining within the nucleus for TCF1, and the average permanent red staining within the cell for CD103. By applying the two criteria simultaneously to a single image, each cell was classified into four subgroups: CD8-/TCF1-, CD8+/TCF1-, CD8-/TCF1+, and CD8+/TCF1 + or CD8-/CD103-, CD8+/CD103-, CD8-/CD103+, and CD8+/CD103+. Throughout the image analyses, the quantitative analysis included immune cells located within the tumor tissues (TIL), and the calculated mean number of positive cells per mm 2 , including the mean number of double-positive (CD8+/TCF1 + or CD8+/CD103+) cells, was obtained for further statistical analyses. The study population was dichotomized using various densities of positive cells (mean number of positive cells per mm 2 ) obtained from IHC image analysis. The third quartile (Q3) values were used as thresholds to dichotomize the populations. The criteria for dichotomy included CD8 + cell density, TCF1 + cell density, and CD103 + cell density based on the analysis results of single IHC; CD8+/TCF1 + cell density, CD8+/CD103 + cell density, CD8+/TCF1 + to CD8 + ratio, and CD8+/CD103 + to CD8 + ratio from the analysis results of double IHC. Statistical analyses and graph preparation were performed using R software (version 4.2.2, R Foundation for Statistical Computing, Vienna, Austria). Associations between immune cell density and clinicopathologic parameters were assessed using the Wilcoxon rank-sum test and chi-square test, as appropriate. Kaplan–Meier curves and log-rank tests were used to compare the OS. For both univariable and multivariable analyses, hazard ratios (HRs) and 95% confidence intervals (CIs) were calculated using the Cox proportional hazards model. In the survival analysis using Kaplan-Meier curves, univariable analysis, and multivariable analysis, 380 cases were analyzed because follow-up data for two cases were unavailable. Statistical significance was set at P < 0.05. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Marginal adaptation analysis of CAD/CAM resin crown with non-invasive methods | 9526047f-5868-486c-8842-0717eefd787c | 11861017 | Dentistry[mh] | In the rapidly advancing field of modern dentistry, CAD/CAM technology has significantly enhanced aesthetic restorations . This innovative technique not only meets patients’ high aesthetic expectations but also offers superior light transmission, closely mimicking the color and translucency of natural teeth . The success of these restorations depends critically on their marginal adaptations. Poor adaptation can lead to a cascade of issues, including cement deterioration, microleakage, tissue inflammation, recurrent decay, and ultimately, restoration failure . Prior research has outlined several approaches for measuring marginal adaptation, including the direct-view technique, cross-sectioning technique, replica method, profile projection, digital impression, and microcomputed tomography . The silicone replica method is one of the techniques most commonly used for this purpose. The replica method produces a silicone duplicate of the space between the dental prosthesis and the prepared tooth . The replica was then dissected and examined under a microscope to evaluate the prosthesis alignment and precision. Optical coherence tomography (OCT) is a noninvasive imaging method that utilizes light reflection to generate high-resolution cross-sectional images of internal structures . Swept-source OCT (SS-OCT), an advanced version, employs a wavelength-tuned laser source and offers enhanced resolution and faster scanning . OCT operates based on low-coherence interferometry principles and provides detailed depth information . This technique has been applied in various dental diagnostics including caries and periodontal tissues . In addition, literature indicates that OCT can be used to assess composite restorations, adhesives and indirect restorations . The accuracy of the OCT images can vary depending on the angle of the sample relative to the light source, as the vertical length changes with different cross-sectional angles . Therefore, it is crucial to investigate the precision of the OCT images when the samples are positioned at various angles relative to the OCT laser light source. Numerous factors affect the marginal adaptation of CAD/CAM restorations, including margin design, die spacer thickness, cement type, and cementation method employed . In this study, marginal adaptation was defined as the distance between the outermost points of the abutment and the crown. McLean et al. reported a maximum clinically acceptable marginal gap of 120 𝓊m, while Boening et al. described a clinically acceptable marginal gap of 100 𝓊m to 200 𝓊m. Both the distance between the OCT’s near-infrared (NIR) light source and the incident angle of the light upon the specimen are statistically significantly related to the OCT images . Increasing the angle of incidence lengthens the sample’s light propagation path, reducing imaging resolution. Geometric distortion occurs when the incidence angle changes the image’s transverse-to-longitudinal ratio . This study aimed to compare the silicone replica method with the SS-OCT method for analyzing marginal adaptation and to investigate the impact of the SS-OCT light incidence angle on this analysis. The null hypothesis was that the SS-OCT method would not differ from the silicone replica method in measuring marginal adaptation and that the angle of light incidence in SS-OCT would not affect the analysis of marginal adaptation in CAD/CAM-fabricated resin crowns. Schematic illustration for this study is presented in Fig. . Specimen preparation A plastic mandibular right first molar die for a jacket crown (A55A-461, Nissin, Kyoto, Japan) was mounted on a typodont standard model (500 H-1 M, Nissin, Kyoto, Japan) and scanned using an intraoral scanner (Trios 3, 3Shape, Copenhagen, Denmark). The crown shape was then designed using CAD software (S-WAVE Dental System, Shofu, Kyoto, Japan) by selecting an appropriate library template, with the cement space set at 150 μm for the occlusal surface and axial wall, and 50 μm for the margin. Fourteen crowns were fabricated using CAD/CAM resin blocks (Katana Avencia P Block, Kuraray Noritake Dental, Tokyo, Japan) and milled using a 5-axis milling machine (Dental Mill, DWX-50, Roland, Osaka, Japan) according to the manufacturer’s specifications. Measurement of marginal adaptation using silicone replica method Each crown was filled with light-body occlusal contact and fit-checking material (GC Blue Silicone, GC, Tokyo, Japan) and then placed onto the abutment die. A 2 kg weight was applied from the occlusal direction for 5 min. Once the light-body silicone was set, the resin crown was removed, leaving a thin film of light-body silicone on the die. Heavy-body hydrophilic vinyl polysiloxane (Exafine Regular Hard Type, GC, Tokyo, Japan) was then applied over the light-body silicone film as an outer layer. After the heavy-body vinyl polysiloxane had set, the two-layer replica was removed from the die, and the heavy-body vinyl polysiloxane was placed into the intaglio surface of the two-layer replica until it set. The three-layered silicone replica was segmented buccolingually and mesiodistally by using a razor blade (FAS-10; Feather, Osaka, Japan). Each segment of the silicone replica was photographed using an optical microscope (SMZ1000, Nikon, Tokyo, Japan) at 28x magnification to obtain cross-sectional images of the buccal, lingual, mesial, and distal points (Fig. ). The buccolingual and mesiodistal cutting lines were passed through the midpoint of the die’s occlusal surface and were perpendicular to each other, bisecting the buccal, mesial, distal, and lingual sides. SS-OCT Observation The SS-OCT system (IVS-2000, Santec, Aichi, Japan) utilized a central wavelength ranging from 1,260 nm to 1,360 nm, centered at 1,310 nm, and generated NIR light at a sweep rate of 20 kHz. In the air medium, the device achieved a depth resolution of less than 7 μm, while the axial and lateral resolutions were 11 μm and 17 μm, respectively. The probe connected to the interferometer had a power of 5mW, which was within the safety limit of the American Standard Institute. The laser source emitted from the probe was directed onto the sample at the desired location in the X and Z dimensions. The backscattered light carrying information from each single scan point of the sample was returned to the system, digitized on a time scale, and analyzed in the Fourier domain to disclose the depth information (A-scan) of the sample. The A-line and 2D frame A-scan rates are 20,000 lines/second and 260 lines/frame. By combining a series of A-scans in a linear fashion across the sample, a cross-section (B-scan) was obtained. Finally, cross-sectional images could be created by converting the B-scan raw data into a greyscale image with 2001 × 1019 pixels . The crown and abutment dies were observed in an uncemented state. The crown was connected to the abutment die using a small amount of clear vinyl polysiloxane (Exaclear, GC), ensuring that the material did not encroach the observation area while still providing stability to the crown and abutment die. Wedge-shaped supports with inclinations of 15° and 30° were used to vary the light incidence angle. The handheld scanning probe fixed on the OCT device was 5 cm away from the specimen and aligned perpendicularly, and the probe could only be moved in the vertical direction during scanning . Subsequently, all samples were observed using the SS-OCT system with a scanning range of 8 × 8 mm from the buccal, lingual, mesial, and distal sides at three different light incidence angles: 60°, 75°, and 90° (Fig. ), at an intensity of − 10 dB and an A-scan average of one. A tailored pedestal was employed to support the abutment die and to denote the orientation for the different cross sections, short lines were drawn with a marker pen in all four directions of the abutment die in accordance with the orientation indicated on the pedestal. Each sample was moved in a mid-range direction across the laser beam until it aligned with the short line marker on the abutment die, at which point the clearest cross-sectional B-scan image was selected and captured from a series of real-time B-scan previews. Image processing All two-dimensional (2D) SS-OCT images were saved in CSV format at a pixel size of 16,000 × 7,481 μm. ImageJ (version 13.0.6, National Institutes of Health, Bethesda, MD, USA) was used to analyze all microscopic and 2D SS-OCT images. Marginal discrepancy was defined as the distance between the outermost points of the abutment and restoration (Fig. ). Statistical analysis The number of CAD/CAM resin crown ( n = 14) were determined by data of the pilot study with statistical software (G*power 3.1.9.2) and the α error and statistical power (1-β error) were set to 0.05 and 0.8, respectively. After the normality and variance of the SS-OCT data were confirmed using the Shapiro-Wilk test and Levene’s test, respectively, two-way ANOVA and t-tests with Bonferroni correction were applied. Subsequently, comparisons between the replica method and SS-OCT at each degree and position were conducted using t-tests. Statistical analyses were conducted at a significance level of 0.05, using statistical software (SPSS ver. 27.0 for Windows, IBM, Chicago, IL, USA). A plastic mandibular right first molar die for a jacket crown (A55A-461, Nissin, Kyoto, Japan) was mounted on a typodont standard model (500 H-1 M, Nissin, Kyoto, Japan) and scanned using an intraoral scanner (Trios 3, 3Shape, Copenhagen, Denmark). The crown shape was then designed using CAD software (S-WAVE Dental System, Shofu, Kyoto, Japan) by selecting an appropriate library template, with the cement space set at 150 μm for the occlusal surface and axial wall, and 50 μm for the margin. Fourteen crowns were fabricated using CAD/CAM resin blocks (Katana Avencia P Block, Kuraray Noritake Dental, Tokyo, Japan) and milled using a 5-axis milling machine (Dental Mill, DWX-50, Roland, Osaka, Japan) according to the manufacturer’s specifications. Each crown was filled with light-body occlusal contact and fit-checking material (GC Blue Silicone, GC, Tokyo, Japan) and then placed onto the abutment die. A 2 kg weight was applied from the occlusal direction for 5 min. Once the light-body silicone was set, the resin crown was removed, leaving a thin film of light-body silicone on the die. Heavy-body hydrophilic vinyl polysiloxane (Exafine Regular Hard Type, GC, Tokyo, Japan) was then applied over the light-body silicone film as an outer layer. After the heavy-body vinyl polysiloxane had set, the two-layer replica was removed from the die, and the heavy-body vinyl polysiloxane was placed into the intaglio surface of the two-layer replica until it set. The three-layered silicone replica was segmented buccolingually and mesiodistally by using a razor blade (FAS-10; Feather, Osaka, Japan). Each segment of the silicone replica was photographed using an optical microscope (SMZ1000, Nikon, Tokyo, Japan) at 28x magnification to obtain cross-sectional images of the buccal, lingual, mesial, and distal points (Fig. ). The buccolingual and mesiodistal cutting lines were passed through the midpoint of the die’s occlusal surface and were perpendicular to each other, bisecting the buccal, mesial, distal, and lingual sides. The SS-OCT system (IVS-2000, Santec, Aichi, Japan) utilized a central wavelength ranging from 1,260 nm to 1,360 nm, centered at 1,310 nm, and generated NIR light at a sweep rate of 20 kHz. In the air medium, the device achieved a depth resolution of less than 7 μm, while the axial and lateral resolutions were 11 μm and 17 μm, respectively. The probe connected to the interferometer had a power of 5mW, which was within the safety limit of the American Standard Institute. The laser source emitted from the probe was directed onto the sample at the desired location in the X and Z dimensions. The backscattered light carrying information from each single scan point of the sample was returned to the system, digitized on a time scale, and analyzed in the Fourier domain to disclose the depth information (A-scan) of the sample. The A-line and 2D frame A-scan rates are 20,000 lines/second and 260 lines/frame. By combining a series of A-scans in a linear fashion across the sample, a cross-section (B-scan) was obtained. Finally, cross-sectional images could be created by converting the B-scan raw data into a greyscale image with 2001 × 1019 pixels . The crown and abutment dies were observed in an uncemented state. The crown was connected to the abutment die using a small amount of clear vinyl polysiloxane (Exaclear, GC), ensuring that the material did not encroach the observation area while still providing stability to the crown and abutment die. Wedge-shaped supports with inclinations of 15° and 30° were used to vary the light incidence angle. The handheld scanning probe fixed on the OCT device was 5 cm away from the specimen and aligned perpendicularly, and the probe could only be moved in the vertical direction during scanning . Subsequently, all samples were observed using the SS-OCT system with a scanning range of 8 × 8 mm from the buccal, lingual, mesial, and distal sides at three different light incidence angles: 60°, 75°, and 90° (Fig. ), at an intensity of − 10 dB and an A-scan average of one. A tailored pedestal was employed to support the abutment die and to denote the orientation for the different cross sections, short lines were drawn with a marker pen in all four directions of the abutment die in accordance with the orientation indicated on the pedestal. Each sample was moved in a mid-range direction across the laser beam until it aligned with the short line marker on the abutment die, at which point the clearest cross-sectional B-scan image was selected and captured from a series of real-time B-scan previews. All two-dimensional (2D) SS-OCT images were saved in CSV format at a pixel size of 16,000 × 7,481 μm. ImageJ (version 13.0.6, National Institutes of Health, Bethesda, MD, USA) was used to analyze all microscopic and 2D SS-OCT images. Marginal discrepancy was defined as the distance between the outermost points of the abutment and restoration (Fig. ). The number of CAD/CAM resin crown ( n = 14) were determined by data of the pilot study with statistical software (G*power 3.1.9.2) and the α error and statistical power (1-β error) were set to 0.05 and 0.8, respectively. After the normality and variance of the SS-OCT data were confirmed using the Shapiro-Wilk test and Levene’s test, respectively, two-way ANOVA and t-tests with Bonferroni correction were applied. Subsequently, comparisons between the replica method and SS-OCT at each degree and position were conducted using t-tests. Statistical analyses were conducted at a significance level of 0.05, using statistical software (SPSS ver. 27.0 for Windows, IBM, Chicago, IL, USA). Figure shows the buccal, lingual, mesial, and distal images of the silicone replica and SS-OCT methods with 60°, 75°, and 90° light incidences. Table shows the results of the marginal discrepancy between the silicone replica and SS-OCT methods with 60°, 75°, and 90° light incidence. On 60° SS-OCT, the marginal discrepancy values were significantly larger than those on the silicone replica method at the buccal, lingual, and mesial points ( p < 0.05). On 75° SS-OCT, only the lingual point showed significantly larger values than the silicone replica method ( p < 0.05); the other three points showed no differences ( p > 0.05). For 90° SS-OCT, there were no statistically significant differences between the silicone replica and SS-OCT methods at any point ( p > 0.05). At the buccal, lingual, mesial, and distal points, the SS-OCT values tended to increase as the angle changed from 90° to 75° to 60°. At the buccal and lingual points, there were significant differences in the marginal discrepancy data between 60° and 75°, and between 60° and 90° on SS-OCT ( p < 0.05). When comparing the four points (buccal, lingual, mesial, and distal) at 60°, 75°, and 90° of SS-OCT, there were no significant differences among the four points at 75° and 90° ( p > 0.05). At 60° SS-OCT, while there were no significant differences between buccal and lingual and between mesial and distal ( p > 0.05), significant differences were found in all other combinations ( p < 0.05). The purpose of this experiment was to evaluate the performance of OCT in measuring the marginal adaptation of indirect restorations. The film thickness and marginal adaptation significantly affect the longevity and durability of indirect restorations . Inadequate marginal fit and gaps in the crown leads to cement dissolution, microbial colonization, deposition of dental biofilms, gingival inflammation, recurrent caries, increased periodontal pockets, and bone loss . Kokubo et al. emphasize that clinicians must be aware that the position of the crown and the type of restoration will affect a considerable range of marginal fit. A proper marginal fit ensures long-term results of an indirect restoration’s length of service and persistence. In clinical situations, it is difficult to directly determine the film thickness of indirect restorations. OCT can noninvasively create cross-sectional images of biomaterials and organs using light. This non-ionizing imaging method has shown significant potential in dentistry, with applications ranging from the mineral content estimation of hard-tooth structures to incomplete crown cracks . The backscattered light intensity was recorded based on the depth (axial position) of the tissue. Low-coherence interferometry is employed to capture the intensity of backscattered light, providing positional information with a resolution of up to 20 microns . Each substance, whether solid, liquid, or gas, has a refractive index. Successful imaging relies on the incident light reflected by SS-OCT between two media with different refractive indices . When the refractive index of an object is greater than one, it appears enlarged in the direction of the NIR light incidence in an SS-OCT cross-sectional image . However, when investigating the marginal adaptation, the measurement points were located at the edges of the abutment tooth and restoration. Consequently, the refractive index had a smaller impact on these specific points. In SS-OCT observations, the scanning angle relative to the sample influences the length and width of the resulting image . In clinical situations, when the anatomical shape of the tooth prevents the perpendicular NIR light, an image is captured at an oblique angle. When using SS-OCT with an incident angle of < 90°, the NIR light must travel a longer path to pass through the edge of the resin crown. This increased distance also makes it more challenging to obtain a clear image because the edge of the resin crown is farther from the light source than at a 90° angle. The anatomical shape of the tooth and length of the restoration contour also affected the SS-OCT image. The abutment die used in the experiment was a right mandibular first molar, accompanied by 14 CAD/CAM fabricated resin crowns; each sample was scanned from four directions. The resin crowns were similar in shape to natural teeth, with the mesial and distal sides having a shorter and straighter occlusal-cervical profile than the buccal and lingual sides (Fig. ). The angle between the incident light and surface of the object affects the direction of light refraction. The distance that the NIR light must travel is greater when passing through the edge of a restoration at an obtuse angle than when passing through the edge of a restoration at an acute angle. This implies that the distance between the outermost point of the restoration and the outermost point of the abutment increases . As a result, the buccal and lingual sides on 60° SS-OCT showed larger values than those obtained using the silicone replica method and 75° and 90° SS-OCT in this experiment. However, there was no statistically significant difference between the results of the SS-OCT and silicone replica methods when the incident light was perpendicular to the sample surface. This finding underscores the distinctive efficacy of the 90° incidence of the SS-OCT method, as it yielded outcomes that surpassed those of the other SS-OCT groups and attained an accuracy level comparable to that of the silicone replica method (Table ). The null hypothesis was partially rejected based on the points discussed above. The mean values of marginal adaptation in this study were within the acceptable range, except for the buccal and lingual values at a 60° angle of incidence in SS-OCT observation. The angle of incidence of the NIR light significantly affected the measurements. In the clinical setting, determining the tooth axis and probe tilt is crucial for accurate marginal adaptation assessment using OCT. As noted by Kikuchi et al. , it would be desirable to have software that can correct the measured length based on the angle of incidence of NIR light, which can be used to improve the measurement accuracy, particularly for teeth with large tilt angles. This feature should be considered in clinical OCT systems. In this in vitro experiment, the SS-OCT probe was temporarily immobilized above a horizontal sample stage that could be moved up and down, left and right, and forward and backward. The specimen loaded in the pedestal is placed on the sample stage for observation. The customized pedestal of the abutment die ensures that the NIR light is perpendicular to the specimen’s surface, and the short line marker ensures no translation or rotation of the specimen while observing the four target cross-sections. In addition, the method of using feature point matching for image correction in oral digital impressions has potential to help correct OCT data affected by incidence angle differences. Although the silicone replica method is widely used, it has several limitations in assessing marginal adaptation . It relies on two-dimensional (2D) analysis with limited measurement points, typically only two to four per sample . Thin silicone films may deform or tear during removal, affecting the measurement accuracy . Moreover, the silicone material type and measurement process can affect the method’s accuracy . Currently, there are no absolute methods for measuring marginal discrepancies in restorations. The lack of standardization makes inter-study comparisons difficult . In this study, the results of the marginal discrepancy measurements were consistent between the replica and SS-OCT methods at 90° incidence, both of which are recognized as noninvasive techniques. These methods are promising and effective tools for detecting marginal adaptation between crowns and abutment teeth. Based on the results of this experiment, the following conclusions can be drawn: The SS-OCT method can serve as a viable alternative to the silicone replica method for assessing marginal adaptation when the observation incidence is set at 90° towards the CAD/CAM resin crown. However, it is important to note that the angle of near-infrared light incidence significantly affects the precision of the marginal adaptation analysis. Furthermore, the marginal discrepancy in the CAD/CAM resin crowns demonstrated good consistency across the four observed positions. |
Learning curves of novice residents on cataract surgery simulator: the E3CAPS pedagogic study | ae3c9921-5084-48ae-be59-8dc1a5bbeec6 | 11443795 | Ophthalmology[mh] | Cataract surgery is the most frequent surgical procedure worldwide: approximately 1 million procedures are performed in France each year and about 80 million people have moderate or severe distance vision impairment or blindness due to cataract . This procedure under operating microscope demands a high level of expertise due to the risk of surgical complications, notably rupture of the posterior capsule, which is the main vision-threatening intraoperative complication . In fact, the incidence of posterior capsular rupture is higher in patients operated on by ophthalmology residents (5 to 19%) than by experienced surgeons (0.5 to 3.5%) . While these complication rates seem relatively minor, they finally impact many patients. This partly explains the difficulties in accessing real-life surgical training. In two surveys involving hundreds of residents from more than 30 European countries, 42% were completely dissatisfied with the surgical skills achieved and more than 25% had not undergone any live cataract surgery training by the end of residency . Many residents must therefore continue their training after the end of their residency. Alternative teaching methods for cataract surgery are: theoretical learning, observation of surgeons in real life and manipulation of porcine eyes or artificial eyes. Rehearsing surgical steps on porcine eyes or artificial eyes is useful but insufficient because of significant differences in tissue thickness and texture and resistance compared to human eye. In France, cataract surgery is taught throughout the 4-year residency, in successive stages: theoretical training, practical work on pig’s eyes, artificial eyes and virtual simulator, then gradual training in real life one surgical step at a time, under the supervision of a senior surgeon. Full surgeries are then performed under supervision once each step has been mastered individually, and unsupervised surgeries are possible after the end of the 4-year residency. Using cataract surgery simulators is a valuable approach to moderate the risks associated with training and support the “never the first time on a patient” principle set by the French National Health Agency (ANSM) . The EyeSi ® surgical simulator (VRMagic, Mannheim, Germany) holds the highest usage rate in France: it enables to virtually execute the majority of the steps involved in cataract surgery, adjusting the complexity level and providing the student with both qualitative and quantitative evaluations for each surgical step. The benefits of using the EyeSi ® in resident training have been shown: the score achieved on the simulator correlates with various real-life surgical skill scores; the simulator distinguishes between skilled and novice operators, enhances surgical skill scores of both novice and experienced surgeons and decreases the occurrence of surgical complications . However, access to the simulator is hampered by many factors: expensive and limited equipment, travel time and cost, limited availability of the residents and teachers also taken up by other essential learning activities . Organizing rigorous simulation training in all residency training centers before the first surgery on a patient is needed but challenging: determining the minimum number of training sessions is needed to lay the foundations of an effective and realistic resident training program. In France, the “Collège des Ophtalmologistes Universitaires de France” (COUF) recommends undergoing 4 simulation sessions, each lasting 2 h, during the initial year of residency. The aim of the present study is to evaluate the relevance of the French training program through analysis of the learning curve of novice ophthalmology residents undergoing 4 simulation sessions. We hypothesize that it will improve the training program by determining whether additional training sessions are needed and which surgical steps are the most difficult for students to master. Design and intervention This prospective interventional study corresponds to Axis 1 of the E3CAPS pedagogic study (ClinicalTrials registration number: NCT05722080 (first submitted 22/12/2022, first posted 10/02/2023). The methodology of the study and the technical specifications of the EyeSi simulator have been previously described in detail . Briefly, The EyeSi surgical simulator is a high-fidelity virtual reality simulator that includes a platform on which a patient’s head dummy is placed. The resident inserts two handpieces into a digital box positioned in place of the eye and uses a realistic two-axis phaco foot pedal to interact with the virtual operating field created by stereoscopy via operating microscope eyepieces placed above the platform. This installation provides a highly immersive reproduction of real-life conditions. Training modules include navigation training exercises and most steps of cataract surgery: capsulorhexis, hydromaneuver, phacoemulsification, irrigation and aspiration, intraocular lens (IOL) insertion and posterior capsular rupture management. Throughout the simulation, a digital interface provides real-time feedback and assigns positive points for completed tasks and negative points for errors, inappropriate or dangerous gestures, eye tissue injury or eye shift. Finally, the simulator provides a score for each attempt. Each resident completed four two-hour training sessions over a period of 4 consecutive days under the supervision of an experienced cataract surgery educator. To increase the session’s relevance and manage resident fatigue, each two-hour session was divided into four 30-minute training periods, followed by four 30-minute recovery or observation periods, alternating with another resident. To measure the learning curve, each resident performed a standardized assessment at the beginning of the first session (A0), then repeated at the end of the first (A1), second (A2), third (A3) and fourth (A4) sessions. Additional assessments of the capsulorhexis step alone were performed at the beginning of the second (pre-A2), third (pre-A3) and fourth (pre-A4) sessions: this served to assess any loss since the end of the previous session. To avoid inducing fatigue or reducing the length of the training session, the capsulorhexis step was chosen because it is short, difficult to master and responsible for critical surgical complications. The following data were collected for each resident: age, sex and dominant hand. To account for any population heterogeneity, real-life surgical practice experience was assessed using the number of attempts of any task performed in the operating room (incision, capsulorhexis, hydromaneuver, phaco-emulsification, irrigation and aspiration, IOL insertion, viscous removal, stromal hydratation for incision closure). This clinical study was approved by the Ethics Committee of Nantes University on October 17th, 2022. The clinical study was conducted in accordance with the French Public Health Code, national and international Good Clinical Practice (GCP) guidelines, and the Declaration of Helsinki, each in the applicable version. Participant and sample size Given the exploratory nature of this study, the sample selected was a convenience sample, represented by the 16 newly nominated ophthalmology residents in the participating University Hospitals of Nantes, Tours, Angers and Rennes. All study participants provided written informed consent to participate. The only exclusion criterion was the absence of written consent. Outcomes A standardized assessment took approximately 20–30 min and consisted of the following exercises: capsulorhexis level 1, hydromaneuver level 2, phacoemulsification divide and conquer level 6, irrigation and aspiration level 3 and IOL insertion level 3. Exercises were chosen and placed in the correct order to simulate a complete standard cataract surgery. For each attempt, the following data were collected: EyeSi score (ranging from 0 to 100), distance traveled in the eye by the instruments (odometer in millimeters), completion time, posterior capsular rupture (during phacoemulsification and irrigation and aspiration) and amount of energy delivered by ultrasounds (during phacoemulsification). To ensure that the same instructions were given to all residents, standardized instructions were given orally by a Senior Surgeon Investigator (JBD) based on a written document. Assessments were performed without any student present in the room to avoid competition bias. The results of each attempt remained anonymous and confidential. They were given privately to each resident after the completion of the Axis 1 study. Data analysis Quantitative variables were described by their median and interquartile range (IQR). For categorical variables, the frequencies and percentages for each modality were displayed. A learning curve was derived from the plot of the score against the number of sessions. The percentage of residents reaching the performance threshold (success defined as: 80/100 for each surgical step, 400/500 for the overall procedure) at the end of each attempt and its 95% confidence interval were estimated. To assess the impact of previous real-life surgical practice experience, overall scores of residents with more than 10 real-life attempts of any surgical step were compared to the scores of residents with strictly less than 10 at A0 and A4 (Wilcoxon rank-sum test for independent data). An alpha risk of 5% was used. Statistical analyses were performed using R (version 4.0.2) . Figures were drawn using the ggplot2 package . This prospective interventional study corresponds to Axis 1 of the E3CAPS pedagogic study (ClinicalTrials registration number: NCT05722080 (first submitted 22/12/2022, first posted 10/02/2023). The methodology of the study and the technical specifications of the EyeSi simulator have been previously described in detail . Briefly, The EyeSi surgical simulator is a high-fidelity virtual reality simulator that includes a platform on which a patient’s head dummy is placed. The resident inserts two handpieces into a digital box positioned in place of the eye and uses a realistic two-axis phaco foot pedal to interact with the virtual operating field created by stereoscopy via operating microscope eyepieces placed above the platform. This installation provides a highly immersive reproduction of real-life conditions. Training modules include navigation training exercises and most steps of cataract surgery: capsulorhexis, hydromaneuver, phacoemulsification, irrigation and aspiration, intraocular lens (IOL) insertion and posterior capsular rupture management. Throughout the simulation, a digital interface provides real-time feedback and assigns positive points for completed tasks and negative points for errors, inappropriate or dangerous gestures, eye tissue injury or eye shift. Finally, the simulator provides a score for each attempt. Each resident completed four two-hour training sessions over a period of 4 consecutive days under the supervision of an experienced cataract surgery educator. To increase the session’s relevance and manage resident fatigue, each two-hour session was divided into four 30-minute training periods, followed by four 30-minute recovery or observation periods, alternating with another resident. To measure the learning curve, each resident performed a standardized assessment at the beginning of the first session (A0), then repeated at the end of the first (A1), second (A2), third (A3) and fourth (A4) sessions. Additional assessments of the capsulorhexis step alone were performed at the beginning of the second (pre-A2), third (pre-A3) and fourth (pre-A4) sessions: this served to assess any loss since the end of the previous session. To avoid inducing fatigue or reducing the length of the training session, the capsulorhexis step was chosen because it is short, difficult to master and responsible for critical surgical complications. The following data were collected for each resident: age, sex and dominant hand. To account for any population heterogeneity, real-life surgical practice experience was assessed using the number of attempts of any task performed in the operating room (incision, capsulorhexis, hydromaneuver, phaco-emulsification, irrigation and aspiration, IOL insertion, viscous removal, stromal hydratation for incision closure). This clinical study was approved by the Ethics Committee of Nantes University on October 17th, 2022. The clinical study was conducted in accordance with the French Public Health Code, national and international Good Clinical Practice (GCP) guidelines, and the Declaration of Helsinki, each in the applicable version. Given the exploratory nature of this study, the sample selected was a convenience sample, represented by the 16 newly nominated ophthalmology residents in the participating University Hospitals of Nantes, Tours, Angers and Rennes. All study participants provided written informed consent to participate. The only exclusion criterion was the absence of written consent. A standardized assessment took approximately 20–30 min and consisted of the following exercises: capsulorhexis level 1, hydromaneuver level 2, phacoemulsification divide and conquer level 6, irrigation and aspiration level 3 and IOL insertion level 3. Exercises were chosen and placed in the correct order to simulate a complete standard cataract surgery. For each attempt, the following data were collected: EyeSi score (ranging from 0 to 100), distance traveled in the eye by the instruments (odometer in millimeters), completion time, posterior capsular rupture (during phacoemulsification and irrigation and aspiration) and amount of energy delivered by ultrasounds (during phacoemulsification). To ensure that the same instructions were given to all residents, standardized instructions were given orally by a Senior Surgeon Investigator (JBD) based on a written document. Assessments were performed without any student present in the room to avoid competition bias. The results of each attempt remained anonymous and confidential. They were given privately to each resident after the completion of the Axis 1 study. Quantitative variables were described by their median and interquartile range (IQR). For categorical variables, the frequencies and percentages for each modality were displayed. A learning curve was derived from the plot of the score against the number of sessions. The percentage of residents reaching the performance threshold (success defined as: 80/100 for each surgical step, 400/500 for the overall procedure) at the end of each attempt and its 95% confidence interval were estimated. To assess the impact of previous real-life surgical practice experience, overall scores of residents with more than 10 real-life attempts of any surgical step were compared to the scores of residents with strictly less than 10 at A0 and A4 (Wilcoxon rank-sum test for independent data). An alpha risk of 5% was used. Statistical analyses were performed using R (version 4.0.2) . Figures were drawn using the ggplot2 package . Study population All sixteen newly appointed ophthalmology residents in November 2022 were included during their third month of residency. The first resident was enrolled on January 9th, 2023, and the last resident ended their training program on January 30th, 2023. Residents’ median age was 25.5 [25.0; 26.0] and the proportion of women was 44% (Table ). Surgical practice experience was low: fifteen (94%) had never performed real-life corneal incisions, capsulorhexis, hydromaneuver and phacoemulsification. Some had made a few attempts at irrigation and aspiration (4/16, 25%) and IOL insertion (6/16, 37%). EyeSi scores The multidimensional curves of overall progression show a linear increase in score and decrease in completion time and odometer, associated with a decrease in variance without a plateau (Fig. ). Throughout the training sessions, the median score increased progressively from 95 [IQR 53; 147] at A0 to 425 [411; 451] at A4 (E-Table 1). No significant difference in overall score was observed between residents who had more than 10 real-life attempts of any surgical step and residents with fewer than 10 at A0 (100 [IQR 86.5; 152] versus 69 [IQR 39; 141] respectively, p = 0.60) and A4 (445 [IQR 400; 454] versus 422 [IQR 418; 444] respectively, p = 0.63). The “capsulorhexis” step showed a linear increase in score with reduction in variance without reaching a plateau. Completion time and odometer decreased linearly without a plateau or reduction in variance. Additional “capsulorhexis” assessments at the beginning of each training session revealed high inter-individual and intra-individual variability during the first 3 sessions with a tendency to decrease compared with the previous session (E-Figure 1). The “hydromaneuver” step had the least valid learning curve (E-Figure 2). The score at the first assessment before any training (A0) was unusually high and represented the only step for which the median A0 score was higher than 0 (36 [0; 75]). The scores for the “emulsification” step were the lowest with a lower interquartile < 80 (86 [60; 94] at A4. While the increase in score for the “emulsification” step was linear, it did not reach a plateau and was not associated with a reduction in completion time and odometer of ultrasounds delivered. The learning curves of the “irrigation and aspiration” step had a negative exponential shape and reached a plateau with the highest decrease in variance. This was the step for which the residents had the highest final scores (median 98 [96; 100]). The “IOL insertion” step showed a linear increase in score with reduction in variance without reaching a plateau. Completion time and odometer did not decrease throughout the training program (E-Figure 2). Success rates The overall average success rate progressively increased throughout the training sessions from 0.0% [0.0; 24.1] at A0 to 75.0% [47.4; 91.7] at A4 (Fig. and E-Table 2). Although the residents’ scores progressed for the 5 steps, progression was found to be heterogeneous: slow for the “emulsification” step for which the success rate at A4 was 56.2% [30.6; 79.2] and rapid for “irrigation and aspiration”, for which the success rate was 87.5% [60.4; 97.8] at A3 and 93.8% [67.7–99.7] at A4. Moreover, the percentage of residents who consecutively succeeded each of the 5 steps at A4 was 25.0% [8.3; 52.6]. Posterior capsular rupture The average rates of posterior capsular rupture decreased from 75% (12/16) at A0 to 12% (2/16) at A4 during the “emulsification” step and from 69% (11/16) at A0 to 0% at A4 during the “irrigation and aspiration” step (E-Table 1). The decrease was linear and continuous throughout the training program (Fig. ). All sixteen newly appointed ophthalmology residents in November 2022 were included during their third month of residency. The first resident was enrolled on January 9th, 2023, and the last resident ended their training program on January 30th, 2023. Residents’ median age was 25.5 [25.0; 26.0] and the proportion of women was 44% (Table ). Surgical practice experience was low: fifteen (94%) had never performed real-life corneal incisions, capsulorhexis, hydromaneuver and phacoemulsification. Some had made a few attempts at irrigation and aspiration (4/16, 25%) and IOL insertion (6/16, 37%). The multidimensional curves of overall progression show a linear increase in score and decrease in completion time and odometer, associated with a decrease in variance without a plateau (Fig. ). Throughout the training sessions, the median score increased progressively from 95 [IQR 53; 147] at A0 to 425 [411; 451] at A4 (E-Table 1). No significant difference in overall score was observed between residents who had more than 10 real-life attempts of any surgical step and residents with fewer than 10 at A0 (100 [IQR 86.5; 152] versus 69 [IQR 39; 141] respectively, p = 0.60) and A4 (445 [IQR 400; 454] versus 422 [IQR 418; 444] respectively, p = 0.63). The “capsulorhexis” step showed a linear increase in score with reduction in variance without reaching a plateau. Completion time and odometer decreased linearly without a plateau or reduction in variance. Additional “capsulorhexis” assessments at the beginning of each training session revealed high inter-individual and intra-individual variability during the first 3 sessions with a tendency to decrease compared with the previous session (E-Figure 1). The “hydromaneuver” step had the least valid learning curve (E-Figure 2). The score at the first assessment before any training (A0) was unusually high and represented the only step for which the median A0 score was higher than 0 (36 [0; 75]). The scores for the “emulsification” step were the lowest with a lower interquartile < 80 (86 [60; 94] at A4. While the increase in score for the “emulsification” step was linear, it did not reach a plateau and was not associated with a reduction in completion time and odometer of ultrasounds delivered. The learning curves of the “irrigation and aspiration” step had a negative exponential shape and reached a plateau with the highest decrease in variance. This was the step for which the residents had the highest final scores (median 98 [96; 100]). The “IOL insertion” step showed a linear increase in score with reduction in variance without reaching a plateau. Completion time and odometer did not decrease throughout the training program (E-Figure 2). The overall average success rate progressively increased throughout the training sessions from 0.0% [0.0; 24.1] at A0 to 75.0% [47.4; 91.7] at A4 (Fig. and E-Table 2). Although the residents’ scores progressed for the 5 steps, progression was found to be heterogeneous: slow for the “emulsification” step for which the success rate at A4 was 56.2% [30.6; 79.2] and rapid for “irrigation and aspiration”, for which the success rate was 87.5% [60.4; 97.8] at A3 and 93.8% [67.7–99.7] at A4. Moreover, the percentage of residents who consecutively succeeded each of the 5 steps at A4 was 25.0% [8.3; 52.6]. The average rates of posterior capsular rupture decreased from 75% (12/16) at A0 to 12% (2/16) at A4 during the “emulsification” step and from 69% (11/16) at A0 to 0% at A4 during the “irrigation and aspiration” step (E-Table 1). The decrease was linear and continuous throughout the training program (Fig. ). In the present study, we provide valid and valuable learning curves of residents during a training program including four two-hour EyeSi simulator sessions, conducted over four consecutive days. First, “irrigation and aspiration” was the only step mastered by residents at the end of the training program. Given the risk of posterior capsular rupture during this surgical step, the learning curve for “irrigation and aspiration” is particularly reassuring for skill transfer in the operating room. Second, the “emulsification” step was the most difficult to perform for students who had a large number of insufficient scores at the end of the training program (i.e. 56.2% success rate, for more information see E-Table 3) with no reduction in completion time and odometer. Third, because the “hydromaneuver” step has the least valid learning curve, it cannot be used to assess a student’s progression. Fourth, the rate of posterior capsular rupture decreased linearly throughout the training program, but remained abnormally high at the end when compared to the incidence reported in the literature . The dynamics of the curves and capsular rupture rate suggest that more than four EyeSi training sessions would be beneficial for obtaining better scores and fewer surgical complications. Aditya et al. analyzed heart rate variations in residents during real-life cataract surgery and discovered that emulsification was the most stressful step for residents and therefore should benefit from special preparation , as we also observed. Belyea et al. found that resident training on the Eyesi Surgical Simulator resulted in a reduction in time to perform phacoemulsification with a reduction in the use of ultrasound power. As we did not observe reduction in emulsification completion time or in ultrasound power used, this result supports our conclusion that more than four training sessions are required to observe further progression . Al-Jindan et al. assessed 200 real-life surgeries performed by 22 residents: the steps that most frequently required assistance, in descending order, were emulsification, capsulorhexis, and irrigation and aspiration. Conversely, the steps where surgical complications most commonly occurred were, in descending order, capsulorhexis, irrigation and aspiration, and emulsification . Taken together, these findings support the strong need for preparation with respect to the following steps: capsulorhexis, irrigation and aspiration, and emulsification. Therefore, the current training program could be of particular benefit to the “capsulorhexis” and “irrigation and aspiration” steps for which the final scores were high, with a decrease in the occurrence of posterior capsular rupture. A growing effort is being made to define the best way to integrate the EyeSi simulator into surgical training programs. Thomsen et al. aimed to define the proficiency level required before surgery by evaluating the ability of all EyeSi modules to discriminate between novice and expert surgeons: capsulorhexis and emulsification discriminated the best, whereas hydromaneuver did not discriminate at all, with similar mean scores between novices and experts . In total, they selected the 7 most discriminating training exercises (capsulorhexis, emulsification and five navigation training exercises) to be part of an evaluation for which the pass/fail threshold was the discriminating score between novice and experts (422/700). This pass/fail score was probably underestimated because expert surgeons only had 10 min to familiarize themselves with the EyeSi simulator, so we can assume that their score would have been higher with more training. The number of training sessions required to reach this threshold was not determined, but Bergkvist et al. demonstrated that repeated training with EyeSi simulator improved scores: ten novice residents with four training sessions outperformed ten other residents with only two training sessions . They were unable to determine how many sessions there should be in a training program and advised further research into learning curves, as we did in our study. The EyeSi simulator comprises a standard training program divided into four modules, which include navigation training exercises and surgical steps (A, B, C for fundamental skills and standard cataract surgery and module D for complex cases and complication management): before moving to the next task the resident must achieve the minimum score three times in a row at each step (minimum score of 50 for category A, 70 for category B and 85 for category C). Montrisuksirikun et al. found that completing modules A, B and C prior to the first surgery decreases the total rate of complications of resident-performed phacoemulsification and shortens the learning curve for cataract surgery training. However no data on the time required to complete the simulation program was available . As recommended, modules A and B must be completed before the first surgery in the United Kingdom . McClay and Lockington analyzed 103 resident accounts on the EyeSi simulator in Scotland: the average time to complete each module was 2h18m ± 1h08 for module A, 3h40m ± 1h37m for module B and 6h03m ± 3h48m for module C . The number of training sessions required to complete modules A, B and C was not available: it was certainly spread over more than four sessions while these times are only related to “time in the eye” and do not include set-up times and time spent reading instructions. Even though these modules and minimum scores are not comparable to our standardized assessments, these data corroborate our results, which suggest that more than 4 training sessions are necessary to obtain sufficiently high scores on the simulator. The main limitations of our study were related to external constraints and the need to produce a realistic study: the relatively small sample size was due to the need for a homogenous population of novice residents without surgical experience. Including additional undergraduate students or residents from other specialties would have induced a significant bias due to differences in age and motivation. Extending the study over several years would have delayed dissemination of the results and monopolized major research efforts on the part of students and teachers. Despite the precaution of including novices during their third month of residency, some had already performed several surgical steps in the operating room: in the hypothesis that this bias artificially improved their scores and progression, it would reinforce our findings that more than 4 sessions would be beneficial for novice residents. The rigor of our methodology, combined with the properties of the EyeSi simulator, resulted in valid learning curves that are in line with the state of the art . The validity of the y-axis (score, completion duration and odometer) is supported by the extensive literature available relating to the benefits for residents and patients of using the EyeSi simulator, the reproducibility and the correlation of score with real-life skills . The high consistency of the methodology supports the validity of the x-axis, which measures the learning effort of residents: each resident was given strictly equal units of time spent practicing on the same simulator with a homogeneous group of instructors and the assessments were standardized and consistent over time. The curves had the expected shape with a negative exponential relationship (progressively decreasing slope), a decrease in variance and no ceiling or floor effect which would have indicated overly easy or hard assessments . We conducted the study under reproducible and immersive conditions during a single month with a homogenous population. Fatigue bias was avoided by multiple breaks during the half-day training session and a half-day’s rest before the next session. Competition bias was prevented by conducting confidential assessments, with no other resident present, and by keeping the results anonymous, even after the study was completed. In conclusion, our study finds that a training program, consisting of four two-hour sessions on the EyeSi simulator over four consecutive days, effectively enhances the surgical skills of ophthalmology residents. However, the high incidence of complications and low performance scores, particularly at the emulsification step, indicate that the skills acquired may not yet be at an acceptable level for performing surgery in the operating room. This suggests a need for further research to optimize the training program. The learning curves that we provided can be a valuable tool in daily practice for a personalized training perspective. They can reassure ophthalmology residents who are progressing normally and help identify those who require additional, tailored training. These findings could serve as a basis for the development of a national certification or ‘license to operate’ in the field. Supplementary Material 1: E-Table 1. Scores, completion time, odometer throughout the training program. E-Table 2. Posterior capsular rupture throughout the training program. E-Table 3. Success rate for each step throughout the training program. Supplementary Material 2: E-Figure 1. Overlaid individual learning curves for the capsulorhexis step. The grey lines represent the score changes for each resident. The dotted horizontal line indicates the performance threshold (80/100). The bold black line shows the evolution of the median score. Supplementary Material 3: E-Figure 2. Multidimensional learning curves showing changes in score, completion time and odometer for the hydromaneuver and intraocular lens insertion steps. In the box plots (columns 1, 3 and 4), the edge of the box indicates the 25th and 75th percentiles, the black line within the box indicates the median. The upper whiskers extend from the edges of the box to the highest and lowest values up to 1.5 times the IQR. Data beyond the end of the whiskers are plotted individually as grey circles. The blue lines connect the medians across sessions. In column 2, the individual overlaid curves show each ophthalmology resident’s score changes across sessions (grey lines). The bold black line shows the evolution of the median score across sessions. |
Evaluation of musculoskeletal complaints, treatment approaches, and patient perceptions in family medicine clinics in a tertiary center in Jordan: a cross-sectional study | 5c42cff3-b43f-4d09-9aa9-0c40ff10d9ad | 11748281 | Family Medicine[mh] | Orthopedic conditions are among the most common health complaints encountered globally, affecting millions of individuals of various ages and backgrounds . Conditions such as back pain, neck pain, and joint disorders, including osteoarthritis and tendonitis, are frequent presentations in medical clinics . These musculoskeletal (MSK) complaints often lead to discomfort, reduced mobility, and a decreased quality of life . Due to the high prevalence of these conditions, patients frequently seek medical advice to alleviate symptoms such as pain, stiffness, and functional limitations. Consequently, musculoskeletal disorders represent a significant burden on healthcare systems worldwide, highlighting the importance of effective management strategies . In many cases, family medicine or primary care clinics serve as the initial point of contact for patients with MSK complaints . This can present a significant challenge for primary healthcare providers, as they must be equipped to manage a wide variety of musculoskeletal conditions. The diversity of orthopedic presentations, combined with the complexity of differentiating between acute, chronic, and potentially serious conditions, places substantial pressure on family medicine systems . Primary care physicians are tasked with providing accurate diagnoses, implementing appropriate management plans, and determining when to refer patients to specialists. These challenges underscore the critical role of family medicine in the early diagnosis and management of orthopedic conditions. Given the high volume of musculoskeletal complaints seen in family medicine clinics, it is essential to evaluate how these conditions are managed and the effectiveness of the treatment approaches employed . Equally important is the assessment of patient perceptions regarding the quality of care they receive. Patient satisfaction and their perception of the quality of care are vital components of healthcare delivery, as they directly influence patient compliance and health outcomes . Understanding the patient experience, particularly in relation to musculoskeletal disorders, can provide valuable insights into the effectiveness of current healthcare practices and highlight areas for improvement. While global studies highlight the prevalence and impact of MSK disorders, there is limited evidence on how these conditions are managed in primary care, particularly in family medicine clinics. This is especially true in developing regions, such as the Middle East, where healthcare systems face unique challenges, including resource constraints and inconsistent service delivery . Existing research provides valuable insights into MSK management in high-income countries, but it does not account for the socio-economic, cultural, and systemic factors that influence care in resource-limited settings. Despite these challenges, the extent to which MSK care is optimized in primary care, especially in addressing gender-specific needs and patient perceptions, remains unclear. Furthermore, there is a significant gap in understanding how treatment pathways and patient outcomes are shaped by these factors in developing countries, leaving a critical grey area in the evidence. Addressing these unknowns is essential to bridge the gap between global standards of care and region-specific needs. This study hypothesizes that variations in the presentation, management, and patient perceptions of musculoskeletal (MSK) complaints exist within family medicine clinics, influenced by factors such as gender, psychosocial considerations, and healthcare delivery practices. By investigating these variations in a tertiary center in Jordan, a developing Middle Eastern country, we aim to address gaps in the literature and provide insights into the complex interplay of these factors. The findings from this study are intended to inform strategies for improving the quality of MSK care and optimizing patient outcomes in resource-constrained healthcare systems. This study was prepared following the STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guideline for cross-sectional studies . Study design We employed a cross-sectional descriptive design using a convenience sampling method. The objective was to evaluate the presentation, management, and perceptions of musculoskeletal (MSK) complaints in family medicine clinics. A comprehensive approach was taken to assess demographic variables, health profiles, comorbidities, visit-specific characteristics, healthcare-related factors, psychological dimensions, and occupational influences. This design provided a snapshot of patient care and the interplay of these factors within the study period. Setting The study was conducted in the family medicine clinics of a central teaching hospital in Amman, Jordan. The clinics, comprising approximately 10 units, operate throughout the week and serve a diverse patient population from urban and rural areas. Data were collected over six months, from January 1st, 2024, to June 30th, 2024, ensuring seasonal variability was captured. The research team consisted of family medicine residents trained in standardized data collection methods and supervised by 2–3 family medicine consultants. Data were gathered during routine clinical visits, ensuring real-time documentation of patient complaints and management pathways. Participants The study’s inclusion criteria targeted adults aged 18 years or older presenting with musculoskeletal (MSK) complaints. These complaints were defined as disorders involving muscles, bones, tendons, ligaments, and connective tissues, typically manifesting as pain, stiffness, or functional impairment. To participate, individuals needed to provide informed consent and agree to interviews and data collection using their authorized medical records. This approach ensured a comprehensive understanding of the participants’ MSK conditions while maintaining ethical standards. Exclusion criteria were implemented to minimize confounding factors and ensure the reliability of the study findings. Patients with non-MSK complaints, cognitive impairments, severe trauma requiring emergency care, recent orthopedic surgeries, or advanced comorbidities such as cancer were excluded. Additionally, pregnant women were not included due to the unique nature of pregnancy-related MSK conditions. Overall, 500 participants were carefully selected, reflecting the diverse spectrum of MSK conditions commonly encountered in primary care settings. Variables The study variables were structured to analyze factors influencing outcomes such as patient-reported satisfaction, perceptions of care, and quality of life (QoL). Exposures included demographic factors (age, gender, socioeconomic status), health profiles, and types of musculoskeletal (MSK) complaints. Predictors and confounders, such as medical comorbidities (e.g., hypertension, diabetes), employment status, psychological variables, and work-related factors, were considered to ensure a thorough and unbiased assessment of relationships between exposures and outcomes. Data collection and measurement Data collection followed a structured protocol combining personalized patient interviews and reviews of electronic health records to ensure comprehensive and accurate data capture. Trained family medicine residents conducted the interviews using a standardized questionnaire specifically developed for this study. The questionnaire was designed to capture demographic, clinical, and psychosocial variables, drawing from validated tools and existing literature on musculoskeletal complaints. Content validity was ensured through expert review, and a pilot test with 20 eligible patients refined its clarity and structure. Reliability was assessed with Cronbach’s alpha, achieving a value above 0.7, and test-retest reliability showed a strong intraclass correlation coefficient (ICC) for key variables. These steps confirmed the questionnaire’s validity and reliability for the study. The interviews, conducted during routine clinic visits, gathered self-reported information on demographic, psychological, and occupational factors, while electronic health records provided clinical data on comorbidities and treatment approaches. Variables such as pain intensity and functional status were assessed using validated scales (e.g., a 0–10 pain scale). All data were recorded electronically, consolidated into a single dataset, and cross-verified for accuracy. The process adhered strictly to the predefined study timeline to maintain consistency and reliability. Study size A sample size of 500 was determined using a power analysis conducted in G*Power software (version 3.1.9.7), ensuring sufficient statistical power to detect meaningful associations between variables. The calculation was based on a 95% confidence level, a 5% margin of error, and an expected effect size of 0.3, derived from prior studies in musculoskeletal health research . Bias Efforts were made to minimize bias throughout the study. To address selection bias, convenience sampling was conducted across various clinic sessions to ensure the inclusion of a representative patient sample. Information bias was mitigated by adhering to a standardized data collection protocol, with trained interviewers ensuring consistency and uniformity in data recording. Additionally, recall bias was minimized by focusing the data collection on recent patient experiences, reducing the likelihood of inaccuracies related to memory. Ethical considerations In conducting this study, all ethical considerations were carefully addressed to ensure the protection and privacy of the participants. Approval was obtained from the institutional review board (IRB) of the teaching hospital where the research was conducted, IRB approval No. (10/2024/25/22). Informed consent was obtained from all participants prior to their inclusion in the study, ensuring that they fully understood the purpose of the research. Patient confidentiality was strictly maintained by anonymizing all data, and access to medical records was restricted to authorized personnel directly involved in the study. Furthermore, the study was conducted in accordance with the principles outlined in the Declaration of Helsinki, ensuring that all participants were treated with respect and dignity throughout the research process. Statistical analysis For statistical analysis, descriptive statistics were used to summarize demographic characteristics and clinical outcomes. Categorical variables were analyzed using Chi-square tests, while continuous variables were compared using t-tests or Mann-Whitney U tests, depending on the data distribution. Odds ratios (OR) with 95% confidence intervals (CI) were calculated to explore associations between variables. All analyses were conducted using the Statistical Package for Social Sciences (SPSS version 23) and Microsoft Excel, with a p -value of less than 0.05 considered statistically significant. Data were compiled into a single electronic file for analysis to ensure consistency and accuracy throughout the study. Dependent variables in this study included patient satisfaction, pain scores, functional status, and quality of life metrics. Independent variables comprised demographic factors (e.g., age, gender, socioeconomic status), clinical factors (e.g., MSK complaint type, comorbidities, and treatment pathways), and psychosocial factors (e.g., coping strategies, anxiety levels, and fear of disability). Variables were categorized based on their role in influencing outcomes, allowing for a structured approach to statistical analysis. Descriptive statistics summarized these variables, and inferential tests examined associations between independent variables and outcomes. This framework ensured a comprehensive understanding of the factors impacting musculoskeletal health and patient experiences. The study methodology is illustrated in Fig. . We employed a cross-sectional descriptive design using a convenience sampling method. The objective was to evaluate the presentation, management, and perceptions of musculoskeletal (MSK) complaints in family medicine clinics. A comprehensive approach was taken to assess demographic variables, health profiles, comorbidities, visit-specific characteristics, healthcare-related factors, psychological dimensions, and occupational influences. This design provided a snapshot of patient care and the interplay of these factors within the study period. The study was conducted in the family medicine clinics of a central teaching hospital in Amman, Jordan. The clinics, comprising approximately 10 units, operate throughout the week and serve a diverse patient population from urban and rural areas. Data were collected over six months, from January 1st, 2024, to June 30th, 2024, ensuring seasonal variability was captured. The research team consisted of family medicine residents trained in standardized data collection methods and supervised by 2–3 family medicine consultants. Data were gathered during routine clinical visits, ensuring real-time documentation of patient complaints and management pathways. The study’s inclusion criteria targeted adults aged 18 years or older presenting with musculoskeletal (MSK) complaints. These complaints were defined as disorders involving muscles, bones, tendons, ligaments, and connective tissues, typically manifesting as pain, stiffness, or functional impairment. To participate, individuals needed to provide informed consent and agree to interviews and data collection using their authorized medical records. This approach ensured a comprehensive understanding of the participants’ MSK conditions while maintaining ethical standards. Exclusion criteria were implemented to minimize confounding factors and ensure the reliability of the study findings. Patients with non-MSK complaints, cognitive impairments, severe trauma requiring emergency care, recent orthopedic surgeries, or advanced comorbidities such as cancer were excluded. Additionally, pregnant women were not included due to the unique nature of pregnancy-related MSK conditions. Overall, 500 participants were carefully selected, reflecting the diverse spectrum of MSK conditions commonly encountered in primary care settings. The study variables were structured to analyze factors influencing outcomes such as patient-reported satisfaction, perceptions of care, and quality of life (QoL). Exposures included demographic factors (age, gender, socioeconomic status), health profiles, and types of musculoskeletal (MSK) complaints. Predictors and confounders, such as medical comorbidities (e.g., hypertension, diabetes), employment status, psychological variables, and work-related factors, were considered to ensure a thorough and unbiased assessment of relationships between exposures and outcomes. Data collection followed a structured protocol combining personalized patient interviews and reviews of electronic health records to ensure comprehensive and accurate data capture. Trained family medicine residents conducted the interviews using a standardized questionnaire specifically developed for this study. The questionnaire was designed to capture demographic, clinical, and psychosocial variables, drawing from validated tools and existing literature on musculoskeletal complaints. Content validity was ensured through expert review, and a pilot test with 20 eligible patients refined its clarity and structure. Reliability was assessed with Cronbach’s alpha, achieving a value above 0.7, and test-retest reliability showed a strong intraclass correlation coefficient (ICC) for key variables. These steps confirmed the questionnaire’s validity and reliability for the study. The interviews, conducted during routine clinic visits, gathered self-reported information on demographic, psychological, and occupational factors, while electronic health records provided clinical data on comorbidities and treatment approaches. Variables such as pain intensity and functional status were assessed using validated scales (e.g., a 0–10 pain scale). All data were recorded electronically, consolidated into a single dataset, and cross-verified for accuracy. The process adhered strictly to the predefined study timeline to maintain consistency and reliability. A sample size of 500 was determined using a power analysis conducted in G*Power software (version 3.1.9.7), ensuring sufficient statistical power to detect meaningful associations between variables. The calculation was based on a 95% confidence level, a 5% margin of error, and an expected effect size of 0.3, derived from prior studies in musculoskeletal health research . Efforts were made to minimize bias throughout the study. To address selection bias, convenience sampling was conducted across various clinic sessions to ensure the inclusion of a representative patient sample. Information bias was mitigated by adhering to a standardized data collection protocol, with trained interviewers ensuring consistency and uniformity in data recording. Additionally, recall bias was minimized by focusing the data collection on recent patient experiences, reducing the likelihood of inaccuracies related to memory. In conducting this study, all ethical considerations were carefully addressed to ensure the protection and privacy of the participants. Approval was obtained from the institutional review board (IRB) of the teaching hospital where the research was conducted, IRB approval No. (10/2024/25/22). Informed consent was obtained from all participants prior to their inclusion in the study, ensuring that they fully understood the purpose of the research. Patient confidentiality was strictly maintained by anonymizing all data, and access to medical records was restricted to authorized personnel directly involved in the study. Furthermore, the study was conducted in accordance with the principles outlined in the Declaration of Helsinki, ensuring that all participants were treated with respect and dignity throughout the research process. For statistical analysis, descriptive statistics were used to summarize demographic characteristics and clinical outcomes. Categorical variables were analyzed using Chi-square tests, while continuous variables were compared using t-tests or Mann-Whitney U tests, depending on the data distribution. Odds ratios (OR) with 95% confidence intervals (CI) were calculated to explore associations between variables. All analyses were conducted using the Statistical Package for Social Sciences (SPSS version 23) and Microsoft Excel, with a p -value of less than 0.05 considered statistically significant. Data were compiled into a single electronic file for analysis to ensure consistency and accuracy throughout the study. Dependent variables in this study included patient satisfaction, pain scores, functional status, and quality of life metrics. Independent variables comprised demographic factors (e.g., age, gender, socioeconomic status), clinical factors (e.g., MSK complaint type, comorbidities, and treatment pathways), and psychosocial factors (e.g., coping strategies, anxiety levels, and fear of disability). Variables were categorized based on their role in influencing outcomes, allowing for a structured approach to statistical analysis. Descriptive statistics summarized these variables, and inferential tests examined associations between independent variables and outcomes. This framework ensured a comprehensive understanding of the factors impacting musculoskeletal health and patient experiences. The study methodology is illustrated in Fig. . Demographic and medical characteristics The questionnaire was completed by a total of 500 patients, with a mean age of 46.11 ± 15.32 years. Of these, 305 patients (61.0%) were female. In terms of education, 332 respondents (66.3%) were university graduates. The majority of participants resided in urban areas (376, 75.2%), were married (342, 68.7%), and reported an average economic status (421, 84.4%). Regarding medical history, hypertension (HTN) was present in 126 patients (25.2%), diabetes mellitus (DM) in 109 patients (21.8%), and cardiovascular disease (CVD) in 53 patients (10.6%). Additionally, 147 patients (29.4%) were smokers. Table provides a detailed summary of the demographic and medical characteristics of the participants. Musculoskeletal pain, treatment pathways, and patient experience in family clinics The most frequently reported musculoskeletal complaints were low back pain (23.8%), knee pain (23.4%), and foot and ankle pain (16.9%). The median pain score was 7 (IQR 2) on a scale of 10, with females showing significantly higher odds of presenting with hip pain (OR = 1.650, p = 0.008) and a higher prevalence of low back pain compared to males (61.9% vs. 38.1%, OR = 1.12, p = 0.024). Pain scores also differed significantly between genders, with females reporting higher scores ( p < 0.001). Most patients (98.4%) received analgesics as initial management, with no significant gender difference. Only 18.1% were referred to orthopedic specialists, while 57.3% reported receiving counseling on musculoskeletal health, and just 41.5% underwent diagnostic tests during their visits, highlighting gaps in assessment. Patients expressed high satisfaction with their clinic visits, with a median satisfaction score of 8 (IQR 3) and counseling adequacy rated similarly at 8 (IQR 2). However, disability and functional status showed gender-specific differences: males reported higher disability scores (median 8, IQR 4) compared to females (median 4, IQR 4), while females demonstrated better functional status in daily activities (median 8, IQR 3 vs. males at 6, IQR 2). These findings underscore overall positive patient experiences alongside gender-related variations in pain, disability, and functional capabilities, summarized in Tables and . Patient perceptions and confidence in family medicine healthcare Gender differences in healthcare access and perceptions revealed notable trends. Most participants reported easy access to healthcare facilities, with females slightly more likely to report difficulties, though this was not statistically significant ( p = 0.087). Health insurance coverage was predominantly partial for both genders, with no significant differences in type. Males reported greater access to social support (OR = 1.309, p = 0.039), while fear of disability or loss of independence was more common among females (OR = 0.992, p = 0.048), highlighting distinct psychosocial concerns. Males also expressed significantly higher confidence in the healthcare system’s capacity to manage musculoskeletal issues (OR = 1.926, p = 0.035). Despite these variations, satisfaction with healthcare services was high for both genders (median score 8), and females perceived clinics as busier, though this was not statistically significant ( p = 0.082). These findings emphasize nuanced gender differences in social support, confidence in healthcare, and psychosocial concerns, as detailed in Table . Gender differences in mental health and quality of life related to musculoskeletal conditions Gender differences in the psychosocial impact of musculoskeletal conditions reveal significant trends. Males report greater concerns about employment and income loss (58.0% vs. 42.0%, OR = 1.525, p = 0.032) and are more likely to use coping strategies (OR = 1.363, p = 0.036), while females experience higher anxiety about disease progression (62.2% vs. 37.8%, OR = 1.22, p = 0.045). Males also report better mental health (median score 9 vs. 7, p = 0.036), sleep quality (median 8 vs. 6, p = 0.044), and overall quality of life (median 8 vs. 6, p = 0.019), highlighting distinct gender disparities. These findings underscore the need for gender-sensitive approaches to address coping strategies, anxiety, and quality of life in managing musculoskeletal conditions, as detailed in Table . Occupational factors, work-related support, and safety concerns in patients attending a family medicine clinic Gender-related differences in occupational factors impacting musculoskeletal health indicate that males experience a significantly higher workload, with 54.4% of males reporting high workload compared to 45.6% of females (OR = 1.564, p = 0.020). Males are also far more likely to report work-related injuries, accounting for 82.8% of those injured (OR = 8.623, p < 0.001). Access to recreational facilities for physical activity is notably higher for males (52.4% vs. 47.6% for females, OR = 1.956, p < 0.001), and males are more frequently advised to adjust their work patterns, with 48.5% of males versus 51.5% of females receiving such recommendations (OR = 1.679, p = 0.020). These findings, summarized in Table , emphasize that males face a greater occupational burden. The questionnaire was completed by a total of 500 patients, with a mean age of 46.11 ± 15.32 years. Of these, 305 patients (61.0%) were female. In terms of education, 332 respondents (66.3%) were university graduates. The majority of participants resided in urban areas (376, 75.2%), were married (342, 68.7%), and reported an average economic status (421, 84.4%). Regarding medical history, hypertension (HTN) was present in 126 patients (25.2%), diabetes mellitus (DM) in 109 patients (21.8%), and cardiovascular disease (CVD) in 53 patients (10.6%). Additionally, 147 patients (29.4%) were smokers. Table provides a detailed summary of the demographic and medical characteristics of the participants. The most frequently reported musculoskeletal complaints were low back pain (23.8%), knee pain (23.4%), and foot and ankle pain (16.9%). The median pain score was 7 (IQR 2) on a scale of 10, with females showing significantly higher odds of presenting with hip pain (OR = 1.650, p = 0.008) and a higher prevalence of low back pain compared to males (61.9% vs. 38.1%, OR = 1.12, p = 0.024). Pain scores also differed significantly between genders, with females reporting higher scores ( p < 0.001). Most patients (98.4%) received analgesics as initial management, with no significant gender difference. Only 18.1% were referred to orthopedic specialists, while 57.3% reported receiving counseling on musculoskeletal health, and just 41.5% underwent diagnostic tests during their visits, highlighting gaps in assessment. Patients expressed high satisfaction with their clinic visits, with a median satisfaction score of 8 (IQR 3) and counseling adequacy rated similarly at 8 (IQR 2). However, disability and functional status showed gender-specific differences: males reported higher disability scores (median 8, IQR 4) compared to females (median 4, IQR 4), while females demonstrated better functional status in daily activities (median 8, IQR 3 vs. males at 6, IQR 2). These findings underscore overall positive patient experiences alongside gender-related variations in pain, disability, and functional capabilities, summarized in Tables and . Gender differences in healthcare access and perceptions revealed notable trends. Most participants reported easy access to healthcare facilities, with females slightly more likely to report difficulties, though this was not statistically significant ( p = 0.087). Health insurance coverage was predominantly partial for both genders, with no significant differences in type. Males reported greater access to social support (OR = 1.309, p = 0.039), while fear of disability or loss of independence was more common among females (OR = 0.992, p = 0.048), highlighting distinct psychosocial concerns. Males also expressed significantly higher confidence in the healthcare system’s capacity to manage musculoskeletal issues (OR = 1.926, p = 0.035). Despite these variations, satisfaction with healthcare services was high for both genders (median score 8), and females perceived clinics as busier, though this was not statistically significant ( p = 0.082). These findings emphasize nuanced gender differences in social support, confidence in healthcare, and psychosocial concerns, as detailed in Table . Gender differences in the psychosocial impact of musculoskeletal conditions reveal significant trends. Males report greater concerns about employment and income loss (58.0% vs. 42.0%, OR = 1.525, p = 0.032) and are more likely to use coping strategies (OR = 1.363, p = 0.036), while females experience higher anxiety about disease progression (62.2% vs. 37.8%, OR = 1.22, p = 0.045). Males also report better mental health (median score 9 vs. 7, p = 0.036), sleep quality (median 8 vs. 6, p = 0.044), and overall quality of life (median 8 vs. 6, p = 0.019), highlighting distinct gender disparities. These findings underscore the need for gender-sensitive approaches to address coping strategies, anxiety, and quality of life in managing musculoskeletal conditions, as detailed in Table . Gender-related differences in occupational factors impacting musculoskeletal health indicate that males experience a significantly higher workload, with 54.4% of males reporting high workload compared to 45.6% of females (OR = 1.564, p = 0.020). Males are also far more likely to report work-related injuries, accounting for 82.8% of those injured (OR = 8.623, p < 0.001). Access to recreational facilities for physical activity is notably higher for males (52.4% vs. 47.6% for females, OR = 1.956, p < 0.001), and males are more frequently advised to adjust their work patterns, with 48.5% of males versus 51.5% of females receiving such recommendations (OR = 1.679, p = 0.020). These findings, summarized in Table , emphasize that males face a greater occupational burden. The main findings of this study reveal distinct gender differences across multiple dimensions of musculoskeletal health. Females reported a higher prevalence of conditions such as low back and hip pain, greater anxiety about disease progression, and better functional status in daily activities, but experienced higher pain levels and poorer quality of life indicators, including sleep and mental health. In contrast, males demonstrated higher confidence in the healthcare system, greater use of coping strategies, and more concerns about employment and income loss related to their conditions. These gender-specific patterns highlight the diverse experiences of patients with musculoskeletal complaints, emphasizing the importance of tailoring healthcare approaches to address the unique needs and challenges of both genders. Greater prevalence of pain in females Our findings indicate that female patients are significantly more likely to report pain than male patients, with a particular prevalence of low back and hip pain. Low back pain was notably more common among females, while hip pain was reported exclusively by female patients, underscoring a clear gender-specific pattern. These results align with previous studies suggesting that biological, hormonal, and psychosocial factors contribute to the higher pain prevalence and sensitivity observed in females. Research consistently supports the greater prevalence of low back pain (LBP) in women compared to men. For instance, Wáng et al. and Brady et al. found that females exhibit higher rates of low back pain across all age groups, with this gender difference widening post-menopause . Schneider et al. reported a 40% prevalence of back pain among women in Germany, notably higher than in men, attributing this disparity to hormonal changes, psychological influences, and anatomical differences . Furthermore, Chenot et al. observed that women not only have lower functional capacity but also higher rates of chronic LBP and a greater likelihood of experiencing depression related to LBP . Our findings, consistent with these studies, underscore the need for gender-sensitive pain management approaches to address the specific pain patterns and experiences of female patients effectively. Notably, our study observed a higher prevalence of hip pain among female patients compared to males. This finding may be attributed to a higher incidence of hip pathologies, such as hip dysplasia, prevalent within our population . However, it is important to highlight that previous studies have not specifically examined the prevalence of hip pain among patients visiting family or orthopedic clinics without a prior diagnosis of hip pathology. This gap underscores the unique contribution of our study in highlighting hip pain patterns in a general clinical population, pointing to a need for further research on undiagnosed hip pain, especially in primary care settings. Gender differences in diagnostic and treatment approaches for musculoskeletal pain Our study found that female patients were more likely than males to receive analgesia, referrals to physical therapy, and diagnostic testing during clinic visits. This trend may reflect the higher prevalence and intensity of musculoskeletal pain reported by females, especially in low back and hip pain, which likely prompts more extensive management and evaluation. Additionally, healthcare providers may view female patients as needing broader diagnostic and therapeutic support due to gender-based differences in pain presentation and reporting patterns. These findings suggest that clinicians may adopt a more comprehensive approach to meet the specific healthcare needs of female patients, integrating diverse diagnostic and treatment measures. Our findings align with Barr et al., who observed that females generally use more pain-relieving medications, though it remains uncertain whether this is due to actual usage differences or potential reporting bias . In contrast, Weimer et al. and Procento et al. found that females are less frequently prescribed strong opioids compared to males , while Richardson reported that women are more likely to visit emergency departments for pain-related complaints, leading to higher rates of diagnostic evaluations . Our results align with these findings, demonstrating that female patients not only receive more referrals to physical therapy and diagnostic evaluations but also utilize a broader range of pain management strategies compared to their male counterparts. Greater disability, reduced functional status, and employment anxiety in male patients Our study found that male patients with musculoskeletal (MSK) conditions report higher levels of disability, lower functional status, and greater anxiety about potential loss of employment or income compared to female patients. This suggests that MSK conditions may affect men differently, likely due to societal expectations that emphasize physical capacity and occupational stability. Physical limitations from MSK conditions can directly impact male patients’ job performance, particularly in physically demanding roles, potentially explaining the increased levels of disability and functional impairment we observed. Additionally, as males often carry primary financial responsibilities, their concerns about job and income stability may be heightened. These findings highlight the importance of integrated MSK management that includes occupational support to address both physical limitations and economic concerns specific to male patients. Our findings align with gender-based patterns reported in prior research on MSK disorders, with notable similarities and contrasts. Consistent with our results, Wolf et al. and Raina et al. observed that males with MSK conditions experience greater disability, reduced functional status, and higher employment anxiety . Conversely, Holland et al. and Stubbs et al. reported that females often experience more pain-related disability and are more likely to leave employment, potentially reflecting a different coping mechanism compared to males . Supporting our observation of functional impairment in male patients, Zink et al. found severe disability in males with specific MSK conditions like ankylosing spondylitis , while Wijnhoven et al. identified greater work-related disability in men with low back pain . Additionally, Bailey et al. noted that men are more frequently referred to mental health services for work-related issues, suggesting added psychological strain in employment settings . Together, these studies underscore the complex employment-related challenges faced by male MSK patients and the need for occupational and psychological support tailored to their specific needs. Lower social support, higher anxiety, and coping challenges in female patients Our study found that female patients with musculoskeletal (MSK) conditions reported lower levels of social support, a greater fear of disability and loss of independence, fewer coping mechanisms, and increased anxiety about disease progression compared to their male counterparts. These findings may reflect social and cultural dynamics in which females have limited access to social resources or perceive less support in managing chronic health conditions. This reduced social support, combined with an elevated fear of disability, could contribute to heightened anxiety, as managing chronic pain without sufficient support or effective coping strategies can intensify concerns about the condition’s impact on their future independence. These observations underscore the need for integrated support systems and psychosocial interventions that enhance coping skills and directly address the unique challenges faced by female MSK patients. Previous research supports these findings and offers further insight into the coping challenges and psychosocial dynamics specific to female MSK patients. Grossi et al. observed that females with musculoskeletal pain report lower coping capacity, which correlates with higher levels of emotional distress and greater disability . Similarly, Soares et al. found that lower self-esteem among female patients with MSK pain is associated with increased anxiety and depression, while positive self-esteem aligns with active coping mechanisms . Espwall et al. noted that women with undefined MSK disorders receive less emotional support than those with non-MSK conditions, potentially exacerbating their psychosocial burdens . Further, Cheng et al. reported that females with chronic MSK pain experience worse physical functioning than males . Together, these studies and our findings highlight the importance of tailored psychosocial support to bolster coping strategies and enhance social support for female MSK patients, aiming to improve both mental health and physical outcomes. Challenges in mental health and quality of life for female patients with MSK disorders Our study found that female patients with musculoskeletal (MSK) conditions experience a greater impact on emotional well-being, with lower reported quality of mental health, poorer sleep quality, and a reduced overall quality of life compared to males. This pattern may reflect the compounded effect of higher pain prevalence, reduced social support, and fewer coping mechanisms among female patients, contributing to increased emotional and physical strain. These findings emphasize the need for comprehensive care approaches that address mental health, improve sleep quality, and support overall quality of life in female MSK patients. Previous studies reinforce our findings on the emotional and quality-of-life impacts of MSK conditions in female patients. Björnsdóttir et al. similarly reported that chronic musculoskeletal pain is linked to poor mental health, diminished quality of life, and disrupted sleep in women , mirroring the challenges we observed. Heikkinen et al. also found that common musculoskeletal conditions are associated with worsened mental health, especially in adults over 45 , aligning with our study’s results on mental health impact in female MSK patients. Additionally, Molina et al. observed significant psychological distress and reduced quality of life in adolescents with idiopathic MSK pain, suggesting that these effects span across ages . Together, these studies and our findings underscore the need for targeted mental health and quality-of-life interventions for female MSK patients. Gender-based disparities in occupational strain and workplace support among MSK patients Our study reveals notable gender-based differences in occupational strain and workplace support for patients with musculoskeletal (MSK) conditions. Male patients reported significantly higher workloads and more frequent work-related injuries, likely due to greater engagement in physically demanding roles, which may exacerbate MSK-related risks and severity. Conversely, female patients reported less access to recreational activities, received less workplace support, and demonstrated a stronger interest in modifying their work environments or patterns. This disparity suggests that female-dominated roles may offer fewer opportunities for physical respite and support, which could intensify MSK-related strain. Addressing these differences through targeted workplace interventions—such as injury prevention programs for men and increased support and recreation access for women—may help alleviate the occupational burden of MSK conditions for both genders. Previous research on the occupational impact of MSK complaints is sparse. However, Wolf et al. reported similar findings, noting that men with MSK conditions face higher workloads and more work-related injuries, while women have less access to recreational activities and workplace support, and show more interest in changing work environments . These findings underscore the importance of further research into the occupational ramifications of MSK conditions to develop effective, gender-sensitive interventions. Integrating patient perspectives and multidisciplinary approaches in musculoskeletal pain management Addressing musculoskeletal (MSK) complaints effectively requires integrating patient perspectives and expectations into care strategies, as this approach has been shown to enhance satisfaction and treatment outcomes . Understanding individual experiences enables clinicians to align treatment goals with patient needs, fostering trust and improving adherence to management plans. Additionally, managing MSK conditions through a multidisciplinary team, including physicians, nurses, and physiotherapists, has demonstrated superior outcomes by addressing the complex and multifaceted nature of these conditions . This collaborative approach ensures comprehensive assessments, personalized interventions, and enhanced patient satisfaction. Together, these strategies highlight the importance of patient-centered and team-based care in optimizing MSK management and improving healthcare delivery. Need for policy development and research in Jordan and the broader middle east Our study highlights a critical need for targeted health policy development in Jordan and the broader Middle East to address the gender-specific impacts of musculoskeletal (MSK) conditions. Findings from our research reveal notable differences in pain prevalence, treatment approaches, occupational strain, and psychosocial effects between male and female MSK patients. However, there is a distinct lack of research investigating these issues within Middle Eastern populations. Existing studies on MSK conditions are primarily focused on Western contexts, where health policies, social norms, and workplace practices differ considerably from those in the Middle East, leaving critical gaps in region-specific knowledge. Addressing these gaps requires comprehensive, gender-sensitive research across the Middle East to understand the unique social, cultural, and occupational factors influencing MSK conditions. Such studies could inform the development of policies tailored to the needs of Middle Eastern populations, ultimately guiding healthcare practices, workplace safety standards, and psychosocial support mechanisms for MSK patients. Implementing these policies would promote more equitable healthcare access, safer work environments, and stronger support systems, enhancing quality of life for MSK patients in Jordan and the broader region. Strength and limitations Our study provides valuable insights into musculoskeletal (MSK) complaints and their management in a Jordanian tertiary care setting, with notable strengths and limitations. A key strength is the focus on gender-specific differences, addressing significant gaps in regional literature, while the urban hospital setting allowed us to capture a broad and diverse patient profile reflective of similar healthcare contexts. The use of a structured protocol ensured reliable data collection, and the findings offer a foundation for future research and policy development. However, the cross-sectional design limits longitudinal analysis, and the use of a self-created questionnaire may reduce comparability with validated tools. Additionally, reliance on self-reported data introduces potential subjective bias, and the biological or social mechanisms behind observed gender differences were not explored. Despite these limitations, the study provides essential data to guide clinical practices and inform healthcare strategies in the region. Our findings indicate that female patients are significantly more likely to report pain than male patients, with a particular prevalence of low back and hip pain. Low back pain was notably more common among females, while hip pain was reported exclusively by female patients, underscoring a clear gender-specific pattern. These results align with previous studies suggesting that biological, hormonal, and psychosocial factors contribute to the higher pain prevalence and sensitivity observed in females. Research consistently supports the greater prevalence of low back pain (LBP) in women compared to men. For instance, Wáng et al. and Brady et al. found that females exhibit higher rates of low back pain across all age groups, with this gender difference widening post-menopause . Schneider et al. reported a 40% prevalence of back pain among women in Germany, notably higher than in men, attributing this disparity to hormonal changes, psychological influences, and anatomical differences . Furthermore, Chenot et al. observed that women not only have lower functional capacity but also higher rates of chronic LBP and a greater likelihood of experiencing depression related to LBP . Our findings, consistent with these studies, underscore the need for gender-sensitive pain management approaches to address the specific pain patterns and experiences of female patients effectively. Notably, our study observed a higher prevalence of hip pain among female patients compared to males. This finding may be attributed to a higher incidence of hip pathologies, such as hip dysplasia, prevalent within our population . However, it is important to highlight that previous studies have not specifically examined the prevalence of hip pain among patients visiting family or orthopedic clinics without a prior diagnosis of hip pathology. This gap underscores the unique contribution of our study in highlighting hip pain patterns in a general clinical population, pointing to a need for further research on undiagnosed hip pain, especially in primary care settings. Our study found that female patients were more likely than males to receive analgesia, referrals to physical therapy, and diagnostic testing during clinic visits. This trend may reflect the higher prevalence and intensity of musculoskeletal pain reported by females, especially in low back and hip pain, which likely prompts more extensive management and evaluation. Additionally, healthcare providers may view female patients as needing broader diagnostic and therapeutic support due to gender-based differences in pain presentation and reporting patterns. These findings suggest that clinicians may adopt a more comprehensive approach to meet the specific healthcare needs of female patients, integrating diverse diagnostic and treatment measures. Our findings align with Barr et al., who observed that females generally use more pain-relieving medications, though it remains uncertain whether this is due to actual usage differences or potential reporting bias . In contrast, Weimer et al. and Procento et al. found that females are less frequently prescribed strong opioids compared to males , while Richardson reported that women are more likely to visit emergency departments for pain-related complaints, leading to higher rates of diagnostic evaluations . Our results align with these findings, demonstrating that female patients not only receive more referrals to physical therapy and diagnostic evaluations but also utilize a broader range of pain management strategies compared to their male counterparts. Our study found that male patients with musculoskeletal (MSK) conditions report higher levels of disability, lower functional status, and greater anxiety about potential loss of employment or income compared to female patients. This suggests that MSK conditions may affect men differently, likely due to societal expectations that emphasize physical capacity and occupational stability. Physical limitations from MSK conditions can directly impact male patients’ job performance, particularly in physically demanding roles, potentially explaining the increased levels of disability and functional impairment we observed. Additionally, as males often carry primary financial responsibilities, their concerns about job and income stability may be heightened. These findings highlight the importance of integrated MSK management that includes occupational support to address both physical limitations and economic concerns specific to male patients. Our findings align with gender-based patterns reported in prior research on MSK disorders, with notable similarities and contrasts. Consistent with our results, Wolf et al. and Raina et al. observed that males with MSK conditions experience greater disability, reduced functional status, and higher employment anxiety . Conversely, Holland et al. and Stubbs et al. reported that females often experience more pain-related disability and are more likely to leave employment, potentially reflecting a different coping mechanism compared to males . Supporting our observation of functional impairment in male patients, Zink et al. found severe disability in males with specific MSK conditions like ankylosing spondylitis , while Wijnhoven et al. identified greater work-related disability in men with low back pain . Additionally, Bailey et al. noted that men are more frequently referred to mental health services for work-related issues, suggesting added psychological strain in employment settings . Together, these studies underscore the complex employment-related challenges faced by male MSK patients and the need for occupational and psychological support tailored to their specific needs. Our study found that female patients with musculoskeletal (MSK) conditions reported lower levels of social support, a greater fear of disability and loss of independence, fewer coping mechanisms, and increased anxiety about disease progression compared to their male counterparts. These findings may reflect social and cultural dynamics in which females have limited access to social resources or perceive less support in managing chronic health conditions. This reduced social support, combined with an elevated fear of disability, could contribute to heightened anxiety, as managing chronic pain without sufficient support or effective coping strategies can intensify concerns about the condition’s impact on their future independence. These observations underscore the need for integrated support systems and psychosocial interventions that enhance coping skills and directly address the unique challenges faced by female MSK patients. Previous research supports these findings and offers further insight into the coping challenges and psychosocial dynamics specific to female MSK patients. Grossi et al. observed that females with musculoskeletal pain report lower coping capacity, which correlates with higher levels of emotional distress and greater disability . Similarly, Soares et al. found that lower self-esteem among female patients with MSK pain is associated with increased anxiety and depression, while positive self-esteem aligns with active coping mechanisms . Espwall et al. noted that women with undefined MSK disorders receive less emotional support than those with non-MSK conditions, potentially exacerbating their psychosocial burdens . Further, Cheng et al. reported that females with chronic MSK pain experience worse physical functioning than males . Together, these studies and our findings highlight the importance of tailored psychosocial support to bolster coping strategies and enhance social support for female MSK patients, aiming to improve both mental health and physical outcomes. Our study found that female patients with musculoskeletal (MSK) conditions experience a greater impact on emotional well-being, with lower reported quality of mental health, poorer sleep quality, and a reduced overall quality of life compared to males. This pattern may reflect the compounded effect of higher pain prevalence, reduced social support, and fewer coping mechanisms among female patients, contributing to increased emotional and physical strain. These findings emphasize the need for comprehensive care approaches that address mental health, improve sleep quality, and support overall quality of life in female MSK patients. Previous studies reinforce our findings on the emotional and quality-of-life impacts of MSK conditions in female patients. Björnsdóttir et al. similarly reported that chronic musculoskeletal pain is linked to poor mental health, diminished quality of life, and disrupted sleep in women , mirroring the challenges we observed. Heikkinen et al. also found that common musculoskeletal conditions are associated with worsened mental health, especially in adults over 45 , aligning with our study’s results on mental health impact in female MSK patients. Additionally, Molina et al. observed significant psychological distress and reduced quality of life in adolescents with idiopathic MSK pain, suggesting that these effects span across ages . Together, these studies and our findings underscore the need for targeted mental health and quality-of-life interventions for female MSK patients. Our study reveals notable gender-based differences in occupational strain and workplace support for patients with musculoskeletal (MSK) conditions. Male patients reported significantly higher workloads and more frequent work-related injuries, likely due to greater engagement in physically demanding roles, which may exacerbate MSK-related risks and severity. Conversely, female patients reported less access to recreational activities, received less workplace support, and demonstrated a stronger interest in modifying their work environments or patterns. This disparity suggests that female-dominated roles may offer fewer opportunities for physical respite and support, which could intensify MSK-related strain. Addressing these differences through targeted workplace interventions—such as injury prevention programs for men and increased support and recreation access for women—may help alleviate the occupational burden of MSK conditions for both genders. Previous research on the occupational impact of MSK complaints is sparse. However, Wolf et al. reported similar findings, noting that men with MSK conditions face higher workloads and more work-related injuries, while women have less access to recreational activities and workplace support, and show more interest in changing work environments . These findings underscore the importance of further research into the occupational ramifications of MSK conditions to develop effective, gender-sensitive interventions. Addressing musculoskeletal (MSK) complaints effectively requires integrating patient perspectives and expectations into care strategies, as this approach has been shown to enhance satisfaction and treatment outcomes . Understanding individual experiences enables clinicians to align treatment goals with patient needs, fostering trust and improving adherence to management plans. Additionally, managing MSK conditions through a multidisciplinary team, including physicians, nurses, and physiotherapists, has demonstrated superior outcomes by addressing the complex and multifaceted nature of these conditions . This collaborative approach ensures comprehensive assessments, personalized interventions, and enhanced patient satisfaction. Together, these strategies highlight the importance of patient-centered and team-based care in optimizing MSK management and improving healthcare delivery. Our study highlights a critical need for targeted health policy development in Jordan and the broader Middle East to address the gender-specific impacts of musculoskeletal (MSK) conditions. Findings from our research reveal notable differences in pain prevalence, treatment approaches, occupational strain, and psychosocial effects between male and female MSK patients. However, there is a distinct lack of research investigating these issues within Middle Eastern populations. Existing studies on MSK conditions are primarily focused on Western contexts, where health policies, social norms, and workplace practices differ considerably from those in the Middle East, leaving critical gaps in region-specific knowledge. Addressing these gaps requires comprehensive, gender-sensitive research across the Middle East to understand the unique social, cultural, and occupational factors influencing MSK conditions. Such studies could inform the development of policies tailored to the needs of Middle Eastern populations, ultimately guiding healthcare practices, workplace safety standards, and psychosocial support mechanisms for MSK patients. Implementing these policies would promote more equitable healthcare access, safer work environments, and stronger support systems, enhancing quality of life for MSK patients in Jordan and the broader region. Our study provides valuable insights into musculoskeletal (MSK) complaints and their management in a Jordanian tertiary care setting, with notable strengths and limitations. A key strength is the focus on gender-specific differences, addressing significant gaps in regional literature, while the urban hospital setting allowed us to capture a broad and diverse patient profile reflective of similar healthcare contexts. The use of a structured protocol ensured reliable data collection, and the findings offer a foundation for future research and policy development. However, the cross-sectional design limits longitudinal analysis, and the use of a self-created questionnaire may reduce comparability with validated tools. Additionally, reliance on self-reported data introduces potential subjective bias, and the biological or social mechanisms behind observed gender differences were not explored. Despite these limitations, the study provides essential data to guide clinical practices and inform healthcare strategies in the region. Based on our study findings, several clinical implications emerge. Gender differences in pain prevalence and intensity, particularly the higher rates of low back and hip pain among female patients, emphasize the importance of adopting individualized pain management strategies. Tailored evaluations and treatments that consider gender-specific pain patterns are essential for improving patient outcomes and enhancing treatment adherence. The study also highlights the need for integrated psychosocial support services. Female patients reported a greater impact on emotional well-being, lower mental health scores, and increased anxiety about disease progression. To address this, clinics should incorporate routine mental health screenings and psychosocial support into musculoskeletal (MSK) care. Providing resources for coping strategies, emotional support, and stress management can significantly reduce mental health burdens and improve the overall quality of life for these patients. Workplace-focused interventions are crucial for addressing the higher incidence of work-related injuries and physically demanding workloads among male patients. Collaborations with occupational health programs can provide injury prevention guidance, ergonomic recommendations, and workplace safety consultations. Such initiatives are especially beneficial for patients engaged in physically intensive roles. Proactive monitoring and follow-up care are also essential, particularly for female patients who received more diagnostic tests and physical therapy referrals. Establishing structured follow-up systems ensures comprehensive care continuity and enables timely reassessment of pain and functional status. This approach is particularly beneficial for high-risk groups, optimizing long-term outcomes and preventing the progression of MSK conditions. Lastly, the findings reveal the need for strengthening social and mental health support for female patients. Many reported lower social support and higher anxiety related to disease progression and disability. Clinics should prioritize developing support networks and mental health resources tailored to women. Initiatives such as peer support groups, community resources, anxiety management workshops, and individual counseling can help patients manage fears about future disability, foster resilience, and improve emotional well-being. In conclusion, our study reveals significant gender-based differences in musculoskeletal complaints, treatment approaches, and patient perceptions in a Jordanian tertiary care setting. Female patients experienced higher rates of low back and hip pain, more frequent diagnostic testing and physical therapy referrals, as well as greater anxiety about disease progression, lower social support, and reduced quality of life. In contrast, male patients reported higher occupational workloads, more work-related injuries, increased disability, and greater anxiety about income loss due to MSK conditions. These findings highlight the importance of gender-sensitive approaches in MSK management that address both physical and psychosocial needs to improve patient outcomes and quality of life. |
A Retrospective Cohort Study on Scan Quality of Implant Scanbodies Matched With | 1cf499ee-dbdb-47b3-b1c7-8a0b40f47e98 | 11841021 | Dentistry[mh] | Introduction Intraoral scanners are commonly used in the treatment of short‐span edentulous areas for both tooth‐ and implant‐supported fixed dental prostheses (FDP) . However, the digitization of dental implants significantly differs from tooth surfaces not in scanning principles but in requirements for appropriate data acquisition. This is basically related to the necessity to accurately transfer the 3‐D spatial position of the implant in the jaw. To facilitate this, a transfer device known as scanbody (SB) is used to capture and integrate the implant's position into the dental arch scan . Fundamentally, this is a data exchange process carried out in the dental CAD software interface to initialize restorative design. SBs are characterized by a variety of features that are related to both hardware and software specifications. The hardware‐related specifications of a SB can be described in three sections. The scan region and the base present the functionality, which the former serves as the primary reference area for dental CAD software matching, and the latter connects SB either to implant or abutment. Third, the body provides the connection between the scan region and the base . The software specification feature involves activating the SB library through precise alignment between the scan file and its geometrically perfect CAD file . Subsequent to automated virtual 3D implant positioning, restoration design can be executed utilizing the relevant library elements. User defined factors affecting the accuracy of intraoral scanning data are fundamentally scanning strategy , ambient lighting conditions , and operator experience . In addition to these general factors, the impact of SB hardware specifications coupled with implant location, angulation, and number on scanning accuracy remains debatable. SBs can be designed as either single‐piece or two‐piece configurations. Single‐piece designs are typically composed of metal or PEEK, whereas two‐piece designs involve a combination of two materials, often with a metal implant connection in such designs. The stability of the implant‐SB connection has been hypothesized to influence 3D positional transfer . Conversely, Sawyers and Baig reported that reuse of single‐piece PEEK SBs may not compromise registration accuracy. Apart from this, it has been documented that the machinability of the material utilized in production influences the accuracy of alignment between the SB scan file and the CAD library file . Moreover, SBs can exhibit a variety of geometric configurations including cylindrical, conical, stepped, rectangular, or combinations thereof . It has been documented that SBs with simpler geometric shapes are easier to scan, minimizing the potential scanning errors , and leading to a manageable processability of the acquired images . Scientific knowledge on this topic is mostly derived from experimental studies lacking in simulating intraoral conditions. Therefore, optimal design regarding the geometrical features of a SB for accurate scanning remains unclear. The SB scan file, simultaneously created by the intraoral scanner's algorithm, is matched with the library file in the dental CAD software using a best‐fit algorithm. The amount of surface available for the best fit can be decided and adjusted by a special function in order to improve the overlap. Depending upon the area for best fit alignment, the position of the virtual analogue may change. Beyond this, as intraoral scanning of an SB technically presents imperfections in quality, the overlapping of the generated stl file in comparison to its library CAD stl file would likely be affected. The current literature lacks information on the scan quality of SBs matched with the corresponding digital libraries. This retrospective cohort study, therefore, aimed to evaluate the scan quality of the SBs matched with their designated libraries in understanding the vulnerability in SB scanning. The null hypothesis of the study posits that the parameters of SB type, edentulous site, and restoration type do not affect the scan quality. Materials and Methods 2.1 Study Design This retrospective cohort study was designed to assess the surface scan quality of library‐matched SBs for single tooth implant crowns (iC) or implant‐fixed dental prostheses (iFDP) with two implants. The evaluation was categorically performed in monochrome view on post‐processed data using the interface of the scanner (3Shape A/S v22.1 TRIOS3, Copenhagen, Denmark) and conducted by the same researcher. 2.2 Data Collection In the study, SBs connected to the bone‐level dental implants (Straumann, BLT/BL RC/NC, Straumann Holding AG, Basel, Switzerland) placed in the maxilla and mandible for the treatment of short edentulous spans with iC and iFDPs between January 2017 and January 2021 were considered. The included SBs were digitized following a 2‐stage scanning protocol applying cut‐out and rescan procedure, and matched with the appropriate library in dental CAD software (3Shape Dental System v2020, 3Shape A/S) (Figure ). Intraoral scans were performed by the senior researcher (KA), while the evaluation of SB scan quality was carried out by the other researcher (FD). Prior to quality assessment, randomly selected SB scans ( n = 50) not included in the study were exercised between the researchers for intra‐observer (FD) consistency. For the SBs enrolled in the study, the cases that remained with uncertainty were resolved with inter‐observer discussion. 2.3 Data Variables SB type, edentulous site, and restoration type were the considered independent variables. Two different SBs were assessed: a one‐piece PEEK original SB (CARES RC/NC Mono Scanbody; Institut Straumann AG) and a one‐piece aluminum nonoriginal SB (Scanbody; 3Shape A/S) compatible with the implant (Figure ). Edentulous site was identified as maxilla and mandible, either with anterior or posterior location, and their treatment was categorized restoration types as iC and iFDP (Figure ). 2.4 Data Evaluation To appraise SB scan quality, three categorical domains were defined: SB‐reference area, SB‐scan, and SB‐representation (Figure ). In the qualitative evaluation, to begin with, the scanning in the SB‐reference region was recorded as either complete or incomplete. In addition, the SB‐scan assessment was carried‐out for two sections of SB: the body and the base. Scanning in the body was recorded as complete or incomplete, whereas the base was described with regard to the merging of the rescan into the first scan sufficiently or not. Furthermore, SB‐representation was evaluated in terms of surface reconstruction utilizing the image stitching algorithm. This comprised geometry and texture evaluation as paired or impaired for the former and smooth or rough for the latter. Definitions for the evaluation of SB scan are described in Table . 2.5 Statistical Analysis Chi‐squared test was applied to compare the frequencies of the clinical variables, and logistic regression models were constructed to find the factor that affects SB scan quality. The candidate variables for multiple logistic regression models were specified based on univariate logistic regression models with p value ≤ 0.20. With those candidate variables, backward elimination was applied to present the final model. In final models, the odds ratio (OR) with a 95% confidence interval (CI) was represented. Kappa statistics were applied to identify inter‐rater reliability. Statistical analysis was performed using a statistical software program (SPSS Statistics, v23.0; IBM Corp, New York, USA). The significance level was set at p < 0.05. Study Design This retrospective cohort study was designed to assess the surface scan quality of library‐matched SBs for single tooth implant crowns (iC) or implant‐fixed dental prostheses (iFDP) with two implants. The evaluation was categorically performed in monochrome view on post‐processed data using the interface of the scanner (3Shape A/S v22.1 TRIOS3, Copenhagen, Denmark) and conducted by the same researcher. Data Collection In the study, SBs connected to the bone‐level dental implants (Straumann, BLT/BL RC/NC, Straumann Holding AG, Basel, Switzerland) placed in the maxilla and mandible for the treatment of short edentulous spans with iC and iFDPs between January 2017 and January 2021 were considered. The included SBs were digitized following a 2‐stage scanning protocol applying cut‐out and rescan procedure, and matched with the appropriate library in dental CAD software (3Shape Dental System v2020, 3Shape A/S) (Figure ). Intraoral scans were performed by the senior researcher (KA), while the evaluation of SB scan quality was carried out by the other researcher (FD). Prior to quality assessment, randomly selected SB scans ( n = 50) not included in the study were exercised between the researchers for intra‐observer (FD) consistency. For the SBs enrolled in the study, the cases that remained with uncertainty were resolved with inter‐observer discussion. Data Variables SB type, edentulous site, and restoration type were the considered independent variables. Two different SBs were assessed: a one‐piece PEEK original SB (CARES RC/NC Mono Scanbody; Institut Straumann AG) and a one‐piece aluminum nonoriginal SB (Scanbody; 3Shape A/S) compatible with the implant (Figure ). Edentulous site was identified as maxilla and mandible, either with anterior or posterior location, and their treatment was categorized restoration types as iC and iFDP (Figure ). Data Evaluation To appraise SB scan quality, three categorical domains were defined: SB‐reference area, SB‐scan, and SB‐representation (Figure ). In the qualitative evaluation, to begin with, the scanning in the SB‐reference region was recorded as either complete or incomplete. In addition, the SB‐scan assessment was carried‐out for two sections of SB: the body and the base. Scanning in the body was recorded as complete or incomplete, whereas the base was described with regard to the merging of the rescan into the first scan sufficiently or not. Furthermore, SB‐representation was evaluated in terms of surface reconstruction utilizing the image stitching algorithm. This comprised geometry and texture evaluation as paired or impaired for the former and smooth or rough for the latter. Definitions for the evaluation of SB scan are described in Table . Statistical Analysis Chi‐squared test was applied to compare the frequencies of the clinical variables, and logistic regression models were constructed to find the factor that affects SB scan quality. The candidate variables for multiple logistic regression models were specified based on univariate logistic regression models with p value ≤ 0.20. With those candidate variables, backward elimination was applied to present the final model. In final models, the odds ratio (OR) with a 95% confidence interval (CI) was represented. Kappa statistics were applied to identify inter‐rater reliability. Statistical analysis was performed using a statistical software program (SPSS Statistics, v23.0; IBM Corp, New York, USA). The significance level was set at p < 0.05. Results Table presents the descriptive statistics of 243 SBs enrolled in the cohort. The categorical assessments of SB scan quality and Cohen's κ‐coefficients are shown in Table . SB scan quality at SBs' three major sections, reference area, body, and base, was exceptionally high 97,5%, 92,6%, and 100%, respectively. Undecided SB representation geometry and texture resolved upon inter‐observer discussion were six and one, respectively. The logistic regression analysis outcomes are displayed at Table . The generated models revealed that SB type significantly affects the SB scan quality in the SB representation domain for both geometry (< 0.001) and texture (< 0.001), and the relationship between jaw and SB representation in geometry and texture is significant as well (0.003 and < 0.001, respectively). Restoration type, crown versus FDP, was significantly effective on scan quality at SB's scan at the body region (0.004). In evaluation of SB‐type variable, cross tabulation presented that 79.5% and 75% of the impairment in geometry and roughness in texture, respectively, is related to original SBs' scan quality. Further comprehension along with the logistic regression model is that the risk of impaired geometry for original SBs is 4.462 times higher than for nonoriginal SBs, and the occurrence of surface roughness is 6.663 times higher as well (Table ). Regarding the edentulous‐site variable, SB scan quality is being negatively affected in the mandible than in the maxilla by 3.378 (1/0.296) and 4.081 (1/0.245) times for representation in geometry and texture, respectively (Table ). Taking the restoration‐type variable into consideration, 83.3% of incompleteness of SB scanning in the body region for iCs compared to 16.7% for iFDPs is noticeable with cross tabulation. This remained statistically significant in the logistic regression model, with the odds ratio indicating that the risk of incompleteness for iCs was 6.598 times higher compared with iFDPs (Table ). Discussion In this retrospective cohort study, the effect of SB type, edentulous site, and restoration type on scan quality of SB‐reference area, SB‐scan, and SB‐representation was categorically evaluated. As the outcomes revealed that at least one domain in the assessment of SB scan quality was significantly affected by one of the variables, the null hypothesis was rejected. The eligible SBs for scan quality assessment were scanned with a staged approach in which implant digitalization was finalized following implant region cut‐out from the initial arch scan, and then rescan to merge SB scan into the arch scan. Outcomes of the studies on scanning accuracy for cut‐out and rescan protocol are inconclusive . In the current study, all scan quality evaluations for SBs' base region presented sufficient integration between the two scans for the SB type variable. Therefore, we may imply that SBs differing in material, original SB with peek versus non‐original SB with aluminum, are predictably scannable in terms of the SB base to follow the 2‐stage scan protocol. SB representation was the most affected domain in scan quality evaluation as roughed texture and impaired geometry, which were recorded at 44.4% and 16%, respectively. In essence, it is not surprising due to challenges, particularly for the image‐stitching algorithm in intraoral scanning of geometric and monochromatic surfaces. With this, one may assume SB representation would be poorer in cases with extended edentulous spans, literally presenting reduced commonalities for the image‐processing algorithm. It has been presented that precision had been significantly affected by the SB design and geometry under extraoral scanning conditions . Moreover, a recent systematic review based on in vitro studies reported that SB geometry affected scanning accuracy . In this cohort, impaired geometry in SB representation was significantly different for the SB type variable and roughed texture as well. Accordingly, various geometrical features in the non‐original SB were helpful for deformation‐free SB scan in comparison to the original SB, which is limited to cylindrical shape with beveled reference scan region (Figure ). Authors believe that the irregularities, even geometrical, facilitate image stitching in the algorithm with the scanner using optical sectioning technology. Park and Choi addressed deficiencies in SB scanning in an in vitro study. The SB scan data was modified to simulate incomplete scanning prior to surface alignment with the CAD library. They concluded that based on the created defect size, the implant positioning accuracy in the dental CAD software decreases. Finally, they posited that the restorations fabricated upon that data would remain beyond clinically acceptable limits. In the current study, SB scan quality was categorically evaluated, and incomplete reference and scan‐body scanning clinically reduced the scan quality of the SBs by 9.9%. Furthermore, the outcomes clearly demonstrated that SB scan quality would be dramatically decreased when the challenges in representation for deformation‐free SB scanning are considered. Therefore, simply matching the SB scan with its library CAD file may not assure predictable restorative outcomes. As the focus on the target for the current study was to understand how clinical variables interact with SB scan quality, further studies are needed to explore the interplay between SC scan quality and restorative outcomes. The effect of the restoration type variable on scan quality categorically for SB scan at body was remarkable with the studied regression model. It was predicted that incompleteness in SB scan at body is 6.598 times more prevalent for iCs compared with iFDPs. This outcome may be explained by the limitation of the scanner's field of view due to the proximity of SB to the adjacent teeth for iC restorations. Having significantly favorable outcomes at SB representation for non‐original SB, we recommend the use of a SB with geometrical features for single‐tooth spaces in clinical practice. It is essential to acknowledge that intraoral scanners have the potential to introduce scanning errors in the stitching of images due to a lack of stable reference points . This fact can explain why the mandible demonstrated a significantly higher risk for roughed texture at SB representation. This challenge may become even more complicated in cases with free‐end edentulism . Remembering that the scanning of non‐anatomical surfaces couples the defined difficulties, it is recommended to use appropriate tools to move away movable tissues such as the buccal and lingual areas from the teeth and to keep the scanning field as dry as possible. For such challenging cases, the use of SBs with geometrical features may lower the probability of reduced scan quality specifically for SB representation. The non‐homogeneity in the number of SBs between anterior and posterior edentulous sites represents a potential limitation of this study. Another limitation may be addressed as information lacking in stability of SB to implant connection with regard to multiple use of SBs. Furthermore, additional information on how scan quality affects the restorative outcomes would be invaluable for correct clinical interpretations. Conclusion Scanbody‐, restoration‐type, and edentulous site are the clinical variables presenting significant influence on SB scan quality in which SB representation is the most affected. It may be suggested to prefer an SB with distinct geometrical variations for an implant when there are options to choose from. K.A. conceived the ideas, administered the project, wrote the original draft, reviewed, and edited the manuscript, approval of article. F.D.B. collected the data, wrote the draft, reviewed, and edited the manuscript. K.A. and F.D.B. analyzed and interpreted the data. This study was approved by the NonInterventional Clinical Research Ethics Committee of Hacettepe University (Protocol Code: GO 21/848). The authors declare no conflicts of interest. |
Evaluation of the Effect Coronavirus Lockdown had on Chronic Disease Management Care in Pediatrics: A Survey of Jordanian Pediatricians | d54cc164-16cd-4677-b103-850144f17d5b | 9560848 | Pediatrics[mh] | The initial discovery of coronavirus, also known as (SARS-COV-2), began from patients suffering from pneumonia. The first case of coronavirus was reported in December 2019 in Hubei province, China. The World Health Organization (WHO) pronounced the spread of this virus on 30 th January 2020 and later in March 2020, and the disease was declared as a global pandemic . By April 2022, over six million individuals died with COVID-19. Lockdown and social distancing were the major practices implemented in an effort to contain the pandemic. This has led to drastic changes in daily routines, eating habits, and physical activities . Caregivers of children suffering from chronic diseases are challenged with their daily routines of exercise, diet, and the need for regular follow-up. Strict lockdown and social distancing related to COVID-19 have rendered coping with existing chronic diseases much more challenging. This is related to the lack of access to health facilities and healthcare providers, especially in developing countries . The influence of COVID-19 on the healthcare system was tremendous and multifaced. There was an overwhelming demand for healthcare system services, workers, and health facilities. During the different phases of complete and partial lockdown, healthcare access was compromised. While some of the policies adopted by the governments were strict, they affected the unique patient population. This is especially true for children with chronic diseases who found it difficult to reach their caring specialists or health care providers . On the other hand, indirect effects of epidemics such as in 2003, the Severe Acute Respiratory Syndrome (SARS) epidemic , and the Ebola virus epidemic of 2014 in West Africa were seen through history . Meanwhile the pediatric patients with chronic diseases have shown some deterioration in management and follow up during lockdown . The long-lasting impact of the disruption of care of patients with chronic diseases is likely to surpass the duration of the COVID-19 pandemic. World Health Organization (WHO) reported that irrespective of the disease prognosis, patients presenting with different co-morbidities have a high risk for severe COVID-19 disease course and complications . In pediatric patients with chronic diseases such as chronic lung disease, primary immune deficiency, chronic renal disease, and neurological disorders, the risk for morbidity and mortality of COVID-19 disease is increased . In order to provide the optimum management for those children, the necessary precautions should be planned for, and follow-up pathways should be specified . The current study was aimed to assess the effects of lockdown on maintaining an acceptable chronic disease follow up in pediatric population. This subgroup of patients might face a more difficult course in the management and follow up especially taking into consideration that telemedicine is less reliable in this population . The studies in this subgroup are scarce and more focused on adult population rather than pediatrics. . This study should establish how coronavirus lockdown was impacting chronic disease management in pediatric patients admitted to hospitals in a developing country setting. 2.1. Research Design A cross-sectional study based on online survey. 2.2. Sampling All Jordanian pediatricians ( n = 840), who are registered in the Medical Union and the Jordanian Pediatric Society, were contacted through their registered official e-mail and phone number. Registration in the union is an obligation to practice pediatrics in Jordan. The sample included mainly general pediatricians from all medical sectors; governmental entities, universities, and the private sectors. Pediatricians with subspeciality are scares in Jordan leading to the fact that most children with chronic diseases are followed up by general pediatricians. Moreover, pediatricians in Jordan are subdivided into residents, who are under training (yet in the COVID pandemic compromised the majority of working physicians), specialists, and consultants. Pediatric specialists and consultants carry out the same duties but with difference in years of experience where consultants should have more than 10 years of experience in their field. The sample size of this study was calculated through the sample size calculator. A representative sample size of pediatricians in Jordan was calculated to be 264 participants providing 95% confidence interval and 5% margin error for a population of 840 active pediatricians. 2.3. Instruments and Data Collection The study instrument was pilot tested on a sample of 10 pediatricians. Statisticians approved the questionnaire after the pilot testing. The ten pilot test participants were included in the study sample as there were no changes done to the questionnaire. The study was an open-ended questionnaire study comprised of valid cross-examinations to capture the responses from selected research contributors. The questionnaire was constructed using Google Forms. An online link to the survey was sent to registered pediatricians through their official emails and phone numbers. The questionnaire was distributed on June 16, 2020, and the form was closed to responses when we reached the desired sample size on September 30, 2020. The response rate was 31.4%. Data were retrieved in Excel and analyzed using SPSS version 25 (SPSS Inc. Chicago, Illinois, USA). 2.4. Ethical Considerations This research activity was revised then attained an endorsement from the IRB committee, Faculty of Medicine at Hashemite University, Jordan. The objectives of the study were explained at the beginning of the questionnaire to all participating pediatricians. 2.5. Data Analysis Data captured during this study were inspected using both quantitative and qualitative methods. Quantification of the research was carried out through expressing responders' answers by frequencies and percentages. Univariate analysis was carried out to test the association between the level of practice of responders, and characteristics of pediatric health services provided to patients during the pandemic. In cases where the chi-square test for association showed statistically significant relationships ( p < 0.05), Cramer's V coefficient was used to assess the magnitude and strength of nominal-by-nominal relationship. The following “crude estimate” values were considered for interpreting the strengths of relationships. The absolute value of the coefficient of <0.19 was considered a negligible relationship, 0.2–0.29 was considered a weak relationship, and 0.30–0.39 was considered a moderate relationship. In cases where a statistically significant association and moderate strength relationships were demonstrated, further post hoc analysis was carried out through a cell-by-cell comparison approach. The adjusted standardized residual was used to provide post hoc evidence of association. If an adjusted standardized residual is positive, it indicates that there are more observed frequencies than expected frequencies. If an adjusted standardized residual is negative, it indicates that there are less observed frequencies than expected frequencies given the null hypothesis of independence. If the standardized residual had an absolute value > 3, then the lack of independence is further approved. This indicates that the variables tested are either positively or negatively associated as confirmed by the post hoc evidence of association. A cross-sectional study based on online survey. All Jordanian pediatricians ( n = 840), who are registered in the Medical Union and the Jordanian Pediatric Society, were contacted through their registered official e-mail and phone number. Registration in the union is an obligation to practice pediatrics in Jordan. The sample included mainly general pediatricians from all medical sectors; governmental entities, universities, and the private sectors. Pediatricians with subspeciality are scares in Jordan leading to the fact that most children with chronic diseases are followed up by general pediatricians. Moreover, pediatricians in Jordan are subdivided into residents, who are under training (yet in the COVID pandemic compromised the majority of working physicians), specialists, and consultants. Pediatric specialists and consultants carry out the same duties but with difference in years of experience where consultants should have more than 10 years of experience in their field. The sample size of this study was calculated through the sample size calculator. A representative sample size of pediatricians in Jordan was calculated to be 264 participants providing 95% confidence interval and 5% margin error for a population of 840 active pediatricians. The study instrument was pilot tested on a sample of 10 pediatricians. Statisticians approved the questionnaire after the pilot testing. The ten pilot test participants were included in the study sample as there were no changes done to the questionnaire. The study was an open-ended questionnaire study comprised of valid cross-examinations to capture the responses from selected research contributors. The questionnaire was constructed using Google Forms. An online link to the survey was sent to registered pediatricians through their official emails and phone numbers. The questionnaire was distributed on June 16, 2020, and the form was closed to responses when we reached the desired sample size on September 30, 2020. The response rate was 31.4%. Data were retrieved in Excel and analyzed using SPSS version 25 (SPSS Inc. Chicago, Illinois, USA). This research activity was revised then attained an endorsement from the IRB committee, Faculty of Medicine at Hashemite University, Jordan. The objectives of the study were explained at the beginning of the questionnaire to all participating pediatricians. Data captured during this study were inspected using both quantitative and qualitative methods. Quantification of the research was carried out through expressing responders' answers by frequencies and percentages. Univariate analysis was carried out to test the association between the level of practice of responders, and characteristics of pediatric health services provided to patients during the pandemic. In cases where the chi-square test for association showed statistically significant relationships ( p < 0.05), Cramer's V coefficient was used to assess the magnitude and strength of nominal-by-nominal relationship. The following “crude estimate” values were considered for interpreting the strengths of relationships. The absolute value of the coefficient of <0.19 was considered a negligible relationship, 0.2–0.29 was considered a weak relationship, and 0.30–0.39 was considered a moderate relationship. In cases where a statistically significant association and moderate strength relationships were demonstrated, further post hoc analysis was carried out through a cell-by-cell comparison approach. The adjusted standardized residual was used to provide post hoc evidence of association. If an adjusted standardized residual is positive, it indicates that there are more observed frequencies than expected frequencies. If an adjusted standardized residual is negative, it indicates that there are less observed frequencies than expected frequencies given the null hypothesis of independence. If the standardized residual had an absolute value > 3, then the lack of independence is further approved. This indicates that the variables tested are either positively or negatively associated as confirmed by the post hoc evidence of association. A total of two hundred sixty-four participants filled the online survey, providing 95% CI and 5% margin error for 840 active Jordanian pediatricians. illustrates a summary of the basic demographic characteristics of the participants. Participants were almost equally distributed across different levels of practice. Consultants were 95 (36%), specialists were 81 (30.7%), and residents were 88 (33.3%). The most frequent age category from the study was 31–40 years ( n = 120, 45.5%). The number of male participants was 148 (56.1%). Respondents from Ministry of Health hospitals n = 126 (44.7%) returned to most of the polls. Residents were the largest group of responders from the Ministry of Health (79.5%). Respondents from the private sector constituted 28.4% ( n = 75), while from the Royal Medical Services 14.8% ( n = 39) responded to the survey. Only n = 24 (9.1%) of the responses came from university hospitals. Most of the responders practiced pediatrics in the capital city-Amman (71.2%, and Supplementary ). Specific effects of COVID-19 lockdown procedures in relation to level of practice are summarized in . Due to official COVID-19 related procedures, 26.9% ( n = 71) of the respondents had their facility of practice exclusively dedicated for treating COVID-19 patients. Meanwhile, most pediatricians n = 217 (82.2%) continued practicing during the lockdown period, and 20.5% ( n = 54) of the pediatricians had COVID-19 positive patients to attend to. About, 55.6% of all patients who were diagnosed with COVID-19 came from the Ministry of Health hospitals. The Ministry of Health had the highest burden of patients during the lockdown. For example, the Ministry of Health's proportion was 75% among all other sectors for managing more than 30 patients per pediatrician per day. Residents (48.9%) reported a significantly ( p < 0.001) higher proportion providing face-to-face services compared with consultants (17.9%) and specialists (13.6%). In comparison, 34.7% of consultants provided remote services compared with 16.1% of specialists, and only 1.1% of the residents ( p < 0.001, ). Regarding the care of chronically ill children, 79% of pediatricians treated at least one child with a chronic condition, and all of them had at least one patient with a status change during the lockdown period. Almost half of such patients were stable yet needed closer follow-up and approximately 15% of patients with chronic diseases visiting pediatricians were uncontrolled and had emerging complications. The emerging complications were related to the chronic diseases the patients suffered from either flare-ups or complications due to not adhering to management. Of respondent pediatricians, 30% felt comfortable contacting the primary care physician. Among those, a good connection with the primary care team was observed, as 41.3% of respondents contacted the primary care team to manage patient care. Moreover, 43.2% of respondents reported that it was easy to contact the primary care physician. Unfortunately, according to pediatricians, 84% of caregivers faced difficulties in getting a patient prescription. Only 4.9% of caregivers were never reluctant to provide medical care during the lockdown. Even in emergencies, only 8.7% of caregivers had never avoided going to the emergency department during the lockdown . About 31% of pediatricians participating in this survey reported a loss of a chronically ill patient, not due to COVID-19 diagnosis, and approximately 9% of these pediatricians have lost more than five patients. After the experience with COVID-19 in Jordan, 40% of active pediatricians disagreed with choosing tertiary hospitals as quarantine hospitals for COVID-19 patients, and 63.3% recommended trying to pick other establishments outside large cities with less negative impacts on patients. In comparison, 21.2% agreed with the same procedures. The effect of levels of experience was studied in this questionnaire. Statistically significant differences in responses to the questionnaire were noticed in three questions. However, the magnitude and strength of association between answers and the level of specialty were weak. Female specialists were significantly more than males. Male residents were significantly more than females. However, the strength of such association was negligible ( χ 2 (4) = 18.06, Cramer's V = 0.185, p = 0.001, Supplementary ). A significantly higher proportion of all levels of specialty practiced pediatrics during the lockdown period. However, the association between variables was negligible, Cramer's V = 0.171. The association with practice level was significant χ 2 (4) = 15.62, p = 0.016. On the other hand, a higher proportion of respondents confirmed that patients were stable yet needed closer follow up; however, the association was negligible, Cramer's V = 0.172, Supplementary . A moderate strengh statistically signficant association between the place of practice and the level of practice was noticed ( p < 0.001). Residents (79.5%) at the Ministry of Health hospitals were more than the expected frequencies (Standardized Residual = 4.3). The observed frequencies at the private sector were less than expected (Standardized Residual = −3). The highest proportion of consultants was noticed at the private sector (38.9% of consultants). The lowest proportion of specialists was noticed at university hospitals (6.2%), Supplementary Table . Among all levels of practice, residents reported significantly ( p < 0.001) higher proportion (28.4%) of seeing more than 30 patients per day compared with specialists (6.2%) and consultants (6.3%). The observed frequencies were more than the expected frequencies (Standardized Residual = 3.8, Supplementary ). Delivering services by consultants through remote means, such as phone or Facebook, showed the highest level of association compared with resident and specialists ( p < 0.001, Cramer's V = 0.33 and the Standardized Residual = 3.9, Supplementary ). Despite relatively weak association (Cramer's V = 0.236), residents reported the highest frequency (19.3%) for fatalities, where consultants and specialists reported 3.2% and 3.7%, respectively. The observed frequencies were more than the expected frequencies (Standardized Residual = 3.4, Supplementary ). In Jordan, as in many other developing countries, healthcare is provided by the public and private sectors, yet the public sector carries the heaviest burden; thus, most of the respondent pediatricians were from the public sector as well as 30% of COVID-positive patients at the time of the study. In Jordan, a total of 106 public and private hospitals are equipped with more than 12,000 beds. About 67% of these beds are provided by the public sector . When COVID-19 posed a considerable burden on many countries, governments worldwide had to face the challenge and flatten the infection curve. These efforts were aimed towards minimizing the direct effect of the rapidly spreading pandemic without overwhelming the already stretched health care system. Governments should not overlook other health services, mainly as pandemics are the threat of today's world. Notably, in the countries with strict policies and lockdowns, there were reports of disrupted primary healthcare facilities such as labor and delivery services, immunization, HIV and TB care, dialysis, and cancer treatment . One of the most troublesome indirect effects of lockdown was the restriction on routine physician consultations. Consequently, children suffering from chronic illnesses, even if stable, could not access the usual values of care due to the restrictions of movement and deployment of healthcare personnel to the frontline of the COVID-19 contamination. These strict governmental policies impacted significantly the patients suffering from chronic diseases that require revisits as well as prescription refills. These impacts denied access to healthcare facilities and attending doctors . As a sequence of these policies, pediatricians who had to follow these chronically ill patients apart from their treating physicians reported that around 80% of these chronically ill children had a change of status. Although 40% of the pediatricians in this study were able to contact the subspecialists responsible for the chronically ill children in their care, 30% reported a loss of at least one of these children. Moreover, around 9% reported the loss of more than five children with chronic illnesses not due to COVID-19 infection but due to the improper management of these children in relation to the lack of the specialized facilities as these facilities were locked down for quarantine for COVID-19 patients. Multiple studies documented that complicated COVID-19 cases had pre-existing medical conditions such as chronic lung disease, cardiovascular disease, immunosuppression, obesity, and neurologic conditions. The risk of COVID-19 infection and the severity of illness in children with pre-existing conditions can be greatly reduced via abiding and adhering to proper physical distancing, hand hygiene, mask use, attending to underlying medical conditions, and management of surfacing complication under doctors' guidance . During the lockdown drastic measures, one of the solutions for establishing patient-physician contact was telemedicine. Among participants of this study, only 18% of physicians reported using social media or telemedicine to contact their patients. Virtual visits have been approved to decrease personal medical visit up to about 50% across specialties. Aydemir and colleagues reported in their study that despite poor telemedicine experience, there was an overall patient and physician satisfaction with using telemedicine in follow-up visits of chronically ill children . Virtual consultations reduce deaths besides lessening the number of days a patient stays in the hospital even if the patient is in life-threatening care situations. In developed countries, the presence of a practical infrastructure led to well-established app-based medical services . On the other hand, Mubaraki and colleagues addressed the issue of telemedicine disadvantages in their research and reported that it was difficult to reach a definitive diagnosis through remote consultations. They also reported that the fear of sudden practice changes caused by the COVID-19 was an issue that led the physicians to be prudent while using telemedicine in their practice during the pandemic . The change in the health status of these children could be attributed to one of the following factors during the lockdown; (1) patients and caregivers face difficulties reaching emergency rooms, (2) they fear that the children might get infected, or (3) they are unable to get their prescription medications, and most of the time a combination of all. Myonihan and colleagues highlighted the long-term impacts of missed care of those without COVID-19 disease during the pandemic. It was recommended that public campaigns should urge public and caregivers to seek medical care when needed as evidence by excess population mortality that was demonstrated in several studies . The direct and indirect effects of lockdown and quarantine policies should always be thoroughly studied before imposing strict policies and defense orders. Sixty-three percent of the participant pediatricians disagreed with choosing large, specialized tertiary hospitals for quarantine of patients simply because they have a positive COVID-19 test result. This is because choosing large, specialized tertiary hospitals for quarantine has a significant negative impact on the continuity of care for the chronically ill children who were being followed in these facilities. COVID-19 is likely to leave behind a long-term legacy and sequel that can only be revealed in the future. However, learning about failing policies along the pathway might encourage better healthcare-oriented decisions to minimize the impact of this aggressive pandemic and control its direct and indirect damages. Governments should always consider the indirect and remote consequences of their health policies when imposing strict measures during extraordinary situations, as the indirect impact of these policies might prove to be more devastating than the pandemic itself. To minimize these indirect sequelae of lockdown, state agencies in developing nations should recognize and legitimize remote healthcare distribution in addition to virtual consultation. The limitations of this study can be attributed as the cross-sectional design of the survey, which was directed at Jordanian pediatricians and the fact that there were no previous studies on the health care services status in Jordanian Hospitals for purposes of comparison. The health care policies and decisions taken to overcome the sequels of pandemics must always take into consideration preserving health care services for patients with chronic diseases who depend on these health care facilities for a better quality of life. |
Sensitivity and applications of the PCR Single-Strand Conformation Polymorphism method | b9ff3af4-04a6-4f5b-8974-ee085356872d | 8065318 | Pathology[mh] | The SSCP method follows the steps of DNA extraction, PCR amplification of the target fragment and finally denaturation of the double-stranded PCR product by heat and formamide and electrophoresis on a non-denaturing polyacrylamide gel. During electrophoresis, single-stranded DNAs are converted to stable conformations (there is none theory can predict the exact folded structure of ssDNA) depending on their major sequence. The SSCP method is based on the ability of single-stranded DNAs to fall into unique conformations depending on their primary sequence whose structures are stabilized by intramolecular interactions, under non-denaturing conditions. As a result, even a base change can lead to a modulation change, which can be detected by the altered mobility of the single-stranded DNA molecule in the SSCP method. Therefore, for each ssDNA fragment, the number of constant configurations that create bands of different mobility during SSCP electrophoresis must be determined experimentally under strictly controlled conditions. Several parameters have been found empirically to affect the sensitivity of SSCP analysis . Among them is (i) a type of mutation (base substitutions, small insertions, deletions and rearrangements); (ii) DNA fragment size (no longer than 300 bp); (iii) G and C fragment; (iv) polyacrylamide (use of low percentage of crosslinker) or other gel matrix composition (addition of glycerol enhances the sensitivity); (v) gel size and potential; (vi) electrophoresis gel temperature (ssDNA have more compact conformation at lower temperatures); (vii) DNA concentration; (viii) buffer composition, including ionic strength and pH (low pH buffer can improve the sensitivity) and (ix) buffer additives. All these parameters are described in detail by Kakavas et al. . A detailed comparison of these methods is made in the work of Hashim et al. . Both of them have advantages and disadvantages. On the one hand PCR-SSCP can detect unknown mutations and is rapid, inexpensive and convenient on the other hand PCR-RLFP is fast and can detect only known SNPs. We can’t claim that one method is better than the other. Detection of somatic mutations in cancer, genes responsible for hereditary diseases, polymorphisms and other applications are described in detail at Kakavas et al. . Recent researches in the last 4 years shows dispersion of SSCP method in the above applications: on cancer prognosis , on asthma disease , on blood groups , on gilbert’s syndrome , on diabetes disease , on respiratory distress syndrome , on infertile men with varicocele , on gastric mucosa , on traditional Chinese medicine , detection of bacterial DNA , identification of trichomonas vaginalis . Animals PCR-SSCP has been used for several traits of animals such as wool characteristics , milk production traits , profile of sheep fatty acids , detection of Escherichia coli in the black swan , growth and carcass traits in lambs , reproductive traits in cattle , egg production traits in hens , body weight traits on chicken , gene study for fat-tailed and nonfat-tailed sheep , serotyping dichelobacter nodosus , mitochondrial diversity role in the productivity of quails , miR-9 gene affects litter size in goats , mutations in porcine , prolactin gene and association with egg production in ducks , wool fibre diameter in ewes , lactoferrin gene affects milk content in buffaloes , milk traits in yaks , association between MyoG gene and egg quality . Birds Genes identification in ostrich populations and mtDNA identification of Alectoris rufa populations . Fishes Identification of shrimp species , SNPs and white spot syndrome virus resistance in black tiger shrimp . Amphibians Caretta-caretta mtDNA polymorphisms detection . Plants PCR-SSCP could be used for the authentication of snapper species by SSCP , the population genetic structure of zeugodacus tau species complex , for the genetic diversity of citrus trees , identification of phytophthora genes polymorphism of potato , identification of genetic diversity of lemon and mandarin varieties , detection of the genetic variation of alfalfa gene , prediction of the chloroplast gene polymorphism in barley varieties and bacteria identification in wood tick . Microbial organisms Molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust , analysis of the fungal flora in environmental dust samples , development of methods to analyze bacterial species diversity in freshwater and soil ecosystems , impact of Fe oxides mineralogy on iron solubilization and associated microbial communities , difference in some biological properties of soils , gene comparison from culturable denitrifying bacteria , food waste composting and microbial community structure profiling . Food Microbial communities identification in cheese and for food authenticity . Applications in forensic medicine and biochemistry Usually, a big number of biological samples used, are found in a crime because SSCP uses mtDNA for it΄s different variations . An application with coronavirus A very interesting research work concerned the cat infection with coronaviruses by Battilani et al. . This study investigated the biological importance of the mutations detected in the genome of feline coronaviruses from naturally infected cats . All the applications of SSCP analyzed above are shown in Table and Fig. . An exhaustive search in the literature determined that the SSCP method was first used in 1989, it reached its peak in the year 1999 and by 2021 demonstrated a downward trend with spread to applications of environmental, veterinary and forensic medicine interest. The total of the research works amounts to 9.944 in a depth of 32 years (Fig. ). This may encourage us to use SSCP in routine analyses in clinical, environmental, veterinary, microbiological, and forensic medicine laboratories. PCR-SSCP has been used for several traits of animals such as wool characteristics , milk production traits , profile of sheep fatty acids , detection of Escherichia coli in the black swan , growth and carcass traits in lambs , reproductive traits in cattle , egg production traits in hens , body weight traits on chicken , gene study for fat-tailed and nonfat-tailed sheep , serotyping dichelobacter nodosus , mitochondrial diversity role in the productivity of quails , miR-9 gene affects litter size in goats , mutations in porcine , prolactin gene and association with egg production in ducks , wool fibre diameter in ewes , lactoferrin gene affects milk content in buffaloes , milk traits in yaks , association between MyoG gene and egg quality . Genes identification in ostrich populations and mtDNA identification of Alectoris rufa populations . Identification of shrimp species , SNPs and white spot syndrome virus resistance in black tiger shrimp . Caretta-caretta mtDNA polymorphisms detection . PCR-SSCP could be used for the authentication of snapper species by SSCP , the population genetic structure of zeugodacus tau species complex , for the genetic diversity of citrus trees , identification of phytophthora genes polymorphism of potato , identification of genetic diversity of lemon and mandarin varieties , detection of the genetic variation of alfalfa gene , prediction of the chloroplast gene polymorphism in barley varieties and bacteria identification in wood tick . Molecular epidemiological studies on the exposure of farm children to bacteria in environmental dust , analysis of the fungal flora in environmental dust samples , development of methods to analyze bacterial species diversity in freshwater and soil ecosystems , impact of Fe oxides mineralogy on iron solubilization and associated microbial communities , difference in some biological properties of soils , gene comparison from culturable denitrifying bacteria , food waste composting and microbial community structure profiling . Microbial communities identification in cheese and for food authenticity . Usually, a big number of biological samples used, are found in a crime because SSCP uses mtDNA for it΄s different variations . A very interesting research work concerned the cat infection with coronaviruses by Battilani et al. . This study investigated the biological importance of the mutations detected in the genome of feline coronaviruses from naturally infected cats . All the applications of SSCP analyzed above are shown in Table and Fig. . An exhaustive search in the literature determined that the SSCP method was first used in 1989, it reached its peak in the year 1999 and by 2021 demonstrated a downward trend with spread to applications of environmental, veterinary and forensic medicine interest. The total of the research works amounts to 9.944 in a depth of 32 years (Fig. ). This may encourage us to use SSCP in routine analyses in clinical, environmental, veterinary, microbiological, and forensic medicine laboratories. The PCR-SSCP method is widely used in the detection of mutations in both basic and applied biological and environmental science. The separation of mutated sequences could be further improved by using a gel matrix rather than a polyacrylamide one or adding agents that can affect the folding of single-stranded DNA. Thus, PCR-SSCP is considered still modern as a method not only to screen sequence variations but also to identify new mutations. Among the various methods used for the detection of mutations, SSCP is one of the simplest and most sensitive methods, thus making it attractive for use in the clinical diagnostic, veterinary, environmental, microbiological, food and forensic medicine laboratories. Everyone’s expectation for the future application of SSCP technique in genotyping experiments is very high especially with the detection of different and new coronavirus (SARS-CoV-2) strains. |
Comparing perioperative outcomes after transmetatarsal amputation in patients with or without peripheral vascular disease | 2a27ee25-a1ca-48ad-9ec9-20edc636a7f9 | 11807761 | Surgical Procedures, Operative[mh] | INTRODUCTION Transmetatarsal amputation (TMA) remains a commonly utilized procedure for management of gangrene and infection in the setting of diabetes and peripheral vascular disease. Compared to below knee amputation, it provides a less complex procedure, less blood loss, and preservation of the ankle joint . Furthermore, TMA can serve as a treatment modality for limb salvage, potentially increasing the chance of eventual ambulation in the appropriate setting . Nevertheless, there is significant morbidity after TMA, particularly in patients with underlying medical comorbidities. Several studies have demonstrated a high rate of surgical complications and overall morbidity after these procedures . Wound healing complications, recurrent infection, and dehiscence can necessitate hospital readmission, revision procedures, delayed ambulation, and a significant burden of morbidity for patients . Conversion rates to higher levels of amputation, including below or above knee amputation, are significant . Patient selection, surgical indication, and amputation level selection is crucial to minimize complications and maximize function, particularly in patients with complex medical conditions such as diabetes mellitus and peripheral vascular disease. Despite the frequency in which TMA procedures are performed and the significant risk of postoperative complications, there is a lack of large population studies investigating complications after TMA for different surgical indications. The purpose of this study is to utilize a validated national surgical database to investigate the incidence of and risk factors for short‐term complications, namely reoperation, after TMA in patients with or without peripheral arterial disease. MATERIALS AND METHODS 2.1 Data source All data were extracted from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) surgical database . ACS NSQIP reports over 150 variables, including morbidity and mortality outcome measures, after commonly performed outpatient and inpatient procedures from participating institutions . Operating room logs are audited to ensure correct sampling of cases, and the results of these audits have yielded an overall disagreement rate of about 2% for all variables . The database has been utilized in numerous studies investigating surgical outcomes . 2.2 Data extraction The ACS NSQIP database was queried to identify patients undergoing TMA between January 1, 2015 and December 31, 2020 for all indications using Current Procedural Terminology (CPT) code 28805–Amputation Foot, Transmetatarsal. The International Classification of Disease 9 and 10 (ICD‐9, ICD‐10) codes were then independently screened to identify the specific surgical indication for each case. All revision procedures, traumatic indications, and cases with concurrent procedures were excluded from the analysis. Demographic and patient‐specific variables of interested were reported, including sex, age, body mass index (BMI), medical comorbidities, and American Society of Anesthesiologist (ASA) classification. Surgical variables of interest included surgical indication and whether sepsis or septic shock were present preoperatively. Surgical indications were grouped into infectious wounds (including diabetic foot wounds), peripheral vascular disease, and tumor/other indications. American Society of Anesthesiologist classes were defined as previously described: Class I is a normal, healthy patient; Class II is a patient with mild systemic disease; Class III is a patient with severe systemic disease; Class IV is a patient with a severe systemic disease that constantly threatens life; and Class V is a moribund patient that is not expected to survive for 24 h with or without operation . The cases were grouped into two major groups of interest: those undergoing surgery for primarily infectious/diabetic wounds versus peripheral vascular disease. Grouping was based on the reported ICD‐9 or ICD‐10 codes for the primary diagnosis, as reported in the database. Thirty‐day outcome measures included mortality, reoperation, readmission, nonhome discharge, and various complications. We defined surgical complications as those clearly related directly to surgical intervention: superficial surgical site infection (SSI), deep SSI, wound infection, dehiscence, and bleeding requiring transfusion. We defined medical complications as other complications that may be indirectly related to surgery, anesthesia, inpatient hospitalization, or baseline comorbidities. These complications included septic shock, systemic sepsis, deep venous thromboembolism, myocardial infarction (MI), cardiac arrest, cerebrovascular accident, urinary tract infection (UTI), renal insufficiency, renal failure, pneumonia, pulmonary embolism, unplanned intubation, and prolonged ventilator use for more than 48 h. 2.3 Statistical analysis Chi‐squared analysis was used to compare categorical variables–including patient demographics, surgical variables, and outcome measures–between (i) patients with and without a 30‐day reoperation after the index procedure and (ii) patients undergoing surgery for infectious/diabetic versus vascular indications. A series of stepwise binary logistic regressions were then utilized to identify variables that were independently associated with each dependent variable (e.g., outcome measure of interest), namely mortality, reoperation, readmission, nonhome discharge, surgical complications, and medical complications. The independent variables included all potentially clinically relevant variables: age, sex, BMI, medical comorbidities, ASA class, and surgical indication. Statistical significance was defined as p < 0.05. All statistical analyses were completed using International Business Machines (IBM) SPSS Version 24.0 (Armonk, NY. IBM Corp). Data source All data were extracted from the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) surgical database . ACS NSQIP reports over 150 variables, including morbidity and mortality outcome measures, after commonly performed outpatient and inpatient procedures from participating institutions . Operating room logs are audited to ensure correct sampling of cases, and the results of these audits have yielded an overall disagreement rate of about 2% for all variables . The database has been utilized in numerous studies investigating surgical outcomes . Data extraction The ACS NSQIP database was queried to identify patients undergoing TMA between January 1, 2015 and December 31, 2020 for all indications using Current Procedural Terminology (CPT) code 28805–Amputation Foot, Transmetatarsal. The International Classification of Disease 9 and 10 (ICD‐9, ICD‐10) codes were then independently screened to identify the specific surgical indication for each case. All revision procedures, traumatic indications, and cases with concurrent procedures were excluded from the analysis. Demographic and patient‐specific variables of interested were reported, including sex, age, body mass index (BMI), medical comorbidities, and American Society of Anesthesiologist (ASA) classification. Surgical variables of interest included surgical indication and whether sepsis or septic shock were present preoperatively. Surgical indications were grouped into infectious wounds (including diabetic foot wounds), peripheral vascular disease, and tumor/other indications. American Society of Anesthesiologist classes were defined as previously described: Class I is a normal, healthy patient; Class II is a patient with mild systemic disease; Class III is a patient with severe systemic disease; Class IV is a patient with a severe systemic disease that constantly threatens life; and Class V is a moribund patient that is not expected to survive for 24 h with or without operation . The cases were grouped into two major groups of interest: those undergoing surgery for primarily infectious/diabetic wounds versus peripheral vascular disease. Grouping was based on the reported ICD‐9 or ICD‐10 codes for the primary diagnosis, as reported in the database. Thirty‐day outcome measures included mortality, reoperation, readmission, nonhome discharge, and various complications. We defined surgical complications as those clearly related directly to surgical intervention: superficial surgical site infection (SSI), deep SSI, wound infection, dehiscence, and bleeding requiring transfusion. We defined medical complications as other complications that may be indirectly related to surgery, anesthesia, inpatient hospitalization, or baseline comorbidities. These complications included septic shock, systemic sepsis, deep venous thromboembolism, myocardial infarction (MI), cardiac arrest, cerebrovascular accident, urinary tract infection (UTI), renal insufficiency, renal failure, pneumonia, pulmonary embolism, unplanned intubation, and prolonged ventilator use for more than 48 h. Statistical analysis Chi‐squared analysis was used to compare categorical variables–including patient demographics, surgical variables, and outcome measures–between (i) patients with and without a 30‐day reoperation after the index procedure and (ii) patients undergoing surgery for infectious/diabetic versus vascular indications. A series of stepwise binary logistic regressions were then utilized to identify variables that were independently associated with each dependent variable (e.g., outcome measure of interest), namely mortality, reoperation, readmission, nonhome discharge, surgical complications, and medical complications. The independent variables included all potentially clinically relevant variables: age, sex, BMI, medical comorbidities, ASA class, and surgical indication. Statistical significance was defined as p < 0.05. All statistical analyses were completed using International Business Machines (IBM) SPSS Version 24.0 (Armonk, NY. IBM Corp). RESULTS A total of 3392 cases were included in the final analysis. Table summarizes patient demographics, medical comorbidities, and surgical variables across the entire cohort. The majority of patients were between 50 and 69 years of age (55.1%), with BMI between 18.5 and 29.9 (54.0%), and ASA class 3 (64.7%). Table provides detailed information regarding demographics and baseline medical comorbidities of the cohort. There was an overall high prevalence of medical comorbidities, including diabetes (76.7%), smoking (27.5%), dialysis use or renal failure (21.8%), and hypertension (75.8%). Infection/diabetic wounds were the most common indication (82.9%) followed by peripheral vascular disease (16.3%) and tumor/other (0.8%). Sepsis was present in 7.0% of patients at the time of surgery. Septic shock was present in 0.9% of patients at the time of surgery. Table provides a summary of the common CPT and ICD codes defined within the cohort. Table summarizes 30‐day outcome measures across the entire cohort. There was a mortality rate of 2.9%, reoperation rate of 13.8%, hospital readmission rate of 16.8%, and nonhome discharge rate of 41.9%. The rate of overall surgical complications was 22.2%. The rate of overall medical complications was 15.8%. Table summarizes a comparison of patient and surgical variables between patients with and without a 30‐day reoperation after TMA. There were 468 patients (13.8%) requiring reoperation. Patients requiring reoperation were more likely to have an abnormal BMI, ascites, renal failure, dialysis use, bleeding disorder, and an ASA class of 4 or 5. Patients requiring reoperation were also more likely to have undergone surgery for a vascular indication and to have sepsis present preoperatively. Table summarizes a comparison of patient and surgical variables between patients undergoing TMA surgery for infectious/diabetic wounds versus vascular indications. Patients undergoing surgery for peripheral vascular disease had more female patients, more patients in the 70–79 year, and 80+ year groups, more underweight patients, and less obese class I, II, and III patients. The vascular cohort also had more patients with Chronic Obstructive Pulmonary Disease, ascites, CHF, dialysis use, and chronic steroid use, and less patients with bleeding disorders. The vascular cohort had less patients with ASA Class 2 and 3, and more patients with ASA Class 4. The vascular cohort was also less likely to have sepsis present preoperatively. Table and Figure summarize a comparison of 30‐day outcomes between patients undergoing TMA surgery for infection/diabetic wounds versus vascular indications. Patients undergoing surgery for peripheral vascular disease were more likely to experience 30‐day mortality (2.4% vs. 5.4%, p < 0.001), reoperation (13.0% vs. 18.3%, p = 0.001), hospital readmission (15.5% vs. 23.5%, p < 0.001), and nonhome discharge (40.3% vs. 50.8%, p < 0.001). Patients undergoing surgery for peripheral vascular disease were also more likely to have various medical complications, including pneumonia (2.3% vs. 4.7%, p = 0.002), reintubation (1.3% vs. 3.6%, p < 0.001), failure to wean intubation (1.0% vs. 2.0%, p = 0.036), UTI (0.5% vs. 1.4%, p = 0.017), cardiac arrest (1.0% vs. 2.2%, p = 0.015), and MI (1.1% vs. 3.1%, p < 0.001). Patients undergoing surgery for infectious/diabetic foot wounds were more likely to have a deep SSI (5.8% vs. 3.5%, p = 0.025) and post‐surgical systemic sepsis (9.1% vs. 6.1%, p = 0.025). Table summarizes results from the multivariate logistic regression identifying independent risk factors for various 30‐day outcome measures. Advanced age, underweight BMI, CHF, diabetes, bleeding disorder, and septic shock present at the time of surgery were all independent predictors of mortality. Dialysis use, bleeding disorders, sepsis present at the time of surgery, and a vascular surgery indication were all independently associated with reoperation. Advanced age, hypertension, dialysis use, and sepsis present preoperatively were all associated with readmission. Advanced age, CHF, dialysis use, and sepsis present preoperatively were independently associated with nonhome discharge. Diabetes, dialysis use, and sepsis or septic shock present at the time of surgery were independently associated with overall surgical complications. Advanced age, hypertension, renal failure, dialysis use, chronic steroid use, bleeding disorders, and vascular surgery indication were independently associated with overall medical complications. DISCUSSION TMA is commonly performed for a variety of indications, most notably for gangrene or wounds in the setting of diabetes or peripheral vascular disease. The population undergoing these procedures often has a high burden of medical comorbidities. Furthermore, these patients may have systemic signs of illness including sepsis from the underlying infection. Surgical and medical complications are common after these procedures. Despite these considerations, there is a lack of research investigating perioperative complications, medical optimization, and the risk for reoperation after TMA for different patient populations. The purpose of this study was to utilize a large national dataset to investigate perioperative complications after TMA for specific surgical indications. 4.1 Mortality after TMA A 30‐day mortality rate of nearly 3% was reported in this cohort. Older age, underweight BMI, congestive heart failure, dialysis use, bleeding disorders, and septic shock being present preoperatively were all independent risk factors for 30‐day mortality. Surgical indication was not associated with mortality on regression analysis. These findings underscore the importance of medical optimization prior to surgical intervention, when possible, particularly in a population with a high degree of underlying comorbidities. There is room for improvement in the management of these patients in the perioperative period to help reduce overall mortality. However, it should also be noted that many of these procedures are done on an urgent or even emergent basis, often for serious infections, and preoperative medical optimization may not always be feasible. Nonetheless, these data provide a framework to inform patients of risks after TMA for different indications. Pollard et al. analyzed outcomes after TMA in a cohort of 90 patients and reported two patient deaths within 30 days postoperatively . The authors found that end‐stage renal disease and nonpalpable pedal pulses were independently associated with poor healing . Adams et al. similarly reported on outcomes after nontraumatic TMA in a cohort of 375 patients. The authors reported that 36.3% of patients had died within 3 years postoperatively and 36.8% had required a more proximal limb amputation . Only 22.1% had healed without surgical complications . End‐stage renal disease was independently associated with mortality–these patients were nearly 3 times more likely to die within 3 years postoperatively . Hill et al. compared the estimated probability of mortality after TMA compared to other commonly performed surgical procedures using a national surgical database–reporting that TMA had one of the highest risks of associated mortality within 30 days of the index procedure . While multiple studies have demonstrated that end‐stage renal disease is a risk factor for mortality after TMA, our study also found that advanced age (70+ years), and underweight BMI, CHF, and bleeding disorders were also independently associated with 30‐day mortality after TMA. To our knowledge, these risk factors for mortality after TMA were not previously described. Patients with any of these risk factors should be counseled appropriately and medically optimized prior to any intervention, particularly if surgery is being done on a nonurgent basis. 4.2 Reoperation after TMA The risk of reoperation after TMA in our cohort was substantial. Nearly 14% of patients underwent a reoperation within 30 days of the index surgery. Thorud et al. published a metaanalysis investigating reoperation and reamputation after TMA, reporting a reoperation rate of 26.9%, a reamputation rate of 29.7% and a major amputation rate of 33.2% of patients . Jupiter et al. reported that minor amputations, such as TMA, were 2.5 times more likely to require short‐term irrigation and debridement compared with major amputations . However, minor amputation patients were significantly less likely to require a blood transfusion or develop UTI after surgery . Identifying the appropriate patients for TMA remains paramount to optimizing patient outcomes and minimizing excessive healthcare utilization after failed minor amputation procedures. 4.3 Risk factors for complications after TMA Identifying risk factors for poor outcomes is an important step in defining appropriate surgical indications. Ammendola et al. published a systematic review investigating the use of TMA for diabetic foot gangrene and found that data regarding patient selection, specific surgical indications, and contraindications were sparse in the current literature . Landry et al. assessed multiple variables as potential predictors of wound healing after TMA, including demographic characteristics, preoperative vascular status, and several perioperative variables. The authors reported a high rate of poor wound healing after TMA (14%) but could not identify any independent variables associated with this outcome . Our study harnessed the statistical power of a large national database. We specifically identified that a vascular indication for surgery was an independent risk factor for short‐term reoperation after TMA. Additionally, dialysis dependence, the presence of preoperative sepsis, and bleeding disorders were independent risk factors for reoperation. The conclusions regarding any association of peripheral arterial disease with risk of reoperation or reamputation in prior studies have been conflicting, however . Nguyen et al. reported that ankle‐to‐brachial index (ABI) ratios were similar between patients with failed or successful TMA procedures . Others have reported that ischemia is directly associated with failed TMA . Interestingly, Shi et al. reported that the timing of any vascular surgery intervention–performed either before or after TMA–was not associated with limb loss or wound healing . Nonetheless, the risk of reoperation or reamputation should be considered carefully in patients undergoing TMA, particularly with underlying vascular disease. 4.4 Influence of renal function on outcomes after TMA Poor renal function is also a known risk factor for reoperation after TMA . Ahn et al. reported that elevated serum creatinine, blood urea nitrogen, dialysis use, and the stage of Chronic Kidney Disease (CKD) were associated with reamputation . Patients with CKD stage IV‐V had markedly increased odds of reamputation after TMA . A minor amputation in patients with advanced CKD is unlikely to be a viable long‐term solution. Although a higher level amputation can result in more functional impairment, unsuccessful attempts at limb salvage in patients with poor wound healing potential creates a significant burden on the patient’s health and the healthcare system itself. 4.5 Readmission after TMA Hospital readmission remains an important metric for quality improvement. Unplanned hospital readmission yields a significant burden to the patient, increases the risk for hospital‐acquired infections and iatrogenic injuries, and burdens the healthcare system by increasing costs and utilization. We found advanced age, hypertension, dialysis use, and the presence of sepsis preoperatively for all independent predictors for 30‐day hospital readmission in this cohort. Interestingly, patients undergoing surgery for a vascular indication were not at higher risk of unplanned hospital readmission. Similar to our findings, Casciato et al. recently reported that geriatric patients were at a significantly increased risk of unplanned hospital readmission after outpatient TMA . However, they did not find that any other patient demographic variables, past medical history, or surgical presentation were associated with readmission . Beaulieu et al. reported on the incidence and predictors of readmission after minor lower extremity amputations more generally in the vascular surgery population; they found that elective admission, peripheral arterial disease, and chronic renal insufficiency were associated with readmission . Furthermore, reamputation occurred in 95% of patients readmitted to the hospital, and 64% underwent major limb amputation (below knee, through knee, or above knee amputation) . 4.6 Comparing outcomes after TMA for diabetic versus peripheral vascular disease indications Patients undergoing TMA for infections with underlying diabetes and/or peripheral vascular disease are clearly different patient populations with unique risk factors for complication. However, there is currently a lack of research directly comparing outcomes after TMA for these two distinct patient populations. Kanter et al. previously reported that patients undergoing TMA for diabetic foot wounds had a short‐term wound complication rate of nearly 11.9%, and obesity was associated with higher wound complications in that cohort . Younger et al. previously reported the elevated risk of TMA failure in patients with hemoglobin a1c (HbA1c) values greater than 8–concluding that surgery in patients with HbA1c values less than 8 should not be performed unless the indication is to save life or limb . Our nonvascular cohort also had a particularly high incidence of wound complications (16.4%)–including infection and dehiscence. Notably, the deep SSI rate in the nonvascular cohort was nearly double that of the vascular cohort–5.8% versus 3.4%, respectively. Compared to the infectious/diabetic wound group, the peripheral vascular disease cohort had unique complication profiles after surgery. Shi et al. reported that patients undergoing TMA for peripheral arterial disease had an overall 44% rate of eventual limb loss, and that the time between vascular intervention and TMA had no association with wound healing or limb loss . However, there is a lack of existing data comparing outcomes after TMA for patients with or without peripheral vascular disease. Notably, our cohort found that patients undergoing TMA for peripheral arterial disease were at increased risk of many short‐term complications, including mortality, reoperation, hospital readmission, nonhome discharge, and several medical complications, as described previously. The vascular surgery cohort had more than double the mortality rate compared to the diabetic cohort, 5.4% versus 2.4%, respectively. This increased risk of serious perioperative complications must be considered when electing to proceed with limb salvage operations like TMA in patients with peripheral vascular disease. 4.7 Limitations There are limitations of this study that must be discussed in the context of the conclusions herein. The retrospective nature and data source of the study introduces inherent risk of selection bias. The participating institutions included in the ACS NSQIP database have a trend toward more academic, tertiary care centers, which may limit generalizability of the findings. The database also lacks specific surgical variables that may be important for determining the risk of complications, such as the overall severity of the arterial disease, the severity or acuity of the infectious wounds, and surrounding soft tissue integrity. Outcomes data are only reported for the first 30 days postoperatively, so mid‐ and long‐term outcomes cannot be assessed. Additionally, the specific indications for reoperation are not provided and therefore cannot be reported in this study. Lastly, the authors acknowledge that there is often overlap between patients with diabetes and peripheral vascular disease. As summarized in the supplemental data, nearly 60% of patients undergoing surgical intervention for peripheral vascular disease had been diagnosed with diabetes. The reported primary ICD‐9/10 code was used to define the main indication for surgery (i.e., diabetic wounds vs. peripheral vascular disease). Multivariate logistic regression was used to help control for potential confounding variables but this limitation must be considered in the context of the results. Nonetheless, the database provides a large validated dataset with significant statistical power, which is particularly useful for assessing differences in less common complications, which smaller sample studies may not be adequately powered to detect. Mortality after TMA A 30‐day mortality rate of nearly 3% was reported in this cohort. Older age, underweight BMI, congestive heart failure, dialysis use, bleeding disorders, and septic shock being present preoperatively were all independent risk factors for 30‐day mortality. Surgical indication was not associated with mortality on regression analysis. These findings underscore the importance of medical optimization prior to surgical intervention, when possible, particularly in a population with a high degree of underlying comorbidities. There is room for improvement in the management of these patients in the perioperative period to help reduce overall mortality. However, it should also be noted that many of these procedures are done on an urgent or even emergent basis, often for serious infections, and preoperative medical optimization may not always be feasible. Nonetheless, these data provide a framework to inform patients of risks after TMA for different indications. Pollard et al. analyzed outcomes after TMA in a cohort of 90 patients and reported two patient deaths within 30 days postoperatively . The authors found that end‐stage renal disease and nonpalpable pedal pulses were independently associated with poor healing . Adams et al. similarly reported on outcomes after nontraumatic TMA in a cohort of 375 patients. The authors reported that 36.3% of patients had died within 3 years postoperatively and 36.8% had required a more proximal limb amputation . Only 22.1% had healed without surgical complications . End‐stage renal disease was independently associated with mortality–these patients were nearly 3 times more likely to die within 3 years postoperatively . Hill et al. compared the estimated probability of mortality after TMA compared to other commonly performed surgical procedures using a national surgical database–reporting that TMA had one of the highest risks of associated mortality within 30 days of the index procedure . While multiple studies have demonstrated that end‐stage renal disease is a risk factor for mortality after TMA, our study also found that advanced age (70+ years), and underweight BMI, CHF, and bleeding disorders were also independently associated with 30‐day mortality after TMA. To our knowledge, these risk factors for mortality after TMA were not previously described. Patients with any of these risk factors should be counseled appropriately and medically optimized prior to any intervention, particularly if surgery is being done on a nonurgent basis. Reoperation after TMA The risk of reoperation after TMA in our cohort was substantial. Nearly 14% of patients underwent a reoperation within 30 days of the index surgery. Thorud et al. published a metaanalysis investigating reoperation and reamputation after TMA, reporting a reoperation rate of 26.9%, a reamputation rate of 29.7% and a major amputation rate of 33.2% of patients . Jupiter et al. reported that minor amputations, such as TMA, were 2.5 times more likely to require short‐term irrigation and debridement compared with major amputations . However, minor amputation patients were significantly less likely to require a blood transfusion or develop UTI after surgery . Identifying the appropriate patients for TMA remains paramount to optimizing patient outcomes and minimizing excessive healthcare utilization after failed minor amputation procedures. Risk factors for complications after TMA Identifying risk factors for poor outcomes is an important step in defining appropriate surgical indications. Ammendola et al. published a systematic review investigating the use of TMA for diabetic foot gangrene and found that data regarding patient selection, specific surgical indications, and contraindications were sparse in the current literature . Landry et al. assessed multiple variables as potential predictors of wound healing after TMA, including demographic characteristics, preoperative vascular status, and several perioperative variables. The authors reported a high rate of poor wound healing after TMA (14%) but could not identify any independent variables associated with this outcome . Our study harnessed the statistical power of a large national database. We specifically identified that a vascular indication for surgery was an independent risk factor for short‐term reoperation after TMA. Additionally, dialysis dependence, the presence of preoperative sepsis, and bleeding disorders were independent risk factors for reoperation. The conclusions regarding any association of peripheral arterial disease with risk of reoperation or reamputation in prior studies have been conflicting, however . Nguyen et al. reported that ankle‐to‐brachial index (ABI) ratios were similar between patients with failed or successful TMA procedures . Others have reported that ischemia is directly associated with failed TMA . Interestingly, Shi et al. reported that the timing of any vascular surgery intervention–performed either before or after TMA–was not associated with limb loss or wound healing . Nonetheless, the risk of reoperation or reamputation should be considered carefully in patients undergoing TMA, particularly with underlying vascular disease. Influence of renal function on outcomes after TMA Poor renal function is also a known risk factor for reoperation after TMA . Ahn et al. reported that elevated serum creatinine, blood urea nitrogen, dialysis use, and the stage of Chronic Kidney Disease (CKD) were associated with reamputation . Patients with CKD stage IV‐V had markedly increased odds of reamputation after TMA . A minor amputation in patients with advanced CKD is unlikely to be a viable long‐term solution. Although a higher level amputation can result in more functional impairment, unsuccessful attempts at limb salvage in patients with poor wound healing potential creates a significant burden on the patient’s health and the healthcare system itself. Readmission after TMA Hospital readmission remains an important metric for quality improvement. Unplanned hospital readmission yields a significant burden to the patient, increases the risk for hospital‐acquired infections and iatrogenic injuries, and burdens the healthcare system by increasing costs and utilization. We found advanced age, hypertension, dialysis use, and the presence of sepsis preoperatively for all independent predictors for 30‐day hospital readmission in this cohort. Interestingly, patients undergoing surgery for a vascular indication were not at higher risk of unplanned hospital readmission. Similar to our findings, Casciato et al. recently reported that geriatric patients were at a significantly increased risk of unplanned hospital readmission after outpatient TMA . However, they did not find that any other patient demographic variables, past medical history, or surgical presentation were associated with readmission . Beaulieu et al. reported on the incidence and predictors of readmission after minor lower extremity amputations more generally in the vascular surgery population; they found that elective admission, peripheral arterial disease, and chronic renal insufficiency were associated with readmission . Furthermore, reamputation occurred in 95% of patients readmitted to the hospital, and 64% underwent major limb amputation (below knee, through knee, or above knee amputation) . Comparing outcomes after TMA for diabetic versus peripheral vascular disease indications Patients undergoing TMA for infections with underlying diabetes and/or peripheral vascular disease are clearly different patient populations with unique risk factors for complication. However, there is currently a lack of research directly comparing outcomes after TMA for these two distinct patient populations. Kanter et al. previously reported that patients undergoing TMA for diabetic foot wounds had a short‐term wound complication rate of nearly 11.9%, and obesity was associated with higher wound complications in that cohort . Younger et al. previously reported the elevated risk of TMA failure in patients with hemoglobin a1c (HbA1c) values greater than 8–concluding that surgery in patients with HbA1c values less than 8 should not be performed unless the indication is to save life or limb . Our nonvascular cohort also had a particularly high incidence of wound complications (16.4%)–including infection and dehiscence. Notably, the deep SSI rate in the nonvascular cohort was nearly double that of the vascular cohort–5.8% versus 3.4%, respectively. Compared to the infectious/diabetic wound group, the peripheral vascular disease cohort had unique complication profiles after surgery. Shi et al. reported that patients undergoing TMA for peripheral arterial disease had an overall 44% rate of eventual limb loss, and that the time between vascular intervention and TMA had no association with wound healing or limb loss . However, there is a lack of existing data comparing outcomes after TMA for patients with or without peripheral vascular disease. Notably, our cohort found that patients undergoing TMA for peripheral arterial disease were at increased risk of many short‐term complications, including mortality, reoperation, hospital readmission, nonhome discharge, and several medical complications, as described previously. The vascular surgery cohort had more than double the mortality rate compared to the diabetic cohort, 5.4% versus 2.4%, respectively. This increased risk of serious perioperative complications must be considered when electing to proceed with limb salvage operations like TMA in patients with peripheral vascular disease. Limitations There are limitations of this study that must be discussed in the context of the conclusions herein. The retrospective nature and data source of the study introduces inherent risk of selection bias. The participating institutions included in the ACS NSQIP database have a trend toward more academic, tertiary care centers, which may limit generalizability of the findings. The database also lacks specific surgical variables that may be important for determining the risk of complications, such as the overall severity of the arterial disease, the severity or acuity of the infectious wounds, and surrounding soft tissue integrity. Outcomes data are only reported for the first 30 days postoperatively, so mid‐ and long‐term outcomes cannot be assessed. Additionally, the specific indications for reoperation are not provided and therefore cannot be reported in this study. Lastly, the authors acknowledge that there is often overlap between patients with diabetes and peripheral vascular disease. As summarized in the supplemental data, nearly 60% of patients undergoing surgical intervention for peripheral vascular disease had been diagnosed with diabetes. The reported primary ICD‐9/10 code was used to define the main indication for surgery (i.e., diabetic wounds vs. peripheral vascular disease). Multivariate logistic regression was used to help control for potential confounding variables but this limitation must be considered in the context of the results. Nonetheless, the database provides a large validated dataset with significant statistical power, which is particularly useful for assessing differences in less common complications, which smaller sample studies may not be adequately powered to detect. CONCLUSION TMAs are commonly performed in a high‐risk population, with a significant risk of perioperative morbidity and mortality. The risk of short‐term reoperation is substantial, particularly in patients undergoing surgery in the setting of peripheral vascular disease. Dialysis use, bleeding disorders, a vascular surgery indication, and sepsis being present preoperatively were all independently associated with 30‐day reoperation in this population. A number of risk factors associated with poor 30‐day outcome measures were identified and described herein. Understanding these risks is important when indicating patients for surgery, choosing the appropriate amputation procedure, and ensuring patients have informed consent prior to surgery. Further investigation with prospective cohorts can help expand these findings. M.A.P: Manuscript writing; manuscript editing; data/statistical analysis; conceptualization; and methodology. R.B.: Manuscript writing; manuscript editing; and data/statistical analysis. E.G.: Manuscript editing and methodology. M.M.: Conceptualization; manuscript editing; and supervision. M.P.: Conceptualization; manuscript editing; and supervision. A.R.K.: Conceptualization; manuscript editing; supervision; and project administration. M.A.P: No conflicts of interest to disclose. R.B.: No conflicts of interest to disclose. E.G.: No conflicts of interest to disclose. M.M.: No conflicts of interest to disclose. M.P.: No conflicts of interest to disclose. A.R.K.: ‐ Consulting for Arthrex, Inc. ‐ Royalty payments and inventor share from Acumed, Limited Liability Company and DePuy Orthopedics, Inc. The data used herein were obtained from the American College 1 Surgeons National Surgical Quality Improvement Program. The data are deidentified prior to use by the authors. The research does therefore not involve “human subjects”. Tables S1–S3 |
Cholesterol-Lowering Bioactive Foods and Nutraceuticals in Pediatrics: Clinical Evidence of Efficacy and Safety | c3bc46a8-b2b2-435c-8d6b-f83af8385004 | 11123713 | Pediatrics[mh] | Optimal low-density lipoprotein cholesterol and/or triglyceride plasma levels significantly protect against cardiovascular disease (CVD) development, and higher levels are associated with an increased risk . The optimal values are defined based on age and ethnicity. Dyslipidemias are largely prevalent disorders of lipoprotein metabolism that can result in abnormal lipid and lipoprotein values. The prevalence of dyslipidaemias is increasing all over the world, even in Mediterranean countries where the diet should be qualitatively healthier. A recent Italian study showed a prevalence of suboptimal cholesterolemia (total cholesterol–TC > 170 mg/dL) in 78% of children and adolescents . Recent studies suggest that long-term mild exposure during childhood to even a mild suboptimal LDL-C level is associated with an increased risk of coronary artery disease. International guidelines suggest screening children and adolescents for lipid levels, especially in families with dyslipidemia and/or atherosclerotic CVD (ASCVD) . In a large Australian cohort, subjects who had incident non-high density lipoprotein-cholesterol (non-HDL-C) dyslipidemia from childhood to adulthood and those with persistent dyslipidemia had dramatic increased risks of cardiovascular events (Hazard Ratio 2.17 [95% Confidence Interval (CI) 1.00–4.69] and Hazard Ratio 5.17 [95% CI 2.80–9.56], respectively), when compared with those whose non-HDL-C levels remained within the guideline-recommended range in childhood and adulthood. Then, participants who had high non-HDL-C in childhood but whose non-HDL-C levels were within the guideline-recommended range in adulthood did not have a significantly increased risk (Hazard Ratio 1.13 (95%CI 0.50–2.56)) . Thus, improvements in physical activity and some dietary behaviors that ensure all necessary nutrients for adequate growth during childhood are advisable , although specific lipid-lowering pharmacological treatment is needed to manage secondary and severe genetic dyslipidemias . Some nutraceuticals have clearly demonstrated cholesterol-lowering activity in adults. Experts recommend their utilization in managing individuals who have a low estimated risk of developing ASCVD, but who show an insufficient metabolic response to dietary changes. Additionally, nutraceuticals may be considered for some low-risk patients with statin-intolerance when combined with ezetimibe . According to the American Academy of Pediatrics (AAP) and European Atherosclerosis Society (EAS), the first step in the management of adult and pediatric patients with hypercholesterolemia is nutritional approach and life-style management, followed by drug therapy . Functional foods or “nutraceuticals” have been used as adjunct treatment for adult patients, and are suggested for pediatric patients with familial hypercholesterolaemia (FH) . Studies on cholesterol-lowering nutraceuticals in children and adolescents are relatively limited, as only a few short-term, randomized, controlled studies have been performed. These are mainly concerned with the administration of (soluble) fibers and plant sterols/stanols, whereas sporadic reports have tested the efficacy and tolerability of standardized red yeast rice extract, soy proteins, probiotics, and Omega-3 and Omrega-6 polyunsaturated fatty acids (PUFAs). This review article aimed to critically summarize the scientific evidence supporting cholesterol-lowering dietary supplements and nutraceuticals in managing children and adolescents with dyslipidemia. We focused on the most widely used nutraceuticals in dyslipidemia management, especially those that have shown evidence of modifying plasma levels of LDL-C and triglycerides. Additionally, we also examined the impact of these nutraceuticals on TC and HDL-C levels, to provide clinicians comprehensive objective information to guide their decision-making process. A detailed literature search was performed in this narrative review on PubMed using the following keywords: “Children”, “Adolescent”, “Pediatric”, “Hypercholesterolemia”, “Hypertriglyceridemia”, “Dyslipidemia”, “Dietary supplement”, “Nutraceutical”, “Efficacy”, “Tolerability”, “Safety”, “Cholesterol-lowering” and “Lipid-lowering”. Preference was given to placebo-controlled randomized clinical trials. The collected articles underwent independent review by two authors. Inclusion criteria were established prior to article review, and were as follows: (i) all published scientific papers that describe dietary supplement, nutraceuticals and lipid-lowering agents in pediatric population; and (ii) high-quality systematic research, randomized control trials, study cohorts and cross-sectional studies that were published in English. We excluded studies that were not reported in English, and those that exclusively focused on adults (age ≥ 19 years). Findings were classified by the main mechanisms of action (i.e., cholesterol absorption inhibitors from the bowel, LDL synthesis inhibition by the liver, and a mixed mechanism of action), and summarized in tables and figures. The review tables included nutraceutical, study type, study aim, participants, intervention, intolerance, compliance and observed effects. Several natural compounds can interfere with cholesterol absorption, significantly improving cholesterolemia in children and adolescents. 3.1. Soluble Fibers Fibers are edible parts of plants that pass through the small intestine relatively unchanged in humans, and include complex carbohydrates such as non-starch polysaccharides (pectins, gums, cellulose, hemicellulose, oat bran and wheat bran), oligosaccharides (inulin and fructooligosaccharides), and lignin (i.e., the non-carbohydrate fraction of the dietary fiber). Fibers intake is linked to positive health outcomes, including a lower risk of developing ASCVD and obesity . Fibers naturally contained in cereals, vegetables and fruits (as part of a balanced dietary pattern) ameliorate the lipid profile in adults , and reduce concentrations of TC and Low Density Lipoprotein-Cholesterol (LDL-C) by 5–15% and 9–22%, respectively . These beneficial effects have led regulatory agencies to issue health claims for the intake of fibers , oat β-glucan and its LDL-C lowering effect or ASCVD risk reduction . A large cohort study on 5873 Japanese children (10–11 years) highlighted the presence of an inverse association between the consumption of dietary fibers and plasma concentrations of TC, and the presence of overweight and obesity, confirming data from clinical trials in adults . Although dietary fibers help maintain good health, their quantitative need in children has not been defined yet . Food and Drug Administration (FDA) guidelines relate it to the need for calorie intake (12 g/1000 calories), while the American Academy of Pediatrics guidelines relate it to weight or age . Pediatric recommendations widely vary across countries, being also influenced by the available evidence . In general practice, many pediatricians follow a formula that involves adding 5 g/day to the child’s age (for children older than 3 years) . Even if this guideline is often considered when advocating for a Mediterranean diet or a diet rich in vegetables, it is seldom met in clinical practice. Children living in Western countries typically consume fewer vegetables and fruits, contributing to a lower dietary fiber intake, high calorie dense food, and highly refined high-fat diet . The consequences of such an incorrect dietary intake include metabolic changes and hyperlipidemia, which are sometimes associated with overweight/obesity. In this context, a positive effect of dietary fibers on blood lipids with monounsaturated FAs (MUFAs) was shown in children in the Healthy Start Preschool Study of Cardiovascular Disease Risk Factors and Diet . Soluble fibers include psyllium (viscous and non-fermentable fiber), glucomannan, oat, pectin, and guar gum (viscous and fermentable fiber) and are commercially available as unprocessed fibers that can be added to food or used as flavored powders or capsules. Psyllium, derived from the seed husk of Plantago ovata , is one of the richest sources of soluble mucilaginous dietary fiber, acting as a gel-forming polysaccharide, similarly to pectin and guar gum . Locust bean gum (that is, a galactomannan from the carob tree) is a white and odorless powder extracted from the endosperm of beans without a distinctive taste. Pectin is less viscous than other fibers, but similar in ash and more palatable . Glucomannan, the main polysaccharide obtained from the Asian tuber Amorphophallus konjac, is a palatable, highly viscous soluble fiber. Its chemical structure consists of a mannose (8): glucose (5) ratio linked by b-glycosidic bonds. Glucomannan has the highest molecular weight and viscosity among all dietary fibers . Oat seeds are also an important source of the viscous soluble fiber beta-glucan . The ability of fibers to lower plasma lipids relies on their physicochemical properties and viscosity. Soluble fiber acts mainly to form viscous solutions that slow gastric emptying and reduce fat absorption, thereby modulating lipoprotein metabolism. In the small intestine, the gelling process binds to dietary fats and hinders the absorption of cholesterol, and the reabsorption of bile acids increases their excretion in feces. It follows that there is a reduced uptake of intestinal cholesterol and a reduced circulation of chylomicrons. Of consequence is that the synthesis of bile in the liver increases and LDL-C levels decrease. Another cholesterol-lowering mechanism involves bacterial fermentation in the colon (except for lignin), which leads to production of short-chain FAs (acetate, propionate, and butyrate). Propionate inhibits cholesterol synthesis . A relatively small number of short-term randomized clinical trials have investigated the cholesterol-lowering effect of fibers in children and adolescents with largely variable results, ranging from no effect to a 30% reduction in LDL-C plasma levels. The most frequently studied fiber is psyllium, followed by glucomannan, oats, and gum, which are usually added to STEP I (daily fat intake < 30%, saturated FAs < 10%, cholesterol < 300 mg) or STEP II (saturated FAs 7%, cholesterol < 200 mg), now indicated as CHILD I and CHILD II diet . Balancing the fiber intake from food and nutraceuticals or fiber-added food is relevant to compliance and outcomes. A very restricted diet—as required by the STEP II diet—even if safe, is not always well accepted by children . Combining the STEP I diet with food-enriched or capsule-containing fiber is often more effective in reducing LDL-C levels. Many studies have demonstrated the efficacy of psyllium . A 12-week randomized controlled study on 50 children with mild hypercholesterolemia on a STEP I diet supplemented with psyllium (3.2 g/daily) showed an additional 8.9% LDL-C decrease compared with the controls . Further, in 36 children with familial combined hyperlipoproteinemia, supplementation with psyllium (2.5–10 g, depending on the age) to a STEP I diet led to a TC and LDL-C level reduction of 11.9% and 13.8%, respectively . Psyllium supplementation also improved the LDL-C lowering effect of the STEP II diet in children with hyperlipidemia , different from what was previously observed in a randomized, double-blinded, placebo-controlled, cross-over study employing 6 g/day of psyllium in 20 children with mild hypercholesterolemia, already on the STEP II diet . Glucomannan supplementation was successfully tested in 36 children with hyperlipidemia who underwent a double-blinded, randomized, placebo-controlled cross-over trial that lasted 24 weeks. This cohort, affected by primary dyslipidemia, was fed a CHILD I diet for ≥1 month. Capsules containing glucomannan (500 mg) were administered at a dose of 1000–1500 mg/day depending on the proband’s weight. TC, LDL-C, and non-HDL-C levels decreased significantly by 5.1%, 7.3%, and 7.2%, respectively . Consistent with previous findings, these results were more pronounced in females than males . However, two meta-analyses of the LDL-C-lowering effect of glucomannan supplementation in children did not confirm any positive effect of this fiber on LDL-C levels . Among other fibers, oat bran supplementation has been tested in children in several clinical trials . For instance, oat bran significantly increased HDL-C levels and reduced LDL-C levels after 7 months of consumption (dosage: 1 g/kg body weight/day) compared with soy derivatives in 20 children with hypercholesterolemia (5–12 years) . Furthermore, locust bean gum (Carruba) showed a significant 11–19% LDL-C level decrease when comparing active and placebo groups in a 16-week cross-over controlled trial, including 11 children with familial combined hyperlipidemia, 10 controls, and 17 adults who consumed locust bean gum (8–30 g/daily) . Among these interventions , psyllium consistently showed the highest reduction in LDL-C levels, ranging from 6.8% to 23%. This was followed by gum interventions, which resulted in a reduction of LDL-C levels ranging from 11% to 19%. Pectin interventions demonstrated a significant reduction of 17% in LDL-C levels. Lastly, Glucomannan interventions combined with Chromium polynicotinate or policosanols showed moderate reductions, ranging from 7.3% to 16% in LDL-C levels. It is important to note that these interventions were effective in lowering LDL-C levels in pediatric populations, but the extent of reduction varied across studies. The compliance was overall good, even if some children refused to follow the prescribed diet or take the capsules. Even if a fiber rich diet is always to be preferred, when the intake is not sufficient, supplemented fibers are usually safe and well tolerated. Mild intestinal discomfort has been reported in clinical trials. 3.2. Plant Sterols Plant sterols, also known as phytosterols or non-cholesterol sterols, are natural compounds found in plants, and are commonly consumed through foods like vegetable oils and nuts. These compounds are ingested in amounts comparable to cholesterol intake (200–400 mg/day), which cannot be synthesized by the human body. Plant sterols effectively and safely lower serum cholesterol levels by hindering cholesterol absorption . Since 2001, plant sterol-enriched foods have been recommended by the National Cholesterol Education Program Guidelines as part of dietary strategies to reduce LDL-C levels . Non-cholesterol sterols, or stanols (in the form of sterol esters), are available commercially, and are added to various foods such as bread, cereals, salad dressings, milk, margarine, and yogurt, often with different flavors and a good taste . Studies have shown that, in adults, incorporating stanols into the milk matrix yielded better results compared to cereals, with LDL-C levels decreasing by 15.9% versus 5.4%, respectively . While stanols were more effective in reducing cholesterol levels compared to sterols, most studies administered sterols at varying doses ranging from 1.6 to 2 g daily. Plant sterols work by inhibiting cholesterol absorption in the intestines, leading to a reduction in serum cholesterol concentration . Phytosterols, particularly sitostanol, compete with cholesterol for absorption in the intestines and displace cholesterol from micelles . Phytosterols are more hydrophobic than cholesterol, making them more susceptible to mixed micelles. Cholesterol and phytosterols rely on Niemann–Pick C1-Like 1 (NPC1L1) protein for absorption into enterocytes. Once absorbed, non-esterified cholesterol and phytosterols are transported back into the intestinal lumen through the action of the ABCG5/G8 gene. Approximately 50% of the cholesterol, but less than 5% of plant sterols, is ultimately absorbed . Phytosterols, when in their free form, are absorbed at low rates (less than 10%), while stanols are not absorbed physiologically . The reduced uptake of intestinal cholesterol and its transport via chylomicrons to the liver result in decreased levels of intermediate-density lipoproteins in addition to LDL-C . In several randomized clinical trials, plant sterols significantly decreased cholesterolemia in children with mild hypercholesterolemia and FH . Dietary supplementation with 1.2–2.0 g/day sterols has been mainly tested in children with FH who had already been on STEP I or II, showing a further LDL-C lowering effect of ~10% in 2–12 months . In children with FH, a daily intake of 2.3 g phytosterols significantly decreased TC (−11%) and LDL-C (−14%) levels compared with placebo spread , whereas higher decreases were observed in children undergoing stanol-added diet (3 g/day) . Apolipoprotein B (Apo-B) levels were also significantly reduced by plant sterols (7–10%) . The efficacy of phytosterols was further demonstrated in non-FH children on a STEP II diet and with mild hypercholesterolemia (mean TC > 197 mg and LDL-C > 125 mg/dL). The daily intake of 1.2 g plant sterol in two doses reduced TC (from −7% to −11%) and LDL-C (from −9% to −14%) levels, respectively, compared with the control group . Margarine containing 1.6 g/day plant sterols or plant stanol ester reduced TC (−9%) and LDL-C (−12%) in children with FH after for 5–6 weeks , while in the STRIP study, 6-year-olds with mild hypercholesterolemia significantly decreased TC and LDL-C, respectively, by −5.4% and −7.5% . Then, plant sterol supplementation could safely reduce LDL-C by roughly 10% and without significantly affecting other lipoprotein levels . Remarkably, the Apo E4 or E3 genotypes were not reported to influence the biochemical effects of sterol addiction in children . The administration of milk, yogurt and margarine frequently could influence the cholesterol-lowering effect of phytosterols , whereas the lipid drop seems independent of baseline levels, being the maximum effect usually reached in a short time (2 weeks, usually) . An additive benefit of the above-mentioned changes is the significant decrease in small dense LDL-C levels after the daily dietary supplementation of 2 g plant sterols in children and adolescents with dyslipidemia . It must, however, be recognized that TG and HDL-C concentrations in plasma are usually unaffected by phytosterol supplementation , as well as the endothelial function . Children undergoing statin therapy show homeostatic changes characterized by increased cholesterol absorption and plant sterol levels. Phytosterol supplementation reverses these changes, and should be considered advantageous . Phytosterols are usually safe and well-tolerated. Variations in carotenoids and fat-soluble vitamins have been reported by studies that used plant sterol- or stanol ester-enriched spreads in adults and children. In children with FH, lipid-adjusted lycopene levels decreased by 8.1% ( p = 0.015) during the stanol period; however, this reduction was not significant at the 6-month follow-up. In addition, alfa- and beta-carotene levels significantly decreased by 17.4% and 10.9%, respectively, in children with FH after the daily consumption of 1.2 g plant sterols for 2 months, recovering at the 6-month follow-up . In the Special Turku Coronary Risk Factor Intervention Project for children (STRIP study), the dietary supplementation of 1.5 g phytosterols in children with mild hypercholesterolemia was associated to a decrease (−19%; p = 0.003) in serum beta-carotene to LDL-C ratio, while the alpha-tocopherol to LDL-C ratio remained unchanged . Moreover, no changes were observed in the levels of the other carotenoids or fat-soluble vitamins . To the extent that there is little data, improving vegetables and fruits intake in children should be suggested as add-on to phytosterol-added dietary regimen, to compensate for any possible reduction in carotenoid also related to seasonal dietary variations. Long-term safety was questioned as phytosterol plasma levels increased the incidence of atherosclerosis , and should be related to an increased risk of cardiovascular events, as described in the large epidemiological cohorts of the PROCAM and MONICA/KORA studies . Premature atherosclerosis has also been observed in the rare autosomal recessive familial form of sitosterolemia . However, campesterol and sitosterol under physiological conditions do not exceed 1% of the total serum sterols, whereas cholesterol accounts for >99% of serum sterols. Moreover, lathosterol was not modified over a 12-week period, proving that the inhibition of cholesterol absorption by phytosterols does not cause an increased cholesterol synthesis . 3.3. Probiotics Probiotics have a limited evidence of cholesterol-lowering effects in adults. This effect results from cholesterol absorption and bile salts hydrolysis (BSH) . The first mechanism, activated by lactic acid bacteria, suppresses the reabsorption of cholesterol in the intestines, while the second mechanism affects the balance of bile salts, resulting in a decrease in plasma LDL-C levels. Moreover, certain strains of bifidobacteria improve blood lipid levels by converting linoleic acid (LA) into conjugated linoleic acid (CLA) . A recent umbrella systematic review of 38 meta-analyses concluded that the probiotics supplementation was effective in reducing TC (effect size [ES], −0.46 mg/dL; 95%CI, −0.61, −0.30; p < 0.001), TG (ES, −0.13 mg/dL; 95%CI, −0.23, −0.04; p = 0.006), and LDL-C levels (ES, −0.29 mg/dL; 95%CI, −0.40, −0.19; p < 0.001), without affecting HDL-C The evidence in children is rare. A 32-week-long, double-blinded, randomized, placebo-controlled, cross-over trial was conducted involving children whose TC levels exceeded the 90th percentile for their age and sex. Administering a mixture of three bifidobacterium strains, selected for characteristics that ameliorated the lipid profile, such as BSH activity, cholesterol adsorption, and CLA production, mildly but significantly improved TC (3.4%), LDL-C (3.8%), and TG (1.9%) levels, and increased HDL-C (1.7%) levels . The effect seems less impressive than that in adults; however, the effect could depend on the tested probiotic formulation, as the probiotic action depends on the strain, strain mix, dosage, and administration medium. Probiotic supplementation is usually safe and well-tolerated. The available clinical data regarding probiotics in adults is inconclusive, and there is limited information available regarding their effects in children. Therefore, it would be premature to definitively state that probiotics have a significant lipid-lowering effect, given the limited evidence available. Fibers are edible parts of plants that pass through the small intestine relatively unchanged in humans, and include complex carbohydrates such as non-starch polysaccharides (pectins, gums, cellulose, hemicellulose, oat bran and wheat bran), oligosaccharides (inulin and fructooligosaccharides), and lignin (i.e., the non-carbohydrate fraction of the dietary fiber). Fibers intake is linked to positive health outcomes, including a lower risk of developing ASCVD and obesity . Fibers naturally contained in cereals, vegetables and fruits (as part of a balanced dietary pattern) ameliorate the lipid profile in adults , and reduce concentrations of TC and Low Density Lipoprotein-Cholesterol (LDL-C) by 5–15% and 9–22%, respectively . These beneficial effects have led regulatory agencies to issue health claims for the intake of fibers , oat β-glucan and its LDL-C lowering effect or ASCVD risk reduction . A large cohort study on 5873 Japanese children (10–11 years) highlighted the presence of an inverse association between the consumption of dietary fibers and plasma concentrations of TC, and the presence of overweight and obesity, confirming data from clinical trials in adults . Although dietary fibers help maintain good health, their quantitative need in children has not been defined yet . Food and Drug Administration (FDA) guidelines relate it to the need for calorie intake (12 g/1000 calories), while the American Academy of Pediatrics guidelines relate it to weight or age . Pediatric recommendations widely vary across countries, being also influenced by the available evidence . In general practice, many pediatricians follow a formula that involves adding 5 g/day to the child’s age (for children older than 3 years) . Even if this guideline is often considered when advocating for a Mediterranean diet or a diet rich in vegetables, it is seldom met in clinical practice. Children living in Western countries typically consume fewer vegetables and fruits, contributing to a lower dietary fiber intake, high calorie dense food, and highly refined high-fat diet . The consequences of such an incorrect dietary intake include metabolic changes and hyperlipidemia, which are sometimes associated with overweight/obesity. In this context, a positive effect of dietary fibers on blood lipids with monounsaturated FAs (MUFAs) was shown in children in the Healthy Start Preschool Study of Cardiovascular Disease Risk Factors and Diet . Soluble fibers include psyllium (viscous and non-fermentable fiber), glucomannan, oat, pectin, and guar gum (viscous and fermentable fiber) and are commercially available as unprocessed fibers that can be added to food or used as flavored powders or capsules. Psyllium, derived from the seed husk of Plantago ovata , is one of the richest sources of soluble mucilaginous dietary fiber, acting as a gel-forming polysaccharide, similarly to pectin and guar gum . Locust bean gum (that is, a galactomannan from the carob tree) is a white and odorless powder extracted from the endosperm of beans without a distinctive taste. Pectin is less viscous than other fibers, but similar in ash and more palatable . Glucomannan, the main polysaccharide obtained from the Asian tuber Amorphophallus konjac, is a palatable, highly viscous soluble fiber. Its chemical structure consists of a mannose (8): glucose (5) ratio linked by b-glycosidic bonds. Glucomannan has the highest molecular weight and viscosity among all dietary fibers . Oat seeds are also an important source of the viscous soluble fiber beta-glucan . The ability of fibers to lower plasma lipids relies on their physicochemical properties and viscosity. Soluble fiber acts mainly to form viscous solutions that slow gastric emptying and reduce fat absorption, thereby modulating lipoprotein metabolism. In the small intestine, the gelling process binds to dietary fats and hinders the absorption of cholesterol, and the reabsorption of bile acids increases their excretion in feces. It follows that there is a reduced uptake of intestinal cholesterol and a reduced circulation of chylomicrons. Of consequence is that the synthesis of bile in the liver increases and LDL-C levels decrease. Another cholesterol-lowering mechanism involves bacterial fermentation in the colon (except for lignin), which leads to production of short-chain FAs (acetate, propionate, and butyrate). Propionate inhibits cholesterol synthesis . A relatively small number of short-term randomized clinical trials have investigated the cholesterol-lowering effect of fibers in children and adolescents with largely variable results, ranging from no effect to a 30% reduction in LDL-C plasma levels. The most frequently studied fiber is psyllium, followed by glucomannan, oats, and gum, which are usually added to STEP I (daily fat intake < 30%, saturated FAs < 10%, cholesterol < 300 mg) or STEP II (saturated FAs 7%, cholesterol < 200 mg), now indicated as CHILD I and CHILD II diet . Balancing the fiber intake from food and nutraceuticals or fiber-added food is relevant to compliance and outcomes. A very restricted diet—as required by the STEP II diet—even if safe, is not always well accepted by children . Combining the STEP I diet with food-enriched or capsule-containing fiber is often more effective in reducing LDL-C levels. Many studies have demonstrated the efficacy of psyllium . A 12-week randomized controlled study on 50 children with mild hypercholesterolemia on a STEP I diet supplemented with psyllium (3.2 g/daily) showed an additional 8.9% LDL-C decrease compared with the controls . Further, in 36 children with familial combined hyperlipoproteinemia, supplementation with psyllium (2.5–10 g, depending on the age) to a STEP I diet led to a TC and LDL-C level reduction of 11.9% and 13.8%, respectively . Psyllium supplementation also improved the LDL-C lowering effect of the STEP II diet in children with hyperlipidemia , different from what was previously observed in a randomized, double-blinded, placebo-controlled, cross-over study employing 6 g/day of psyllium in 20 children with mild hypercholesterolemia, already on the STEP II diet . Glucomannan supplementation was successfully tested in 36 children with hyperlipidemia who underwent a double-blinded, randomized, placebo-controlled cross-over trial that lasted 24 weeks. This cohort, affected by primary dyslipidemia, was fed a CHILD I diet for ≥1 month. Capsules containing glucomannan (500 mg) were administered at a dose of 1000–1500 mg/day depending on the proband’s weight. TC, LDL-C, and non-HDL-C levels decreased significantly by 5.1%, 7.3%, and 7.2%, respectively . Consistent with previous findings, these results were more pronounced in females than males . However, two meta-analyses of the LDL-C-lowering effect of glucomannan supplementation in children did not confirm any positive effect of this fiber on LDL-C levels . Among other fibers, oat bran supplementation has been tested in children in several clinical trials . For instance, oat bran significantly increased HDL-C levels and reduced LDL-C levels after 7 months of consumption (dosage: 1 g/kg body weight/day) compared with soy derivatives in 20 children with hypercholesterolemia (5–12 years) . Furthermore, locust bean gum (Carruba) showed a significant 11–19% LDL-C level decrease when comparing active and placebo groups in a 16-week cross-over controlled trial, including 11 children with familial combined hyperlipidemia, 10 controls, and 17 adults who consumed locust bean gum (8–30 g/daily) . Among these interventions , psyllium consistently showed the highest reduction in LDL-C levels, ranging from 6.8% to 23%. This was followed by gum interventions, which resulted in a reduction of LDL-C levels ranging from 11% to 19%. Pectin interventions demonstrated a significant reduction of 17% in LDL-C levels. Lastly, Glucomannan interventions combined with Chromium polynicotinate or policosanols showed moderate reductions, ranging from 7.3% to 16% in LDL-C levels. It is important to note that these interventions were effective in lowering LDL-C levels in pediatric populations, but the extent of reduction varied across studies. The compliance was overall good, even if some children refused to follow the prescribed diet or take the capsules. Even if a fiber rich diet is always to be preferred, when the intake is not sufficient, supplemented fibers are usually safe and well tolerated. Mild intestinal discomfort has been reported in clinical trials. Plant sterols, also known as phytosterols or non-cholesterol sterols, are natural compounds found in plants, and are commonly consumed through foods like vegetable oils and nuts. These compounds are ingested in amounts comparable to cholesterol intake (200–400 mg/day), which cannot be synthesized by the human body. Plant sterols effectively and safely lower serum cholesterol levels by hindering cholesterol absorption . Since 2001, plant sterol-enriched foods have been recommended by the National Cholesterol Education Program Guidelines as part of dietary strategies to reduce LDL-C levels . Non-cholesterol sterols, or stanols (in the form of sterol esters), are available commercially, and are added to various foods such as bread, cereals, salad dressings, milk, margarine, and yogurt, often with different flavors and a good taste . Studies have shown that, in adults, incorporating stanols into the milk matrix yielded better results compared to cereals, with LDL-C levels decreasing by 15.9% versus 5.4%, respectively . While stanols were more effective in reducing cholesterol levels compared to sterols, most studies administered sterols at varying doses ranging from 1.6 to 2 g daily. Plant sterols work by inhibiting cholesterol absorption in the intestines, leading to a reduction in serum cholesterol concentration . Phytosterols, particularly sitostanol, compete with cholesterol for absorption in the intestines and displace cholesterol from micelles . Phytosterols are more hydrophobic than cholesterol, making them more susceptible to mixed micelles. Cholesterol and phytosterols rely on Niemann–Pick C1-Like 1 (NPC1L1) protein for absorption into enterocytes. Once absorbed, non-esterified cholesterol and phytosterols are transported back into the intestinal lumen through the action of the ABCG5/G8 gene. Approximately 50% of the cholesterol, but less than 5% of plant sterols, is ultimately absorbed . Phytosterols, when in their free form, are absorbed at low rates (less than 10%), while stanols are not absorbed physiologically . The reduced uptake of intestinal cholesterol and its transport via chylomicrons to the liver result in decreased levels of intermediate-density lipoproteins in addition to LDL-C . In several randomized clinical trials, plant sterols significantly decreased cholesterolemia in children with mild hypercholesterolemia and FH . Dietary supplementation with 1.2–2.0 g/day sterols has been mainly tested in children with FH who had already been on STEP I or II, showing a further LDL-C lowering effect of ~10% in 2–12 months . In children with FH, a daily intake of 2.3 g phytosterols significantly decreased TC (−11%) and LDL-C (−14%) levels compared with placebo spread , whereas higher decreases were observed in children undergoing stanol-added diet (3 g/day) . Apolipoprotein B (Apo-B) levels were also significantly reduced by plant sterols (7–10%) . The efficacy of phytosterols was further demonstrated in non-FH children on a STEP II diet and with mild hypercholesterolemia (mean TC > 197 mg and LDL-C > 125 mg/dL). The daily intake of 1.2 g plant sterol in two doses reduced TC (from −7% to −11%) and LDL-C (from −9% to −14%) levels, respectively, compared with the control group . Margarine containing 1.6 g/day plant sterols or plant stanol ester reduced TC (−9%) and LDL-C (−12%) in children with FH after for 5–6 weeks , while in the STRIP study, 6-year-olds with mild hypercholesterolemia significantly decreased TC and LDL-C, respectively, by −5.4% and −7.5% . Then, plant sterol supplementation could safely reduce LDL-C by roughly 10% and without significantly affecting other lipoprotein levels . Remarkably, the Apo E4 or E3 genotypes were not reported to influence the biochemical effects of sterol addiction in children . The administration of milk, yogurt and margarine frequently could influence the cholesterol-lowering effect of phytosterols , whereas the lipid drop seems independent of baseline levels, being the maximum effect usually reached in a short time (2 weeks, usually) . An additive benefit of the above-mentioned changes is the significant decrease in small dense LDL-C levels after the daily dietary supplementation of 2 g plant sterols in children and adolescents with dyslipidemia . It must, however, be recognized that TG and HDL-C concentrations in plasma are usually unaffected by phytosterol supplementation , as well as the endothelial function . Children undergoing statin therapy show homeostatic changes characterized by increased cholesterol absorption and plant sterol levels. Phytosterol supplementation reverses these changes, and should be considered advantageous . Phytosterols are usually safe and well-tolerated. Variations in carotenoids and fat-soluble vitamins have been reported by studies that used plant sterol- or stanol ester-enriched spreads in adults and children. In children with FH, lipid-adjusted lycopene levels decreased by 8.1% ( p = 0.015) during the stanol period; however, this reduction was not significant at the 6-month follow-up. In addition, alfa- and beta-carotene levels significantly decreased by 17.4% and 10.9%, respectively, in children with FH after the daily consumption of 1.2 g plant sterols for 2 months, recovering at the 6-month follow-up . In the Special Turku Coronary Risk Factor Intervention Project for children (STRIP study), the dietary supplementation of 1.5 g phytosterols in children with mild hypercholesterolemia was associated to a decrease (−19%; p = 0.003) in serum beta-carotene to LDL-C ratio, while the alpha-tocopherol to LDL-C ratio remained unchanged . Moreover, no changes were observed in the levels of the other carotenoids or fat-soluble vitamins . To the extent that there is little data, improving vegetables and fruits intake in children should be suggested as add-on to phytosterol-added dietary regimen, to compensate for any possible reduction in carotenoid also related to seasonal dietary variations. Long-term safety was questioned as phytosterol plasma levels increased the incidence of atherosclerosis , and should be related to an increased risk of cardiovascular events, as described in the large epidemiological cohorts of the PROCAM and MONICA/KORA studies . Premature atherosclerosis has also been observed in the rare autosomal recessive familial form of sitosterolemia . However, campesterol and sitosterol under physiological conditions do not exceed 1% of the total serum sterols, whereas cholesterol accounts for >99% of serum sterols. Moreover, lathosterol was not modified over a 12-week period, proving that the inhibition of cholesterol absorption by phytosterols does not cause an increased cholesterol synthesis . Probiotics have a limited evidence of cholesterol-lowering effects in adults. This effect results from cholesterol absorption and bile salts hydrolysis (BSH) . The first mechanism, activated by lactic acid bacteria, suppresses the reabsorption of cholesterol in the intestines, while the second mechanism affects the balance of bile salts, resulting in a decrease in plasma LDL-C levels. Moreover, certain strains of bifidobacteria improve blood lipid levels by converting linoleic acid (LA) into conjugated linoleic acid (CLA) . A recent umbrella systematic review of 38 meta-analyses concluded that the probiotics supplementation was effective in reducing TC (effect size [ES], −0.46 mg/dL; 95%CI, −0.61, −0.30; p < 0.001), TG (ES, −0.13 mg/dL; 95%CI, −0.23, −0.04; p = 0.006), and LDL-C levels (ES, −0.29 mg/dL; 95%CI, −0.40, −0.19; p < 0.001), without affecting HDL-C The evidence in children is rare. A 32-week-long, double-blinded, randomized, placebo-controlled, cross-over trial was conducted involving children whose TC levels exceeded the 90th percentile for their age and sex. Administering a mixture of three bifidobacterium strains, selected for characteristics that ameliorated the lipid profile, such as BSH activity, cholesterol adsorption, and CLA production, mildly but significantly improved TC (3.4%), LDL-C (3.8%), and TG (1.9%) levels, and increased HDL-C (1.7%) levels . The effect seems less impressive than that in adults; however, the effect could depend on the tested probiotic formulation, as the probiotic action depends on the strain, strain mix, dosage, and administration medium. Probiotic supplementation is usually safe and well-tolerated. The available clinical data regarding probiotics in adults is inconclusive, and there is limited information available regarding their effects in children. Therefore, it would be premature to definitively state that probiotics have a significant lipid-lowering effect, given the limited evidence available. Other dietary and food components with cholesterol-lowering actions beyond inhibiting cholesterol absorption from the bowel have also been tested in children and/or adolescents. These trials were usually small, short-term, and limited to specific settings , suggesting the need for further confirming larger and long-term studies. 4.1. Nuts Nuts (i.e., almonds, hazelnuts, pistachios, walnuts, macadamia nuts and peanuts) are classified as dry fruits. In recent decades, the cardioprotective and health-promoting qualities of nuts—particularly walnuts—have been extensively demonstrated in epidemiological studies. These positive effects are due to their composition rich in bioactives, such as monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), tocopherols (vitamin E), antioxidants, phytosterols, fibers and polyphenols, with hazelnuts being especially noteworthy in this regard . Additionally, hazelnuts stand out for having the highest content of MUFAs among all nuts . Regular hazelnut intake also seems to improve microbiota composition and reduce the intestinal concentration of short-chain fatty acids (SCFAs) . Moreover, polyphenols influence cholesterol absorption, TG synthesis and secretion and exert antioxidant effects . To the best of our knowledge, only a few small trials until now have investigated the effects of hazelnuts on plasma lipids in children. Hazelnuts peeled or with the skin (0.43 g/kg of body weight, 15–30 g portions) were shown to significantly reduce LDL-C, while the ratio HDL-C/LDL-C increased compared to a control group receiving the STEP I diet. Moreover, their intake increased the MUFA/saturated fatty acids (SFA) ratio in red blood cells and lowered the endogenous and oxidative induced DNA damage . 4.2. Soy Soy contains several bioactive compounds that are supposed to improve plasma lipid levels in humans (i.e., isoflavones, phytosterols and specific peptides, which can promote LDL-C receptor expression in liver cells) . A recent meta-analysis showed that a median intake of 25 g/day of soy proteins during a median follow-up of 6 weeks decreased LDL-C plasma levels by 3–4% in adults (−4.8 mg/dL; 95%CI −6.7, −2.8 mg/dL, p < 0.0001) . However, only few studies involving children exist. In a pilot study testing the cholesterol-lowering effect of soy or milk protein in children with FH, TC and LDL-C levels did not significantly change though TG and HDL-C improved . Different results were obtained in a prospective study conducted on 16 children with FH. TC, LDL-C, and ApoB significantly improved (−7.7%, −6.4% and −12.6%, respectively) after a 3-month period in which soy proteins were incorporated into the Step I diet, being administered in the form of soy-based dairy-free milk at a dosage of 0.25–0.5 g/kg body weight . This study represents an extended examination of soy protein in pediatric populations. Interestingly, even if children were generally adherent to the program, not all participants (4 out of 16) exhibited the desired response, despite having similar characteristics at entry. Overall, most studies concur on the efficacy of soy protein; however, safety concerns—such as allergic reactions or the potential effects of dietary phytoestrogens (i.e., isoflavones)—remain debatable, especially in the youngest participants . A 13-week randomized controlled clinical trial has been recently launched to address the effect of a soy-rich diet compared to a low-fat diet and a control diet in children with FH. After 7 weeks from randomization, the reduction in LDL-C levels was notably greater in the soy group (155 ± 29 mg/dL) compared to the control group (176 ± 28 mg/dL; P for comparison = 0.038), with a similar trend observed at 13-week follow-up (LDL-C = 180 ± 42 mg/dL in the control group and 155 ± 30 mg/dL in the soy group; P for comparison = 0.089). The relative decrease in LDL-C levels was significantly associated with plasma isoflavone concentrations (specifically daidzein and genistein), as measured at week 7 . Moreover, it must be acknowledged that soy proteins are usually well tolerated, and the occurrence of acute reactions depends on the individual hypersensitivity. However, total protein intake must be balanced with soy intake, and safety of the phytoestrogens must be confirmed on the long term. 4.3. Polyunsaturated Fatty Acids (PUFAs) The dietary fats composition is a crucial determinant of lipid concentrations in plasma . Nonetheless, the dietary intake of polyunsaturated fatty acids (PUFAs)—including Omega-3 and Omega-6—is frequently insufficient in children . Dietary supplementation of PUFAs have shown to impact cardiovascular risk markers, such as TG, LDL-C and adhesion molecules, while also possessing anti-inflammatory properties . In a randomized double-blinded placebo-controlled clinical trial involving 107 healthy children, PUFAs and low saturated fatty acids were able to significantly reduce markers of endothelial cell activation (i.e., adhesion molecules, E-selectin, ICAM-1 and lymphocyte levels), while increasing plasma concentration of Docosahexaenoic acid (DHA) after receiving a 5-month daily intake of a milk enriched product . Another functional food that has been studied in this context is the hempseed oil, which is notably rich in essential fatty acids, including Omega-3 PUFA α-linolenic acid (ALA) and Omega-6 PUFA linoleic acid (LA), with an LA/ALA ratio ranging between 2:1 and 3:1 . The cholesterol-lowering effect of hempseed oil has been extensively assessed in animals and adult humans. However, a recent 8-week randomized controlled trial showed that HSO increases the content of total n-3 and n-6 PUFAs in red blood cells and improves the Omega-3 index, even in children with hyperlipidemia. Moreover, according to the findings of the study, hempseed oil exerted significant reductions in LDL-C (−14%) compared to the control . Overall, emerging observations offer valuable insights for enhancing and complementing food intake with PUFAs. However, further studies are warranted to validate preliminary data. 4.4. Red Yeast Rice Red yeast rice is a widely used and clinically tested cholesterol-lowering nutraceutical derived from the fermentation of standard rice by specific mycelia (usually Monascus purpureus Went) with the production of a pigment (making the rice red) and some bioactive compounds, among which monacolins are reversible inhibitors of 3-hydroxy-3-methyl-glutaril Coenzyme A reductase . The most bioactive compound was monacolin K, which is chemically analogous to lovastatin. In adults, monacolin K significantly reduces cholesterolemia while maintaining an acceptable safety profile . To date, only one study has been conducted in children at increased cardiovascular risk, including those with familial hypercholesterolemia (FH) and familial combined hyperlipidemia, while following a step II diet . The study, designed as a double-blinded, randomized cross-over trial, spanned 6 months in total, during which all participants completed the trial without experiencing any notable adverse effects. After 8 weeks of treatment, there was a significant reduction in TC, LDL-C, and ApoB levels by −18.5%, −25.1%, and −25.3%, respectively, while HDL-C remained unchanged. These findings were remarkable in terms of compliance, tolerability, and efficacy. Notably, the administered dose of Monacolin K was 3 mg/day, demonstrating impressive results comparable to those achieved with pravastatin (10 mg/day or more), indicating a potential synergistic effect with other bioactive components. A recent change in European Union (EU) regulations forbids the use of red yeast rice in children and adolescents because of the presumed (but not demonstrated) risk to health . Nuts (i.e., almonds, hazelnuts, pistachios, walnuts, macadamia nuts and peanuts) are classified as dry fruits. In recent decades, the cardioprotective and health-promoting qualities of nuts—particularly walnuts—have been extensively demonstrated in epidemiological studies. These positive effects are due to their composition rich in bioactives, such as monounsaturated fatty acids (MUFAs), polyunsaturated fatty acids (PUFAs), tocopherols (vitamin E), antioxidants, phytosterols, fibers and polyphenols, with hazelnuts being especially noteworthy in this regard . Additionally, hazelnuts stand out for having the highest content of MUFAs among all nuts . Regular hazelnut intake also seems to improve microbiota composition and reduce the intestinal concentration of short-chain fatty acids (SCFAs) . Moreover, polyphenols influence cholesterol absorption, TG synthesis and secretion and exert antioxidant effects . To the best of our knowledge, only a few small trials until now have investigated the effects of hazelnuts on plasma lipids in children. Hazelnuts peeled or with the skin (0.43 g/kg of body weight, 15–30 g portions) were shown to significantly reduce LDL-C, while the ratio HDL-C/LDL-C increased compared to a control group receiving the STEP I diet. Moreover, their intake increased the MUFA/saturated fatty acids (SFA) ratio in red blood cells and lowered the endogenous and oxidative induced DNA damage . Soy contains several bioactive compounds that are supposed to improve plasma lipid levels in humans (i.e., isoflavones, phytosterols and specific peptides, which can promote LDL-C receptor expression in liver cells) . A recent meta-analysis showed that a median intake of 25 g/day of soy proteins during a median follow-up of 6 weeks decreased LDL-C plasma levels by 3–4% in adults (−4.8 mg/dL; 95%CI −6.7, −2.8 mg/dL, p < 0.0001) . However, only few studies involving children exist. In a pilot study testing the cholesterol-lowering effect of soy or milk protein in children with FH, TC and LDL-C levels did not significantly change though TG and HDL-C improved . Different results were obtained in a prospective study conducted on 16 children with FH. TC, LDL-C, and ApoB significantly improved (−7.7%, −6.4% and −12.6%, respectively) after a 3-month period in which soy proteins were incorporated into the Step I diet, being administered in the form of soy-based dairy-free milk at a dosage of 0.25–0.5 g/kg body weight . This study represents an extended examination of soy protein in pediatric populations. Interestingly, even if children were generally adherent to the program, not all participants (4 out of 16) exhibited the desired response, despite having similar characteristics at entry. Overall, most studies concur on the efficacy of soy protein; however, safety concerns—such as allergic reactions or the potential effects of dietary phytoestrogens (i.e., isoflavones)—remain debatable, especially in the youngest participants . A 13-week randomized controlled clinical trial has been recently launched to address the effect of a soy-rich diet compared to a low-fat diet and a control diet in children with FH. After 7 weeks from randomization, the reduction in LDL-C levels was notably greater in the soy group (155 ± 29 mg/dL) compared to the control group (176 ± 28 mg/dL; P for comparison = 0.038), with a similar trend observed at 13-week follow-up (LDL-C = 180 ± 42 mg/dL in the control group and 155 ± 30 mg/dL in the soy group; P for comparison = 0.089). The relative decrease in LDL-C levels was significantly associated with plasma isoflavone concentrations (specifically daidzein and genistein), as measured at week 7 . Moreover, it must be acknowledged that soy proteins are usually well tolerated, and the occurrence of acute reactions depends on the individual hypersensitivity. However, total protein intake must be balanced with soy intake, and safety of the phytoestrogens must be confirmed on the long term. The dietary fats composition is a crucial determinant of lipid concentrations in plasma . Nonetheless, the dietary intake of polyunsaturated fatty acids (PUFAs)—including Omega-3 and Omega-6—is frequently insufficient in children . Dietary supplementation of PUFAs have shown to impact cardiovascular risk markers, such as TG, LDL-C and adhesion molecules, while also possessing anti-inflammatory properties . In a randomized double-blinded placebo-controlled clinical trial involving 107 healthy children, PUFAs and low saturated fatty acids were able to significantly reduce markers of endothelial cell activation (i.e., adhesion molecules, E-selectin, ICAM-1 and lymphocyte levels), while increasing plasma concentration of Docosahexaenoic acid (DHA) after receiving a 5-month daily intake of a milk enriched product . Another functional food that has been studied in this context is the hempseed oil, which is notably rich in essential fatty acids, including Omega-3 PUFA α-linolenic acid (ALA) and Omega-6 PUFA linoleic acid (LA), with an LA/ALA ratio ranging between 2:1 and 3:1 . The cholesterol-lowering effect of hempseed oil has been extensively assessed in animals and adult humans. However, a recent 8-week randomized controlled trial showed that HSO increases the content of total n-3 and n-6 PUFAs in red blood cells and improves the Omega-3 index, even in children with hyperlipidemia. Moreover, according to the findings of the study, hempseed oil exerted significant reductions in LDL-C (−14%) compared to the control . Overall, emerging observations offer valuable insights for enhancing and complementing food intake with PUFAs. However, further studies are warranted to validate preliminary data. Red yeast rice is a widely used and clinically tested cholesterol-lowering nutraceutical derived from the fermentation of standard rice by specific mycelia (usually Monascus purpureus Went) with the production of a pigment (making the rice red) and some bioactive compounds, among which monacolins are reversible inhibitors of 3-hydroxy-3-methyl-glutaril Coenzyme A reductase . The most bioactive compound was monacolin K, which is chemically analogous to lovastatin. In adults, monacolin K significantly reduces cholesterolemia while maintaining an acceptable safety profile . To date, only one study has been conducted in children at increased cardiovascular risk, including those with familial hypercholesterolemia (FH) and familial combined hyperlipidemia, while following a step II diet . The study, designed as a double-blinded, randomized cross-over trial, spanned 6 months in total, during which all participants completed the trial without experiencing any notable adverse effects. After 8 weeks of treatment, there was a significant reduction in TC, LDL-C, and ApoB levels by −18.5%, −25.1%, and −25.3%, respectively, while HDL-C remained unchanged. These findings were remarkable in terms of compliance, tolerability, and efficacy. Notably, the administered dose of Monacolin K was 3 mg/day, demonstrating impressive results comparable to those achieved with pravastatin (10 mg/day or more), indicating a potential synergistic effect with other bioactive components. A recent change in European Union (EU) regulations forbids the use of red yeast rice in children and adolescents because of the presumed (but not demonstrated) risk to health . Multiple nutraceuticals have shown efficacy in reducing lipid levels, as evidenced by the available clinical trials. However, it is important to note that no single dietary supplement can replace the importance of proper dietary counseling. Lifestyle changes and adherence to a correct Mediterranean diet showed a mean decrease of 9.5% in TC, 13.5% in LDL-C, and −10.9% in non-HDL-C plasma levels in a recent large retrospective study conducted on children with polygenic and familial hypercholestremia . This may be sufficient to manage mild polygenic hypercholesterolemia. Phytosterols and fiber-enriched foods are usually shown to be effective in reducing TC and LDL-C levels in children with FH or polygenic hypercholesterolemia or children affected by other secondary dyslipidaemias, especially when combined with a STEP I (daily fat intake < 30%, saturated FAs < 10%, cholesterol < 300 mg) or STEP II (saturated FAs 7%, cholesterol < 200 mg) diet. ApoB is also ameliorated after phytosterol intake, while contrasting results were observed after fiber intake. In contrast, no favorable variations have been observed in the endothelial function of phytosterol/sterol addition, and only two studies concerning this topic are inconclusive. Plant sterol/stanol and fiber were well received, with high compliance observed, particularly in the short term. Some gastrointestinal side effects would be expected if phytosterols are assumed with addition of artificial sweeteners. Noteworthy is the absence of significant adverse effects; nonetheless, abdominal discomfort or diarrhea were commonly reported symptoms with fiber supplementation. They could be even more frequent and severe when fibers are assumed with addition of artificial sweeteners. Functional foods incorporating phytosterols/stanols were associated with a notable decline in carotenoids, but not other vitamins, highlighting the importance of maintaining an adequate intake of vegetables and fruits to prevent nutritional deficiencies. While randomized and controlled studies focusing on robust endpoints in children are lacking and infrequent, certain benefits have been noted, particularly in the short term, primarily relating to plant sterol/stanol and fibers. Many other bioactive compounds have demonstrated efficacy as cholesterol-lowering agents in adults (red yeast rice, berberine, bergamot polyphenol fraction, and artichoke extracts), but not in children. Presently, no dietary supplements have been shown to significantly reduce lipoprotein (a) levels, beyond L-carnitine and Coenzyme Q10, but this effect has never tested in children . The European Atherosclerosis Society Consensus Panel recommended that functional foods containing phytosterols to be considered for children with familial hypercholesterolemia, and as dietary additive supplementation rather than independent pharmaceutical treatment, especially with lifestyle modification . Despite the limitation on the available evidence of dyslipidemia in pediatric age group, guidelines and evidence suggest that nutraceuticals, particularly fibers and phytosterols, can be utilized when combined with appropriate diet in children with genetic dyslipidemias starting from the age of 6 years old . However, it could make sense if the treatment with cholesterol-lowering nutraceuticals are adequately dosed, long-term and effective. Lifestyle and dietary modifications are recommended for any child or adolescent presenting with mild to moderate dyslipidemia. Phytosterols and fibers are deemed safe, while the other mentioned nutraceuticals may serve as possible efficacious additions to dietary treatment when combined with appropriate diet regimen. It is important to note that nutraceuticals should not be viewed as substitutes for diet or statins when they are medically indicated, as advised by the main international guidelines. |
Clinicopathological Significance of Claudin-6 Immunoreactivity in Low-grade, Early-stage Endometrioid Endometrial Carcinoma | eeb36903-b79f-40e8-bbf8-36d632b270d1 | 11705117 | Anatomy[mh] | Case selection. This study protocol was reviewed and approved by the Institutional Review Board (IRB) of the Samsung Medical Center (Seoul, Republic of Korea; protocol number: 2024-09-073). Owing to the retrospective nature of this study, the IRB waived the requirement for the investigators to obtain signed informed consent. We searched patients who underwent surgery for primary EEC in our institutional databases. All available slides were examined by a single gynecological pathologist (H.-S.K.). Diagnoses were established based on the 2020 World Health Organization Classification of Female Genital Tumors . The inclusion criteria were International Federation of Gynecology and Obstetrics (FIGO) grade 1 or 2, initial FIGO stage I or II, and wild-type p53 expression. Finally, we included 118 patients who underwent hysterectomy for primary LGES-EEC at the Samsung Medical Center (Seoul, Republic of Korea). Data collection. The following clinicopathological information was collected from the electronic medical records and pathology reports: patient age at initial diagnosis, FIGO grade, initial FIGO stage, lymphovascular space invasion (LVSI), postoperative recurrence, survival status, and follow-up period between the time of surgery and the last follow-up. As the initial stages of all patients were determined based on the 2009 FIGO staging system, they were restaged according to the updated 2023 FIGO staging system . The extent of LVSI was categorized into focal (<5 vessels) and substantial (≥5 vessels) groups to classify the cases into 2023 FIGO stages IB and IIB, respectively . Immunostaining. Formalin-fixed, paraffin-embedded tissue blocks were cut into 5-μm-thick sections, deparaffinized in xylene, and rehydrated using graded alcohols. Sections were then incubated with hydrogen peroxide to block endogenous peroxidase activity. After antigen retrieval, the sections were incubated with a CLDN6 antibody (dilution 1:100, clone EPR28103-113, Abcam, Cambridge, UK). Immunostaining was performed using a Bond-RX automated stainer (Leica Biosystem, Melbourne, Australia) using a Bond Polymer Refine Detection System (DS9800; Vision Biosystems, Melbourne, Australia) . After chromogenic visualization, the slides were counterstained with hematoxylin. Positive and negative controls were stained concurrently. Human testicular seminoma exhibiting membranous CLDN6 immunoreactivity served as a positive control , whereas non-immune serum substituted for the primary antibody was used as a negative control, resulting in undetectable staining. Staining intensity was designated as negative (score 0), weak (score 1), moderate (score 2), or strong (score 3). Staining proportions were determined in increments of 5% across a 0-100% range. Histoscore was calculated as the sum of the proportion of stained area at each intensity level multiplied by the weighted staining intensity . All immunostained slides were scored by a single board-certified pathologist (Y.L.) who was blinded to the patients’ identities. Statistical analysis. Pearson’s chi-squared test or Fisher’s exact test was used to examine the association between CLDN6 expression and the clinicopathological characteristics of patients with LGES-EEC. All statistical analyses were performed using an IBM SPSS Statistics for Windows (version 23.0; IBM Corp., Armonk, NY, USA). Statistical significance was set at p <0.05. Baseline characteristics. summarizes the clinico-pathological characteristics of the 118 patients with LGES-EEC. Patients’ ages ranged from 30 to 88 years (median=57 years; mean=57.1 years). Thirty-three (28.0%) and 85 (72.0%) patients were diagnosed with grade 1 and 2 EECs, respectively. Seventy-eight (66.1%) tumors invaded less than half of the myometrium, whereas 40 (33.9%) tumors involved equal to or more than half of the myometrium. Cervical stromal extension was identified in 20 (16.9%) patients. The distribution of initial FIGO stages was as follows: IA1 (6/118; 5.1%), IA2 (36/118; 30.5%), 1A3 (3/118; 2.5%), IB (33/118; 28.0%), IIA (18/118; 15.3%), and IIB (22/118; 18.6%). LVSI was absent in more than half of the patients (65/118, 55.1%), whereas 22 (18.6%) patients showed substantial LVSI. In particular, 13 of 22 patients whose tumors invaded less than half of the myometrium without cervical stromal extension (2009 FIGO stage IA) were upstaged to IIB because of the presence of substantial LVSI. Areas of mucinous and squamous differentiation were identified in 15 (12.7%) and 29 patients (24.6%), respectively. Five patients (4.2%) experienced recurrence. Three patients (2.5%) died due to disease progression. CLDN6 immunoreactivity. A illustrates the typical morphology of LGES-EEC. Figure 1B-E illustrates the immunoreactivity of CLDN6, including focal weak (histoscore 3; Figure 1B), focal strong (histoscore 30; Figure 1C), and diffuse strong (histoscore 210; Figure 1D and E). CLDN6 was primarily localized along the membranes of tumor cells, and the staining intensities and proportions varied among cases. We considered tumors showing both a staining proportion of ≥5% and an intensity of ≥2 (resulting in histoscore ≥10) as CLDN6-positive ones. Based on semi-quantification using the histoscore , 26 of 118 cases (22.0%) were positive for CLDN6 expression. In this group, 15 (12.7%), 5 (4.2%), and 6 (5.1%) patients had histoscores of 15-30, 60-90, and 120-210, respectively. CLDN6 reacted with more than half of the tumor cells in five cases (4.2%). Regarding staining intensity, 39 cases (33.1%) exhibited moderate to strong immunoreactivity for CLDN6. CLDN6 exhibited intratumoral heterogeneity in some cases, in which CLDN6-positive and CLDN6-negative subpopulations were observed in adjacent glands (Figure 1F). CLDN6 was localized within the invasive tumor front in a few cases, whereas in other cases, its expression was observed in areas of endometrial intraepithelial neoplasia. Cytoplasmic and nuclear CLDN immunoreactivity was observed in two (1.7%) and one (0.8%) cases, respectively, while in another case (0.8%), CLDN6 was localized on the luminal surface of the neoplastic glands. Clinicopathological significance of CLDN6 expression. To reveal the clinical relevance of CLDN6 expression in patients with LGES-EEC, we investigated whether there is a significant relationship between CLDN6 expression and clinicopathological parameters, including age at diagnosis, histological grade, myometrial invasion, cervical stromal extension, stage, LVSI, mucinous and squamous differentiation, and recurrence. As shown in , positive CLDN6 expression was significantly associated with deeper myometrial invasion ( p =0.001), higher stage ( p =0.015), and substantial LVSI ( p =0.018). Patients with CLDN6-low tumors tended to be less prone to recurrence, but the difference was not significant ( p =0.070). In early-stage (stage I) tumors, a significant relationship between positive CLDN6 expression and deeper myometrial invasion was observed ( p =0.012; ). In this study, we demonstrated that aberrant CLDN6 expression in LGES-EEC tissues, in which the positive CLDN6 immunoreactivity on the cell membranes was observed in 22.0% (26/118) when a histoscore cutoff value of 10 was applied, was significantly associated with deeper myometrial invasion, higher initial stage, and substantial LVSI. Our results are consistent with those of Kojima et al . , who reported that high CLDN6 expression is related to stage III-IV, non-endometrioid type, grade 3, LVSI, lymph node metastasis, and distant metastasis. Although we did not examine the prognostic significance of CLDN6 expression, they found that CLDN6 expression is an independent prognostic factor for overall survival in patients with EC. Similarly, Cao et al . analyzed RNA sequencing data obtained from The Cancer Genome Atlas database and reported that CLDN6 is over-expressed in EC tissues. They observed that increased CLDN6 expression levels correlate with non-endometrioid type, higher grade, higher stage, and worse overall survival in patients with EC. In addition, Zhang et al . analyzed RNA sequencing data and relevant clinical data downloaded from the TCGA and Genotype-Tissue Expression databases and noted that CLDN6 expression is higher in patients older than 60 years, with stage III-IV disease, grade 3, and serous type, and that a high CLDN6 expression level is an independent risk factor for worse disease-specific survival and shorter progression-free interval in variable clinical subgroups of EC. Although previous studies have been conducted using both low- and high-grade ECs of various histological types and stages, we focused only on LGES-EECs and obtained concordant results. Collectively, these data underscore the significance of CLDN6 in EC progression. It is unknown how high CLDN6 expression leads to tumor progression and poor prognosis in patients with EC. Previous studies have assessed the biological function of CLDN6 and the molecular mechanisms underlying altered CLDN6 expression in EC. Cao et al . transfected small interfering RNA targeting CLDN6 into an EC cell line to establish CLDN6-knockdown EC cells and noted that the knockdown of CLDN6 significantly inhibited cellular proliferation and colony formation and restrained the invasive and migratory abilities of EC cells. They also found that the inhibition of CLDN6 remarkably decreased the expression levels of phosphorylated Akt, phosphorylated phosphatidylinositol 3-kinase (PI3K), and mammalian target of rapamycin (mTOR) in EC cells, indicating that the up-regulation of CLDN6 expression results in a malignant phenotype mediated through PI3K/Akt/mTOR signaling in EC. Sugimoto et al . demonstrated that CLDN6 recruits and activates Src family kinases (SFKs), which, in turn, phosphorylate CLDN6 and propagate the PI3K/Akt pathway. They showed that the CLDN6/SFK/PI3K/Akt signaling axis targets retinoic acid receptor γ and estrogen receptor (ER)-α and stimulates their activities. In addition, Kojima et al . revealed that CLDN6/SFK/PI3K-dependent Akt signaling stimulates the transcriptional activity of ER-α in EC cells, thereby promoting tumor progression. Taken together with the notion that ER acts as a master transcription factor in EC , aberrant CLDN6 signaling may promote the malignant behavior of EC cells by hijacking the CLDN6/ER pathway. Accumulating evidence has shown that increased CLDN6 expression contributes to the malignant progression of EC and that CLDN6 may be a promising therapeutic target for patients with EC. It would also be interesting to determine whether the link between cell adhesion and nuclear receptor signaling regulates tumor progression in EC. Study limitations. First, our cohort was obtained from a single institution, which limits the reproducibility of the data. Second, owing to the limited number of deaths and recurrences, statistical analysis of survival differences was not feasible. Further studies using more extensive prognostic data from larger cohorts are warranted. Third, molecular analyses of the mechanisms underlying CLDN6 aberrations were beyond the scope of this study. Finally, we did not examine the clinical implications of intratumoral CLDN6 heterogeneity observed in a small number of our cases. Since spatial heterogeneity in biomarker expression may have prognostic significance and influence therapeutic response, further studies are required to clarify the role of intratumoral heterogeneity in CLDN6 expression in EC. We demonstrated aberrant CLDN6 immunoreactivity in tumor cell membranes in LGES-EEC tissues. Positive CLDN6 expression is significantly associated with deeper myometrial invasion, a higher initial stage, and substantial LVSI in patients with LGES-EEC. In stage I tumors, a significant association was also observed between positive CLDN6 expression and deeper myometrial invasion. CLDN6 may be involved in tumor progression in EC; thus, CLDN6 may be a promising therapeutic target for patients with EC. Further studies are warranted to clarify the molecular mechanisms underlying the alteration of CLDN6 expression and its biological function in EC. All Authors have no conflicts of interest or financial ties to declare that are relevant to the content of this article. All Authors made substantial contributions to the conception and design of this work, acquisition and interpretation of data, drafting and critical revision of the manuscript for important intellectual content, and approval of the final version to be published. This study was supported by the Samsung Medical Center Grant (SMO1240641) and the National Research Foundation of Korea (NRF) grant funded by the Korean Government (MSIT) (2023R1A2C2006223). |
Postmortem histological freeze–thaw artifacts: a case report of a frozen infant and literature review | dfd5fa9d-940f-4309-a9d7-e76236e8a14f | 11790699 | Pathology[mh] | Freezing and thawing have the potential to alter the gross and histological appearance of tissues, damaging individual cells and disrupting the overall architecture. However, the exact mechanism and influencing factors of this damage are not yet fully understood . It is believed that the formation and rupture of cell wall pores result from fluid shifts that take place during the freezing and thawing process. As the extracellular water freezes, fluid moves out from the cells and pours into the extracellular space. This movement of liquid leads to increased intracellular solute concentration, causing cell shrinkage and the disruption of cellular junctions. When the tissue thaws, the fluid returns to the cells, resulting in changes in volume that can rupture weakened cell walls. These combined effects are considered to be the main driving force behind the observed changes, rather than physical damage caused by ice crystals . The outcome of the cells is highly dependent on the rate at which the tissue has been frozen. Depending on the freezing rate and the permeability of the cell, water will either flow out of the cell, which will shrink, or stay inside, forming intracellular ice crystals . In forensic investigations, freezing and thawing can play a crucial role in cases of unknown cause of death. Perpetrators may employ freezing preservation to conceal the body or obscure the time of death. Freezing can also occur naturally when a body is exposed to the elements, and it may even be a cause of death itself. Determining whether a body has been frozen can help corroborate or challenge a suspect’s statements and ultimately determine if any laws have been violated . In this paper, we present a case report in which an autopsy was performed on an infant, who had died of natural causes, after undergoing freezing and thawing. The objective of this study was to reveal several histological artifacts in different tissues resulting from the freeze–thaw process. The authors compare and discuss their findings with the literature. Medical history The infant was a 1-month-old male, born at full term with anthropometrical parameters within the standard range for age: weight 3970 g, 51 cm in crown-heel length, and head circumference of 36 cm. The pregnancy was unremarkable, with normal periodic ultrasound scans, and the delivery was spontaneous and uncomplicated. Maternal vaginal and rectal swabs were negative, and she received intrapartum antibiotic prophylaxis. Approximately 1 month after birth, the infant presented persistent cough, and aerosol therapy was promptly administered. However, after 3 days, he progressively became drowsy and unresponsive. The parents quickly took him to the hospital, but he was already in cardiopulmonary arrest. Cardiopulmonary resuscitation (CPR) was immediately started: oxygen was given through a bag valve mask, and then, an injection of adrenaline was necessary. Despite all attempts, CPR was stopped after 35 min due to pulseless electrical activity (PEA) and the infant was pronounced dead. Subsequent nasal and pharyngeal swabs revealed positive results for respiratory syncytial virus A (RSV), Haemophilus influenzae , and Staphylococcus aureus . Autopsy findings An autopsy was requested to better understand the cause of death. However, in that period COVID-19 was initially spreading, and the lack of medical resources made it necessary to temporarily store the infant’s body at − 10 °C in the freezer of the morgue. Then, due to the health-related challenges arising from the COVID-19 pandemic, the autopsy was delayed for 21 days. Before the autopsy was performed, the body was thawed at room temperature (20 °C) for 12 h. External examination showed growth parameters within the normal limits for 1 month of age: weight 4301 g (50th percentile), crown-heel length 55 cm (50th percentile), and head circumference 37 cm (25th percentile) . The face was regular with no dysmorphisms. Grossly, no anomalies were identified. Internal examination evidenced diffuse congestion of the organs, but no anatomical anomalies. The lungs displayed the expected lobes, with three on the right and two on the left. The right lung weighed 61 g and the left 50 g. Segmental sequential analysis of the heart (weight 29 g) revealed atrioventricular and ventriculoarterial concordance and regular systemic and pulmonary venous returns. The coronary arteries presented a regular course. The other organ weights were as follows: liver 173 g, right kidney 20.8 g, left kidney 22.4 g, spleen 16.4 g, brain 489 g. Histological examination of the lungs revealed bilateral extensive acute necrotizing bronchitis and bronchiolitis with focal hyaline membrane formation and pulmonary edema (Fig. ). Scattered cells with enlarged nuclei due to RSV infection were seen (Fig. ). Minimal diffuse ischemic changes were observed in the myocardium and brain. Both kidneys showed acute tubular necrosis, tubular dilatation, and occasional microcalcifications. No significant histopathological findings were observed in the other organs. The integration of clinical, autopsy, and histological data identified the cause of death as acute respiratory failure resulting from bilateral and diffuse acute necrotizing bronchitis and bronchiolitis. This was caused by RSV type A complicated by bacterial over-infection with Haemophilus influenzae and Staphylococcus aureus . Histological freeze–thaw artifacts Evidence of histological freeze–thaw artifacts was present in many organs. The brain disclosed ice crystal artifacts with multiple clefts within the parenchyma (Fig. ). The lungs, apart from histological evidence of infection, exhibited alveolar distension (Fig. ). The heart presented marked extracellular separation (Fig. ). Abnormal dilatation of lymphatic or capillary vessels was excluded by the regular expression of CD31, which is a marker for endothelial cells, in the vascular structures located between the bundles of cardiomyocytes. Moreover, the artifactual clefts were devoid of endothelial lining (Fig. ). The liver showed shrinkage of the hepatocellular laminae with marked expansion of the sinusoidal space (Fig. ). The kidneys presented unusual cystic lacunae, probably due to expanded extracellular spaces, with tubules strained and flattened (Fig. ). The thymus structure was almost fully preserved (Fig. A). The thyroid presented clefts mostly along the septa, and the cell nuclei appeared more basophilic (Fig. B). The acinar architecture of the pancreas was maintained, with a few artifactual lacunae within the septa and the islets were still recognizable (Fig. C). On the whole, the cell nuclei appeared shrunk and strongly basophilic. The spleen displayed some cystic slightly eosinophilic spaces devoid of epithelium (Fig. D). The brown adipose tissue was mainly preserved, but focal cellular shrinkage was seen in the most eosinophilic cells (Fig. ). The white adipose tissue and the skeletal muscle were virtually unaffected by artifacts (Fig. ). The infant was a 1-month-old male, born at full term with anthropometrical parameters within the standard range for age: weight 3970 g, 51 cm in crown-heel length, and head circumference of 36 cm. The pregnancy was unremarkable, with normal periodic ultrasound scans, and the delivery was spontaneous and uncomplicated. Maternal vaginal and rectal swabs were negative, and she received intrapartum antibiotic prophylaxis. Approximately 1 month after birth, the infant presented persistent cough, and aerosol therapy was promptly administered. However, after 3 days, he progressively became drowsy and unresponsive. The parents quickly took him to the hospital, but he was already in cardiopulmonary arrest. Cardiopulmonary resuscitation (CPR) was immediately started: oxygen was given through a bag valve mask, and then, an injection of adrenaline was necessary. Despite all attempts, CPR was stopped after 35 min due to pulseless electrical activity (PEA) and the infant was pronounced dead. Subsequent nasal and pharyngeal swabs revealed positive results for respiratory syncytial virus A (RSV), Haemophilus influenzae , and Staphylococcus aureus . An autopsy was requested to better understand the cause of death. However, in that period COVID-19 was initially spreading, and the lack of medical resources made it necessary to temporarily store the infant’s body at − 10 °C in the freezer of the morgue. Then, due to the health-related challenges arising from the COVID-19 pandemic, the autopsy was delayed for 21 days. Before the autopsy was performed, the body was thawed at room temperature (20 °C) for 12 h. External examination showed growth parameters within the normal limits for 1 month of age: weight 4301 g (50th percentile), crown-heel length 55 cm (50th percentile), and head circumference 37 cm (25th percentile) . The face was regular with no dysmorphisms. Grossly, no anomalies were identified. Internal examination evidenced diffuse congestion of the organs, but no anatomical anomalies. The lungs displayed the expected lobes, with three on the right and two on the left. The right lung weighed 61 g and the left 50 g. Segmental sequential analysis of the heart (weight 29 g) revealed atrioventricular and ventriculoarterial concordance and regular systemic and pulmonary venous returns. The coronary arteries presented a regular course. The other organ weights were as follows: liver 173 g, right kidney 20.8 g, left kidney 22.4 g, spleen 16.4 g, brain 489 g. Histological examination of the lungs revealed bilateral extensive acute necrotizing bronchitis and bronchiolitis with focal hyaline membrane formation and pulmonary edema (Fig. ). Scattered cells with enlarged nuclei due to RSV infection were seen (Fig. ). Minimal diffuse ischemic changes were observed in the myocardium and brain. Both kidneys showed acute tubular necrosis, tubular dilatation, and occasional microcalcifications. No significant histopathological findings were observed in the other organs. The integration of clinical, autopsy, and histological data identified the cause of death as acute respiratory failure resulting from bilateral and diffuse acute necrotizing bronchitis and bronchiolitis. This was caused by RSV type A complicated by bacterial over-infection with Haemophilus influenzae and Staphylococcus aureus . Evidence of histological freeze–thaw artifacts was present in many organs. The brain disclosed ice crystal artifacts with multiple clefts within the parenchyma (Fig. ). The lungs, apart from histological evidence of infection, exhibited alveolar distension (Fig. ). The heart presented marked extracellular separation (Fig. ). Abnormal dilatation of lymphatic or capillary vessels was excluded by the regular expression of CD31, which is a marker for endothelial cells, in the vascular structures located between the bundles of cardiomyocytes. Moreover, the artifactual clefts were devoid of endothelial lining (Fig. ). The liver showed shrinkage of the hepatocellular laminae with marked expansion of the sinusoidal space (Fig. ). The kidneys presented unusual cystic lacunae, probably due to expanded extracellular spaces, with tubules strained and flattened (Fig. ). The thymus structure was almost fully preserved (Fig. A). The thyroid presented clefts mostly along the septa, and the cell nuclei appeared more basophilic (Fig. B). The acinar architecture of the pancreas was maintained, with a few artifactual lacunae within the septa and the islets were still recognizable (Fig. C). On the whole, the cell nuclei appeared shrunk and strongly basophilic. The spleen displayed some cystic slightly eosinophilic spaces devoid of epithelium (Fig. D). The brown adipose tissue was mainly preserved, but focal cellular shrinkage was seen in the most eosinophilic cells (Fig. ). The white adipose tissue and the skeletal muscle were virtually unaffected by artifacts (Fig. ). An electronic search was performed in three databases (PubMed, Scopus, and Web of Science), and keywords related to the study aim and included in the search string were (freezing OR frosty OR thawing) AND (forensic OR autopsy OR histological OR histology. The English language and time interval of publication, from January 1960 to March 2023, were applied as filters and inclusion criteria. The literature review showed that nine articles reported histological features connected to tissue freezing and thawing . All the related data are summarized in Table and discussed in the “ ” section. Histological artifacts in the freezing–thawing process of tissue have not been widely reported in the literature. However, some major histological changes caused by freezing have been noted, including loss of staining, extracellular fluid accumulation, cell shrinkage, fractures, hemolysis, and hematin formation, and minor changes include loss of bronchial cilia, prominence of collagen in alveolar septa and meninges, and intracellular vacuolization of epithelial cells. In the respective studies, despite these artifacts, adequate visualization of the tissues was possible, allowing diagnosis . The most common histological findings in the freeze–thaw process were the expansion of the extracellular spaces and cell shrinkage, and the heart and the liver were the most widely affected organs. In general, in most tissues, strong basophilic nuclear staining and hemolysis were common . In a few reported cases of frozen newborns, cardiac and hepatic artifactual features were the most evident, with marked separation of the myocardiocytes and the sinusoidal spaces, respectively . In the brain, pseudobubbles or linear freeze artifacts were described . In another study, skeletal muscle was reported to be preserved, but in the case of two freeze–thaw cycles, fibers underwent separation due to large crystal formation . A further study showed that if a body is frozen soon after death, autolysis and putrefaction are less evident, as the cold reduces the activity of enteric microorganisms, and anaerobic decomposition is much less strong than in fresh bodies . In forensic investigations, the freezing and thawing of bodies can be critical in cases of unknown cause of death, as histological artifacts are produced in the process. The mechanisms underlying the extended extracellular areas are still unclear . Freezing of organic tissues begins in the extracellular spaces with ice crystal formation and fixing of water. Thawing melts the ice, resulting in tissue damage due to the changes in osmotic pressure between the cells and the extracellular space . We described a case of an infant who died of respiratory failure caused by bronchitis and bronchiolitis in RSV infection. The autopsy was delayed, and the body was kept frozen in the morgue at − 10 °C for 3 weeks. It was then thawed for 12 h at room temperature (20 °C) before dissection. The infant presented the most common histological features mentioned in the literature . However, freezing artifacts in brain tissue were only mentioned in one book in our review and were described as parenchymal clefts, as we found in our case . Extracellular space expansion, tissue clefts, cell shrinkage, and deep staining nuclei were found in almost every organ. Ice crystal artifacts were evident in the brain as parenchymal clefts and in the heart and liver as marked expansion of the extracellular spaces. Freeze–thaw artifacts may also show some features of congenital diseases. The abnormal cardiac structure raised the suspicion of dilatated lymphatic or vascular channels, but immunostaining for CD31 highlighted their regular location among the bundles of cardiomyocytes. The suspicion of ventricular non-compaction was ruled out, as endocardial fibroelastosis was absent . The hepatic sinusoidal dilatation could mimic hepatic peliosis, a feature of X-linked myotubular myopathy, but liver immunohistochemistry for smooth muscle actin was weakly expressed and not increased . The renal lacunar cystic changes could resemble polycystic kidney disease, but these spaces were devoid of epithelial lining . In our study, the adipose tissue and the skeletal muscle were the only two tissues without artifacts. Only one experimental study described artifacts in skeletal muscle, but they appeared after two freeze–thaw cycles . The absence of artifacts in our case aligns with the fact that the body underwent a single freeze–thaw cycle. The effects of freezing and thawing on adipose tissue have not yet been documented. Our case exhibited white and brown adipose tissues virtually unaffected by freezing and thawing, because they are rich in fat and scarce in water, and ice crystal formation is limited. The brown adipose tissue showed minimal cell shrinkage and deep staining nuclei, due to its composition, as the cytoplasm is packed with multiple lipid droplets and water concentration is higher than in white adipose cells . Therefore, the lack of crystals should not be considered a confounding factor in interpreting freeze–thaw artifacts. In forensic investigations, indirect indicators of freezing and thawing can provide valuable circumstantial evidence to aid forensic pathologists in understanding the sequence of events. Freezing of a body can be connected to criminal activities, such as attempts to conceal a body or manipulate the time of death, as well as natural weather conditions when the body is exposed to the elements. Ice crystal identification can offer insights into the rate at which the body froze and, when combined with other circumstantial data, becomes a significant source of information. The findings of our study can have significant implications in the field of forensic pathology. Specifically, when it is known that a body has been frozen, awareness of these potential artifacts can help forensic pathologists avoid misdiagnoses by considering the possibility of their formation. On the other hand, the presence of these alterations indirectly suggests that the body had been previously frozen and might have already thawed at the time of discovery. Freezing and thawing may disrupt the tissue architecture due to ice crystal formation and subsequent melting. Histological artifacts are mostly evident in the brain, heart, liver, and kidneys. Recognition of histological artifacts may help in forensic analysis whether the body is known to have been frozen or not. In infants, histological freeze–thaw artifacts may mimic congenital diseases. White adipose tissue is unaffected by these artifacts as it contains little water. |
Minimal Common Oncology Data Elements Genomics Pilot Project: Enhancing Oncology Research Through Electronic Health Record Interoperability at Vanderbilt University Medical Center | c3cd7c2b-5e9f-4b83-95f7-9e70a76382ec | 11371088 | Internal Medicine[mh] | As the adoption of electronic health records (EHRs) continues to rise, health care providers, medical practitioners, researchers, and patients are experiencing unparalleled levels of data access. The increasing prominence of agile and scalable cloud services for health care expands the potential of EHRs as a cornerstone for data-driven clinical research. Despite their potential, the current landscape of EHRs, especially data on patients with cancer, faces the barrier of limited interoperability. Most EHRs and home health infrastructures today are constructed atop various systems with overlapping functionalities. This has led to the complex challenge of synchronizing data across these systems, resulting in redundancy and inconsistency in data storage. , Data integration and standardization in health systems are required to solve the interoperability problem. CONTEXT Key Objective How does the integration of Epic-based electronic health records (EHRs) with the Minimal Common Oncology Data Elements (mCODE) standard, using the Fast Healthcare Interoperability Resources framework, represent a novel approach to improving data interoperability and transmission in cancer patient care at Vanderbilt University Medical Center? Knowledge Generated Our project elucidates the transformative potential of integrating mCODE standards with EHR systems, showcasing advancements in data interoperability that can streamline clinical workflows and enhance personalized oncology care. It offers a blueprint for health care institutions on leveraging standardized data to accelerate cancer research and improve patient outcomes through informed, precision medicine approaches. Relevance (F.P.-Y. Li) Implementing a common data standard, such as mCODE as demonstrated in this study, can reduce the interoperability barrier when building digital applications to explore institutional EHR data, thereby facilitating the reuse of data for meaningful secondary analyses.* *Relevance section written by JCO Clinical Cancer Informatics Associate Editor Frank P.-Y. Lin, PhD, MBChB, FRACP, FAIDH. Historically, standards like Health Level 7 (HL7), including HL7v2 and v3, were developed as a solution aiming to promote seamless integration between different health systems. While HL7 has effectively facilitated integration, it struggles with achieving complete data standardization and interoperability given the complexity and lack of commonly used web standards. To address these issues, the use of Fast Healthcare Interoperability Resources (FHIR), developed by HL7, emerged to leverage advanced web standards and provide a comprehensive solution to interoperability. Focusing on uniform data structures and HTTP-based Representational State Transfer (RESTful) application programming interfaces (APIs) offer a progressive approach to the system thereby making the implementation more accessible. Building on the FHIR framework, the Minimal Common Oncology Data Elements (mCODE) initiative was launched in 2019 to promote interoperability in the field of oncology research and take advantage of the powerful features of FHIR like API management. Developed by a group of oncologists, informaticians, researchers, and experts in terminologies and standards, mCODE standardizes 23 profiles composed of 90 data elements spanning six primary domains, including patient, laboratory/vital, cancer, genomics, treatment, and outcome. The mCODE project has the potential to provide a more structured and standardized way to share cancer patient data and facilitate cancer care delivery and research. Furthermore, recent federal mandates dictate that EHR vendors must adopt APIs compliant with FHIR Release 4.0.1 to improve patient data access. This move, acknowledging the widespread benefits of implementation on FHIR, further demonstrates a promising outlook for the broader adoption of mCODE in the specific field of oncology research. Nevertheless, the practical application of genomic profiles within mCODE is not widespread among institutions. This limitation is primarily due to the unavailability of computable genomic data within EHRs, thereby presenting obstacles when evaluating the practicality and identifying the advantages of integrating mCODE within the industry. In this study, we used genomic data from the EHR database of Vanderbilt University Medical Center (VUMC), one of the few distinguished institutions with computable genomic data. We examined the practicality of implementing mCODE genomics profiles using EHR data. To further showcase the potential of mCODE standards for future application integrations, we designed a web application that maximizes the utilization of our institution's genomic data to enhance accessibility. We also provided data visualization to better assist researchers in extracting valuable insights from cancer patient data. Key Objective How does the integration of Epic-based electronic health records (EHRs) with the Minimal Common Oncology Data Elements (mCODE) standard, using the Fast Healthcare Interoperability Resources framework, represent a novel approach to improving data interoperability and transmission in cancer patient care at Vanderbilt University Medical Center? Knowledge Generated Our project elucidates the transformative potential of integrating mCODE standards with EHR systems, showcasing advancements in data interoperability that can streamline clinical workflows and enhance personalized oncology care. It offers a blueprint for health care institutions on leveraging standardized data to accelerate cancer research and improve patient outcomes through informed, precision medicine approaches. Relevance (F.P.-Y. Li) Implementing a common data standard, such as mCODE as demonstrated in this study, can reduce the interoperability barrier when building digital applications to explore institutional EHR data, thereby facilitating the reuse of data for meaningful secondary analyses.* *Relevance section written by JCO Clinical Cancer Informatics Associate Editor Frank P.-Y. Lin, PhD, MBChB, FRACP, FAIDH. Data Selection and Preparation VUMC uses the Epic Clarity database to store EHRs. Our data set included over 12,000 real cancer patient EHRs extracted from the VUMC database that contained patient demographic information and their genomic test results. In total, we collected data on 1,055 unique gene alterations, 45,279 amino acid changes, and 10,417 cancer diagnoses. To decrease complications associated with the data migration process and to avoid contaminating the VUMC EHR database, we constructed a sandbox environment in Azure to store the VUMC Clarity patient data. We will refer to this created database as the Temporary Database for simplicity, but it is important to note that it is the same structure but not the same EHR database used by VUMC. The Temporary Database in Azure was architected to minimize any impact on the primary Production reporting database of VUMC. No transformation occurred at this stage. Deploying FHIR Server As mCODE extends from FHIR R4 standards and uses a similar set of API calls, we deployed the open-source FHIR server developed by Microsoft on Azure to serve as our FHIR server. An Azure SQL database that follows the entity attribute value (EAV) model was automatically deployed during the process. All interactions with this database, as well as any internal structural alterations, were managed by the FHIR server, and there is no direct access available to interfere with it. The Azure FHIR server supports all API calls stated within the FHIR R4 IG. After mapping the patient data from the Temporary Database to the new mCODE standard, we loaded all mCODE profiles through this Azure FHIR server and connected our web application to the server to query data stored in the Azure database. Data Mapping This project focused on transforming the EHRs in Temporary Database to implement the Cancer Patient profile and the Genomics profile defined in the mCODE standard. The progression of data from collection to application use is depicted in Figure . Genomic results, initially communicated through HL7 messages, are ingested by Epic's Chronicles database, a transactional database that serves as a digital ledger for storing and managing patient transactions. From Chronicles, the data were transferred to the Clarity database, where it is structured into tables and columns suitable for analysis. In the real-world production process, data transformation into compatible mCODE profiles would happen directly if the Chronicle database contained the necessary FHIR profiles for full mCODE implementation. However, to avoid any possible contamination of the production database, we created a sandbox environment that only extracted a subset of the Clarity database to test the validity of mCODE implementation. Thus, no data transformation was made when moving the patient data from Clarity database to the Temporary Database. An extract, transform, load (ETL) tool encompassing three pivotal steps was used to transition the raw Clarity data in the Temporary Database into the structured mCODE format: (1) extracting the EHRs from the Temporary Database; (2) transforming extracted EHRs into mCODE profiles; and (3) loading the transformed mCODE profiles to the Azure FHIR server which automatically validates the profile and stores them into EAV format in its accompanied database. Subsequently, a clinical application is connected to the FHIR server to demonstrate the transformation step is successfully implemented. In particular, the transformation step required mapping the selected data entries of the Clarity EHRs to their corresponding mCODE data fields for the Cancer Patient profile and Genomic profiles, which included four subprofiles: Genomics Report Profile, Genomic Variant Profile, Genomic Region Studied Profile, and Genomic Specimen Profile. To implement those profiles, we located the required data fields within the Clarity database according to mCODE standards and extracted these to a separate relational database system. Next, we mapped the selected data entries to their corresponding mCODE fields and created the various mCODE profiles. If no matching data could be found to fill in an mCODE Must Support data field, a data absent reason was loaded as a placeholder. For important patient data from the Temporary Database that had no specific location to store in an mCODE profile, we included such information as extensions of their related data field. The last step for data mapping used the bulk import feature of the Azure FHIR server to accomplish loading constructed data. Website After the data migration process, we started designing the front-end side of the web application. The final deliverables of the web application consisted of the following pages: (1) a home page that visualizes all the Phecode diagnoses and genes present in the Temporary Database (Appendix Fig A ); (2) a summary page for requesting and downloading patient data for VUMC users (Fig ); and (3) two pages for evaluating the risk of cancer incidence based on identified genomic variations, one calculated based on the Temporary Database and the other based on the dataset acquired from The Cancer Genome Atlas (TCGA) Program , (Figs and ). A reference page was also included that presents all documentation related to this study, including a brief introduction and in-depth analysis of both FHIR and mCODE standards (Appendix Fig A ). The primary objective for this web application was twofold. First, the summary page offers users the ability to view and download deidentified patient information. It provides the functionality to sort the patient database by criteria such as race, sex, age, and genomic alteration. Each row corresponds to a single gene variation of a patient, with details like DNA change, DNA change type, genomic source class, amino change, allelic frequency, and clinical significance, allowing for the exploration of specific relationships between certain genomic mutations and demographics. This feature can only be accessed by users connected to the VUMC network for security reasons. Second, with the extensive data in the Temporary Database and TCGA program, we computed the likelihood of developing one of the 33 cancer types profiled by the TCGA program, given the presence of a known gene variant. This functionality enables a more nuanced understanding of the genomic underpinnings of cancer, paving the way for targeted diagnostics and personalized treatment strategies. The underlying logic was based on Bayes' Theorem, commonly used to calculate the probability of an event occurring on the basis of previous conditions. In the context of our research, we applied Bayes' Theorem to determine the conditional probability of being diagnosed with one of the cancer types on the basis of the presence of a specific gene variant. The equation, as applied in our analysis, is shown in Figure . VUMC uses the Epic Clarity database to store EHRs. Our data set included over 12,000 real cancer patient EHRs extracted from the VUMC database that contained patient demographic information and their genomic test results. In total, we collected data on 1,055 unique gene alterations, 45,279 amino acid changes, and 10,417 cancer diagnoses. To decrease complications associated with the data migration process and to avoid contaminating the VUMC EHR database, we constructed a sandbox environment in Azure to store the VUMC Clarity patient data. We will refer to this created database as the Temporary Database for simplicity, but it is important to note that it is the same structure but not the same EHR database used by VUMC. The Temporary Database in Azure was architected to minimize any impact on the primary Production reporting database of VUMC. No transformation occurred at this stage. As mCODE extends from FHIR R4 standards and uses a similar set of API calls, we deployed the open-source FHIR server developed by Microsoft on Azure to serve as our FHIR server. An Azure SQL database that follows the entity attribute value (EAV) model was automatically deployed during the process. All interactions with this database, as well as any internal structural alterations, were managed by the FHIR server, and there is no direct access available to interfere with it. The Azure FHIR server supports all API calls stated within the FHIR R4 IG. After mapping the patient data from the Temporary Database to the new mCODE standard, we loaded all mCODE profiles through this Azure FHIR server and connected our web application to the server to query data stored in the Azure database. This project focused on transforming the EHRs in Temporary Database to implement the Cancer Patient profile and the Genomics profile defined in the mCODE standard. The progression of data from collection to application use is depicted in Figure . Genomic results, initially communicated through HL7 messages, are ingested by Epic's Chronicles database, a transactional database that serves as a digital ledger for storing and managing patient transactions. From Chronicles, the data were transferred to the Clarity database, where it is structured into tables and columns suitable for analysis. In the real-world production process, data transformation into compatible mCODE profiles would happen directly if the Chronicle database contained the necessary FHIR profiles for full mCODE implementation. However, to avoid any possible contamination of the production database, we created a sandbox environment that only extracted a subset of the Clarity database to test the validity of mCODE implementation. Thus, no data transformation was made when moving the patient data from Clarity database to the Temporary Database. An extract, transform, load (ETL) tool encompassing three pivotal steps was used to transition the raw Clarity data in the Temporary Database into the structured mCODE format: (1) extracting the EHRs from the Temporary Database; (2) transforming extracted EHRs into mCODE profiles; and (3) loading the transformed mCODE profiles to the Azure FHIR server which automatically validates the profile and stores them into EAV format in its accompanied database. Subsequently, a clinical application is connected to the FHIR server to demonstrate the transformation step is successfully implemented. In particular, the transformation step required mapping the selected data entries of the Clarity EHRs to their corresponding mCODE data fields for the Cancer Patient profile and Genomic profiles, which included four subprofiles: Genomics Report Profile, Genomic Variant Profile, Genomic Region Studied Profile, and Genomic Specimen Profile. To implement those profiles, we located the required data fields within the Clarity database according to mCODE standards and extracted these to a separate relational database system. Next, we mapped the selected data entries to their corresponding mCODE fields and created the various mCODE profiles. If no matching data could be found to fill in an mCODE Must Support data field, a data absent reason was loaded as a placeholder. For important patient data from the Temporary Database that had no specific location to store in an mCODE profile, we included such information as extensions of their related data field. The last step for data mapping used the bulk import feature of the Azure FHIR server to accomplish loading constructed data. After the data migration process, we started designing the front-end side of the web application. The final deliverables of the web application consisted of the following pages: (1) a home page that visualizes all the Phecode diagnoses and genes present in the Temporary Database (Appendix Fig A ); (2) a summary page for requesting and downloading patient data for VUMC users (Fig ); and (3) two pages for evaluating the risk of cancer incidence based on identified genomic variations, one calculated based on the Temporary Database and the other based on the dataset acquired from The Cancer Genome Atlas (TCGA) Program , (Figs and ). A reference page was also included that presents all documentation related to this study, including a brief introduction and in-depth analysis of both FHIR and mCODE standards (Appendix Fig A ). The primary objective for this web application was twofold. First, the summary page offers users the ability to view and download deidentified patient information. It provides the functionality to sort the patient database by criteria such as race, sex, age, and genomic alteration. Each row corresponds to a single gene variation of a patient, with details like DNA change, DNA change type, genomic source class, amino change, allelic frequency, and clinical significance, allowing for the exploration of specific relationships between certain genomic mutations and demographics. This feature can only be accessed by users connected to the VUMC network for security reasons. Second, with the extensive data in the Temporary Database and TCGA program, we computed the likelihood of developing one of the 33 cancer types profiled by the TCGA program, given the presence of a known gene variant. This functionality enables a more nuanced understanding of the genomic underpinnings of cancer, paving the way for targeted diagnostics and personalized treatment strategies. The underlying logic was based on Bayes' Theorem, commonly used to calculate the probability of an event occurring on the basis of previous conditions. In the context of our research, we applied Bayes' Theorem to determine the conditional probability of being diagnosed with one of the cancer types on the basis of the presence of a specific gene variant. The equation, as applied in our analysis, is shown in Figure . Data Migration To facilitate the transfer of VUMC EHR data from VUMC Epic Clarity database into mCODE profiles, we used the developed ETL tool that extracted data from Clarity, compiled corresponding data into mCODE profiles, and uploaded in bulk the constructed profiles to the Azure FHIR server. Data from 12,000 patients with over 80,000 variant profiles were loaded onto the Azure FHIR server. Concurrently, each profile underwent validation at the time of being loaded into the FHIR server. The migration process took roughly a day to complete; during the process, a bottleneck of the migration step was detected, which was the step of making external API calls to the FHIR server from the ETL tool. To circumvent this issue, a multithread processing feature in Spring Boot was used that significantly improved the speed of uploading profiles to the Azure FHIR server by allowing simultaneous and nonblocking posting of constructed profiles to the server. Web Application The finished website application has five main functionalities implemented on five separated pages. An account registered with a VUMC email is required to access any content on the website other than login, ensuring data security and preventing patient data breach (Appendix Fig A ). The home page displays the distribution of cancers and genes within the database as two colored pie charts, shown in Figure . The chart on the left displays the most common Phecode diagnosis and their respective percentage within our Azure database; the chart on the right shows the most common gene variations and their percentages. It is worth noting that since there is a large number of diseases and gene variations in the database, only the top 30 are shown on the home page for more concise visualization. The cancer incidence calculator is presented on two separate web pages: one uses the Temporary Database and the other is based on data from the TCGA program. Figures and provide illustrations of the calculator's application using the Temporary Database. The summary page in Figure displays a summary table of genomic profiles with a focus on gene variation. Before loading the page, an alert message will appear that asks whether the user is on the VUMC virtual net. Users who are not connected to the virtual network will be restricted to view only the patient data to protect patient privacy. The summary page first presents a variation of 10 patients in the database; users have the choice to load a custom number of patients following the initial 10 patients up to 200 patients. A user can choose to load all patients, but this option will trigger a warning message about possible waiting times. To facilitate the transfer of VUMC EHR data from VUMC Epic Clarity database into mCODE profiles, we used the developed ETL tool that extracted data from Clarity, compiled corresponding data into mCODE profiles, and uploaded in bulk the constructed profiles to the Azure FHIR server. Data from 12,000 patients with over 80,000 variant profiles were loaded onto the Azure FHIR server. Concurrently, each profile underwent validation at the time of being loaded into the FHIR server. The migration process took roughly a day to complete; during the process, a bottleneck of the migration step was detected, which was the step of making external API calls to the FHIR server from the ETL tool. To circumvent this issue, a multithread processing feature in Spring Boot was used that significantly improved the speed of uploading profiles to the Azure FHIR server by allowing simultaneous and nonblocking posting of constructed profiles to the server. The finished website application has five main functionalities implemented on five separated pages. An account registered with a VUMC email is required to access any content on the website other than login, ensuring data security and preventing patient data breach (Appendix Fig A ). The home page displays the distribution of cancers and genes within the database as two colored pie charts, shown in Figure . The chart on the left displays the most common Phecode diagnosis and their respective percentage within our Azure database; the chart on the right shows the most common gene variations and their percentages. It is worth noting that since there is a large number of diseases and gene variations in the database, only the top 30 are shown on the home page for more concise visualization. The cancer incidence calculator is presented on two separate web pages: one uses the Temporary Database and the other is based on data from the TCGA program. Figures and provide illustrations of the calculator's application using the Temporary Database. The summary page in Figure displays a summary table of genomic profiles with a focus on gene variation. Before loading the page, an alert message will appear that asks whether the user is on the VUMC virtual net. Users who are not connected to the virtual network will be restricted to view only the patient data to protect patient privacy. The summary page first presents a variation of 10 patients in the database; users have the choice to load a custom number of patients following the initial 10 patients up to 200 patients. A user can choose to load all patients, but this option will trigger a warning message about possible waiting times. A key decision made during the project was to avoid directly interacting with the Clarity database at VUMC because of the additional oversight and complexity that accompany production environment modifications. For this reason, a sandbox environment was established, including the Temporary Database, which allowed for developmental flexibility and testing without the risk of compromising the stability of the primary health record system. The central challenge in the data mapping and transformation process is the necessity to map data from the Epic Clarity relational database to the EAV-styled mCODE format. This task becomes particularly formidable because of the absence of FHIR resources at VUMC's production environment and the widespread lack of implementation in many institutions across the country. The unavailability of FHIR resources complicates the exploration of the path for transforming Clarity data into mCODE profiles. Within this context, challenges arise, including disparities in naming conventions, variations in code systems, and the need to transform table-based data structures into profile-based formats. These factors collectively contribute to the complexity of the data migration process. One of the first challenges faced during the data extraction stage was the discrepancy in naming conventions and code systems. The same data field may be named differently in the Clarity database and in the mCODE format, which required a careful search within the official documentation of Epic Clarity databases and the mCODE implementation guide to find matches. Medical professionals were also consulted to assist in connecting the lines between the data. Another challenge encountered while transforming and compiling patient data into mCODE profiles was assembling data scattered across various tables into the six proposed mCODE profiles. The structure of various mCODE profiles and their dependency relationships had to be fully understood to ensure the data were placed correctly. Although all must-support elements in mCODE profiles can be implemented, as data absent reason elements can be placed in fields that lack corresponding information in the EHR database, there remain many parts in genomics profiles that the Clarity database only provide empty value, causing some profiles to be only complete in structure but lacking important contents. The integration of FHIR aimed to promote data interoperability and advance oncology research. However, our experience highlighted that stemming from the design of FHIR, FHIR tends to be better suited for managing individual patient-level data rather than population-level data. As a result, employing FHIR for analytics presented challenges, especially in contexts requiring comprehensive data aggregation and analysis at the population level. To generalize this project for other institutions, it is essential to recognize that while the Epic Clarity database maintains a high level of structural uniformity across sites, minor schema variations can occur with different Epic versions. Additionally, some institutions may opt out of extracting certain tables for their FHIR resource construction, though these variances are minor. Although our project targets VUMC Clarity data, the transformation process is designed to be adaptable. Besides, direct transformation from Clarity is not the sole method; institutions with comprehensive FHIR profiles for mCODE can directly apply mCODE transformations to these profiles. One priority when working with EHR and patient data is to ensure data safety and protect patient privacy. For these purposes, the web application has multiple layers of protection implemented. However, not all fields require multiple layers of authorization. Most data visualization pages do not require users to be on the VUMC virtual net. The homepage pie charts draw from precalculated statistics stored in static Azure FHIR database tables to lower querying overhead. Other features like the PanCanAtlas and VUMC genomic calculators also bypass the private server for data retrieval. Although most planned functionalities were accomplished, several problems were encountered that stopped the team from implementing more advanced features. The current search function only scans static content for titles and links without text highlighting capabilities. Additionally, owing to Azure FHIR API constraints, data loading on the summary page is capped at 200 rows to prevent delays, and the FHIR server's limit of 1,000 profiles necessitates multiple API calls for comprehensive data, leading to significant wait times. In conclusion, our project has illuminated several key insights regarding the application of mCODE. First, by migrating VUMC's EHR data into an Azure FHIR database, we have demonstrated the potential of cloud-based EHR data management. The transition to a cloud environment not only provides secure storage but also allows for the integration of vital authentication services. Furthermore, this project has shown the possibility of building mCODE genomic profiles on existing real-world EHRs. However, this integration necessitates consideration of database structures to align with the specifics of mCODE genomic data. Presently, incongruities exist between the mCODE standard and prevailing EHR data standards, mandating a manual data mapping process to transition between those two standards. Moreover, our efforts culminated in developing a web application utilizing mCODE FHIR APIs. Yet, we encountered limitations in statistical methodologies, with many FHIR APIs lacking support for such analytical techniques. This underscores the need for updates and enhancements to the existing FHIR API standard, particularly focusing on bolstering support for statistical methodologies, and consequently optimizing the application of FHIR data for medical research purposes. Despite the mentioned challenges, our project still affirms the applicability of the mCODE standard in constructing applications centered around genomics and oncology data. This affirmation holds great promise, particularly if future updates enhance mCODE and FHIR standard databases to better support data retrieval for statistical analysis, further amplifying its potential impact in the domain of medical research and care. |
Correlation Between the Clinical and Histopathological Results in Experimental Sciatic Nerve Defect Surgery | db8408ba-ed10-476d-a87d-125d9b6b19ec | 11857492 | Biochemistry[mh] | Injured nerves require a longer time to regenerate, due to Wallerian degeneration of the axons . After an initial lesion, the nerves begin to grow at a specific speed of 1 mm/day . In order to aid this process, manufactured tubes can be used for nerve gaps up to 70 mm . Furthermore, there has been significant technological development in this field—biocompatible 3D-printed devices or textile conduits can now be used to bridge nerve gaps . Generally, nerve conduits offer a good alternative to grafts, alone or in combination with different tissues or substances (stem cells and PRP) . Nerve regeneration can be properly assessed by clinical evaluation, imagistic investigations and histopathological examination. While clinical evaluation may be difficult to perform, the pathological examination (gross, as well as microscopic) can become a useful tool for evaluating nerve regeneration. There are three types of nerve injuries in the Seddon classification (neurapraxia, axonotmesis, neurotmesis), while Sunderland uses a 5-degree injury classification . The regeneration process after Wallerian degeneration requires a favorable environment as well as a “clear way” along which the axonal sprouts can grow. The Schwann cells, differentiated into Büngner bands, are responsible for directing the axonal growth . There are several new molecules or drugs that aid nerve regeneration: growth factors, PRP and stem cells , as well as novel synthetic conduits capable of directing the axonal growth of the nerve. These modern techniques have the potential to replace nerve grafts . Depending on the level of injury, the axonal growth requires different periods of time for regeneration . The more proximal the lesion, the longer it will take for the nerve to grow—this nerve growth rate is estimated to be 1 mm/day in humans and 1.5–2 mm/day in rats. There are several methods of analyzing nerve regeneration in the setting of a previous injury. Clinical evaluation involves assessing both motor and sensitive functions. The motor function can be evaluated using a footprint test and calculating a sciatic functional index (SFI). The quality of the nerve growth can also be evaluated by directly assessing the nerve at the level of injury. Apart from the conventional histopathology analysis, there are several other valuable immunohistochemistry (IHC) markers that can be used in both qualitative and quantitative studies. Reconstruction after nerve defects does not have an ideal solution and therefore multiple options should be considered and tested in order to discover which one offers the best results. 2.1. Ethical Acknowledgement and Animal Lot Description Forty male Wistar rats weighing 250–300 g and aged between 55–60 days were divided into 4 groups to assess 4 different techniques used for the repair of a 0.5 cm iatrogenic nerve defect on the right lower limb: (1) nerve graft–control group, (2) empty aortic conduit, (3) aortic conduit filled with platelet-rich plasma (PRP) and (4) aortic conduit filled with mesenchymal stem cells (MSC cell line). Another 2 rats with the same characteristics were sacrificed to obtain the aortic conduits and the PRP for the operations. The minimal number of animals was used and postoperative analgesia was given to reduce animal suffering. The international protocols for euthanasia were also respected. Animal care and use were performed according to the laws regarding the well-being of the animal and the project was approved by the Local Ethics Committee (approval no. 1738/17.04.2019). The experiment was conducted according to international guidelines and regulations. The animals were provided proper lodging in standard cages and fed with normative murine food and water ad libitum. The acclimatization conditions involved 3 spacious rooms dedicated to this experiment, sheltered from outside noise. All rats benefitted from the same lodging conditions, same food and same climate. 2.2. Operation and Postoperative Protocol The rats were anesthetized using a mixture of ketamine and xylazine (75 mg/kg and 10 mg/kg) injected intraperitoneally. All rats were operated on the right lower limb under the same circumstances (microscopic magnification, sterile conditions) and a number of 4–5 stitches were performed for each anastomosis. All rats received the same anesthesia from a veterinarian and were operated on by the same plastic surgeon, assisted by a colleague to ensure sterile conditions. The postoperative care implied enroxil 0.003 mg/kg s.c. and meloxicam 1 mg/kg s.c. for 3 days. The operated rats were placed in separate cages for a few days to avoid cannibalism. Body temperature as well as clinical recovery was monitored after each operation for 3 h. The wounds were treated locally in case of dehiscence with Bacitracin/Neomycin ointment. All rats were assessed for 12 weeks postoperative to observe the nerve regeneration. 2.3. Follow-Up Protocol The team that monitored the rats after the operation involved 2 veterinarians, the surgeon and the assistant who operated the rats and several students who provided logistic support. The clinical evaluations were performed by the assistant, while the nerve dissection and nerve excision were performed at the end of 12 weeks after rat euthanasia by the surgeon. The footprint test and the SFI were calculated by a medical engineer. The histopathology and IHC were performed by a team of pathologists. All data were recorded and interpreted by a statistician. Two rats in the nerve graft group (1) were excluded, one due to postoperative death (the dead rat was then used for aorta harvesting for the second batch) and the other one due to nerve rupture. 2.3.1. Clinical Evaluation The remaining 38 rats were assessed clinically every other week in terms of sensitivity (pinch test), mobility and footprint test. The sensitivity test was graded on a scale from 0–3 as follows: No reaction at any level—0 points; Pinch at calf level determines retraction of the limb—1 point; Pinch at ankle level determines retraction of the limb—2 points; Pinch at metatarsal level determines retraction of the limb—3 points. The mobility test had a scale of 0–3 points: No movement—0 points; Minimal movement/flexion—1 point; Abduction of the fingers—2 points; Abduction and extension of the fingers–3 points. The footprint test was performed after the rat passed onto a sponge filled with ink, leaving behind on a graded piece of paper a line of footprints. The SFI after the Bain and MacKinnon formula was then calculated for each trial. This index takes into consideration 3 parameters: plantar length (PL) (between the calcaneus and the longest finger), intermediary interdigital length (IT) (between digits 2 and 5) and maximal interdigital length (TS) (between the 1st and 5th digit) . The calculus of this index takes into account the operated as well as the non-operated limb, using the formula (the operated are with E, non-operated with N in front of the above-mentioned parameters): SFI = −38.3 × EPL-NPL/NPL + 109.5 × ETS-NTS/NTS + 13.3 × EIT-NIT/NIT − 8.8 This formula attributes a null value for the uninjured limb and −100 for total loss of function. 2.3.2. Paraclinic Evaluation At 12 weeks, after the final clinical evaluation, the euthanasia of the rats was performed using a mixture of 225 mg/kg ketamine and 30 mg/kg xylazine administered intraperitoneally followed by an intracardiac injection of 80 mg/kg of KCl. The consequent hyperkaliemia-induced arrhythmia caused cardiac failure and exitus . After the euthanasia, the right sciatic nerve was dissected and examined. The macroscopic examination at the anastomosis site was based on 2 characteristics: Diameter at the anastomotic site: normal (1 point), narrow/reduced diameter (0 points); Aspect of the nerve: opaque (1 point), translucent (0 points). After gross examination, the nerves were excised and assessed microscopically using conventional Hematoxylin and Eosin staining (HE), special stains and immunohistochemistry (IHC). The nerve regeneration was graded based on HE on a scale from 0–3 points accordingly: No regeneration (ruptured nerve)—0 points; Less than 50% normal nerve cells/many visible gaps between the nerve fibers—1 point; 50–75% normal nerve cells and few visible gaps between the nerve fibers—2 points; >75% of the normal and small gaps between the nerve fibers—3 points. The data were analyzed using IBM SPSS Statistics 26. To establish a correlation between the histopathology and clinical findings (taking into consideration the type of variables-nominal and continuous), Cramer’s V. correlation coefficient and Pearson’s correlation coefficient were used. The nerves were also evaluated using immunohistochemistry and special stains. The following antibodies were used for immunolabeling: S100 (4C4.9 clone, Cell Marque, Rocklin, CA, USA), PGP9.5 (RP clone, Cell Marque), CD34 (QBEnd/10 clone, Cell Marque) and Ku80 (EPR3468 clone, Abcam, Cambridge, UK). The special stains included Masson’s trichrome stain (Diapath, Martinengo, Italy) and Gömori silver stain (Bio-Optica, Milan, Italy). Forty male Wistar rats weighing 250–300 g and aged between 55–60 days were divided into 4 groups to assess 4 different techniques used for the repair of a 0.5 cm iatrogenic nerve defect on the right lower limb: (1) nerve graft–control group, (2) empty aortic conduit, (3) aortic conduit filled with platelet-rich plasma (PRP) and (4) aortic conduit filled with mesenchymal stem cells (MSC cell line). Another 2 rats with the same characteristics were sacrificed to obtain the aortic conduits and the PRP for the operations. The minimal number of animals was used and postoperative analgesia was given to reduce animal suffering. The international protocols for euthanasia were also respected. Animal care and use were performed according to the laws regarding the well-being of the animal and the project was approved by the Local Ethics Committee (approval no. 1738/17.04.2019). The experiment was conducted according to international guidelines and regulations. The animals were provided proper lodging in standard cages and fed with normative murine food and water ad libitum. The acclimatization conditions involved 3 spacious rooms dedicated to this experiment, sheltered from outside noise. All rats benefitted from the same lodging conditions, same food and same climate. The rats were anesthetized using a mixture of ketamine and xylazine (75 mg/kg and 10 mg/kg) injected intraperitoneally. All rats were operated on the right lower limb under the same circumstances (microscopic magnification, sterile conditions) and a number of 4–5 stitches were performed for each anastomosis. All rats received the same anesthesia from a veterinarian and were operated on by the same plastic surgeon, assisted by a colleague to ensure sterile conditions. The postoperative care implied enroxil 0.003 mg/kg s.c. and meloxicam 1 mg/kg s.c. for 3 days. The operated rats were placed in separate cages for a few days to avoid cannibalism. Body temperature as well as clinical recovery was monitored after each operation for 3 h. The wounds were treated locally in case of dehiscence with Bacitracin/Neomycin ointment. All rats were assessed for 12 weeks postoperative to observe the nerve regeneration. The team that monitored the rats after the operation involved 2 veterinarians, the surgeon and the assistant who operated the rats and several students who provided logistic support. The clinical evaluations were performed by the assistant, while the nerve dissection and nerve excision were performed at the end of 12 weeks after rat euthanasia by the surgeon. The footprint test and the SFI were calculated by a medical engineer. The histopathology and IHC were performed by a team of pathologists. All data were recorded and interpreted by a statistician. Two rats in the nerve graft group (1) were excluded, one due to postoperative death (the dead rat was then used for aorta harvesting for the second batch) and the other one due to nerve rupture. 2.3.1. Clinical Evaluation The remaining 38 rats were assessed clinically every other week in terms of sensitivity (pinch test), mobility and footprint test. The sensitivity test was graded on a scale from 0–3 as follows: No reaction at any level—0 points; Pinch at calf level determines retraction of the limb—1 point; Pinch at ankle level determines retraction of the limb—2 points; Pinch at metatarsal level determines retraction of the limb—3 points. The mobility test had a scale of 0–3 points: No movement—0 points; Minimal movement/flexion—1 point; Abduction of the fingers—2 points; Abduction and extension of the fingers–3 points. The footprint test was performed after the rat passed onto a sponge filled with ink, leaving behind on a graded piece of paper a line of footprints. The SFI after the Bain and MacKinnon formula was then calculated for each trial. This index takes into consideration 3 parameters: plantar length (PL) (between the calcaneus and the longest finger), intermediary interdigital length (IT) (between digits 2 and 5) and maximal interdigital length (TS) (between the 1st and 5th digit) . The calculus of this index takes into account the operated as well as the non-operated limb, using the formula (the operated are with E, non-operated with N in front of the above-mentioned parameters): SFI = −38.3 × EPL-NPL/NPL + 109.5 × ETS-NTS/NTS + 13.3 × EIT-NIT/NIT − 8.8 This formula attributes a null value for the uninjured limb and −100 for total loss of function. 2.3.2. Paraclinic Evaluation At 12 weeks, after the final clinical evaluation, the euthanasia of the rats was performed using a mixture of 225 mg/kg ketamine and 30 mg/kg xylazine administered intraperitoneally followed by an intracardiac injection of 80 mg/kg of KCl. The consequent hyperkaliemia-induced arrhythmia caused cardiac failure and exitus . After the euthanasia, the right sciatic nerve was dissected and examined. The macroscopic examination at the anastomosis site was based on 2 characteristics: Diameter at the anastomotic site: normal (1 point), narrow/reduced diameter (0 points); Aspect of the nerve: opaque (1 point), translucent (0 points). After gross examination, the nerves were excised and assessed microscopically using conventional Hematoxylin and Eosin staining (HE), special stains and immunohistochemistry (IHC). The nerve regeneration was graded based on HE on a scale from 0–3 points accordingly: No regeneration (ruptured nerve)—0 points; Less than 50% normal nerve cells/many visible gaps between the nerve fibers—1 point; 50–75% normal nerve cells and few visible gaps between the nerve fibers—2 points; >75% of the normal and small gaps between the nerve fibers—3 points. The data were analyzed using IBM SPSS Statistics 26. To establish a correlation between the histopathology and clinical findings (taking into consideration the type of variables-nominal and continuous), Cramer’s V. correlation coefficient and Pearson’s correlation coefficient were used. The nerves were also evaluated using immunohistochemistry and special stains. The following antibodies were used for immunolabeling: S100 (4C4.9 clone, Cell Marque, Rocklin, CA, USA), PGP9.5 (RP clone, Cell Marque), CD34 (QBEnd/10 clone, Cell Marque) and Ku80 (EPR3468 clone, Abcam, Cambridge, UK). The special stains included Masson’s trichrome stain (Diapath, Martinengo, Italy) and Gömori silver stain (Bio-Optica, Milan, Italy). The remaining 38 rats were assessed clinically every other week in terms of sensitivity (pinch test), mobility and footprint test. The sensitivity test was graded on a scale from 0–3 as follows: No reaction at any level—0 points; Pinch at calf level determines retraction of the limb—1 point; Pinch at ankle level determines retraction of the limb—2 points; Pinch at metatarsal level determines retraction of the limb—3 points. The mobility test had a scale of 0–3 points: No movement—0 points; Minimal movement/flexion—1 point; Abduction of the fingers—2 points; Abduction and extension of the fingers–3 points. The footprint test was performed after the rat passed onto a sponge filled with ink, leaving behind on a graded piece of paper a line of footprints. The SFI after the Bain and MacKinnon formula was then calculated for each trial. This index takes into consideration 3 parameters: plantar length (PL) (between the calcaneus and the longest finger), intermediary interdigital length (IT) (between digits 2 and 5) and maximal interdigital length (TS) (between the 1st and 5th digit) . The calculus of this index takes into account the operated as well as the non-operated limb, using the formula (the operated are with E, non-operated with N in front of the above-mentioned parameters): SFI = −38.3 × EPL-NPL/NPL + 109.5 × ETS-NTS/NTS + 13.3 × EIT-NIT/NIT − 8.8 This formula attributes a null value for the uninjured limb and −100 for total loss of function. At 12 weeks, after the final clinical evaluation, the euthanasia of the rats was performed using a mixture of 225 mg/kg ketamine and 30 mg/kg xylazine administered intraperitoneally followed by an intracardiac injection of 80 mg/kg of KCl. The consequent hyperkaliemia-induced arrhythmia caused cardiac failure and exitus . After the euthanasia, the right sciatic nerve was dissected and examined. The macroscopic examination at the anastomosis site was based on 2 characteristics: Diameter at the anastomotic site: normal (1 point), narrow/reduced diameter (0 points); Aspect of the nerve: opaque (1 point), translucent (0 points). After gross examination, the nerves were excised and assessed microscopically using conventional Hematoxylin and Eosin staining (HE), special stains and immunohistochemistry (IHC). The nerve regeneration was graded based on HE on a scale from 0–3 points accordingly: No regeneration (ruptured nerve)—0 points; Less than 50% normal nerve cells/many visible gaps between the nerve fibers—1 point; 50–75% normal nerve cells and few visible gaps between the nerve fibers—2 points; >75% of the normal and small gaps between the nerve fibers—3 points. The data were analyzed using IBM SPSS Statistics 26. To establish a correlation between the histopathology and clinical findings (taking into consideration the type of variables-nominal and continuous), Cramer’s V. correlation coefficient and Pearson’s correlation coefficient were used. The nerves were also evaluated using immunohistochemistry and special stains. The following antibodies were used for immunolabeling: S100 (4C4.9 clone, Cell Marque, Rocklin, CA, USA), PGP9.5 (RP clone, Cell Marque), CD34 (QBEnd/10 clone, Cell Marque) and Ku80 (EPR3468 clone, Abcam, Cambridge, UK). The special stains included Masson’s trichrome stain (Diapath, Martinengo, Italy) and Gömori silver stain (Bio-Optica, Milan, Italy). Variable nerve regeneration was observed in all batches, both from the clinical and histopathological perspectives. There have been positive statistical correlations, showing robust nerve healing in batches (3) and (4), good healing in batch (1) and lesser results in batch (2). 3.1. Macroscopic Examination The pictures depict the gross examination of the sciatic nerves 3 months after the operation (photos were taken in situ). Some nerves presented a robust, opaque texture, while others had visible narrowing or translucent appearance . 3.2. Microscopic Examination Microscopically, the nerve regeneration was assessed using conventional HE staining, special stains and IHC, shown in the following images ( , , , , and ). 3.2.1. HE Stain On HE, all batches showed variable nerve regeneration. Groups 2–4 showed some periarterial expansion. Batch 2 displayed moderate nervous growth, but the intraluminal fibers exhibited frequent vacuolizations and areas of atrophy. 3.2.2. IHC The cytoplasmic expression of PGP 9.5 was seen in all 4 groups, with a higher intensity and a broader distribution in batches 3 and 4. Moreover, in batch 2, the staining showed moderate intensity and displayed a heterogeneous pattern. S100 expression paralleled that of PGP 9.5. In batch 3, CD34 highlighted numerous newly formed capillaries, proving that there was a rich blood supply. In batch 4, it was focally positive in the stellate mesenchymal cells, highly suggestive of their stem phenotype. In batches 2–4, the aortic conduit showed no staining due to endothelial denudation. Batch 4 showed Ku80 loss. 3.2.3. Special Stains Masson’s trichrome stain underlined prominent perineural fibrosis in batch 2 while the other groups showed variable collagen deposition around the suture site. In terms of myelin sheaths, batches 1 and 2 displayed faint staining, whereas the staining in batches 3 and 4 was more intense, although discontinuous. Gömori silver stain showed distorted reticulin meshwork in batch 2, due to prominent intercellular edema. In batch 3, numerous reticulin fibers were seen around the newly formed capillaries. Batch 4 showed delicate reticulin fibers extending into the loose mesenchymal tissue as scaffolds for the sprouting nerve fibers and capillaries. 3.3. Statistical Analysis From a clinical point of view, all 38 rats had a full recovery in terms of sensitivity at 12 weeks (noted with 3 points). Therefore, the correlation between the histopathology and the sensitivity test was achieved using the sensitivity test performed at 10 weeks. For the other two clinical indicators (SFI and motor test), the values obtained in the last test (at 12 weeks) were used for the correlation with the histopathology tests. The statistical correlations between the clinical and pathological parameters are as follows: There was no correlation between the macroscopic examination and the sensitivity test at 10 weeks ( p = 0.12). There was a positive correlation between the macroscopic examination and the motor test at 12 weeks ( p = 0.038). There was a positive correlation between the macroscopic examination and the SFI at 12 weeks ( p = 0.001). There was a positive correlation between the microscopic examination and the sensitivity test at 10 weeks ( p = 0.031). There was a positive correlation between the microscopic examination and the motor test at 12 weeks ( p = 0.002). There was a positive correlation between the microscopic examination and the SFI at 12 weeks ( p < 0.001). There was a positive correlation between the micro- and macroscopic examinations ( p < 0.001). The histology image of the nerve with nerve fibers occupying >75% of the sectioned nerve with small gaps between the nerve fibers (a microscopic indicator of a robust nerve regeneration) correlated with good results in all three of the tests (sensitivity test at 12 weeks, motor test at 12 weeks and SFI at 12 weeks). In contrast, fibers occupying <25% of the nerve circumference with large gaps in between (an indicator of poor nerve regeneration) correlated with overall poor test results regarding sensitivity, motor function and SFI. The pictures depict the gross examination of the sciatic nerves 3 months after the operation (photos were taken in situ). Some nerves presented a robust, opaque texture, while others had visible narrowing or translucent appearance . Microscopically, the nerve regeneration was assessed using conventional HE staining, special stains and IHC, shown in the following images ( , , , , and ). 3.2.1. HE Stain On HE, all batches showed variable nerve regeneration. Groups 2–4 showed some periarterial expansion. Batch 2 displayed moderate nervous growth, but the intraluminal fibers exhibited frequent vacuolizations and areas of atrophy. 3.2.2. IHC The cytoplasmic expression of PGP 9.5 was seen in all 4 groups, with a higher intensity and a broader distribution in batches 3 and 4. Moreover, in batch 2, the staining showed moderate intensity and displayed a heterogeneous pattern. S100 expression paralleled that of PGP 9.5. In batch 3, CD34 highlighted numerous newly formed capillaries, proving that there was a rich blood supply. In batch 4, it was focally positive in the stellate mesenchymal cells, highly suggestive of their stem phenotype. In batches 2–4, the aortic conduit showed no staining due to endothelial denudation. Batch 4 showed Ku80 loss. 3.2.3. Special Stains Masson’s trichrome stain underlined prominent perineural fibrosis in batch 2 while the other groups showed variable collagen deposition around the suture site. In terms of myelin sheaths, batches 1 and 2 displayed faint staining, whereas the staining in batches 3 and 4 was more intense, although discontinuous. Gömori silver stain showed distorted reticulin meshwork in batch 2, due to prominent intercellular edema. In batch 3, numerous reticulin fibers were seen around the newly formed capillaries. Batch 4 showed delicate reticulin fibers extending into the loose mesenchymal tissue as scaffolds for the sprouting nerve fibers and capillaries. On HE, all batches showed variable nerve regeneration. Groups 2–4 showed some periarterial expansion. Batch 2 displayed moderate nervous growth, but the intraluminal fibers exhibited frequent vacuolizations and areas of atrophy. The cytoplasmic expression of PGP 9.5 was seen in all 4 groups, with a higher intensity and a broader distribution in batches 3 and 4. Moreover, in batch 2, the staining showed moderate intensity and displayed a heterogeneous pattern. S100 expression paralleled that of PGP 9.5. In batch 3, CD34 highlighted numerous newly formed capillaries, proving that there was a rich blood supply. In batch 4, it was focally positive in the stellate mesenchymal cells, highly suggestive of their stem phenotype. In batches 2–4, the aortic conduit showed no staining due to endothelial denudation. Batch 4 showed Ku80 loss. Masson’s trichrome stain underlined prominent perineural fibrosis in batch 2 while the other groups showed variable collagen deposition around the suture site. In terms of myelin sheaths, batches 1 and 2 displayed faint staining, whereas the staining in batches 3 and 4 was more intense, although discontinuous. Gömori silver stain showed distorted reticulin meshwork in batch 2, due to prominent intercellular edema. In batch 3, numerous reticulin fibers were seen around the newly formed capillaries. Batch 4 showed delicate reticulin fibers extending into the loose mesenchymal tissue as scaffolds for the sprouting nerve fibers and capillaries. From a clinical point of view, all 38 rats had a full recovery in terms of sensitivity at 12 weeks (noted with 3 points). Therefore, the correlation between the histopathology and the sensitivity test was achieved using the sensitivity test performed at 10 weeks. For the other two clinical indicators (SFI and motor test), the values obtained in the last test (at 12 weeks) were used for the correlation with the histopathology tests. The statistical correlations between the clinical and pathological parameters are as follows: There was no correlation between the macroscopic examination and the sensitivity test at 10 weeks ( p = 0.12). There was a positive correlation between the macroscopic examination and the motor test at 12 weeks ( p = 0.038). There was a positive correlation between the macroscopic examination and the SFI at 12 weeks ( p = 0.001). There was a positive correlation between the microscopic examination and the sensitivity test at 10 weeks ( p = 0.031). There was a positive correlation between the microscopic examination and the motor test at 12 weeks ( p = 0.002). There was a positive correlation between the microscopic examination and the SFI at 12 weeks ( p < 0.001). There was a positive correlation between the micro- and macroscopic examinations ( p < 0.001). The histology image of the nerve with nerve fibers occupying >75% of the sectioned nerve with small gaps between the nerve fibers (a microscopic indicator of a robust nerve regeneration) correlated with good results in all three of the tests (sensitivity test at 12 weeks, motor test at 12 weeks and SFI at 12 weeks). In contrast, fibers occupying <25% of the nerve circumference with large gaps in between (an indicator of poor nerve regeneration) correlated with overall poor test results regarding sensitivity, motor function and SFI. The positive correlations (macroscopic examination and the sensitivity test; macroscopic examination and the SFI; microscopic examination and the sensitivity test; microscopic examination and the motor test; microscopic examination and the SFI) indicate that histopathology findings (both microscopic and macroscopic) are tightly connected with the results of the clinical evaluation at the end of the experiment. Histopathology and immunohistochemistry are valuable investigations that can be used for ex vivo evaluations of nerve regeneration. Microsurgery requires skills and training in order to perform correct nerve anastomosis. Proper preoperative preparation and a good surgical technique are important to prevent complications. Chlorhexidine is a potent disinfectant, which can be used at the surgical site with maximal efficiency to prevent infections, while wound dehiscence could be treated in specific cases conservatory . Postoperative care has also great significance for the outcome. Meloxicam prevents inflammation, reducing pain in rats . Blythe et al. have used the rat grimace scale to evaluate the effects of Meloxicam on neuropathic cervical pain caused by cervical root compression and demonstrated that NSAID reduces this pain . In terms of clinical examination, the SFI has been a standard measurement for evaluating nerve regeneration after different types of sciatic nerve injury. However, due to its possible subjectivity, new methods such as video gait analysis and quantitative measurement of isometric tetanic muscle force have been suggested . Aside from the pinch test, Wang et al. used novel assessment techniques: laser Doppler perfusion imaging, walking track analysis and transmission electron microscopy (the latter being able to show unmyelinated axons in the injured sciatic nerve) . Extensive work on peripheral nerve defect repair in rats was performed by Siemionow et al. In her studies, she evaluated the rats using functional (pinprick, toe-spread), neurosensory (somatosensory-evoked potentials) and histomorphometric evaluations. Siemionow evaluated the results at 3, 6 and 12 weeks . In the same study, the authors showed that the epineural sheath graft technique (a flat rectangular epineural sheath created after removing the fascicles from the excised nerve) was a good method for nerve regeneration . Another study conducted by Siemionow et al. showed that epineural tubes containing isogenic bone marrow stromal cells (BMSC) could also be a good alternative to autograft repair . One more study conducted by Nijhuis et al. used the toe spread and pinprick test to evaluate motor and sensory function for a 15 mm sciatic nerve defect repaired in three different ways: nerve autograft (group 1), a vein filled with muscle and bone marrow stromal cells BMSC (group 2) and a vein filled just with muscle (group 3). In this study, group 2 presented more Schwann cells compared to group 3, proving that BMSC have a beneficial effect when used in nerve conduits . Another study conducted by Nijhuis showed that a vein graft supported with isogenic bone marrow stromal cells provides better nerve regeneration for 20 mm nerve gaps in comparison to an empty venous graft or a vein graft filled with saline . Nerve allografts from cadavers have also been considered for nerve defects; however, these are rapidly rejected if proper immunosuppression is not achieved . Aside from clinical tests, histopathological examination employing both classical HE and special stains, as well as novel immunohistochemistry markers, can tremendously aid in regeneration quantification. Morphologic features that correlated with better nerve regeneration on HE staining were intact myelin sheaths, thick continuous fibers, little to no nerve vacuolizations and intercellular edema, newly formed capillaries, minimal perineural fibrosis, an optimal amount of reticulin and collagen meshwork. The correlations between clinical and histological outcomes showed that the opaque robust nerve texture (a macroscopic indicator of optimal nerve regeneration) correlated with good results of the motor test and of the SFI at 12 weeks, but not with the optimal results of the sensitivity 10-week test. In opposition, the translucent aspect of a narrow nerve (a macroscopic indicator of poor nerve regeneration) correlated with poor motor test and SFI results but did not correlate with the results of the sensitivity test. Similar to our research, another study conducted by Pavic et al. used immunohistochemical methods in a sciatic nerve crush injury model. They showed changes in sensory and motor axons in the spinal cord segment L3–L6 on the injured side, corresponding to the plantar test results, by using antibodies for Myelin-associated glycoprotein (MAG) and gangliosides GD1a and GT1b on the aforesaid part of the spinal cord . Immunohistochemistry was used in our study to increase the accuracy of the evaluation. PGP 9.5 is a general neural marker. A positive reaction with anti-PGP9.5 antibodies is seen in neurons, nerve fibers and neuroendocrine cells . In our study, the intensity and cytoplasmic distribution of PGP 9.5 were proportional to nerve regeneration. On one hand, the heterogeneous pattern of PGP 9.5 in batches 1 and 2 was consistent with focal nerve damage. On the other hand, the higher intensity and the broader distribution of PGP 9.5 expression in batches 3 and 4 indicated better nerve regeneration in these two groups. S100 represents a calcium-binding cytosolic protein family. Neurons, Schwann cells and dendritic cells exhibit S100 positivity. In damaged nerve fibers, the staining might be decreased or even absent . In our study, its expression paralleled that of PGP9.5, showing better regeneration in batches 3 and 4 and patchy positivity, with varying intensity, in batches 1 and 2. CD34 is a transmembrane glycoprotein expressed in a variety of cells. A positive reaction with anti-CD34 antibody is seen in stem cells and endothelial cells in a membranous pattern . The positivity of CD34 in batch 3 indicated that the PRP used was effective in generating new blood vessels, therefore improving overall nerve regeneration. Although it was expected for CD34 to be positive in the batches in which an aortic conduit was used, the negative staining in these cases could be attributed to the denuded endothelium and its degenerative changes. Ku80 is a nuclear protein that promotes the repair of double-strand DNA breaks. Its absence is correlated with tumorigenesis and premature degenerative processes . Ku80 was used in batch 4 to assess the quality of nerve regeneration. Although the nerve fibers were morphologically intact in batch 4, the loss of Ku80 staining suggests that DNA repair might be impaired in the long run. Special stains were used to better assess the architecture and associated collagen and reticulin meshworks. Gömori silver stain highlights the reticulin fibers, which are argyrophilic and therefore appear black . In our study, the reticulin meshwork was prominent in batches 3 and 4, directly proportional to the nerve regeneration. By contrast, batches 1 and 2 displayed distorted reticulin fibers due to poorer nerve regeneration. Masson’s trichrome stain is a special stain that highlights connective tissue fibers. In normal peripheral nerves, the associated collagen fibers of the epi-, peri- and endoneurium appear blue, whereas the myelin sheaths stain purple–red. In the case of myelin sheath damage, the stain becomes faint or is absent . In our study, there was considerable perineural fibrosis in batches 1 and 2. This finding was associated with inferior nerve regeneration. Myelin sheath staining was more intense in batches 3 and 4, a feature that correlated with optimal nervous growth. In another study with transected rat sciatic nerves, some repaired with tubular collagen nerve sleeves, others without, the histologic examination of nerve regeneration observed three aspects: neuroma formation, connective tissue proliferation and axonal regrowth. When comparing the wrapped to non-wrapped repaired nerves, Kim et al. showed that significantly more connective tissue formation was present in non-wrapped nerves, but there was no neuroma formation and no significant difference in axonal growth . Evaluation of nerve regeneration can be performed using different techniques: in situ high-resolution ultrasound (HRUS) magnetic resonance microscopy (MRM), histological cross-sections (HCS) and optical projection tomography (OPT) . Furthermore, stem cells constitute an innovative solution not only for peripheral nerve regeneration but also for obtaining functional neurons in vitro. Soni A. et al. have developed an efficient method for converting neural progenitor cells into functional neurons, which are then available without further animal sacrifice . Regarding the loss of Ku80 protein expression by immunohistochemistry in batch 4, it would be interesting to explore this phenomenon at the molecular level—some studies indicate that Wnt and COX2 signaling pathways are involved in its up- and downregulation in nerve defects inflicted in mice. As electron microscopy and immunofluorescence studies are required to further investigate this hypothesis, we hope to look into it in a future experimental setting . A limitation of our study is that the results after PRP and stem cell treatment could have been better in clinical evaluation compared to batch 2, but more rats should have been included in the study for a significant statistical result. Nerve regeneration was observed in all batches, both from the clinical and histopathological perspectives. Positive correlations between the clinical and the histopathological evaluations were observed in the study. Promising results were obtained in batches where PRP and stem cells were used, followed by nerve autograft. Superior nerve growth correlated with diffuse and intense expression of the neural markers PGP9.5 and S100. The vascular marker CD34 was expressed in batches 3 and 4, due to rich neovascularization, which was also linked to better nervous regeneration. Denser perineural fibrosis and poorer nerve regeneration were seen in batch 2. The markers used in the last batch showed excellent nerve regeneration. In conclusion, immunohistochemistry and special staining are useful tools in the assessment of nerve regeneration and could be corroborated with other investigations in experimental projects. |
Long-Term Evaluation of Ocular Surface and Meibomian Gland Function after Laser-Assisted in situ Keratomileusis Surgery | 0a64da25-aa94-4f1d-9d45-417a6f52ea09 | 11844694 | Surgical Procedures, Operative[mh] | Laser-assisted in situ keratomileusis (LASIK) is one of the most common refractive surgeries. It applies an excimer laser to create a hinged corneal flap from the epithelium, Bowman’s membrane, and the superficial stromal layer . Corneal sensitivity is reduced following LASIK due to the damage of the subbasal nerve plexus and might result in neurotrophic keratitis. Dry eye symptoms following LASIK are related to impaired corneal blink reflex, lacrimal gland secretion, and reduced tear secretion . Despite intracorneal nerve regeneration usually occurring between 3 and 6 months postoperatively, chronic dry eye symptoms are reported in post-LASIK patients . Ocular surface characteristics during preoperative examination might predict chronic dry eye development in LASIK. The pathophysiology of long-term dry eye symptoms in post-LASIK eyes remains imcompletely understood . Dry eye disease is a multifactorial disease involving the tear film and ocular surface that result in ocular discomfort, visual disturbance, and tear film instability . It imposes disruptive effects on the quality of life if not properly treated and managed . There are different hypotheses for chronic dry eye symptoms following LASIK. Corneal hypoesthesia resulting from the damage to the sub-basilar never plexus can contribute to reduced tear production from sensory input disruption . Post-LASIK eyes were found to have a decreased blinking rate of up to 40% and thus prone to developing dry eyes . The risk of chronic dry eye after LASIK was found higher among Asian than Caucasian patients . A reduction of functional meibomian was observed in post-LASIK eyes . Despite the intriguing relationships that have been observed, the mechanism of chronic dry eye symptoms in post-LASIK eyes is not completely understood. In this study, we evaluate and report the ocular surface changes, partial blinking (PB) rate, and both the functional and structural meibomian gland (MG) statuses in patients who underwent LASIK for at least 48 months. This is a cross-sectional, matched case-control comparison study. This study was approved by the Institutional Review Board (CUHK-NTEC CREC 2020.34). Written informed consent was obtained from every individual participant, and all study procedures conformed to the tenets of the Declaration of Helsinki. We included adult patients (18 years old or above) who underwent LASIK for myopia at least 48 months ago. All healthy controls were sex-, age-, smoking-, and axial length-matched. Both post-LASIK patients and healthy controls with a history of inflammatory disease, neoplastic disease, ophthalmic disease, and ocular treatment, including any surgery, topical eyedrops, or contact lenses in the past 12 months, were excluded from this study. Post-myopic LASIK patients who fulfilled the inclusion criteria and healthy controls were recruited from a community eye screening program at The Chinese University of Hong Kong between July 2022 and December 2022 . Ocular Surface Evaluation The slit-lamp examination was performed by one of the 2 masked ophthalmologists on 9 MG dysfunction items: corneal fluorescein staining, lid telangiectasia, lid margin thickness, lid margin irregularity, lid-parallel conjunctival folds, papillae, MG plugging, MG expressibility, and meibum quality . Oxford Scheme was used to grade corneal fluorescein staining . Lid margin telangiectasia was graded on a scale from 0 to 3: 0 = no lid margin redness or telangiectasia, 1 = redness in the lid margin and no telangiectasia crossing the MG orifices, 2 = redness in lid margin and telangiectasia crossing MG orifices, moderate telangiectasia or redness, and 3 = severe telangiectasia or redness . Lid margin thickness was graded on a scale from 0 to 2, i.e., 0 = no thickening, 1 = lid margin thickening with or without localized rounding, and 2 = lid margin thickening with diffuse rounding . Eyelid margin irregularity was graded from 0 to 2: 0 = no irregularity, 1 = fewer than 3 lid margin irregularities and shallow notching, and 2 = three or more lid margin irregularities or deep notching . Lid-parallel bulbar conjunctival fold was graded on a scale from 0 to 3: 0 = without fold, 1 = single small fold, 2 = more than 2 folds and not higher than the tear meniscus height (TMH), and 3 = multiple folds and higher than the tear meniscus . Papillae were graded using the papillae-limbal corneal epithelial score . MG plugging was assessed using the scale from 0 to 3: 0 = no plugging, 1 = fewer than 3 pluggings of gland orifices, 2 = three or more pluggings of gland orifices with a distribution of less than half of the full length of the eyelid, and 3 = three or more pluggings of gland orifices with a distribution of half or more of the full length of the lid . MG expressibility was graded from 0 to 3 based on the central 5 MGs of the lower eyelid: 0 = all glands expressible, 1 = 3–4 glands expressible, 2 = 1–2 glands expressible, and 3 = no gland expressible . Meibum quality was graded on a scale of 3: 0 = clear, 1 = cloudy, 2 = cloudy with debris, and 3 = inspissated, toothpaste-like . Subjective dry eye symptom was measured using the ocular surface disease index (OSDI) 12-item questionnaire, which had been translated into Chinese version . Baseline tear production was measured by Schirmer’s test without topical anesthetia . The noninvasive tear break-up time and TMH were measured using a corneal topographer with a built-in real-time keratometer (Oculus Keratograph 5M, Oculus Inc., Arlington, WA, USA) . Lipid layer thickness (LLT), PB rates, and meibography were obtained using the tear interferometer (LipiViewII, TearScience Inc., Morrisville, NC, USA). The MG dropout was measured from the meibograph using the ImageJ software. The area covered by tarsal conjunctiva and MG dropout was segmented using a freehand tool by two independently trained masked investigators, and the average was used for data analysis. The degree of MG dropout was measured by dividing the MG dropout area by the total area of the tarsal conjunctiva . Schirmer test was performed without anesthesia. Numerical results were presented as mean ± standard deviation and range unless otherwise stated. The comparison of ocular surface evaluation was made only on the right eye between post-LASIK patients and healthy controls, using the paired t test. All statistical analyses were performed using SPSS statistical software package (Windows version 24.0; IBM Corp., Armonk, NY, USA). The slit-lamp examination was performed by one of the 2 masked ophthalmologists on 9 MG dysfunction items: corneal fluorescein staining, lid telangiectasia, lid margin thickness, lid margin irregularity, lid-parallel conjunctival folds, papillae, MG plugging, MG expressibility, and meibum quality . Oxford Scheme was used to grade corneal fluorescein staining . Lid margin telangiectasia was graded on a scale from 0 to 3: 0 = no lid margin redness or telangiectasia, 1 = redness in the lid margin and no telangiectasia crossing the MG orifices, 2 = redness in lid margin and telangiectasia crossing MG orifices, moderate telangiectasia or redness, and 3 = severe telangiectasia or redness . Lid margin thickness was graded on a scale from 0 to 2, i.e., 0 = no thickening, 1 = lid margin thickening with or without localized rounding, and 2 = lid margin thickening with diffuse rounding . Eyelid margin irregularity was graded from 0 to 2: 0 = no irregularity, 1 = fewer than 3 lid margin irregularities and shallow notching, and 2 = three or more lid margin irregularities or deep notching . Lid-parallel bulbar conjunctival fold was graded on a scale from 0 to 3: 0 = without fold, 1 = single small fold, 2 = more than 2 folds and not higher than the tear meniscus height (TMH), and 3 = multiple folds and higher than the tear meniscus . Papillae were graded using the papillae-limbal corneal epithelial score . MG plugging was assessed using the scale from 0 to 3: 0 = no plugging, 1 = fewer than 3 pluggings of gland orifices, 2 = three or more pluggings of gland orifices with a distribution of less than half of the full length of the eyelid, and 3 = three or more pluggings of gland orifices with a distribution of half or more of the full length of the lid . MG expressibility was graded from 0 to 3 based on the central 5 MGs of the lower eyelid: 0 = all glands expressible, 1 = 3–4 glands expressible, 2 = 1–2 glands expressible, and 3 = no gland expressible . Meibum quality was graded on a scale of 3: 0 = clear, 1 = cloudy, 2 = cloudy with debris, and 3 = inspissated, toothpaste-like . Subjective dry eye symptom was measured using the ocular surface disease index (OSDI) 12-item questionnaire, which had been translated into Chinese version . Baseline tear production was measured by Schirmer’s test without topical anesthetia . The noninvasive tear break-up time and TMH were measured using a corneal topographer with a built-in real-time keratometer (Oculus Keratograph 5M, Oculus Inc., Arlington, WA, USA) . Lipid layer thickness (LLT), PB rates, and meibography were obtained using the tear interferometer (LipiViewII, TearScience Inc., Morrisville, NC, USA). The MG dropout was measured from the meibograph using the ImageJ software. The area covered by tarsal conjunctiva and MG dropout was segmented using a freehand tool by two independently trained masked investigators, and the average was used for data analysis. The degree of MG dropout was measured by dividing the MG dropout area by the total area of the tarsal conjunctiva . Schirmer test was performed without anesthesia. Numerical results were presented as mean ± standard deviation and range unless otherwise stated. The comparison of ocular surface evaluation was made only on the right eye between post-LASIK patients and healthy controls, using the paired t test. All statistical analyses were performed using SPSS statistical software package (Windows version 24.0; IBM Corp., Armonk, NY, USA). A total of 48 right eyes of 48 post-LASIK Chinese patients (39 females, 2 smokers) were analyzed with 48 right eyes of 48 matched healthy controls. The age on ocular surface examination was 50.0 ± 11.0 years for post-LASIK patients and 51.1 ± 11 years for healthy controls. The duration between LASIK and examination was 177 ± 68 (range, 48–240) months. The axial length was 26.0 ± 1.0 mm for post-LASIK eyes and 25.4 ± 1.2 mm for healthy eyes. The demographic and clinical data of post-LASIK patients and healthy controls are summarized in . Grading of the 9 MG dysfunction items on anterior segment examination was compared between post-LASIK eyes and healthy eyes. Post-LASIK eyes were found to have a higher grade of meibum quality (1.3 vs. 0.9, p = 0.008) compared to healthy controls. Other anterior segment data were comparable between the 2 groups . Post-LASIK eyes were associated with a shorter Schirmer test (10.8 vs. 14.1, p = 0.03). The OSDI score was higher in post-LASIK patients (20.8 vs. 7.5, p = 0.00001), with 75% of post-LASIK patients scored over 12. PB rate, average noninvasive tear break-up time, MG dropout, LLT, and TMH were comparable between the 2 groups . In this cross-sectional, matched case-control comparison study, the post-LASIK patients were associated with a greater degree of dry eye symptoms, and up to 75% of them scored over 12 on the OSDI questionnaire, which indicates the presence of dry eye symptoms. Despite the post-LASIK eyes being found to have a reduction of aqueous production on the Schirmer test score, the TMH and average tear break-up time were not reduced. LLT and MG dropouts were comparable with the healthy controls. The percentage of PB was similar between the 2 groups. Dry eye disease is the most common complication following LASIK, and several hypotheses were proposed in the pathophysiology of post-LASIK dry eye including the loss of corneal sensitivity, impaired blinking mechanism, altered tear film biomarkers, and MG dysfunction . The sensory automatic neural reflex loop between the corneal surface and lacrimal gland is important to maintain the tear film stability, as lacrimal glands secrete a mixture of aqueous, mucus, lipids, antibodies, and enzymes. A longitudinal study reported that the Schirmer test score was reduced at 3 months following LASIK . Another Greece study showed that the Schirmer test was decreased at 1 and 3 months following LASIK but restored by the 6th month . Lee et al. reported that corneal nerve regeneration in post-LASIK eyes is inferior to that in laser-assisted epithelial keratomileusis eyes and does not return to the preoperative level even after 6 months. Lee et al. reported that the number of subbasal and stromal nerve fiber bundles decreased by 90% after LASIK and gradually returned at 1 year despite remaining less than half of the baseline. Our study showed a reduced Schirmer test in post-LASIK eyes. However, both the tear break-up time and TMH were not significantly affected. A long-term longitudinal study would be warranted to study the regeneration of corneal nerves in LASIK patients. A normal blinking motion protects the ocular surface and serves as a pumping force to secrete the meibum to form the lipid tear layer . Jung et al. reported that a reduction in LLT was associated with the history of refractive surgery, and a subsequent study showed a reduction of the functional MG in post-LASIK eyes . An impaired blinking mechanism may result from the disruption of corneal sensation that leads to obstructive MG obstruction . The impaired corneal nerves may affect the sensory feedback to the lacrimal gland and also reduce blinking in post-LASIK patients . The reduction of blinking can lead to increase of tear hyperosmolarity and upregulation of tear inflammatory cytokines . The meibum quality is worse with more cloudy debris in post-LASIK eyes, and we postulate that the changes in tear film dynamics may lead to an increased MG stress, causing the meibum to stagnate, become thicker, and more turbid . Notably, both the patrial blinking rates and MG dropout were comparable between post-LASIK eyes and healthy controls in our study. It is known that the Chinese population has a higher percentage of baseline MG dysfunction when compared to other ethnic groups, and no exaggeration of MG dropout was observed from our patients in contrast to Caucasian patients . Subjective dry eye symptoms are commonly reported in post-LASIK patients, and they usually improve 6 months after LASIK . Heightened parasympathetic tone and prolonged pain sensitivity in patients measured before LASIK predicted greater dry eye symptoms after the surgery . A higher OSDI was found in the post-LASIK patients in our study, which was consistent with most reported studies and not explained by the ocular surface parameters. Review study suggested the corneal nerve damage produced by LASIK may resemble the pathologic neuroplasticity associated with other forms of persistent postoperative pain . Spierer et al. recently reported that clinically significant asymptomatic preoperative MG dysfunction was not found to have a significant impact on LASIK outcomes, especially the dry eye complaints at 1 month. In contrary, our findings indicate the need for periodic comprehensive ocular surface evaluation and appropriate treatment in post-LASIK patients with chronic dry eye symptoms. There are several limitations in the current study. First, this is a cross-sectional study without follow-up data. The longitudinal development of this study cohort will have to be monitored. Second, the LASIK procedures were not standardized in every patient, and the surgical techniques may confound our data. Third, the associations that may affect the degree of ocular surface parameters, such as corneal sensation, nerve evaluation, corneal biomechanics, and tear composition, were not evaluated. Fourth, the history of MG dysfunction treatment was not documented. Our data serve as a reference for future prospective longitudinal studies to understand the chronic dry eye characteristics in post-LASIK patients. In summary, up to 75% of post-LASIK patients had symptomatic dry eye disease on the OSDI questionnaire. Long-term post-LASK eyes showed reduction of aqueous tear production, but without difference in the ocular surface parameters. Post-LASIK patients with chronic dry eye symptoms are therefore recommended to have comprehensive ocular surface evaluation in regular follow-up. Treatment for dry eye disease should be continued in patients with poor tear film stability. Further studies are warranted to evaluate and understand the ocular surface changes in long-term post-LASIK eyes. This study was approved by the Institutional Review Board (Chinese University of Hong Kong – New Territories East Cluster Clinical Research Ethics Committee 2020.34). Written informed consent was obtained from any adults participating in this study. No conflicting relationship exists for any author. The work in this paper was supported in part by a research grant from the Food and Health Bureau (FHB) – Health and Medical Research Fund (HMRF) (Project No. 08190266 to K.K.L.C.). K.K.H.L., Z.H., F.A., J.T.C., and K.K.L.C. designed the study. J.U.S., G.P.M.C., W.W.K.Y., A.L.Y., and K.K.L.C. collected recruited patients and recorded clinical data. K.K.H.L., F.M.A.A.A., J.T.C., and Z.H. reviewed clinical data. K.K.H.L. wrote the first draft of the manuscript. K.K.H.L, F.M.A.A.A., J.T.C., Z.H., G.P.M.C., W.W.K.Y., A.L.Y., C.C.Y.T., C.C.P.P., and K.K.L.C. interpreted the data and reviewed the manuscript. All authors have read and approved the final manuscript. |
Variation in cystectomy pathology reporting practice—results from an international survey of 212 pathologists | dcdd8ad0-9e0c-4a23-938d-42a1677948fe | 11564217 | Pathology[mh] | Radical cystectomy with preceding neoadjuvant cisplatin-based chemotherapy is recommended by the European Urological Association as definitive treatment for muscle-invasive bladder cancer (MIBC) in eligible patients . The pathological assessment of cystectomy specimens is important for confirming the presence of remaining tumour, accurate assessment of tumour and nodal stage, diagnosing subtype histology and examination of resection margins. These parameters have impacts on post-operative/adjuvant therapy decisions. For example, the PD-L1 inhibitor Nivolumab is licensed for use in the UK for patients with ypT2 + /ypN + MIBC who have received neoadjuvant chemotherapy (NAC) or patients with pT3 + /pN + MIBC who did not have neoadjuvant treatment . Pathological assessment of cystectomy specimens poses some unique challenges not encountered in other cancer resections. Most patients will have had a trans-urethral resection of bladder tumour (TURBT) as a diagnostic and potentially therapeutic procedure early in their management pathway. As a result, there may be no macroscopic tumour present in the bladder and, in approximately 10% of cases, no tumour is found microscopically (pT0) . In a proportion of patients, this may be partially attributable to neoadjuvant chemotherapy; however, patients who have received solely TURBT may also achieve pT0 at cystectomy. In the post-NAC setting, an assessment of treatment effect is also necessary. Pathological down-staging to any of < ypT2, ypTis, ypTa or ypT0 has been used in clinical trials of neoadjuvant chemotherapy and, more recently, neoadjuvant immunotherapy. Downstaging correlates with survival in the neoadjuvant setting and is thus a useful surrogate end point which can give an earlier signal of treatment effect than waiting for follow-up data to mature. An attempt has been made to standardise the assessment and reporting of response to NAC in cystectomies , similar to semi-quantitative systems used in breast and colorectal cancer. However, this approach has not seen widespread adoption in national guidelines for bladder cancer diagnosis and management. Despite the importance of pathological assessment of cystectomy specimens, there is a surprising lack of evidence to support current approaches to practice . If there is variation in practice, this could contribute to variation in the information provided by pathologists. In turn, this could affect clinically important parameters such as pathological response to NAC or accurate staging. The pathological assessment process has many points where variation can occur, from methods of fixation and dissection to tissue block selection and the use of scoring systems for treatment response. In this study, we evaluated this variation through an international survey of pathologists who report cystectomy specimens. We designed an 18-question survey which was distributed electronically as a Google form by the British Association of Urological Pathologists (BAUP), the International Society of Uropathology (ISUP), the Genito-Urinary Pathology Society (GUPS) and the Working group Uropathology of the German Society of Pathology (DGP) via their mailing lists. The survey was open to receive responses from 14th August to 25th September 2023 and a reminder email was sent halfway through this period. The participants were independently practicing pathologists with an interest in uropathology (consultant/attending level). All participants gave informed consent and the study received ethical approval from the University of Sheffield (UK) Ethics Committee on 24th July 2023 (approval number: 054611). The survey questions covered practice across the entire cystectomy specimen journey and included questions about fixation methods, dissection and sampling, microscopy and molecular and digital pathology. We also asked specific questions about the assessment of cystectomies following neoadjuvant chemotherapy (NAC). The full questionnaire, study information and consent form are available in . Questionnaire data was collected in Excel (Microsoft Corporation, Redmond, WA, USA) and analysed in R version 4.0.3 (R Foundation, Indianapolis, IN, USA) . Categorical data are presented as proportional waffle plots. Upset plot is used to represent combinations of answers. Chi-squared test was used to assess statistical significance of categorical variables. Questionnaire cohort demographics A total of 212 pathologists from 49 countries completed the online survey. The commonest countries were USA ( n = 49, 23%), UK ( n = 18, 8%), India ( n = 10, 5%) and Canada ( n = 10, 5%) (Fig. a). Some 36% of pathologists were from centres that performed more than 50 cystectomies per year (Fig. b). Experience of reporting radical cystectomies was assessed as 5-year groupings and there was an even distribution of participants across the categories 1–5 years’ experience to 15–20 years’ experience. Altogether, these groups comprised 81% of pathologists. The remaining 19% had more than 20 years’ reporting experience (Fig. c). Together, these results show that the questionnaire responses captured a wide representation of geography, reporting activity and reporting experience. Approach to fixation and sampling of routine cystectomy specimens We next asked about pathologists’ approach to fixation, processing and sampling of cystectomies. Methods of fixation varied: 67% of respondents incised the bladder anteriorly and submerged the entire specimen in formalin to fix, 16% bisected the specimen into two halves and placed in formalin to fix and 14% inflated the bladder with formalin via the urethra (Fig. a). Most (93%) pathologists did not routinely perform fresh sampling of cystectomy specimens. Two situations are commonly encountered when sampling cystectomy specimens. There may be a scarred area with no tumour present owing to previous TURBT, intra-vesical therapy and/or neoadjuvant chemotherapy. Alternatively, macroscopic tumour may remain in the bladder. We asked how pathologists approached these situations and found that, when no tumour was visible macroscopically, most respondents (81%) sampled the entire scarred area. Some 12% of respondents would take representative sections of the scar and 6% would put the entire bladder into blocks (Fig. b). In the second situation of a cystectomy specimen containing residual macroscopic tumour, 75% of pathologists would take representative tumour sections and sample the background bladder whereas 19% would block the entire tumour and sample the background bladder. Four respondents (2%) would block the entire bladder. The remaining nine respondents gave descriptions where their sampling strategy varied based on the size of the tumour (Fig. c). Many situations in diagnostic pathology require additional work after an initial microscopic assessment of tissue sections. We recognised this as a possible scenario in the cystectomy setting when no tumour is identified in the initial tissue sampling. Respondents were asked about their approach in this context. Some 45% of pathologists would sample the rest of the bladder if their initial blocks showed no tumour. However, half of the respondents would not take this approach (Fig. d). To further probe microscopic assessment of cystectomies, we asked if pathologists routinely used levels or step sections to examine tissue from cystectomies in general. The majority (90%) did not routinely examine tissue at multiple levels (Fig. e). When asked if they used levels on a case-by-case basis, 196 pathologists gave evaluable answers. Some 64% did use levels on a case-by-case basis and listed reasons including dealing with technical issues such as requiring full-face sections, closer examination of the scarred area, assessment of margins and in cases where tumour was equivocal at a stage boundary. Assessment of response to neoadjuvant chemotherapy in cystectomies Complete response to neoadjuvant chemotherapy is a good surrogate marker of cancer-specific and overall survival. Furthermore, the rate of complete response or downstaging from muscle invasive to non-muscle invasive bladder cancer is frequently used as an endpoint in neoadjuvant chemotherapy and immune-therapy trials. How response to neoadjuvant chemotherapy is assessed is therefore important as variation in assessment could impact on the predictive ability of this metric. To investigate the approach to cystectomy assessment following NAC, we first asked pathologists to estimate what proportion of patients received NAC at their institution. Interestingly, 32% of respondents did not know. Two respondents stated that patients did not receive NAC at their centre. The remaining respondents reported an approximately even distribution across the quintiles 1–20%, 21–40%, 41–60% and 61–80% of patients receiving NAC. Only 7/212 respondents indicated that greater than 80% of patients received NAC. Next, we asked how pathologists approached reporting cystectomy specimens following NAC. Overall, 192/212 (90%) pathologists stated they would use the ypT nomenclature when reporting pathological complete response. In total, 19% of respondents reported using a response score such as that described by Fleischmann et al. . Interestingly, all of these pathologists were from institutions outside of the USA (41/122 from non-USA institutions vs. 0/49 from USA institutions, p < 0.001, c 2 test). As participants could select more than one option when asked how they reported pathologic response, we next investigated if there were common combinations of reporting practice. Most respondents (71, 33.5%) used the ypT0 nomenclature together with a qualitative description of the response to neoadjuvant therapy. The next commonest reporting combination was to use only ypT0 nomenclature with no qualitative description, quantification or scoring system to characterise the response to neoadjuvant treatment ( n = 61, 28.8%) (Fig. ). We also asked if NAC changed the pathologists’ approach to reporting cystectomies. Some 56% said they would take more blocks in this scenario whilst the remaining 44% would not change their approach. The reported routine use of levels or step sections was significantly higher in post-NAC cystectomies with 18% of respondents using routine levels after NAC compared to 10% in when NAC had not been given ( p = 0.03, c 2 test). Use of digital and molecular pathology in reporting cystectomies As digital and molecular pathology have become established facets of modern pathology practice, we sought understand how these tools are used in the assessment of cystectomies. Some 86% of respondents report cystectomies using traditional glass slides. By contrast, only 8% and 6% of pathologists reported using digital slides for all or some of their cystectomy work respectively. Of the 29 pathologists of who use digital slides, 14 (48%) reported using digital tools such as digital measuring in their assessment of cystectomies. Finally, we asked about molecular pathology reporting practice. There were 157 pathologists who answered the question and, of these, 52% preferred to perform molecular tests on the TURBT specimen. Thirty-five percent of respondents would do molecular tests on the cystectomy specimen if there was macroscopic or microscopic tumour present whereas 6% of pathologists would use the cystectomy but only if macroscopic tumour was visible (Fig. c). In the last question, we asked if NAC would change the molecular testing approach. The majority (140/181, 77%) of pathologists indicated that NAC would not change their approach to the choice of specimen for molecular testing. Some 20% of respondents would not use the cystectomy specimen for molecular testing if the patient had received NAC. A total of 212 pathologists from 49 countries completed the online survey. The commonest countries were USA ( n = 49, 23%), UK ( n = 18, 8%), India ( n = 10, 5%) and Canada ( n = 10, 5%) (Fig. a). Some 36% of pathologists were from centres that performed more than 50 cystectomies per year (Fig. b). Experience of reporting radical cystectomies was assessed as 5-year groupings and there was an even distribution of participants across the categories 1–5 years’ experience to 15–20 years’ experience. Altogether, these groups comprised 81% of pathologists. The remaining 19% had more than 20 years’ reporting experience (Fig. c). Together, these results show that the questionnaire responses captured a wide representation of geography, reporting activity and reporting experience. We next asked about pathologists’ approach to fixation, processing and sampling of cystectomies. Methods of fixation varied: 67% of respondents incised the bladder anteriorly and submerged the entire specimen in formalin to fix, 16% bisected the specimen into two halves and placed in formalin to fix and 14% inflated the bladder with formalin via the urethra (Fig. a). Most (93%) pathologists did not routinely perform fresh sampling of cystectomy specimens. Two situations are commonly encountered when sampling cystectomy specimens. There may be a scarred area with no tumour present owing to previous TURBT, intra-vesical therapy and/or neoadjuvant chemotherapy. Alternatively, macroscopic tumour may remain in the bladder. We asked how pathologists approached these situations and found that, when no tumour was visible macroscopically, most respondents (81%) sampled the entire scarred area. Some 12% of respondents would take representative sections of the scar and 6% would put the entire bladder into blocks (Fig. b). In the second situation of a cystectomy specimen containing residual macroscopic tumour, 75% of pathologists would take representative tumour sections and sample the background bladder whereas 19% would block the entire tumour and sample the background bladder. Four respondents (2%) would block the entire bladder. The remaining nine respondents gave descriptions where their sampling strategy varied based on the size of the tumour (Fig. c). Many situations in diagnostic pathology require additional work after an initial microscopic assessment of tissue sections. We recognised this as a possible scenario in the cystectomy setting when no tumour is identified in the initial tissue sampling. Respondents were asked about their approach in this context. Some 45% of pathologists would sample the rest of the bladder if their initial blocks showed no tumour. However, half of the respondents would not take this approach (Fig. d). To further probe microscopic assessment of cystectomies, we asked if pathologists routinely used levels or step sections to examine tissue from cystectomies in general. The majority (90%) did not routinely examine tissue at multiple levels (Fig. e). When asked if they used levels on a case-by-case basis, 196 pathologists gave evaluable answers. Some 64% did use levels on a case-by-case basis and listed reasons including dealing with technical issues such as requiring full-face sections, closer examination of the scarred area, assessment of margins and in cases where tumour was equivocal at a stage boundary. Complete response to neoadjuvant chemotherapy is a good surrogate marker of cancer-specific and overall survival. Furthermore, the rate of complete response or downstaging from muscle invasive to non-muscle invasive bladder cancer is frequently used as an endpoint in neoadjuvant chemotherapy and immune-therapy trials. How response to neoadjuvant chemotherapy is assessed is therefore important as variation in assessment could impact on the predictive ability of this metric. To investigate the approach to cystectomy assessment following NAC, we first asked pathologists to estimate what proportion of patients received NAC at their institution. Interestingly, 32% of respondents did not know. Two respondents stated that patients did not receive NAC at their centre. The remaining respondents reported an approximately even distribution across the quintiles 1–20%, 21–40%, 41–60% and 61–80% of patients receiving NAC. Only 7/212 respondents indicated that greater than 80% of patients received NAC. Next, we asked how pathologists approached reporting cystectomy specimens following NAC. Overall, 192/212 (90%) pathologists stated they would use the ypT nomenclature when reporting pathological complete response. In total, 19% of respondents reported using a response score such as that described by Fleischmann et al. . Interestingly, all of these pathologists were from institutions outside of the USA (41/122 from non-USA institutions vs. 0/49 from USA institutions, p < 0.001, c 2 test). As participants could select more than one option when asked how they reported pathologic response, we next investigated if there were common combinations of reporting practice. Most respondents (71, 33.5%) used the ypT0 nomenclature together with a qualitative description of the response to neoadjuvant therapy. The next commonest reporting combination was to use only ypT0 nomenclature with no qualitative description, quantification or scoring system to characterise the response to neoadjuvant treatment ( n = 61, 28.8%) (Fig. ). We also asked if NAC changed the pathologists’ approach to reporting cystectomies. Some 56% said they would take more blocks in this scenario whilst the remaining 44% would not change their approach. The reported routine use of levels or step sections was significantly higher in post-NAC cystectomies with 18% of respondents using routine levels after NAC compared to 10% in when NAC had not been given ( p = 0.03, c 2 test). As digital and molecular pathology have become established facets of modern pathology practice, we sought understand how these tools are used in the assessment of cystectomies. Some 86% of respondents report cystectomies using traditional glass slides. By contrast, only 8% and 6% of pathologists reported using digital slides for all or some of their cystectomy work respectively. Of the 29 pathologists of who use digital slides, 14 (48%) reported using digital tools such as digital measuring in their assessment of cystectomies. Finally, we asked about molecular pathology reporting practice. There were 157 pathologists who answered the question and, of these, 52% preferred to perform molecular tests on the TURBT specimen. Thirty-five percent of respondents would do molecular tests on the cystectomy specimen if there was macroscopic or microscopic tumour present whereas 6% of pathologists would use the cystectomy but only if macroscopic tumour was visible (Fig. c). In the last question, we asked if NAC would change the molecular testing approach. The majority (140/181, 77%) of pathologists indicated that NAC would not change their approach to the choice of specimen for molecular testing. Some 20% of respondents would not use the cystectomy specimen for molecular testing if the patient had received NAC. To our knowledge, this is the first international survey of cystectomy dissection and reporting practice. We found variation in practice across the entire specimen journey. Most pathologists open the bladder anteriorly and fix by submersion in formalin. This has the advantage of allowing fresh tissue sampling prior to fixation and is the method currently in use by the INVEST window of opportunity trial . Formalin inflation via a catheter is also described in best practice guidelines ; however, our data shows this not widely used. To our knowledge, there has not been a rigorous, head-to-head comparison of the two techniques. Proponents of formalin inflation claim that the urothelium undergoes rapid fixation, presumably with better resulting tissue preservation and microscopic morphology. However, this and the effect of each method on molecular testing have not been formally evaluated. In MIBC, any gains in diagnostic fidelity may be negligible when assessing grossly evident tumour. However, it is conceivable that microscopic foci of tumour following neoadjuvant therapy or in cystectomies for non-muscle invasive bladder cancer with prior intravesical BCG instillation may have variable appearances depending on fixation method. There was broad consensus regarding block taking regardless of the presence of macroscopic tumour in the bladder. A small but significant minority of respondents described taking representative sections of scarred areas. A smaller proportion of pathologists indicated that they examined the entire bladder. A previous study showed that this approach did not change the detection of prognostically important parameters such as tumour stage . Coupled with our findings, this suggests that representative sections and sampling of the entire scarred area where applicable is sufficient. We recognise that the wording of this part of the questionnaire does not allow us to distinguish between scenarios where the urothelium appears completely normal macroscopically or where a scarred area is present. Following fixation, it can be difficult to identify subtle macroscopic changes and so the distinction between normality and scar may not be reliable. This specific situation warrants further investigation. It may be that pathologists, when faced with a completely normal bladder macroscopically, are more likely to submit the entire specimen for microscopic evaluation. Whilst this was not an explicit option in the questionnaire, we did not receive feedback regarding this in the free text comments section. In addition, we not ask specifically about how pathologists used radiology to guide their sampling or the perceived benefits of radiological-pathological correlation. From the authors’ experience, pre-operative CT or MRI scans of the bladder can be useful in identifying the site of tumour after apparently complete TURBT. We also assessed pathologist approach to resampling a specimen after initial microscopic assessment and found equipoise between the approaches of further sampling and no further sampling where no tumour was found in initial sections. This is a potential source of variability and should be evaluated prospectively. Furthermore, we demonstrated variability in the use of levels/step sections with more than half of pathologists using these on a case-by-case basis. It is important to note that extra sampling and levels still only provide a representative sample of the tissue examined and sampling error cannot be entirely excluded. However, there is likely to be a point at which sampling and levels approaches the limit of the useful information that could be achieved by complete examination. As complete examination (e.g. complete embedding and complete sectioning) is not feasible, exploration of the utility of sampling and levels is required. Our data show variation in the application of these tools, implying the optimum approach is not yet known. Ours is the second survey to evaluate pathologists’ approach to post-NAC cystectomy specimens. Saunders et al. surveyed 55 pathologists practicing in the USA via X (formerly Twitter). In agreement with our data, they also found that most pathologists submit the entire tumour bed area for assessment. Interestingly, respondents estimated tumour bed or scar only was a situation encountered in 71% of cases which is significantly higher than reported ypT0 rates following NAC . Whilst presence of tumour bed or scar only does not directly translate to the absence of microscopic tumour, the discrepancy between macroscopic and microscopic impression implies a significant proportion of patients with microscopic-only residual tumour. Furthermore, ypT0 could represent complete resection by TURBT and limited contribution of NAC. These situations have not been evaluated in the literature to date and merit further study. Our study adds to the evidence base of post-NAC cystectomy assessment. Interestingly, a third of respondents did not know what proportion of patients received NAC in their centre. We found that the complete absence of tumour in the bladder (ypT0) was the preferred definition of complete pathologic response (pCR); however, some pathologists also regarded downstaging as a pathologic response. We identified factors that could lead to variation in reporting pCR including variability in whether NAC changed pathologists’ approach to sampling and tissue submission. We also identified combinations of reporting practices when describing response to NAC. Nearly 20% of pathologists use the tumour response score described by Fleischmann et al. ; however, this was used exclusively by pathologists from outside of the USA. This system has been validated to predict overall survival following NAC; however, we could only find its inclusion in one recently published guideline from the Brazilian Societies of Pathology, Urology and Clinical Oncology . Our data suggest that the uptake of this system has been low. This may reflect uncertainty over how this score might be used in clinical practice and if it would influence post-operative decision-making around adjuvant therapy. pCR is widely used as a surrogate outcome measure in clinical trials of neoadjuvant therapies. Our survey results suggest that there is variation in how pCR is assessed and reported. This may in turn introduce variation into clinical trial results where pCR is an outcome. Recently, completed trials of neoadjuvant therapy in MIBC where pCR or pathological downstaging was an outcome include NEOBLADE , ABACUS and NCT02812420 . These trials define pCR as pT0 and used downstaging to < pT2 as a secondary outcome measure. However, protocols for fixation, processing and assessment of cystectomies were not documented in detail, and no central pathology review of pCR was mandated. Given the variability in practice we have highlighted in our survey responses, it is possible that these trial outcomes include variation from pathology practice that could mask or alter true therapeutic effect. Indeed, a recent position statement from the Society for Immunotherapy of Cancer and the International Bladder Cancer Group suggested that pT0/pCR may not be an appropriate sole primary endpoint in neoadjuvant trials . Our study has some limitations. We used an electronic survey distributed via four major urological pathology societies. Whilst this resulted in the largest cohort of pathologists giving their opinion on cystectomy reporting to date, this approach may also have self-selection bias. This has previously been noted in patient surveys and citizen science projects. This bias should be taken into account when considering the generalisability of our findings. A further limitation was highlighted by the free comments section of the survey. Respondents suggested further areas for scrutiny including approaches to prostate sampling in cysto-prostatectomy, uterus and vaginal wall sampling in anterior exenteration, lymph node sampling and approaches to sampling cystectomies for non-muscle invasive bladder cancer. Our survey included 18 questions. Consideration of these additional areas may have made the survey more difficult to complete and affected the number of responses. We suggest that these areas are included in future work in this area. In summary, we have demonstrated variability in cystectomy pathology reporting practices using an international survey of more than 200 pathologists. Clinical trials often use pathological measures of response to therapy as a surrogate endpoint but the variability of reporting practice could have an effect on the veracity of these measures and consequently the conclusions of clinical trials. The evidence base around cystectomy pathology reporting needs development and we have identified key research questions (Table ). A Delphi survey would be a reasonable next step using the responses described in our questionnaire to inform statement design and expert discussion/ consensus. Delphi studies are underutilised in pathology but can provide useful information about current and best practice, and highlight areas for future research . This could be particularly useful for standardising the approach to histological response to NAC and this will become more important with greater use of neoadjuvant immunotherapy and small molecule inhibitors and ongoing trials investigating bladder sparing approaches . Recently, there has been renewed interest in the evidence base underpinning macroscopic evaluation and specimen dissection and sampling . Development of evidence-based macroscopy and assessment of pCR have had clinical impact in colorectal and breast cancer . We believe similar development of the evidence base for cystectomy sampling and reporting could be similarly useful. Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 20 KB) |
Electrophysiology lab efficiency comparison between cryoballoon and point-by-point radiofrequency ablation: a German sub-analysis of the FREEZE Cohort study | 8de61f16-42ee-4ede-a5ef-fc29797ce7d6 | 9830778 | Physiology[mh] | Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is an increasingly pressing issue for health care systems in Western Europe countries due to the ageing population. Currently, at least 8 million inhabitants of the European Union suffer from AF, and the revised lifetime risk for AF in individuals over 55 years old increased from 1 out of 4 to 1 out of 3 . According to the European Society of Cardiology guidelines, AF catheter ablation is a well-established treatment for the prevention of AF recurrences and is recommended to improve AF symptoms in patients with paroxysmal or persistent AF . The cornerstone of AF catheter ablation is the complete pulmonary vein isolation (PVI), and can be accomplished with radiofrequency point-by-point circumferential lesions or single-shot ablation devices like the cryoballoon . In Germany, an estimated number of 92,220 ablations were performed in 2018, an increase of more than 5% within one year, according to a yearly survey among several hundred electrophysiology units . Out of 237 responding ablation centres, 27% of all facilities performed up to 100 ablations per year, and 17.6% more than 500. The increasing number of patients with AF is a challenge for physicians, hospitals and the healthcare system in Germany. Therefore, procedural efficiency is a key element to manage resources consumption in hospitals. The aim of this analysis is to assess the hospital efficiency of single-shot cryoballoon ablation (CBA) and irrigated tip point-by-point radiofrequency ablation (RFA), based on the FREEZE Cohort study. FREEZE Cohort study design The FREEZE Cohort study (NCT01360008) was a prospective, non-randomized, observational cluster cohort study that compared the effectiveness and safety of RFA and CBA, conducted in 42 experienced hospitals across eight countries . Each centre was assigned as a RFA or a CBA study site, chosen according to the centre’s experience and preference. Patients who received a first ablation for symptomatic paroxysmal or drug-refractory persistent AF were eligible to participate. All enrolled patients were intended to be treated by the designated technique. The study design and the main results of the intention-to-treat analysis were presented in a previous publication . The present analysis evaluates the consequences of differently distributed procedure times between CBA and RFA on schedules and utilisation of resources and staff. Only the procedures undergone in the participating centres across Germany were included as they included all RFA procedures and most of CBA procedures. The calculated metrics and model assumptions were adapted to be illustrative of German hospitals. Analysis population Patients who underwent a PVI-only approach were included in this analysis; PVI procedures with additional ablation lesions (e.g. linear lesions, ablation of complex fractionated atrial electrograms, right atrial flutter, and ablation at the AV node) were excluded. Focal RF touch-ups or treatment with two sizes of the cryoballoon needed in some CBA cases were not excluded. The treatment groups were defined according to whether CBA or RFA was utilized for the initial ablation, as procedure times should be ascribed to the procedures that were actually completed to reflect clinical reality and to avoid an artificial setting. The crossover rate to any other technique was very low in both groups . Discrete event simulation model A discrete event simulation (DES) model was used to evaluate electrophysiology (EP) lab efficiency based on PVI procedure times reported in the FREEZE Cohort. EP lab efficiency for this analysis is defined as improvements in utilization gained from shorter and more predictable procedure times, which includes fewer cumulative overtime hours, more days where overtime is avoided and more days with remaining time for additional EP lab usage. DES is a method of simulating the behaviour and performance of a real-life process, facility or system, and has been used to model the efficient use of resources in various healthcare settings. These simulations are based on a stochastic time series of individual, granular events representative of realistic occurrences. Individuals and critical resources in the DES are treated distinctly from each other, having unique characteristics and memory, and are drawn from a detailed probabilistic characterization derived from real-world data and experience. SIMUL8 professional version 26.0 (SIMUL8 Corporation) was used for DES in this analysis. DES model inputs This DES aimed to model the impact of operational changes on German EP lab occupancy based on the variability of the ablation procedure start and stop times. In the model, the first PVI case was scheduled at 7:30 am, and three PVI cases were scheduled in a lab day using either the CBA system (Arctic Front™ Cardiac Cryoablation system, Medtronic, Inc.) or an irrigated point-by-point RFA system. The EP lab was modelled fully flexible scheduling: each procedure started as soon as the lab opened or the previous case was over . Operational delays were assumed in the model, including: (1) room turnover time of 20 min and (2) patient delays between 0 and 15 min. No delay associated to the electrophysiologist was assumed in this model. EP lab overtime was counted when PVI procedures extended after 6:00 pm. Figure provides a schematic diagram of the model and Table provides the input values used in the model. The model used lab occupancy time, defined as the time the patient enters the EP lab until the patient exits it. Lab occupancy time includes both the ablation procedure time (sheath in/out) and non-procedural time during which the patient is in the EP lab, but the procedure is not ongoing. Procedure times were extracted from the FREEZE Cohort study and were separated between CBA and RFA. Non-procedural times were derived from the FAST-PVI study and were similar for both CBA and RFA. Gamma curves were used to fit all procedure time probability distribution data. DES model statistics The length of the base case simulation was set to 1000 lab days to get a procedure time distribution in the model that follows the original one used, from the FREEZE Cohort study. A simulation over 1000 lab days results in an overall average procedure time at ± 1.5% from the FREEZE Cohort mean procedure time, with 95% confidence. The 1000 lab days simulation was run repeatedly with different random combinations of number seeds, and the final metrics reported represent the overall average of means from these individual runs to ensure the results were not dependent on an arbitrary set of random numbers. Model outputs were reported as the percent of days leading to overtime, the percent of days with lab time remaining at the end of the planned schedule and the cumulative overtime (hours) over the study period. EP labs have variability in operating parameters such as staff begin and end time, when overtime starts, and room turnover time. For the purposes of a consistent analysis, we have selected parameters generally representative of EP labs and created a simulation model representing efficiency gains in this illustrative EP lab operation. In addition, sensitivity analyses were performed by individually varying single model inputs (while keeping all other inputs constant), including: (1) lab occupancy time, (2) room turnover time, (3) EP lab shift end, and (4) mean patient delay. Variable values used in the sensitivity analyses are provided in Additional file : Table S1. As the DES is stochastic in nature, a separate probabilistic sensitivity analysis was not performed. Descriptive statistics Binary and categorical data are reported as absolute numbers and frequencies, mean and standard deviation or median and quartiles are presented for continuous characteristics of patients treated with CBA and RFA. Procedure time is displayed as a box plot and histogram for both cohorts and the distributions are compared between patient groups by Wilcoxon rank-sum test. The treatment groups are also compared with regard to patient characteristics calculating p-values by Pearson chi-squared test and odds ratios with 95%-confidence intervals for binary variables and using Wilcoxon rank-sum test for ordinal and metrical variables. All tests are two-sided and statistical significance was defined as p < 0.05. The association of patient characteristics with the procedure duration is assessed for CBA and RFA separately by comparing patients above and below a cut-off near the median. Standardized mean differences are calculated showing the direction and magnitude of the imbalance (Austin, Balance diagnostics). The documentation is more than 95% complete with regard to the main variables. No imputation of or adjustment for missing data was performed, with the number of available cases indicated as denominator of rates. Correlation within centre clusters was not taken into consideration. These statistical computations were performed using SAS version 9.4 (Cary, USA). The FREEZE Cohort study (NCT01360008) was a prospective, non-randomized, observational cluster cohort study that compared the effectiveness and safety of RFA and CBA, conducted in 42 experienced hospitals across eight countries . Each centre was assigned as a RFA or a CBA study site, chosen according to the centre’s experience and preference. Patients who received a first ablation for symptomatic paroxysmal or drug-refractory persistent AF were eligible to participate. All enrolled patients were intended to be treated by the designated technique. The study design and the main results of the intention-to-treat analysis were presented in a previous publication . The present analysis evaluates the consequences of differently distributed procedure times between CBA and RFA on schedules and utilisation of resources and staff. Only the procedures undergone in the participating centres across Germany were included as they included all RFA procedures and most of CBA procedures. The calculated metrics and model assumptions were adapted to be illustrative of German hospitals. Patients who underwent a PVI-only approach were included in this analysis; PVI procedures with additional ablation lesions (e.g. linear lesions, ablation of complex fractionated atrial electrograms, right atrial flutter, and ablation at the AV node) were excluded. Focal RF touch-ups or treatment with two sizes of the cryoballoon needed in some CBA cases were not excluded. The treatment groups were defined according to whether CBA or RFA was utilized for the initial ablation, as procedure times should be ascribed to the procedures that were actually completed to reflect clinical reality and to avoid an artificial setting. The crossover rate to any other technique was very low in both groups . A discrete event simulation (DES) model was used to evaluate electrophysiology (EP) lab efficiency based on PVI procedure times reported in the FREEZE Cohort. EP lab efficiency for this analysis is defined as improvements in utilization gained from shorter and more predictable procedure times, which includes fewer cumulative overtime hours, more days where overtime is avoided and more days with remaining time for additional EP lab usage. DES is a method of simulating the behaviour and performance of a real-life process, facility or system, and has been used to model the efficient use of resources in various healthcare settings. These simulations are based on a stochastic time series of individual, granular events representative of realistic occurrences. Individuals and critical resources in the DES are treated distinctly from each other, having unique characteristics and memory, and are drawn from a detailed probabilistic characterization derived from real-world data and experience. SIMUL8 professional version 26.0 (SIMUL8 Corporation) was used for DES in this analysis. This DES aimed to model the impact of operational changes on German EP lab occupancy based on the variability of the ablation procedure start and stop times. In the model, the first PVI case was scheduled at 7:30 am, and three PVI cases were scheduled in a lab day using either the CBA system (Arctic Front™ Cardiac Cryoablation system, Medtronic, Inc.) or an irrigated point-by-point RFA system. The EP lab was modelled fully flexible scheduling: each procedure started as soon as the lab opened or the previous case was over . Operational delays were assumed in the model, including: (1) room turnover time of 20 min and (2) patient delays between 0 and 15 min. No delay associated to the electrophysiologist was assumed in this model. EP lab overtime was counted when PVI procedures extended after 6:00 pm. Figure provides a schematic diagram of the model and Table provides the input values used in the model. The model used lab occupancy time, defined as the time the patient enters the EP lab until the patient exits it. Lab occupancy time includes both the ablation procedure time (sheath in/out) and non-procedural time during which the patient is in the EP lab, but the procedure is not ongoing. Procedure times were extracted from the FREEZE Cohort study and were separated between CBA and RFA. Non-procedural times were derived from the FAST-PVI study and were similar for both CBA and RFA. Gamma curves were used to fit all procedure time probability distribution data. The length of the base case simulation was set to 1000 lab days to get a procedure time distribution in the model that follows the original one used, from the FREEZE Cohort study. A simulation over 1000 lab days results in an overall average procedure time at ± 1.5% from the FREEZE Cohort mean procedure time, with 95% confidence. The 1000 lab days simulation was run repeatedly with different random combinations of number seeds, and the final metrics reported represent the overall average of means from these individual runs to ensure the results were not dependent on an arbitrary set of random numbers. Model outputs were reported as the percent of days leading to overtime, the percent of days with lab time remaining at the end of the planned schedule and the cumulative overtime (hours) over the study period. EP labs have variability in operating parameters such as staff begin and end time, when overtime starts, and room turnover time. For the purposes of a consistent analysis, we have selected parameters generally representative of EP labs and created a simulation model representing efficiency gains in this illustrative EP lab operation. In addition, sensitivity analyses were performed by individually varying single model inputs (while keeping all other inputs constant), including: (1) lab occupancy time, (2) room turnover time, (3) EP lab shift end, and (4) mean patient delay. Variable values used in the sensitivity analyses are provided in Additional file : Table S1. As the DES is stochastic in nature, a separate probabilistic sensitivity analysis was not performed. Binary and categorical data are reported as absolute numbers and frequencies, mean and standard deviation or median and quartiles are presented for continuous characteristics of patients treated with CBA and RFA. Procedure time is displayed as a box plot and histogram for both cohorts and the distributions are compared between patient groups by Wilcoxon rank-sum test. The treatment groups are also compared with regard to patient characteristics calculating p-values by Pearson chi-squared test and odds ratios with 95%-confidence intervals for binary variables and using Wilcoxon rank-sum test for ordinal and metrical variables. All tests are two-sided and statistical significance was defined as p < 0.05. The association of patient characteristics with the procedure duration is assessed for CBA and RFA separately by comparing patients above and below a cut-off near the median. Standardized mean differences are calculated showing the direction and magnitude of the imbalance (Austin, Balance diagnostics). The documentation is more than 95% complete with regard to the main variables. No imputation of or adjustment for missing data was performed, with the number of available cases indicated as denominator of rates. Correlation within centre clusters was not taken into consideration. These statistical computations were performed using SAS version 9.4 (Cary, USA). Patient characteristics In total, 1743 patients enrolled in German hospitals were treated by CBA and 1829 by RFA between April 2011 and February 2016. In CBA group, 138 (7.9%) received PVI plus additional lesions and 382 (20.9%) in RFA group. In RFA group, 67 patients treated with the anatomically-designed PVAC Gold RF catheter were excluded, as this analysis aims to compare point-by-point RFA to CBA. Moreover, 45 (CBA) and 36 (RFA) patients were not available for the analysis because of missing documentation of procedure time. Altogether, 1560 CBA patients and 1344 RFA patients from 30 experienced German centres were included in the analysis. Relevant patient baseline characteristics are shown in Table . The proportion of persistent AF was significantly higher in RFA cohort, and these patients presented with more frequent and more severe symptoms and have more often AF as the current rhythm. Generally, individuals were slightly older, and coronary artery disease, valve disease, and heart failure were more commonly documented as a concomitant disease in this cohort, resulting in a higher risk profile as expressed by the CHA 2 DS 2 -VASc score. On the other hand, CBA patients were more often suffering from hypertensive heart disease. No baseline patient characteristic consistently predicted the duration of RFA or CBA procedure (Additional file : Table S2). Therefore, the analysis of the procedure durations was not adjusted for differences in baseline patient characteristics. Procedural characteristics The cryoablation catheter system (Medtronic, Inc.) was used in all CBA cases, 17% from the first generation (Arctic Front), 78% from the second generation (Arctic Front Advance), and 5% from the third generation (Arctic Front Advance ST). The cryoablation protocol (number of freeze cycles, freeze time, use real-time PV potential recordings) was left to the treating physician. In RFA cases, the manufacturers of the RF catheters were Biosense Webster (77.3%), St. Jude (20.8%), Biotronik (1.3%) and others (0.5%). In RFA and CBA groups, 3D electroanatomical mapping was used in 99.5% and 0.1%, respectively ( p < 0.0001). Intracardiac echocardiography was used in 5.6% and 47.8% of the procedures ( p < 0.0001), X-ray duration lasted 19 and 17 min ( p < 0.001) and the dose-area product was 2187 and 1736 cGy*cm 2 ( p < 0.001) in average, for RFA and CBA procedures respectively. No information was available on the rate of contact-force-sensing catheters in RFA group . The majority of patients were treated without any sedation or under analgo-sedation in CBA and RFA groups (99.7% vs 99.9% respectively, p = 0.53), and only a few patients were treated under endotracheal anaesthesia (0.3% and 0.1% respectively). Figure provides details on the distribution of CBA and RFA procedure durations in the FREEZE Cohort sample. The mean procedure time in CBA cohort was 38 min shorter than in RFA cohort, with smaller standard deviation (122.2 ± 39.4 vs 160.3 ± 53.5 min respectively, p < 0.0001). There was also less variability in procedure times between centres undertaking CBA than those with RFA (Fig. ). Finally, CBA mean procedure time decreased by about an hour over time during the FREEZE Cohort study, whereas RFA mean procedure time seemed fairly steady over the study period (Fig. ). DES model: base case results Results have been consolidated through the repeated run of 1000 lab days simulation for both CBA and RFA. With 3 cases per day simulated for each ablation technique, 3000 PVI using CBA and 3000 using RFA were simulated within the model. A sample of simulated case time distributions is represented in Fig. . The DES model demonstrated that of the 1000 simulated Cryo lab days, 257 (25.7%) days required overtime to complete the three PVI cases. In contrast, 707 (70.7%) of the simulated RF lab days required overtime to complete the three PVI cases. In total, the cumulative overtime associated with CBA was 253 h during the studied period whereas 1285 h of overtime were required to complete RFA PVI procedures. Overall, RFA was associated with a more than five-fold increase of cumulative overtime compared to CBA over the course of 1000 PVI cases. The model also demonstrated that CBA was associated with 478 days with a remaining hour at the end of the EP lab shift, compared to 115 days with a remaining hour at the end of the lab day when using RFA (47.8% vs 11.5% days with one remaining hour, respectively). These results are represented in Fig. . DES model: sensitivity analyses results The sensitivity analyses explored varying the simulation inputs. The results illustrated that the model outputs are most sensitive to procedure time. In all input scenarios, the advantage of CBA as compared to RFA was preserved for all model outputs. All sensitivity analyses and the results are available in the Additional file . In total, 1743 patients enrolled in German hospitals were treated by CBA and 1829 by RFA between April 2011 and February 2016. In CBA group, 138 (7.9%) received PVI plus additional lesions and 382 (20.9%) in RFA group. In RFA group, 67 patients treated with the anatomically-designed PVAC Gold RF catheter were excluded, as this analysis aims to compare point-by-point RFA to CBA. Moreover, 45 (CBA) and 36 (RFA) patients were not available for the analysis because of missing documentation of procedure time. Altogether, 1560 CBA patients and 1344 RFA patients from 30 experienced German centres were included in the analysis. Relevant patient baseline characteristics are shown in Table . The proportion of persistent AF was significantly higher in RFA cohort, and these patients presented with more frequent and more severe symptoms and have more often AF as the current rhythm. Generally, individuals were slightly older, and coronary artery disease, valve disease, and heart failure were more commonly documented as a concomitant disease in this cohort, resulting in a higher risk profile as expressed by the CHA 2 DS 2 -VASc score. On the other hand, CBA patients were more often suffering from hypertensive heart disease. No baseline patient characteristic consistently predicted the duration of RFA or CBA procedure (Additional file : Table S2). Therefore, the analysis of the procedure durations was not adjusted for differences in baseline patient characteristics. The cryoablation catheter system (Medtronic, Inc.) was used in all CBA cases, 17% from the first generation (Arctic Front), 78% from the second generation (Arctic Front Advance), and 5% from the third generation (Arctic Front Advance ST). The cryoablation protocol (number of freeze cycles, freeze time, use real-time PV potential recordings) was left to the treating physician. In RFA cases, the manufacturers of the RF catheters were Biosense Webster (77.3%), St. Jude (20.8%), Biotronik (1.3%) and others (0.5%). In RFA and CBA groups, 3D electroanatomical mapping was used in 99.5% and 0.1%, respectively ( p < 0.0001). Intracardiac echocardiography was used in 5.6% and 47.8% of the procedures ( p < 0.0001), X-ray duration lasted 19 and 17 min ( p < 0.001) and the dose-area product was 2187 and 1736 cGy*cm 2 ( p < 0.001) in average, for RFA and CBA procedures respectively. No information was available on the rate of contact-force-sensing catheters in RFA group . The majority of patients were treated without any sedation or under analgo-sedation in CBA and RFA groups (99.7% vs 99.9% respectively, p = 0.53), and only a few patients were treated under endotracheal anaesthesia (0.3% and 0.1% respectively). Figure provides details on the distribution of CBA and RFA procedure durations in the FREEZE Cohort sample. The mean procedure time in CBA cohort was 38 min shorter than in RFA cohort, with smaller standard deviation (122.2 ± 39.4 vs 160.3 ± 53.5 min respectively, p < 0.0001). There was also less variability in procedure times between centres undertaking CBA than those with RFA (Fig. ). Finally, CBA mean procedure time decreased by about an hour over time during the FREEZE Cohort study, whereas RFA mean procedure time seemed fairly steady over the study period (Fig. ). Results have been consolidated through the repeated run of 1000 lab days simulation for both CBA and RFA. With 3 cases per day simulated for each ablation technique, 3000 PVI using CBA and 3000 using RFA were simulated within the model. A sample of simulated case time distributions is represented in Fig. . The DES model demonstrated that of the 1000 simulated Cryo lab days, 257 (25.7%) days required overtime to complete the three PVI cases. In contrast, 707 (70.7%) of the simulated RF lab days required overtime to complete the three PVI cases. In total, the cumulative overtime associated with CBA was 253 h during the studied period whereas 1285 h of overtime were required to complete RFA PVI procedures. Overall, RFA was associated with a more than five-fold increase of cumulative overtime compared to CBA over the course of 1000 PVI cases. The model also demonstrated that CBA was associated with 478 days with a remaining hour at the end of the EP lab shift, compared to 115 days with a remaining hour at the end of the lab day when using RFA (47.8% vs 11.5% days with one remaining hour, respectively). These results are represented in Fig. . The sensitivity analyses explored varying the simulation inputs. The results illustrated that the model outputs are most sensitive to procedure time. In all input scenarios, the advantage of CBA as compared to RFA was preserved for all model outputs. All sensitivity analyses and the results are available in the Additional file . Procedure times illustrated in this report from the FREEZE Cohort study demonstrate that PVI using CBA is a significantly shorter, more predictable and more efficient procedure than ablation using RFA. Furthermore, procedure times were more consistent across centres with CBA and so the impact of variability on EP lab efficiency was less pronounced. Also, CBA trend of decreasing procedure time over years during FREEZE Cohort study period was striking, and this characteristic is still observed today, even after many years of CBA use . These findings are consistent with previously published studies . Recent studies have used DES to model AF ablation. Kowalski used lab occupancy times from the VALUE PVI trial to assess the economic impact of procedural efficiencies between CBA and RFA in the United States (US). In this analysis, CBA for paroxysmal AF was associated with a reduction of 36.2% in days with overtime, 92.7% less cumulative overtime hours, and an increase of 46.7% in days with time for an additional EP lab usage. Monnickendam also compared the impact on average procedure consumptions of AF ablation with CBA and RFA via a DES model, using data from a single surgical centre in Belgium. The study demonstrated that 28.6% of RFA procedures are over 30 min longer than the median duration, compared to only 4.4% of CBA procedures. The authors concluded that the choice of AF ablation technology has a significant impact on the operating room efficiency and should be considered in decision-making. Kowalski evaluated EP lab utilization with a DES model using the single-arm STOP Persistent AF trial data in the US, and demonstrated that using CBA to treat persistent AF patients confers EP lab efficiencies that can support an additional third PVI case in a lab day, as opposed to the standard of two in the US. The FREEZE Cohort analysis results are consistent with these previously published DES analyses: hospitals benefit from CBA through efficient use of the EP lab resources, including remaining time for physicians and staff, for the same volume of patients treated. While the extra hours preserved by using CBA instead of RFA are represented here by avoidance of overtime, it may not be overtime avoidance for every centre. However, completing PVI cases more quickly is likely to lead to meaningful advantages, whether it be for avoiding overtime or freeing up lab capacities. With more efficient CBA procedures particularly with streamlined protocols and the latest generation of the cryoballoon , there is opportunity for more than 3 PVI procedures to be undergone during an EP lab day. This analysis used German centers procedural data and applied an illustrative EP lab case. The efficiency profile of CBA proved by the present and previously published studies provides a reliable trend to other hospitals with a fully flexible scheduling, from geographies beyond Germany. Unpredictable and long duration PVI procedures cause overtime which can be associated with an under-utilization of EP lab time and less optimal use of staff resources. Moreover, overtime can be a human resource issue for some hospitals, where the scheduled staff shifts cannot cover the overtime hours, particularly given the low physician and nurse to hospital bed ratios in Germany . The additional hours of overtime with RFA compared to CBA could also represent incremental costs to the hospitals that remunerate overtime hours at a higher tarif than usual. Finally, overtime may contribute to a high turnover of EP lab staff and reduce the efficiency and effectiveness of the workforce in delivering high quality and safe care . In addition to ensuring optimal patient care, employee satisfaction is an important consideration for the employers in a context of shortage of skilled health workers . A previously published analysis of FIRE and ICE trial (NCT01490814) estimated that CBA was associated with meaningful and persistent cost-savings across multiple different healthcare systems, including a total cost-saving of €245,000 during the trial period for German system, due to fewer rehospitalizations, cardioversions and repeat ablations compared to RFA . The FREEZE Cohort study also demonstrated fewer all-cause rehospitalizations and repeat ablations with CBA compared to RFA in real-world settings . Beyond substantial cost-savings for German payers, CBA is also a time-efficient procedure to treat AF patients, with an efficient use of EP lab resources in hospitals in the German healthcare setting. Given the increasing prevalence of AF, it is important that the clinical community increases access to AF ablation and delivers rhythm control therapies in a timely fashion, particularly, if one considers that today only 5% of all AF patients undergo catheter ablation. The EAST-AFNET trial demonstrated that early rhythm control improves patient outcomes . Moreover, the EARLY-AF , STOP AF First and CRYO-First trials illustrate that an early AF ablation with CBA is superior to anti-arrhythmic drug therapy in drug-naïve patients regarding recurrence of atrial tachyarrhythmia after one year. This evidence suggests that the demand for AF ablation is likely to increase even faster than AF prevalence. This additional strain on the healthcare system will reinforce the need to streamline all aspects of AF ablation procedures in order to ensure that patients are treated as efficiently, effectively and safely as possible. Limitations Firstly, the FREEZE Cohort study was not randomized, and patient groups were different at the baseline for some parameters. However, no baseline patient characteristic consistently predicted the duration of RFA or CBA procedure. Also, basing DES analysis on a real-world settings study data provides an added value for simulation results robustness. Secondly, there was no collected data from the FREEZE Cohort study to undertake direct comparisons of the measures predicted by the model: cumulative overtime, days with overtime and days with a remaining hour. Although, DES modelling benefit is to understand impacts that are not easily measured in the real world. This analysis did not address additional measures of EP lab efficiency, such as staffing levels and equipment logistics. The specific gains in efficiency for centres that have different operating parameters than what was chosen for this analysis might deviate, but the sensitivity analyses demonstrate that the gains are likely to be preserved in some form for all reasonable variations of the operating parameters. Thirdly, the FREEZE cohort study did not collect different levels of operators involved in the procedure. Level of experience can be a factor for the duration of the procedure, with less experience resulting in longer procedure times. This effect was addressed in the sensitivity analysis; the impact of reasonable variations in procedure times is explored in the Additional file : Fig. S4. The main results of the study remained true even with this variation. Fourthly, only German centres procedural data were included in this analysis to complete the simulation of one representative EP lab case. The vast majority of patients in both groups were treated under analgo-sedation or without any sedation. It remains unclear if the results can be transferred to settings with general anaesthesia. In addition, there are other procedure details that were not explicitly taken into account in the model. However, the variability in procedure times due to these details were reflected in the model, and the efficiency profile of CBA in this model is consistent with previously published studies . Thus, this report results are expected to provide a reliable trend to other hospitals with a fully-flexible scheduling, from geographies beyond Germany. Also, radiation exposure was not included in the DES model, but the associated results from FREEZE Cohort were previously published . Procedure times were collected from the FREEZE Cohort study, and are expected to be lower today because of the current efficiency of AF ablation procedures and based on the advanced ablation technology. The number of PVI per EP lab day, directly linked to the EP lab time per case, is also expected to be higher in most of the centres, however the EP lab efficiency benefit of CBA over RFA is expected to remain. Finally, no information on the rate of contact-force-sensing catheters applied in the RF group, which might potentially influence procedural parameters as well as acute and clinical outcomes, can be provided in our analysis. Firstly, the FREEZE Cohort study was not randomized, and patient groups were different at the baseline for some parameters. However, no baseline patient characteristic consistently predicted the duration of RFA or CBA procedure. Also, basing DES analysis on a real-world settings study data provides an added value for simulation results robustness. Secondly, there was no collected data from the FREEZE Cohort study to undertake direct comparisons of the measures predicted by the model: cumulative overtime, days with overtime and days with a remaining hour. Although, DES modelling benefit is to understand impacts that are not easily measured in the real world. This analysis did not address additional measures of EP lab efficiency, such as staffing levels and equipment logistics. The specific gains in efficiency for centres that have different operating parameters than what was chosen for this analysis might deviate, but the sensitivity analyses demonstrate that the gains are likely to be preserved in some form for all reasonable variations of the operating parameters. Thirdly, the FREEZE cohort study did not collect different levels of operators involved in the procedure. Level of experience can be a factor for the duration of the procedure, with less experience resulting in longer procedure times. This effect was addressed in the sensitivity analysis; the impact of reasonable variations in procedure times is explored in the Additional file : Fig. S4. The main results of the study remained true even with this variation. Fourthly, only German centres procedural data were included in this analysis to complete the simulation of one representative EP lab case. The vast majority of patients in both groups were treated under analgo-sedation or without any sedation. It remains unclear if the results can be transferred to settings with general anaesthesia. In addition, there are other procedure details that were not explicitly taken into account in the model. However, the variability in procedure times due to these details were reflected in the model, and the efficiency profile of CBA in this model is consistent with previously published studies . Thus, this report results are expected to provide a reliable trend to other hospitals with a fully-flexible scheduling, from geographies beyond Germany. Also, radiation exposure was not included in the DES model, but the associated results from FREEZE Cohort were previously published . Procedure times were collected from the FREEZE Cohort study, and are expected to be lower today because of the current efficiency of AF ablation procedures and based on the advanced ablation technology. The number of PVI per EP lab day, directly linked to the EP lab time per case, is also expected to be higher in most of the centres, however the EP lab efficiency benefit of CBA over RFA is expected to remain. Finally, no information on the rate of contact-force-sensing catheters applied in the RF group, which might potentially influence procedural parameters as well as acute and clinical outcomes, can be provided in our analysis. FREEZE Cohort analysis confirms in real-world settings that CBA is faster and more predictable than RFA, and enables improvements in EP lab efficiency. The DES of the FREEZE Cohort data demonstrated the benefits of AF ablation procedures using the cryoballoon system, including fewer cumulative overtime hours, more days where overtime is avoided and more days with remaining time for the staff or for any EP lab usage. Additional file 1 . Figure S1 . Procedure time distribution for Cryoballoon ablation (CBA) procedures (FREEZE Cohort, German CBA centres). Figure S2 . Procedure time distribution for point-by-point radiofrequency ablation (RFA) procedures (FREEZE Cohort, German RFA centres). Figure S3 . Plot gamma curves for procedure time distribution (FREEZE Cohort, German centres). Table S1 . DES model parameters used for the study (Base Case) and the sensitivity analyses. Figure S4 . Sensitivity analyses results. Table S2 . Patients baseline characteristics below and above median procedure time for CBA and RFA. |
Participatory development and implementation of inclusive digital health communication on COVID-19 with homeless people | bd06257d-1f6e-43ce-976e-344fee8e6ece | 9687377 | Health Communication[mh] | People experiencing homelessness (PEH) are disproportionally affected by the COVID-19 pandemic. Precarious living conditions on the street, in encampments and cramped shelters, limited access to hygienic supplies and prevention measures, stigmatization, marginalization from social, political and economic resources as well as exclusion from health services result in high rates of underlying health conditions and a high risk of SARS-CoV-2-infection . The prevalence among homeless individuals may be similar to that found in the general population, however, the increased risk of outbreaks with high infections rates has to be considered . Also, social determinants and pre-existing health conditions place PEH at higher risk of severe COVID-19 infection . However, studies to properly assess the outcome of SARS-CoV-2 infection in PEH are still required . Since the beginning of the COVID-19 pandemic, the need to consider the living conditions of PEH when implementing measures of infection control and prevention (IPC) has been addressed by the German national working group on homelessness services (Bundesarbeitsgemeinschaft Wohnungslosenhilfe, BAG W) . In July 2021, the Robert Koch Institute, the national public health institute in Germany, published targeted recommendations for COVID-19 in the context of homelessness that were developed together with experts from the field . Many good practice solutions were implemented locally to protect PEH during the pandemic, such as provision of adequate isolation and quarantine options addressing possible complex needs of PEH, 24/7 accommodation with single rooms, regular voluntary universal testing for SARS-CoV-2, and mobile vaccination campaigns . However, in many places, isolation, access to vaccination and testing as well as targeted information remained a challenge. This is particularly critical because it is known from past respiratory viral outbreaks that these control measures are crucial in managing epidemics or pandemics . In our opinion, PEH in Germany are to date not sufficiently addressed in the pandemic response including information campaigns. With the onset of the pandemic, digitalization was discussed more publicly than before as it affected everyone to a substantial extent. Being digitalized took on a new relevance, as it was, for example, a prerequisite for the digital EU certificate, which allowed unrestricted access to public buildings, use of transport and freedom to travel between international borders . Multiple problems faced by PEH result from their precarious socioeconomic situation which also affects the ability to maintain a digital device and to have access to internet-based services . Digital inequalities result in further social exclusion as it limits career opportunities, represents a barrier to maintaining social and service-related contacts, causes financial hardship and are a determinant of health . At the same time, digitalization can be an opportunity for better social inclusion . In a previous COVID-19 project among PEH, we identified the necessity to address language barriers, to include digital information formats, and to use participatory approaches considering homeless people's needs and life situation . This follow-up project aimed a participatory development of inclusive health communication on COVID-19 to strengthen options for IPC for PEH. We describe the development, implementation, and evaluation of targeted digital as well as non-digital (hybrid) health communication material (videos and posters). This project was conducted over a period of 11 months, from October 2020 to August 2021 in Berlin, Germany. According to the principle that in participatory projects participants should benefit directly from the research process , the perspective of PEH represented the basis for all project steps, and all PEH had decision-making power and were paid for their work effort. Study team and recruitment of community partners The study was initiated and supervised by two experts from the fields of medicine and public health. The study team also included a clinician, a health scientist, two social workers, two student assistants and a professional communications designer. Four of the team members had long-term work experience with PEH. The PEH who participated as community partners were actively involved in the planning, production, evaluation and decision-making processes. They were the protagonists with one main actor for the videos and 12 further actors for portraits (vignettes) in the videos and posters. Three PEH had reservations to be filmed or photographed, and were involved in translations, sound recordings or evaluation rounds. Community partners were recruited in a social facility for PEH operated by the Berliner Stadtmission in Berlin. The facility included a 24/7 shelter, a medical outpatient clinic and a clothing store, where people could easily be approached during the day and in the waiting areas. Some members of the study team were already known to the PEH from their (voluntary) work at this facility, which formed the trust base to approach people directly. We showed a short film clip (mood board) to the PEH in order to introduce them to the project. The communication designer created the clip especially for recruitment purposes. Inclusion criteria were age above 18 years, current or previous homelessness and written informed consent. We aimed at partnering with PEH from different age groups, genders, (dis-)abilities, languages and country of origin to reflect the diverse image of PEH in Berlin and to create material that is easier to identify with. We consulted the following stakeholders for external supervision: the German national working group on homelessness services (BAG W), a self-organization (Selbstvertretung wohnungsloser Menschen e. V.), the coordination group for homeless shelters in Berlin (Koordinierungsstelle Berliner Kältehilfe), the Robert Koch Institute as well as staff of the Berliner Stadtmission . Videos Scripts in German language were created for two videos with general information about COVID-19 and SARS-CoV-2 testing. Expertise of the medical specialists and social workers and the experience of the community partners were combined to gain a better understanding of the health, personal needs and coping strategies of PEH during the pandemic. The scripts were edited in a multi-step revision process. After they were shared with and adapted by all stakeholders, the main protagonist translated them to a simple and clear language. Native speakers translated the scripts into four other languages that were identified in the previous project we performed . Quality control was performed by professional translators. The main actor speaks German in the videos. Three PEH and one professional actor have dubbed the clips into Russian, Polish, Romanian and English. Posters Information posters with precise key messages on access to vaccination were developed and provided in a digital and printed version to support the public COVID-19 vaccination campaign for PEH that started in Berlin in March 2021. To identify main questions or concerns of PEH regarding vaccinations, we consulted PEH and staff of service providers, as well as our stakeholders. Participation and participatory decision making The shooting locations were chosen together with the protagonists. The aim was to find locations that would create a pleasant working environment and at the same time provide images that PEH could identify with and which would not reproduce stereotypes. For the whole process, the individual needs of the protagonists were considered. This included for example a transport service if needed, food and drinks, as well as access to barrier-free sanitary facilities at all locations. The selection and editing of the video material and photo motifs took place during several feedback rounds with community partners. The videos were watched together several times on a screen in the common room of the homeless shelter. Attending community partners and other PEH were asked for their opinion and criticism, e.g., about the content and the locations. Stakeholders who were not directly involved in the production were also asked for feedback. Dissemination of the information material On April 2021, a website was created in order to provide open access to the videos and posters, as well as to provide further information about the project . Furthermore, the material was disseminated through various social media channels (Twitter, Facebook and Instagram of the participating institutions). The printed posters were sent to all homeless service providers that were part of the Kältehilfe Network in Berlin (shelters, soup kitchens, warm rooms, medical facilities for people without health insurance and counseling centers). The German national working group on homelessness services informed service providers all over Germany through their network about the project and the offer to order the posters free of charge. Evaluation and data analysis All institutions that received material were listed. A telephone survey with randomly selected homeless service providers who had received the printed posters was conducted. Recruitment for the survey took place via email. After written informed consent, a semi-structured telephone interview was conducted for 15 min addressing the practical implementation and perception of the posters and the videos. A social worker interviewed PEH in two shelters of the Berliner Stadtmission to determine how the material was perceived by PEH. Participants were randomly approached during the service hours of the shelters. After written informed consent, a semi-structured interview was conducted for 20 min. The data analysis was based on written notes taken during the qualitative phone interviews and face to face interviews. A systematic qualitative content analysis was undertaken . After reviewing the data material, it was coded by an inductive procedure and summarized in categories. Furthermore, the usage of the project website with the number of video views was measured. Ethics This study was approved by the Ethics Committee of the Charité – Universitätsmedizin Berlin (No.: EA2/168/21). All PEH who contributed to the implementation of the project were paid for their work. The study was explained to PEH in the preferred language, and written informed consent was provided for participation. The scope and time frame of the project were transparently communicated, as well as the possibility to withdraw participation at any point of the project without repercussions. As PEH are a particularly vulnerable group, privacy, data security and a familiar atmosphere (to avoid any discomfort) were taken into careful consideration during the interviews. The study was initiated and supervised by two experts from the fields of medicine and public health. The study team also included a clinician, a health scientist, two social workers, two student assistants and a professional communications designer. Four of the team members had long-term work experience with PEH. The PEH who participated as community partners were actively involved in the planning, production, evaluation and decision-making processes. They were the protagonists with one main actor for the videos and 12 further actors for portraits (vignettes) in the videos and posters. Three PEH had reservations to be filmed or photographed, and were involved in translations, sound recordings or evaluation rounds. Community partners were recruited in a social facility for PEH operated by the Berliner Stadtmission in Berlin. The facility included a 24/7 shelter, a medical outpatient clinic and a clothing store, where people could easily be approached during the day and in the waiting areas. Some members of the study team were already known to the PEH from their (voluntary) work at this facility, which formed the trust base to approach people directly. We showed a short film clip (mood board) to the PEH in order to introduce them to the project. The communication designer created the clip especially for recruitment purposes. Inclusion criteria were age above 18 years, current or previous homelessness and written informed consent. We aimed at partnering with PEH from different age groups, genders, (dis-)abilities, languages and country of origin to reflect the diverse image of PEH in Berlin and to create material that is easier to identify with. We consulted the following stakeholders for external supervision: the German national working group on homelessness services (BAG W), a self-organization (Selbstvertretung wohnungsloser Menschen e. V.), the coordination group for homeless shelters in Berlin (Koordinierungsstelle Berliner Kältehilfe), the Robert Koch Institute as well as staff of the Berliner Stadtmission . Scripts in German language were created for two videos with general information about COVID-19 and SARS-CoV-2 testing. Expertise of the medical specialists and social workers and the experience of the community partners were combined to gain a better understanding of the health, personal needs and coping strategies of PEH during the pandemic. The scripts were edited in a multi-step revision process. After they were shared with and adapted by all stakeholders, the main protagonist translated them to a simple and clear language. Native speakers translated the scripts into four other languages that were identified in the previous project we performed . Quality control was performed by professional translators. The main actor speaks German in the videos. Three PEH and one professional actor have dubbed the clips into Russian, Polish, Romanian and English. Information posters with precise key messages on access to vaccination were developed and provided in a digital and printed version to support the public COVID-19 vaccination campaign for PEH that started in Berlin in March 2021. To identify main questions or concerns of PEH regarding vaccinations, we consulted PEH and staff of service providers, as well as our stakeholders. The shooting locations were chosen together with the protagonists. The aim was to find locations that would create a pleasant working environment and at the same time provide images that PEH could identify with and which would not reproduce stereotypes. For the whole process, the individual needs of the protagonists were considered. This included for example a transport service if needed, food and drinks, as well as access to barrier-free sanitary facilities at all locations. The selection and editing of the video material and photo motifs took place during several feedback rounds with community partners. The videos were watched together several times on a screen in the common room of the homeless shelter. Attending community partners and other PEH were asked for their opinion and criticism, e.g., about the content and the locations. Stakeholders who were not directly involved in the production were also asked for feedback. On April 2021, a website was created in order to provide open access to the videos and posters, as well as to provide further information about the project . Furthermore, the material was disseminated through various social media channels (Twitter, Facebook and Instagram of the participating institutions). The printed posters were sent to all homeless service providers that were part of the Kältehilfe Network in Berlin (shelters, soup kitchens, warm rooms, medical facilities for people without health insurance and counseling centers). The German national working group on homelessness services informed service providers all over Germany through their network about the project and the offer to order the posters free of charge. All institutions that received material were listed. A telephone survey with randomly selected homeless service providers who had received the printed posters was conducted. Recruitment for the survey took place via email. After written informed consent, a semi-structured telephone interview was conducted for 15 min addressing the practical implementation and perception of the posters and the videos. A social worker interviewed PEH in two shelters of the Berliner Stadtmission to determine how the material was perceived by PEH. Participants were randomly approached during the service hours of the shelters. After written informed consent, a semi-structured interview was conducted for 20 min. The data analysis was based on written notes taken during the qualitative phone interviews and face to face interviews. A systematic qualitative content analysis was undertaken . After reviewing the data material, it was coded by an inductive procedure and summarized in categories. Furthermore, the usage of the project website with the number of video views was measured. This study was approved by the Ethics Committee of the Charité – Universitätsmedizin Berlin (No.: EA2/168/21). All PEH who contributed to the implementation of the project were paid for their work. The study was explained to PEH in the preferred language, and written informed consent was provided for participation. The scope and time frame of the project were transparently communicated, as well as the possibility to withdraw participation at any point of the project without repercussions. As PEH are a particularly vulnerable group, privacy, data security and a familiar atmosphere (to avoid any discomfort) were taken into careful consideration during the interviews. Participatory production Sixteen PEH participated actively in the production of the information material. All participants were experiencing homelessness at the time of the study. Ten protagonists were recruited in the 24/7 shelter, 3 in the clothing store and 3 were recruited in the streets during the shooting days. Four of the participants were women and 12 were men, aged between 25 and 75 years from 6 countries speaking 8 different languages, two of them were in a wheelchair. We produced two multilingual COVID-19 information videos under the slogan “We keep Corona off the streets”. The protagonist changed some parts of the script for a better understanding. For example, regarding information about the opioid substitution programme in the quarantine facility, “ substitution is provided ” was replaced by “ you can stay there, also if you consume ”. The 5-day shooting of the videos took place at 15 locations. The videos were produced with 13 protagonists. Six protagonists chose spots on the grounds of the Berliner Stadtmission or in the near vicinity as shooting locations, while 7 chose spots around the main railway station, a nearby park and public places. The first video clip (duration 3 min 13 s) contained general information about COVID-19 . It explained the transmission modes of SARS-CoV-2, symptoms, increased risk of infection among PEH and strategies for self-protection (hygiene measures). The second video clip (duration 1 min 14 s) contained details about COVID-19 testing and the proceeding after a positive test result for PEH who lack the possibility of self-isolation at home . Thus, the video talks about the possibility of isolation in quarantine accommodations that consider the needs of PEH in a sensitive way. In the second step, we designed multilingual posters with seven different motifs . With two versions, we have covered a total of nine languages identified as most relevant in a previous local project : - 1st language version: German, Polish, English, Farsi, Russian - 2nd language version: German, Romanian, Bulgarian, Arabic, French Out of the 13 protagonists in the videos, three participated in the production of the posters. Two more were recruited in the 24/7 shelter. Portraits were taken on two shooting days. The main questions or uncertainties about the SARS-CoV-2 vaccination among PEH were identified through discussions with our community partners and staff of homeless service providers. These were having access to vaccination without a permanent address, documents or health insurance, as well as implications of drug use. Accordingly, the posters were designed with the slogan “ You can get vaccinated. Even with no fixed address, no health insurance, no documents. Get informed in the social or health care services you know ”. In another poster version we included the sentence “ Even if you use drugs ”. The posters that address drug use do not contain portraits of PEH to avoid stigmatization . As a result of the feedback rounds, the hybrid nature of the posters was extended by including a QR-code linking the poster with the project website. Dissemination The videos were launched on February 11, 2021 during a hybrid (online and in presence) event. It was screened in the homeless shelter of Berliner Stadtmission under COVID-19 hygiene measures to allow community partners without internet access or mobile devices to participate. Over 100 participants joined online from various fields such as homeless services, politics, research and community. By May 2022, a total of 1,754 posters were sent to 163 institutions and facilities in 53 cities within Germany . Whereas a set of posters was automatically sent out to all 91 institutions that were part of the Berlin Kältehilfe list, the others (72) received the material upon request. The institutions included facilities for PEH such as clothing facilities, day care centers, consulting services, hygiene facilities, medical facilities and night shelters (146), facilities for drug users (9), facilities for refugees (2) and municipalities and public facilities such as a library and public authorities (6). The distribution period lasted from February 2021 until May 2022. The dissemination via the social media channel of the Charité – Universitätsmedizin Berlin took place in February 2021. Between February 8, 2021 and May 31, 2022, the videos have been viewed 2,064 times via the project website. We registered peaks in the numbers of requests of the posters at the beginning of the vaccination campaigns in spring 2021 and then again in winter 2021. At that time, the booster vaccinations started, the night shelters opened for winter season, and the institutions were informed about the material by email. Evaluation Out of 20 homeless service providers that were invited to the evaluation, 12 agreed to participate in a telephone interview which were conducted by two of our study team members in August 2021. At the same time, another team member interviewed 8 PEH on 2 days in front of a shelter and in a day center. All were experiencing homelessness at the time of the study. Out of the notes that were taken during the interviews, thirteen categories of different topics were identified . Not all categories were addressed by everyone. There was a general appreciation of how the information was presented in terms of acceptability and sensitivity toward the targeted population. All service providers approved that the posters were widely used for the vaccination campaigns. All 3 respondents who knew the videos considered them helpful for providing targeted information and allowing PEH to feel addressed directly. However, the streaming of the videos in the services was reported to be difficult due to lack of digital devices. Only 1 institution reported to have used the videos within the counseling context. Eight out of 12 service providers pointed out the digital challenges for PEH, e.g., owning and maintaining a mobile or smart phone, and the lacking digital infrastructure in services for PEH. It was acknowledged that digital tools and offers can be a chance to provide better health and social care for PEH, also those living in hidden homelessness. All respondents spoke about the lack of targeted information material for PEH in general. It was suggested to address a wide range of further topics with targeted digital information material. Out of 8 PEH, 3 had seen the posters in relevant facilities of homelessness services in Berlin. None had seen the videos before. Participants appreciated that PEH were protagonists of the videos and posters, but even more diversity would have been appreciated. They could identify with the material and pointed out that mentioning consuming drugs and alcohol was crucial. It was reported that the posters encouraged PEH to think about a vaccination. Precise information on vaccination offers in the respective locations would have been helpful. Both, dissemination of information through posters and videos was appreciated. It was pointed out, that visually and auditorily appealing videos could be suitable especially for illiterate people. Respondents appreciated the idea of disseminating information via videos. One person addressed the digital gap, saying that it was only useful if you had the convenience of owning a digital device. Sixteen PEH participated actively in the production of the information material. All participants were experiencing homelessness at the time of the study. Ten protagonists were recruited in the 24/7 shelter, 3 in the clothing store and 3 were recruited in the streets during the shooting days. Four of the participants were women and 12 were men, aged between 25 and 75 years from 6 countries speaking 8 different languages, two of them were in a wheelchair. We produced two multilingual COVID-19 information videos under the slogan “We keep Corona off the streets”. The protagonist changed some parts of the script for a better understanding. For example, regarding information about the opioid substitution programme in the quarantine facility, “ substitution is provided ” was replaced by “ you can stay there, also if you consume ”. The 5-day shooting of the videos took place at 15 locations. The videos were produced with 13 protagonists. Six protagonists chose spots on the grounds of the Berliner Stadtmission or in the near vicinity as shooting locations, while 7 chose spots around the main railway station, a nearby park and public places. The first video clip (duration 3 min 13 s) contained general information about COVID-19 . It explained the transmission modes of SARS-CoV-2, symptoms, increased risk of infection among PEH and strategies for self-protection (hygiene measures). The second video clip (duration 1 min 14 s) contained details about COVID-19 testing and the proceeding after a positive test result for PEH who lack the possibility of self-isolation at home . Thus, the video talks about the possibility of isolation in quarantine accommodations that consider the needs of PEH in a sensitive way. In the second step, we designed multilingual posters with seven different motifs . With two versions, we have covered a total of nine languages identified as most relevant in a previous local project : - 1st language version: German, Polish, English, Farsi, Russian - 2nd language version: German, Romanian, Bulgarian, Arabic, French Out of the 13 protagonists in the videos, three participated in the production of the posters. Two more were recruited in the 24/7 shelter. Portraits were taken on two shooting days. The main questions or uncertainties about the SARS-CoV-2 vaccination among PEH were identified through discussions with our community partners and staff of homeless service providers. These were having access to vaccination without a permanent address, documents or health insurance, as well as implications of drug use. Accordingly, the posters were designed with the slogan “ You can get vaccinated. Even with no fixed address, no health insurance, no documents. Get informed in the social or health care services you know ”. In another poster version we included the sentence “ Even if you use drugs ”. The posters that address drug use do not contain portraits of PEH to avoid stigmatization . As a result of the feedback rounds, the hybrid nature of the posters was extended by including a QR-code linking the poster with the project website. The videos were launched on February 11, 2021 during a hybrid (online and in presence) event. It was screened in the homeless shelter of Berliner Stadtmission under COVID-19 hygiene measures to allow community partners without internet access or mobile devices to participate. Over 100 participants joined online from various fields such as homeless services, politics, research and community. By May 2022, a total of 1,754 posters were sent to 163 institutions and facilities in 53 cities within Germany . Whereas a set of posters was automatically sent out to all 91 institutions that were part of the Berlin Kältehilfe list, the others (72) received the material upon request. The institutions included facilities for PEH such as clothing facilities, day care centers, consulting services, hygiene facilities, medical facilities and night shelters (146), facilities for drug users (9), facilities for refugees (2) and municipalities and public facilities such as a library and public authorities (6). The distribution period lasted from February 2021 until May 2022. The dissemination via the social media channel of the Charité – Universitätsmedizin Berlin took place in February 2021. Between February 8, 2021 and May 31, 2022, the videos have been viewed 2,064 times via the project website. We registered peaks in the numbers of requests of the posters at the beginning of the vaccination campaigns in spring 2021 and then again in winter 2021. At that time, the booster vaccinations started, the night shelters opened for winter season, and the institutions were informed about the material by email. Out of 20 homeless service providers that were invited to the evaluation, 12 agreed to participate in a telephone interview which were conducted by two of our study team members in August 2021. At the same time, another team member interviewed 8 PEH on 2 days in front of a shelter and in a day center. All were experiencing homelessness at the time of the study. Out of the notes that were taken during the interviews, thirteen categories of different topics were identified . Not all categories were addressed by everyone. There was a general appreciation of how the information was presented in terms of acceptability and sensitivity toward the targeted population. All service providers approved that the posters were widely used for the vaccination campaigns. All 3 respondents who knew the videos considered them helpful for providing targeted information and allowing PEH to feel addressed directly. However, the streaming of the videos in the services was reported to be difficult due to lack of digital devices. Only 1 institution reported to have used the videos within the counseling context. Eight out of 12 service providers pointed out the digital challenges for PEH, e.g., owning and maintaining a mobile or smart phone, and the lacking digital infrastructure in services for PEH. It was acknowledged that digital tools and offers can be a chance to provide better health and social care for PEH, also those living in hidden homelessness. All respondents spoke about the lack of targeted information material for PEH in general. It was suggested to address a wide range of further topics with targeted digital information material. Out of 8 PEH, 3 had seen the posters in relevant facilities of homelessness services in Berlin. None had seen the videos before. Participants appreciated that PEH were protagonists of the videos and posters, but even more diversity would have been appreciated. They could identify with the material and pointed out that mentioning consuming drugs and alcohol was crucial. It was reported that the posters encouraged PEH to think about a vaccination. Precise information on vaccination offers in the respective locations would have been helpful. Both, dissemination of information through posters and videos was appreciated. It was pointed out, that visually and auditorily appealing videos could be suitable especially for illiterate people. Respondents appreciated the idea of disseminating information via videos. One person addressed the digital gap, saying that it was only useful if you had the convenience of owning a digital device. The participatory approach involves all levels of knowledge A key factor for the realization of the videos and posters was the set-up of an interdisciplinary team together with community partners and thus bridging the gap between research, practice and community. Of particular significance were good contacts of the study team with homeless service providers and PEH. Furthermore, it was crucial to involve various local and national stakeholders right from the beginning of the project for advice, support and distribution of the materials. The participatory approach enables active generation of knowledge together with practice and communities . Accordingly, we addressed hierarchies transparently and valued PEH's own life experiences as equivalent to knowledge from professionals within the fields of social work and public health. A challenge was to deal with poverty and precarious living conditions in a sensitive way. Poverty was neither to be tabooed nor trivialized. The goal was to show a realistic picture of the social system and living environment of PEH without reproducing stereotypes or exposing people. The image and portrayal of homelessness in times of the pandemic was therefore to be determined primarily by the community partners themselves. The main protagonist decided to participate by stating “ I think I'm someone people accept ”. In front of the camera, most PEH placed emphasis on a proud attitude showing an active and upright posture. For the evaluation, 8 PEH and 12 service providers were interviewed. They acknowledged the sensitive implementation of the project. The consideration of the concerns and the diverse presentation of PEH was perceived as particularly important. In this manuscript we have chosen the term “people experiencing homelessness” because it presents homelessness as one aspect among others and is less generalizing than the term “homeless people”. Participation and its limitations To enable participation according to the needs of the community partners, work conditions were defined and arranged together. For example, the main protagonist's condition for participation was the assurance of permanent access to barrier-free sanitary facilities. He knew from personal experience that this would be a main challenge, especially during the pandemic when sanitary facilities were even less available for PEH. This highlights one of the fundamental problems that people living in the streets face on a daily basis. For some people there were barriers that made participation difficult or impossible such as hidden homelessness, illegalization, and precarious employment. We have tried to enable safe participation for them as well. For some, a solution was to participate by dubbing the videos, translating the scripts or by attending the feedback sessions. Others chose to not participate altogether. Our impression was that it has been easier for people to participate if there was pre-existing contact with project staff or other PEH who had already taken part. In regard to the evaluation, we found it easier to find interview partners in the day center which provided a safe setting for the interview compared to the recruitment in front of the shelter where the interviews were performed outdoors. It is crucial to be aware of and respect the right to non-participation. We did not collect socio demographic data from the PEH who participated in the production and evaluation of the materials. All were experiencing homelessness at the time of the study. Some had shelter available. Similar to varying infection risks, differences in the participation and responses between sheltered and unsheltered individuals are possible, but this could be not further analyzed in this study . Impact of the information material Despite the mainly positive feedback in the evaluation, the impact of the videos could only be verified to a limited extent. Most of the homeless service providers were unable to show the videos on their premises, due to technical and spatial limitations. The videos and posters were widely distributed via social media and email lists. It remains unclear to what extent they reached the actual target group. However, considering the important role that social media has in shaping people's state of information and attitudes toward public health interventions such as vaccination campaigns, efforts should be increased to utilize these means of communication . The posters seem to have been particularly useful for the vaccination campaign according to the feedback of service providers and the number of poster-orders throughout Germany. Addressing the specific questions and concerns of PEH—e.g., having access to vaccination without a permanent address, documents or health insurance, as well as implications of drug use—may be one approach to increase vaccination coverage . The type of institutions that ordered the posters reflect the broad range of services that address the complex needs of PEH . Among them were services targeting people without official documents (e.g., passport, citizenship, health insurance), people living in poverty, people using drugs, as well as services exclusively provided for women. The way how people are approached as well as the information that is provided must be tailored to PEH's situation and needs. The videos and posters of this project demonstrate a step into this direction. A concise point that was emphasized by staff in homeless service providers is that no information material can replace personal contact and face-to-face conversation. Relationships to social workers and medical staff remain essential—especially when people are in precarious situations, access to information is difficult and social contacts are limited. Limitations of the information material In the evaluation, it was critically mentioned that the material provided only general information without any details on how to get access to vaccinations. As the provision of tests and vaccinations were locally organized in various different ways, specific information could not be integrated into the material. Some institutions therefore added distinct information for the local context directly on the posters. The material intended to encourage testing and vaccination. One of the statements was that both is possible despite alcohol, other substance usage, and without official documents. Although this information was in line with official regulations, we could not guarantee appropriate implementation. In fact, reports from homeless service providers and PEH showed that people lacking identification documents had difficulties accessing public COVID-19 test and vaccination centers . The material also stated that people with positive test results would be cared for according to their needs. However, it was frequently reported to us that quarantine and isolation capacities for PEH were insufficient, and that substitution or medical care was only partially offered. Benefit and challenges of digital inclusion among PEH During the pandemic, interpersonal contact became limited and digitalization crucial. Most of the population has been able to continuously access updated information about the pandemic, whereas many PEH were excluded from this information flow due to technical and socio-economic reasons that make it difficult to acquire or maintain digital devices. Even with an internet-enabled device, access to information remains difficult with a lack of free Wi-Fi and a limited access to electricity. When fighting a pandemic, it is important to reach all people in society equally, albeit through different channels and in different languages. To ensure this, digitalization has to be facilitated for social and health institutions as well as for PEH . The digital gap also had to be considered in the planning and implementation of our project. Our community partners without mobile- or smartphones were visited personally by our study team for recruitment and further collaboration. People who were temporarily sheltered in a 24/7 facility were easier to locate than people living on the streets. Creative and individual ways had to be identified to stay in contact with all community partners, regardless of their housing or digital situation. This shows the daily challenges of PEH without digital access, that also affects the interaction with friends and relatives, employers, social and medical service providers as well as with public institutions. However, it has to be noted that PEH are not per se excluded from digitalization. A considerable number of PEH manage to purchase and maintain a mobile phone or smartphone and find ways of charging it and accessing internet. Progressive health communication There was a wide range of institutions that ordered the elaborated posters, including a library, facilities for people who use drugs as well as different facilities for specific populations. This demonstrates that PEH can and need to be reached in different ways that consider their complex needs and situations including hidden homelessness. Particularly with a group in which digitalization is highly variable, hybrid material enables analog and digital use simultaneously. For personal contact with medical professionals or social workers, the integration of digital formats such as (anonymous) online counseling should also be considered. Recommendations Valuable lessons were learned through the project, that can help to strengthen participation of PEH and to consider their perspectives in health communication strategies . Engaging marginalized populations as community experts and partners in the planning, implementation and evaluation of projects is crucial to adequately grasp their situation and needs. Strong links to the community, trust and the involvement of relevant stakeholders are indispensable when working with PEH. Progressive health communication in terms of hybrid information material (analog and digital) considers the heterogeneity of the target group, identifies the specific needs and addresses them concretely but sensitively, without stereotypes. The material should be readily accessible, multi-lingual with a simple and clear message, addressing taboo topics while being designed in a discrimination-sensitive way. In this project people were addressed in their various spoken languages, including minority languages and a clear and simple language for functional illiterates. The experiences and feedback of peers and communities must be included in the development of information material. Access to mobile and digital devices positively impacts the daily lives and health of PEH. Sustained use of the devices requires access to electricity and Wi-Fi as well as safe storage facilities. Social institutions should receive financial and professional support to develop a digital infrastructure and have it available for its users. Exclusion from (digital) information, on the other hand, is a major factor in the structural marginalization of PEH. Closing the digital gap can be a contribution to counteracting social and health inequalities. A key factor for the realization of the videos and posters was the set-up of an interdisciplinary team together with community partners and thus bridging the gap between research, practice and community. Of particular significance were good contacts of the study team with homeless service providers and PEH. Furthermore, it was crucial to involve various local and national stakeholders right from the beginning of the project for advice, support and distribution of the materials. The participatory approach enables active generation of knowledge together with practice and communities . Accordingly, we addressed hierarchies transparently and valued PEH's own life experiences as equivalent to knowledge from professionals within the fields of social work and public health. A challenge was to deal with poverty and precarious living conditions in a sensitive way. Poverty was neither to be tabooed nor trivialized. The goal was to show a realistic picture of the social system and living environment of PEH without reproducing stereotypes or exposing people. The image and portrayal of homelessness in times of the pandemic was therefore to be determined primarily by the community partners themselves. The main protagonist decided to participate by stating “ I think I'm someone people accept ”. In front of the camera, most PEH placed emphasis on a proud attitude showing an active and upright posture. For the evaluation, 8 PEH and 12 service providers were interviewed. They acknowledged the sensitive implementation of the project. The consideration of the concerns and the diverse presentation of PEH was perceived as particularly important. In this manuscript we have chosen the term “people experiencing homelessness” because it presents homelessness as one aspect among others and is less generalizing than the term “homeless people”. To enable participation according to the needs of the community partners, work conditions were defined and arranged together. For example, the main protagonist's condition for participation was the assurance of permanent access to barrier-free sanitary facilities. He knew from personal experience that this would be a main challenge, especially during the pandemic when sanitary facilities were even less available for PEH. This highlights one of the fundamental problems that people living in the streets face on a daily basis. For some people there were barriers that made participation difficult or impossible such as hidden homelessness, illegalization, and precarious employment. We have tried to enable safe participation for them as well. For some, a solution was to participate by dubbing the videos, translating the scripts or by attending the feedback sessions. Others chose to not participate altogether. Our impression was that it has been easier for people to participate if there was pre-existing contact with project staff or other PEH who had already taken part. In regard to the evaluation, we found it easier to find interview partners in the day center which provided a safe setting for the interview compared to the recruitment in front of the shelter where the interviews were performed outdoors. It is crucial to be aware of and respect the right to non-participation. We did not collect socio demographic data from the PEH who participated in the production and evaluation of the materials. All were experiencing homelessness at the time of the study. Some had shelter available. Similar to varying infection risks, differences in the participation and responses between sheltered and unsheltered individuals are possible, but this could be not further analyzed in this study . Despite the mainly positive feedback in the evaluation, the impact of the videos could only be verified to a limited extent. Most of the homeless service providers were unable to show the videos on their premises, due to technical and spatial limitations. The videos and posters were widely distributed via social media and email lists. It remains unclear to what extent they reached the actual target group. However, considering the important role that social media has in shaping people's state of information and attitudes toward public health interventions such as vaccination campaigns, efforts should be increased to utilize these means of communication . The posters seem to have been particularly useful for the vaccination campaign according to the feedback of service providers and the number of poster-orders throughout Germany. Addressing the specific questions and concerns of PEH—e.g., having access to vaccination without a permanent address, documents or health insurance, as well as implications of drug use—may be one approach to increase vaccination coverage . The type of institutions that ordered the posters reflect the broad range of services that address the complex needs of PEH . Among them were services targeting people without official documents (e.g., passport, citizenship, health insurance), people living in poverty, people using drugs, as well as services exclusively provided for women. The way how people are approached as well as the information that is provided must be tailored to PEH's situation and needs. The videos and posters of this project demonstrate a step into this direction. A concise point that was emphasized by staff in homeless service providers is that no information material can replace personal contact and face-to-face conversation. Relationships to social workers and medical staff remain essential—especially when people are in precarious situations, access to information is difficult and social contacts are limited. In the evaluation, it was critically mentioned that the material provided only general information without any details on how to get access to vaccinations. As the provision of tests and vaccinations were locally organized in various different ways, specific information could not be integrated into the material. Some institutions therefore added distinct information for the local context directly on the posters. The material intended to encourage testing and vaccination. One of the statements was that both is possible despite alcohol, other substance usage, and without official documents. Although this information was in line with official regulations, we could not guarantee appropriate implementation. In fact, reports from homeless service providers and PEH showed that people lacking identification documents had difficulties accessing public COVID-19 test and vaccination centers . The material also stated that people with positive test results would be cared for according to their needs. However, it was frequently reported to us that quarantine and isolation capacities for PEH were insufficient, and that substitution or medical care was only partially offered. During the pandemic, interpersonal contact became limited and digitalization crucial. Most of the population has been able to continuously access updated information about the pandemic, whereas many PEH were excluded from this information flow due to technical and socio-economic reasons that make it difficult to acquire or maintain digital devices. Even with an internet-enabled device, access to information remains difficult with a lack of free Wi-Fi and a limited access to electricity. When fighting a pandemic, it is important to reach all people in society equally, albeit through different channels and in different languages. To ensure this, digitalization has to be facilitated for social and health institutions as well as for PEH . The digital gap also had to be considered in the planning and implementation of our project. Our community partners without mobile- or smartphones were visited personally by our study team for recruitment and further collaboration. People who were temporarily sheltered in a 24/7 facility were easier to locate than people living on the streets. Creative and individual ways had to be identified to stay in contact with all community partners, regardless of their housing or digital situation. This shows the daily challenges of PEH without digital access, that also affects the interaction with friends and relatives, employers, social and medical service providers as well as with public institutions. However, it has to be noted that PEH are not per se excluded from digitalization. A considerable number of PEH manage to purchase and maintain a mobile phone or smartphone and find ways of charging it and accessing internet. There was a wide range of institutions that ordered the elaborated posters, including a library, facilities for people who use drugs as well as different facilities for specific populations. This demonstrates that PEH can and need to be reached in different ways that consider their complex needs and situations including hidden homelessness. Particularly with a group in which digitalization is highly variable, hybrid material enables analog and digital use simultaneously. For personal contact with medical professionals or social workers, the integration of digital formats such as (anonymous) online counseling should also be considered. Valuable lessons were learned through the project, that can help to strengthen participation of PEH and to consider their perspectives in health communication strategies . Engaging marginalized populations as community experts and partners in the planning, implementation and evaluation of projects is crucial to adequately grasp their situation and needs. Strong links to the community, trust and the involvement of relevant stakeholders are indispensable when working with PEH. Progressive health communication in terms of hybrid information material (analog and digital) considers the heterogeneity of the target group, identifies the specific needs and addresses them concretely but sensitively, without stereotypes. The material should be readily accessible, multi-lingual with a simple and clear message, addressing taboo topics while being designed in a discrimination-sensitive way. In this project people were addressed in their various spoken languages, including minority languages and a clear and simple language for functional illiterates. The experiences and feedback of peers and communities must be included in the development of information material. Access to mobile and digital devices positively impacts the daily lives and health of PEH. Sustained use of the devices requires access to electricity and Wi-Fi as well as safe storage facilities. Social institutions should receive financial and professional support to develop a digital infrastructure and have it available for its users. Exclusion from (digital) information, on the other hand, is a major factor in the structural marginalization of PEH. Closing the digital gap can be a contribution to counteracting social and health inequalities. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving human participants were reviewed and approved by the Ethics Committee of the Charité – Universitätsmedizin Berlin (No.: EA2/168/21). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article. AS: conceptualization, methodology, investigation, and writing original draft. NS: conceptualization, methodology, and scientific advice. TL, TH, MH, and MW: methodology, investigation, and formal analysis. GE and JS: scientific advice and supervision. AL: project administration, conceptualization, and supervision. All authors: writing—review and editing. All authors contributed to the article and approved the submitted version. This study was funded by Bundesministerium für Bildung und Forschung/Federal Ministry of Education and Research: NaFoUniMedCovid19 (FKZ: 01KX2021) B-FAST. The funder had no role in the design and conduct of the study, analysis, interpretation of data, or in writing of the manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. |
Ready to leave? – Adolescents’ and parents’ perceptions of transition from paediatric to adult rheumatology care | 98ee75c4-40cc-44a7-a888-14c7539309a5 | 11234774 | Internal Medicine[mh] | Juvenile Idiopathic Arthritis (JIA) is a heterogeneous group of disorders categorized into seven subgroups, all of which have inflammatory arthritis as a common denominator . JIA can arise at any time during childhood, and girls are more likely than boys to be affected . Yearly, 150–200 children in Sweden are diagnosed with JIA and around 1 500–2 000 children live with the disease . Approximately half of the adolescents with JIA continue to have an active disease as they enter adulthood, and need to transfer to adult care . In Sweden, most adolescents will be transferred to adult care at the age of 18 years. The transfer from paediatric to adult rheumatology care is, for some, perceived as difficult, and can cause anxiety among adolescents and their parents . There is a distinction between transfer and transition. The term ‛transfer’ refers to an occurrence or series of events when adolescents with ongoing physical and medical issues move from receiving care in a paediatric setting to an adult medical setting. ‘Transition’, on the other hand, is a process that occurs throughout adolescence and aims to educate adolescents to manage their lives and health in relation to their chronic illness. The learning process should start before the adolescent enters puberty, and last until they are old enough to take care of themselves . The Society for Adolescent Medicine defines transitional care as “the purposeful, planned movement of adolescents and young adults with chronic physical and medical conditions from child-centred to adult-oriented healthcare systems” (p.570) . Previous research shows that the transition process gives the best results if initiated as soon as the child enters adolescence. It can begin as early as 11 years of age but not later than the age of 14 . The necessity of starting the process in early adolescence was also demonstrated by McDonagh and colleagues, who examined a coordinated evidence-based approach. Twelve months following the program’s implementation, the youngest group (aged 11) displayed significant gains in knowledge of arthritis, an increase in self-medication, and increased satisfaction with rheumatology care . In the transition process, it is important to include the parents and to help them adjust to their new role as they will no longer be the ones who have the main responsibility for the adolescent’s healthcare . During the transition process, healthcare professionals (HCPs) should try to strengthen the adolescent’s independence without undermining the parent’s role . HCPs, adolescents, and parents must try to create a common view on how the transition process facilitates and strengthens the adolescent’s independence . There are several methods for measuring readiness for transition targeting adolescents, parents, and HCPs. In the literature, both quantitative and qualitative methods are used. In Sweden the Readiness for Transition Questionnaire (RTQ) has recently been used . Results from previous research measuring readiness show that both adolescents and parents need more knowledge about transition. In a study with 49 patients suffering from JIA and 103 parents, Matsumoto (2021) shows that over half of the adolescents and about one-third of their parents had limited knowledge about what transitional care was, and over half of the adolescents and nearly four-fifths of the parents felt worried to transfer to adult care . This study also showed that about half of both the adolescents and the parents were not given the opportunity to talk to the doctor about the upcoming transfer to adult care. A major worry was that the medical doctors in adult care would not have adequate knowledge about JIA . In another study by Sömnez (2021), of 157 patients with different rheumatology diagnoses and their parents, half of the adolescents and almost all the parents wanted to stay in paediatric care and were, accordingly, not sufficiently ready for transfer. Moreover, age seems to be an important determinator for perceptions of readiness for transfer from paediatric rheumatology . Bingham and colleagues found that older children had higher self-reported autonomy in most questions asked regarding accessing medical care . Other patient characteristics associated with high self-perceived autonomy included having a family member with a similar disease, having a younger parent, and longer disease duration . Transfer to adult rheumatology is under-researched . In Sweden, no studies have been made on readiness for transfer to adult care among adolescents with JIA and there is no structured transition program or guidelines for this patient group. In order to tailor and improve transitional care for adolescents with JIA, it is essential to know how ready adolescents and their parents feel about different aspects of the transition and, ultimately, the transfer from paediatric to adult care. Investigation of adolescents’ and parents’ readiness could facilitate understanding of the transition process and create foundations for individualized support. Aims The aim of this study was to investigate Swedish adolescents with JIA and their parents’ perceptions of readiness for transition from paediatric to adult rheumatology care. The aim of this study was to investigate Swedish adolescents with JIA and their parents’ perceptions of readiness for transition from paediatric to adult rheumatology care. Study design A cross-sectional quantitative study. Participants Data were collected from March 2020 to March 2022. Patients at the paediatric rheumatology clinic at a university hospital in Stockholm and members of the Swedish National Organization for Young Rheumatics were invited, together with their parents, to participate in the study. Inclusion criteria Confirmed Juvenile Idiopathic Arthritis, adolescents turning 14 the year of inclusion and adolescents up to 18 years. Exclusion criteria Uncertain paediatric rheumatic disease. Non-Swedish speaking adolescents/parents. Questionnaire The readiness for transition questionnaire (RTQ) was originally developed and validated by Gilleland for patient groups with different chronic diagnoses, and is also available in a parental version . The questionnaire has been translated and culturally adapted into Swedish using scientific guidelines . The first part of the RTQ covers four questions about responsibility for healthcare . Furthermore, the RTQ covers adolescent responsibility and parental involvement in different healthcare-related behaviours, as well as final questions about overall transition readiness and overall readiness for responsibility for healthcare . Included healthcare-related behaviours are, for example, regular blood samples, taking medications, and being in contact with the clinic. The questions about healthcare-related behaviours have five possible answers for adolescent responsibility: 1 = Not responsible at all; 2 = Sometimes responsible; 3 = Often responsible; 4 = Almost always responsible; and 5 = Not relevant. The same behaviours are also investigated in the section about parental involvement, with response options as follows: 1 = Not involved at all; 2 = Sometimes involved; 3 = Often involved; 4 = Almost always involved; and 5 = Not relevant. The response option Not relevant was added for questions related to healthcare behaviours during the adaptation process . Response alternatives to the final overall questions are: Not at all ready; Somewhat ready; Mostly ready; and Completely ready. In the present study, two minor adjustments were made to the Swedish version , in both the adolescent and parent versions: 1) the word “daily” was excluded from the question about daily medications since not all medications are taken daily within paediatric rheumatology, and 2) the example of type of clinics in the question about scheduling specialty care appointments was replaced by clinics more relevant to the patient group. Furthermore, questions ( n = 5) about health were excluded due to ambiguities in what adolescents include in the health concept, and that the original questionnaire focused on healthcare and not health. Data collection In total, 225 members of the Swedish National Organization for Young Rheumatics and 110 patients from the paediatric rheumatology clinic were invited to participate in the study by a research invitation letter sent to their home addresses. The research invitation letter included information explaining the purpose of the study and separate QR codes for adolescents and parents to access the digital anonymous RTQ. One reminder was sent to the intended study participants at one point. Ethical approval declarations The study was approved by the Ethical Review Board in Sweden Dnr 2019–01540. Data analysis Data were analysed with descriptive statistical methods calculating percentages, medians, ranges, means, and standard deviations. Comparative analyses were made using non-parametric tests (Mann Whitney U test) with a significance level of 0.05. The two parts concerning adolescent responsibility and parental involvement for different healthcare-related behaviours were combined to obtain a measure of the adolescent’s level of “independent responsibility”. In the combined measure, no distinction was made between the two response options “Often” and “Almost always” since we found it difficult to distinguish between them. The independent responsibility measures are described below: No responsibility – Adolescent not responsible at all. Minor responsibility – Adolescent sometimes responsible. Shared responsibility – Adolescent often responsible with parents often involved. Major responsibility – Adolescent often responsible with parents sometimes or not at all involved. Factor analysis (Varimax rotated principal component analysis) was used to investigate the dimensions of the items. Logistic regression was used to analyse what factor(s) explain adolescents feeling almost fully or fully ready to transfer to adult care. A cross-sectional quantitative study. Data were collected from March 2020 to March 2022. Patients at the paediatric rheumatology clinic at a university hospital in Stockholm and members of the Swedish National Organization for Young Rheumatics were invited, together with their parents, to participate in the study. Confirmed Juvenile Idiopathic Arthritis, adolescents turning 14 the year of inclusion and adolescents up to 18 years. Uncertain paediatric rheumatic disease. Non-Swedish speaking adolescents/parents. The readiness for transition questionnaire (RTQ) was originally developed and validated by Gilleland for patient groups with different chronic diagnoses, and is also available in a parental version . The questionnaire has been translated and culturally adapted into Swedish using scientific guidelines . The first part of the RTQ covers four questions about responsibility for healthcare . Furthermore, the RTQ covers adolescent responsibility and parental involvement in different healthcare-related behaviours, as well as final questions about overall transition readiness and overall readiness for responsibility for healthcare . Included healthcare-related behaviours are, for example, regular blood samples, taking medications, and being in contact with the clinic. The questions about healthcare-related behaviours have five possible answers for adolescent responsibility: 1 = Not responsible at all; 2 = Sometimes responsible; 3 = Often responsible; 4 = Almost always responsible; and 5 = Not relevant. The same behaviours are also investigated in the section about parental involvement, with response options as follows: 1 = Not involved at all; 2 = Sometimes involved; 3 = Often involved; 4 = Almost always involved; and 5 = Not relevant. The response option Not relevant was added for questions related to healthcare behaviours during the adaptation process . Response alternatives to the final overall questions are: Not at all ready; Somewhat ready; Mostly ready; and Completely ready. In the present study, two minor adjustments were made to the Swedish version , in both the adolescent and parent versions: 1) the word “daily” was excluded from the question about daily medications since not all medications are taken daily within paediatric rheumatology, and 2) the example of type of clinics in the question about scheduling specialty care appointments was replaced by clinics more relevant to the patient group. Furthermore, questions ( n = 5) about health were excluded due to ambiguities in what adolescents include in the health concept, and that the original questionnaire focused on healthcare and not health. In total, 225 members of the Swedish National Organization for Young Rheumatics and 110 patients from the paediatric rheumatology clinic were invited to participate in the study by a research invitation letter sent to their home addresses. The research invitation letter included information explaining the purpose of the study and separate QR codes for adolescents and parents to access the digital anonymous RTQ. One reminder was sent to the intended study participants at one point. The study was approved by the Ethical Review Board in Sweden Dnr 2019–01540. Data were analysed with descriptive statistical methods calculating percentages, medians, ranges, means, and standard deviations. Comparative analyses were made using non-parametric tests (Mann Whitney U test) with a significance level of 0.05. The two parts concerning adolescent responsibility and parental involvement for different healthcare-related behaviours were combined to obtain a measure of the adolescent’s level of “independent responsibility”. In the combined measure, no distinction was made between the two response options “Often” and “Almost always” since we found it difficult to distinguish between them. The independent responsibility measures are described below: No responsibility – Adolescent not responsible at all. Minor responsibility – Adolescent sometimes responsible. Shared responsibility – Adolescent often responsible with parents often involved. Major responsibility – Adolescent often responsible with parents sometimes or not at all involved. Factor analysis (Varimax rotated principal component analysis) was used to investigate the dimensions of the items. Logistic regression was used to analyse what factor(s) explain adolescents feeling almost fully or fully ready to transfer to adult care. Out of 335 patients receiving the invitation, including an invitation to their parents, 106 adolescents (girls n = 85, boys n = 20, one missing) and 96 parents answered the questionnaire. The response rate was 32% for adolescents but unknown for parents since information about the number of parents in each family receiving the invitation is missing. Adolescents’ ages ranged from 13 to 18 years old (Fig. ) with a median age of 16 years (participants who were 13 years old were turning 14 the same year). Sociodemographic questions in RTQ include age and gender but not diagnosis subgroup. Therefore, we do not know which subgroup of JIA the participants had. The clinic treats patients with all types of JIA and therefore it can be assumed, but not certain, that there also were a spread of JIA sub group diagnosis among the respondents. Responsibility and involvement in different healthcare-related behaviours Adolescents and parents were asked about how much responsibility the adolescents took for different healthcare-related behaviours, and how much the parents were involved in, for example, taking medication, renewing prescriptions, and attending medical appointments (Table ). In general, 16–18-year-old adolescents reported taking more responsibility and having lower parental involvement than 13–15-year-olds. For several behaviours, there were significant differences between the two age groups of adolescents (Table ). When analysing gender, one significant difference emerged; namely, girls reported more responsibility for communication with the clinic on the phone compared to boys (p value = 0.045) (Table ). The adolescents reported themselves as taking greater responsibility than the parents reported them as doing. However, both adolescents and parents reported equally that the adolescents took less responsibility for tasks involving direct contact with the clinic (Table ). Both adolescents and parents reported that the parents were largely involved in healthcare-related behaviours. The younger adolescents (13–15 years) reported that their parents were more involved in healthcare-related behaviours than the older adolescents (16–18 years) reported (Table ). Adolescents’ and parents' perception of independent responsibility regarding different healthcare-related behaviours In the combined measure of responsibility with different levels of parental involvement, the analysis showed that direct contact with the healthcare system was most challenging for adolescents (Fig. ). In many of the other healthcare-related behaviours, for example, taking medication, getting regular labs, and explaining the disease to others, the adolescents perceived themselves as taking major responsibility, while perceiving parents as often involved. A small proportion of adolescents perceived themselves as taking major responsibility without parental involvement. The substantial progress in responsibility, compared between the younger and older adolescent groups, was for getting regular labs and attending medical appointments. For the more challenging activities, there was progress between the younger and older age groups, but still, less than 25% of the older adolescents perceived major responsibility, with parents often/sometimes involved. Adolescents’ and parents’ perceptions of overall readiness to transfer to adult care There was a significant difference ( p value = 0.005) (Table ) between the age groups when reporting perceptions of overall readiness to transfer to adult care. In the younger group (13–15 years), 2% perceived that they were fully ready to transfer to adult care compared to 7% of the adolescents in the older group (16–18 years) (Table ). Moreover, in the younger age group (13–15 years), 48% perceived that they were not ready to transfer to adult care compared to 24% in the older age group (16–18 years). There were no significant gender differences when adolescents reported perceptions on how fully ready they were to transfer to adult care. Parents’ perceptions are congruent with the younger age group of adolescents. Three percent reported that they perceived their adolescents as being fully ready to transfer and half (49%) of the parental group reported that they did not perceive their adolescents as ready to transfer (Table ). Factors influencing adolescents’ and parents’ perception of overall readiness to transfer to adult care A factor analysis revealed two factors with eigenvalues above 1, explaining 63.9% of the variance (Table ). Factor 1 includes variables that have to do with bookings and communication by telephone, conceptualized as “Administration”. Factor 2 includes handling labs/medications and communicating in person, which seem related to engagement and routines of the healthcare, conceptualized as “Engagement”. The engagement factor gets high scores in the group that feels not ready for transfer and increases for the group that feels rather ready. Thus, it seems that this factor includes the first steps to independent responsibility for healthcare. Adolescents’ and parents’ results are in reasonable agreeance for Factor 2 (Table ). The administrative factor is continuously increasing in the three levels of readiness, and there is a bigger gap between the group that feels rather ready and the group that feels almost/fully ready. Parents especially don’t seem to judge their adolescents as fully ready for transfer until they also show independent responsibility for administrative matters. Separate logistic regressions for adolescents and parents showed that only Factor 1 (administrative) was significant for explaining being almost/fully ready for transfer when also controlling for Factor 2 (engagement) (Table ). Adolescents and parents were asked about how much responsibility the adolescents took for different healthcare-related behaviours, and how much the parents were involved in, for example, taking medication, renewing prescriptions, and attending medical appointments (Table ). In general, 16–18-year-old adolescents reported taking more responsibility and having lower parental involvement than 13–15-year-olds. For several behaviours, there were significant differences between the two age groups of adolescents (Table ). When analysing gender, one significant difference emerged; namely, girls reported more responsibility for communication with the clinic on the phone compared to boys (p value = 0.045) (Table ). The adolescents reported themselves as taking greater responsibility than the parents reported them as doing. However, both adolescents and parents reported equally that the adolescents took less responsibility for tasks involving direct contact with the clinic (Table ). Both adolescents and parents reported that the parents were largely involved in healthcare-related behaviours. The younger adolescents (13–15 years) reported that their parents were more involved in healthcare-related behaviours than the older adolescents (16–18 years) reported (Table ). In the combined measure of responsibility with different levels of parental involvement, the analysis showed that direct contact with the healthcare system was most challenging for adolescents (Fig. ). In many of the other healthcare-related behaviours, for example, taking medication, getting regular labs, and explaining the disease to others, the adolescents perceived themselves as taking major responsibility, while perceiving parents as often involved. A small proportion of adolescents perceived themselves as taking major responsibility without parental involvement. The substantial progress in responsibility, compared between the younger and older adolescent groups, was for getting regular labs and attending medical appointments. For the more challenging activities, there was progress between the younger and older age groups, but still, less than 25% of the older adolescents perceived major responsibility, with parents often/sometimes involved. There was a significant difference ( p value = 0.005) (Table ) between the age groups when reporting perceptions of overall readiness to transfer to adult care. In the younger group (13–15 years), 2% perceived that they were fully ready to transfer to adult care compared to 7% of the adolescents in the older group (16–18 years) (Table ). Moreover, in the younger age group (13–15 years), 48% perceived that they were not ready to transfer to adult care compared to 24% in the older age group (16–18 years). There were no significant gender differences when adolescents reported perceptions on how fully ready they were to transfer to adult care. Parents’ perceptions are congruent with the younger age group of adolescents. Three percent reported that they perceived their adolescents as being fully ready to transfer and half (49%) of the parental group reported that they did not perceive their adolescents as ready to transfer (Table ). A factor analysis revealed two factors with eigenvalues above 1, explaining 63.9% of the variance (Table ). Factor 1 includes variables that have to do with bookings and communication by telephone, conceptualized as “Administration”. Factor 2 includes handling labs/medications and communicating in person, which seem related to engagement and routines of the healthcare, conceptualized as “Engagement”. The engagement factor gets high scores in the group that feels not ready for transfer and increases for the group that feels rather ready. Thus, it seems that this factor includes the first steps to independent responsibility for healthcare. Adolescents’ and parents’ results are in reasonable agreeance for Factor 2 (Table ). The administrative factor is continuously increasing in the three levels of readiness, and there is a bigger gap between the group that feels rather ready and the group that feels almost/fully ready. Parents especially don’t seem to judge their adolescents as fully ready for transfer until they also show independent responsibility for administrative matters. Separate logistic regressions for adolescents and parents showed that only Factor 1 (administrative) was significant for explaining being almost/fully ready for transfer when also controlling for Factor 2 (engagement) (Table ). This cross-sectional quantitative study’s analysis reveals that many adolescents with JIA were ill-prepared to transfer to adult care. The same issue was reported by their parents. Parents and adolescents alike stated that it was difficult for the adolescents to take responsibility for several healthcare-related behaviours connected to adolescents’ direct interaction with the HCPs at the paediatric rheumatology clinic. It was evident that the adolescents who perceived they were ready to take responsibility for the aspects related to direct interaction with HCPs were more ready to be transferred to adult care. As mentioned above, challenging healthcare-related behaviours for adolescents included them having direct contact with HCPs, e.g., calling to book an appointment or renew prescriptions. The same results have been shown in other studies . The reasons for these results are not clear. One could argue that the feeling of uncertainty and the fear of making mistakes could be one explanation. Research has shown that adolescents transitioning from adolescent to adult care felt anxious, uncertain, and fearful , which could reinforce the fear of making mistakes. The two items that involve using the phone – to communicate with HCPs and renew prescriptions – seem especially difficult for adolescents. There may be several reasons why young people find it challenging to contact healthcare providers by phone. One reason may be that the phone hours are during the day when they are at school, which makes it difficult for them to handle the contact on their own without having to leave classes. Another reason may be that adolescents rarely make telephone calls, and are especially uncomfortable talking to adults on the phone. On the other hand, adolescents are used to using their mobile phones for many other things and it is an environment they feel safe in, which could be argued would perhaps contribute to them feeling comfortable to contact healthcare providers. Healthcare clinics caring for adolescents must fulfill their task to be adolescent-friendly and customize accessibility according to adolescents’ preferences. Using digital platforms, including communication pathways such as chats, could be one solution and would increase flexibility regarding contact times with HCPs. The digital opportunities that exist today could, perhaps, also be utilized in the transition work itself. In a study by Miller , it was shown that adolescents who were given the opportunity to use digital transition support by an app on their phone increased their self-confidence in taking care of their illness, and the proportion of those who took responsibility for booking visits to the healthcare system. They also used the app to increase their knowledge of their disease . The present study also provides us with the knowledge that this group of adolescents did not feel ready to transfer to adult care. Only a small percent (5%) of the adolescents reported that they were fully ready to transfer. Our results demonstrate that neither does it seem to be enough to take responsibility for some possibly simpler behaviours to feel fully ready. We speculate that the challenge is, perhaps above all, to start taking responsibility for ‛adult’ things, like booking and calling, i.e., the behaviours included in factor 1. However, it may be difficult for adolescents to evaluate whether they are ready to be transferred, as the idea of it may be abstract and they may not know what to expect. They might not know geographically where they are going, which medical doctor to see, and how the care is conducted there. Despite years of increased study and policy focus on the topic of transition, there are still unmet requirements for adolescents and their families. As a crucial component of an adolescent’s development, health transitions take place concurrently with, and in relation to, a variety of other significant transitions, like transitioning from childhood to being an adolescent, that has an impact on many different facets of life , and which may complicate the process further. Additionally, our analysis reveals that the levels of readiness increase with the age of the adolescents. This result was expected and has been described by others , and is most likely associated with the developmental process of going from adolescence to adulthood . In this study, parents graded the adolescents less ready for the transfer than the adolescents graded themselves. Similar results have been reported in other studies investigating transition among adolescent’s with chronic diseases . Only 13% of parents reported that their adolescent was almost or fully ready to transfer, which is low even compared to the youngest age group’s own perception of readiness. The results could be related to a variety factors. One possibility is that adults and adolescents interpret “fully ready” different. Some adolescents may, for example, express readiness without realizing the impact of more independent responsibility that their parents may include in their interpretation of readiness. However, the results may also be an effect of parents underestimating the adolescent’s knowledge and ability. This could mean that adolescents never develop abilities to take responsibility if parents, for example, continue to administer medications and communicate with healthcare providers . It is therefore important for HCPs to enable adolescents to increase their abilities and put them to use to support positive adolescent development. For instance, adolescents could practice asking questions about the care or sensitive topics if they are given the chance to do so while their parents are present. Although the group of boys is small, the results show that boys seem to perceive themselves as ready for transfer to a greater degree than girls. In the present study population, this perhaps can be explained by gender roles and that girls are a little more open about their lack of abilities, knowledge, and about expressing worries about the transfer. However, the girls reported to a larger extent than boys that they take responsibility for talking on the phone with HCPs. In a study by Eaton et al., the opposite was shown since they concluded that girls were more ready to transfer and had less parent involvement than boys . We speculate that this might be due to culture and/or contextual differences, which makes measuring readiness among adolescents in specific contexts and cultures extra important, to enable support to be tailored according to specific needs. In a Swedish study by Burström et al. , the aims were to investigate levels of readiness for transition in adolescents with congenital heart disease and to compare adolescents’ levels with their parents’ assessments. Similarly to our study, they demonstrated that adolescents scored higher on overall readiness than their parents. However, they did not compare girls’ and boys’ readiness but investigated differences in perceptions between mothers and fathers. The results show that parents, regardless of gender, perceive adolescents’ responsibility equally. However, perceptions of parental involvement differed between parental genders, meaning that mothers to a greater extent than fathers, perceived themselves as involved . We would argue that this again indicates that differences in perceptions of transition readiness depend on contexts, and that it is important to study the specific population in order to offer tailored support. Another interesting question about concepts that might have influenced our results, is how the participants interpreted responsibility. If a parent asks an adolescent to call and renew a prescription and the adolescent does so, the adolescent will probably feel that they have taken responsibility, but in fact, the parent was the one responsible for checking that the prescription needed to be renewed and arranging for it to be done. The meaning and interpretation of responsibility for different age groups and parents would be interesting for further studies, to get deeper knowledge about how to communicate about and support independent responsibility for adolescents. As a final remark, we would argue that to meet adolescents’ and parents’ needs for transition during adolescence, HCPs in both child and adult healthcare must have adequate training. The HCPs need to have good knowledge of adolescents’ normal development and be able to use it in relation to the difficulties that can arise if they also have a chronic illness. It is also important that HCPs are willing to bring up and talk about sensitive topics, for example, sex, alcohol, and relationships. Studies show that structured transition programs can increase both adolescents’ and parents’ confidence regarding the transfer to adult care . This means that introducing a person-centred transition program does not only mean educating patients and parents, but also ensuring that HCPs have adequate knowledge to enable transition in an optimal and positive way. Methodological considerations This study is based on an anonymous survey sent out to adolescents with JIA, with encouragement for their parents to respond to the parent version. However, we do not have information on whether the adolescent lived with a single parent or both parents. The anonymous feature of the survey makes it impossible to link an adolescent to his or her parent(s). Some adolescents may therefore have answered without any parent answering the parent questionnaire and vice versa. Consequently, we do not know how the parents of a specific adolescent responded, and we cannot determine if there is agreement between adolescents and their parents on an individual level. However, our findings indicate that the responses at the group level are consistent. There is also a possibility that both parents chose to participate and answered one questionnaire each. This makes it impossible to calculate a correct response rate for parents as well as compare the results from the adolescent’s and parent’s perspectives in the same family. On the other hand, the advantage of anonymity is that respondents hopefully felt confident to give truthful answers. When asking the parents about age and gender, some of the parents reported their age and some reported the adolescent’s age. The reports of age and gender are therefore not reliable for parents and not used in the analysis. Furthermore, we did not ask about the age of the adolescent in the parent questionnaire. This means we cannot ensure that the parents’ results correspond to the same age distribution among adolescents as the adolescents’ results. Another limitation is that background information other than gender and age is not present. This means that we do not know what kind of JIA the patient had. It might have been interesting to be able to see if patients with a milder disease are more prepared for transferring to adult care than those who have a more severe disease or contrariwise. It would have been valuable to ascertain the duration of the adolescents' diagnoses, as this factor might influence their level of responsibility for managing their disease and their readiness for transitioning to adult healthcare. Additionally, investigating the impact of various family structures on the adolescents' ability to assume responsibility for their disease would have been insightful. For instance, the presence of multiple siblings within a family could potentially affect this ability. However, such analyses were not possible in this study due to the lack of relevant background information. As described in the method section, the decision was made to exclude the questions about responsibility for health in the questionnaire. In the original, Gilleland and colleagues use healthcare, and in Burström’s translated Swedish questionnaire, the word health is used . Since we did not conduct cognitive interviews about what adolescents with JIA refer to and include when thinking about health, we assessed it was more scientifically rigorous to exclude the five questions about health. In future studies, it would be interesting to explore this further in cognitive interviews with adolescents, and thereby find out if it is most suitable to ask about health versus healthcare. In the combined measure of independent responsibility, no distinction was made between the parent being involved sometimes or not at all. The reason for this is that it seems reasonable that parents are sometimes involved in visits to the clinic or talking to the staff, even if the adolescent takes most of the responsibility. The factor analysis and the two latent factors are based on summaries of the scores (1–4) of the items in each factor. Summarizing ordinal data is not optimal but is used here to give a rough picture of the two different factors in this rather small amount of material. In a future larger study, Rasch analysis could be used to develop a validated measurement scale for independent responsibility. Lastly, we would like to point out that it is difficult to state the results in the sudy are representative of the population concerned since we have no data on JIA subtypes, for example. Moreover, it is probable that the participants who responded are those who felt most concerned or more comfortable with the subject. However, the results in this study based on the rather large sample of 106 adolescents shows strong indications of levels of readiness in this population. In future research stratified sampling could be used to ensure that each subgroup is adequately represented. Clinical implications The results from this study can be used as a foundation for structuring a transition program for adolescents with JIA, including tailoring transition care and creating opportunities for HCPs to focus on the parts of the transition that are perceived as challenging by the adolescents. Based on the findings of the present study, it is evident that HCPs working in paediatric care have to provide the adolescents and their parents with information and knowledge so that they can feel safe when transferring to adult care. To guarantee an optimal transition from paediatric to adult care, it is imperative to comprehend the special needs of each adolescent, and acknowledge cultural and contextual differences. The RTQ can be used as a screening tool to discover individual needs. This study is based on an anonymous survey sent out to adolescents with JIA, with encouragement for their parents to respond to the parent version. However, we do not have information on whether the adolescent lived with a single parent or both parents. The anonymous feature of the survey makes it impossible to link an adolescent to his or her parent(s). Some adolescents may therefore have answered without any parent answering the parent questionnaire and vice versa. Consequently, we do not know how the parents of a specific adolescent responded, and we cannot determine if there is agreement between adolescents and their parents on an individual level. However, our findings indicate that the responses at the group level are consistent. There is also a possibility that both parents chose to participate and answered one questionnaire each. This makes it impossible to calculate a correct response rate for parents as well as compare the results from the adolescent’s and parent’s perspectives in the same family. On the other hand, the advantage of anonymity is that respondents hopefully felt confident to give truthful answers. When asking the parents about age and gender, some of the parents reported their age and some reported the adolescent’s age. The reports of age and gender are therefore not reliable for parents and not used in the analysis. Furthermore, we did not ask about the age of the adolescent in the parent questionnaire. This means we cannot ensure that the parents’ results correspond to the same age distribution among adolescents as the adolescents’ results. Another limitation is that background information other than gender and age is not present. This means that we do not know what kind of JIA the patient had. It might have been interesting to be able to see if patients with a milder disease are more prepared for transferring to adult care than those who have a more severe disease or contrariwise. It would have been valuable to ascertain the duration of the adolescents' diagnoses, as this factor might influence their level of responsibility for managing their disease and their readiness for transitioning to adult healthcare. Additionally, investigating the impact of various family structures on the adolescents' ability to assume responsibility for their disease would have been insightful. For instance, the presence of multiple siblings within a family could potentially affect this ability. However, such analyses were not possible in this study due to the lack of relevant background information. As described in the method section, the decision was made to exclude the questions about responsibility for health in the questionnaire. In the original, Gilleland and colleagues use healthcare, and in Burström’s translated Swedish questionnaire, the word health is used . Since we did not conduct cognitive interviews about what adolescents with JIA refer to and include when thinking about health, we assessed it was more scientifically rigorous to exclude the five questions about health. In future studies, it would be interesting to explore this further in cognitive interviews with adolescents, and thereby find out if it is most suitable to ask about health versus healthcare. In the combined measure of independent responsibility, no distinction was made between the parent being involved sometimes or not at all. The reason for this is that it seems reasonable that parents are sometimes involved in visits to the clinic or talking to the staff, even if the adolescent takes most of the responsibility. The factor analysis and the two latent factors are based on summaries of the scores (1–4) of the items in each factor. Summarizing ordinal data is not optimal but is used here to give a rough picture of the two different factors in this rather small amount of material. In a future larger study, Rasch analysis could be used to develop a validated measurement scale for independent responsibility. Lastly, we would like to point out that it is difficult to state the results in the sudy are representative of the population concerned since we have no data on JIA subtypes, for example. Moreover, it is probable that the participants who responded are those who felt most concerned or more comfortable with the subject. However, the results in this study based on the rather large sample of 106 adolescents shows strong indications of levels of readiness in this population. In future research stratified sampling could be used to ensure that each subgroup is adequately represented. The results from this study can be used as a foundation for structuring a transition program for adolescents with JIA, including tailoring transition care and creating opportunities for HCPs to focus on the parts of the transition that are perceived as challenging by the adolescents. Based on the findings of the present study, it is evident that HCPs working in paediatric care have to provide the adolescents and their parents with information and knowledge so that they can feel safe when transferring to adult care. To guarantee an optimal transition from paediatric to adult care, it is imperative to comprehend the special needs of each adolescent, and acknowledge cultural and contextual differences. The RTQ can be used as a screening tool to discover individual needs. The results of this study show that adolescents need more support to feel ready to take responsibility for specific healthcare-related behaviours and transfer to adult care. The results indicated that the healthcare behaviours most difficult to take responsibility for included adolescents having to make direct contact with healthcare. The parents also perceived that this was the area that was most difficult for the adolescents to take responsibility for. It is important to pay attention to possible gender differences as well as contextual and cultural differences. The RTQ may be a relevant tool to screen for individual needs during the transition process. With the results from this study, we can customize, and thus optimize, transitional care in Sweden for adolescents with JIA. |
SARS-CoV-2 infection: advocacy for training and social distancing in healthcare settings | 4e6157b2-f078-42d4-aa9a-93463145a3b0 | 7414384 | Preventive Medicine[mh] | |
Profile of patients with essential blepharospasm and hemifacial spasm
in the two largest ophthalmology reference centers in Brazil | 6173af80-5305-40f6-8c8f-9998d91d0ec8 | 11630479 | Ophthalmology[mh] | Essential blepharospasm, the most frequent cranial dystonia, and hemifacial spasm are movement disorders affecting the facial muscles. These conditions usually develop and progress slowly over several years . Essential blepharospasm is associated with bilateral and involuntary eyelid spasms, resulting in intermittent periods of forced eyelid closure . In many cases, there are precipitating factors, such as stress, bright light, emotion, and anxiety-provoking social situations . Patients may adopt sensory “tricks”, purposeful maneuvers which, according to patients, temporarily reduce the spasms. Such “tricks” include local compression, singing, and deep breathing to reduce or mask the symptoms . Blepharospasm associated with dystonic movements of other muscle groups in the face, neck, or limbs is known as Meige’s syndrome . Hemifacial spasm is not considered a form of dystonia but a peripheral movement disorder . It is characterized by irregular involuntary tonic and/or clonic contractions of muscles innervated by the facial nerve . Typically, unilateral facial muscle twitching initially affects the orbicularis oculi muscle. After months or years, the paranasal and perioral muscles become affected . In contrast to essential blepharospasm, hemifacial spasms do not fade when the patient is asleep. Emotion and stress are also described as aggravating factors of the disease . Regarding epidemiologic features, essential blepharospasm has its peak onset in the sixth decade and occurs more frequently in women . Women may also have a higher symptom frequency and severity . Many environmental risk factors are thought to be involved in the onset of blepharospasm, such as urbanization, highly demanding jobs, and a stressful lifestyle . Hemifacial spasm commonly has an insidious or subacute onset, usually earlier than essential blepharospasm, with a peak incidence in middle age and affects women more frequently than men . Although life expectancy seems unaffected in patients with essential blepharospasm and hemifacial spasm, the disease notably affects their quality of life . Large-series data are lacking regarding patients with essential blepharospasm and hemifacial spasm in Brazil. The present study aimed to assess the clinical profile of patients with these conditions followed up in Brazil’s two largest ophthalmology services. Apart from demographic and clinical features, past stressful events related to the first symptoms (triggering event), aggravating factors, sensory tricks, and other ameliorating factors for the eyelid spasms were assessed in the two centers. This study complied with the ethical principles of the Declaration of Helsinki and was approved by the institutions’ review boards. We included patients with essential blepharospasm, hemifacial spasm, and Meige’s syndrome, followed up at the Departments of Ophthalmology at Universidade Federal de São Paulo and Universidade de São Paulo . The exclusion criteria included patients with less than 1-year follow-up, patients lost to follow-up, and those with secondary disorders. The data examined were age, sex, diagnosis, age at onset of symptoms, comorbidities, and follow-up time. Additionally, participants underwent a structured interview in which they were asked about major stressful life events that had caused significant personal distress (such as the death of a loved one, divorce, or job loss), the presence of aggravating factors for the eyelid spasms, and the presence of sensory tricks or other ameliorating factors. The study included 102 patients with essential blepharospasm, hemifacial spasm, and Meige’s syndrome, followed up at the two centers. Essential blepharospasm was the most frequent disorder [51/102 (50%) patients]. Hemifacial spasm was found in 46 (45%) patients, while Meige’s syndrome composed a smaller part of the sample [5 (5%) patients] . Concerning the sex of the patients, 33 were male (32.3%) and 69 were female (67.7%). Most patients with essential blepharospasm (84.3%) and Meige’s syndrome (80%) were female . The patients’ ages ranged from 48 to 100 years, with a mean value of 70.63 ± 10.66 years. The mean age of patients with hemifacial spasm, blepharospasm, and Meige’s syndrome was 65.80, 74.22, and 78.40 years, respectively . The mean age at the onset of symptoms was 58.15 ± 12.18 years. The mean age at the onset of symptoms was 54.12, 61.00, and 64.25 years for hemifacial spasm, blepharospasm, and Meige’s syndrome, respectively . The majority of patients (76.5%) had other comorbid chronic diseases; 50 patients (49.0%) had systemic hypertension, 16 (15.7%) had diabetes mellitus, 8 (7.8%) had a thyroid condition, 10 (9.8%) had high cholesterol, 4 (3.9%) had a heart disease, 7 (6.9%) had depression, 6 (5.9%) had glaucoma, and 17 (16.7%) had other comorbidities. Several patients had more than one comorbid disease. The mean duration of disease was 12.30 ± 9.18 (3-40) years. The mean duration for each condition was 11.38 ± 9.76, 12.96 ± 8.33, and 14.75 ± 13.70 years for hemi facial spasm, essential blepharospasm, and Meige’s syndrome, respectively. The follow-up of the patients in the two centers varied between 14 months to 30 years, with a mean value of 7.53 ± 6.16 years. Regarding triggering events, 65 (63.5%) of the patients noted the onset of the disorder was associated with a past stressful event. Other reported events were prior diagnosis of another disease (21.6%), family disagreement (13.7%), death of a loved one (10%), and financial difficulty (7.8%). The presence of triggering events was reported by most patients with hemifacial spasm (74%), essential blepharospasm (55%), and Meige’s syndrome (60%) . Ameliorating factors were reported by 76.5% of patients (80.4% for those with blepharospasm, 82.6% for those with hemifacial spasm, and 60% for those with Meige’s syndrome) . Rest (45.1%), local compression (34.3%), and cold water (9.8%) were the most frequently cited. Apart from local compression, additional 13 (12.8%) patients reported employing a sensory trick as an ameliorating factor, including singing (4.9%), concentrating on a pleasant activity (3.9%), speaking (2%), and whistling (2%). Approximately 87% of the patients confirmed the presence of an aggravating factor. Stress was the most common [52 (51%) patients]; light and fatigue were reported by 26 (25.5%) and 8 (7.8%) patients, respectively. The present study analyzed the clinical features of esse ntial blepharospasm and hemifacial spasm in Brazil’s two largest ophthalmology services. Essential blepharospasm was the most common disorder and was observed in 50% of our patients, corroborating with previous studies . Women most frequently have cranial dystonias, especially in their fifties and sixties . Our results agree with the literature, as we observed a female predominance (67.7%), and the mean age at symptom onset was earlier in hemifacial spasm than in essential blepharospasm (54.12 vs. 61.00 years) . Comorbid chronic diseases were similar to those found in a previous study, with a higher prevalence of hypertension, followed by diabetes and hypercholesterolemia . Moreover, almost 6% of patients in this series had glaucoma (3% with essential blepharospasm and 3% with hemifacial spasm). Patients with essential blepharospasm and hemifacial spasm (on the affected side) have sustained abnormal eyelid tension from involuntary eyelid spasms . Forced eyelid closure may lead to intraocular pressure peaks . Glaucomatous optic neuropathy and corresponding visual field defects were observed in patients with essential blepharospasm. Furthermore, glaucoma-associated morphological findings were observed on the affected side in longlasting hemifacial spasm patients, suggesting that chronic repeated orbicularis contractions may be associated with glaucoma susceptibility . The prevalence of a major stressful triggering event preceding symptoms development was found in 63.5% of our patients. Prior studies reported rates varying from 24% to 72% . Anderson et al. and Johnson et al. also reported the occurrence of a stressful event before the onset of symptoms. Johnson et al. observed that both types of facial spasms began within 1 year of a notably stressful life event in 70% of cases. Major life stressors and grief or depression might play a role in the pathogeneses of essential blepharospasm and hemifacial spasm in genetically susceptible patients . Sensory tricks are significant clinical features of essential blepharospasm . Kilduff et al. and Loyola et al. observed that patients with hemifacial spasm also benefit from these alleviating maneuvers. Although little is known regarding the mechanism, sensory tricks act as relieving factors for the spasms . Several sensory tricks have been described . Local compression is one of the most frequently described tricks and was reported by 34.3% of our patients. Fantato et al. , based on local compression as an alleviating maneuver for eyelid spasms, conducted a study that demonstrated an adjuvant effect of a simple spectacle-mounted device ( Pressop ) in those with essential blepharospasm. In our study, 47% of patients reported using a sensory trick. Kilduff et al. found that 52.7% of patients with essential blepharospasm and 44.6% with hemifacial spasm used ameliorating maneuvers in their series. Almost 87% of the patients from this series confirmed the presence of an aggravating factor for the spasms. Stress was the most common, followed by light and fatigue. Anderson et al. reported that bright light might trigger or exacerbate symptoms in nearly 80% of patients with essential blepharospasm. In another study, patients with hemifacial spasm reported worse spasms in situations of fatigue and anxiety . The main limitation of this study is related to possible recall bias. Because these are chronic conditions, information regarding symptom durations might be inaccurate. In addition, analysis regarding treatment was not performed in the present study because of the different types of botulinum toxins used in the two centers. Moreover, the types of botulinum toxin received in each center varied periodically and precluded any meaningful comparisons. In conclusion, the present study provides information regarding the clinical features of patients followed up at the two largest ophthalmology reference centers in Brazil. Future studies analyzing additional demographics of this population, such as stressful jobs, long working schedules, and information about rural vs. urban living, would be of value to better clarify the role of stressful routines as triggering/ aggravating factors in these patients. |
Whole Slide Imaging (WSI) in Pathology: Current Perspectives and Future Directions | ba040193-ff95-4f7e-a6a0-07be52f2b568 | 7522141 | Pathology[mh] | Digital imaging is widely used by pathologists for creation of static images using microscope-dedicated optical cameras and, more recently, using smartphones . The introduction of whole slide imaging (WSI) in 1999 provided the opportunity of digitally converting the entire tissue on glass slide into a high-resolution virtual slide (VS). In the last two decades, we have witnessed an exponential growth in technology of acquiring virtual slide as well as its applications in various subspecialties of pathology . Development and refinement of artificial intelligence (AI) and machine learning algorithms have been an area of intense research in both radiology and pathology for automated or computer-aided diagnosis. WSI is a promising tool to develop and utilize such algorithms to be used in diagnostic pathology. There has been an ever-increasing demand for telemedicine which surged remarkably in times of lockdowns of the current corona pandemic throughout the globe. As the country prepares itself for telemedicine under the guidance of Medical Council of India , the authors have reviewed the existing literature as a part of preparedness for adopting the new technology in routine pathology, with special reference to cytopathology. Since majority of the available studies pertain to application of WSI in surgical pathology, its utility in cytopathology has not been established well . The authors of this review have been practicing cytopathology for last 3–4 decades and their personal experience has shown that conventional cytology slides pose difficulty in conversion to a good-quality WSI due to the presence of single cells and cell groups in different planes which necessitates repeated focusing. Hence, this review paper, besides encompassing the various aspects of WSI in diagnostic pathology in general, lays a special focus on the available literature on its applications and limitations in cytopathology subspecialty. This review summarizes the technical aspects of WSI, status of current applications, and potential future applications in pathology. The benefits, limitations, and various challenges related to adoption of this technology in routine practice are discussed based on both review of the literature and personal experience of the authors . This review is targeted at medical students, residents, and budding pathologists to apprise them of the basics of WSI technology and accompanying informatics. This is likely to help them make an informed decision related to WSI implementation. Technical Requirements for WSI WSI includes four sequential processes: image acquisition, storage, processing, and visualization. The hardware components of the device required for image acquisition comprise of two systems: image capture and image display. Image capture is performed by a digital scanner, which is basically a trinocular microscope with robotic control of illumination intensity, mechanical stage, objectives, and coarse and fine focusing facilities and is equipped with a high-resolution camera . Unlike the still microscopic images, WSI scanners capture sequential images either in a tiled or line-scanning manner which are subsequently assembled or stitched into a VS, an exact replica of the glass slide . For surgical pathology, the section thickness, placement of section in the center of slide, away from the coverslip edges, avoidance of artifacts of microtomy or mounting have to be optimized . Thick slides or broken slides may not be scanned automatically, since most of the scanners accept only one slide-thick stacks. In this regard, use of liquid-based cytology (LBC) smear or cell blocks offer an advantage due to the standardized approach in preparation and staining. Apart from the x - and y -axes, digital scanners are required to utilize the z -axis of the slide in order to provide an image that appears similar to what we view through the microscope directly. The commercially available scanners differ in their methodology of z-focusing—from every tile to selected tiles or focus points . The highest quality results in WSI are achieved by focusing on every tile during image acquisition, which is a time-consuming strategy leading to compromise in the throughput of the scanner. The resolution of VS depends on the magnification used during scanning. Scanning at × 20 magnification is considered suitable for routine surgical pathology and immunohistochemistry slides . However, the same may not hold true for cytology slides. A study evaluating WSI in cervico-vaginal cytology reported higher diagnostic accuracy with × 40 or × 40 z-stack scanning . For cytological samples, advanced cell layer topographical analysis can allow the samples to be selected for scanning based on the cell distribution on the smear. Such a triage helps both in speeding up the workflow and feedback to the laboratory for modification in collection of certain samples . Continuous technological advances have made available high-throughput scanners (handling up to 400 slides), reduced scanning times (30 s to several minutes), and specialization for digitizing whole tissue mounts, larger glass slides, fluorescent-labeled sections, or smears . The viewing and managing of VS is dictated by the intended use of the WSI system. Different vendors offer differing options such as image viewing software installed locally or as a part of software suite installed on network servers. These image viewers often provide the ability to annotate the image and export to other file formats. A few advanced image viewers such as Surface slide, Aperio ImageScope, and PathXL offer the ability of finger touch annotation using an onscreen virtual keyboard . Use of devices such as smartphones, XDesk, or Microsoft Surfaces provides a large touch screen for interactive discussion of VS images in large meetings and conferences. Applications of WSI in Pathology Health care facilities are witnessing tremendous digitization with inclusion of digital imaging in medical specialities such as radiology connected to hospital information systems (HIS), laboratory information systems (LIS), picture archiving, and communication systems. Pathology laboratories equipped with WSI facility would gel well in such a setting with varied applications in diagnosis, education, and research. Digital Diagnosis, Teleconsultation, Ancillary Studies, Quality Assurance Programs, and Archiving Services Telepathology has been one of the first uses of WSI allowing a pathologist in a centralized laboratory to support peripheral centers in specialized sign-outs, and offering the sender pathologist an opportunity to seek experts’ opinion on a case without incurring the expense or delay in international shipping . Telepathology has been validated for second opinion in challenging cases of surgical pathology, cytopathology, and immunohistochemistry with the American Telemedicine Association issuing guidelines for the adoption of telepathology in patient care . As a step forward, the United States’ Food and Drug Administration (FDA) granted its approval, in 2017 to the first WSI (Philips IntelliSite Pathology Solution™) for primary diagnosis in surgical pathology on the basis of non-inferiority of WSI vis-à-vis glass slide with respect to diagnostic concordance and the reproducibility of repeated scanning . However, a review from the Digital Pathology Committee of the College of American Pathologists highlighted some key issues like the need for laboratories to internally validate WSI in their clinical practice and the opportunity for the pathologists to render diagnosis from locations other than their offices . These queries need to be addressed by the regulatory authorities to ensure a responsible use of WSI with a view to strengthen the place of pathology in the modern era of precision medicine. The other diagnostic applications of WSI in pathology have included intra-operative frozen section diagnosis through remote telepathology consultation with experts. A high concordance rate between WSI-based frozen section and permanent section diagnosis or on-site interpretation has been demonstrated in a number of studies . However, further studies on a range of pathologies from various organ systems are required to validate the utility and limitations of WSI in this field. WSI offers added advantages in enhancing objectivity in the interpretation of immunohistochemistry and electron microscopy used in tumor diagnosis, prognosis, and evaluation of biomarkers for targeted therapy. A concordance of 90% between WSI and glass slides of Her2/neu expression in breast cancer was reported in a recent study . Application of automated image analysis with algorithm-based scoring for the prognostic markers can assist in improving the scoring protocols and thereby enhance the efficacy of targeted therapies . In electron microscopy, virtual ultrathin slide allows the pathologists to navigate the slide in their office while noting the exact location of the specific features. Apart from this, WSI technology can be valuable for obtaining consultation on ultrathin sections from experts located in higher centers . WSI can also facilitate preparation and conduct of tumor boards through obviating the need of a mutiheaded microscope or microscope with projection attachment or acquisition of multiple static images of a case . The use of this technology in quality assurance (QA) programs in surgical pathology and cytopathology can help in cost cutting and overcoming transportation difficulties, as also minimizing the potential second-reviewer bias by hiding the initial diagnosis . Studies have also demonstrated the ease of same-day QA reviews with > 90% diagnostic agreement . The constant and ever-increasing requirement of physical space for storage of glass slides can also be taken care of with WSI and creation of VS. The routine H&E slides as well as special stains, immunochemistry, or fluorescent-labeled slides can be digitally archived while still fresh and free from artifacts. Digital images, if linked to the HIS, shall be readily available in the electronic health record of a patient . Similarly, WSI provides a permanent digital record for cases sent physically for consultation or tissue sent elsewhere for molecular testing, as also for medico-legal and forensic purposes . Pathology Education, Training, and Competency Testing The traditional practice of pathology training and examination through physical microscope-based sessions is witnessing a sea change with recent focus on the ability to diligently identify and interpret rather than the skill of using the microscope . Since glass slides are a scarce resource with the case range dependent on the laboratory or center, WSI provides an opportunity of standardization of pathology education across the country with the same slides being viewed simultaneously by all students. WSI also allows the experts to impart training in interpretation of immunohistochemistry or electron microscopy to pathology residents in remote areas having limited or no exposure to these specialized fields. The WSI images can also be annotated and linked with relevant clinical and/or radiological data so as to provide a holistic view of the diagnostic approach to the students. Use of WSI also obviates the need of having large classrooms with numerous microscopes or multi-headers for teaching the students . WSI offers many benefits to learners as well such as flexibility to engage at their own pace, compare normal and abnormal even on the same screen and foster a team-based learning spirit. The WSI collections can also be employed for pathology examinations and proficiency testing. For instance, the American Board of Pathology utilized 25 VS along with 120 static digital images during a computer-based anatomic pathology examination . Online WSI resources such as CAP Virtual Slide Box, Digital Pathology Association-hosted repository, and the Cancer Digital Slide Archive offer virtual slide sets for training and learning purposes . Virtual slides are also being used in pathology conferences and meetings to promote interactive learning and provide ease of visualization of multiple images of different stains in conjunction with relevant clinical material . Electronic publication of text books and articles in scientific journals has also opened new vistas of scientific communication . Utilization of WSI-generated VS has proven to be the single most upgrade for pathology journals, thus empowering the readers to be involved in a scientifically based diagnostic approach to the lesion described . Research WSI has attracted the attention of biotechnology and pharmaceutical companies because of the opportunity to understand spatial relationship of various biological phenotypes and assistance in development of IHC-based biomarkers that can be utilized further in translational research studies . In conjunction with tissue microarrays, WSI with image analysis tools allows the researchers to assess and score the biomarkers across all specimens quickly and objectively. In instances of possible biomarker heterogeneity, fluorescent WSI or multispectral imaging facilitates multiplexed analysis and supports further biomarker or drug discovery . This technology can also be employed in development of oncologic biomarker strategies with augmented throughput and quantitative accuracy, hence supporting drug discovery . Since advanced WSI scanners can function with transmitted light as well as fluorescent modes, their range of applications in research is radical. WSI includes four sequential processes: image acquisition, storage, processing, and visualization. The hardware components of the device required for image acquisition comprise of two systems: image capture and image display. Image capture is performed by a digital scanner, which is basically a trinocular microscope with robotic control of illumination intensity, mechanical stage, objectives, and coarse and fine focusing facilities and is equipped with a high-resolution camera . Unlike the still microscopic images, WSI scanners capture sequential images either in a tiled or line-scanning manner which are subsequently assembled or stitched into a VS, an exact replica of the glass slide . For surgical pathology, the section thickness, placement of section in the center of slide, away from the coverslip edges, avoidance of artifacts of microtomy or mounting have to be optimized . Thick slides or broken slides may not be scanned automatically, since most of the scanners accept only one slide-thick stacks. In this regard, use of liquid-based cytology (LBC) smear or cell blocks offer an advantage due to the standardized approach in preparation and staining. Apart from the x - and y -axes, digital scanners are required to utilize the z -axis of the slide in order to provide an image that appears similar to what we view through the microscope directly. The commercially available scanners differ in their methodology of z-focusing—from every tile to selected tiles or focus points . The highest quality results in WSI are achieved by focusing on every tile during image acquisition, which is a time-consuming strategy leading to compromise in the throughput of the scanner. The resolution of VS depends on the magnification used during scanning. Scanning at × 20 magnification is considered suitable for routine surgical pathology and immunohistochemistry slides . However, the same may not hold true for cytology slides. A study evaluating WSI in cervico-vaginal cytology reported higher diagnostic accuracy with × 40 or × 40 z-stack scanning . For cytological samples, advanced cell layer topographical analysis can allow the samples to be selected for scanning based on the cell distribution on the smear. Such a triage helps both in speeding up the workflow and feedback to the laboratory for modification in collection of certain samples . Continuous technological advances have made available high-throughput scanners (handling up to 400 slides), reduced scanning times (30 s to several minutes), and specialization for digitizing whole tissue mounts, larger glass slides, fluorescent-labeled sections, or smears . The viewing and managing of VS is dictated by the intended use of the WSI system. Different vendors offer differing options such as image viewing software installed locally or as a part of software suite installed on network servers. These image viewers often provide the ability to annotate the image and export to other file formats. A few advanced image viewers such as Surface slide, Aperio ImageScope, and PathXL offer the ability of finger touch annotation using an onscreen virtual keyboard . Use of devices such as smartphones, XDesk, or Microsoft Surfaces provides a large touch screen for interactive discussion of VS images in large meetings and conferences. Health care facilities are witnessing tremendous digitization with inclusion of digital imaging in medical specialities such as radiology connected to hospital information systems (HIS), laboratory information systems (LIS), picture archiving, and communication systems. Pathology laboratories equipped with WSI facility would gel well in such a setting with varied applications in diagnosis, education, and research. Digital Diagnosis, Teleconsultation, Ancillary Studies, Quality Assurance Programs, and Archiving Services Telepathology has been one of the first uses of WSI allowing a pathologist in a centralized laboratory to support peripheral centers in specialized sign-outs, and offering the sender pathologist an opportunity to seek experts’ opinion on a case without incurring the expense or delay in international shipping . Telepathology has been validated for second opinion in challenging cases of surgical pathology, cytopathology, and immunohistochemistry with the American Telemedicine Association issuing guidelines for the adoption of telepathology in patient care . As a step forward, the United States’ Food and Drug Administration (FDA) granted its approval, in 2017 to the first WSI (Philips IntelliSite Pathology Solution™) for primary diagnosis in surgical pathology on the basis of non-inferiority of WSI vis-à-vis glass slide with respect to diagnostic concordance and the reproducibility of repeated scanning . However, a review from the Digital Pathology Committee of the College of American Pathologists highlighted some key issues like the need for laboratories to internally validate WSI in their clinical practice and the opportunity for the pathologists to render diagnosis from locations other than their offices . These queries need to be addressed by the regulatory authorities to ensure a responsible use of WSI with a view to strengthen the place of pathology in the modern era of precision medicine. The other diagnostic applications of WSI in pathology have included intra-operative frozen section diagnosis through remote telepathology consultation with experts. A high concordance rate between WSI-based frozen section and permanent section diagnosis or on-site interpretation has been demonstrated in a number of studies . However, further studies on a range of pathologies from various organ systems are required to validate the utility and limitations of WSI in this field. WSI offers added advantages in enhancing objectivity in the interpretation of immunohistochemistry and electron microscopy used in tumor diagnosis, prognosis, and evaluation of biomarkers for targeted therapy. A concordance of 90% between WSI and glass slides of Her2/neu expression in breast cancer was reported in a recent study . Application of automated image analysis with algorithm-based scoring for the prognostic markers can assist in improving the scoring protocols and thereby enhance the efficacy of targeted therapies . In electron microscopy, virtual ultrathin slide allows the pathologists to navigate the slide in their office while noting the exact location of the specific features. Apart from this, WSI technology can be valuable for obtaining consultation on ultrathin sections from experts located in higher centers . WSI can also facilitate preparation and conduct of tumor boards through obviating the need of a mutiheaded microscope or microscope with projection attachment or acquisition of multiple static images of a case . The use of this technology in quality assurance (QA) programs in surgical pathology and cytopathology can help in cost cutting and overcoming transportation difficulties, as also minimizing the potential second-reviewer bias by hiding the initial diagnosis . Studies have also demonstrated the ease of same-day QA reviews with > 90% diagnostic agreement . The constant and ever-increasing requirement of physical space for storage of glass slides can also be taken care of with WSI and creation of VS. The routine H&E slides as well as special stains, immunochemistry, or fluorescent-labeled slides can be digitally archived while still fresh and free from artifacts. Digital images, if linked to the HIS, shall be readily available in the electronic health record of a patient . Similarly, WSI provides a permanent digital record for cases sent physically for consultation or tissue sent elsewhere for molecular testing, as also for medico-legal and forensic purposes . Pathology Education, Training, and Competency Testing The traditional practice of pathology training and examination through physical microscope-based sessions is witnessing a sea change with recent focus on the ability to diligently identify and interpret rather than the skill of using the microscope . Since glass slides are a scarce resource with the case range dependent on the laboratory or center, WSI provides an opportunity of standardization of pathology education across the country with the same slides being viewed simultaneously by all students. WSI also allows the experts to impart training in interpretation of immunohistochemistry or electron microscopy to pathology residents in remote areas having limited or no exposure to these specialized fields. The WSI images can also be annotated and linked with relevant clinical and/or radiological data so as to provide a holistic view of the diagnostic approach to the students. Use of WSI also obviates the need of having large classrooms with numerous microscopes or multi-headers for teaching the students . WSI offers many benefits to learners as well such as flexibility to engage at their own pace, compare normal and abnormal even on the same screen and foster a team-based learning spirit. The WSI collections can also be employed for pathology examinations and proficiency testing. For instance, the American Board of Pathology utilized 25 VS along with 120 static digital images during a computer-based anatomic pathology examination . Online WSI resources such as CAP Virtual Slide Box, Digital Pathology Association-hosted repository, and the Cancer Digital Slide Archive offer virtual slide sets for training and learning purposes . Virtual slides are also being used in pathology conferences and meetings to promote interactive learning and provide ease of visualization of multiple images of different stains in conjunction with relevant clinical material . Electronic publication of text books and articles in scientific journals has also opened new vistas of scientific communication . Utilization of WSI-generated VS has proven to be the single most upgrade for pathology journals, thus empowering the readers to be involved in a scientifically based diagnostic approach to the lesion described . Research WSI has attracted the attention of biotechnology and pharmaceutical companies because of the opportunity to understand spatial relationship of various biological phenotypes and assistance in development of IHC-based biomarkers that can be utilized further in translational research studies . In conjunction with tissue microarrays, WSI with image analysis tools allows the researchers to assess and score the biomarkers across all specimens quickly and objectively. In instances of possible biomarker heterogeneity, fluorescent WSI or multispectral imaging facilitates multiplexed analysis and supports further biomarker or drug discovery . This technology can also be employed in development of oncologic biomarker strategies with augmented throughput and quantitative accuracy, hence supporting drug discovery . Since advanced WSI scanners can function with transmitted light as well as fluorescent modes, their range of applications in research is radical. Telepathology has been one of the first uses of WSI allowing a pathologist in a centralized laboratory to support peripheral centers in specialized sign-outs, and offering the sender pathologist an opportunity to seek experts’ opinion on a case without incurring the expense or delay in international shipping . Telepathology has been validated for second opinion in challenging cases of surgical pathology, cytopathology, and immunohistochemistry with the American Telemedicine Association issuing guidelines for the adoption of telepathology in patient care . As a step forward, the United States’ Food and Drug Administration (FDA) granted its approval, in 2017 to the first WSI (Philips IntelliSite Pathology Solution™) for primary diagnosis in surgical pathology on the basis of non-inferiority of WSI vis-à-vis glass slide with respect to diagnostic concordance and the reproducibility of repeated scanning . However, a review from the Digital Pathology Committee of the College of American Pathologists highlighted some key issues like the need for laboratories to internally validate WSI in their clinical practice and the opportunity for the pathologists to render diagnosis from locations other than their offices . These queries need to be addressed by the regulatory authorities to ensure a responsible use of WSI with a view to strengthen the place of pathology in the modern era of precision medicine. The other diagnostic applications of WSI in pathology have included intra-operative frozen section diagnosis through remote telepathology consultation with experts. A high concordance rate between WSI-based frozen section and permanent section diagnosis or on-site interpretation has been demonstrated in a number of studies . However, further studies on a range of pathologies from various organ systems are required to validate the utility and limitations of WSI in this field. WSI offers added advantages in enhancing objectivity in the interpretation of immunohistochemistry and electron microscopy used in tumor diagnosis, prognosis, and evaluation of biomarkers for targeted therapy. A concordance of 90% between WSI and glass slides of Her2/neu expression in breast cancer was reported in a recent study . Application of automated image analysis with algorithm-based scoring for the prognostic markers can assist in improving the scoring protocols and thereby enhance the efficacy of targeted therapies . In electron microscopy, virtual ultrathin slide allows the pathologists to navigate the slide in their office while noting the exact location of the specific features. Apart from this, WSI technology can be valuable for obtaining consultation on ultrathin sections from experts located in higher centers . WSI can also facilitate preparation and conduct of tumor boards through obviating the need of a mutiheaded microscope or microscope with projection attachment or acquisition of multiple static images of a case . The use of this technology in quality assurance (QA) programs in surgical pathology and cytopathology can help in cost cutting and overcoming transportation difficulties, as also minimizing the potential second-reviewer bias by hiding the initial diagnosis . Studies have also demonstrated the ease of same-day QA reviews with > 90% diagnostic agreement . The constant and ever-increasing requirement of physical space for storage of glass slides can also be taken care of with WSI and creation of VS. The routine H&E slides as well as special stains, immunochemistry, or fluorescent-labeled slides can be digitally archived while still fresh and free from artifacts. Digital images, if linked to the HIS, shall be readily available in the electronic health record of a patient . Similarly, WSI provides a permanent digital record for cases sent physically for consultation or tissue sent elsewhere for molecular testing, as also for medico-legal and forensic purposes . The traditional practice of pathology training and examination through physical microscope-based sessions is witnessing a sea change with recent focus on the ability to diligently identify and interpret rather than the skill of using the microscope . Since glass slides are a scarce resource with the case range dependent on the laboratory or center, WSI provides an opportunity of standardization of pathology education across the country with the same slides being viewed simultaneously by all students. WSI also allows the experts to impart training in interpretation of immunohistochemistry or electron microscopy to pathology residents in remote areas having limited or no exposure to these specialized fields. The WSI images can also be annotated and linked with relevant clinical and/or radiological data so as to provide a holistic view of the diagnostic approach to the students. Use of WSI also obviates the need of having large classrooms with numerous microscopes or multi-headers for teaching the students . WSI offers many benefits to learners as well such as flexibility to engage at their own pace, compare normal and abnormal even on the same screen and foster a team-based learning spirit. The WSI collections can also be employed for pathology examinations and proficiency testing. For instance, the American Board of Pathology utilized 25 VS along with 120 static digital images during a computer-based anatomic pathology examination . Online WSI resources such as CAP Virtual Slide Box, Digital Pathology Association-hosted repository, and the Cancer Digital Slide Archive offer virtual slide sets for training and learning purposes . Virtual slides are also being used in pathology conferences and meetings to promote interactive learning and provide ease of visualization of multiple images of different stains in conjunction with relevant clinical material . Electronic publication of text books and articles in scientific journals has also opened new vistas of scientific communication . Utilization of WSI-generated VS has proven to be the single most upgrade for pathology journals, thus empowering the readers to be involved in a scientifically based diagnostic approach to the lesion described . WSI has attracted the attention of biotechnology and pharmaceutical companies because of the opportunity to understand spatial relationship of various biological phenotypes and assistance in development of IHC-based biomarkers that can be utilized further in translational research studies . In conjunction with tissue microarrays, WSI with image analysis tools allows the researchers to assess and score the biomarkers across all specimens quickly and objectively. In instances of possible biomarker heterogeneity, fluorescent WSI or multispectral imaging facilitates multiplexed analysis and supports further biomarker or drug discovery . This technology can also be employed in development of oncologic biomarker strategies with augmented throughput and quantitative accuracy, hence supporting drug discovery . Since advanced WSI scanners can function with transmitted light as well as fluorescent modes, their range of applications in research is radical. Compared with surgical pathology, the application of WSI in cytopathology is inherently difficult due to the three-dimensional nature of the cytology smears. Hence, it is imperative to integrate z-stacking or multiplane scanning into the systems intended for use in cytopathology . The z-stacking can be circumvented by multiplane scanning and use of the best focused image at each tile into the final file. Another approach that has been recently attempted includes the conversion of z-stacks of images into video frames and storing the stack as a high-efficiency video coding file. Subsequent video compression was shown to exceed the JPEG compression with comparable image quality . There have been a few studies on the use of WSI in cytopathology. A comparison of conventional glass slides and WSI in 10 cervical and 20 non-gynecologic cytology cases showed similar diagnostic concordance between the two modalities among reviewing cytopathologists . Another recent study comparing WSI with glass slides of thin-layer cervical specimens demonstrated 95.3% concordance rates, paving the way for WSI use in routine cytologic diagnosis . Wright et al., in their study, evaluated the efficiency of WSI in cervico-vaginal cytology and highlighted issues such as a lack of familiarity with the technology, difficulty for the WSI in detecting few abnormal cells in the smears, problems with hyperchromatic crowded groups, and enormous image file size leading to increased scanning times. Another issue that is probably unique and intrinsic to cytology is the inability of the whole slide scanners to scan the edges of the coverslip . The quality of WSI images when applied to cytology smears is fraught with certain problems that are not encountered in tissue sections, such as (a) presence of dense tissue fragments making it difficult for scanners to focus on the cells, (b) red cell contamination of the smear leading the scanner to focus on red cells in the background rather than the cells of interest, (c) smears with scant cellularity making z-stacking difficult, and (d) need to remove the screening marks/dots before scanning (for which keeping a photographic record of the diagnostic screening marks is recommended) . Pap-stained smears, due to their wet fixation, often have cells in multiple planes and thus require z-scanning to obtain a crisp and good-quality WSI image. On the other hand, air-dried Romanowsky-stained smears can be scanned with only x - and y -axes, since air drying flattens the cells thus minimizing the requirement of z-stacking . WSI, with all its aforementioned advantages, holds promise in various arenas of pathology. However, it is prudent to acknowledge that many unresolved issues, as outlined below, still need to be addressed before WSI finds its place in routine application across the wide specialty of pathology. Cost The cost of procurement, implementation, and operational costs of WSI may be prohibitive, especially for small pathology laboratories due to huge initial cost of the scanners (US $100,000 to US $1,500,000 per piece) and additional hidden costs of training of staff and pathologists, technical support, digital slide storage systems, and regulatory or licensing costs . Technological support for telepathology further compounds these costs. A recently published cost-benefit analysis at a large-volume academic center with slides in excess of 1.5 million showed a projected $1.3 million savings over a 5-year period . However, the same analysis needs to be undertaken for smaller laboratories and low-resource settings. Technological Glitches While considering the implementation of WSI, it must be kept in mind that the WSI images would be only as good as the original glass slide. Scanning the whole slide/smear is a tedious and time-consuming process at present. Scanning times can vary from 1 to 5 min for a small biopsy to 5–20 min for a surgical specimen and 3–5 min for a liquid-based cytology smear . This time can further go up to hour(s) for multiplane or z-stacked scanning. Another limitation with currently available scanners is the requirement of massive data storage capacity. Scanning at × 40 magnification of a 1-mm 2 area results in a file size of 48 megabytes. Hence, majority of the WSI systems incorporate image compression algorithms (JPEG, JPEG 2000, LZW) to reduce the file size. However, image compression introduces image artifacts . Some scanners offer the ability of multi-resolution representation (pyramid representation) where the field of view on the screen is inversely proportional to the magnification being viewed . Majority of the WSI systems utilize a content management system (CMS) with specific programming in order to display the virtual slides in a consistent and specific manner . Currently, there are vendor-dependent limitations with WSI systems. Some vendors use proprietary modules with limited scope of cross-browser compatibility or seamless execution on multiple devices. However, there are some open-source WSI systems such as cuMicroscope, Digital Slide Archive, the Sedeen viewer, and QuPath that support invocation of image analysis software . Storage and confidentiality issues of the VS, especially when linked to the patients’ metadata and clinical information, also need to be given due consideration. If retention of WSI data is necessary, reliable and safe archival with backup strategy such as off-site storage, RAID (redundant array of independent disks) storage, optical storage, or some combinations should be available . Professional Barriers Unlike radiology where digital systems obviate the need of making films, WSI in pathology does not reduce the laboratory’s workload since glass slides still need to be prepared to be scanned. However, WSI does allow for streamlined navigation of the slides at various magnifications without the fear of accidentally breaking a slide at the microscope. The current WSI systems allow for batch-wise scanning of slides, thus improving the efficiency of the laboratory . Other commonly encountered issues include available bandwidth of the network at the pathologists’ workplace, security issues related to information technology, and installation of compatible browsers. However, with progress in information technology, the systems shall continue to be upgraded for improved speed and compatibility with browsers. The USFDA approval of WSI in primary surgical pathology diagnosis does open up the issue of legal implications for the reporting pathologists, as discussed earlier. The relevant regulatory agencies (such as CLIA) need to put forth their guidelines in light of the expected changes with adoption of WSI by pathologists. Regulatory Issues Though USFDA has accorded its approval for use of WSI in surgical pathology practice, the other subspecialties of pathology still have a long path to tread towards this goal. At the same time, validation of WSI for introduction into the surgical pathology practice is still merely a recommendation of College of American Pathologists . Regulations also need to be put in place regarding the archiving, retrieval, and access rights of the virtual slide library so formed. Hence, the choice of the WSI system for a laboratory should consider factors such as the desired application, pertinent resolution to work upon, the time-to-view (time from placing the slide on the scanner to viewing of the VS) and throughput, image analysis options, and the possibility of multiplane scanning and/or integration with the HIS or patient’s electronic health record. The cost of procurement, implementation, and operational costs of WSI may be prohibitive, especially for small pathology laboratories due to huge initial cost of the scanners (US $100,000 to US $1,500,000 per piece) and additional hidden costs of training of staff and pathologists, technical support, digital slide storage systems, and regulatory or licensing costs . Technological support for telepathology further compounds these costs. A recently published cost-benefit analysis at a large-volume academic center with slides in excess of 1.5 million showed a projected $1.3 million savings over a 5-year period . However, the same analysis needs to be undertaken for smaller laboratories and low-resource settings. While considering the implementation of WSI, it must be kept in mind that the WSI images would be only as good as the original glass slide. Scanning the whole slide/smear is a tedious and time-consuming process at present. Scanning times can vary from 1 to 5 min for a small biopsy to 5–20 min for a surgical specimen and 3–5 min for a liquid-based cytology smear . This time can further go up to hour(s) for multiplane or z-stacked scanning. Another limitation with currently available scanners is the requirement of massive data storage capacity. Scanning at × 40 magnification of a 1-mm 2 area results in a file size of 48 megabytes. Hence, majority of the WSI systems incorporate image compression algorithms (JPEG, JPEG 2000, LZW) to reduce the file size. However, image compression introduces image artifacts . Some scanners offer the ability of multi-resolution representation (pyramid representation) where the field of view on the screen is inversely proportional to the magnification being viewed . Majority of the WSI systems utilize a content management system (CMS) with specific programming in order to display the virtual slides in a consistent and specific manner . Currently, there are vendor-dependent limitations with WSI systems. Some vendors use proprietary modules with limited scope of cross-browser compatibility or seamless execution on multiple devices. However, there are some open-source WSI systems such as cuMicroscope, Digital Slide Archive, the Sedeen viewer, and QuPath that support invocation of image analysis software . Storage and confidentiality issues of the VS, especially when linked to the patients’ metadata and clinical information, also need to be given due consideration. If retention of WSI data is necessary, reliable and safe archival with backup strategy such as off-site storage, RAID (redundant array of independent disks) storage, optical storage, or some combinations should be available . Unlike radiology where digital systems obviate the need of making films, WSI in pathology does not reduce the laboratory’s workload since glass slides still need to be prepared to be scanned. However, WSI does allow for streamlined navigation of the slides at various magnifications without the fear of accidentally breaking a slide at the microscope. The current WSI systems allow for batch-wise scanning of slides, thus improving the efficiency of the laboratory . Other commonly encountered issues include available bandwidth of the network at the pathologists’ workplace, security issues related to information technology, and installation of compatible browsers. However, with progress in information technology, the systems shall continue to be upgraded for improved speed and compatibility with browsers. The USFDA approval of WSI in primary surgical pathology diagnosis does open up the issue of legal implications for the reporting pathologists, as discussed earlier. The relevant regulatory agencies (such as CLIA) need to put forth their guidelines in light of the expected changes with adoption of WSI by pathologists. Though USFDA has accorded its approval for use of WSI in surgical pathology practice, the other subspecialties of pathology still have a long path to tread towards this goal. At the same time, validation of WSI for introduction into the surgical pathology practice is still merely a recommendation of College of American Pathologists . Regulations also need to be put in place regarding the archiving, retrieval, and access rights of the virtual slide library so formed. Hence, the choice of the WSI system for a laboratory should consider factors such as the desired application, pertinent resolution to work upon, the time-to-view (time from placing the slide on the scanner to viewing of the VS) and throughput, image analysis options, and the possibility of multiplane scanning and/or integration with the HIS or patient’s electronic health record. As outlined in the present review, WSI is an exciting and promising technology with various advantages and a few challenges to be considered by the interested stakeholders. To overcome the biggest limitation, i.e., the cost or investment factor, numerous attempts have been made to develop cost-effective WSI systems. For instance, a low-cost and high-throughput WSI (OpenWSI) built with open-source hardware and using single-frame autofocusing has been developed . A scalable WSI with smartphones mounted on optical microscopes was developed for android as well as iPhone smartphones . However, before recommending for use, these low-cost systems need to be validated for their quality, scanning reproducibility, and diagnostic accuracy on WSI images. Another challenge posed by vendor-specific modules can potentially hinder optimum utilization of this technology in applications such as telepathology. This can potentially be circumvented by use of open-source software such as Google Earth that requires minimal technical skills or assistance. The future of WSI lies in having an ideal vendor-neutral archive wherein a single software-hardware solution allows single viewing, storage, and retrieval with no barriers of the data source. Validation of the WSI systems in different subspecialties of pathology and conduct of research with adequate sample size inclusive of all organ systems is also the need of the hour. Studies conducted so far have taken into account either a specific organ system or a limited number of cases of more than one body site. For laboratories with intention of incorporating WSI in routine surgical pathology sign-outs, it would be a good practice to undertake a validation study with a mix of the range of body sites and cases (benign and malignant) received by the laboratory. Computer algorithms utilizing image analysis to recognize tumor areas, quantifying tissue features, delineating the tissue architecture, and cellular relationships in WSI images need to be developed and refined to offer computer-assisted diagnosis (CAD). Though CAD has been in vogue for cervical cytology since the late nineties with a number of commercially available systems offering screening and flagging of smears for pathologists’ review , recent international initiatives utilized WSI with CADS in clinical diagnosis, such as Camelyon 17 challenge for a fully automated method to detect nodal metastasis of breast cancer in WSI images or the NIH-led Kidney Precision Medicine Project . WSI in Pathology: Where Does Future Lie? Availability of high-resolution 3-dimensional imaging, especially for tumors, would improve the use of this technology with correlation between radiologic imaging and WSI. Multispectral imaging, when applied to WSI, would offer the ability to characterize chromatic properties and support color-based classification and multi-labeling studies. Adoption of DICOM (Digital Imaging and Communications in Medicine) standards by the WSI vendors would allow vendor-neutral interoperability. Refinement of artificial intelligence and machine learning algorithms would allow the pathologists contribute in a larger role in improving patient management and outcomes. Availability of high-resolution 3-dimensional imaging, especially for tumors, would improve the use of this technology with correlation between radiologic imaging and WSI. Multispectral imaging, when applied to WSI, would offer the ability to characterize chromatic properties and support color-based classification and multi-labeling studies. Adoption of DICOM (Digital Imaging and Communications in Medicine) standards by the WSI vendors would allow vendor-neutral interoperability. Refinement of artificial intelligence and machine learning algorithms would allow the pathologists contribute in a larger role in improving patient management and outcomes. Virtual microscopy using whole slide scanning is an area of profound and rapid technologic development with far reaching and overarching applications in the field of pathology. Despite the advantages and claims of its non-inferiority compared with conventional microscopy, the adoption of this technique has been rather slow even in the developed nations. The barriers referred to in this paper currently preclude the wide application of whole slide scanning in the resource constrained medical institutions of the developing world. Some of these impediments may be overcome by collaborations between a reference laboratory equipped with a WSI system and smaller laboratories, through a hub-and-spoke model. Apart from the technical and cost-related issues, regulatory and validation requirements also need to be adequately addressed, especially for the developing nations. Nevertheless, virtual microscopy does provide a golden opportunity for pathologists to guide its evolution, standardization, and implementation by playing a key role in defining/refining guidelines, designing the resource specific digital pathology laboratories, and propagating standardized educational modules to train the next generation of virtual pathologists. |
A New Perspective on Gas Chromatography-Mass Spectrometry Urinary Metabolomic Analysis and Efficient Risk Assessment of Urolithiasis: Morning Urine Organic Acid Profiles | 9512b32c-fd5f-4d80-b763-3ec60406b8d5 | 11844692 | Biochemistry[mh] | Urolithiasis, also known as urinary stones, is a common urological disorder that affects approximately 5–13% of the global population and is associated with metabolic disorders . The incidence of urolithiasis ranges from 6.4% to 11.6% in China, with higher rates observed in specific regions, reaching up to 22% . Urolithiasis can lead to severe complications such as urinary tract obstruction, hydronephrosis, hematuria, urinary tract infection, and sepsis . In addition, urolithiasis has considerable recurrence with lifetime rates estimated from 60% up to 80% if left untreated, leading to a substantial disease burden . Multiple clinical guidelines recommend conducting 24-h urine metabolic evaluations in patients to prevent future stones . However, many studies indicate limited predictive efficacy of the existing method based on supersaturation alone and insufficient to fully explain the pathology and risk of urolithiasis . Thus, effective preventive strategies, including the identification of new biomarkers based on proper metabolic profiles and the development of effective risk assessment models, are crucial in preventing stone formation and recurrence . Organic acids (OAs) are a common class of small molecule metabolites found in urine. These compounds, which can reflect metabolic characteristics under physiological or pathological conditions, have garnered significant interest as potential biomarkers in urolithiasis research . Oxalate, urate, and citrate have been reported to be closely associated with urolithiasis . In addition, several untargeted metabolomic studies have shown that other urinary OAs are strongly associated with the development of urolithiasis. An NMR-based metabolomic study found that hippuric acid and citrate were significantly different in the 24-h urine of patients with kidney stones compared to controls . Metabolomic analysis based on UPLC-Q-TOF/MS identified 18 differential metabolites from 24-h urine samples, of which OAs accounted for about half (7/18), and most of the OAs had good diagnostic power (AUC >0.7) for calcium oxalate urolithiasis . Untargeted urinary metabolomic analysis of the bladder stone rabbit model found a differentially expressed OA, and subsequently, in vitro experiments demonstrated that it effectively inhibited crystal nucleation . Therefore, OA profiles may provide a more comprehensive stone pathology and help improve the metabolic evaluation of urolithiasis. While untargeted metabolomic studies have provided valuable insights into the association between various OAs and urolithiasis, there is a lack of targeted metabolomic studies specifically focusing on urinary OA markers for urolithiasis. Gas chromatography-mass spectrometry (GC-MS) is a suitable method for the simultaneous detection of hundreds of OAs in urine. In addition, the data generated from metabolomic experiments exhibit high dimensionality with numerous variables and limited samples . It promotes a high demand for analysis methods that enable the full utilization of information-rich metabolic profiles. Machine learning (ML) algorithms provide an approach to guide metabolic prediction by learning experimental datasets, where proper feature selection helps in the removal of unnecessary, redundant attributes from the small-sample disease dataset which, in turn, gives robust and reliable biomarkers and classifies diseases . Based on ML, the robust and effective features are accurately screened out, which can further transform the interpretation of the pathology by the metabolomic features. In this study, we utilized GC-MS to quantitatively detect the profiles of OAs in the morning urine samples obtained from both urolithiasis patients and healthy controls. Through ML algorithms, we aimed to identify potential OA biomarkers and develop a urolithiasis risk prediction model. The findings of this study may facilitate noninvasive assessment of urolithiasis risks and gain insights into the metabolic pathways associated with the pathogenesis of the disease. Ultimately, these findings may improve the early detection, risk assessment, and management of urolithiasis. Sample Collection Patients who had an active episode of urolithiasis were given the option to participate in the current study. We recruited control subjects without a history of urolithiasis. Urine samples were obtained before the first surgery after diagnosis in all patients, who were not under treatment for urolithiasis prior to collection. Between June 2022 and October 2022, we collected a total of 209 samples from two medical institutions: The Eighth Affiliated Hospital of Sun Yat-sen University and the South China Hospital Affiliated to Shenzhen University. Out of these samples, 124 were obtained from patients clinically diagnosed with urolithiasis, while 85 samples were collected from healthy individuals without a history of stones. We divided these samples into a model training set and a test set to develop and evaluate our classification model. In November 2022, we collected an additional 64 samples from the same medical institutions. This set included 32 healthy individuals without a history of stones and 32 patients with clinically confirmed urolithiasis. We designated these newly collected samples as the external validation set, allowing us to independently assess the performance of the developed model. In the collected samples, 46 patients underwent percutaneous nephrolithotomy to obtain their stones. All stones were crushed into powder and analyzed using infrared spectroscopy to determine the main components of the stones. The predominant stone compositions were identified as calcium oxalate (30, 65.22%), carbonate apatite (8, 17.39%), and uric acid (8, 17.38%). We collected morning urine samples from all subjects without the addition of any reagents. All samples were promptly stored at −20°C to ensure sample preservation and stability for subsequent analysis and testing. Metabolite Detection For this study, we conducted data acquisition using a gas chromatograph tandem quadrupole mass spectrometer (GC-MS-QP2030, Shimadzu, Japan) equipped with an autosampler and open split injector. The internal standard solution employed consisted of tetracosane (0.5 mg/mL), margaric acid (MGA, 0.5 mg/mL), and tropic acid (1.0 mg/mL) dissolved in ethyl acetate as the solvent. Following sample treatment with the derivatization reagent BSTFA +1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide plus 1% trimethylchlorosilane, ANPEL, Shanghai, China), a reaction took place for 30 min at a constant temperature of 70°C before cooling and subsequent transfer to the machine. Helium served as the carrier gas, while the chromatographic column maintained a constant flow rate of 1.1 mL/min. During the electron ionization process, we set the ion source temperature to 200°C, the interface temperature to 280°C, and subjected the electron beam to bombardment with 70 eV to generate fragment ions. Mass spectra were obtained within the mass-to-charge ratio (m/z) range of 50 to 500. Quality control materials were used to reflect the reproducibility of the experimental phase. To determine the urine creatinine value, we utilized an ACR-300 (Lifotronic, CN) automatic urine microalbumin creatinine analyzer. Creatinine was used to correct metabolites to reduce the effect of urine concentration or dilution on metabolites. Furthermore, the detection of citric acid was incorporated into the OA detection protocol, and we established a standard curve to accurately obtain the quantitative value of citric acid. We employed ion chromatography (ICS-600, Thermo Fisher Scientific, USA) along with specific reagents to collect and measure the concentrations of sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate, phosphate, and oxalic acid. After filtration, the urine samples underwent extraction and enrichment using an IC-RP solid-phase extraction column, followed by elution. The eluted samples were then subjected to detection by the ion chromatography, where separation occurred on the ion chromatographic column, enabling the quantitative determination of each component. The biochemical method was employed for the determination of urea. Data Preprocessing and Statistical Analysis The collected data were randomly divided into a training set and a test set at a ratio of 7:3. The training set was utilized for bootstrap resampling, model development, and fitting, while the test set was used for preliminary evaluation of the model’s performance. The ensemble learning strategy with resampling methods was used to generate multiple perturbed data sets, which are similar in size and approximate to the original training set, and establish feature selectors. Additionally, a validation set consisting of samples collected at a later stage was used for the final evaluation of the model’s performance and its actual predictive ability (shown in ). Three classifiers, namely, logistic regression (LR), support vector machines (SVMs), and partial least squares discriminant analysis (PLS-DA), were used. LR is a multivariate method used to establish the relationship between multiple independent variables and categorical variables , and the least absolute shrinkage and selection operator (LASSO) was used to shrink the regression coefficients to screen for features . SVM is a classification algorithm that utilizes support vectors, or hyperplanes, to distinguish between two categories . In this study, the widely used radial basis function kernel was employed for SVM, and recursive feature elimination was employed to generate a feature weight sorting list, retaining the largest weight feature until the optimal feature subset was identified . PLS is a projection method that models the relationship between high-dimensional data and a dependent variable by projecting the data into a lower dimensional space known as a latent variable. This helps capture relevant information while reducing dimensionality . PLS-DA is suitable for handling highly correlated metabolic data , which is widely used in metabolomic research because of its ability to understand the causes of discrimination directly via the informative vector, such as variable importance in the projection (VIP) . Demographic and biochemical index characteristics between patients with urolithiasis and healthy controls were compared using the Mann-Whitney U test or independent chi-square test. The data were summarized and analyzed using the median and interquartile range or frequency (n) and fraction proportion . The significance threshold for statistical tests was set at 0.05, considering results with a p value below 0.05 as statistically significant. The evaluation of the classifiers involved comprehensive indicators such as the receiver operating characteristic curve (ROC curve), area under the receiver operating characteristics curve (AUC), accuracy (ACC), sensitivity, and specificity. The implementation of these algorithms was carried out using R software (version 4.2.1) and packages were glmnet, e1071, ropls, ROCR, caret, ggplot2 and tidyverse. Patients who had an active episode of urolithiasis were given the option to participate in the current study. We recruited control subjects without a history of urolithiasis. Urine samples were obtained before the first surgery after diagnosis in all patients, who were not under treatment for urolithiasis prior to collection. Between June 2022 and October 2022, we collected a total of 209 samples from two medical institutions: The Eighth Affiliated Hospital of Sun Yat-sen University and the South China Hospital Affiliated to Shenzhen University. Out of these samples, 124 were obtained from patients clinically diagnosed with urolithiasis, while 85 samples were collected from healthy individuals without a history of stones. We divided these samples into a model training set and a test set to develop and evaluate our classification model. In November 2022, we collected an additional 64 samples from the same medical institutions. This set included 32 healthy individuals without a history of stones and 32 patients with clinically confirmed urolithiasis. We designated these newly collected samples as the external validation set, allowing us to independently assess the performance of the developed model. In the collected samples, 46 patients underwent percutaneous nephrolithotomy to obtain their stones. All stones were crushed into powder and analyzed using infrared spectroscopy to determine the main components of the stones. The predominant stone compositions were identified as calcium oxalate (30, 65.22%), carbonate apatite (8, 17.39%), and uric acid (8, 17.38%). We collected morning urine samples from all subjects without the addition of any reagents. All samples were promptly stored at −20°C to ensure sample preservation and stability for subsequent analysis and testing. For this study, we conducted data acquisition using a gas chromatograph tandem quadrupole mass spectrometer (GC-MS-QP2030, Shimadzu, Japan) equipped with an autosampler and open split injector. The internal standard solution employed consisted of tetracosane (0.5 mg/mL), margaric acid (MGA, 0.5 mg/mL), and tropic acid (1.0 mg/mL) dissolved in ethyl acetate as the solvent. Following sample treatment with the derivatization reagent BSTFA +1% TMCS (N, O-bis (trimethylsilyl) trifluoroacetamide plus 1% trimethylchlorosilane, ANPEL, Shanghai, China), a reaction took place for 30 min at a constant temperature of 70°C before cooling and subsequent transfer to the machine. Helium served as the carrier gas, while the chromatographic column maintained a constant flow rate of 1.1 mL/min. During the electron ionization process, we set the ion source temperature to 200°C, the interface temperature to 280°C, and subjected the electron beam to bombardment with 70 eV to generate fragment ions. Mass spectra were obtained within the mass-to-charge ratio (m/z) range of 50 to 500. Quality control materials were used to reflect the reproducibility of the experimental phase. To determine the urine creatinine value, we utilized an ACR-300 (Lifotronic, CN) automatic urine microalbumin creatinine analyzer. Creatinine was used to correct metabolites to reduce the effect of urine concentration or dilution on metabolites. Furthermore, the detection of citric acid was incorporated into the OA detection protocol, and we established a standard curve to accurately obtain the quantitative value of citric acid. We employed ion chromatography (ICS-600, Thermo Fisher Scientific, USA) along with specific reagents to collect and measure the concentrations of sodium, ammonium, potassium, magnesium, calcium, chloride, sulfate, phosphate, and oxalic acid. After filtration, the urine samples underwent extraction and enrichment using an IC-RP solid-phase extraction column, followed by elution. The eluted samples were then subjected to detection by the ion chromatography, where separation occurred on the ion chromatographic column, enabling the quantitative determination of each component. The biochemical method was employed for the determination of urea. The collected data were randomly divided into a training set and a test set at a ratio of 7:3. The training set was utilized for bootstrap resampling, model development, and fitting, while the test set was used for preliminary evaluation of the model’s performance. The ensemble learning strategy with resampling methods was used to generate multiple perturbed data sets, which are similar in size and approximate to the original training set, and establish feature selectors. Additionally, a validation set consisting of samples collected at a later stage was used for the final evaluation of the model’s performance and its actual predictive ability (shown in ). Three classifiers, namely, logistic regression (LR), support vector machines (SVMs), and partial least squares discriminant analysis (PLS-DA), were used. LR is a multivariate method used to establish the relationship between multiple independent variables and categorical variables , and the least absolute shrinkage and selection operator (LASSO) was used to shrink the regression coefficients to screen for features . SVM is a classification algorithm that utilizes support vectors, or hyperplanes, to distinguish between two categories . In this study, the widely used radial basis function kernel was employed for SVM, and recursive feature elimination was employed to generate a feature weight sorting list, retaining the largest weight feature until the optimal feature subset was identified . PLS is a projection method that models the relationship between high-dimensional data and a dependent variable by projecting the data into a lower dimensional space known as a latent variable. This helps capture relevant information while reducing dimensionality . PLS-DA is suitable for handling highly correlated metabolic data , which is widely used in metabolomic research because of its ability to understand the causes of discrimination directly via the informative vector, such as variable importance in the projection (VIP) . Demographic and biochemical index characteristics between patients with urolithiasis and healthy controls were compared using the Mann-Whitney U test or independent chi-square test. The data were summarized and analyzed using the median and interquartile range or frequency (n) and fraction proportion . The significance threshold for statistical tests was set at 0.05, considering results with a p value below 0.05 as statistically significant. The evaluation of the classifiers involved comprehensive indicators such as the receiver operating characteristic curve (ROC curve), area under the receiver operating characteristics curve (AUC), accuracy (ACC), sensitivity, and specificity. The implementation of these algorithms was carried out using R software (version 4.2.1) and packages were glmnet, e1071, ropls, ROCR, caret, ggplot2 and tidyverse. Univariate Data Analysis Urine biochemical analysis was performed to assess the concentrations of urinary OAs and ionic compounds in both the urolithiasis group and the healthy control group. summarizes the population characteristics of the patient and control groups. In the urolithiasis patient group for the training set and in the validation set, males were included in 71.00% and 59.00%, respectively. In the control group, males represented 25.00% and 44.00%, respectively. Baseline statistics (shown in ) reveal that the control and patient groups show differences in sex ( p value <0.001). To determine whether it has an effect on the metabolites and whether it would significantly bias the classification results, sufficient evidence was collected to exclude this possibility of interference from age differences (shown in online suppl. Fig. S1, S2, S3; for all online suppl. material, see https://doi.org/10.1159/000542263 ). Visualization of the metabolic features by principal component analysis (PCA) for the training set for all acids is shown in . The figure revealed that PCA cannot distinguish between urolithiasis patient (patients) and healthy control (controls) groups because the new component variables with the largest variance produced may not be consistent with the final feature variables. However, it did identify some outliers, suggesting that there was a difference between the two groups but the PCA cannot clearly separate them. Identification of Differential Biomarkers The parameter λ for Lasso regression was determined through cross-validation, and factors with non-zero coefficients were selected. a and b display that the number of high-frequency characteristic markers is nine, including urine pH, calcium ion, sulfate ion, 5- l -pyroglutamic acid-2, palmitic acid-1, 3-hydroxy-phenylacetic acid-2, 2-hydroxy-butyric acid-2, 2-ketoglutaric acid-OX-2(1), 4-hydroxy-phenyllactic acid-3. Recursive feature elimination employed the radial basis kernel function (svmRadial). After parameter adjustment using 10-fold cross-validation, the highest ACC and Kappa values were achieved by selecting 4 traditional risk factors and 9 OA risk factors (shown in c, d). In total, 13 characteristic markers were identified, including calcium ion, chloride ion, sulfate ion, pH, palmitic acid-1, 4-hydroxyphenyllactic acid-3, 5- l -pyroglutamic acid-2, glyoxalic acid-OX-2, pyruvic acid-OX-2, 2-hydroxy-hippuric acid-2, 2-hydroxy-butyric acid-2, pimelic acid-2, 3-hydroxy-isobutyric acid-2. VIP was employed to select deep feature variables as markers from the pool of 132 OA variables and 13 traditional variables, using VIP value >1 as the criteria. e depicts the VIP scores of the variables, where dark green indicates variables with VIP >1 and yellow represents variables with VIP <1. Among the identified markers, there were 25 OAs and 4 traditional factors (shown in ). Notably, OAs such as palmitic acid-1, 2-ketoglutaric acid OX-2(1), 3-hydroxyphenylacetic acid-2, and 5- l -pyroglutamic acid-2 have been confirmed to be associated with stone formation and exhibit significant metabolic mechanisms. Additionally, the markers include classic influencing factors such as calcium ions, pH value, and sulfate. Construction of Risk Assessment Models After selecting the feature variables, an optimal combination was used in the construction of risk assessment model. Compared to the PCA score plot, the PLS-DA score plot based on these distinctive features demonstrated that the urolithiasis patients and healthy control subjects could be well separated, both clustered within a 95% confidence interval (R 2 X (cum) = 0.509, R 2 Y (cum) = 0.736, Q 2 (cum) = 0.633) (shown in a). The first two components explained a cumulative variance of 47%, with PLS-1 (PC1) accounting for 32% and PLS-2 (PC2) accounting for 15% of the variance. PC1 axis included factors such as palmitic acid, pH, calcium ion, sulfate ion, glyoxylic acid, pyruvic acid, and l -pyroglutamic acid. PC2 axis primarily included sex and age, while PC3 axis included glycolic acid, glutaric acid, and others. The six metabolites with high VIP scores, namely, palmitate, ketoglutaric acid, hydroxyphenylacetic acid, l -pyroglutamic acid, pyruvate, and glyoxylate, showed significant differences in urolithiasis patients compared to controls (shown in b, p value <0.001). To address the common issue of data overfitting in establishing PLS-DA models, a permutation test was conducted. In this test, the classification labels of each sample were randomly rearranged, and the model was repeatedly simulated and predicted. c shows 20 random permutations, with the original PLS-DA values displayed as R 2 Y = 0.736 (gray) and Q 2 Y = 0.633 (black). The permutation test values (bottom left) were notably lower than the corresponding original values (top right), the scatter points represent the Q 2 Y and R 2 Y values of the model after randomly permuting the data, with each point assigned a corresponding color. It can be observed that all scatter points are distributed below the horizontal line and do not exceed the Q 2 Y and R 2 Y values of the original model. This confirms the effectiveness of the model and eliminates concerns of overfitting. LR and SVM models were established in the training set. SVM hyperparameter adjustment was performed, resulting in parameters gamma = 2 and cost = 0.2, achieving a lower error value (0.075). The ROC curves of LR, SVM, and PLS-DA in the validation set were compared, yielding corresponding AUC values of 0.899, 0.912, and 0.925, while ACC values were 0.813, 0.844, and 0.859, respectively (shown in d). The sensitivity and specificity of the PLS-DA model were 87.50% and 84.38%, while LR and SVM models achieved 100.00% sensitivity and 68.75% specificity (shown in ). The PLS-DA model demonstrated sensitivity and specificity exceeding 80%, establishing it as a superior discriminator for urolithiasis patients compared to healthy individuals. Notably, the PLS-DA model using only traditional risk factors showed suboptimal performance (AUC = 0.686), whereas the inclusion of OA variables significantly improved the AUC of the model (AUC = 0.925). Reliability Testing Reliability testing was carried out to evaluate the consistency and reliability of the urolithiasis risk assessment using the PLS-DA model. For this purpose, risk assessment scores were calculated for morning urine samples collected from four individuals (1 patient and 3 healthy individuals) over a consecutive 5-day period. These scores were then represented as a line graph (shown in a). The intraclass correlation coefficient is commonly employed to measure the reliability of an indicator across multiple measurements. In the case of the urolithiasis risk assessment, the intraclass correlation coefficient for the repeated measurements over the 5-day period was 0.895. The 95% confidence interval ranged from 0.636 to 0.992, and the p value <0.01. These findings suggest that the risk assessment scores demonstrated moderately high reliability and consistency among the tested healthy individuals and patients. Metabolic Pathway Analysis In the metabolic pathway analysis using the KEGG database (shown in b), we have identified several prominent metabolic pathways that are closely linked to the presence of urolithiasis. These pathways encompass arginine and proline metabolism, fatty acid degradation, glycine, serine, and threonine metabolism, glyoxylate and dicarboxylic acid metabolism, propionate metabolism, purine metabolism, pyrimidine metabolism, and tyrosine metabolism. The significance of metabolite enrichment was determined based on adjusted p values. The intensity of color represents the degree of enrichment, with a darker red indicating a higher level of significance. Notably, among the identified pathways, the fatty acid metabolism pathway exhibited a significant enrichment of the screened differential metabolites. Regarding their association with urolithiasis, the pathways that displayed the strongest correlation, ranked in order of significance, were fatty acid degradation, arginine and proline metabolism, and propionate metabolism. Urine biochemical analysis was performed to assess the concentrations of urinary OAs and ionic compounds in both the urolithiasis group and the healthy control group. summarizes the population characteristics of the patient and control groups. In the urolithiasis patient group for the training set and in the validation set, males were included in 71.00% and 59.00%, respectively. In the control group, males represented 25.00% and 44.00%, respectively. Baseline statistics (shown in ) reveal that the control and patient groups show differences in sex ( p value <0.001). To determine whether it has an effect on the metabolites and whether it would significantly bias the classification results, sufficient evidence was collected to exclude this possibility of interference from age differences (shown in online suppl. Fig. S1, S2, S3; for all online suppl. material, see https://doi.org/10.1159/000542263 ). Visualization of the metabolic features by principal component analysis (PCA) for the training set for all acids is shown in . The figure revealed that PCA cannot distinguish between urolithiasis patient (patients) and healthy control (controls) groups because the new component variables with the largest variance produced may not be consistent with the final feature variables. However, it did identify some outliers, suggesting that there was a difference between the two groups but the PCA cannot clearly separate them. The parameter λ for Lasso regression was determined through cross-validation, and factors with non-zero coefficients were selected. a and b display that the number of high-frequency characteristic markers is nine, including urine pH, calcium ion, sulfate ion, 5- l -pyroglutamic acid-2, palmitic acid-1, 3-hydroxy-phenylacetic acid-2, 2-hydroxy-butyric acid-2, 2-ketoglutaric acid-OX-2(1), 4-hydroxy-phenyllactic acid-3. Recursive feature elimination employed the radial basis kernel function (svmRadial). After parameter adjustment using 10-fold cross-validation, the highest ACC and Kappa values were achieved by selecting 4 traditional risk factors and 9 OA risk factors (shown in c, d). In total, 13 characteristic markers were identified, including calcium ion, chloride ion, sulfate ion, pH, palmitic acid-1, 4-hydroxyphenyllactic acid-3, 5- l -pyroglutamic acid-2, glyoxalic acid-OX-2, pyruvic acid-OX-2, 2-hydroxy-hippuric acid-2, 2-hydroxy-butyric acid-2, pimelic acid-2, 3-hydroxy-isobutyric acid-2. VIP was employed to select deep feature variables as markers from the pool of 132 OA variables and 13 traditional variables, using VIP value >1 as the criteria. e depicts the VIP scores of the variables, where dark green indicates variables with VIP >1 and yellow represents variables with VIP <1. Among the identified markers, there were 25 OAs and 4 traditional factors (shown in ). Notably, OAs such as palmitic acid-1, 2-ketoglutaric acid OX-2(1), 3-hydroxyphenylacetic acid-2, and 5- l -pyroglutamic acid-2 have been confirmed to be associated with stone formation and exhibit significant metabolic mechanisms. Additionally, the markers include classic influencing factors such as calcium ions, pH value, and sulfate. After selecting the feature variables, an optimal combination was used in the construction of risk assessment model. Compared to the PCA score plot, the PLS-DA score plot based on these distinctive features demonstrated that the urolithiasis patients and healthy control subjects could be well separated, both clustered within a 95% confidence interval (R 2 X (cum) = 0.509, R 2 Y (cum) = 0.736, Q 2 (cum) = 0.633) (shown in a). The first two components explained a cumulative variance of 47%, with PLS-1 (PC1) accounting for 32% and PLS-2 (PC2) accounting for 15% of the variance. PC1 axis included factors such as palmitic acid, pH, calcium ion, sulfate ion, glyoxylic acid, pyruvic acid, and l -pyroglutamic acid. PC2 axis primarily included sex and age, while PC3 axis included glycolic acid, glutaric acid, and others. The six metabolites with high VIP scores, namely, palmitate, ketoglutaric acid, hydroxyphenylacetic acid, l -pyroglutamic acid, pyruvate, and glyoxylate, showed significant differences in urolithiasis patients compared to controls (shown in b, p value <0.001). To address the common issue of data overfitting in establishing PLS-DA models, a permutation test was conducted. In this test, the classification labels of each sample were randomly rearranged, and the model was repeatedly simulated and predicted. c shows 20 random permutations, with the original PLS-DA values displayed as R 2 Y = 0.736 (gray) and Q 2 Y = 0.633 (black). The permutation test values (bottom left) were notably lower than the corresponding original values (top right), the scatter points represent the Q 2 Y and R 2 Y values of the model after randomly permuting the data, with each point assigned a corresponding color. It can be observed that all scatter points are distributed below the horizontal line and do not exceed the Q 2 Y and R 2 Y values of the original model. This confirms the effectiveness of the model and eliminates concerns of overfitting. LR and SVM models were established in the training set. SVM hyperparameter adjustment was performed, resulting in parameters gamma = 2 and cost = 0.2, achieving a lower error value (0.075). The ROC curves of LR, SVM, and PLS-DA in the validation set were compared, yielding corresponding AUC values of 0.899, 0.912, and 0.925, while ACC values were 0.813, 0.844, and 0.859, respectively (shown in d). The sensitivity and specificity of the PLS-DA model were 87.50% and 84.38%, while LR and SVM models achieved 100.00% sensitivity and 68.75% specificity (shown in ). The PLS-DA model demonstrated sensitivity and specificity exceeding 80%, establishing it as a superior discriminator for urolithiasis patients compared to healthy individuals. Notably, the PLS-DA model using only traditional risk factors showed suboptimal performance (AUC = 0.686), whereas the inclusion of OA variables significantly improved the AUC of the model (AUC = 0.925). Reliability testing was carried out to evaluate the consistency and reliability of the urolithiasis risk assessment using the PLS-DA model. For this purpose, risk assessment scores were calculated for morning urine samples collected from four individuals (1 patient and 3 healthy individuals) over a consecutive 5-day period. These scores were then represented as a line graph (shown in a). The intraclass correlation coefficient is commonly employed to measure the reliability of an indicator across multiple measurements. In the case of the urolithiasis risk assessment, the intraclass correlation coefficient for the repeated measurements over the 5-day period was 0.895. The 95% confidence interval ranged from 0.636 to 0.992, and the p value <0.01. These findings suggest that the risk assessment scores demonstrated moderately high reliability and consistency among the tested healthy individuals and patients. In the metabolic pathway analysis using the KEGG database (shown in b), we have identified several prominent metabolic pathways that are closely linked to the presence of urolithiasis. These pathways encompass arginine and proline metabolism, fatty acid degradation, glycine, serine, and threonine metabolism, glyoxylate and dicarboxylic acid metabolism, propionate metabolism, purine metabolism, pyrimidine metabolism, and tyrosine metabolism. The significance of metabolite enrichment was determined based on adjusted p values. The intensity of color represents the degree of enrichment, with a darker red indicating a higher level of significance. Notably, among the identified pathways, the fatty acid metabolism pathway exhibited a significant enrichment of the screened differential metabolites. Regarding their association with urolithiasis, the pathways that displayed the strongest correlation, ranked in order of significance, were fatty acid degradation, arginine and proline metabolism, and propionate metabolism. The objective of this study is to elucidate the potential OA biomarkers and the associated metabolic pathways of urolithiasis. To compare the urine samples from the patients with urolithiasis to healthy individuals, in addition to the 13 traditional risk factors recommended by EAU guidelines, we also employed a GC-MS metabolomic approach to quantify the 132 urinary OAs. Moreover, we employed three different ML algorithms to develop the optimal model that could predict the classes of suspected samples. Our findings indicated that when compared to the model built solely using traditional risk factors (AUC = 0.69), the performance of the combined model based on traditional risk factors and OA difference factors improved significantly (AUC = 0.925), suggesting that OAs improved the ACC of predicting urolithiasis. Moreover, the combined model demonstrated good sensitivity (87.50%) and specificity (84.38%), demonstrating its ability to distinguish between samples from patients and healthy individuals with a reasonable level of ACC. Urolithiasis is widely recognized as a condition associated with systemic metabolic abnormalities, which can manifest through urinary metabolites . Multiple clinical guidelines recommend conducting metabolic assessments for patients, particularly through evaluation of 24-h urine supersaturation . However, the pathological mechanisms underlying urolithiasis are intricate, and multiple studies and reviews have indicated that differences in urine supersaturation between patients and healthy individuals are not significant and cannot reliably predict urolithiasis recurrence . Identifying additional potential markers for urolithiasis can contribute to the development of novel preventive strategies and treatment targets for the disease. Urinary OAs, which are abundant metabolites reflecting the activity of major metabolic pathways, have been utilized in assessing health status, nutritional status, vitamin deficiencies, and responses to exogenous substances, all of which are closely linked to the formation of stones . The improvement in model performance observed in this study underscores the value of expanding metabolite analysis, emphasizing the importance of OAs as complementary biomarkers for a more comprehensive characterization of urolithiasis risk. Twenty-five key metabolites (VIP >1) were significantly altered in morning urine samples of urolithiasis patients, involving several key metabolic pathways, including arginine and proline metabolism, fatty acid degradation, glycine, serine, and threonine metabolism, and so on. These pathways are likely implicated in the pathophysiological processes underlying stone formation. The results of the Mann-Whitney U test revealed that patients with urolithiasis showed a significant upregulation of palmitic acid, pyruvic acid, glyoxylic acid, and l -pyroglutamic acid, while 3-hydroxy-phenylacetic acid and 2-ketoglutaric acid were downregulated compared to the healthy control group ( p value <0.01). Among the upregulated OAs, palmitic acid is a well-documented negative factor in human health. Although no studies have directly linked palmitic acid to urolithiasis, excessive amounts of palmitic acid in the body have been associated with metabolic syndrome, cardiovascular diseases, and inflammation . A sedentary lifestyle can contribute to the accumulation of palmitic acid, leading to dyslipidemia, hyperglycemia, and ectopic fat deposition, indirectly affecting crystal deposition in the body . Palmitic acid may also impact kidney function . In a mouse model, it was observed that podocytes exhibited sensitivity to palmitic acid, resulting in depletion of Ca 2+ in the endoplasmic reticulum of podocytes. This subsequently caused changes in cytosolic and mitochondrial matrix Ca 2+ levels, ultimately leading to apoptosis . l -pyroglutamic acid was identified as a metabolite associated with stone formation, possibly related to disturbed glutamate and glutathione metabolism . The pyruvic acid, an important participant in the tricarboxylic acid cycle, is in dynamic equilibrium with citric acid and 2-ketoglutaric acid. Among the downregulated OAs, 2-ketoglutaric acid is a downstream product of citric acid in the tricarboxylic acid cycle . In vitro experiments have indicated that 2-ketoglutaric acid is more effective in dissolving urinary phosphate stones than citric acid . Glutamate metabolism produces α-ketoglutarate and ammonium, and if the conversion of glutamine to α-ketoglutarate is impaired, it will lead to reduced ammonia production in the kidney and increase the possibility of uric acid stones . Additionally, 2-ketoglutaric acid acts as a clearance agent for ammonium ions in the body, reducing toxic levels and serving as a renal function protector . 3-hydroxy-phenylacetic acid, a flavonoid metabolite formed by human gut microbes , has been shown to possess numerous biological activities, including blood pressure reduction, antioxidant, and anti-apoptosis effects , and a decrease in its levels may lead to abnormalities of in-vivo metabolism in humans. At the same time, we conducted a preliminary investigation using existing data on different types of kidney stones to explore the levels of OA metabolites and ionic compounds in morning urine samples across different stone types. Among them, significant differences ( p < 0.05) were observed in the levels of 16 OAs and 4 ionic compounds among different stone types (shown in online suppl. Fig. S4, S5). Changes in these metabolites may be related to the occurrence and development of urolithiasis and reflect their impact on overall human health status or kidney function. However, further research utilizing metabolic network analysis and animal models is necessary to elucidate the specific functional roles and mechanisms of these metabolites in the pathological process. The selected OA biomarkers and enriched metabolic pathways identified in our study demonstrate certain distinctions compared to previous untargeted metabolomic investigations on urolithiasis. First, there is a variance in the scope of metabolites investigated. Our study primarily focuses on the analysis of urinary OAs, whereas other studies encompass the detection and analysis of additional metabolites. Second, different experimental techniques were employed. In our study, we utilized GC-MS analysis, which may differ in terms of separation capability, sensitivity, and qualitative/quantitative ability when compared to techniques such as NMR or UPLC-Q-TOF-MS, especially regarding the preference for target compounds. The choice of distinct techniques can contribute to disparities in metabolite detection and analysis. Lastly, our study employed human morning urine samples, which may yield inconsistent results when compared to previous studies utilizing model animal urine or 24-h human urine. While the assessment, detection, and research of urinary metabolism related to urological stones traditionally rely on 24-h urine samples, there is a growing interest in exploring more convenient alternatives such as morning urine or even random urine. Research by Porowski and Rodriguez have suggested the existence of a “peak period” of stone formation risk within a day, with morning urine potentially providing a more sensitive indication of risk compared to 24-h urine. Although these studies primarily focus on urinary supersaturation, considering the diurnal variation in urine composition among individuals prone to stone formation, it appears that targeted urine analysis at specific times of the day may offer more valuable insights than a comprehensive 24-h urine analysis. Some recent consensus statements advocate for further research in this area . In our study, morning urine was chosen for analysis due to its convenient sample collection and the potential for providing more sensitive risk indications. In this study, we employed an innovative approach utilizing GC-MS detection and interpretable ML algorithms to investigate the variations in OAs between patients with urolithiasis and healthy controls. As a result, several potential OA markers were identified, and a robust model for urolithiasis risk assessment was constructed. This study contributes to an improved understanding of the underlying pathology and the development of more effective prevention strategies for urolithiasis. Additionally, it facilitates the screening and risk assessment of urolithiasis. Nevertheless, it is important to acknowledge the limitations of this study. To further validate the candidate markers and the predictive model, a larger sample size validation is warranted in future investigations. The association between stone components and metabolites also needs further exploration. Furthermore, conducting multicenter prospective cohort studies and exploring the potential of other complementary risk markers, such as body mass index, risk factors in blood, and imaging data, are essential steps in enhancing the comprehensive understanding of urolithiasis. Similarly, comparing the differences among morning urine, random spot urine, and 24-h urine samples is necessary to develop optimal strategies for sample collection. These avenues of research will contribute to the advancement of our knowledge in this field. This research offers valuable insights into the detection method, potential pathogenesis, and risk assessment of urolithiasis. Furthermore, the approach of ML algorithm has highlighted a new perspective of in morning urine GC-MS OA profiles linking of metabolomics with urolithiasis for detailed and comprehensive understanding of the involvement of metabolic perturbations in the biological pathways. It contributes significantly to the progress of clinical translation in the field of urolithiasis and establishes a foundation for future studies and enhancements in risk assessment methodologies. This study was conducted in accordance with the Declaration of Helsinki and approved by the Eighth Affiliated Hospital of Sun Yat-sen University Institutional Review Board (Approval No. 2021-050-01) and the South China Hospital Affiliated to Shenzhen University Institutional Review Board (Approval No. HNLS20220624001-A). Written informed consent was obtained from all the participants in this study. The authors have no conflicts of interest to declare. This study was supported by Shenzhen Science and Technology Program (JCYJ20220818100015031), National Natural Science Foundation of China (No. 21803009), the Science Foundation of Educational Commission of Hubei Province of China (No. T2020008), the Shenzhen Futian District Public Health Research Project (No. FTWS2021066), Guangdong Basic and Applied Basic Research Foundation (2021A1515110402), and Shenzhen Scientific and Technological Research Program (JSGG20201103153801005). Project development, data collection, and material preparation: J.T.Y., D.F.Z., H.X.M., S.W., H.M.L., H.X.Z., J.X.C., and C.L.Z.; data analysis: Y.L., C.Q.Z., P.P.J., and Q.Y.; manuscript writing: J.T.Y., H.M.L., Y.L., P.P.J., and L.P.W.; manuscript reviewing and editing: J.T.Y., D.F.Z., Q.F.L., Z.H.H., Q.Y., H.M.L., Y.L., P.P.J., L.P.W., R.Q.Y., and Y.L.Y; and reading and approval of the final version of the manuscript: all authors. |
Lights, Facts, and Goals: A Novel Framework to Enhance Community Health Messaging Campaign Design, Implementation, and Assessment | b33e948c-cee2-481b-b288-97b590ea024f | 11370212 | Health Communication[mh] | Introduction The fundamental objective of public health messaging is to improve individual and community health. To achieve this goal, health messaging often seeks to promote specific health behaviors. However, the complexity of the health information environment challenges health officials’ ability to affect behavior change. Defeating Health Misinformation Insufficient communication from trusted sources contributed to the success of misinformation during the COVID-19 pandemic . During ongoing and future health threats, doctors, scientists, and public health officials must facilitate health communication from trusted sources to defeat misinformation. Importance of Community Engagement As emphasized in the U.S. Surgeon General’s 2021 report on Confronting Health Misinformation , the employment of “trusted messengers” to reach and communicate with specific populations is growing in use in public health . Community-based organizations (CBOs), with strong ties to community members, are key partners in implementation of effective public health messaging . To standardize the critical role of CBOs in community communication, a model for health communication needs to include CBOs centrally as health communicators. As shown in , we propose the “Source-Communicator-User” model, which builds on previous models of communication but highlights the more active role CBOs can fulfill in effective health communication . In other communication models , CBOs might appear as “channels” for information dissemination. However, CBOs can tailor health information by wording, framing, and translating content to be more usable and relevant to their communities, which gives them a significant role within the health communication process as “communicators.” “Sources,” such as doctors, scientists, and public health officials, can communicate more effectively when amplified through trusted messengers and CBOs . We also deliberately chose the term “user” rather than “receiver,” as in the communication model, to describe community members engaged with health messaging. A user of health information suggests the more active rather than passive role community members have in learning from and acting on health information. The “Source-Communicator-User” model solidifies the necessary relationships scientists, community leaders, and community members should maintain in health messaging campaigns. Need for Messaging-Specific Planning Frameworks Communication professionals in fields like marketing rely on communication frameworks to plan and implement messaging, such as “See, Think, Do” which models consumer behavior to generate purchases. Public health communicators could similarly employ several established public health intervention planning frameworks for messaging to improve health. The PRECEDE-PROCEED model is widely used in public health intervention planning and describes steps to ensure interventions address a population’s needs and tie outcomes to health improvement . The six-step intervention mapping model links intervention design to a specific and modifiable health issue that can be measured and affected through behavioral, environmental, or structural change . Both the U.S. Centers for Disease Control and Prevention (CDC) and the National Cancer Institute (NCI) developed guidance on communication plans and messaging content through Crisis & Emergency Risk Communication (CERC) and Making Health Communications Programs Work or “the pink book” . These frameworks and communication guidance are valuable for public health communicators. However, these resources were primarily developed for use by doctors and officials from health agencies, rather than CBO leaders and trusted messengers who specialize in community engagement. To empower trusted messengers, health messaging requires a framework to guide both health messaging campaign design and messaging content creation that is as simple and self-explanatory as marketing’s “See, Think, Do” framework. While “See, Think, Do” focuses on consumers seeing an advertisement, thinking it is relevant, and doing or making a purchase, a health messaging campaign equivalent should focus on empowering community members with information to make improvements to their health in an authentic way that promotes mutual trust. Thus, we created the “Lights, Facts, and Goals” framework as a succinct planning, implementation, and assessment tool specific for health messaging campaigns, complementary to other established public health frameworks, and easily employed by partnered messaging groups. The fundamental objective of public health messaging is to improve individual and community health. To achieve this goal, health messaging often seeks to promote specific health behaviors. However, the complexity of the health information environment challenges health officials’ ability to affect behavior change. Insufficient communication from trusted sources contributed to the success of misinformation during the COVID-19 pandemic . During ongoing and future health threats, doctors, scientists, and public health officials must facilitate health communication from trusted sources to defeat misinformation. As emphasized in the U.S. Surgeon General’s 2021 report on Confronting Health Misinformation , the employment of “trusted messengers” to reach and communicate with specific populations is growing in use in public health . Community-based organizations (CBOs), with strong ties to community members, are key partners in implementation of effective public health messaging . To standardize the critical role of CBOs in community communication, a model for health communication needs to include CBOs centrally as health communicators. As shown in , we propose the “Source-Communicator-User” model, which builds on previous models of communication but highlights the more active role CBOs can fulfill in effective health communication . In other communication models , CBOs might appear as “channels” for information dissemination. However, CBOs can tailor health information by wording, framing, and translating content to be more usable and relevant to their communities, which gives them a significant role within the health communication process as “communicators.” “Sources,” such as doctors, scientists, and public health officials, can communicate more effectively when amplified through trusted messengers and CBOs . We also deliberately chose the term “user” rather than “receiver,” as in the communication model, to describe community members engaged with health messaging. A user of health information suggests the more active rather than passive role community members have in learning from and acting on health information. The “Source-Communicator-User” model solidifies the necessary relationships scientists, community leaders, and community members should maintain in health messaging campaigns. Communication professionals in fields like marketing rely on communication frameworks to plan and implement messaging, such as “See, Think, Do” which models consumer behavior to generate purchases. Public health communicators could similarly employ several established public health intervention planning frameworks for messaging to improve health. The PRECEDE-PROCEED model is widely used in public health intervention planning and describes steps to ensure interventions address a population’s needs and tie outcomes to health improvement . The six-step intervention mapping model links intervention design to a specific and modifiable health issue that can be measured and affected through behavioral, environmental, or structural change . Both the U.S. Centers for Disease Control and Prevention (CDC) and the National Cancer Institute (NCI) developed guidance on communication plans and messaging content through Crisis & Emergency Risk Communication (CERC) and Making Health Communications Programs Work or “the pink book” . These frameworks and communication guidance are valuable for public health communicators. However, these resources were primarily developed for use by doctors and officials from health agencies, rather than CBO leaders and trusted messengers who specialize in community engagement. To empower trusted messengers, health messaging requires a framework to guide both health messaging campaign design and messaging content creation that is as simple and self-explanatory as marketing’s “See, Think, Do” framework. While “See, Think, Do” focuses on consumers seeing an advertisement, thinking it is relevant, and doing or making a purchase, a health messaging campaign equivalent should focus on empowering community members with information to make improvements to their health in an authentic way that promotes mutual trust. Thus, we created the “Lights, Facts, and Goals” framework as a succinct planning, implementation, and assessment tool specific for health messaging campaigns, complementary to other established public health frameworks, and easily employed by partnered messaging groups. Framework Introduction The process of designing a public health messaging campaign can be described in three segments: “Lights, Facts, and Goals.” “Lights” refers to methods of community engagement that illuminate key elements of health information important to a supported population. Lights could include specific trusted messengers like barbers or stylists talking to community members, billboard advertisements staged in public areas, or text messages sent directly to community members. “Facts” are sourced and concise scientific information that are relevant to a specific and modifiable health threat, such as the increased risk of cardiovascular disease (CVD) in people who smoke. “Goals” are the pertinent, culturally relevant, and realistic behavior changes community members or users of health information can undertake to improve their health, such as quitting smoking or increasing physical activity. Below, we describe how a CBO or health communication team can employ the “Lights, Facts, and Goals” framework in both planning and implementing a health messaging campaign. In planning, the CBO starts with goal-setting, aligns facts to support those goals, and finally chooses the lights or methods of engagement to communicate the facts and goals. As shown in , the CBO, in the “communicator” role, translates prepared content for implementation through specific framing to enhance usability by the supported population. During implementation of the campaign, the CBO evaluates success or failure of the health messaging campaign from both a short- and long-term perspective. Short-term evaluation focuses on message receipt and engagement by the users. Long-term evaluation focuses on metrics of individual or collective behavior change and differences in community health outcomes. Using the Framework—Starting With Goals The “Lights, Facts, and Goals” framework requires initial focus on a desired health objective, such as reduced measures of cardiovascular disease in a specific community. Goals, like increasing physical activity or cutting down on smoking, represent changes to behavior that make individuals more likely to achieve the campaign’s intended improved health outcome. Goals should be specific with research-backed metrics. For example, defining increased activity by minutes allows individuals to guide their own behaviors. Defining goals thus requires significant research to understand the needs and capabilities of a supported community; methods like the BEHAVE framework—which identifies who you are trying to reach, what you want them to do, what factors influence their behavior, and which actions will most effectively address these factors—can help define meaningful and measurable behavioral change goals . As intended through the self-explanatory nature of the “Lights, Facts, and Goals” framework, defining goals translates quickly to what is communicated by the CBO to its users. A clear and concise goal could be set off simply as “ Goal: Replace unhealthy snacks with vegetables and nuts.” Prior to implementation, a CBO could frame this message for their community by using research-supported metrics and other specific wording to increase its relevance and usability. A framed message could read “ Goal: Ditch the chips and candy bars! Try snacking on fresh vegetables and nuts from the downtown farmers markets 2 to 3 times a week instead.” Goal-setting is an established method of behavior change with increasing usage in public health; communicating goals directly to community members allows for measurable outcomes of change and transparency in communication . Aligning Facts to Support Goals Facts provide the meaning and purpose behind the goals included in a “Lights, Facts, and Goals” messaging campaign. Facts must be straightforward, understandable, and supported by verifiable scientific evidence. Through research and consultation with experts, the CBO should define facts relevant both to the specific behavior change and strategic community health objective. Facts can be communicated as simply as “ Fact: Smoking increases your risk for heart disease.” Like the goal previously described, a CBO could use more relevant wording around smoking and increased risk to frame this fact for the community. Using community-level data, a CBO could communicate “ Fact: Among everyone in our county, people who smoke are 2x as likely to have a heart attack.” Choosing “Lights” or Engagement Methods to Reach the Community While facts and goals are communicated explicitly to community members, the lights portion of the “Lights, Facts, and Goals” framework represents different methods of engagement, rather than basic messaging content. Community members could encounter a billboard, read an online advertisement, or receive a text message with a fact and a goal related to a specific health threat. Defining lights to reach the supported community requires analysis of settings, or places community members encounter health information like on public transportation, and channels, or the communication methods used like radio, television, online, or cellular . The CBO or health communication team must ensure selected lights can reach as much of the supported community or users as possible. Specific trusted messengers, like local leaders, clergy members, or barbers and stylists with direct access to their communities, represent effective lights as they can ensure message receipt more directly than an advertisement or a text message . Short- and Long-Term Assessment Assessment of public health communication campaigns in general can be summarized with two questions focused separately on process and effect: “are we doing things right?” and “are we doing the right things?” . For short-term assessment specifically in health messaging, the process question of “are we doing things right?” can be asked as “did the supported community (or users) engage with the messages?” This question addresses the effectiveness of the lights or methods of engagement in the messaging campaign. Engagement can be measured by views, clicks, shares, and response rates or through feedback surveys that rate message utility, relevance, and trustworthiness. The effect question of “are we doing the right things” has both short- and long-term aspects. The short-term aspect of the desired effect is behavior change: “did the supported community (or users) make the desired behavior change?” Since individual adherence to chosen goals generally cannot be measured directly, surveys on behavior intention and behavior can substitute for direct measurement of behavior change. These surveys also can define changes in health literacy and trust in health messaging, as a further assessment of messaging engagement. Both surveys and engagement data, such as views, clicks, and shares, facilitate in-stride assessment of messaging effectiveness, which allows the CBO or health communication team to enhance messaging content and maximize community engagement. The long-term aspect of “are we doing the right things” focuses on the health outcome: “did the supported community (or users) achieve a better health outcome?” This question aligns more with the fundamental objective of public health messaging: to improve individual and community health. For a CBO, attempting to measure a novel aspect of a community health outcome could be costly and difficult; however, seeking to improve a health outcome tracked regularly by a local or regional Department of Health allows for a CBO to follow changes over time. Even if only through correlation, the true success or failure of a public health messaging intervention lies within changes in these specific health outcomes. The process of designing a public health messaging campaign can be described in three segments: “Lights, Facts, and Goals.” “Lights” refers to methods of community engagement that illuminate key elements of health information important to a supported population. Lights could include specific trusted messengers like barbers or stylists talking to community members, billboard advertisements staged in public areas, or text messages sent directly to community members. “Facts” are sourced and concise scientific information that are relevant to a specific and modifiable health threat, such as the increased risk of cardiovascular disease (CVD) in people who smoke. “Goals” are the pertinent, culturally relevant, and realistic behavior changes community members or users of health information can undertake to improve their health, such as quitting smoking or increasing physical activity. Below, we describe how a CBO or health communication team can employ the “Lights, Facts, and Goals” framework in both planning and implementing a health messaging campaign. In planning, the CBO starts with goal-setting, aligns facts to support those goals, and finally chooses the lights or methods of engagement to communicate the facts and goals. As shown in , the CBO, in the “communicator” role, translates prepared content for implementation through specific framing to enhance usability by the supported population. During implementation of the campaign, the CBO evaluates success or failure of the health messaging campaign from both a short- and long-term perspective. Short-term evaluation focuses on message receipt and engagement by the users. Long-term evaluation focuses on metrics of individual or collective behavior change and differences in community health outcomes. The “Lights, Facts, and Goals” framework requires initial focus on a desired health objective, such as reduced measures of cardiovascular disease in a specific community. Goals, like increasing physical activity or cutting down on smoking, represent changes to behavior that make individuals more likely to achieve the campaign’s intended improved health outcome. Goals should be specific with research-backed metrics. For example, defining increased activity by minutes allows individuals to guide their own behaviors. Defining goals thus requires significant research to understand the needs and capabilities of a supported community; methods like the BEHAVE framework—which identifies who you are trying to reach, what you want them to do, what factors influence their behavior, and which actions will most effectively address these factors—can help define meaningful and measurable behavioral change goals . As intended through the self-explanatory nature of the “Lights, Facts, and Goals” framework, defining goals translates quickly to what is communicated by the CBO to its users. A clear and concise goal could be set off simply as “ Goal: Replace unhealthy snacks with vegetables and nuts.” Prior to implementation, a CBO could frame this message for their community by using research-supported metrics and other specific wording to increase its relevance and usability. A framed message could read “ Goal: Ditch the chips and candy bars! Try snacking on fresh vegetables and nuts from the downtown farmers markets 2 to 3 times a week instead.” Goal-setting is an established method of behavior change with increasing usage in public health; communicating goals directly to community members allows for measurable outcomes of change and transparency in communication . Facts provide the meaning and purpose behind the goals included in a “Lights, Facts, and Goals” messaging campaign. Facts must be straightforward, understandable, and supported by verifiable scientific evidence. Through research and consultation with experts, the CBO should define facts relevant both to the specific behavior change and strategic community health objective. Facts can be communicated as simply as “ Fact: Smoking increases your risk for heart disease.” Like the goal previously described, a CBO could use more relevant wording around smoking and increased risk to frame this fact for the community. Using community-level data, a CBO could communicate “ Fact: Among everyone in our county, people who smoke are 2x as likely to have a heart attack.” While facts and goals are communicated explicitly to community members, the lights portion of the “Lights, Facts, and Goals” framework represents different methods of engagement, rather than basic messaging content. Community members could encounter a billboard, read an online advertisement, or receive a text message with a fact and a goal related to a specific health threat. Defining lights to reach the supported community requires analysis of settings, or places community members encounter health information like on public transportation, and channels, or the communication methods used like radio, television, online, or cellular . The CBO or health communication team must ensure selected lights can reach as much of the supported community or users as possible. Specific trusted messengers, like local leaders, clergy members, or barbers and stylists with direct access to their communities, represent effective lights as they can ensure message receipt more directly than an advertisement or a text message . Assessment of public health communication campaigns in general can be summarized with two questions focused separately on process and effect: “are we doing things right?” and “are we doing the right things?” . For short-term assessment specifically in health messaging, the process question of “are we doing things right?” can be asked as “did the supported community (or users) engage with the messages?” This question addresses the effectiveness of the lights or methods of engagement in the messaging campaign. Engagement can be measured by views, clicks, shares, and response rates or through feedback surveys that rate message utility, relevance, and trustworthiness. The effect question of “are we doing the right things” has both short- and long-term aspects. The short-term aspect of the desired effect is behavior change: “did the supported community (or users) make the desired behavior change?” Since individual adherence to chosen goals generally cannot be measured directly, surveys on behavior intention and behavior can substitute for direct measurement of behavior change. These surveys also can define changes in health literacy and trust in health messaging, as a further assessment of messaging engagement. Both surveys and engagement data, such as views, clicks, and shares, facilitate in-stride assessment of messaging effectiveness, which allows the CBO or health communication team to enhance messaging content and maximize community engagement. The long-term aspect of “are we doing the right things” focuses on the health outcome: “did the supported community (or users) achieve a better health outcome?” This question aligns more with the fundamental objective of public health messaging: to improve individual and community health. For a CBO, attempting to measure a novel aspect of a community health outcome could be costly and difficult; however, seeking to improve a health outcome tracked regularly by a local or regional Department of Health allows for a CBO to follow changes over time. Even if only through correlation, the true success or failure of a public health messaging intervention lies within changes in these specific health outcomes. “Lights, Facts, and Goals” Summary The “Lights, Facts, and Goals” framework can be implemented by any organization aiming to improve health communication. The use of established health messaging principles in the creation of the “Lights, Facts, and Goals” framework, such as goal-setting, authenticity, and transparency, suggests its success in implementation during health messaging campaigns. Defining goals and facts initially in planning focuses a health communication team on a specific and modifiable health outcome important to the users or supported community. Using multiple lights or methods of engagement ensures that a messaging campaign will reach its users. Finally, the “Lights, Facts, and Goals” framework translates to content creation more directly than other public health intervention models. CBOs and trusted messengers, specialists in community engagement, can improve community health more effectively through this simple messaging framework and the larger scientific community in a strictly supporting role. The “Lights, Facts, and Goals” framework can be implemented by any organization aiming to improve health communication. The use of established health messaging principles in the creation of the “Lights, Facts, and Goals” framework, such as goal-setting, authenticity, and transparency, suggests its success in implementation during health messaging campaigns. Defining goals and facts initially in planning focuses a health communication team on a specific and modifiable health outcome important to the users or supported community. Using multiple lights or methods of engagement ensures that a messaging campaign will reach its users. Finally, the “Lights, Facts, and Goals” framework translates to content creation more directly than other public health intervention models. CBOs and trusted messengers, specialists in community engagement, can improve community health more effectively through this simple messaging framework and the larger scientific community in a strictly supporting role. |
Complex challenges of estimating the age and vitality of muscle wounds: a study with matrix metalloproteinases and their inhibitors on animal and human tissue samples | 299e1f2d-919c-40d2-a904-917d99185422 | 8354971 | Pathology[mh] | Reconstructing events of physical violence by evaluating the point of time when wounds have been inflicted is a recurring task in forensic routine work. Not surprisingly, wound age estimation has been a central field of forensic research for a long time. Once injured by force, the body tissues respond with numerous molecular and cellular reactions in order to fix the damage: Wound healing is activated. The chronological course of wound healing can be divided in different phases, which are characterized by various processes and mediators . Estimating the wound age and/or the vitality of wounds is based on identifying the phases of wound healing by detecting these processes/mediators and putting them into a temporal context. Besides evaluating the macroscopic appearance of an injury, wound age estimation also comprises an assessment of microscopic and molecular alterations. A recent review by Li et al. stated that progress towards a more precise estimation of vitality and the age of wounds has been made during the last years. However, the authors also point out that an unrestrictedly reliable marker or set of markers has not been identified yet. Research problems are mainly seen in the availability and quantity of human tissue samples with sufficient information on wound age and wound vitality, as well as the results’ reproducibility, the examiner’s experience and methodological limitations. Casse et al. arrive at a similar conclusion. According to their study, research on wound age estimation and wound vitality demands the consideration of various factors to ensure not only high specificity and sensitivity, but also the reproducibility of the results. They underline the necessity of control groups and the differentiation between antemortem and postmortem wounds with a simultaneously adequate number of samples. This necessity derives from the phenomenon of the so-called biological death, meaning the death of each single cell a certain time after the death of the organism, the “individual death”. In 2010, Alaeddini et al. stated that the interval of survival varies in different body tissues due to their own survival mechanisms. In this time span, some physiologic processes might still go on and bias the estimation of wound age. They might even suggest vitality of an injury that has actually been inflicted postmortem. A review by Dunjić et al. also suggests that the activity of cells after the individual death depends on the type of tissue. Consequently, transferring research findings from a specific type of wound to other body tissues is nearly impossible . A recent study of our own research group on myocardial infarctions came up with positive immunohistochemical staining for matrix metalloproteases (MMP) 2 and 9 and their inhibitor TIMP-1 (tissue inhibitor of matrix metalloproteases) not only in ischemic areas, but also adjacent to wounds inflicted mechanically by electrodes or vessel ligations in rats’ hearts . In contrast, myocardial samples without an injury, ischemia, or other determinable harms (increased workload due to pulmonary embolism, cardiac resuscitation) represented no (increased) staining. MMPs are zinc-requiring proteolytic enzymes that are synthetized as zymogens, meaning in a proactive form, and are activated after an injury . In healthy tissue, MMPs are expressed continuously on a low level. Their expression increases when tissues are being remodeled , in physiologic as well as pathologic processes . They play a central role especially in the early phase of wound healing by degrading extracellular matrix (EM) . In humans, more than twenty different, class-divided MMPs have been identified , one important sub-group are the gelatinases (MMP-2 and MMP-9). Due to repeats homologous to fibronectin type II in their catalytic domains, they specifically degrade different types of collagens in the EM . High levels of MMP-2 and MMP-9 were detected in wounds after surgery and in chronic wounds . Furthermore, studies on skin samples and studies on ovarian carcinoma cells have shown that MMP-9 influences wound healing by activating TGF-beta via proteolysis and inducing the expression of vascular endothelial growth factor (VEGF) . A study on MMP-2 knockout mice was able to show that MMP-2 plays a key role in angiogenesis and tumor progression . The activation of MMPs is complex and strictly regulated on multiple stages . The primary control instance are TIMPs, a family of four enzymes (TIMP-1 to TIMP-4). They inhibit MMPs and thus inhibit the degradation of EM. TIMP-1 has a high affinity to MMP-9 and its inactive form, progelatinase B . The interaction of MMPs and TIMPs seems to be important for wound healing . In a review, Conlon et al. stated that MMPs not only act as proteolytic enzymes and inductors of the expression of signal molecules; moreover, they induce the activation of other MMPs. The different ways of MMP activation seem to intertwine. Previous studies also indicate that the ratio between MMPs and TIMPs is of great importance for wound healing. Ladwig et al. showed that the ratio of MMP-9 and TIMP-1 could be used as an indicator of wound healing in wound fluid of pressure ulcers. A dysregulation of MMPs and TIMPs seems to be one of the reasons why wound healing is defective in chronic wounds . In a study on dermal wounds, Gillard et al. discovered elevated expression of MMP-9, MMP-2, and TIMP-1 especially in early phases of wound healing, concluding that MMP-9 might be important for angiogenesis, whereas MMP-2 might play a role in tissue transformation. In addition to our own research results , all these findings suggest that a closer look on the applicability of MMPs and TIMPs in the context of wound age estimation and wound vitality could be worthwhile. Though promising, our findings in the preceding study only gave a hint that MMPs and TIMPS can be detected immunohistochemically in the early phase of wound healing of myocardial tissue—other important questions, however, remained unanswered: Can MMP-2, MMP-9, and TIMP-1 be detected immunohistochemically not only in injured myocardium, but also in skeletal muscle? If so, does their occurrence depend on the “age” of the examined wounds? Are there differences between the two types of muscle tissue? Can the markers help to differentiate between vital and postmortem-inflicted wounds? We aimed on addressing these questions with a broad and complex approach. The immunohistochemical detectability of MMP-2, MMP-9, and TIMP-1 was examined in two types of injured muscle tissue, myocardium and skeletal muscle. Moreover, we worked with postmortem drawn, human tissue samples and with an animal model, the isolated perfused Langendorff heart. This model allowed us to generate myocardial wounds with a defined “age” as well as postmortem-inflicted wounds. Animal experiments were performed in accordance with the German legislation on protection of animals and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). The protocol for the Langendorff system was approved by the local Animal Ethics Committee (project no. O 27/11). The examination of human myocardium and skeletal muscle samples drawn during autopsies was approved by the ethical committee of the Medical Faculty of the Heinrich-Heine-University Düsseldorf (project no. 5833). Human study samples A total of 208 tissue samples of muscle wounds, skeletal muscle (140 samples) and myocardium (68 samples), were selected from 141 autopsies at the Department of Legal Medicine at the University Hospital Düsseldorf, Germany, in the period from 2006 to 2017. We included different types of violence (strangulation, blunt force, sharp force, polytrauma, myocardial injuries due to surgery, and myocardial injuries due to infarction). The age of the tissue donors ranged between 16 months and 94 years and both sexes were included. In a first step, wound age of each sample was estimated roughly according to the available data (assumed time period between infliction of wound and death of the individual). The estimate was refined by additionally considering hematoxylin&eosin (HE) staining results and classifying the findings according to Cummings et al. : A: very short survival time, few min max.—no signs of inflammation, no neutrophilic infiltration B: few min up to 4 h—single perivascular neutrophils C: 4 h up to 8 h—enhanced neutrophilic infiltration D: 8 h up to 12 h—infiltration of neutrophils, macrophages, and fibroblasts Isolated perfused Langendorff heart We used white male Wistar rats aged 2–3 months. The weight ranged between 250 and 350 g. The preparation of the rats’ hearts was performed as described before : The rats were kept on a 12:12 light/dark schedule (lights on at 0600 h) with food and water ad libitum. The animals were anesthetized by intraperitoneal injection of Pentobarbital (90 mg kg −1 ) and Heparin (0.2 ml). The depth of sedation was verified by the absence of reactions to pain. In this state, the rats were decapitated, an immediate thoracotomy was conducted and hearts were excised and mounted onto the Langendorff system. The hearts were perfused with modified Krebs–Henseleit-Buffer: 118 mM sodium chloride (VWR Chemicals Prolabo) 4.7 mM potassium chloride (Fluka) 1.2 mM magnesium sulfate hepta-hydrate (Sigma-Aldrich) 1.2 mM potassium dihydrogen phosphate (Merck) 25 mM sodium hydrogen carbonate (Roth) 0.5 mM ethylenediaminetetraacetic acid (Roth) 11 mM D-glucose (VWR Life Science and Roth) 1 mM L-lactic acid sodium salt (Serva) 2.25 mM calcium chloride (Merck) Heart function was monitored by observing heart rate, intraventricular pressure, and electrocardiogram (ECG). For data digitalization, we used an analog to digital converter (PowerLab/8SP, ADInstruments Pty Ltd., Castle hill, Australia) with a sampling rate of 500 Hz. Data documentation was carried out frequently by using Chart for Windows v5.0 (AD-Instruments). Rats’ hearts with vital wounds After a stabilization period of about 20 min on the Langendorff system, 16 hearts were injured by stabbing the wall of the left chamber with a scalpel. After defined time intervals of 5, 10, 15, 30, 60, 120, 180, and 240 min, the hearts were removed from the Langendorff system and directly immersed in 4% formalin. An overview of the study protocol is given in Fig. . Rats’ hearts with postmortem-inflicted wounds Eight hearts were excised after decapitation without being attached to the Langendorff system. After a defined time interval, they were injured by stabbing the wall of the left chamber with a scalpel: Two hearts each were stitched immediately, 10 min and 180 min after they had stopped beating; after another 180 min, the six hearts were fixed in 4% formalin. Furthermore, two hearts were injured 20 min after they had stopped beating and were fixed in 4% formalin after another 240 min (see also Fig. ). Immunohistochemical analysis Identical staining methods were used for both human study samples and rat study samples and were performed as described before by Mayer et al. : Tissue sections were deparaffinized, washed in distilled water three times for 5 min and washed in TBS buffer with 0.5% Tween 20 two times for 5 min. MMP-2: Slides were boiled in citrate buffer pH 6.0 for 10–15 min, cooled, and then washed in distilled water two times for 5 min. Primary antibody against MMP-2 (Medac, rabbit, E 18,012) was used in a concentration of 1:200, and the slides were incubated over night at + 4 C. Slides were washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 20–25 min. MMP-9: Slides were treated with proteinase K (Dako, S3020) for 7 min and washed two times in TBS buffer with 0.5% Tween 20 for 5 min. Primary antibody against MMP-9 (Biorbyt orb, rabbit, 13,583) was used in a concentration of 1:300, and the slides were incubates over night at + 4 C. Slides were washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 10–15 min. TIMP-1: Primary antibody against TIMP-1 (Biorbyt orb, rabbit, 195,994) was used in a concentration of 1:300, and the slides were incubated over night at + 4 C. Slides were then washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 10–15 min. After blocking of endogenous peroxidase, all slides were washed in distilled water two times for 5 min and then in TBS buffer with 0.5% Tween 20 two times for 5 min. Afterwards, all slides were incubated with a peroxidase-marked polymer (Medac, Histofine1 Simple Stain MAX PO against rabbit, 414,142) for 30 min. Slides were stained with AEC (3-Amino-9-Ethylcarbazole, Cohesion Biosciences) and counterstained with Mayers hematoxylin (Merck, HX87717149). Evaluation system for immunohistochemical analysis To standardize the results of the immunohistochemical analysis, we used the following evaluation system as published before : MMP-9 and TIMP-1: 0: No visible staining I: Positive staining of single cells II: Positive staining of cell groups III: Positive staining of large tissue areas MMP-2: 0: No visible staining I: Positive staining of EM in the perivascular regions II: Positive staining of EM in larger areas III: Positive staining not only of EM but also of intracellular A total of 208 tissue samples of muscle wounds, skeletal muscle (140 samples) and myocardium (68 samples), were selected from 141 autopsies at the Department of Legal Medicine at the University Hospital Düsseldorf, Germany, in the period from 2006 to 2017. We included different types of violence (strangulation, blunt force, sharp force, polytrauma, myocardial injuries due to surgery, and myocardial injuries due to infarction). The age of the tissue donors ranged between 16 months and 94 years and both sexes were included. In a first step, wound age of each sample was estimated roughly according to the available data (assumed time period between infliction of wound and death of the individual). The estimate was refined by additionally considering hematoxylin&eosin (HE) staining results and classifying the findings according to Cummings et al. : A: very short survival time, few min max.—no signs of inflammation, no neutrophilic infiltration B: few min up to 4 h—single perivascular neutrophils C: 4 h up to 8 h—enhanced neutrophilic infiltration D: 8 h up to 12 h—infiltration of neutrophils, macrophages, and fibroblasts We used white male Wistar rats aged 2–3 months. The weight ranged between 250 and 350 g. The preparation of the rats’ hearts was performed as described before : The rats were kept on a 12:12 light/dark schedule (lights on at 0600 h) with food and water ad libitum. The animals were anesthetized by intraperitoneal injection of Pentobarbital (90 mg kg −1 ) and Heparin (0.2 ml). The depth of sedation was verified by the absence of reactions to pain. In this state, the rats were decapitated, an immediate thoracotomy was conducted and hearts were excised and mounted onto the Langendorff system. The hearts were perfused with modified Krebs–Henseleit-Buffer: 118 mM sodium chloride (VWR Chemicals Prolabo) 4.7 mM potassium chloride (Fluka) 1.2 mM magnesium sulfate hepta-hydrate (Sigma-Aldrich) 1.2 mM potassium dihydrogen phosphate (Merck) 25 mM sodium hydrogen carbonate (Roth) 0.5 mM ethylenediaminetetraacetic acid (Roth) 11 mM D-glucose (VWR Life Science and Roth) 1 mM L-lactic acid sodium salt (Serva) 2.25 mM calcium chloride (Merck) Heart function was monitored by observing heart rate, intraventricular pressure, and electrocardiogram (ECG). For data digitalization, we used an analog to digital converter (PowerLab/8SP, ADInstruments Pty Ltd., Castle hill, Australia) with a sampling rate of 500 Hz. Data documentation was carried out frequently by using Chart for Windows v5.0 (AD-Instruments). After a stabilization period of about 20 min on the Langendorff system, 16 hearts were injured by stabbing the wall of the left chamber with a scalpel. After defined time intervals of 5, 10, 15, 30, 60, 120, 180, and 240 min, the hearts were removed from the Langendorff system and directly immersed in 4% formalin. An overview of the study protocol is given in Fig. . Eight hearts were excised after decapitation without being attached to the Langendorff system. After a defined time interval, they were injured by stabbing the wall of the left chamber with a scalpel: Two hearts each were stitched immediately, 10 min and 180 min after they had stopped beating; after another 180 min, the six hearts were fixed in 4% formalin. Furthermore, two hearts were injured 20 min after they had stopped beating and were fixed in 4% formalin after another 240 min (see also Fig. ). Identical staining methods were used for both human study samples and rat study samples and were performed as described before by Mayer et al. : Tissue sections were deparaffinized, washed in distilled water three times for 5 min and washed in TBS buffer with 0.5% Tween 20 two times for 5 min. MMP-2: Slides were boiled in citrate buffer pH 6.0 for 10–15 min, cooled, and then washed in distilled water two times for 5 min. Primary antibody against MMP-2 (Medac, rabbit, E 18,012) was used in a concentration of 1:200, and the slides were incubated over night at + 4 C. Slides were washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 20–25 min. MMP-9: Slides were treated with proteinase K (Dako, S3020) for 7 min and washed two times in TBS buffer with 0.5% Tween 20 for 5 min. Primary antibody against MMP-9 (Biorbyt orb, rabbit, 13,583) was used in a concentration of 1:300, and the slides were incubates over night at + 4 C. Slides were washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 10–15 min. TIMP-1: Primary antibody against TIMP-1 (Biorbyt orb, rabbit, 195,994) was used in a concentration of 1:300, and the slides were incubated over night at + 4 C. Slides were then washed in TBS buffer with 0.5% Tween 20 two times for 5 min. Endogenous peroxidase was blocked with 0.03% H2O2 for 10–15 min. After blocking of endogenous peroxidase, all slides were washed in distilled water two times for 5 min and then in TBS buffer with 0.5% Tween 20 two times for 5 min. Afterwards, all slides were incubated with a peroxidase-marked polymer (Medac, Histofine1 Simple Stain MAX PO against rabbit, 414,142) for 30 min. Slides were stained with AEC (3-Amino-9-Ethylcarbazole, Cohesion Biosciences) and counterstained with Mayers hematoxylin (Merck, HX87717149). To standardize the results of the immunohistochemical analysis, we used the following evaluation system as published before : MMP-9 and TIMP-1: 0: No visible staining I: Positive staining of single cells II: Positive staining of cell groups III: Positive staining of large tissue areas MMP-2: 0: No visible staining I: Positive staining of EM in the perivascular regions II: Positive staining of EM in larger areas III: Positive staining not only of EM but also of intracellular General observations Whereas positive staining for MMP-9 and TIMP-1 was found strictly intracellular, positive staining reactions for MMP-2 were also found in the EM. These findings correlate with the results of Mayer et al. . Slides from human samples that had been stored in formalin for a longer time showed less distinct staining results compared to those samples collected more recently (2015–2017). In order to exclude a relevant influence of storage time on our results, we evaluated the slides of the “older” cases separately and compared them to the “younger” ones without finding any differences. We also checked, if the causative type of violence has an impact on the occurrence of the markers, again, no differences were found. Therefore, the results in this publication comprise all collected samples without separating them into the different types of violence for a clearer depiction. Human skeletal muscle Table is enclosed for detailed results; examples for staining results are presented in Fig. . MMP-9Positive staining results for MMP-9 were found in all wound age groups, even in samples with very short survival times. The intensity of staining was mostly equivalent to grade II or even III. However, there was also a considerable number of samples showing no positive staining at all. The share of samples with negative staining was especially large in wound age group D. TIMP-1The majority of samples of wound age groups A and B presented positive staining results with an intensity according to grade II or III. In wound age groups C and especially D, the share of samples with negative staining was larger. MMP-2Strong positive staining results for MMP-2 according to grades II and III were found in all samples regardless of the wound age group. Negative staining was found in age groups B to D but their share was rather small. Human myocardium Table presents the detailed results for human myocardium injuries. Examples for staining results can be found in Fig. . MMP-9A high number of samples in wound age group A showed staining results with intensities grade II and III. The share of grade III was especially high in wounds that followed an infarction. However, nearly half of the samples in group A showed no positive staining. Similar findings could be observed for group B. The single sample in group D presented no positive staining. TIMP-1The majority of samples of wounds in group A showed results with staining intensities grade II and III. Again, the share of grade III was especially high in wounds that followed an infarction. Also, a considerable number of samples showed no positive staining at all. The same accounted for samples in group B. The one sample in group D presented a staining intensity grade I. MMP-2Staining results in group A mainly presented intensities grade II and III with a considerable high share of grade III in infarction-derived wounds. In group B, the share of samples with a staining intensity grade I was greater. The sample in group D also presented positive staining with intensity grade I. Negative staining results for MMP-2 were only found in single cases. Rats’ hearts—vital wounds Table shows the detailed results of rat hearts with vital wounds. MMP-9 Only two positive staining results with intensity grade I were observed in one heart with a survival time of 30 min and in one heart with a survival time of 3 h. In all other cases, staining was negative. TIMP-1 There was only one heart with a survival time of 1 h that showed discreet positive staining. All other hearts showed no positive staining at all. MMP-2 Nine hearts showed positive staining results with intensities grade I and especially grade II. The shortest survival time with positive staining results was 15 min. From the hearts with longer survival times, only one heart with a survival time of 1 h stained positive. Rats’ hearts—postmortem-inflicted wounds Table shows the detailed results of rats’ hearts with postmortem-inflicted wounds, and examples of staining results are presented in Fig. . MMP-9 Three hearts presented positive staining results with intensities grade I and II. The time spans between the end of heartbeat and the infliction of the wounds varied between 0 min and 3 h In all cases, and the time span after wound infliction was 3 h. MMP-2 Three hearts presented negative staining results, including the two hearts with wounds inflicted 20 min after the heart had stopped beating. All the other hearts showed positive staining with intensities grade I and especially grade II. TIMP-1 None of the hearts presented positive staining results. Whereas positive staining for MMP-9 and TIMP-1 was found strictly intracellular, positive staining reactions for MMP-2 were also found in the EM. These findings correlate with the results of Mayer et al. . Slides from human samples that had been stored in formalin for a longer time showed less distinct staining results compared to those samples collected more recently (2015–2017). In order to exclude a relevant influence of storage time on our results, we evaluated the slides of the “older” cases separately and compared them to the “younger” ones without finding any differences. We also checked, if the causative type of violence has an impact on the occurrence of the markers, again, no differences were found. Therefore, the results in this publication comprise all collected samples without separating them into the different types of violence for a clearer depiction. Table is enclosed for detailed results; examples for staining results are presented in Fig. . MMP-9Positive staining results for MMP-9 were found in all wound age groups, even in samples with very short survival times. The intensity of staining was mostly equivalent to grade II or even III. However, there was also a considerable number of samples showing no positive staining at all. The share of samples with negative staining was especially large in wound age group D. TIMP-1The majority of samples of wound age groups A and B presented positive staining results with an intensity according to grade II or III. In wound age groups C and especially D, the share of samples with negative staining was larger. MMP-2Strong positive staining results for MMP-2 according to grades II and III were found in all samples regardless of the wound age group. Negative staining was found in age groups B to D but their share was rather small. Table presents the detailed results for human myocardium injuries. Examples for staining results can be found in Fig. . MMP-9A high number of samples in wound age group A showed staining results with intensities grade II and III. The share of grade III was especially high in wounds that followed an infarction. However, nearly half of the samples in group A showed no positive staining. Similar findings could be observed for group B. The single sample in group D presented no positive staining. TIMP-1The majority of samples of wounds in group A showed results with staining intensities grade II and III. Again, the share of grade III was especially high in wounds that followed an infarction. Also, a considerable number of samples showed no positive staining at all. The same accounted for samples in group B. The one sample in group D presented a staining intensity grade I. MMP-2Staining results in group A mainly presented intensities grade II and III with a considerable high share of grade III in infarction-derived wounds. In group B, the share of samples with a staining intensity grade I was greater. The sample in group D also presented positive staining with intensity grade I. Negative staining results for MMP-2 were only found in single cases. Table shows the detailed results of rat hearts with vital wounds. MMP-9 Only two positive staining results with intensity grade I were observed in one heart with a survival time of 30 min and in one heart with a survival time of 3 h. In all other cases, staining was negative. TIMP-1 There was only one heart with a survival time of 1 h that showed discreet positive staining. All other hearts showed no positive staining at all. MMP-2 Nine hearts showed positive staining results with intensities grade I and especially grade II. The shortest survival time with positive staining results was 15 min. From the hearts with longer survival times, only one heart with a survival time of 1 h stained positive. Table shows the detailed results of rats’ hearts with postmortem-inflicted wounds, and examples of staining results are presented in Fig. . MMP-9 Three hearts presented positive staining results with intensities grade I and II. The time spans between the end of heartbeat and the infliction of the wounds varied between 0 min and 3 h In all cases, and the time span after wound infliction was 3 h. MMP-2 Three hearts presented negative staining results, including the two hearts with wounds inflicted 20 min after the heart had stopped beating. All the other hearts showed positive staining with intensities grade I and especially grade II. TIMP-1 None of the hearts presented positive staining results. We addressed the challenges that go along with forensic wound age estimation as illustrated above by a unique sample collection comprised in the study: Not only were we able to include two different types of tissues (skeletal muscle and myocardium) from two species (humans, rats), the sample set also includes wounds with a defined wound age and reliably postmortem-inflicted wounds thanks to the Langendorff system. Thus, we not only took advantage of controlled experimental conditions in the rat model but also examined the applicability of the results gained on human tissue samples. Despite this broad approach, the presented results are very inhomogeneous and show great “scattering”. We found positive staining for all the tested markers in a considerable number of human samples regardless of their origin and the wound age. However, the same accounts for negative staining results (see Fig. for examples of human skeletal muscle). If any, there was a slight tendency of more intense staining results towards cases with a “younger” wound age: Apparently, the number of samples showing no positive staining or less intense staining increased with higher wound age, implying that the markers we evaluated occur shortly after the infliction of a wound and disappear rather fast. Similar findings have also been published by Wang et al. : In skin wounds of mice, high levels of MMP-9/MMP-2 seem to suggest an earlier stage of wound healing. The authors came to the conclusion that an increased expression and activation of MMP-2 might be important for the inflammation phase following an injury rather than being crucial for the process of wound healing. Regarding the results of human samples with wound age group A (very short survival time, few min max.), the share of those with staining intensities grade III was slightly higher in heart muscle samples than in skeletal muscle samples. This might lead to the assumption that wound healing and the emergence of the evaluated markers kick in faster in myocardium. However, a closer look on the myocardium samples revealed that the high staining intensities are mainly found in infarction-derived injuries. Though the differences were rather discreet, it is still obvious that staining of MMP-2, MMP-9, and TIMP-1 in younger wound age groups was more intense in cases with an underlying infarction compared to those with injuries of other origins. Therefore, we assume that in infarctions, the already existing inflammation might have caused an earlier expression and/or activation of the markers, which goes along with other research findings . Since particularly inner organs, but also skin in certain cases, might present acute or chronic illnesses, statements on wound age based on the detection of an inflammation-related marker have to be made cautiously. Referring to the review by Li et al. , it appears that “[…] wound age estimation is an intricate and multifactorial problem […]” which means that numerous intrinsic and extrinsic factors must be taken into account when trying to determine the age and vitality of a wound. Compared to human samples, rat hearts with vital wounds presented rather few positive staining results especially for markers MMP-9 and TIMP-1. In addition, positive staining was found earliest in cases with a wound age of 15 min. In many of the human cases, a shorter survival time has to be assumed. At least in part this alleged contradiction might be connected to the fact that tissues/cells can “survive” the death of the individual for some time. In case of the rats’ hearts, “wound age” refers to the time between infliction of wounds and fixation of the hearts in formalin. Under such circumstances, the death of all cells occurs almost simultaneously and at the same point of time as the death of the individual, i.e. the heart. Since such a sudden stop of all intracellular activities does not apply for the human cases, wound age processes that have been triggered at or shortly before the time of death might still have proceeded and caused the emergence of the investigated markers some time later—simulating a faster occurrence in human tissue. In addition, the lack of blood in the Langendorff system might result in a delayed activation and/or expression of MMPs and TIMPs since relevant mediators might be missing. Dunjic et al. already stated that after the individual death of a person, some cells are still active. Referring to this review, the time span during which the cells are active also seems to depend upon the type of tissue. According to Tsujimoto et al. , the time of cell death is also influenced by ATP levels. Fibroblasts in human skin samples could be analyzed several days postmortem . In this context, supravital reactions even several hours after the individual death can be explained. White blood cells seem to remain active for up to 12 h postmortem, which questions the presence of an inflammation as a vital reaction after injury. Additionally, Alaeddini et al. and Jennings et al. also described different intervals of survival due to different tissue mechanisms and stated that necrosis starts in defined regions of every organ, such as the subendocardial regions in the human heart. Compared to other organs, skeletal muscle tissue seems to show postmortem ultrastructural changes quite late . In a study on lamb muscle, Sylvestre et al. were able to provide evidence of postmortem activity of MMP-2. High levels of pro-MMP-2 and of active MMP-2, but also of active MMP-2, were detected not only on the day of slaughter, but also 21 days later in samples that had been stored at 4 °C. The high levels of MMP-2 led to the assumption that MMP-2 is involved in the degradation of the tissue. The problem of distinguishing between vital wounds and postmortem-inflicted wounds was also described in a review by Cecchi et al. in 2010 who pointing out that a variability in their detection makes many markers unreliable when it comes to this question. Furthermore, the methods used for detecting a marker, e.g. polymerase chain reaction or IHC, seem to have an impact upon the results. Against this background, the behavior of the markers tested in our study is not surprising. The occurrence of MMP-2 and MMP-9 in vital and postmortem-inflicted wounds does not show obvious differences. Merely for TIMP-1, some interesting results were obtained: Although there were many positive staining results in human samples, rats’ hearts with vital wounds presented positive staining for TIMP-1 only in one case with a wound age of 1 h. Furthermore, there were no positive staining results for TIMP-1 in rats’ hearts with postmortem-inflicted wounds. The overall picture of these findings suggests that TIMP-1 is not as sensitive as MMP-2 and MMP-9, implying a possible use as a vitality marker. To verify this hypothesis, TIMP-1 needs to be tested on human muscle samples with reliably postmortem-inflicted wounds. Unfortunately, such samples are difficult to obtain and were therefore not comprised in the study at hand. Our study is subjected to some limitations: Our collected samples of human tissue only include wounds with a wound age up to 12 h. The maximum post-infliction time span of rats’ hearts accounted for 240 min. We therefore have no information about the behavior of the markers when used on wounds that are days or even weeks old. In addition, the available data for the tissue samples drawn during autopsies underlie some uncertainties, especially with a view to the exact time of the infliction of the wounds. Uninjured control samples were already included in the preceding study , whereas samples of human skeletal muscle with postmortem-inflicted wounds and a reasonable postmortem interval are difficult to obtain and therefore could not be examined. Furthermore, the number of rat tissue samples examined in this study might seem comparably low. We resigned from increasing the number due to ethical aspects. Our study again demonstrates the challenges that go along with forensic wound age estimation and the establishment of new markers. Despite our complex approach of examining MMP-9, MMP-2, and TIMP-1 on both human and rat muscle tissue, as well as on vital and postmortem-inflicted wounds, we were faced with disappointing results. Though unexpected findings in a preceding study on rat hearts were quite promising, the results of the far more comprehensive sample collection show a very inhomogeneous picture leading to the conclusion that the markers do not meet the complex requirements of forensic wound age and wound vitality estimation. Only TIMP-1 might be of use when trying to differentiate between vital and postmortem-inflicted wounds but it needs to be tested on postmortem-inflicted wounds of human muscle samples. Overall, it became clear again that a profound understanding of the usefulness of potential markers can only be achieved by examining a variety of samples. The sample collection needs to include vital wounds and postmortem-inflicted wounds. When working with an animal model, human control samples are indispensable; otherwise, the transferability of results remains questionable. The same accounts for different types of tissues. Finally, potential influences of acute or chronic illnesses have to be kept in mind when interpreting analytical results. |
Review of robotic surgery platforms and end effectors | 42c8980f-f1bb-498a-8344-fcb0cd836d5c | 10864559 | Gynaecology[mh] | The advantages of robotic surgery in terms of safety, precision, and accuracy have made it more and more popular in recent years . Today the market is offering a wide range of surgical robots. The competition among surgical robot producers helps us to raise standards and improve their value/cost ratio. The review aims to describe the robotics devices produced by some of the most popular manufacturing companies. Special attention is devoted to the end effectors, key components essential for performing the surgical task . The dexterity of end effectors is crucial for the correct execution of surgical procedures. End-effectors have the task of manipulating tissues and instruments. They can have small dimensions and an extremely fine tip, to reach and grip tissues that sometimes are very delicate. Reusable end effectors can be built using titanium or stainless steel, which are resistant to corrosion and degradation. Disposables are often made of 304 stainless steel, while reusable of 316L or equivalent. Both 304 and 316L are suitable for pressing and sintering in metal injection molding (MIM), while 316L has the highest level of corrosion resistance and is stronger at high temperatures. Abrasive materials allow the tip of the end effectors to have a better grasp. The most common shapes of end effectors are: Maryland : these are used to grasp and manipulate delicate tissues, such as blood vessels and nerves. Curved : these are used to reach hard-to-access areas. Traumatic : they have a claw-shaped tip, which allows you to grip and hold tissues. They are used to manipulate delicate tissues, such as the skin or blood vessels. Fenestrated : they have a sharp tip with a set of teeth, allowing you to grip and hold tissues more securely. They are used to manipulate thicker tissues, such as muscles or tendons. Pre-tensioned needle holders : they are used to suture tissues or to grasp needles and other instruments. Microsurgery end effectors are used in a variety of surgeries, including: Plastic surgery : for the correction of cosmetic imperfections or the reconstruction of damaged tissues. Orthopedic surgery : for the repair of bones, joints, or tendons. Ophthalmic surgery : for the correction of visual defects or the removal of cataracts. Dental surgery : for the removal of cavities or the implantation of artificial teeth. Neurosurgery : to treat nervous diseases like brain tumors. Microsurgery end effectors are essential tools for precision surgery. Thanks to their extremely fine tip and robust construction, they allow you to perform delicate and complex surgeries with the utmost precision. Open surgery, compared to minimally invasive surgery, allows more freedom of movement, and better feedback of the scene. Patients subjected to a minimally invasive approach enjoy shorter recovery time (smaller incisions), less infection risk, lower pain, and less blood loss. The minimally invasive approach is relatively expensive in terms of equipment cost, setup time and surgeon training. Surgeons operating with minimally invasive robots suffer less fatigue but may be more stressed, due to the loss of direct contact with the body of the patient. Hands tremor filtering, view magnifications and motion scaling are nice features that raise the price of minimally robotic surgery compared to minimally invasive surgery. The dexterity of robotic surgery tools is high, while their control can be less intuitive/direct than the handheld laparoscopic surgery tools. Minimally invasive robotic surgery allows the best precision and the ability to access remote places inside the human body. The objective of the research is to collect, in a single place, a selection of surgical robots. Due to space constraints, only a few robots are recalled. For each platform, the main features are mentioned and compared. Robots' accuracy and sensitivity are crucial. A discussion about disposable, reusable and reposable instruments is reported: there is a trade-off between the number of usages and instruments cost. Most advanced robots feature tremor control and Stereo-optics. To enhance the instrument’s dexterity a wrist or a snake articulation is provided. Commercial products This section of the review examines the offerings of some of the most well-known manufacturers of surgical robots. The choice of the right surgical platform is not easy. Several aspects need to be considered. Each product has its characteristics. Each robot can perform specific surgical procedures. Moreover, according to the size and dexterity of the end effector, the same procedure may be executed differently. The accuracy of the execution of the task is also a specific characteristic to consider. Patient safety is always of paramount importance; safety measures can vary among surgical platforms. There is always an important trade-off between the device’s capabilities and the time the surgeons need to learn and operate. The level of training and support offered is important to optimize the surgeon’s learning curve . The surgical platforms may have different purchase and operating costs. System versatility and availability are also important; some systems may be available only in certain regions while others may be suitable only for a limited set of procedures—statistics on surgical operation success rate and post-recovery time help to consider the patient benefit. According to our perspective, user and patient feedback can be used best to assess the performance of a surgical platform. For each firm considered, location and foundation date are reported (Table ). Some companies were founded many years ago while their first surgical robots were more recently developed. The list of surgical robot manufacturers provided, while not exhaustive, allows us to take an outlook of the sector. The United Kingdom has an old tradition of surgical robotics. The United States is one of the most important players. Europe is showing a growing interest in the field. Asia is also following this global innovation trend. The data of Table finds a graphic representation in Fig. . A description of each of the selected firms is now provided. Their main commercial products are shortly described. Accuray Incorporated Accuray Incorporated focuses on developing cutting-edge medical technology solutions for the treatment of cancer and other illnesses. The business is headquartered in Sunnyvale, California, and was established in 1990. To provide precise and accurate control during radiation therapy procedures, Accuray has created sophisticated robotic systems. The company’s commercial product line-up includes CyberKnife , TomoTherapy system and Radixact Treatment Delivery System . Stereotactic body radiation therapy and stereotactic radiosurgery are two applications for CyberKnife. The system minimizes damage to surrounding healthy tissue, by accurately delivering radiation to malignancies, using cutting-edge imaging technology. With the TomoTherapy system, treatment regimens may be tailored precisely and individually for image-guided intensity-modulated radiation therapy. The Radixact Treatment Distribution System enables precise radiation delivery during treatment. The end effectors made by Accuray are used in radiation treatment procedures in conjunction with their robotic systems. To meet diverse treatment requirements, the end effectors are available in a range of sizes and shapes. Because of its simple interchangeability, procedures can be carried out with more flexibility. Asensus surgical TransEnterix was founded in 2006 in North Carolina. Since February 2021, TransEnterix has changed the name to Asensus Surgical. The international company, formed by over 200 employers, is focused on surgical technology. Senhance Surgical System is the main company product. The laparoscopic system features eye-tracking camera control, haptic sensing, improved ergonomics, an open footprint for clear communication and access and reusable instruments. The Senhance system is studied to have a similar cost to classic laparoscopy. For example, Senhance can be equipped with 3 mm instruments for hernia repair. Pediatric surgical operations are also possible . The robotic platform can be used to make hernia repair, cholecystectomy, gynecologic and colorectal surgery. Auris Health Inc. The company Auris Health Inc., established in California in 2007, produces minimally invasive surgical robots. In 2019 Auris Health was acquired by Ethicon, part of the Johnson & Johnson Medical Devices. The Health Monarch Platform , is used for therapeutic and diagnostic bronchoscopy treatments. The Monarch EBUS , an additional product from Auris Health, detects and stages lung cancer. The platform offers advanced vision and fine control. To meet diverse surgical needs, the end effectors come in different diameters. AVRA Medical Robotics Inc. AVRA Medical Robotics Inc. creates surgical robotic systems for minimally invasive treatments. The company is headquartered in Orlando, Florida, and was launched in 2015. The ARVIS system, Autonomous Robot for Minimally Invasive Surgery, is the only product sold by AVRA Medical Robotics. The ARVIS system is used in laparoscopic surgeries. With the use of proprietary software and cutting-edge imaging and sensing technologies, the system allows the robot to operate on its own, performing surgical tasks without direct human assistance. The ARVIS system is equipped with different end effectors. The end effectors are readily replaceable, providing more flexibility during treatments, and they are available in a variety of sizes and forms to meet varied surgical needs. The end effectors have good dexterity and can accurately and precisely execute a broad variety of surgical activities. BEC medical BEC ROBOTICS is a technology company working on human–robot collaboration in the medical, entertainment and industrial sectors. BEC Medical was founded in 2003. The scope of the company is to provide a safe interaction between humans and robots. The company produces Exacure and guidoo. Exacure is a robot-assisted patient positioning system, specially developed for radiotherapy. The system allows positioning under neutron irradiation in BNCT treatment. Guidoo can be used for percutaneous biopsies and ablative procedures. Guidoo supports the positioning of needles. The system accuracy reduces the control scans. Brainlab Brainlab is a German company founded in 1989. The company produces Loop-X™ a fully robotic intraoperative imaging device for surgery. The platform provides the surgeon with high-end intraoperative 2D and 3D imaging. The patient is lying on a robot bed. The system moves the scan area to the region of interest. The patient's body is scanned. Then, using a laser, Loop-X™ projects both the incision start and end points directly onto the patient’s skin. The system can be used for spinal surgery. Cambridge Medical Robotics Ltd. Cambridge Medical Robotics Ltd. (CMR) is a UK-based business, established in 2014, that designs and produces minimally invasive surgical robots. Versius Surgical Robotic System is the main offering of the company ; the system consists of a portable robot suitable for a variety of surgical operations. Versius is a modular robotic system that performs minimally invasive surgical procedures using several robotic arms. The system's lightweight robotic arm may be readily moved to various locations to carry out various jobs. Moving the system between operating rooms is simple. There are several end effectors included with the Versiu s Surgical System that can be used for different types of surgical operations. During surgery, the end effectors can be simply replaced. Collin medical. The origin of the Collin France company dates to 1820. RobOtol® is a surgical navigator who received a CE mark in 2016. RobOtol® is a mechanical system designed for otologic surgery, offering 7 degrees of freedom: 3 rotations, 3 translations, and one distal movement . The design and ergonomics are adapted to the constraints of the operating room. The system is expected to be used in surgery for otosclerosis, cochlear implants, and drug delivery to the inner ear. The instrument holder arm is designed to assist in the insertion of cochlear implants and allows surgeons to use both hands for endoscopic ear surgery. Distalmotion Distalmotion was founded in 2012 as a spin-off of the Robotics Lab of the Swiss Federal Institute of Technology in Lausanne (EPFL). The company produces the surgical platform Dexter suitable for minimally invasive surgery. A wide set of laparoscopic instruments is available: staplers, vessel sealers and clip appliers. Dexter adopts single-use instruments, removing the complexity of reprocessing. The system is designed to be easily moved from one operating room to another. The Dexter platform is open: surgeons are free to select and use their preferred 3D laparoscopes available in the market. EndoControl SAS Founded in 2006, EndoControl SAS is a medical robotics firm located in France. The business focuses on creating and producing medical robots for endoscopic procedures. Medical robots from EndoControl SAS include wristed tools for robotic-assisted interventions, laparoscopic surgery, and Natural Orifice Transluminal Endoscopic Surgery (NOTES). Among EndoControl SAS's well-known products are: ViKY® and JAIMy® system . ViKY® system is a modular robot-assisted platform for laparoscopic procedures, while the JAIMy® system , is a miniature robot designed for NOTES procedures . A variety of motorized devices are included with the ViKY® system, offering a high degree of flexibility and adaptability that may be readily changed out during surgery. The JAIMy® system has a robotic arm with a 5 mm diameter and 4 degrees of freedom. Corindus Vascular Robotics Inc. Corindus Vascular Robotics Inc., founded in 2002 and headquartered in Massachusetts, produces precision vascular robots. The company’s main products are the CorPath GRX System and the CorPath 200 System . Both systems are used to perform robotic-assisted vascular surgeries such as Percutaneous Coronary Interventions (PCI). The systems are intended to enhance precision, control, and procedural safety for the operator, while also improving clinical outcomes for the patient. CorPath comes with a range of end effectors, including guide catheters, guide wires, and balloon catheters. These end effectors are designed to enable precise control and positioning during procedures, while also providing enhanced visualization and safety features. A wide range of end effectors is available. EndoMaster Surgical System EndoMaster Surgical System is a medical company that develops surgical robots for minimally invasive surgery. EndoMaster Surgical System’s flagship product is the EndoMaster Robotic System. It is a robotic surgical platform specially designed for minimally invasive digestive system procedures. The system is composed of a surgeon console, robotic arm, and endoscopic camera. The three EndoMaster configurations EASE, FLEX and FLIP are designed for specific surgical procedures . The EndoMaster Surgical System is used in endoscopic surgeries, such as gastric bypass, bariatric, and general surgery. The EndoMaster end effectors are highly dexterous and enable precise movement in tight spaces. Human Xtensions Human Xtensions is a medical company born in Israel in 2012. The company's main product is HandX , a surgical device able to extend the human hand’s reach . The handheld robotics device is an affordable laparoscopic instrument. HandX is like a human extension inside the anatomy. The device allows the surgeon to reach ergonomic positioning close to the patients. HandX is the pioneer of a new product segment, positioned between traditional laparoscopy and classic robotic surgery. Intuitive Surgical Inc. Intuitive Surgical Inc. was founded in the US in 1995. The company produces da Vinci surgical systems. The system is used for the following surgical procedures: urologic, gynecologic, thoracic, cardiac, and colorectal. The Sunnyvale company has offices in Europe and Asia. The da Vinci surgical system is made of a surgeon's console, a patient-side cart, and a set of robotic arms. The surgeon, looking inside a high-definition 3D camera, controls the robotic arms. The platform offers vision magnification and translates the surgeon's hand movements into smaller movements of the surgical instruments. Da Vinci Surgical System has been released in different versions: Xi, X, SP, and Si . The platform costs around $2,000,000. The end effectors mimic the human wrist. The surgical instruments can cut, grasp, and suture. Mazor Robotics Ltd. Mazor Robotics Ltd. is a medical robotics company founded in 2001 in Israel, which specializes in the development of surgical guidance systems and robotic-assisted surgery. Mazor Robotics has developed three main products for surgical guidance and robotic-assisted surgery. The Mazor X is a robotic platform used for spine surgery that provides pre-operative planning, intra-operative guidance, and real-time imaging. The Renaissance is a guidance system for the spinal surgery that helps surgeons place screws and implants with high precision . The Mazor Core is a module that integrates with the Mazor X to provide more accurate and efficient surgical planning. The Renaissance Guidance System is used in spine surgeries, such as pedicle screw fixation and interbody fusion. The price is about $ 900,000. Mazor end effectors are specifically designed to handle spinal implants, such as screws and rods. Medrobotics Corporation Medrobotics Corporation Company, founded in 2005 in Massachusetts USA, produces the Flex® Robotic System, designed for minimally invasive surgical operations . The Flex® Robotic System has a flexible robotic arm that performs transoral procedures in hard-to-reach areas. A high-definition camera allows vision. The range of end effectors includes graspers, scissors, and retractors. The modular end effectors can be easily swapped out during a procedure. Flex® Robotic System can be used for tongue resection, polypectomy, and vocal cord procedures. Medtronic Inc. Medtronic Inc., founded in 1949 in Ireland, is a medical company that produces robotic surgical solutions. The company sells globally its products. In December 2018, Medtronic acquired Mazor Robotics for $1.7 billion. The surgeon, thanks to sensors on the end effector, receives real-time feedback, enabling better control. Medtronics produces Mazor X Stealth Edition for spine robotics , O-arm Imaging System, Midas Rex Legend EHS Stylus and Medtronic NIM nerve monitoring system . Medtronic's surgical robots are designed to work with a variety of end effectors, which are specialized instruments used to perform specific surgical tasks, including electrosurgery, suction and irrigation, grasping, cutting and stapling. Meerecompany Inc. Meerecompany is a medical robotics company founded in 2019 and headquartered in Germany. The company specializes in developing and manufacturing surgical robotic systems that enable precision in minimally invasive surgery. The company’s vision is to make surgery less invasive, more efficient, and patient-friendly. Meerecompany surgical robots have a modular design and can be customized for various surgical applications. They use a haptic feedback system to provide surgeons with tactile sensation and precise control during surgery. The Meere Surgical System , from Meerecompany, comprises a surgical console, a robotic arm, and an endoscopic camera . The system suits general, gynecologic, urological, and thoracic procedures. The range of end effectors includes graspers, scissors, dissectors, and suction instruments. Microbot Medical Inc. Microbot Medical Company produces minimally invasive surgery robots. These devices are used for neurosurgery, urology, and gynecology. Some of Microbot Medical Inc.'s commercial products include TipCAT, LIBERTY™ and Self-Cleaning Shunt. TipCAT is a robotic catheter system for the diagnosis and treatment of peripheral arterial disease . LIBERTY™ is a robotic system for endovascular procedures . Self-Cleaning Shunt is a robotic system that treats hydrocephalus . The end effectors can perform aspiration, ablation, and cutting. Microsure Microsure was founded in 2014 in the Netherlands. MUSA-3 is a microsurgical robot made of a surgeon console and a robotic arm cart . Disposable adapters are used to connect the surgeon's instruments. The MUSA-3 dexterity enables very precise suturing. The robot can be used for a variety of surgical operations, since it can carry out complex movements on difficult wound surfaces. Using a digital or hybrid microscope, the surgeon views the screen while seated at the surgeon console. Utilizing MUSA-3 in conjunction with digital microscopes increases the system's ergonomics. The joysticks, which have a large workspace to allow for ample movement, control the robot. To improve accuracy, tremor filtering and movement scaling are applied when transferring joystick movements to the robotic arms. MMI The company MMI was born in Italy in 2015. MMI produces the Symani® Surgical System. The robotic platform is suitable for microsurgery and super microsurgery. Two wristed robotic arms, equipped with the NanoWrist® robotic micro instruments, make up the Symani® Surgical System . The wrist's seven degrees of freedom provide the control and accuracy required for surgeons to manipulate delicate sutures and procedures. The system features tremor filtration and 7–20 × motion scaling. Surgeons can execute precise surgical procedures and scale their hand movements. The surgeon moves the manipulators directly with the ergonomic Symani® Console, just like they would with manual instruments. A heads-up 3D visualization system can be utilized with the console. Momentis Surgical Momentis Surgical was founded in 2013 and is in Israel. The company focuses on creating a surgical robotic system with fingers. Co-founders Dvir Cohen and Nir Shvalb, PhD, have studied how to maneuver instruments within the body, reducing the number of instrument portals needed. The AnovoTM Surgical System has arms that replicate the surgeon's shoulder, elbow, and wrist movements . This allows surgeons to use the fundus-to-cervix technique without multiple abdominal incisions. Because the system is less expensive and has a smaller footprint than traditional robotic systems, more hospitals may use it. Myomo Inc. Myomo Inc. Company produces myoelectric orthotics. This technology helps to enhance the mobility of people with difficult neurological conditions like stroke, sclerosis, and spinal cord injuries. The orthotics allow patients to regain mobility. The patient’s muscle signals are amplified and used to control the movement of the device. Myomo Inc. produces MyoPro and MyoCare . The end effectors can be customized according to the individual patient's needs. Neocis Inc. Neocis Inc. has been founded in Florida in 2014. The company produces robots for dental implants. The Yomi Robotic System, from Neocis Inc., provides real-time visualization and guidance for dental implant procedures . A robot arm guides the dentist's instruments with high precision, improving accuracy and reducing complications. The end effector is a drill controlled by the dentist by a foot pedal. Haptic feedback, detects the presence of hard or soft tissues, allowing for greater accuracy during the implant placement. QUANTUM Surgical QUANTUM Surgical is a company founded in France in 2017. QUANTUM develops robots for hepatology and oncology. Epione® is a robotic-assisted technology for the treatment of tumors. Thanks to a fusion technology, that coordinates the robotic arm and the images, Epione® allows to target tumors . The Epione® device is CE-marked for abdomen and lung indications, and FDA-cleared for abdominal ablation indication. For example, the platform may be used for minimally invasive liver cancer treatment. First, the tumor is seen in 3D. Then the ablation modality is defined. The robot is registered to the patient synchronizing the breath. The robot reaches the planned trajectory and delivers the ablative therapy. Finally, the CT images are used to assess the results. Remebot Remebot is a robotic company founded in China in 2010. The company produces neurosurgery robots having an accuracy of 0,5 mm and a registration time of under two minutes. The Remebot neurosurgical robot is a navigation and orientation robot for neurosurgery . The system integrates image processing and surgical planning, automatic positioning and navigation, and a multi-functional surgical operation platform. It can assist doctors in completing nearly one hundred operations. Some examples are Hematoma Aspiration, Percutaneous Puncture, Craniotomy Navigation and Endoscopic Neurosurgery. Surgery planning software can complete multi-modal fusion including computed tomography (CT) and magnetic resonance imaging (MRI). The optical tracker can automatically identify customized markers, and establish a one-to-one mapping relationship between the three-dimensional model in the software and the real scene to achieve precise navigation and positioning. Renishaw plc Renishaw plc is a global engineering company born in 1973. The company specializes in the development, manufacture, and sale of precision measurement and control equipment, including metrology systems, spectroscopy systems, and motion control systems. Renishaw designs and manufactures Neuromate a surgical robot for neurosurgical procedures. This system can be used for brain biopsies and stimulation. Also, stereotactic neurosurgery can be performed. The surgeon, from a computer console, controls the robotic arm. Neuromate surgical robot comes with a range of end effectors that vary in size and dexterity, with some being designed for fine, delicate work and others for larger, more robust tasks. The Renishaw's products also include Coordinate Measuring Machines (CMMs), laser encoders and additive manufacturing systems. Renishaw offers a range of CMMs including: the Equator™ gaging system , a versatile gaging system that can be configured to match specific inspection needs the REVO® 5-axis scanning system which offers a unique head system that can carry both tactile and non-contact probes. REVO® may be used for prostatectomy and cholecystectomy. The company also provides software solutions for data analysis and visualization. Restoration Robotics Inc. Restoration Robotics Inc., recently merged with Venus Concept, is a medical device company focused on developing and commercializing the ARTAS® Robotic Hair Restoration System . The ARTAS® Robotic Hair Restoration System is a minimally invasive, image-guided robotic system for hair restoration procedures. The system uses advanced algorithms to identify and select the optimal hair follicles for harvesting and then performs follicular unit extraction (FUE) using robotic technology. The system is designed to improve the precision, consistency, and speed of hair restoration procedures while reducing the trauma and scarring associated with traditional manual methods. The ARTAS ® Robotic Hair Restoration System uses a small, motorized tool called a punch to extract individual hair follicles from the scalp. The punch has a diameter of 0.9 mm, which is smaller than traditional manual punches, allowing for more precise and accurate extraction. The system also includes a set of needles that are used to create the recipient sites for the transplanted follicles. The needles are adjustable in size and depth, allowing for customizable hair restoration procedures. Robocath Robocath company was founded in 2009 by Philippe Bencteux. The company produces smart robotic solutions to treat cardiovascular diseases. The robotic approach maximizes the security of coronary angioplasty using robotic assistance. The goal of this medical operation is to revascularize the heart muscle by putting one or more stents into the blood vessels that supply it. The medical robot R-One + ™ comprises a Command and a Robotic Unit . The medical robot has an articulated support and can use guidewires, balloons, or stents. RoboticScope BHS was founded in 2017 in Innsbruck, Austria. The company produces microsurgery devices. RoboticScope® is a microscopic visualization system that uses a head-mounted display (HMD), a robotic arm, and a complete digital camera system to replace the conventional setup of fixed eyepieces and microscopes . The HMD's two digital micro screens provide the surgeon with real-time, three-dimensional (3D) video of the surgical site, sourced from the robotic camera unit. With simple head movements, the surgeons control the camera while maintaining a constant gaze on the surgical field and without taking their hands off it. Rob Surgical Systems Inc. The Rob Surgical spin-off was founded in Barcelona in 2012. The Institute for Bioengineering of Catalonia (IBEC) and the Polytechnic University of Catalonia (UPC) founded Rob Surgical in response to the European Robotic Surgery Project (EuRoSurge), which was funded by the European Commission in the FP7, and the Mayo Clinic in the United States regarding minimally invasive robotic surgery (MIRS). The company’s goal is to make surgical robots more effective so that they can replace more conventional surgical techniques. The company has two products: the Bitrack System and Hybrid Surgery . Bitrack System performs abdominal and pelvic surgical procedures. Hybrid surgery allows robotic and traditional lap instruments to operate simultaneously. Smith & Nephew PLC Smith & Nephew PLC is a British medical technology company that produces various medical devices, including joint replacement systems, sports medicine implants, wound care dressings, and trauma devices. Some of the product lines by Smith & Nephew include JOURNEY II Total Knee System, POLAR3 Total Hip Solution, NAVIO Surgical System, HEALICOIL PK Suture Anchor, PICO Single Use Negative Pressure Wound Therapy System, and DYONICS POWER II Control System . Cutting, coagulation, and suturing end effectors are controlled by surgeons. In February 2022 the company Smith & Nephew announced the launch of the CORI robotic-assisted surgical system for knee arthroplasty . The compact robotic solution includes 3-D intraoperative imaging with a robotic milling tool. Stryker Corporation The Stryker Corporation, founded in 1941 in Michigan produces medical equipment, such as implants, surgical instruments, endoscopy equipment, and neurotechnology products. Some of the company's products are Mako Robotic-Arm Assisted Technology , Neptune Waste Management System and SurgiCount Safety-Sponge System . Mako system is suitable for orthopedic surgeries, and joint replacement surgeries, such as hip and knee replacements. The Mako system costs $1.25 million for the robot, and an added $100,000 service contract is needed every year. Stryker Corporation's Mako Robotic-Arm Assisted Technology is equipped with end effectors that have high precision and accuracy. SurgiScope In 1989, the University of Grenoble and the business AID started developing SurgiScope (ISIS Robotics, Saint Martin d'Hères, France). At least forty SurgiScope robots have been sold and installed. Based on a parallel delta mechanism, this ceiling-mounted 7 DoF robotized manipulator is primarily used for neuro-navigation applications or endoscopy and biopsy procedures . SurgiScope can mount a wide range of end effectors. Think Surgical Inc. Think Surgical, founded in 2006, is a California-based medical technology company that develops and manufactures surgical robots and automation technology for orthopedic surgery. Think Surgical primary product is the TSolution One Surgical System , a robotic platform designed to assist surgeons in total hip arthroplasty procedures . The system includes a 3D preoperative planning workstation that enables surgeons to plan and simulate the surgery in a virtual environment, as well as a robotic arm that assists in the preparation of the hip socket and placement of the implant. The TSolution One Surgical System uses a range of surgical instruments and end effectors, including bone mills, broaches, rasps, and reamers, which are used to prepare the bone and place the hip implant. The system's end effectors are designed to provide precise control and accuracy during surgery, while also reducing the physical strain on the surgeon. TPLAN is a software platform developed by Think Surgical that helps surgeons construct customized 3D surgical plans from CT images of patients. The pre-operative plan, generated by the surgeon in the 3D Planning Workstation, is utilized by the computer-assisted TCAT equipment to prepare the joint surface and bone cavity. Titan Medical Inc. Titan Medical Inc. Company, founded in 2008 in Canada, produces robotic surgical technologies. The company produces the Sport robotic surgical system, a versatile minimally invasive surgical platform suitable for different surgical procedures. In 2020, Titan rebranded its Sport surgical system to the Enos robotic single-access surgical system . The surgeon, thanks to the 3D view, controls multi-articulating instruments. The single-port robot can be used for urology, gynaecology and general surgery . The end effectors are multi-articulated. For example, Sport can be used, for example for cholecystectomy and prostatectomy. Vicarious Surgical Inc. Founded in 2014, Vicarious Surgical Virtual Incision Corporation Virtual Incision Corporation, founded in 2006 produces surgical robotic devices. The company robot MIRA (Miniature In Vivo Robotic Assistant) allows abdominal surgeries using a single, small incision in the patient's belly button . MIRA allows the execution of the following procedures cholecystectomy, colorectal surgery, and appendectomy. The robot's arms can use graspers, scissors, and cautery devices. The surgical instruments are designed to be disposable, reducing the risk of infection and minimizing the need for sterilization. MIRA 's end effectors wish to replicate the same degree of precision and control as traditional laparoscopic instruments. WEGO WEGO, a Chinese enterprise founded in 1988, is the manufacturer of the MicroHand S Surgical Robot System . This apparatus is a minimally invasive surgical robot designed for laparoscopic surgeries. Its robotic arm is flexible enough to execute 540-degree rotations at the end, accurately replicating the surgeon’s hand movements. Additionally, the robot swiftly and automatically stitches wounds and carries out other basic medical activities. A dual CMOS sensor 3D stereoscopic imaging system installed on the endoscope produces high-resolution, real-time images. A study has compared the MicroHand S and the da Vinci surgical robot . The MicroHand S robot is slower to install respect to the da Vinci robot. MicroHand S offers lower total hospital and surgery costs. The operation time is similar. XACT Robotics Ltd. In 2013 was found in Israel the XACT Robotics Ltd. Company. XACT Robotic System is a minimally invasive surgical robot featuring high accuracy . A miniature robot navigates inside the body’s natural pathways. The robot uses small and flexible reusable end effectors. Sensors provide real-time feedback. The robot can be used for the following applications: ablation, biopsy, drainage, coil markers, brachytherapy, pain management and drug delivery. ZEISS ZEISS Company was founded in Germany in 1846. The company has developed Preceyes a robot assistant for retina surgery . The has a precision of 20 μm. This high surgical precision improves treatment outcomes. The System is compatible with a wide range of instruments. Anesthesia, either local or general, is required for patients. Scaling of gestures and filtering of hand tremors allow to increase the surgery precision. The PRECEYES Surgical System has been clinically validated, received a CE mark and is commercially available. Zimmer Biomet Holdings Inc. Zimmer Biomet Holdings Inc. is a global leader in musculoskeletal healthcare that designs, develops, manufactures, and markets orthopedic reconstructive products, spine and trauma devices, dental implants, and related surgical products. Zimmer Biomet offers a wide range of medical devices and surgical products, including joint replacement and reconstruction products, spine and dental implants and surgical instruments. The ROSA ONE® system , from Zimmer Biomet, is used for neurosurgery (such as brain biopsies and electrode placements) and spine surgery . ROSA ONE®, for example, allows pedicle screw fixation and interbody fusion. Some of the notable product names by Zimmer Biomet include the Persona Knee System , the Mobi-C Cervical Disc , the Vanguard 360 Revision Knee System, the Comprehensive Reverse Shoulder System , the Comprehensive Segmental Revision System, and the Taperloc Complete Hip Stem . Zimmer Biomet's surgical instruments and end effectors are designed to help surgeons perform complex procedures with greater precision and accuracy. The company produces different surgical instruments. Some examples are scissors, needle holders, retractors, and bone-cutting tools. Comparison of robotic platforms and end effectors All the robotic end effectors enable the precision execution of procedures. Each end effector is designed for specific procedures. For example, the Stryker Mako system is specifically designed for joint replacement procedures, while the Smith & Nephew NAVIO system is designed for both joint replacement and spine procedures. The surgical procedures performed by some of the main robot end effectors are briefly discussed, in Tab. . Depending on the system and the kind of work being done, robotic surgical systems have varying degrees of accuracy. Designed for joint replacement surgery, the Mako Robotic-Arm Assisted Technology makes use of a 3D model of the patient's anatomy to help plan and carry out the process with extreme precision, leading to better alignment and positioning of the implants. The ROSA ONE® Surgical Robot offers precise and accurate targeting of surgical tools and implant placement. It is used in neurosurgery and spine surgery. Comparably, it has been demonstrated that the ExcelsiusGPS system , which is used in spine surgery, can precisely and accurately target pedicle screws and other spinal instrumentation. Robotic surgical systems are extremely accurate and precise, which can result in better surgical results and quicker patient recovery times. The end effectors range of motion varies greatly depending on the surgical instrument, Tab. : The offer of robotic devices is wide. Each surgeon is interested and specializes in a specific set of procedures. Table has been created to easily find the commercial products that can be used to perform similar procedures. Reusable, disposable and reposable end effectors Surgical robotic systems employ various end effectors to carry out procedures. These end effectors can be reusable, disposable, or deposable. Disposable end effectors are designed for one-time or temporary use. Reposable laparoscopic instruments are formed by reusable and disposable elements: for example, a sterilized handle can be reused, while the interchangeable tips, like scissors, are disposed of. Each family of end effectors can be made of different materials. Unlike disposable instruments, reusable end effectors can be used several times. Stainless steel 316L or titanium are examples of materials used for reusable tools. Disposable tools may be made of 304 stainless. Plastic disposable surgical tools have been successfully produced and tested . Reusable end effectors can be built using titanium or stainless steel, that are resistant to corrosion and degradation. Disposables are often made of 304 stainless steel while reusable of 316L or equivalent. Disposable tools are relatively cheap, do not need sterilization steps and have limited risk of cross-contamination. Some complex geometries of disposable end effectors are made using 3D printing. Both disposable and reusable effectors may break during surgery. When an end effector is used a limited number of times, environmental issues related to waste management arise. Reusable end effectors usually are made of high-quality durable metals like titanium or stainless steel; these tools are machined with tight tolerances. These end effectors are usually expensive but offer a limited cost per use because can be used even a hundred times. Reusable medical end effectors require cleaning and sterilization procedures for each use. Reusable instruments, that can steer in the body, are often difficult to clean due to the internal driving mechanisms that are based on the cable actuation of the tip. Driven by climate issues, groups are working on fundamentally new steering concepts that should foster the introduction of stronger, thinner, and modular instruments that are driven not by cables but by shaft rotations and translations . It is desirable that hospital policies, in future, will increasingly opt for the use of reusable or recyclable instruments for environmental reasons. Hospital waste management is very complex and heavy to sustain. This section of the review examines the offerings of some of the most well-known manufacturers of surgical robots. The choice of the right surgical platform is not easy. Several aspects need to be considered. Each product has its characteristics. Each robot can perform specific surgical procedures. Moreover, according to the size and dexterity of the end effector, the same procedure may be executed differently. The accuracy of the execution of the task is also a specific characteristic to consider. Patient safety is always of paramount importance; safety measures can vary among surgical platforms. There is always an important trade-off between the device’s capabilities and the time the surgeons need to learn and operate. The level of training and support offered is important to optimize the surgeon’s learning curve . The surgical platforms may have different purchase and operating costs. System versatility and availability are also important; some systems may be available only in certain regions while others may be suitable only for a limited set of procedures—statistics on surgical operation success rate and post-recovery time help to consider the patient benefit. According to our perspective, user and patient feedback can be used best to assess the performance of a surgical platform. For each firm considered, location and foundation date are reported (Table ). Some companies were founded many years ago while their first surgical robots were more recently developed. The list of surgical robot manufacturers provided, while not exhaustive, allows us to take an outlook of the sector. The United Kingdom has an old tradition of surgical robotics. The United States is one of the most important players. Europe is showing a growing interest in the field. Asia is also following this global innovation trend. The data of Table finds a graphic representation in Fig. . A description of each of the selected firms is now provided. Their main commercial products are shortly described. Accuray Incorporated focuses on developing cutting-edge medical technology solutions for the treatment of cancer and other illnesses. The business is headquartered in Sunnyvale, California, and was established in 1990. To provide precise and accurate control during radiation therapy procedures, Accuray has created sophisticated robotic systems. The company’s commercial product line-up includes CyberKnife , TomoTherapy system and Radixact Treatment Delivery System . Stereotactic body radiation therapy and stereotactic radiosurgery are two applications for CyberKnife. The system minimizes damage to surrounding healthy tissue, by accurately delivering radiation to malignancies, using cutting-edge imaging technology. With the TomoTherapy system, treatment regimens may be tailored precisely and individually for image-guided intensity-modulated radiation therapy. The Radixact Treatment Distribution System enables precise radiation delivery during treatment. The end effectors made by Accuray are used in radiation treatment procedures in conjunction with their robotic systems. To meet diverse treatment requirements, the end effectors are available in a range of sizes and shapes. Because of its simple interchangeability, procedures can be carried out with more flexibility. TransEnterix was founded in 2006 in North Carolina. Since February 2021, TransEnterix has changed the name to Asensus Surgical. The international company, formed by over 200 employers, is focused on surgical technology. Senhance Surgical System is the main company product. The laparoscopic system features eye-tracking camera control, haptic sensing, improved ergonomics, an open footprint for clear communication and access and reusable instruments. The Senhance system is studied to have a similar cost to classic laparoscopy. For example, Senhance can be equipped with 3 mm instruments for hernia repair. Pediatric surgical operations are also possible . The robotic platform can be used to make hernia repair, cholecystectomy, gynecologic and colorectal surgery. The company Auris Health Inc., established in California in 2007, produces minimally invasive surgical robots. In 2019 Auris Health was acquired by Ethicon, part of the Johnson & Johnson Medical Devices. The Health Monarch Platform , is used for therapeutic and diagnostic bronchoscopy treatments. The Monarch EBUS , an additional product from Auris Health, detects and stages lung cancer. The platform offers advanced vision and fine control. To meet diverse surgical needs, the end effectors come in different diameters. AVRA Medical Robotics Inc. creates surgical robotic systems for minimally invasive treatments. The company is headquartered in Orlando, Florida, and was launched in 2015. The ARVIS system, Autonomous Robot for Minimally Invasive Surgery, is the only product sold by AVRA Medical Robotics. The ARVIS system is used in laparoscopic surgeries. With the use of proprietary software and cutting-edge imaging and sensing technologies, the system allows the robot to operate on its own, performing surgical tasks without direct human assistance. The ARVIS system is equipped with different end effectors. The end effectors are readily replaceable, providing more flexibility during treatments, and they are available in a variety of sizes and forms to meet varied surgical needs. The end effectors have good dexterity and can accurately and precisely execute a broad variety of surgical activities. BEC ROBOTICS is a technology company working on human–robot collaboration in the medical, entertainment and industrial sectors. BEC Medical was founded in 2003. The scope of the company is to provide a safe interaction between humans and robots. The company produces Exacure and guidoo. Exacure is a robot-assisted patient positioning system, specially developed for radiotherapy. The system allows positioning under neutron irradiation in BNCT treatment. Guidoo can be used for percutaneous biopsies and ablative procedures. Guidoo supports the positioning of needles. The system accuracy reduces the control scans. Brainlab is a German company founded in 1989. The company produces Loop-X™ a fully robotic intraoperative imaging device for surgery. The platform provides the surgeon with high-end intraoperative 2D and 3D imaging. The patient is lying on a robot bed. The system moves the scan area to the region of interest. The patient's body is scanned. Then, using a laser, Loop-X™ projects both the incision start and end points directly onto the patient’s skin. The system can be used for spinal surgery. Cambridge Medical Robotics Ltd. (CMR) is a UK-based business, established in 2014, that designs and produces minimally invasive surgical robots. Versius Surgical Robotic System is the main offering of the company ; the system consists of a portable robot suitable for a variety of surgical operations. Versius is a modular robotic system that performs minimally invasive surgical procedures using several robotic arms. The system's lightweight robotic arm may be readily moved to various locations to carry out various jobs. Moving the system between operating rooms is simple. There are several end effectors included with the Versiu s Surgical System that can be used for different types of surgical operations. During surgery, the end effectors can be simply replaced. The origin of the Collin France company dates to 1820. RobOtol® is a surgical navigator who received a CE mark in 2016. RobOtol® is a mechanical system designed for otologic surgery, offering 7 degrees of freedom: 3 rotations, 3 translations, and one distal movement . The design and ergonomics are adapted to the constraints of the operating room. The system is expected to be used in surgery for otosclerosis, cochlear implants, and drug delivery to the inner ear. The instrument holder arm is designed to assist in the insertion of cochlear implants and allows surgeons to use both hands for endoscopic ear surgery. Distalmotion was founded in 2012 as a spin-off of the Robotics Lab of the Swiss Federal Institute of Technology in Lausanne (EPFL). The company produces the surgical platform Dexter suitable for minimally invasive surgery. A wide set of laparoscopic instruments is available: staplers, vessel sealers and clip appliers. Dexter adopts single-use instruments, removing the complexity of reprocessing. The system is designed to be easily moved from one operating room to another. The Dexter platform is open: surgeons are free to select and use their preferred 3D laparoscopes available in the market. Founded in 2006, EndoControl SAS is a medical robotics firm located in France. The business focuses on creating and producing medical robots for endoscopic procedures. Medical robots from EndoControl SAS include wristed tools for robotic-assisted interventions, laparoscopic surgery, and Natural Orifice Transluminal Endoscopic Surgery (NOTES). Among EndoControl SAS's well-known products are: ViKY® and JAIMy® system . ViKY® system is a modular robot-assisted platform for laparoscopic procedures, while the JAIMy® system , is a miniature robot designed for NOTES procedures . A variety of motorized devices are included with the ViKY® system, offering a high degree of flexibility and adaptability that may be readily changed out during surgery. The JAIMy® system has a robotic arm with a 5 mm diameter and 4 degrees of freedom. Corindus Vascular Robotics Inc., founded in 2002 and headquartered in Massachusetts, produces precision vascular robots. The company’s main products are the CorPath GRX System and the CorPath 200 System . Both systems are used to perform robotic-assisted vascular surgeries such as Percutaneous Coronary Interventions (PCI). The systems are intended to enhance precision, control, and procedural safety for the operator, while also improving clinical outcomes for the patient. CorPath comes with a range of end effectors, including guide catheters, guide wires, and balloon catheters. These end effectors are designed to enable precise control and positioning during procedures, while also providing enhanced visualization and safety features. A wide range of end effectors is available. EndoMaster Surgical System is a medical company that develops surgical robots for minimally invasive surgery. EndoMaster Surgical System’s flagship product is the EndoMaster Robotic System. It is a robotic surgical platform specially designed for minimally invasive digestive system procedures. The system is composed of a surgeon console, robotic arm, and endoscopic camera. The three EndoMaster configurations EASE, FLEX and FLIP are designed for specific surgical procedures . The EndoMaster Surgical System is used in endoscopic surgeries, such as gastric bypass, bariatric, and general surgery. The EndoMaster end effectors are highly dexterous and enable precise movement in tight spaces. Human Xtensions is a medical company born in Israel in 2012. The company's main product is HandX , a surgical device able to extend the human hand’s reach . The handheld robotics device is an affordable laparoscopic instrument. HandX is like a human extension inside the anatomy. The device allows the surgeon to reach ergonomic positioning close to the patients. HandX is the pioneer of a new product segment, positioned between traditional laparoscopy and classic robotic surgery. Intuitive Surgical Inc. was founded in the US in 1995. The company produces da Vinci surgical systems. The system is used for the following surgical procedures: urologic, gynecologic, thoracic, cardiac, and colorectal. The Sunnyvale company has offices in Europe and Asia. The da Vinci surgical system is made of a surgeon's console, a patient-side cart, and a set of robotic arms. The surgeon, looking inside a high-definition 3D camera, controls the robotic arms. The platform offers vision magnification and translates the surgeon's hand movements into smaller movements of the surgical instruments. Da Vinci Surgical System has been released in different versions: Xi, X, SP, and Si . The platform costs around $2,000,000. The end effectors mimic the human wrist. The surgical instruments can cut, grasp, and suture. Mazor Robotics Ltd. is a medical robotics company founded in 2001 in Israel, which specializes in the development of surgical guidance systems and robotic-assisted surgery. Mazor Robotics has developed three main products for surgical guidance and robotic-assisted surgery. The Mazor X is a robotic platform used for spine surgery that provides pre-operative planning, intra-operative guidance, and real-time imaging. The Renaissance is a guidance system for the spinal surgery that helps surgeons place screws and implants with high precision . The Mazor Core is a module that integrates with the Mazor X to provide more accurate and efficient surgical planning. The Renaissance Guidance System is used in spine surgeries, such as pedicle screw fixation and interbody fusion. The price is about $ 900,000. Mazor end effectors are specifically designed to handle spinal implants, such as screws and rods. Medrobotics Corporation Company, founded in 2005 in Massachusetts USA, produces the Flex® Robotic System, designed for minimally invasive surgical operations . The Flex® Robotic System has a flexible robotic arm that performs transoral procedures in hard-to-reach areas. A high-definition camera allows vision. The range of end effectors includes graspers, scissors, and retractors. The modular end effectors can be easily swapped out during a procedure. Flex® Robotic System can be used for tongue resection, polypectomy, and vocal cord procedures. Medtronic Inc., founded in 1949 in Ireland, is a medical company that produces robotic surgical solutions. The company sells globally its products. In December 2018, Medtronic acquired Mazor Robotics for $1.7 billion. The surgeon, thanks to sensors on the end effector, receives real-time feedback, enabling better control. Medtronics produces Mazor X Stealth Edition for spine robotics , O-arm Imaging System, Midas Rex Legend EHS Stylus and Medtronic NIM nerve monitoring system . Medtronic's surgical robots are designed to work with a variety of end effectors, which are specialized instruments used to perform specific surgical tasks, including electrosurgery, suction and irrigation, grasping, cutting and stapling. Meerecompany is a medical robotics company founded in 2019 and headquartered in Germany. The company specializes in developing and manufacturing surgical robotic systems that enable precision in minimally invasive surgery. The company’s vision is to make surgery less invasive, more efficient, and patient-friendly. Meerecompany surgical robots have a modular design and can be customized for various surgical applications. They use a haptic feedback system to provide surgeons with tactile sensation and precise control during surgery. The Meere Surgical System , from Meerecompany, comprises a surgical console, a robotic arm, and an endoscopic camera . The system suits general, gynecologic, urological, and thoracic procedures. The range of end effectors includes graspers, scissors, dissectors, and suction instruments. Microbot Medical Company produces minimally invasive surgery robots. These devices are used for neurosurgery, urology, and gynecology. Some of Microbot Medical Inc.'s commercial products include TipCAT, LIBERTY™ and Self-Cleaning Shunt. TipCAT is a robotic catheter system for the diagnosis and treatment of peripheral arterial disease . LIBERTY™ is a robotic system for endovascular procedures . Self-Cleaning Shunt is a robotic system that treats hydrocephalus . The end effectors can perform aspiration, ablation, and cutting. Microsure was founded in 2014 in the Netherlands. MUSA-3 is a microsurgical robot made of a surgeon console and a robotic arm cart . Disposable adapters are used to connect the surgeon's instruments. The MUSA-3 dexterity enables very precise suturing. The robot can be used for a variety of surgical operations, since it can carry out complex movements on difficult wound surfaces. Using a digital or hybrid microscope, the surgeon views the screen while seated at the surgeon console. Utilizing MUSA-3 in conjunction with digital microscopes increases the system's ergonomics. The joysticks, which have a large workspace to allow for ample movement, control the robot. To improve accuracy, tremor filtering and movement scaling are applied when transferring joystick movements to the robotic arms. The company MMI was born in Italy in 2015. MMI produces the Symani® Surgical System. The robotic platform is suitable for microsurgery and super microsurgery. Two wristed robotic arms, equipped with the NanoWrist® robotic micro instruments, make up the Symani® Surgical System . The wrist's seven degrees of freedom provide the control and accuracy required for surgeons to manipulate delicate sutures and procedures. The system features tremor filtration and 7–20 × motion scaling. Surgeons can execute precise surgical procedures and scale their hand movements. The surgeon moves the manipulators directly with the ergonomic Symani® Console, just like they would with manual instruments. A heads-up 3D visualization system can be utilized with the console. Momentis Surgical was founded in 2013 and is in Israel. The company focuses on creating a surgical robotic system with fingers. Co-founders Dvir Cohen and Nir Shvalb, PhD, have studied how to maneuver instruments within the body, reducing the number of instrument portals needed. The AnovoTM Surgical System has arms that replicate the surgeon's shoulder, elbow, and wrist movements . This allows surgeons to use the fundus-to-cervix technique without multiple abdominal incisions. Because the system is less expensive and has a smaller footprint than traditional robotic systems, more hospitals may use it. Myomo Inc. Company produces myoelectric orthotics. This technology helps to enhance the mobility of people with difficult neurological conditions like stroke, sclerosis, and spinal cord injuries. The orthotics allow patients to regain mobility. The patient’s muscle signals are amplified and used to control the movement of the device. Myomo Inc. produces MyoPro and MyoCare . The end effectors can be customized according to the individual patient's needs. Neocis Inc. has been founded in Florida in 2014. The company produces robots for dental implants. The Yomi Robotic System, from Neocis Inc., provides real-time visualization and guidance for dental implant procedures . A robot arm guides the dentist's instruments with high precision, improving accuracy and reducing complications. The end effector is a drill controlled by the dentist by a foot pedal. Haptic feedback, detects the presence of hard or soft tissues, allowing for greater accuracy during the implant placement. QUANTUM Surgical is a company founded in France in 2017. QUANTUM develops robots for hepatology and oncology. Epione® is a robotic-assisted technology for the treatment of tumors. Thanks to a fusion technology, that coordinates the robotic arm and the images, Epione® allows to target tumors . The Epione® device is CE-marked for abdomen and lung indications, and FDA-cleared for abdominal ablation indication. For example, the platform may be used for minimally invasive liver cancer treatment. First, the tumor is seen in 3D. Then the ablation modality is defined. The robot is registered to the patient synchronizing the breath. The robot reaches the planned trajectory and delivers the ablative therapy. Finally, the CT images are used to assess the results. Remebot is a robotic company founded in China in 2010. The company produces neurosurgery robots having an accuracy of 0,5 mm and a registration time of under two minutes. The Remebot neurosurgical robot is a navigation and orientation robot for neurosurgery . The system integrates image processing and surgical planning, automatic positioning and navigation, and a multi-functional surgical operation platform. It can assist doctors in completing nearly one hundred operations. Some examples are Hematoma Aspiration, Percutaneous Puncture, Craniotomy Navigation and Endoscopic Neurosurgery. Surgery planning software can complete multi-modal fusion including computed tomography (CT) and magnetic resonance imaging (MRI). The optical tracker can automatically identify customized markers, and establish a one-to-one mapping relationship between the three-dimensional model in the software and the real scene to achieve precise navigation and positioning. Renishaw plc is a global engineering company born in 1973. The company specializes in the development, manufacture, and sale of precision measurement and control equipment, including metrology systems, spectroscopy systems, and motion control systems. Renishaw designs and manufactures Neuromate a surgical robot for neurosurgical procedures. This system can be used for brain biopsies and stimulation. Also, stereotactic neurosurgery can be performed. The surgeon, from a computer console, controls the robotic arm. Neuromate surgical robot comes with a range of end effectors that vary in size and dexterity, with some being designed for fine, delicate work and others for larger, more robust tasks. The Renishaw's products also include Coordinate Measuring Machines (CMMs), laser encoders and additive manufacturing systems. Renishaw offers a range of CMMs including: the Equator™ gaging system , a versatile gaging system that can be configured to match specific inspection needs the REVO® 5-axis scanning system which offers a unique head system that can carry both tactile and non-contact probes. REVO® may be used for prostatectomy and cholecystectomy. The company also provides software solutions for data analysis and visualization. Restoration Robotics Inc., recently merged with Venus Concept, is a medical device company focused on developing and commercializing the ARTAS® Robotic Hair Restoration System . The ARTAS® Robotic Hair Restoration System is a minimally invasive, image-guided robotic system for hair restoration procedures. The system uses advanced algorithms to identify and select the optimal hair follicles for harvesting and then performs follicular unit extraction (FUE) using robotic technology. The system is designed to improve the precision, consistency, and speed of hair restoration procedures while reducing the trauma and scarring associated with traditional manual methods. The ARTAS ® Robotic Hair Restoration System uses a small, motorized tool called a punch to extract individual hair follicles from the scalp. The punch has a diameter of 0.9 mm, which is smaller than traditional manual punches, allowing for more precise and accurate extraction. The system also includes a set of needles that are used to create the recipient sites for the transplanted follicles. The needles are adjustable in size and depth, allowing for customizable hair restoration procedures. Robocath company was founded in 2009 by Philippe Bencteux. The company produces smart robotic solutions to treat cardiovascular diseases. The robotic approach maximizes the security of coronary angioplasty using robotic assistance. The goal of this medical operation is to revascularize the heart muscle by putting one or more stents into the blood vessels that supply it. The medical robot R-One + ™ comprises a Command and a Robotic Unit . The medical robot has an articulated support and can use guidewires, balloons, or stents. BHS was founded in 2017 in Innsbruck, Austria. The company produces microsurgery devices. RoboticScope® is a microscopic visualization system that uses a head-mounted display (HMD), a robotic arm, and a complete digital camera system to replace the conventional setup of fixed eyepieces and microscopes . The HMD's two digital micro screens provide the surgeon with real-time, three-dimensional (3D) video of the surgical site, sourced from the robotic camera unit. With simple head movements, the surgeons control the camera while maintaining a constant gaze on the surgical field and without taking their hands off it. The Rob Surgical spin-off was founded in Barcelona in 2012. The Institute for Bioengineering of Catalonia (IBEC) and the Polytechnic University of Catalonia (UPC) founded Rob Surgical in response to the European Robotic Surgery Project (EuRoSurge), which was funded by the European Commission in the FP7, and the Mayo Clinic in the United States regarding minimally invasive robotic surgery (MIRS). The company’s goal is to make surgical robots more effective so that they can replace more conventional surgical techniques. The company has two products: the Bitrack System and Hybrid Surgery . Bitrack System performs abdominal and pelvic surgical procedures. Hybrid surgery allows robotic and traditional lap instruments to operate simultaneously. Smith & Nephew PLC is a British medical technology company that produces various medical devices, including joint replacement systems, sports medicine implants, wound care dressings, and trauma devices. Some of the product lines by Smith & Nephew include JOURNEY II Total Knee System, POLAR3 Total Hip Solution, NAVIO Surgical System, HEALICOIL PK Suture Anchor, PICO Single Use Negative Pressure Wound Therapy System, and DYONICS POWER II Control System . Cutting, coagulation, and suturing end effectors are controlled by surgeons. In February 2022 the company Smith & Nephew announced the launch of the CORI robotic-assisted surgical system for knee arthroplasty . The compact robotic solution includes 3-D intraoperative imaging with a robotic milling tool. The Stryker Corporation, founded in 1941 in Michigan produces medical equipment, such as implants, surgical instruments, endoscopy equipment, and neurotechnology products. Some of the company's products are Mako Robotic-Arm Assisted Technology , Neptune Waste Management System and SurgiCount Safety-Sponge System . Mako system is suitable for orthopedic surgeries, and joint replacement surgeries, such as hip and knee replacements. The Mako system costs $1.25 million for the robot, and an added $100,000 service contract is needed every year. Stryker Corporation's Mako Robotic-Arm Assisted Technology is equipped with end effectors that have high precision and accuracy. In 1989, the University of Grenoble and the business AID started developing SurgiScope (ISIS Robotics, Saint Martin d'Hères, France). At least forty SurgiScope robots have been sold and installed. Based on a parallel delta mechanism, this ceiling-mounted 7 DoF robotized manipulator is primarily used for neuro-navigation applications or endoscopy and biopsy procedures . SurgiScope can mount a wide range of end effectors. Think Surgical, founded in 2006, is a California-based medical technology company that develops and manufactures surgical robots and automation technology for orthopedic surgery. Think Surgical primary product is the TSolution One Surgical System , a robotic platform designed to assist surgeons in total hip arthroplasty procedures . The system includes a 3D preoperative planning workstation that enables surgeons to plan and simulate the surgery in a virtual environment, as well as a robotic arm that assists in the preparation of the hip socket and placement of the implant. The TSolution One Surgical System uses a range of surgical instruments and end effectors, including bone mills, broaches, rasps, and reamers, which are used to prepare the bone and place the hip implant. The system's end effectors are designed to provide precise control and accuracy during surgery, while also reducing the physical strain on the surgeon. TPLAN is a software platform developed by Think Surgical that helps surgeons construct customized 3D surgical plans from CT images of patients. The pre-operative plan, generated by the surgeon in the 3D Planning Workstation, is utilized by the computer-assisted TCAT equipment to prepare the joint surface and bone cavity. Titan Medical Inc. Company, founded in 2008 in Canada, produces robotic surgical technologies. The company produces the Sport robotic surgical system, a versatile minimally invasive surgical platform suitable for different surgical procedures. In 2020, Titan rebranded its Sport surgical system to the Enos robotic single-access surgical system . The surgeon, thanks to the 3D view, controls multi-articulating instruments. The single-port robot can be used for urology, gynaecology and general surgery . The end effectors are multi-articulated. For example, Sport can be used, for example for cholecystectomy and prostatectomy. Founded in 2014, Vicarious Surgical Virtual Incision Corporation, founded in 2006 produces surgical robotic devices. The company robot MIRA (Miniature In Vivo Robotic Assistant) allows abdominal surgeries using a single, small incision in the patient's belly button . MIRA allows the execution of the following procedures cholecystectomy, colorectal surgery, and appendectomy. The robot's arms can use graspers, scissors, and cautery devices. The surgical instruments are designed to be disposable, reducing the risk of infection and minimizing the need for sterilization. MIRA 's end effectors wish to replicate the same degree of precision and control as traditional laparoscopic instruments. WEGO, a Chinese enterprise founded in 1988, is the manufacturer of the MicroHand S Surgical Robot System . This apparatus is a minimally invasive surgical robot designed for laparoscopic surgeries. Its robotic arm is flexible enough to execute 540-degree rotations at the end, accurately replicating the surgeon’s hand movements. Additionally, the robot swiftly and automatically stitches wounds and carries out other basic medical activities. A dual CMOS sensor 3D stereoscopic imaging system installed on the endoscope produces high-resolution, real-time images. A study has compared the MicroHand S and the da Vinci surgical robot . The MicroHand S robot is slower to install respect to the da Vinci robot. MicroHand S offers lower total hospital and surgery costs. The operation time is similar. In 2013 was found in Israel the XACT Robotics Ltd. Company. XACT Robotic System is a minimally invasive surgical robot featuring high accuracy . A miniature robot navigates inside the body’s natural pathways. The robot uses small and flexible reusable end effectors. Sensors provide real-time feedback. The robot can be used for the following applications: ablation, biopsy, drainage, coil markers, brachytherapy, pain management and drug delivery. ZEISS Company was founded in Germany in 1846. The company has developed Preceyes a robot assistant for retina surgery . The has a precision of 20 μm. This high surgical precision improves treatment outcomes. The System is compatible with a wide range of instruments. Anesthesia, either local or general, is required for patients. Scaling of gestures and filtering of hand tremors allow to increase the surgery precision. The PRECEYES Surgical System has been clinically validated, received a CE mark and is commercially available. Zimmer Biomet Holdings Inc. is a global leader in musculoskeletal healthcare that designs, develops, manufactures, and markets orthopedic reconstructive products, spine and trauma devices, dental implants, and related surgical products. Zimmer Biomet offers a wide range of medical devices and surgical products, including joint replacement and reconstruction products, spine and dental implants and surgical instruments. The ROSA ONE® system , from Zimmer Biomet, is used for neurosurgery (such as brain biopsies and electrode placements) and spine surgery . ROSA ONE®, for example, allows pedicle screw fixation and interbody fusion. Some of the notable product names by Zimmer Biomet include the Persona Knee System , the Mobi-C Cervical Disc , the Vanguard 360 Revision Knee System, the Comprehensive Reverse Shoulder System , the Comprehensive Segmental Revision System, and the Taperloc Complete Hip Stem . Zimmer Biomet's surgical instruments and end effectors are designed to help surgeons perform complex procedures with greater precision and accuracy. The company produces different surgical instruments. Some examples are scissors, needle holders, retractors, and bone-cutting tools. All the robotic end effectors enable the precision execution of procedures. Each end effector is designed for specific procedures. For example, the Stryker Mako system is specifically designed for joint replacement procedures, while the Smith & Nephew NAVIO system is designed for both joint replacement and spine procedures. The surgical procedures performed by some of the main robot end effectors are briefly discussed, in Tab. . Depending on the system and the kind of work being done, robotic surgical systems have varying degrees of accuracy. Designed for joint replacement surgery, the Mako Robotic-Arm Assisted Technology makes use of a 3D model of the patient's anatomy to help plan and carry out the process with extreme precision, leading to better alignment and positioning of the implants. The ROSA ONE® Surgical Robot offers precise and accurate targeting of surgical tools and implant placement. It is used in neurosurgery and spine surgery. Comparably, it has been demonstrated that the ExcelsiusGPS system , which is used in spine surgery, can precisely and accurately target pedicle screws and other spinal instrumentation. Robotic surgical systems are extremely accurate and precise, which can result in better surgical results and quicker patient recovery times. The end effectors range of motion varies greatly depending on the surgical instrument, Tab. : The offer of robotic devices is wide. Each surgeon is interested and specializes in a specific set of procedures. Table has been created to easily find the commercial products that can be used to perform similar procedures. Surgical robotic systems employ various end effectors to carry out procedures. These end effectors can be reusable, disposable, or deposable. Disposable end effectors are designed for one-time or temporary use. Reposable laparoscopic instruments are formed by reusable and disposable elements: for example, a sterilized handle can be reused, while the interchangeable tips, like scissors, are disposed of. Each family of end effectors can be made of different materials. Unlike disposable instruments, reusable end effectors can be used several times. Stainless steel 316L or titanium are examples of materials used for reusable tools. Disposable tools may be made of 304 stainless. Plastic disposable surgical tools have been successfully produced and tested . Reusable end effectors can be built using titanium or stainless steel, that are resistant to corrosion and degradation. Disposables are often made of 304 stainless steel while reusable of 316L or equivalent. Disposable tools are relatively cheap, do not need sterilization steps and have limited risk of cross-contamination. Some complex geometries of disposable end effectors are made using 3D printing. Both disposable and reusable effectors may break during surgery. When an end effector is used a limited number of times, environmental issues related to waste management arise. Reusable end effectors usually are made of high-quality durable metals like titanium or stainless steel; these tools are machined with tight tolerances. These end effectors are usually expensive but offer a limited cost per use because can be used even a hundred times. Reusable medical end effectors require cleaning and sterilization procedures for each use. Reusable instruments, that can steer in the body, are often difficult to clean due to the internal driving mechanisms that are based on the cable actuation of the tip. Driven by climate issues, groups are working on fundamentally new steering concepts that should foster the introduction of stronger, thinner, and modular instruments that are driven not by cables but by shaft rotations and translations . It is desirable that hospital policies, in future, will increasingly opt for the use of reusable or recyclable instruments for environmental reasons. Hospital waste management is very complex and heavy to sustain. The review has briefly described surgical robots from a selection of companies around the globe. Each robot is designed for performing specific procedures and has varying features and capabilities. Robotic surgery systems offer several advantages over traditional open surgery. Surgical robots can provide surgeons with greater control and precision than human hands, which is essential for delicate procedures. The minimally invasive approach reduces the risk of complications and minimizes the recovery time. Robotic systems provide surgeons with a magnified three-dimensional view of the surgical field, which helps them to better visualize the area. Surgical robots can reduce the surgeon's physical fatigue, as they do not have to hold their arms in one position for long periods. However, robotic surgery also has some limitations. Surgical robots are expensive to purchase, install, and maintain, which can increase the cost of surgical procedures. Robotic surgery requires extensive training for the surgeon and support staff. Only a few robotic surgery systems provide the surgeon with a sense of touch, which can be critical in some procedures . Healthcare professionals, considering incorporating robotic surgery into their practice, can choose from many commercial robots. Market forecasts show, for the future, an increase in the use of service robots . The development of improved user interfaces, powered with artificial intelligence, will enable surgeons to operate more intuitively . Europe has traditionally been an early adopter of robotic surgery technology, with a high concentration of systems per capita. The da Vinci Surgical System by Intuitive Surgical is the most widely used in Europe and has seen significant adoption across the continent. In the United States, robotic surgery systems have also been widely adopted. Other systems, such as the Mako Robotic-Arm Assisted Technology by Stryker and the Rosa Surgical Robot by Zimmer Biomet, have also gained popularity in recent years. In the last years, Asia has shown to be relatively slower in the adoption of robotic surgery systems. Recently countries like China and Japan have increased interest and investment in robotic surgery technology. Systems like the Versius Surgical System by CMR Surgical and the Senhance Surgical System by TransEnterix are gaining traction. Overall, the popularity and the adoption of robotic surgery systems vary depending on region and healthcare system. |
Participation rate in cervical cancer screening in general practice related to the proximity of gynecology care facilities: A 3 year follow-up cohort study | 7b2dec6d-16f2-45ff-a3e8-fbb878327dc0 | 9618859 | Gynaecology[mh] | According to the Global Cancer Observatory (GLOBOCAN) from 2018 estimating cancer data from 185 countries, cervical cancer (CC) was the fourth most common cancer in women worldwide, with a global age-standardized incidence rate (ASIR) of 13.1/100,000 women. This ASIR varied widely among countries ranging from <2 to 75/100,000 . In Europe between 2012 and 2018, the ASIR of CC varied from 13.4 to 13.9/100,000, and in France from 8.0 to 8.4/100,000, and the age-standardized mortality rate (ASMR) from 2.6 to 3.2/100,000, showing an increase after four decades of decrease . In France, there were 2,920 new cases of CC and 1,117 related deaths in 2018 . In Northern France, the incidence rate is 10% higher compared to the country average . CC is always preceded by neoplastic lesions with a long-lasting persisting evolution before reaching a cancerous stage. This offers the opportunity to prevent cancer by screening and early intervention. The classical screening test is the Papanicolaou-test (Pap-test) by cytologic examination of cervical smears, which requires a gynecological examination. To implement cervical cancer screening (CCS), French health authorities recommend a Pap-smear every 3 years in women between 25 and 29 years of age after two annual normal initial Pap-smears. Since 2019, the same authorities recommend a HPV test every 5 years between 30 and 65 years of age; in the case of positive test a cytology must be achieved. In the case of negative cytology, screening must occur again the next year following the same procedure . In France in 2017, CCS was “opportunistic” except in 13 departments testing an experimental organized screening. The screening participation rate is not in accordance with the recommended rate of 80% in the guidelines for women in the target ages, being insufficient for 51.6% of women or too frequent for 40.6% . In high income countries, insufficiently screened women are mainly those who do not use the services of gynecologists for cultural or economic reasons: low level of education or income [consultations with a gynecologist being more expensive than those with a general practitioner (GP)], women with no children, having no partner or being post-menopausal . Most of these women have at least one encounter with their GP over 3 years. In France, 80% of targeted women have previously chosen to be screened by a gynecologist but their numbers are drastically decreasing . In French Flanders, 53.1% of GPs and more recently midwives also perform this procedure . The performance of smears by the GP or the female gender of the GP, described as positive factors for participation in CCS, do not increase the rates significantly . Socioeconomic environmental factors like the European Deprivation Index (EDI) appear significantly and independently associated with these rates, women dwelling in deprived areas being more often insufficiently screened or not screened at all . Another factor described as positive for participation in CCS is the proximity of the office of a gynecologist . Our interest was to investigate the effect of the close proximity of the office of a gynecologist on the CCS participation rates. In our former publications , we acknowledged as main limitations a follow up period of 2 years and not controlling for the influence of the gynecology care facilities. These elements are considered in this paper. Study design As at that time (2017), the recommended interval between two CCS smears was 3 years, a cohort study was undertaken based on a 3 year retrospective follow up of 93,918 female patients aged from 25 to 65 years and their 345 GPs coupled with a telephone survey. Setting This study took place in primary care in French Flanders (Northern France). Data were collected from 2013/01/01 to 2015/12/31 from the Information System of the main mandatory Health Insurance claim database (SIAM) of French Flanders (CPAM). Telephone surveys with all the practicing GPs registered with the CPAM were carried out. Participants Participants were the GPs listed on the registers of the CPAM. Inclusion required that the GPs were practicing in primary care over the 3 year period selected. GPs having another practice outside of primary care were excluded if they had <100 female patients declared on their patient lists, ruling out GPs with complementary medicine practices (for example homeopathy, acupuncture), other practices than primary care (for example sonography and angiology) and GPs with an unbalanced practice (recently established or nearing retirement). GPs who retired during the follow up period and those who refused to answer the telephone surveys were also excluded. For the included GPs, we considered their female patient population aged from 25 to 65 years eligible for cervical cancer screening under French guidelines. Variables The main outcome was the cervical cancer screening participation rate in the eligible female patient population of included GPs, measured by the refunding to female patients of cytological examination of cervical samples by the health insurance fund. Working with claim databases where patients are anonymised and not traceable for regulatory ethic reasons (we only know their gender, their age between 25 and 65 years, the designation of their GP, and the reimbursement of a pap smear), it was not possible to compute the distance between the dwelling place of patients and offices of gynecologists. However, most of the patients are registered on the patient lists of their closest settled GP and share the same environmental characteristics . As a surrogate outcome of the distance between the dwelling place of patients and offices of gynecologists, we computed as our proximity indicator the density of the gynaecologists' offices around GPs' offices within 5, 10, 20, and 40 km. Thus, the predictor was the distance between the office of a gynecologist and each GP office. This variable was computed using geo-tracking of GP offices and the gynaecologists' offices. The confounding variables on the GP level were the gender of the GP (recovered from the SIAM database) and the performance of vaginal samplings (as a binary variable) in the GP office based on telephone surveys as described in a former paper . The European Deprivation Index (EDI) was the socio-economic effect indicator utilized. The EDI is an ecological marker reflecting the individual deprivation experience of the general population in an area based on the census. The determination of EDI started from the construction of an individual deprivation indicator associated with both objective and subjective poverty and following the identification of the basic needs of people. This first part was undertaken using the European survey specifically dedicated to the study of deprivation (EU-SILC: European Union—Statistics on Income and Living Conditions), since there is no gold-standard of deprivation. It was then necessary to identify and dichotomize the variables available and coded in a similar way both at the individual level (EU-SILC) and in the census data. Variables associated with the individual deprivation indicator were then selected and weighted by multivariate logistic regression. The regression coefficients associated with these variables in the final model then became the weights of these 10 variables measured at the aggregate level in the ecological index: overcrowding, no access to a system of central or electrical heating, non-home owner, unemployment, foreign nationality, no access to a car, unskilled worker—farm worker, household with more than six persons, low level of education, single parent household. The EDI is then defined as the weighted sum of these 10 variables quantifying fundamental basic needs associated with both objective and subjective poverty, normalized to the national average and usually divided into quintiles (national or regional). Areas of reference were the smallest available statistical census units in France (IRIS) allowing for an infra-municipal study scale. Each GP surgery was assigned its IRIS and the EDI of the corresponding IRIS was computed. Elsewhere , we have demonstrated the strong association between the EDI and the CCS rate. The EDI has a mediation effect on the CCS uptake. Bias The number of patients managed by the GP was not considered as we have demonstrated that it is not associated with the CC screening rate . The age of the GP has not been considered though it appears to be associated with the screening rate, as it is linked to the age of the patients, and young female patients are more likely to participate in cervical cancer screening compared to older patients . Another reason is that young GPs are more often of female gender compared to older GPs, and the performance of smears is associated with the gender of the GP as demonstrated earlier , though without influence on CCS uptake in a multivariate analysis. The gender of the GP and the performance of smears therefore seemed to be sufficient substitution variables. Study size This study was implemented on a complete population basis without sampling. Statistics and analysis Continuous quantitative variables are expressed as mean ± standard deviation (SD), median [interquartile range (IQR)] and categorical variables are expressed as frequencies and percentages. In this study, there were two hierarchical levels for the data: the individual GP level (GP's gender and performance of smears, and the outcome “the cervical cancer screening participation rate among the GP's listed eligible female patients”) that were nested in the geographical level (variable EDI and number of gynecologists at a given distance) as the patients of GPs practicing in the same area (IRIS) share common characteristics. The association between the CCS rate and the distance from gynaecologists' offices was analyzed using a linear generalized hierarchical mixed model. This statistical model takes into account the hierarchical structure of the data. The analysis was performed without and with adjustment based on the characteristics of the GPs and the socioeconomic level considered as a mediator (EDI). All statistical tests were two-sided and performed at the 0.05 level. Data were analyzed using the SAS software ® version 9.4 (SAS Institute, Cary, NC). Bioethics The protocol of this trial is available on Clinical Trials under the reference NCT02749110. It was approved by the ethics committee North West III of Caen under the reference 2015-23, on 2016/03/02. As at that time (2017), the recommended interval between two CCS smears was 3 years, a cohort study was undertaken based on a 3 year retrospective follow up of 93,918 female patients aged from 25 to 65 years and their 345 GPs coupled with a telephone survey. This study took place in primary care in French Flanders (Northern France). Data were collected from 2013/01/01 to 2015/12/31 from the Information System of the main mandatory Health Insurance claim database (SIAM) of French Flanders (CPAM). Telephone surveys with all the practicing GPs registered with the CPAM were carried out. Participants were the GPs listed on the registers of the CPAM. Inclusion required that the GPs were practicing in primary care over the 3 year period selected. GPs having another practice outside of primary care were excluded if they had <100 female patients declared on their patient lists, ruling out GPs with complementary medicine practices (for example homeopathy, acupuncture), other practices than primary care (for example sonography and angiology) and GPs with an unbalanced practice (recently established or nearing retirement). GPs who retired during the follow up period and those who refused to answer the telephone surveys were also excluded. For the included GPs, we considered their female patient population aged from 25 to 65 years eligible for cervical cancer screening under French guidelines. The main outcome was the cervical cancer screening participation rate in the eligible female patient population of included GPs, measured by the refunding to female patients of cytological examination of cervical samples by the health insurance fund. Working with claim databases where patients are anonymised and not traceable for regulatory ethic reasons (we only know their gender, their age between 25 and 65 years, the designation of their GP, and the reimbursement of a pap smear), it was not possible to compute the distance between the dwelling place of patients and offices of gynecologists. However, most of the patients are registered on the patient lists of their closest settled GP and share the same environmental characteristics . As a surrogate outcome of the distance between the dwelling place of patients and offices of gynecologists, we computed as our proximity indicator the density of the gynaecologists' offices around GPs' offices within 5, 10, 20, and 40 km. Thus, the predictor was the distance between the office of a gynecologist and each GP office. This variable was computed using geo-tracking of GP offices and the gynaecologists' offices. The confounding variables on the GP level were the gender of the GP (recovered from the SIAM database) and the performance of vaginal samplings (as a binary variable) in the GP office based on telephone surveys as described in a former paper . The European Deprivation Index (EDI) was the socio-economic effect indicator utilized. The EDI is an ecological marker reflecting the individual deprivation experience of the general population in an area based on the census. The determination of EDI started from the construction of an individual deprivation indicator associated with both objective and subjective poverty and following the identification of the basic needs of people. This first part was undertaken using the European survey specifically dedicated to the study of deprivation (EU-SILC: European Union—Statistics on Income and Living Conditions), since there is no gold-standard of deprivation. It was then necessary to identify and dichotomize the variables available and coded in a similar way both at the individual level (EU-SILC) and in the census data. Variables associated with the individual deprivation indicator were then selected and weighted by multivariate logistic regression. The regression coefficients associated with these variables in the final model then became the weights of these 10 variables measured at the aggregate level in the ecological index: overcrowding, no access to a system of central or electrical heating, non-home owner, unemployment, foreign nationality, no access to a car, unskilled worker—farm worker, household with more than six persons, low level of education, single parent household. The EDI is then defined as the weighted sum of these 10 variables quantifying fundamental basic needs associated with both objective and subjective poverty, normalized to the national average and usually divided into quintiles (national or regional). Areas of reference were the smallest available statistical census units in France (IRIS) allowing for an infra-municipal study scale. Each GP surgery was assigned its IRIS and the EDI of the corresponding IRIS was computed. Elsewhere , we have demonstrated the strong association between the EDI and the CCS rate. The EDI has a mediation effect on the CCS uptake. The number of patients managed by the GP was not considered as we have demonstrated that it is not associated with the CC screening rate . The age of the GP has not been considered though it appears to be associated with the screening rate, as it is linked to the age of the patients, and young female patients are more likely to participate in cervical cancer screening compared to older patients . Another reason is that young GPs are more often of female gender compared to older GPs, and the performance of smears is associated with the gender of the GP as demonstrated earlier , though without influence on CCS uptake in a multivariate analysis. The gender of the GP and the performance of smears therefore seemed to be sufficient substitution variables. This study was implemented on a complete population basis without sampling. Continuous quantitative variables are expressed as mean ± standard deviation (SD), median [interquartile range (IQR)] and categorical variables are expressed as frequencies and percentages. In this study, there were two hierarchical levels for the data: the individual GP level (GP's gender and performance of smears, and the outcome “the cervical cancer screening participation rate among the GP's listed eligible female patients”) that were nested in the geographical level (variable EDI and number of gynecologists at a given distance) as the patients of GPs practicing in the same area (IRIS) share common characteristics. The association between the CCS rate and the distance from gynaecologists' offices was analyzed using a linear generalized hierarchical mixed model. This statistical model takes into account the hierarchical structure of the data. The analysis was performed without and with adjustment based on the characteristics of the GPs and the socioeconomic level considered as a mediator (EDI). All statistical tests were two-sided and performed at the 0.05 level. Data were analyzed using the SAS software ® version 9.4 (SAS Institute, Cary, NC). The protocol of this trial is available on Clinical Trials under the reference NCT02749110. It was approved by the ethics committee North West III of Caen under the reference 2015-23, on 2016/03/02. Of the 410 GPs registered on the CPAM of Flanders, 52 were excluded as they had <100 female patients on their patient lists, six because they retired before the end of the study period, five because they refused to answer the telephone survey and two because they planned to suspend their activity as primary care practitioners, resulting in 343 included GPs . Among the 343 GPs participating in this study, 269 (78.4%) were men, and 182 GPs (53.0%) performed smears. Characteristics of the listed patients per GP are described in and shows the mean screening participation rate for female patients from 25 to 65 years during the 3 years was 50.1% (SD: 7.5%). The mean number of gynecologists within 5 km of GP surgeries was 5.4 (SD 5.6, median 5, IQR 0–11), between 5 and 10 km was 3.1 (SD 6.2, median 1, IQR 0–3), between 10 and 20 km was 15.2 (SD 21.0, median 7, IQR 0–20) and between 20 and 40 km was 30.8 (SD 28.1, median 15, IQR 10–42) . The association between the cervical screening rate and the distance from gynecology care facilities is shown in . The table presents the unadjusted model, the model adjusted for GP gender, and performance of smears by the GP and the model adjusted for GP gender, performance of smears by the GP and EDI. All models show the impact of the density of gynecologists within specified distances from the GP offices on the CCS rate with the greatest impact being the density within 5 km. The density of gynecologists within 20–40 km of GP surgeries had a smaller significant positive coefficient. We found a significant association between the density of gynecologists within 5 km of the GP's office and the cervical cancer screening participation rate after adjustment for these GP characteristics and the EDI, with the cervical screening rate increasing by 0.31% with every unit increase in the density of gynecologists within 5 km. When not adjusting for EDI, the density of gynecologists within 5 km of GP surgeries had no significant effect on the screening rate. The density of gynecologists between 20 and 40 km also had a significant effect, with the cervical screening rate increasing by 0.09% with every unit increase in the density of gynecologists between 20 and 40 km. After adjusting for the GP's gender, practice of Pap-smears by the doctor and EDI, the association remained significant though the effect size was small. Main findings In the analysis adjusting for EDI, we found that the density of gynecologists within 5 km of GP surgeries had the most significant positive regression coefficient with the CCS rate. We also found that the density of gynecologists within 20–40 km of GP surgeries had a smaller but still significant positive coefficient. The higher the density of gynecologists within 5 km of GP surgeries, the higher the CCS rate: for each supplementary gynecologist, the screening rate was improved by 0.31%. When not adjusting for EDI, the density of gynecologists within 5 km of GP surgeries had no significant effect on the screening rate, reflecting the major influence of socioeconomic determinants on screening behavior. Thus, in disadvantaged areas (like French Flanders: EDI of 2.3 compared to the mean EDI of 0 for France), a higher number of gynecologists does not increase the screening rate in the overall population, unless erasing the influence of the deprivation factor. This reflects the fact that women from deprived areas are not likely to be managed by gynecologists while women from more favored areas are more likely to be so . Study strengths and limitations The claim database of the CPAM of Flanders is reliable and consistent, and the data extracted from this database for a duration of 3 years are considered trustworthy. A participation rate of 98% of the targeted GPs allows us to consider that our study was based on an entire, not sampled population. Including 345 GPs, their almost 94,000 female patients eligible for CCS, and the 149 gynecologists in the area who may have been consulted by these patients, confers to this study a solid internal validity. Studies exploring the association between the density of gynecological care facilities and the CCS participation rate as the main outcome are scarce. No one has previously explored this association based on the ground distance between GPs and gynecologists. Our results match another French country-side study highlighting the same association by another method strengthening the external validity of our finding. In our previous publications, we acknowledged as limits a follow up of only 2 years (as CCS used to be triennial in France) and no consideration of gynecology care facilities as confounding factors . These limits have been addressed in the current paper. This study only investigates the association between CCS rates and the distance from gynecologist offices to GP offices. The global screening rate in French Flanders of 50.2% is lower than the national rate of 62.3% (range 41.6–72.5%) . The density of GPs in French Flanders was slightly lower than in the rest of France (13 vs. 16/10 000) and is even lower now (retirement of GPs from the baby-boom generation). However, the density of gynecologists (2.7/10 000) in this area was not lower than in the rest of France, which does not explain under-screening and our findings regarding the highlighted association. There are many different compulsory health insurance regimes in France depending on the occupational sector of the insured persons. We based our study on the claim database of the CPAM of Flanders representing 80% of insured persons. This means that we missed some occupational sectors like teachers or farmers. This can possibly be considered as a selection bias though there is no reason that the 20% of missed population substantially differs from the general population as described in other contexts . This does not diminish the external validity of our main result. Comparison to literature The only former publications investigating this association are the above cited French study carried out by Araujo in 2010 , and another French study published by Barré in 2017 , which found that a lower CCS participation rate was associated with a lower density of gynecologists in the residence area, matching our findings. A third study, carried out by Grillo in 2012 in Paris, did not find any significant association between the density of GPs and gynecologists in the residence area of women and the CCS participation rates . However, no adjustment was performed to correct for the influence of the overall deprivation rate and the geographical area studied was smaller with more opportunities for public transport. Profound changes in the mindset of women influenced by the social pressure of their deprived neighborhood will be necessary to enhance participation in CCS. The proximity of care facilities has little influence on enhancing screening participation in deprived areas unless community oriented primary care reaches out to concerned people . Education is probably the main solution to solve the lost opportunity associated with underscreening in deprived areas . In the analysis adjusting for EDI, we found that the density of gynecologists within 5 km of GP surgeries had the most significant positive regression coefficient with the CCS rate. We also found that the density of gynecologists within 20–40 km of GP surgeries had a smaller but still significant positive coefficient. The higher the density of gynecologists within 5 km of GP surgeries, the higher the CCS rate: for each supplementary gynecologist, the screening rate was improved by 0.31%. When not adjusting for EDI, the density of gynecologists within 5 km of GP surgeries had no significant effect on the screening rate, reflecting the major influence of socioeconomic determinants on screening behavior. Thus, in disadvantaged areas (like French Flanders: EDI of 2.3 compared to the mean EDI of 0 for France), a higher number of gynecologists does not increase the screening rate in the overall population, unless erasing the influence of the deprivation factor. This reflects the fact that women from deprived areas are not likely to be managed by gynecologists while women from more favored areas are more likely to be so . The claim database of the CPAM of Flanders is reliable and consistent, and the data extracted from this database for a duration of 3 years are considered trustworthy. A participation rate of 98% of the targeted GPs allows us to consider that our study was based on an entire, not sampled population. Including 345 GPs, their almost 94,000 female patients eligible for CCS, and the 149 gynecologists in the area who may have been consulted by these patients, confers to this study a solid internal validity. Studies exploring the association between the density of gynecological care facilities and the CCS participation rate as the main outcome are scarce. No one has previously explored this association based on the ground distance between GPs and gynecologists. Our results match another French country-side study highlighting the same association by another method strengthening the external validity of our finding. In our previous publications, we acknowledged as limits a follow up of only 2 years (as CCS used to be triennial in France) and no consideration of gynecology care facilities as confounding factors . These limits have been addressed in the current paper. This study only investigates the association between CCS rates and the distance from gynecologist offices to GP offices. The global screening rate in French Flanders of 50.2% is lower than the national rate of 62.3% (range 41.6–72.5%) . The density of GPs in French Flanders was slightly lower than in the rest of France (13 vs. 16/10 000) and is even lower now (retirement of GPs from the baby-boom generation). However, the density of gynecologists (2.7/10 000) in this area was not lower than in the rest of France, which does not explain under-screening and our findings regarding the highlighted association. There are many different compulsory health insurance regimes in France depending on the occupational sector of the insured persons. We based our study on the claim database of the CPAM of Flanders representing 80% of insured persons. This means that we missed some occupational sectors like teachers or farmers. This can possibly be considered as a selection bias though there is no reason that the 20% of missed population substantially differs from the general population as described in other contexts . This does not diminish the external validity of our main result. The only former publications investigating this association are the above cited French study carried out by Araujo in 2010 , and another French study published by Barré in 2017 , which found that a lower CCS participation rate was associated with a lower density of gynecologists in the residence area, matching our findings. A third study, carried out by Grillo in 2012 in Paris, did not find any significant association between the density of GPs and gynecologists in the residence area of women and the CCS participation rates . However, no adjustment was performed to correct for the influence of the overall deprivation rate and the geographical area studied was smaller with more opportunities for public transport. Profound changes in the mindset of women influenced by the social pressure of their deprived neighborhood will be necessary to enhance participation in CCS. The proximity of care facilities has little influence on enhancing screening participation in deprived areas unless community oriented primary care reaches out to concerned people . Education is probably the main solution to solve the lost opportunity associated with underscreening in deprived areas . Adjusting for our deprivation indicator (EDI), the density of gynecology care facilities within 5 km of a GP surgery, and to a lesser extent, within 20–40 km of a GP surgery, was significantly associated with a higher CC screening rate. When the effect of deprivation on the screening participation rate is erased by adjusting the model, the density of gynecology care facilities is linked to an increase of the CCS participation rate, meaning a potential decrease of CC. However, this effect is not noticeable when this adjustment is not made probably because women dwelling in deprived areas do not make use of services offered by gynecologists. The reasons women are not screened are complex and this certainly explains why medical demography alone cannot resolve inequalities and social disparities in participation in screening. This seemed to be confirmed by our models, despite its adjustment using the EDI (an aggregate index quantifying fundamental basic needs associated with both objective and subjective poverty). The current setting of midwifes in primary care practices might be a response to this situation that will have to be confirmed by further studies. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The studies involving human participants were reviewed and approved by Ethics Committee North West III of Caen under the reference 2015-23, on 2016/03/02. The patients/participants provided their written informed consent to participate in this study. Conceptualization and funding acquisition: JF, MR, ADu, VD-D, CB, and TR. Methodology, resources, and writing—review and editing: FQ, FS, JF, MR, ADu, EG, ADe, CC, VD-D, CB, and TR. Software and formal analysis: FQ, EG, and VD-D. Validation: JF, MR, EG, VD-D, CB, and TR. Investigation: FQ, FS, ADe, EG, VD-D, and CB. Data curation: EG, ADu, and VD-D. Writing—original draft: FQ and TR. Visualization: FQ, FS, JF, MR, ADe, CB, and TR. Supervision: CB. Project administration: EG, ADu, VD-D, and CB. All authors have read and agreed to the published version of the manuscript. This was an ancillary study of the PaCUDAHL-Gé trial sponsored by the University Hospital of Lille, which evaluates women's interest in cervical cancer screening using a device from their GP for self-collection of vaginal samples and HPV testing. The trial was funded by the French Ministry of Health (PREPS: LIC-14-14-0615, 2014/12/19). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. |
Evaluation of CD3 and CD8 T-Cell Immunohistochemistry for Prognostication and Prediction of Benefit From Adjuvant Chemotherapy in Early-Stage Colorectal Cancer Within the QUASAR Trial | 51c69829-2f30-4375-b84c-b038936bc364 | 11458110 | Anatomy[mh] | Approximately 80% of patients with stage II colorectal cancer (CRC) and 50% of those with stage III disease are cured by surgery alone. Adjuvant chemotherapy improves 5-year disease-free survival (DFS) by around 14 percentage points in stage III disease, whereas controversy remains regarding the more modest benefits in stage II disease. , CONTEXT Key Objective Are CD3 and CD8 tumor infiltrating lymphocyte densities prognostic in early-stage colorectal cancer (CRC), and do they predict benefit from adjuvant chemotherapy? This is the first such predictive evaluation in a randomized data set with observation-only control. Knowledge Generated CD3/CD8 densities were strongly prognostic, with recurrence rates in high-risk groups twice those in low-risk groups. Both high- and low-risk groups saw proportional reductions in recurrence with adjuvant chemotherapy of similar magnitude. CD3 Score combines information from CD3 densities in the core tumor and invasive margin. Number needed to treat with high-risk CD3 Score was 11, compared with 36 for low-risk CD3 Score. Relevance (E.M. O'Reilly) The authors report on a prognostic tool (CD3 Score) using a CD3/CD8 tumor infiltrating lymphocytes artificial intelligence algorithm in a older large randomized trial in early stage CRC. This relatively simple tool has potential clinical application and warrants further investigation.* *Relevance section written by JCO Associate Editor Eileen M. O'Reilly, MD. Various clinicopathological indicators of inferior prognosis—pT4, poor tumor differentiation, lymph node yield <12, lymphovascular invasion, tumor perforation, or bowel obstruction—are used in stage II disease to improve patient selection for chemotherapy. , - However, although these factors are moderately prognostic, they do not predict sensitivity to adjuvant chemotherapy. , - Circulating tumor DNA (ctDNA) postsurgery indicates increased recurrence risk. - However, again, whether chemotherapy benefits only those with detectable ctDNA has not yet been determined. Novel predictors of chemotherapy efficacy are therefore needed. The immune system plays a major role in modulating cancer progression. Colorectal tumor infiltration by a variety of different immune cell types correlates with better prognosis, of which CD3 (universal T-cell marker) and CD8 (cytotoxic) T-cells are the best studied and have provided the most consistent results. Indeed, in a recent meta-analysis, CD3 and CD8 infiltration were associated with superior survival in both the core tumor (CT) and the invasive margin (IM) across all eligible studies and all stages of disease. Mounting evidence suggests that chemotherapy efficacy may in part be mediated through modulation of local and systemic immune mechanisms. , In preclinical models, chemotherapy has been shown to increase activation of immune effector cells, inhibit regulatory T-cells, and increase immunogenicity. - With respect to fluoropyrimidines specifically, selective depletion of immunosuppressive myeloid-derived suppressor cells has been observed in vivo, and after neoadjuvant treatment in rectal cancer, densities of CD3 and CD8 T-cells in the tumor stroma are known to increase. High baseline densities of tumor infiltrating CD3 and CD8 T-cells may therefore be beneficial for chemotherapy efficacy. However, although there is strong evidence for a prognostic effect of these markers, whether they predict sensitivity to adjuvant chemotherapy is unclear—with conflicting results from previous studies in non--randomly assigned cohorts. - The QUASAR trial is the largest trial of adjuvant chemotherapy versus observation in CRC, having randomly assigned 3,239 patients, 91% of whom had stage II disease. It provides an ideal data set in which to test the ability of immune biomarkers to predict benefit from adjuvant chemotherapy. We have previously developed a deep learning cell classification algorithm to compute the densities of CD3 and CD8-stained T-cells within the CT and IM. Using this technology, we present here a prospectively planned, retrospective analysis of the QUASAR trial, investigating the relationship between CD3/CD8 cell densities in the CT/IM and recurrence and whether they predict benefit from adjuvant chemotherapy. Key Objective Are CD3 and CD8 tumor infiltrating lymphocyte densities prognostic in early-stage colorectal cancer (CRC), and do they predict benefit from adjuvant chemotherapy? This is the first such predictive evaluation in a randomized data set with observation-only control. Knowledge Generated CD3/CD8 densities were strongly prognostic, with recurrence rates in high-risk groups twice those in low-risk groups. Both high- and low-risk groups saw proportional reductions in recurrence with adjuvant chemotherapy of similar magnitude. CD3 Score combines information from CD3 densities in the core tumor and invasive margin. Number needed to treat with high-risk CD3 Score was 11, compared with 36 for low-risk CD3 Score. Relevance (E.M. O'Reilly) The authors report on a prognostic tool (CD3 Score) using a CD3/CD8 tumor infiltrating lymphocytes artificial intelligence algorithm in a older large randomized trial in early stage CRC. This relatively simple tool has potential clinical application and warrants further investigation.* *Relevance section written by JCO Associate Editor Eileen M. O'Reilly, MD. In QUASAR, 3,239 patients with CRC (91% stage II) were randomly assigned between chemotherapy and observation across 150 sites in 19 countries between May 1994 and December 2003. Participants had complete primary tumor resection, no distant metastases, and uncertain need for adjuvant chemotherapy. Consenting participants were randomly assigned to 6 months of 5-fluorouracil and folinic acid chemotherapy (n = 1,622) or observation alone (n = 1,617). Radiotherapy for rectal cancers was as per the treating physician. QUASAR was managed by the Universities of Oxford and Birmingham, United Kingdom and overseen by a trial steering committee, adhering to Good Clinical Practice guidelines and the Declaration of Helsinki. Ethical approval for this translational study was granted by the North East York (08/H0903/62) and University of Leeds research ethics committees (MREC 19-018). Eligibility Randomly assigned QUASAR participants with sufficient archival formalin-fixed paraffin-embedded tumor tissue for immunohistochemical (IHC) analysis were eligible. Exclusion criteria were synchronous tumors, nonadenocarcinoma/mucinous carcinoma, and assay failure (tissue loss/artifact not corrected by repeat staining). Outcome Measures For prognostic analyses, the primary outcome was recurrence-free interval (RFI) over the whole follow-up period. To enhance statistical power, recurrence at 2 years was the primary end point for predictive analyses comparing treatment benefit, as the effects of chemotherapy in QUASAR were seen in this period with no subsequent gain or loss of benefit. RFI is defined as the time in days from random assignment to first CRC recurrence or death due to CRC. Deaths from other causes without recorded recurrence were considered censoring events. Patients without events were censored at the date last known to be recurrence free. Immunohistochemistry Three 4-μm tissue sections were cut onto Superfrost Plus slides (VWR, Lutterworth, United Kingdom) and stained separately with anti-CD3 (2GV6; Roche Diagnostics Solutions [RDS], Tucson, AZ) and anti-CD8 (SP239; RDS) rabbit monoclonal antibodies, and hematoxylin and eosin (H&E; Mayer's hematoxylin; Scott's tap water substitute as the bluing reagent). IHC was performed using a BenchMark ULTRA instrument (RDS). Digital Image Analysis Digital slide images were generated using a VENTANA DP200 scanner. Pathologists (RDS), blinded to treatment allocations and outcomes, annotated the CT and IM on CD3-stained sections. CD3 annotations were transferred to adjacent CD8 images via image registration algorithms. A deep learning cell classification algorithm identified CD3 + and CD8 + cells in the CT and IM. Digital analysis reported the tissue area and detected T-cell counts, yielding cell densities (cells/mm 2 ). Statistical Analysis Baseline patient characteristics were compared between treatment arms using two-tailed t -tests (continuous variables) and Mantel-Haenszel tests (categorical variables). Patient characteristics were compared with the whole trial population using the same tests. SAS version 9.2 was used for all analyses (SAS Institute, Cary, NC). Prognostic Analyses This study set out to analyze tissue from 850 QUASAR participants (approximately 520 with colon and 330 with rectal cancer). Before IHC review, patients were randomly partitioned into two equal-sized training and validation subsets. Optimal cutpoints to divide tumors into high- and low-recurrence risk groups by CD3-CT, CD3-IM, CD8-CT, and CD8-IM cell densities were developed in the training set using maximum-likelihood methods. - Cutpoints developed in the training set were then tested in the validation set. For both the training and validation sets, log-rank analyses of the primary end point (RFI) were used to compare the rate of recurrence in high- and low-recurrence risk groups, with a two-fold higher recurrence rate in the high- versus low-risk group considered clinically important. A two-sided P value <.05 was considered significant. With 425 patients in the training set, of whom approximately 24% would have recurred, there was more than 95% power to detect a two-fold recurrence ratio between high- and low-recurrence risk groups, assuming approximately half of the tumors would be categorized as high-risk and half as low-risk. It was decided a priori that, should the prognostic value of the cell densities be similar in both the training and validation sets, the relationship between cell density and rate of recurrence would be reported in both treatment arms combined, allowing the detection of lesser differences in prognosis between the high- and low-risk groups: with 850 patients, there would be 90% power to detect a 50% higher recurrence risk. Log-rank methods were used to investigate whether relationships between cell densities and rate of recurrence remained significant after controlling for individual clinicopathological covariates (age, primary tumor location, nodal involvement, number of nodes examined, pathologic T stage, tumor grade, mismatch repair [MMR] status, lymphatic and/or vascular invasion), with P < .01 considered significant for tests of heterogeneity. Predictive Analyses Cutpoints developed in prognostic analyses were taken forward to predictive analyses. For predictive analyses, the proportional reductions in recurrence (recurrence rate ratio [RR]) were compared between high-risk and low-risk groups, with estimates of absolute chemotherapy benefit at 2 years given for each group. A log-rank test was used to assess the significance of the biomarker-treatment interaction. Sensitivity Analyses Since adjuvant chemotherapy is not recommended in MMR-deficient (dMMR) stage II colon cancer and, in QUASAR, chemotherapy was ineffective in those age 70 years or older, predictive analyses were repeated in the subset of patients age <70 years with MMR-proficient (pMMR) or MMR-unknown tumors. Randomly assigned QUASAR participants with sufficient archival formalin-fixed paraffin-embedded tumor tissue for immunohistochemical (IHC) analysis were eligible. Exclusion criteria were synchronous tumors, nonadenocarcinoma/mucinous carcinoma, and assay failure (tissue loss/artifact not corrected by repeat staining). For prognostic analyses, the primary outcome was recurrence-free interval (RFI) over the whole follow-up period. To enhance statistical power, recurrence at 2 years was the primary end point for predictive analyses comparing treatment benefit, as the effects of chemotherapy in QUASAR were seen in this period with no subsequent gain or loss of benefit. RFI is defined as the time in days from random assignment to first CRC recurrence or death due to CRC. Deaths from other causes without recorded recurrence were considered censoring events. Patients without events were censored at the date last known to be recurrence free. Three 4-μm tissue sections were cut onto Superfrost Plus slides (VWR, Lutterworth, United Kingdom) and stained separately with anti-CD3 (2GV6; Roche Diagnostics Solutions [RDS], Tucson, AZ) and anti-CD8 (SP239; RDS) rabbit monoclonal antibodies, and hematoxylin and eosin (H&E; Mayer's hematoxylin; Scott's tap water substitute as the bluing reagent). IHC was performed using a BenchMark ULTRA instrument (RDS). Digital slide images were generated using a VENTANA DP200 scanner. Pathologists (RDS), blinded to treatment allocations and outcomes, annotated the CT and IM on CD3-stained sections. CD3 annotations were transferred to adjacent CD8 images via image registration algorithms. A deep learning cell classification algorithm identified CD3 + and CD8 + cells in the CT and IM. Digital analysis reported the tissue area and detected T-cell counts, yielding cell densities (cells/mm 2 ). Baseline patient characteristics were compared between treatment arms using two-tailed t -tests (continuous variables) and Mantel-Haenszel tests (categorical variables). Patient characteristics were compared with the whole trial population using the same tests. SAS version 9.2 was used for all analyses (SAS Institute, Cary, NC). Prognostic Analyses This study set out to analyze tissue from 850 QUASAR participants (approximately 520 with colon and 330 with rectal cancer). Before IHC review, patients were randomly partitioned into two equal-sized training and validation subsets. Optimal cutpoints to divide tumors into high- and low-recurrence risk groups by CD3-CT, CD3-IM, CD8-CT, and CD8-IM cell densities were developed in the training set using maximum-likelihood methods. - Cutpoints developed in the training set were then tested in the validation set. For both the training and validation sets, log-rank analyses of the primary end point (RFI) were used to compare the rate of recurrence in high- and low-recurrence risk groups, with a two-fold higher recurrence rate in the high- versus low-risk group considered clinically important. A two-sided P value <.05 was considered significant. With 425 patients in the training set, of whom approximately 24% would have recurred, there was more than 95% power to detect a two-fold recurrence ratio between high- and low-recurrence risk groups, assuming approximately half of the tumors would be categorized as high-risk and half as low-risk. It was decided a priori that, should the prognostic value of the cell densities be similar in both the training and validation sets, the relationship between cell density and rate of recurrence would be reported in both treatment arms combined, allowing the detection of lesser differences in prognosis between the high- and low-risk groups: with 850 patients, there would be 90% power to detect a 50% higher recurrence risk. Log-rank methods were used to investigate whether relationships between cell densities and rate of recurrence remained significant after controlling for individual clinicopathological covariates (age, primary tumor location, nodal involvement, number of nodes examined, pathologic T stage, tumor grade, mismatch repair [MMR] status, lymphatic and/or vascular invasion), with P < .01 considered significant for tests of heterogeneity. Predictive Analyses Cutpoints developed in prognostic analyses were taken forward to predictive analyses. For predictive analyses, the proportional reductions in recurrence (recurrence rate ratio [RR]) were compared between high-risk and low-risk groups, with estimates of absolute chemotherapy benefit at 2 years given for each group. A log-rank test was used to assess the significance of the biomarker-treatment interaction. Sensitivity Analyses Since adjuvant chemotherapy is not recommended in MMR-deficient (dMMR) stage II colon cancer and, in QUASAR, chemotherapy was ineffective in those age 70 years or older, predictive analyses were repeated in the subset of patients age <70 years with MMR-proficient (pMMR) or MMR-unknown tumors. This study set out to analyze tissue from 850 QUASAR participants (approximately 520 with colon and 330 with rectal cancer). Before IHC review, patients were randomly partitioned into two equal-sized training and validation subsets. Optimal cutpoints to divide tumors into high- and low-recurrence risk groups by CD3-CT, CD3-IM, CD8-CT, and CD8-IM cell densities were developed in the training set using maximum-likelihood methods. - Cutpoints developed in the training set were then tested in the validation set. For both the training and validation sets, log-rank analyses of the primary end point (RFI) were used to compare the rate of recurrence in high- and low-recurrence risk groups, with a two-fold higher recurrence rate in the high- versus low-risk group considered clinically important. A two-sided P value <.05 was considered significant. With 425 patients in the training set, of whom approximately 24% would have recurred, there was more than 95% power to detect a two-fold recurrence ratio between high- and low-recurrence risk groups, assuming approximately half of the tumors would be categorized as high-risk and half as low-risk. It was decided a priori that, should the prognostic value of the cell densities be similar in both the training and validation sets, the relationship between cell density and rate of recurrence would be reported in both treatment arms combined, allowing the detection of lesser differences in prognosis between the high- and low-risk groups: with 850 patients, there would be 90% power to detect a 50% higher recurrence risk. Log-rank methods were used to investigate whether relationships between cell densities and rate of recurrence remained significant after controlling for individual clinicopathological covariates (age, primary tumor location, nodal involvement, number of nodes examined, pathologic T stage, tumor grade, mismatch repair [MMR] status, lymphatic and/or vascular invasion), with P < .01 considered significant for tests of heterogeneity. Cutpoints developed in prognostic analyses were taken forward to predictive analyses. For predictive analyses, the proportional reductions in recurrence (recurrence rate ratio [RR]) were compared between high-risk and low-risk groups, with estimates of absolute chemotherapy benefit at 2 years given for each group. A log-rank test was used to assess the significance of the biomarker-treatment interaction. Since adjuvant chemotherapy is not recommended in MMR-deficient (dMMR) stage II colon cancer and, in QUASAR, chemotherapy was ineffective in those age 70 years or older, predictive analyses were repeated in the subset of patients age <70 years with MMR-proficient (pMMR) or MMR-unknown tumors. Study Material Tumor tissue from 531 (61%) colon cancers and 337 (39%) rectal cancers was selected at random from the QUASAR tissue bank (Fig ) and analyzed by CD3 and CD8 IHC (Fig ). In total, 2,358 of 3,239 randomly assigned patients had tissue available. Patients with tissue were slightly older (mean age 62 v 60 years) and more likely to have colon and stage III cancers. Outcomes were slightly better in those patients in the tissue bank (hazard ratio [HR] for recurrence, 0.80 [95% CI, 0.69 to 0.93]; Data Supplement, Table S1, online only). Tissue samples selected were enriched for rectal cancer to increase the power to examine differences by tumor site. No other factors were significantly associated with selection. Outcomes were not different between the two groups (HR for relapse, 1.04 [95% CI, 0.87 to 1.23]; Data Supplement, Table S2). Samples were randomly partitioned into training and validation sets. Demographic and clinical characteristics were balanced between the two groups. CD3-CT cell density was successfully evaluated in 851 (98.0%), CD3-IM in 833 (96.0%), CD8-CT in 849 (97.0%), and CD8-IM in 820 (94.5%) patients. Association of Cell Density Measures With Outcomes Optimal cell density cutpoints were defined in the training set using maximum-likelihood methods. For CD3-CT, this produced a cutpoint of 318 cells/mm 2 , with 172 (40%) patients classified as high recurrence risk. The cutpoint was 798 cells/mm 2 for CD3-IM (290 [69%] patients in high-risk group), 81 cells/mm 2 for CD8-CT (246 [58%] patients in high-risk group), and 186 cells/mm 2 for CD8-IM (259 [63%] patients in high-risk group; Data Supplement, Fig S1). In the training set, the recurrence rate in the high-risk group was at least twice that of the low-risk group for all measures (CD3-CT: RR, 2.00 [95% CI, 1.33 to 2.94], P = .0008; CD3-IM: 2.38 [95% CI, 1.59 to 3.57], P < .00001; CD8-CT: 2.17 [95% CI, 1.59 to 3.57], P = .0001; CD8-IM: 2.13 [95% CI, 1.43 to 3.23], P = .0001; Figs A, C, E, G). The two-fold higher recurrence rate in the training set was closely replicated in the validation set (CD3-CT: RR, 1.96 [95% CI, 1.30 to 2.94], P = .002; CD3-IM: 1.79 [95% CI, 1.18 to 2.70], P = .005; CD8-CT: 1.72 [95% CI, 1.18 to 2.56], P = .005; CD8-IM: 1.72 [95% CI, 1.15 to 2.56], P = .008; Figs B, D, F, H; Data Supplement, Fig S2). As the prognostic effect of each measure was similar in the training and validation sets, the two cohorts were combined to investigate whether any associations between the high-risk and low-risk groups and other patient or tumor characteristics might partly or wholly explain the association with recurrence. The proportions of tumors with high-risk CD3 and CD8 scores were similar in the training and validation sets and in those allocated chemotherapy and control. High-risk scores were, however, somewhat more likely in rectal than colon cancer, in stage III than stage II, in tumors with vascular invasion, and in pMMR than dMMR tumors (Data Supplement, Table S3). There was no significant association between CD3/CD8 densities and sex, T-stage, tumor grade, or RAS or BRAF status. In the combined training and validation sets, the strength of the prognostic effect of each marker in each region was broadly similar, although CD3-IM had the marginally strongest prognostic effect (Chi-square 18.9, P < .0001) and then CD8-CT (18.4, P < .0001), CD3-CT (16.8, P < .0001), and CD8-IM (14.7, P < .0001). The strength of association of CD3-IM with recurrence was also least affected by adjustment for the other density measures (Data Supplement, Fig S3). Figure shows recurrence RRs for high versus low CD3-IM risk groups stratified by various patient and tumor characteristics. The relationship between CD3-IM and RFI remained undiminished and highly significant after adjustment for these potential confounders. The associations of the other cell density measures with RFI were also unaffected by adjustment for other variables (Data Supplement, Figs S4-S6). Predictive Analyses In each of the cell density risk groups, there were similar reductions in 2-year recurrence in those allocated adjuvant chemotherapy compared with those who were not (Fig ). Tests for interaction showed no suggestion of differences in the proportional reduction achieved with chemotherapy (CD3-CT: P interaction = .9; CD3-IM: P interaction = .7; CD8-CT: P interaction = .8; CD8-IM: P interaction = .5). There remained no predictive effect of any marker after exclusion of patients with dMMR disease and those age 70 years or older (data not shown). CD3 Score: Incorporating Information From Multiple Markers All markers were well correlated with each other but, nevertheless, a proportion of patients were classified as having high recurrence risk with one marker but low with others (Data Supplement, Fig S7). We therefore examined the utility of combining information from more than one marker to refine the prognostic effect. Given the similar prognostic strengths of each marker and to generate the most practical test for potential use in routine clinical practice, we chose to combine CD3-IM with CD3-CT (hereafter, CD3 Score), thus requiring use of a single antibody and analysis of a single IHC slide. By assessing patients with high recurrence risk by both markers, high recurrence risk by one but not both markers, and low recurrence risk by both markers, three risk groups were generated, with 306 (37%), 259 (31%), and 267 (32%) of the 832 patients with IHC results classified as high-, intermediate-, and low-risk by CD3 Score, respectively. The 5-year risk of recurrent disease was 10% for the lowest-risk group, 22% for the intermediate-risk group, and 30% for the highest-risk group (Fig A). Given that QUASAR chemotherapy reduced 2-year recurrence by 42%, with similar proportional reductions in the high-, intermediate-, and low-risk groups (Fig ), the number of patients needed to treat (NNT) with adjuvant chemotherapy to prevent one recurrence of disease in the first 2 years was therefore 11 for the high-risk group, 21 for the intermediate-risk group, and 36 for the low-risk group (Table ). The strength of the prognostic effect of the CD3 Score in comparison with other clinicopathological indicators of recurrence risk is summarized in the Data Supplement (Fig S8), showing that only the nodal status had a stronger influence on RFI. The prognostic associations were equally strong when considering only the subgroups of stage II patients with low and high recurrence risk according to routinely reported clinicopathological criteria (Figs B and C). The absolute difference in 5-year recurrence risk between the high- and low-risk CD3 Score groups was 19% (26% v 7%) in stage II patients considered low recurrence risk by standard clinicopathological criteria and 18% (32% v 14%) for those considered high risk. By comparison, the difference in 5-year recurrence risk for stage II patients considered at high and low recurrence risk by standard clinicopathological criteria alone was just 8% (24% v 16%) or 9% (22% v 13%) when patients with rectal cancer were excluded (Data Supplement, Fig S9). Tumor tissue from 531 (61%) colon cancers and 337 (39%) rectal cancers was selected at random from the QUASAR tissue bank (Fig ) and analyzed by CD3 and CD8 IHC (Fig ). In total, 2,358 of 3,239 randomly assigned patients had tissue available. Patients with tissue were slightly older (mean age 62 v 60 years) and more likely to have colon and stage III cancers. Outcomes were slightly better in those patients in the tissue bank (hazard ratio [HR] for recurrence, 0.80 [95% CI, 0.69 to 0.93]; Data Supplement, Table S1, online only). Tissue samples selected were enriched for rectal cancer to increase the power to examine differences by tumor site. No other factors were significantly associated with selection. Outcomes were not different between the two groups (HR for relapse, 1.04 [95% CI, 0.87 to 1.23]; Data Supplement, Table S2). Samples were randomly partitioned into training and validation sets. Demographic and clinical characteristics were balanced between the two groups. CD3-CT cell density was successfully evaluated in 851 (98.0%), CD3-IM in 833 (96.0%), CD8-CT in 849 (97.0%), and CD8-IM in 820 (94.5%) patients. Optimal cell density cutpoints were defined in the training set using maximum-likelihood methods. For CD3-CT, this produced a cutpoint of 318 cells/mm 2 , with 172 (40%) patients classified as high recurrence risk. The cutpoint was 798 cells/mm 2 for CD3-IM (290 [69%] patients in high-risk group), 81 cells/mm 2 for CD8-CT (246 [58%] patients in high-risk group), and 186 cells/mm 2 for CD8-IM (259 [63%] patients in high-risk group; Data Supplement, Fig S1). In the training set, the recurrence rate in the high-risk group was at least twice that of the low-risk group for all measures (CD3-CT: RR, 2.00 [95% CI, 1.33 to 2.94], P = .0008; CD3-IM: 2.38 [95% CI, 1.59 to 3.57], P < .00001; CD8-CT: 2.17 [95% CI, 1.59 to 3.57], P = .0001; CD8-IM: 2.13 [95% CI, 1.43 to 3.23], P = .0001; Figs A, C, E, G). The two-fold higher recurrence rate in the training set was closely replicated in the validation set (CD3-CT: RR, 1.96 [95% CI, 1.30 to 2.94], P = .002; CD3-IM: 1.79 [95% CI, 1.18 to 2.70], P = .005; CD8-CT: 1.72 [95% CI, 1.18 to 2.56], P = .005; CD8-IM: 1.72 [95% CI, 1.15 to 2.56], P = .008; Figs B, D, F, H; Data Supplement, Fig S2). As the prognostic effect of each measure was similar in the training and validation sets, the two cohorts were combined to investigate whether any associations between the high-risk and low-risk groups and other patient or tumor characteristics might partly or wholly explain the association with recurrence. The proportions of tumors with high-risk CD3 and CD8 scores were similar in the training and validation sets and in those allocated chemotherapy and control. High-risk scores were, however, somewhat more likely in rectal than colon cancer, in stage III than stage II, in tumors with vascular invasion, and in pMMR than dMMR tumors (Data Supplement, Table S3). There was no significant association between CD3/CD8 densities and sex, T-stage, tumor grade, or RAS or BRAF status. In the combined training and validation sets, the strength of the prognostic effect of each marker in each region was broadly similar, although CD3-IM had the marginally strongest prognostic effect (Chi-square 18.9, P < .0001) and then CD8-CT (18.4, P < .0001), CD3-CT (16.8, P < .0001), and CD8-IM (14.7, P < .0001). The strength of association of CD3-IM with recurrence was also least affected by adjustment for the other density measures (Data Supplement, Fig S3). Figure shows recurrence RRs for high versus low CD3-IM risk groups stratified by various patient and tumor characteristics. The relationship between CD3-IM and RFI remained undiminished and highly significant after adjustment for these potential confounders. The associations of the other cell density measures with RFI were also unaffected by adjustment for other variables (Data Supplement, Figs S4-S6). In each of the cell density risk groups, there were similar reductions in 2-year recurrence in those allocated adjuvant chemotherapy compared with those who were not (Fig ). Tests for interaction showed no suggestion of differences in the proportional reduction achieved with chemotherapy (CD3-CT: P interaction = .9; CD3-IM: P interaction = .7; CD8-CT: P interaction = .8; CD8-IM: P interaction = .5). There remained no predictive effect of any marker after exclusion of patients with dMMR disease and those age 70 years or older (data not shown). All markers were well correlated with each other but, nevertheless, a proportion of patients were classified as having high recurrence risk with one marker but low with others (Data Supplement, Fig S7). We therefore examined the utility of combining information from more than one marker to refine the prognostic effect. Given the similar prognostic strengths of each marker and to generate the most practical test for potential use in routine clinical practice, we chose to combine CD3-IM with CD3-CT (hereafter, CD3 Score), thus requiring use of a single antibody and analysis of a single IHC slide. By assessing patients with high recurrence risk by both markers, high recurrence risk by one but not both markers, and low recurrence risk by both markers, three risk groups were generated, with 306 (37%), 259 (31%), and 267 (32%) of the 832 patients with IHC results classified as high-, intermediate-, and low-risk by CD3 Score, respectively. The 5-year risk of recurrent disease was 10% for the lowest-risk group, 22% for the intermediate-risk group, and 30% for the highest-risk group (Fig A). Given that QUASAR chemotherapy reduced 2-year recurrence by 42%, with similar proportional reductions in the high-, intermediate-, and low-risk groups (Fig ), the number of patients needed to treat (NNT) with adjuvant chemotherapy to prevent one recurrence of disease in the first 2 years was therefore 11 for the high-risk group, 21 for the intermediate-risk group, and 36 for the low-risk group (Table ). The strength of the prognostic effect of the CD3 Score in comparison with other clinicopathological indicators of recurrence risk is summarized in the Data Supplement (Fig S8), showing that only the nodal status had a stronger influence on RFI. The prognostic associations were equally strong when considering only the subgroups of stage II patients with low and high recurrence risk according to routinely reported clinicopathological criteria (Figs B and C). The absolute difference in 5-year recurrence risk between the high- and low-risk CD3 Score groups was 19% (26% v 7%) in stage II patients considered low recurrence risk by standard clinicopathological criteria and 18% (32% v 14%) for those considered high risk. By comparison, the difference in 5-year recurrence risk for stage II patients considered at high and low recurrence risk by standard clinicopathological criteria alone was just 8% (24% v 16%) or 9% (22% v 13%) when patients with rectal cancer were excluded (Data Supplement, Fig S9). In this study of tissue from the QUASAR trial, high CD3 and CD8 tumor infiltrating lymphocyte (TIL) densities in both the CT and IM were strongly associated with reduced tumor recurrence risk postsurgery. However, in this first rigorous evaluation of the predictive effect of these biomarkers, CD3 and CD8 densities did not predict chemotherapy sensitivity, with the proportional reductions in recurrence with chemotherapy nearly identical in high and low recurrence risk groups. However, as the 5-year recurrence risk in high-risk tumors was twice that in low-risk tumors, the absolute reductions in recurrence with chemotherapy were also about twice as large for high-risk as compared with low-risk tumors. The tumor node metastasis (TNM) classification has been used to guide treatment decisions in CRC for more than 50 years However, significant variability in recurrence risk exists within stage groups. The tumor immune microenvironment plays an important role in modulating CRC progression, suggesting that an accurate assessment of the immune response to tumors might provide useful supplementary information. To date, it has not been possible to agree a unified approach to such assessments, with the main concerns being how best to strike a balance between simplicity, value of information, and reproducibility—although common approaches have been proposed. , In melanoma, quantification of TILs on an H&E-stained slide is already incorporated into the staging system, where a simple three-point scale of absent, nonbrisk, or brisk is used. However, debate continues as to whether this is the optimal method of categorization, noting issues with interobserver variability despite the simplicity of the scale , —an area where artificial intelligence technologies might provide a solution. CD3/CD8 densities have been most studied as prognostic biomarkers in CRC, and results across studies show consistent associations. One well-established IHC-based method of CD3/CD8 TIL assessment is the Immunoscore, which is strongly associated with DFS in stage I-III colon cancer. This study adds to the field by testing the predictive effect of CD3/CD8 densities on benefit from adjuvant treatment in a randomized data set—a necessary step to fully evaluate the clinical applicability of the biomarkers. A previous predictive analysis of Immunoscore in a non--randomly assigned cohort of patients who did/did not receive adjuvant chemotherapy during routine care suggested that high Immunoscore (low recurrence risk), but not low Immunoscore, might be associated with time-to-recurrence benefit from adjuvant chemotherapy. In our analysis, chemotherapy produced similar reductions in recurrence regardless of CD3/CD8 density, suggesting that the earlier study may have had inadequate power to detect chemotherapy benefit in the smaller Immunoscore-low subgroup. Although there are reports to suggest chemotherapy may have either beneficial or deleterious effects on the tumor immune microenvironment, - our findings are consistent with the lack of a clear biological rationale for an interaction between tumor immune cell infiltration and chemotherapy efficacy. There is a clearer rationale and evidence to support an interaction between the tumor immune contexture and immunotherapy efficacy, for which dMMR status is a well-established proxy measure. - CD3/CD8 densities were higher in dMMR than pMMR tumors in this study but, as in earlier studies, , the prognostic effects of CD3/CD8 densities were independent of MMR status. Interestingly, IHC-based measures of interactions between tumor and immune cells are now showing promise in predicting benefit from checkpoint inhibition in CRC, including in pMMR disease, warranting further study. Adjuvant treatment decisions are most complex in stage II pMMR colon cancer, where risks and benefits are finely balanced. Here, the prognostic value of the CD3 Score substantially outperformed that of current standard clinicopathological prognostic factors. A high-risk CD3 Score could therefore be used to identify a subset of patients with stage II pMMR disease and no additional risk factors where adjuvant chemotherapy might have relevance. Conversely, patients with stage II disease and high-risk features or stage III disease (both higher recurrence risk groups) with a low-risk CD3 Score might wish to consider observation, particularly if elderly or frail. Information regarding the tumor immune contexture might similarly complement that obtained from ctDNA testing. In the DYNAMIC trial, 85% of patients were ctDNA negative, and among these, standard clinicopathological risk markers remained strongly prognostic. CD3 Score in addition to ctDNA and clinicopathological assessments might provide optimal risk stratification. A further role for the CD3 Score might be in patient selection for neoadjuvant chemotherapy, a limitation of which is imprecision in radiological staging. Strengths of this study are use of tissue from QUASAR, the largest randomized trial of adjuvant chemotherapy versus observation in CRC, and internal validation using training and validation sets. A limitation is the age of QUASAR, originally published in 2007. Current surgical and pathology standards are superior to those in place at the time of data collection. Indeed, stage II colon cancer recurrence risk was shown to have roughly halved between 2004-2008 and 2014-2019 in a recently published Danish cohort study. With no similar data sets to QUASAR including an observation-only control, an external validation of the findings presented here is not possible. However, the consistency of the prognostic effect of the CD3 Score across both QUASAR treatment groups means the NNT derived here can be appropriately adjusted using more recent nonrandomized data. In summary, CD3/CD8 TIL densities provide a strong indication of recurrence risk after surgery for CRC. Proportional reductions in recurrence achieved with adjuvant chemotherapy are similar in high and low recurrence risk groups. Hence, as the 5-year recurrence risk in high-risk tumors was twice that in low-risk tumors, the number of low-risk patients needed to treat to prevent one recurrence is about twice the number of high-risk patients—information clinicians and patients may find useful in routine practice. |
The behavioral signature of stepwise learning strategy in male rats and its neural correlate in the basal forebrain | 5fa52c7c-85cf-4b6f-bac7-acb4c0c92f92 | 10362048 | Physiology[mh] | Associative learning is essential for survival and allows animals and humans to predict future reward based on environmental stimuli , or their own actions – . Understanding the algorithmic principles of associative learning has been a central question in psychology and neuroscience – , and has broad implications in machine learning and artificial intelligence – . While the learning of stimulus-reward and action-reward associations have been historically studied under the separate labels of Pavlovian , and instrumental , conditioning, most learning scenarios require the synergistic contribution from both types of learning strategies. For example, when a new reward-predicting stimulus is introduced to the environment, the Pavlovian strategy might not be sufficient because oftentimes the reward would not be delivered unless animals take specific actions. In experimental settings, such actions could be a lever press, or a saccade toward a target, or multiple licks before the reward is delivered. In these scenarios, reward is obtained only when sensory stimuli and animals’ actions occur in a specific order as a behavioral sequence , – . Compared to the wealth of knowledge about Pavlovian and instrumental conditioning, little is understood about how animals learn behavioral sequences that contain both stimuli and actions. Converging views from theoretical studies support the idea that reward-predicting behavioral sequences can be efficiently learned using a strategy that we will refer to as stepwise learning: learning starts from the event closest to the reward, while earlier events are learned in later steps. This learning strategy was initially proposed by Skinner and more recently elaborated into formal learning models , . Similar learning dynamics are also predicted by reinforcement learning algorithms, in which states that are closer to the final reward are learned first . The stepwise learning strategy has also been successfully used in various animal training scenarios to incrementally chain single behaviors into long sequences over multiple training steps . The goal of the current study is to test whether animals use the stepwise learning strategy to learn reward-predicting behavioral sequences that contain both stimuli and actions. We seek to identify the behavioral and neural signatures of this learning process that can delineate the discrete steps of learning. A major challenge in understanding this type of learning is that behavioral sequences are controlled not only by the experimenter but also by the animal, which is free to take various actions. We reason that, at the beginning of learning, animals’ actions would be less constrained and therefore would generate a large repertoire of behavioral sequences that may or may not lead to the rewarding outcome. As the stepwise learning process unfolds, the repertoire of behavioral sequences should become increasingly selective as well as more frequently rewarded. Therefore, the behavioral signature of the stepwise learning strategy may reside in how the entire repertoire of behavioral sequences become sequentially refined during the learning process. In the current study, we identified such a behavioral signature, which corresponded to the discrete steps in the stepwise learning process. In order to validate the behavioral signature for the stepwise learning strategy, we focused on a special subset of noncholinergic neurons in the basal forebrain (BF), which are referred to as BF bursting neurons – . Previous studies have found that BF bursting neurons convey a reward-prediction error signal , , , , and show highly robust phasic bursting responses to reward-predicting sensory stimuli irrespective of their sensory modalities – . Moreover, such responses only emerge after reward-based associative learning , and are tightly coupled with behavioral performance and promotes faster decision speeds – . These observations suggest that increased BF bursting neuron activities toward a behavioral event reflects that the event has been learned as a reward predictor. By observing the temporal evolution of BF bursting neuron activities throughout the learning process in the current study, we predict that BF responses should mirror the stepwise learning process: BF activity should first emerge toward the last behavioral event closest to the reward, and subsequently develop toward the earlier behavioral events. Such behavioral and neurophysiological findings will provide important insights on how behavioral sequences are learned. A model for learning behavioral sequences using the stepwise learning strategy To gain intuition about the stepwise learning strategy, we first considered a toy example in which a three-element sequence A-B-C predicted reward (Fig. ). This sequence can be learned using the stepwise strategy in three discrete steps, starting from the event closest to the reward and sequentially incorporating earlier events (Fig. ). As more behavioral events are learned as reward predictors in each step, only behavioral sequences that contain all the learned events would predict reward and therefore preferentially executed, while incompatible sequences that do not contain all the learned events would not predict reward and therefore be eliminated from the behavioral repertoire. As a result, the discrete steps of learning would correspond to the stepwise elimination of non-rewarded behavioral sequences that share subsets of behavioral elements (Fig. ). We hypothesized that this sequential refinement of reward-seeking behaviors might provide a behavioral signature of the stepwise learning strategy. Sequential refinement of reward-seeking behaviors during new learning To test whether animals indeed use the stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions, we trained adult Long-Evans rats in an auditory discrimination task. Rats entered the fixation port to initiate each trial, where they encountered three trial types (S left ; S right ; catch) with equal probabilities that respectively indicated sucrose water reward in the left or right port, or no reward in the case of catch trials (no stimulus) (Fig. ). During the initial auditory discrimination phase, S left and S right were two distinct sound stimuli. After reaching asymptotic performance, rats entered the new learning phase (first new learning session denoted as the D 0 session), in which the S right sound stimulus was switched to a novel light stimulus that minimized sensory generalization from past experience (Fig. and Table ). During the new learning phase, rats maintained stable levels of performance toward the previously learned S left sound stimulus (Fig. ) (94.3 ± 4.8% correct, 109 ± 20 trials per session, mean ± std). Within the first three sessions of the new learning phase, all rats ( N = 7) began responding correctly in the new light trials (the first such session denoted as the D 1 session) and maintained stable levels of >90% correct response rates afterwards (Fig. and Table ). To understand how the repertoire of behavioral sequences evolved during learning, we examined all possible behavioral sequences that the animal might experience (Fig. ). This approach allowed us to identify non-rewarded behavioral sequences that were not associated with specific trial types but were, nonetheless, highly relevant for the learning process. We identified three types of behavioral sequences whose frequencies consistently increased during the new learning phase. These three types of behavioral sequences included the rewarded licking behavior in the new light trials (light licks), as well as two types of non-rewarded behavioral sequences: catch licks and no-fixation licks (Fig. ). Catch licks refers to licking responses toward the right reward port in catch trials when no sensory stimulus was presented. No-fixation licks refers to the situation in which rats directly licked at the right reward port without first entering the center fixation port. All three behavioral sequences shared the common feature of licking the right reward port (lick-right). Both no-fixation licks and catch licks were largely absent before the new learning phase, and emerged and peaked during early sessions of new learning, before subsequently diminished in later sessions (Fig. ). No-fixation licks occurred most frequently at the D 1 session, while catch licks peaked in the same session ( N = 1/7) or later in most animals ( N = 6/7). We will denote the peak of catch licks as the D 2 session in each animal. The consistent temporal order of the D 1 and D 2 sessions within each animal allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The temporal dynamics of the three types of rightward licks during new learning (Fig. ) showed that non-rewarded behaviors were sequentially eliminated while rewarded behaviors were preserved. This temporal dynamics resembled the pattern of sequential refinement of behavioral sequences predicted by the stepwise learning strategy (Fig. ), and likely corresponded to the discrete steps in the underlying learning process. To further test whether such patterns of sequential refinements represent a general feature of behavioral sequence learning, we trained a separate cohort of animals and observed similar behavioral signatures regardless of the sensory modality of the stimulus or the laterality of the new learning side (Fig. ). These observations support the idea that animals do use the stepwise strategy to learn behavioral sequences. In the following analyses, we tested additional predictions of the stepwise learning strategy at both behavioral and neurophysiological levels. Initial learning was characterized by the rapid emergence of reward-seeking behaviors and corresponding increases in BF activities To validate the behavioral observations and understand the underlying neural dynamics, we recorded BF neuronal activity throughout the learning process (Fig. ) and used the consistent S left sound as the control stimulus to identify stable populations of BF bursting neurons (Figs. and ). A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. ). The population response of BF bursting neurons were highly consistent across animals (Fig. ), and remained remarkably stable in S left trials throughout the learning process (Fig. ). The inclusion of S left trials therefore allowed us to record from stable and representative populations of BF bursting neurons throughout the learning process, and to investigate how their activities dynamically evolved in other trial types during learning at single trial resolution (Fig. ). We first applied this approach to understand the behavioral and neural dynamics in the D 1 session because all three types of rightward licks emerged in this session (Fig. , middle panel). Detailed analysis of behavioral responses from a representative session (Fig. ) revealed that the three types of rightward licking behaviors emerged abruptly after a transition point (see Methods for definition). Rightward licking behaviors were mostly absent before the transition point, and rapidly switched to almost 100% licking after the transition point. This pattern of abrupt transition was consistently observed across all animals (Fig. ), while the behavioral performance and BF activities in S left sound trials remained relatively stable (Fig. ). At the neuronal level, there was a corresponding increase in the activity of BF bursting neurons that rapidly emerged after the transition in rightward reward-seeking behaviors (Figs. e and ). This increase in BF activity was most prominent in the epoch before the trial outcome as animals approached the reward port (Fig. ), but this activity was not consistently aligned with intervening behavioral events (Fig. ). In contrast, in trials before the transition point, BF bursting neurons did not show similar activity increases in the corresponding time window after exiting the fixation port (Figs. e and ). We will refer to the maximum BF activity in this window as the BF evaluation response (see Methods for definition) because it reflected animals’ internal evaluation when no sensory stimuli were presented during this epoch and because it was not consistently associated with intervening behavioral events. The emergence and subsequent elimination of non-rewarded behaviors were mirrored by changes in BF activity The stepwise learning model predicted that, as animals learned about the lick-right action as the first reward predictor, animals would initially engage in all three types of rightward licking behaviors because they all contained this reward predictor (Fig. ). We therefore examined the respective prevalence of the three types of rightward licking behaviors after the transition point in the D 1 session (Fig. ). All three types of rightward licking behaviors emerged immediately after the transition point. Moreover, after about 60 trials, no-fixation licks began to decline and occurred less frequently than light licks and catch licks (Fig. ). At the neuronal level, the corresponding prediction was that the lick-right action would be associated with increases in BF activities during the first learning step, which should be similarly present in all three types of rightward licking behaviors. Indeed, BF evaluation responses quickly increased after the transition point in all three types of rightward licking behaviors (Fig. ). The amplitudes of BF evaluation responses were similar between the three types of rightward licking behaviors within the first 60 trials after the transition. Subsequently, the BF evaluation response in no-fixation licks declined in the next 60 trials, relative to the other two types of rightward licking behaviors (Fig. ). These results support that the first step of the stepwise learning process corresponded to the first 60 trials after the transition point in the D 1 session. All three types of rightward licking behaviors were present during this step of learning. BF activity also increased to similar levels in all three types of behavioral sequences whenever animals approached and licked the right reward port, regardless of whether they had exited from the center fixation port. These results further suggest that the second step of learning started at roughly 60 trials after the transition, at which point animals learned about the second reward predictor: exiting the fixation port (Fig. ). As a result, the no-fixation lick sequence was no longer compatible with the learned reward predictors, which resulted in diminished BF evaluation responses and the elimination of this behavior from the behavioral repertoire. The other two types of rightward licks, light licks and catch licks, remained compatible and maintained high levels of BF evaluation responses. BF neurons did not respond to the new light stimulus during initial learning A further prediction of the stepwise learning model was that, at both the first and the second steps of learning, animals had not learned to use the new light stimulus as a reward predictor, and therefore the new light stimulus would not elicit responses in BF bursting neurons during these steps (Fig. ). We tested this prediction by comparing BF activities between light and catch trials in the D 1 session (Fig. ). Indeed, BF activities in light trials were highly similar to those in catch trials (in the absence of the light stimulus) in the epochs before exiting the fixation port. This was true regardless of whether animals subsequently licked at the right reward port. This observation confirmed the prediction that the new light stimulus did not activate BF bursting neurons in the D 1 session, despite the near-perfect behavioral performance in light trials after the behavior transition point. On the other hand, in the epoch after exiting the fixation port, there were similar increases in BF activity when animals licked at the right reward port in both light and catch trials (Fig. ). This increase in BF activity corresponded to the BF evaluation response described earlier (Fig. ), which reliably distinguished between lick and no lick trials, but not between light and catch trials (Fig. ). These observations support the idea that light and catch trials were treated as the same in the D 1 session, and that the light stimulus has not been learned as a reward predictor at this stage of learning. BF responses to light onset emerged later when the light stimulus was used to guide reward-seeking behavior When did the third step of the stepwise learning process take place? Since the third step was when animals learned to use the light stimulus as a reward predictor to guide reward-seeking behaviors, it should correspond to the time point when the behavioral performance in light and catch trials began to diverge. We noted that the behavioral pattern in light and catch trials were highly similar prior to the D 2 session (Fig. ), and the similarity was best illustrated in the fine behavioral and neuronal dynamics within the D 1 session (Fig. ). In contrast, during the D 2 session, the behavioral pattern in light and catch trials began to show a small but significant difference (Fig. ). This pattern suggests that the D 2 session might be when the third step of learning took place. The corresponding prediction at the neuronal level was that BF bursting neurons should begin to respond to the light stimulus in the D 2 session. We tested this prediction by comparing BF activities between light and catch lick trials in the D 2 session, and found significant differences in all epochs, including the presence of a phasic response to the light onset (Fig. ). This observation supports the idea that BF responses to the new light emerged in the D 2 session. To better understand how BF responses to the new light emerged in the D2 session, we further compared BF responses in light lick and catch lick trials between (1) late trials in the D 2 −1 session; (2) early trials in the D 2 session; (3) late trials in the D 2 session (Fig. ). At the end of the D 2 −1 session, BF neurons did not show response to the new light (paired t -test between light lick and catch lick trials, p = 0.51, N = 6). In early D 2 trials, BF phasic responses to the new light were clearly visible in 4 animals. Comparison between late trials in the D 2 −1 session and the early D 2 trials showed a trend toward increasing BF responses (Fig. , significant at p < 0.05 level; Fig. , paired t -test, p = 0.065, N = 6). Moreover, during the D 2 session, BF responses to the new light increased in all animals between early and late trials (Fig. , paired t -test, p = 0.003, N = 7). This increase took place mostly during the sustained phase of the BF response that was better aligned with fixation port exit than with stimulus onset (Fig. ). Together, these observations suggest that BF responses to the new light developed partly offline between the D 2 −1 and D 2 session, and partly strengthened during the D 2 session. Comparing the temporal dynamics of behavioral and BF responses further suggests that the emergence of BF responses to the new light preceded the elimination of catch licks during the third step of learning (Fig. ). Stronger BF responses to the new light reflected better learning and faster decisions After the third step of learning took place in the D 2 session, did the learning about the new light plateau or did the learning continue to progress? At the neuronal level, BF phasic responses to the new light continued to grow stronger after the D 2 session (Fig. ). At first glance, the continual increases in BF responses to the new light stimulus did not match with the hit rates in light trials, which had already plateaued in the D 2 session (Fig. ). However, as we have shown earlier (Figs. and ), hit rates in light trials could be a poor index of learning about the new light stimulus because light licks in the early learning sessions were not driven by the light stimulus but by later events in the behavioral sequence (exiting fixation and lick-right) as reward predictors. Those behavioral events enabled rightward licking responses in the absence of the light stimulus (catch licks and no-fixation licks). Instead of the hit rate in light trials, a better behavioral index for the learning about the light stimulus would be the difference in the levels of behavioral performance between light and catch trials. This behavioral index accounted for the contributions from the later events in the behavioral sequence that were shared between light and catch licks, and isolated the contribution of the light stimulus to the rightward licking behavior. We found that this index of light learning was strongly correlated with the amplitude of BF phasic responses to the light stimulus in individual sessions (Fig. ). This observation therefore supports the idea that the learning about the light stimulus continued to grow stronger after the D 2 session. Another dimension of the learning about the light stimulus was the change in animals’ decision speeds, measured by reaction times (RTs). Previous studies have shown that stronger BF bursting responses are quantitatively coupled with, and causally lead to, faster RTs , . Such observations predicted that there would be corresponding decreases in RTs toward the light stimulus after the D 2 session. In support of this prediction, we found that stronger phasic bursting responses to light onset were coupled with faster RTs in individual sessions (Fig. ). Together, these observations support the idea that the learning about the new light was reflected by the amplitude of BF phasic response to the light stimulus. In addition, BF activities not only reflected the learning about the light stimulus, but also predicted reward-seeking behaviors in the absence of the light stimulus. For example, in catch trials, stronger BF activities after exiting the fixation port predicted rightward licking behavior and discriminated lick trials from no lick trials (Fig. ). Moreover, increased BF activities before the start of licking predicted longer durations of licking in catch lick trials when no reward was delivered (Fig. ). These observations provided additional support for the idea that the activity of BF bursting neurons promoted reward-seeking behaviors. Reward expectations negatively modulated BF responses to trial outcomes A final validation of the learning dynamics came from how BF responses to the trial outcome were modulated by reward expectations and BF activities in earlier epochs. Previous studies have found that the response of BF bursting neurons to the reward was negatively modulated by reward expectation , , , . Such properties would predict that, in the current experiment, BF responses to the reward should decrease throughout the stepwise learning process. Indeed, BF responses to the right reward decreased over trials in the D 1 session (Figs. e2 and ), and continued to decrease over subsequent sessions (Fig. ). These results support that animals learned to better predict the rewarding outcome across different steps of the learning process. We further investigated whether the response of BF bursting neurons to trial outcomes were negatively correlated with BF responses in earlier epochs. Such a negative correlation is a hallmark feature of reward prediction error encoding and has been previously reported in BF bursting neurons , . Indeed, at the per session level, we found that the amplitude of BF responses to the reward in light trials was strongly and negatively correlated with the amplitude of BF phasic bursting response to the light onset (Fig. ). Moreover, in pre-D 2 sessions where BF responses to the new light stimulus had yet to develop, we found that BF responses to the trial outcome were negatively correlated with BF evaluation responses in the same trial (Fig. ). This negative correlation at the single trial level was observed not only when the reward was delivered (light licks) but also when the reward was absent (no-fixation licks and catch licks) (Fig. b, ). The fact that these patterns were observed in catch licks and non-fixation licks supports that animals were expecting to receive reward in those trials, and the extent of reward expectation was similarly reflected in BF evaluation responses and negatively modulated BF responses to the trial outcome. Together, these observations support that BF bursting neurons encoded reward prediction error, and that BF activities in earlier epochs (stimulus onset or evaluation window) reflected animals’ reward expectations. To gain intuition about the stepwise learning strategy, we first considered a toy example in which a three-element sequence A-B-C predicted reward (Fig. ). This sequence can be learned using the stepwise strategy in three discrete steps, starting from the event closest to the reward and sequentially incorporating earlier events (Fig. ). As more behavioral events are learned as reward predictors in each step, only behavioral sequences that contain all the learned events would predict reward and therefore preferentially executed, while incompatible sequences that do not contain all the learned events would not predict reward and therefore be eliminated from the behavioral repertoire. As a result, the discrete steps of learning would correspond to the stepwise elimination of non-rewarded behavioral sequences that share subsets of behavioral elements (Fig. ). We hypothesized that this sequential refinement of reward-seeking behaviors might provide a behavioral signature of the stepwise learning strategy. To test whether animals indeed use the stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions, we trained adult Long-Evans rats in an auditory discrimination task. Rats entered the fixation port to initiate each trial, where they encountered three trial types (S left ; S right ; catch) with equal probabilities that respectively indicated sucrose water reward in the left or right port, or no reward in the case of catch trials (no stimulus) (Fig. ). During the initial auditory discrimination phase, S left and S right were two distinct sound stimuli. After reaching asymptotic performance, rats entered the new learning phase (first new learning session denoted as the D 0 session), in which the S right sound stimulus was switched to a novel light stimulus that minimized sensory generalization from past experience (Fig. and Table ). During the new learning phase, rats maintained stable levels of performance toward the previously learned S left sound stimulus (Fig. ) (94.3 ± 4.8% correct, 109 ± 20 trials per session, mean ± std). Within the first three sessions of the new learning phase, all rats ( N = 7) began responding correctly in the new light trials (the first such session denoted as the D 1 session) and maintained stable levels of >90% correct response rates afterwards (Fig. and Table ). To understand how the repertoire of behavioral sequences evolved during learning, we examined all possible behavioral sequences that the animal might experience (Fig. ). This approach allowed us to identify non-rewarded behavioral sequences that were not associated with specific trial types but were, nonetheless, highly relevant for the learning process. We identified three types of behavioral sequences whose frequencies consistently increased during the new learning phase. These three types of behavioral sequences included the rewarded licking behavior in the new light trials (light licks), as well as two types of non-rewarded behavioral sequences: catch licks and no-fixation licks (Fig. ). Catch licks refers to licking responses toward the right reward port in catch trials when no sensory stimulus was presented. No-fixation licks refers to the situation in which rats directly licked at the right reward port without first entering the center fixation port. All three behavioral sequences shared the common feature of licking the right reward port (lick-right). Both no-fixation licks and catch licks were largely absent before the new learning phase, and emerged and peaked during early sessions of new learning, before subsequently diminished in later sessions (Fig. ). No-fixation licks occurred most frequently at the D 1 session, while catch licks peaked in the same session ( N = 1/7) or later in most animals ( N = 6/7). We will denote the peak of catch licks as the D 2 session in each animal. The consistent temporal order of the D 1 and D 2 sessions within each animal allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The temporal dynamics of the three types of rightward licks during new learning (Fig. ) showed that non-rewarded behaviors were sequentially eliminated while rewarded behaviors were preserved. This temporal dynamics resembled the pattern of sequential refinement of behavioral sequences predicted by the stepwise learning strategy (Fig. ), and likely corresponded to the discrete steps in the underlying learning process. To further test whether such patterns of sequential refinements represent a general feature of behavioral sequence learning, we trained a separate cohort of animals and observed similar behavioral signatures regardless of the sensory modality of the stimulus or the laterality of the new learning side (Fig. ). These observations support the idea that animals do use the stepwise strategy to learn behavioral sequences. In the following analyses, we tested additional predictions of the stepwise learning strategy at both behavioral and neurophysiological levels. To validate the behavioral observations and understand the underlying neural dynamics, we recorded BF neuronal activity throughout the learning process (Fig. ) and used the consistent S left sound as the control stimulus to identify stable populations of BF bursting neurons (Figs. and ). A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. ). The population response of BF bursting neurons were highly consistent across animals (Fig. ), and remained remarkably stable in S left trials throughout the learning process (Fig. ). The inclusion of S left trials therefore allowed us to record from stable and representative populations of BF bursting neurons throughout the learning process, and to investigate how their activities dynamically evolved in other trial types during learning at single trial resolution (Fig. ). We first applied this approach to understand the behavioral and neural dynamics in the D 1 session because all three types of rightward licks emerged in this session (Fig. , middle panel). Detailed analysis of behavioral responses from a representative session (Fig. ) revealed that the three types of rightward licking behaviors emerged abruptly after a transition point (see Methods for definition). Rightward licking behaviors were mostly absent before the transition point, and rapidly switched to almost 100% licking after the transition point. This pattern of abrupt transition was consistently observed across all animals (Fig. ), while the behavioral performance and BF activities in S left sound trials remained relatively stable (Fig. ). At the neuronal level, there was a corresponding increase in the activity of BF bursting neurons that rapidly emerged after the transition in rightward reward-seeking behaviors (Figs. e and ). This increase in BF activity was most prominent in the epoch before the trial outcome as animals approached the reward port (Fig. ), but this activity was not consistently aligned with intervening behavioral events (Fig. ). In contrast, in trials before the transition point, BF bursting neurons did not show similar activity increases in the corresponding time window after exiting the fixation port (Figs. e and ). We will refer to the maximum BF activity in this window as the BF evaluation response (see Methods for definition) because it reflected animals’ internal evaluation when no sensory stimuli were presented during this epoch and because it was not consistently associated with intervening behavioral events. The stepwise learning model predicted that, as animals learned about the lick-right action as the first reward predictor, animals would initially engage in all three types of rightward licking behaviors because they all contained this reward predictor (Fig. ). We therefore examined the respective prevalence of the three types of rightward licking behaviors after the transition point in the D 1 session (Fig. ). All three types of rightward licking behaviors emerged immediately after the transition point. Moreover, after about 60 trials, no-fixation licks began to decline and occurred less frequently than light licks and catch licks (Fig. ). At the neuronal level, the corresponding prediction was that the lick-right action would be associated with increases in BF activities during the first learning step, which should be similarly present in all three types of rightward licking behaviors. Indeed, BF evaluation responses quickly increased after the transition point in all three types of rightward licking behaviors (Fig. ). The amplitudes of BF evaluation responses were similar between the three types of rightward licking behaviors within the first 60 trials after the transition. Subsequently, the BF evaluation response in no-fixation licks declined in the next 60 trials, relative to the other two types of rightward licking behaviors (Fig. ). These results support that the first step of the stepwise learning process corresponded to the first 60 trials after the transition point in the D 1 session. All three types of rightward licking behaviors were present during this step of learning. BF activity also increased to similar levels in all three types of behavioral sequences whenever animals approached and licked the right reward port, regardless of whether they had exited from the center fixation port. These results further suggest that the second step of learning started at roughly 60 trials after the transition, at which point animals learned about the second reward predictor: exiting the fixation port (Fig. ). As a result, the no-fixation lick sequence was no longer compatible with the learned reward predictors, which resulted in diminished BF evaluation responses and the elimination of this behavior from the behavioral repertoire. The other two types of rightward licks, light licks and catch licks, remained compatible and maintained high levels of BF evaluation responses. A further prediction of the stepwise learning model was that, at both the first and the second steps of learning, animals had not learned to use the new light stimulus as a reward predictor, and therefore the new light stimulus would not elicit responses in BF bursting neurons during these steps (Fig. ). We tested this prediction by comparing BF activities between light and catch trials in the D 1 session (Fig. ). Indeed, BF activities in light trials were highly similar to those in catch trials (in the absence of the light stimulus) in the epochs before exiting the fixation port. This was true regardless of whether animals subsequently licked at the right reward port. This observation confirmed the prediction that the new light stimulus did not activate BF bursting neurons in the D 1 session, despite the near-perfect behavioral performance in light trials after the behavior transition point. On the other hand, in the epoch after exiting the fixation port, there were similar increases in BF activity when animals licked at the right reward port in both light and catch trials (Fig. ). This increase in BF activity corresponded to the BF evaluation response described earlier (Fig. ), which reliably distinguished between lick and no lick trials, but not between light and catch trials (Fig. ). These observations support the idea that light and catch trials were treated as the same in the D 1 session, and that the light stimulus has not been learned as a reward predictor at this stage of learning. When did the third step of the stepwise learning process take place? Since the third step was when animals learned to use the light stimulus as a reward predictor to guide reward-seeking behaviors, it should correspond to the time point when the behavioral performance in light and catch trials began to diverge. We noted that the behavioral pattern in light and catch trials were highly similar prior to the D 2 session (Fig. ), and the similarity was best illustrated in the fine behavioral and neuronal dynamics within the D 1 session (Fig. ). In contrast, during the D 2 session, the behavioral pattern in light and catch trials began to show a small but significant difference (Fig. ). This pattern suggests that the D 2 session might be when the third step of learning took place. The corresponding prediction at the neuronal level was that BF bursting neurons should begin to respond to the light stimulus in the D 2 session. We tested this prediction by comparing BF activities between light and catch lick trials in the D 2 session, and found significant differences in all epochs, including the presence of a phasic response to the light onset (Fig. ). This observation supports the idea that BF responses to the new light emerged in the D 2 session. To better understand how BF responses to the new light emerged in the D2 session, we further compared BF responses in light lick and catch lick trials between (1) late trials in the D 2 −1 session; (2) early trials in the D 2 session; (3) late trials in the D 2 session (Fig. ). At the end of the D 2 −1 session, BF neurons did not show response to the new light (paired t -test between light lick and catch lick trials, p = 0.51, N = 6). In early D 2 trials, BF phasic responses to the new light were clearly visible in 4 animals. Comparison between late trials in the D 2 −1 session and the early D 2 trials showed a trend toward increasing BF responses (Fig. , significant at p < 0.05 level; Fig. , paired t -test, p = 0.065, N = 6). Moreover, during the D 2 session, BF responses to the new light increased in all animals between early and late trials (Fig. , paired t -test, p = 0.003, N = 7). This increase took place mostly during the sustained phase of the BF response that was better aligned with fixation port exit than with stimulus onset (Fig. ). Together, these observations suggest that BF responses to the new light developed partly offline between the D 2 −1 and D 2 session, and partly strengthened during the D 2 session. Comparing the temporal dynamics of behavioral and BF responses further suggests that the emergence of BF responses to the new light preceded the elimination of catch licks during the third step of learning (Fig. ). After the third step of learning took place in the D 2 session, did the learning about the new light plateau or did the learning continue to progress? At the neuronal level, BF phasic responses to the new light continued to grow stronger after the D 2 session (Fig. ). At first glance, the continual increases in BF responses to the new light stimulus did not match with the hit rates in light trials, which had already plateaued in the D 2 session (Fig. ). However, as we have shown earlier (Figs. and ), hit rates in light trials could be a poor index of learning about the new light stimulus because light licks in the early learning sessions were not driven by the light stimulus but by later events in the behavioral sequence (exiting fixation and lick-right) as reward predictors. Those behavioral events enabled rightward licking responses in the absence of the light stimulus (catch licks and no-fixation licks). Instead of the hit rate in light trials, a better behavioral index for the learning about the light stimulus would be the difference in the levels of behavioral performance between light and catch trials. This behavioral index accounted for the contributions from the later events in the behavioral sequence that were shared between light and catch licks, and isolated the contribution of the light stimulus to the rightward licking behavior. We found that this index of light learning was strongly correlated with the amplitude of BF phasic responses to the light stimulus in individual sessions (Fig. ). This observation therefore supports the idea that the learning about the light stimulus continued to grow stronger after the D 2 session. Another dimension of the learning about the light stimulus was the change in animals’ decision speeds, measured by reaction times (RTs). Previous studies have shown that stronger BF bursting responses are quantitatively coupled with, and causally lead to, faster RTs , . Such observations predicted that there would be corresponding decreases in RTs toward the light stimulus after the D 2 session. In support of this prediction, we found that stronger phasic bursting responses to light onset were coupled with faster RTs in individual sessions (Fig. ). Together, these observations support the idea that the learning about the new light was reflected by the amplitude of BF phasic response to the light stimulus. In addition, BF activities not only reflected the learning about the light stimulus, but also predicted reward-seeking behaviors in the absence of the light stimulus. For example, in catch trials, stronger BF activities after exiting the fixation port predicted rightward licking behavior and discriminated lick trials from no lick trials (Fig. ). Moreover, increased BF activities before the start of licking predicted longer durations of licking in catch lick trials when no reward was delivered (Fig. ). These observations provided additional support for the idea that the activity of BF bursting neurons promoted reward-seeking behaviors. A final validation of the learning dynamics came from how BF responses to the trial outcome were modulated by reward expectations and BF activities in earlier epochs. Previous studies have found that the response of BF bursting neurons to the reward was negatively modulated by reward expectation , , , . Such properties would predict that, in the current experiment, BF responses to the reward should decrease throughout the stepwise learning process. Indeed, BF responses to the right reward decreased over trials in the D 1 session (Figs. e2 and ), and continued to decrease over subsequent sessions (Fig. ). These results support that animals learned to better predict the rewarding outcome across different steps of the learning process. We further investigated whether the response of BF bursting neurons to trial outcomes were negatively correlated with BF responses in earlier epochs. Such a negative correlation is a hallmark feature of reward prediction error encoding and has been previously reported in BF bursting neurons , . Indeed, at the per session level, we found that the amplitude of BF responses to the reward in light trials was strongly and negatively correlated with the amplitude of BF phasic bursting response to the light onset (Fig. ). Moreover, in pre-D 2 sessions where BF responses to the new light stimulus had yet to develop, we found that BF responses to the trial outcome were negatively correlated with BF evaluation responses in the same trial (Fig. ). This negative correlation at the single trial level was observed not only when the reward was delivered (light licks) but also when the reward was absent (no-fixation licks and catch licks) (Fig. b, ). The fact that these patterns were observed in catch licks and non-fixation licks supports that animals were expecting to receive reward in those trials, and the extent of reward expectation was similarly reflected in BF evaluation responses and negatively modulated BF responses to the trial outcome. Together, these observations support that BF bursting neurons encoded reward prediction error, and that BF activities in earlier epochs (stimulus onset or evaluation window) reflected animals’ reward expectations. Results from the current study support that animals used a stepwise strategy to learn behavioral sequences that contain both sensory stimuli and their own actions. Behavioral events were learned sequentially, starting from the event closest to the reward and sequentially expanded to earlier events (Fig. ). The behavioral signature of this stepwise learning process was the sequential refinement of rightward licking behaviors, in which non-rewarded licking behaviors (no-fixation licks and catch licks) were sequentially eliminated while the rewarded behavior (light licks) was preserved (Fig. ). Learning about each behavioral event as a new reward predictor was accompanied by the emergence of BF responses toward that event, which conveyed animals’ reward prediction toward that event. Increased BF activities first emerged in the epoch before animals entered the reward port (Figs. e and ), while responses to the earlier event (light stimulus) developed later (Figs. and ). The evolution of BF activities mirrored the behavioral response patterns, which was initially increased in all three types of rightward licks (Figs. e and ) and subsequently decreased in non-rewarded licks as those behaviors were eliminated (Figs. b and ). Throughout the learning process, the activity of BF bursting neurons encoded reward prediction error signals (Fig. ) and their increased activities consistently predicted reward-seeking behaviors and faster reaction times (Fig. ). These results therefore identified the behavioral and neurophysiological signatures of the stepwise learning strategy when animals learned behavioral sequences. The current study focused on the learning of behavioral sequences that contain both sensory stimuli and actions, which reflect behavioral contexts that are commonly encountered both in experimental and natural settings. The learning dynamics that we described in this study cannot be easily accounted for using either Pavlovian , or instrumental , conditioning alone. In this regard, stepwise learning provides a new framework to understand the learning of behavioral sequences. Theoretically, long behavioral sequences are difficult to learn because the number of sequence permutations grows exponentially as a function of the sequence length. However, modeling studies have suggested that such learning can be greatly accelerated using the stepwise strategy, which reduces the number of sequence permutations to a linear function of the sequence length . The current study provides behavioral and neurophysiological evidence to support that animals indeed adopt the stepwise learning strategy to learn behavioral sequences. At each step of learning, animals explored the subset of behavioral sequences that shared common sequence elements learned in previous steps (Figs. and ). Such explorations allowed animals to distinguish those sequences, which were initially indistinguishable at the beginning of that learning step, and selectively eliminate subsets of non-rewarded sequences. From this perspective, the stepwise learning strategy offers an intuitive explanation of why animals committed certain types of ‘errors’ (non-rewarded licks), and suggests that those behaviors in fact represented genuine reward-seeking efforts at earlier stages of learning. By learning the associative relationship one step at a time, the stepwise learning strategy likely minimized the cognitive burden of the animal during the learning process and enabled the efficient learning of complex sequences. Our results suggest that stepwise learning is likely a general strategy for learning behavioral sequences because its behavioral and neural signatures were also observed in other learning settings. In a separate cohort of animals, we showed that the behavioral signatures of sequential refinements were similarly observed when the sensory modalities of the stimulus were reversed, and regardless of the laterality of the new learning side (Fig. ). Moreover, even during the learning of the most simple behavioral sequence containing only two elements (stimulus-action), similar behavioral and neural signatures were also observed (Fig. ). It is important to note that, while these results support that stepwise learning is a widely-used strategy for learning behavioral sequences, they do not preclude other possible learning strategies. In particular, if animals had previously encountered similar behavioral events, such experiences could be generalized to the new learning context, and allow animals to use a forward chaining strategy to learn behavioral sequences, instead of the backward chaining order in the stepwise learning strategy . For example, if animals had previously learned that light stimuli could predict reward, they could generalize that experience to the current learning task and view the new light stimulus as a potential reward predictor. Such generalizations would lead animals to engage in reward-seeking behaviors in light trials and quickly discover the light-reward association, bypassing the learning of behavioral events later in the sequence. Such strategies perhaps underlie the accelerated learning dynamics that we observed in one animal (animal #7, Fig. ), which also featured the strongest BF responses to the light stimulus during the initial encounter of the new light (Fig. ). Our results revealed that the true temporal dynamics about the learning of the new stimulus can be very different from the dynamics of behavioral performance levels in the new stimulus trial. We found that, despite the near-perfect behavioral performance in light trials that began after the transition point in the D 1 session (Figs. and ), the learning of the new light stimulus did not begin until the D 2 session (Figs. and ). Moreover, the learning of the new light stimulus continued to grow after the D 2 session despite the plateaued behavioral performance in light trials (Figs. and ). During early stages of learning, while the light stimulus was clearly perceptible and had been consistently paired with reward over many trials, the reward-seeking behavior in light trials was not driven by the light stimulus but by later behavioral events in the sequence (fixation port exit and lick-right). The critical factors that allowed us to reach this conclusion were the analysis of non-rewarded licking responses and the inclusion of catch trials in our task design. If not for these factors, we would incorrectly conclude that the learning about the new light stimulus occurred much earlier. The discrepancy between behavioral performance in light trials and the learning about the light stimulus highlights the potential mismatch in which salient physical events (such as the light stimulus) are not always automatically used by animals to predict reward, especially during the early stages of learning. The light stimulus was only incorporated as a reward predictor in later stages of the learning process, despite its continued presence from the beginning of the new learning. This observation indicates that, even in the absence of changes in reward contingencies in the environment, the learning process can lead to the addition of new reward predictors. Such structural revisions of the internal reward prediction model pose a fundamental challenge to theories and models of learning that assume a static set of reward predictors. Using models with incorrect reward predictors will lead to incorrect interpretations, regardless of how well the model fits the behavioral performance. The current study extends our understanding about the roles of BF bursting neurons in the encoding of reward prediction error. Previous studies have demonstrated that BF responses to rewards are negatively modulated by reward expectation , , and support the idea that BF neurons encode a reward prediction error signal , . The current study further extends this idea and shows that BF bursting neurons similarly encode reward prediction error in the context of new learning, and such encoding is robust even at the single trial level (Fig. ). This robust encoding of reward prediction error by BF bursting neurons quantitatively conveys the amount of reward prediction associated with each behavioral event at each learning step. As a result, the stepwise learning process was mirrored by the activity of BF bursting neurons, which provides a neural correlate of the stepwise learning process that we were able to track throughout the learning process in single trials. Given that reward prediction error information is also conveyed by other neuromodulatory systems, including midbrain dopaminergic neurons , , and BF cholinergic neurons – , BF bursting neurons are likely part of a broader network with partly correlated activities. Whether the other systems also provide similar neural correlates of stepwise learning remains to be determined. Previous studies have also established that BF bursting neurons serve as a bidirectional gain modulation mechanism for reward-seeking behaviors, where increased BF activities promote faster reaction times , while the inhibition of BF activities leads to rapid behavioral stopping . Manipulations of BF activities using electrical stimulation further suggest that BF bursting neurons likely play a causal role that can modulate decision speed when their activities are increased or inhibited . The current study extends this idea to the context of new learning, by showing that increased BF activities were tightly coupled with reward-seeking behaviors at multiple levels throughout the learning process (Figs. – ), and quantitatively predicted faster reaction times (Fig. ) and longer licking durations (Fig. ). The engagement in reward-seeking behaviors was particularly important in the context of learning behavioral sequences, because such explorations were essential for discovering the relationship between earlier events in the behavioral sequence and the rewarding outcome. Taken together, BF bursting neurons may serve the role of transforming the encoding of reward-prediction error into promoting reward-seeking behaviors during the new learning process. Finally, several studies have suggested that BF bursting neurons are likely a subset of GABAergic neurons , , , but their specific cellular marker(s) remains to be determined. Such marker information will be needed to conduct selective manipulation experiments that specifically target BF bursting neurons to directly test their causal role in the learning process. Ethics statement All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals and approved by the National Institute on Aging (NIA) Animal Care and Use Committee and by the Institutional Animal Care and Use Committee at the National Yang Ming Chiao Tung University, Taiwan (NYCU). Subjects Seven male Long-Evans rats (Charles River, NC), aged 3–6 months and weighing 300–400 g were used for the recording experiment. Rats were housed in a 12/12 day/night cycle and were provided with 10–12 dry pellets per day and unrestricted access to water. Rats were trained in daily sessions lasting 60–90 min. A separate cohort of eight male Long Evans rats (National Laboratory Animal Center, Taiwan) were used for behavioral testing (Fig. ) and an additional recording experiment (Fig. ). During training and recording procedures, these rats were water restricted to their 85–90% weight and were trained in a daily 60-min session. Water-restricted rats received 15 min water access at the end of each training day with free access on weekends. Apparatus Plexiglass operant chambers (11″ L × 8 ¼″ W × 13″ H), custom-built by Med Associates Inc. (St. Albans, VT), were contained in sound-attenuating cubicles (ENV-018MD) each with an exhaust fan that helped mask external noise. Each chamber’s front panel was equipped with an illuminated nose-poke port (ENV-114M) located in the center (horizontal axis) as the fixation port, which was equipped with an infrared (IR) sensor to detect the entry of the animals’ snout into the port. On each side of the center nose-poke port there were two reward ports (CT-ENV-251L-P). Two IR sensors were positioned to detect reward-port entry and sipper-tube licking, respectively. Sucrose solution (13.3%) was used as reward and delivered through the sipper tubes located in the reward ports. Reward delivery was controlled by solenoid valves (Parker Hannifin Corp #003-0111-900, Hollis, NH) and calibrated to provide 10 µl of solution per drop. Each chamber was equipped with a ceiling-mounted speaker (ENV-224BM) to deliver auditory stimuli, and a stimulus light (ENV-221) positioned above the center fixation port to serve as the new light stimulus. For the additional behavioral testing (Fig. ), one stimulus light each was added above the left and the right reward ports to serve as the sensory cue in the visual discrimination experiment, and water was used as the reward. Behavioral training protocols were controlled by Med-PC software (Versions IV & V, Med Associates Inc.), which stored all event timestamps at 1 or 2 ms resolution and also sent out TTL signals to the neurophysiology recording systems. Behavioral training procedures Rats were trained in operant chambers that were dimly lit. Rats were first trained in an auditory or visual discrimination task. See Table and Fig. for details of the stimuli used for each animal. Trials were separated by an unsignaled inter-trial interval (ITI) lasting 4–6 s. Fixation or licks during the ITI reset the ITI timer. After the ITI, the center fixation port was illuminated, which was turned off only when rats poked the fixation port. Rats were required to maintain fixation in the center nose-poke port for a variable amount of foreperiod. Four different foreperiods (0.35, 0.5, 0.65, and 0.8 s) were used, pseudorandomly across trials. After the foreperiod, one of three conditions was randomly presented with equal probabilities: a S right stimulus indicated reward on the right port; a different S left stimulus indicated reward on the left port; or the absence of stimulus (catch trial) indicated no reward. An internal timestamp was recorded in catch trials to mark the onset of the would-be stimulus. Early fixation port exit before the end of the foreperiod led to the re-illumination of the center fixation port. Licking in the correct port within a 3 s window after stimulus onset led to three drops of reward, delivered starting at the 3rd lick. The delivery of reward at the 3rd lick created an expectation for trial outcome at this time point, which was dissociated in time from the initiation of licking (1st lick). We will therefore refer to the time point of the 3rd lick also as the trial outcome event. Trials with licking responses ended after 1 s following the last lick, while trials without licking responses ended after the 3 s response window. The ending of each trial started the ITI timer. After reaching asymptotic behavioral performance in the auditory or the visual discrimination task, a new learning phase was introduced by replacing either the S right or S left stimuli by a novel sensory stimulus in a different sensory modality, while all other aspects of the task remained the same. In the group that were initially trained with auditory discrimination, the S right sound stimulus was replaced by the central light above the fixation port to indicate reward on the right port (Table ). In the group there were initially trained with visual discrimination, either the S right or S left light stimuli was replaced by a 6 kHz sound (70 dB) played from a speaker above the center fixation port (Fig. ). Stereotaxic surgery and electrode Surgery was performed under isoflurane anesthesia similar to our earlier study . Multiple skull screws were inserted to anchor the implant, with one screw over the cerebellum serving as the common electrical reference and a separate screw over the opposite cerebellum hemisphere serving as the electrical ground. Craniotomies were opened to target bilateral BF (AP –0.6 mm, ML ± 2.25 mm relative to Bregma) . The electrode contained two bundles of 16 polyimide-insulated tungsten wires (38 µm diameter; California Fine Wire, CA), each bundle ensheathed in a 28-gauge stainless steel cannula and controlled by a precision microdrive. The impedance of individual wire was ~ 0.1 MΩ measured at 1 kHz (niPOD, NeuroNexusTech, MI or Open Ephys Acquisition Board). During surgery, the cannulae were lowered to DV 6.5 mm below cortical surface using a micropositioner (Model 2662, David Kopf Instrument or Robot Stereotaxic, Neurostar GmbH) at a speed of 2–50 µm/s. After reaching target depth, the electrode and screws were covered with dental cement (Hygenic Denture Resin), and electrodes further advanced to 7.5 mm below the cortical surface. Rats received ibuprofen and topical antibiotics after surgery for pain relief and prevention of infection, and were allowed one week to recover with ad libitum food and water. Cannulae and electrode tip locations were verified with cresyl violet staining of histological sections at the end of the experiment. All electrodes were found at expected positions between AP [–0.2, –1.2] mm, ML [1.5, 3] mm, relative to Bregma, and DV [7.5, 8.5] mm relative to cortical surface (Fig. ). Data acquisition and spike sorting Electrical signals were referenced to a common skull screw placed over the cerebellum. Electrical signals were filtered (0.3 Hz to 7.5 kHz) and amplified using Cereplex M digital headstages and recorded using a Neural Signal Processor (Blackrock Microsystems, UT). Single unit activity was further filtered (250 Hz to 5 kHz) and recorded at 30 kHz. Spike waveforms were sorted offline to identify single units using the KlustaKwik sorting algorithm followed by a custom Python GUI (version 2.7) for manual curation. Only single units with clear separation from the noise cluster and with minimal (<0.1%) spike collisions (spikes with less than 1.5 ms interspike interval) were used for further analyses, consistent with previous studies of BF bursting neurons – . Additional cross-correlation analysis was used to remove duplicate units recorded simultaneously across multiple electrodes – . Recording during the new learning phase After surgery, BF neuronal activity was monitored while rats were re-trained in the auditory discrimination task to asymptotic performance level. During this re-training phase, BF electrode depths were adjusted slightly (by advancing electrodes at 125 µm increment) until a stable population of BF single units can be recorded. At this point, the new learning phase with the light as the new S right stimulus was introduced and rats were trained and recorded daily with BF electrodes remained at the same depth. This approach allowed us to monitor the activity of a large population of BF neurons and follow its temporal evolution across sessions. Data analysis Data were analyzed using custom Matlab (R2018b, MATLAB The MathWorks Inc., Natick, MA) scripts. Define different behavioral response types Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. Define the D 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. Identification of the behavioral transition point in the D 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. Identification of BF bursting neurons BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. Population BF responses to behavioral events The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistics Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). Receiver operating characteristic (ROC) and area under curve (AUC) analysis To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Reporting summary Further information on research design is available in the linked to this article. All experimental procedures were conducted in accordance with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals and approved by the National Institute on Aging (NIA) Animal Care and Use Committee and by the Institutional Animal Care and Use Committee at the National Yang Ming Chiao Tung University, Taiwan (NYCU). Seven male Long-Evans rats (Charles River, NC), aged 3–6 months and weighing 300–400 g were used for the recording experiment. Rats were housed in a 12/12 day/night cycle and were provided with 10–12 dry pellets per day and unrestricted access to water. Rats were trained in daily sessions lasting 60–90 min. A separate cohort of eight male Long Evans rats (National Laboratory Animal Center, Taiwan) were used for behavioral testing (Fig. ) and an additional recording experiment (Fig. ). During training and recording procedures, these rats were water restricted to their 85–90% weight and were trained in a daily 60-min session. Water-restricted rats received 15 min water access at the end of each training day with free access on weekends. Plexiglass operant chambers (11″ L × 8 ¼″ W × 13″ H), custom-built by Med Associates Inc. (St. Albans, VT), were contained in sound-attenuating cubicles (ENV-018MD) each with an exhaust fan that helped mask external noise. Each chamber’s front panel was equipped with an illuminated nose-poke port (ENV-114M) located in the center (horizontal axis) as the fixation port, which was equipped with an infrared (IR) sensor to detect the entry of the animals’ snout into the port. On each side of the center nose-poke port there were two reward ports (CT-ENV-251L-P). Two IR sensors were positioned to detect reward-port entry and sipper-tube licking, respectively. Sucrose solution (13.3%) was used as reward and delivered through the sipper tubes located in the reward ports. Reward delivery was controlled by solenoid valves (Parker Hannifin Corp #003-0111-900, Hollis, NH) and calibrated to provide 10 µl of solution per drop. Each chamber was equipped with a ceiling-mounted speaker (ENV-224BM) to deliver auditory stimuli, and a stimulus light (ENV-221) positioned above the center fixation port to serve as the new light stimulus. For the additional behavioral testing (Fig. ), one stimulus light each was added above the left and the right reward ports to serve as the sensory cue in the visual discrimination experiment, and water was used as the reward. Behavioral training protocols were controlled by Med-PC software (Versions IV & V, Med Associates Inc.), which stored all event timestamps at 1 or 2 ms resolution and also sent out TTL signals to the neurophysiology recording systems. Rats were trained in operant chambers that were dimly lit. Rats were first trained in an auditory or visual discrimination task. See Table and Fig. for details of the stimuli used for each animal. Trials were separated by an unsignaled inter-trial interval (ITI) lasting 4–6 s. Fixation or licks during the ITI reset the ITI timer. After the ITI, the center fixation port was illuminated, which was turned off only when rats poked the fixation port. Rats were required to maintain fixation in the center nose-poke port for a variable amount of foreperiod. Four different foreperiods (0.35, 0.5, 0.65, and 0.8 s) were used, pseudorandomly across trials. After the foreperiod, one of three conditions was randomly presented with equal probabilities: a S right stimulus indicated reward on the right port; a different S left stimulus indicated reward on the left port; or the absence of stimulus (catch trial) indicated no reward. An internal timestamp was recorded in catch trials to mark the onset of the would-be stimulus. Early fixation port exit before the end of the foreperiod led to the re-illumination of the center fixation port. Licking in the correct port within a 3 s window after stimulus onset led to three drops of reward, delivered starting at the 3rd lick. The delivery of reward at the 3rd lick created an expectation for trial outcome at this time point, which was dissociated in time from the initiation of licking (1st lick). We will therefore refer to the time point of the 3rd lick also as the trial outcome event. Trials with licking responses ended after 1 s following the last lick, while trials without licking responses ended after the 3 s response window. The ending of each trial started the ITI timer. After reaching asymptotic behavioral performance in the auditory or the visual discrimination task, a new learning phase was introduced by replacing either the S right or S left stimuli by a novel sensory stimulus in a different sensory modality, while all other aspects of the task remained the same. In the group that were initially trained with auditory discrimination, the S right sound stimulus was replaced by the central light above the fixation port to indicate reward on the right port (Table ). In the group there were initially trained with visual discrimination, either the S right or S left light stimuli was replaced by a 6 kHz sound (70 dB) played from a speaker above the center fixation port (Fig. ). Surgery was performed under isoflurane anesthesia similar to our earlier study . Multiple skull screws were inserted to anchor the implant, with one screw over the cerebellum serving as the common electrical reference and a separate screw over the opposite cerebellum hemisphere serving as the electrical ground. Craniotomies were opened to target bilateral BF (AP –0.6 mm, ML ± 2.25 mm relative to Bregma) . The electrode contained two bundles of 16 polyimide-insulated tungsten wires (38 µm diameter; California Fine Wire, CA), each bundle ensheathed in a 28-gauge stainless steel cannula and controlled by a precision microdrive. The impedance of individual wire was ~ 0.1 MΩ measured at 1 kHz (niPOD, NeuroNexusTech, MI or Open Ephys Acquisition Board). During surgery, the cannulae were lowered to DV 6.5 mm below cortical surface using a micropositioner (Model 2662, David Kopf Instrument or Robot Stereotaxic, Neurostar GmbH) at a speed of 2–50 µm/s. After reaching target depth, the electrode and screws were covered with dental cement (Hygenic Denture Resin), and electrodes further advanced to 7.5 mm below the cortical surface. Rats received ibuprofen and topical antibiotics after surgery for pain relief and prevention of infection, and were allowed one week to recover with ad libitum food and water. Cannulae and electrode tip locations were verified with cresyl violet staining of histological sections at the end of the experiment. All electrodes were found at expected positions between AP [–0.2, –1.2] mm, ML [1.5, 3] mm, relative to Bregma, and DV [7.5, 8.5] mm relative to cortical surface (Fig. ). Electrical signals were referenced to a common skull screw placed over the cerebellum. Electrical signals were filtered (0.3 Hz to 7.5 kHz) and amplified using Cereplex M digital headstages and recorded using a Neural Signal Processor (Blackrock Microsystems, UT). Single unit activity was further filtered (250 Hz to 5 kHz) and recorded at 30 kHz. Spike waveforms were sorted offline to identify single units using the KlustaKwik sorting algorithm followed by a custom Python GUI (version 2.7) for manual curation. Only single units with clear separation from the noise cluster and with minimal (<0.1%) spike collisions (spikes with less than 1.5 ms interspike interval) were used for further analyses, consistent with previous studies of BF bursting neurons – . Additional cross-correlation analysis was used to remove duplicate units recorded simultaneously across multiple electrodes – . After surgery, BF neuronal activity was monitored while rats were re-trained in the auditory discrimination task to asymptotic performance level. During this re-training phase, BF electrode depths were adjusted slightly (by advancing electrodes at 125 µm increment) until a stable population of BF single units can be recorded. At this point, the new learning phase with the light as the new S right stimulus was introduced and rats were trained and recorded daily with BF electrodes remained at the same depth. This approach allowed us to monitor the activity of a large population of BF neurons and follow its temporal evolution across sessions. Data were analyzed using custom Matlab (R2018b, MATLAB The MathWorks Inc., Natick, MA) scripts. Define different behavioral response types Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. Define the D 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. Identification of the behavioral transition point in the D 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. Identification of BF bursting neurons BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. Population BF responses to behavioral events The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistics Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). Receiver operating characteristic (ROC) and area under curve (AUC) analysis To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Licking responses were defined for stimulus (S left and S right ) and catch trials if rats licked at least three times in the reward port within the 3 s window after stimulus onset (or the corresponding timestamp for the would-be stimulus in catch trials). Licking responses to the correct reward port were rewarded with three drops of water, delivered starting at the 3rd lick (referred to as trial outcome event). During the new learning phase, licking responses in the new stimulus and catch trials were predominantly to the reward port associated with the new stimulus. No-fixation licks corresponded to licking responses to the reward port associated with the new stimulus that were not preceded by poking the center fixation port. Specifically, no-fixation licks were defined based on three criteria: (1) rats made at least three consecutive licks in the reward port; (2) the interval between the last exit from the fixation port and the first lick must be greater than 2 s; (3) the interval between the last exit from the reward port and the subsequent first lick in the same reward port must be greater than 1 s. These duration thresholds were determined based on the empirical licking patterns across animals. In the analyses of learning dynamics in the D 1 session (Figs. and ), no-fixation licks were treated as rightward licking trials, even though such behaviors were self-initiated and not imposed by the task design. Reaction time (RT) in light trials in a session (Fig. ) was defined as the median of the interval between the onset of the light stimulus and the exit from the fixation port in light lick trials. Lick duration in catch trials in a session (Fig. ) was defined as the median of the interval between the first and the last lick in catch lick trials. 0 , D 1 and D 2 learning landmarks During the new learning phase, three sessions (D 0 , D 1 , D 2 ) were identified in individual animals as landmarks that demarcated distinct stages of new learning (Fig. ). The D 0 session was defined as the very first session the new light stimulus was introduced. The D 1 session was defined as the first session when animals began to respond correctly in the new stimulus trials and obtained reward in the associated reward port in at least three trials. The D 2 session was defined as the session in which catch licks occurred most frequently. The D 1 and D 2 landmarks allowed us to identify similar learning stages across animals despite their individual differences in learning dynamics. The specific timing of the three landmark sessions in each animal are provided in Table (also see Fig. ). One animal (ID#7) with accelerated learning dynamics, in which D 1 and D 2 occurred in the same session, was excluded from analyses of D 1 neural dynamics (Figs. and ) to ensure that neural activities associated with D 2 did not confound the neural dynamics in the D 1 session. The BF neuronal activity in this animal was included in the analysis of D 2 neural dynamics (Fig. ) and showed the strongest phasic response to the light onset among all animals, consistent with its accelerated learning dynamics. 1 session The behavioral transition points in D 1 sessions (Figs. and ) were identified based on behavioral response patterns in three trial types combined: light trials, catch trials and no-fixation licks. The behavioral response pattern in each trial was coded as either 1 or 0 based on whether animals licked in the right reward port in that trial. The behavioral transition point was defined as the point with the largest difference in licking responses between the 20 trials before and the 20 trials after that point. In 5/7 animals, the first trial after the behavioral transition was a rewarded light lick trial. In the other two animals, the transition point was adjusted to the closest light lick trial by 2 or 4 trials, respectively. BF bursting neurons were defined as BF single units whose average firing rates during the [0.05, 0.2]s window after stimulus onset increased by more than 2 spikes/s in the S left sound trials compared to the corresponding window in catch trials (Fig. ). This contrast between sound trials and catch trials was necessary because it removed the nonstationary baseline before stimulus onset and allowed us to ask whether BF neurons truly responded to the sound stimulus. In addition, BF bursting neurons should have baseline firing rates (during the [−1, 0]s window relative to the trial start signal) less than 10 spikes/s. A total of 1453 BF single units were recorded over 45 sessions ( N = 7 rats), of which 70% (1013/1453) were classified as BF bursting neurons based on their stereotypical phasic response to the S left sound (22.5 ± 7.3 neurons per session, mean ± std) (Fig. and Table ). Since BF electrodes remained at the same depth throughout the recording sessions, the same BF single units might be recorded in multiple sessions. The large number of BF bursting neurons recorded in each session allowed us to treat them as a representative sample of all BF bursting neurons, whose responses to the S left sound were highly stable throughout the learning process (Fig. ). This strategy ensured that we were following functionally the same neuronal ensemble and could track how BF bursting neurons acquired responses to the new light during learning, regardless of whether the identities of these BF neurons were exactly the same in each session. One session with only one BF bursting neuron was excluded from the analysis of BF population activities. The spike timestamps of all BF bursting neurons in a single session were pooled together to approximate the population activity of all BF bursting neurons. Population peri-stimulus time histograms (PSTHs) were calculated with 10-ms bins, and normalized by the number of BF bursting neurons in a session. To properly assess whether BF bursting neurons responded to the onset of the new light stimulus (Figs. – ), it was important to disambiguate such stimulus-onset responses from the increased BF activities after fixation port exit (Fig. ). To achieve this goal, PSTHs to the stimulus onset were calculated based only on spikes that occurred before fixation port exit in individual trials, resulting in different interval lengths (between stimulus onset to fixation port exit) across trials. Accordingly, the calculation of the mean PSTH across all trials in a session was adjusted for the different number of trials at different interval lengths. The mean PSTHs were further truncated at the median interval length of that session to reduce noisy estimates of PSTHs at long interval lengths due to lower number of trials. When PSTHs were averaged across animals, the averaged PSTHs were further truncated at the mean of median interval latencies across animals. This truncation procedure resulted in the uneven lengths of PSTHs across individual animals (Figs. , , S and S ) and across sessions (Fig. ). This procedure was also applied to calculating the BF responses before fixation port exit to include only spikes that occurred after stimulus onset (Figs. , and S ), and for calculating BF responses during licking in catch lick trials (Fig. ). The time windows used to quantify average BF activity in different epochs were indicated in respective figures, and corresponded to the following: [0.05, 0.2]s after S left sound onset; [0.1, 0.3]s after S right light stimulus onset; [0.1, 0.3]s after the timestamp for the would-be stimulus in catch trials; [0.05, 0.35]s after the 3rd lick for outcome responses; [−0.3, 0]s and [0, 0.3]s relative to the fixation port exit. The epoch for calculating evaluation response is described below. Evaluation response (Figs. e, b, a and b, ) refers to the increased BF activity after exiting the fixation port and before the trial outcome (3rd lick). The evaluation response reflected animals’ internal evaluation because no additional sensory stimuli were presented during this epoch and this activity was not consistently aligned with intervening behavioral events (Fig. ). Specifically, the evaluation response was calculated in individual trials and defined as the maximum firing rate of any 500 ms window during the evaluation epoch, which corresponded to the interval between [fix-out, outcome], with additional adjustments according to trial types. In light lick and catch lick trials, the evaluation epoch was defined as [fix-out, outcome] in each trial. The epoch durations in light lick and catch lick trials within each session were used as the reference point for other trial types as described next. In no-fixation licks, in which the fix-out event was absent, the duration of evaluation epoch was set as the 95th percentile of the evaluation epoch durations in light lick and catch lick trials, and the epoch should begin at least 0.5 s before reward port entry. In light no lick and catch no lick trials, in which the 3rd lick event was absent, the duration of the evaluation epoch was set as the median of the evaluation epoch durations in light lick and catch lick trials. These adjustments in the definition of evaluation epochs, as well as its calculation of maximum firing rate within the epoch, took into consideration the behavioral variability across trial types, learning stages and individual animals. To evaluate the dynamic changes of BF activities around the transition point in the D 1 session (Figs. e and ), single trial evaluation and outcome responses were smoothed using moving median over 10 trials. The smoothed trends were aligned at the transition point and then averaged across all animals. Only trials with smoothed trend data from at least 4 animals were plotted in the group average (Fig. ). Statistical comparisons were conducted using the Statistics and Machine Learning Toolbox (version 11.3) in MATLAB (R2018a) ( https://www.mathworks.com/ ). Two-sided paired t -test (ttest.m) was used to compare behavioral and neural activity differences between two groups (Figs. d, a, c2, and ). Repeated measures analysis of variance (ranova.m) was used for comparisons involving more than 2 groups, by specifying the appropriate within-subject models (Figs. a3, b3, and ). Comparisons of PSTHs between two groups (Figs. a, b, c1, d, and ) was conducted for each 100 ms sliding window (10 ms step) using two-sided paired t -test. Significance level was set at p < 0.01 for three consecutive bins. Pearson correlation (corrcoef.m) was used to determine the relationship between neuronal activities and/or behavior (Figs. b, c, a, b2, and ). To determine whether the activity of BF bursting neurons differentiated between trial types within each D 1 session (Fig. ), we compared BF activity for each 100 ms sliding window (10 ms step) using the AUC measure of ROC analysis (auc.m by Alois Schloegl). At each sliding window, BF population activity was calculated for each light and catch trial, and distributions of BF activities were compared between light vs catch trials or between lick vs no lick trials. Significance level was set at p < 0.001 using 10,000 trial-shuffled random permutations (two-sided). To determine whether BF activity differentiated between lick and no lick trials within the same trial type (light or catch trials) (Fig. ), we compared BF activity in the [0, 500] ms window after exiting the fixation port. For each session and each trial type, lick and no lick trials must each constitute at least 10% of that trial type to be included in the analysis. Catch trials from all sessions were included in this analysis. Only light trials before the D 2 session (pre-D 2 ) were included in this analysis because BF responses to the onset of the light stimulus had not developed in those sessions. Significance level was set at p < 0.05 using 1000 trial-shuffled random permutations (two-sided). Further information on research design is available in the linked to this article. Supplementary Information Peer Review File Reporting Summary |
Comprehensive Evaluation of Traditional Herbal Medicine Combined With Adjuvant Chemotherapy on Post-Surgical Gastric Cancer: A Systematic Review and Meta-Analysis | 71d15b91-01bf-4226-a54b-ae71f2c3e026 | 10823854 | Pharmacology[mh] | Gastric cancer (GC) is one of the most common cancers in the digestive system. According to the Global Cancer Observatory (GCO), in 2020, gastric cancer had 1 089 103 new cases and resulted in 768 793 deaths, making it the fourth leading cause of mortality and the fifth most common cancer in terms of incidence. Notably, its incidence in men is twice that of women. The management of gastric cancer involves a comprehensive and multidisciplinary approach to diagnosis and treatment. This requires a carefully planned and coordinated combination of various treatment modalities, such as surgery, radiotherapy, chemotherapy, targeted therapy, endocrine therapy, immunotherapy, and interventional therapy. Adjuvant chemotherapy has been shown to improve overall survival and disease-free survival compared to surgery alone. - While treatment guidelines vary by country, gastric cancer treatment generally involves surgery, radiation, and chemotherapy. However, surgery and chemotherapy for gastric cancer can cause patients to experience numerous side effects and complications, , and patients can suffer from psychological symptoms related to tumor diagnosis, postoperative complications, and the side effects of treatment. According to Bang et al’s study, more than half of the patients receiving adjuvant cancer treatment after gastric cancer surgery reported experiencing side effects. These physiological and psychological changes can significantly lower the quality of life after surgery, and the side effects and complications still need to be resolved in clinical practice. Traditional Herbal Medicine (THM) is widely used as a complementary and alternative therapy and can be helpful in the treatment of gastric cancer. , As highlighted by Lu et al’s study, THM is commonly used either on its own or in combination with chemotherapy at various stages of gastric cancer progression. Notably, THM is used to reduce the side effects of chemotherapy, assist in post-surgical recovery, and alleviate symptoms. Liu et al’s study indicates that the group treated with THM in conjunction with chemotherapy experienced fewer side effects from gastric cancer compared to those undergoing chemotherapy alone. Moreover, this study revealed that THM can bolster the immune response in cancer patients, promoting apoptosis and effectively halting the progression of gastric cancer. Fu et al’s study stated that THM effectively reduces toxicity and side effects caused by chemotherapy and improves clinical symptoms and quality of life. Consequently, THM alleviates symptoms in cancer patients and enhances the quality of life and the efficacy of anticancer treatment. Despite the promising potential of THM in gastric cancer treatment, a significant gap exists in the current literature. Few studies have conducted a comprehensive and standardized review that integrates both THM and GC treatments. Hence, this study aims to provide clinical evidence via a meta-analysis, evaluating the efficacy and safety of combining adjuvant chemotherapy with herbal medicine in the treatment of post-surgical gastric cancer. Study Inclusion Criteria The inclusion criteria were limited to randomized controlled trials (RCTs) that enrolled participants diagnosed with stages I to III gastric cancer. These trials must have allocated patients into 2 distinct groups: an intervention group receiving both THM and adjuvant chemotherapy post-surgery, and a control group receiving adjuvant chemotherapy alone. The primary outcome for inclusion was specified as “Tumor Response,” while secondary outcomes included “Quality of Life (QoL),” “Adverse Drug Reactions (ADRs),” “Tumor Marker Levels,” and “Survival Rate.” Non-randomized trials, observational studies, case reports, and other non-randomized comparisons were excluded to ensure a high level of evidence and homogeneity within the analyzed data. Objective and Design The objective of this study is to conduct a meta-analysis to examine the effectiveness and safety of combining THM with adjuvant chemotherapy for the treatment of gastric cancer. The study was carried out in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study protocol has been registered on PROSPERO with the registration number CRD 42022354133. Our approach has included a quantitative synthesis of data through meta-analysis from studies that have met our pre-defined eligibility criteria, specifically those providing sufficient quantitative data and having comparable outcome measures. We have performed meta-analysis when we have found at least 2 studies that are sufficiently homogenous in terms of participants, interventions, comparators, and outcomes, as per our predefined protocol thresholds. Search Strategy The literature search was conducted by utilizing international web databases including KMBASE, KISS, OASIS, RISS, ScienceON, EMBASE, Pubmed, CNKI, Cochrane Library, and CiNii. To ensure a comprehensive coverage of the literature, articles published in English, Chinese, Japanese, and Korean were included. The search period for all databases extended through June 2022. The searching strategy of the PubMed database, which was searched in the corresponding database, detailed search strategy is provided in Table S1 ( Supplemental Material ). This research solely focused on the electronic searches. Selection of Studies The search results were collected using the reference management program Endnote version 20 and duplicates were removed. Two independent researchers (SDK and JHK) evaluated the titles and abstracts of the studies; and excluded those that did not meet the criteria. The full texts were then reviewed and studies meeting the criteria were included, after consultation between the 2 researchers. If there was a disagreement, the issue was resolved by a third researcher (DHK). Excluded studies were recorded with reasons. Data Extraction The data were extracted by 2 independent researchers (SDK and JHK). The extracted data included the first author, study period, year of publication, participants, comparison, interventions in treatment and control groups, outcomes, and adverse events. If any data were missing from the material, the group could discuss the issue among themselves and reach out to the first author by email. Quality Assessment To assess the quality of the selected literature data, we utilized the Cochrane Risk of Bias (ROB) tool. Two researchers (SDK and JHK) used ROB to assess the risk of bias. The risk of trial bias was assessed through a total of 7 items: random sequence generation and allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other biases. If there was a disagreement, the issue was resolved by a third researcher (DHK). Building upon this foundation, our quality of evidence assessment applied the GRADE system to categorize evidence levels as high, moderate, low, or very low. RCTs initially offer high-quality evidence, which was then downgraded based on the presence of serious limitations like risk of bias, inconsistency, indirectness, and imprecision, and evaluated for potential publication bias. Upgrading of evidence was considered for factors such as a large effect size and dose-response relationship. The quality of evidence for each outcome was then summarized in a GRADE evidence profile, providing a clear and systematic assessment of the evidence strength and areas needing further research. Statistical Analysis Review Manager Software 5.4 was used to generate data results. In this study, the risk ratio (RR) with 95% confidence interval (CI) was used to measure dichotomous data. For continuous data, 95% CI with mean differences (MD) were used when measuring treatment outcomes on the same scale. Heterogeneity was calculated using Higgins’ I 2 in this study. An I 2 value greater than 50% has been considered indicative of substantial heterogeneity. In cases of substantial heterogeneity, we have explored potential sources through subgroup analyses and sensitivity analyses, based on predefined study characteristics such as population demographics, different types of herbal medicines, and chemotherapy regimens. Should substantial heterogeneity have remained unexplained, findings have been cautiously interpreted, and we have provided a narrative synthesis discussing the plausible reasons for these discrepancies. The meta-analysis, if conducted, has utilized a random-effects model to account for any identified heterogeneity among the included studies. Frequency Analysis To identify the predominant herbal interventions in the treatment of gastric cancer, a frequency analysis was conducted on the included studies. The frequency analysis aimed to determine the most commonly used herbs in the studies included in the meta-analysis. This analysis was carried out by tallying the occurrences of various herbs across the studies. The inclusion criteria were limited to randomized controlled trials (RCTs) that enrolled participants diagnosed with stages I to III gastric cancer. These trials must have allocated patients into 2 distinct groups: an intervention group receiving both THM and adjuvant chemotherapy post-surgery, and a control group receiving adjuvant chemotherapy alone. The primary outcome for inclusion was specified as “Tumor Response,” while secondary outcomes included “Quality of Life (QoL),” “Adverse Drug Reactions (ADRs),” “Tumor Marker Levels,” and “Survival Rate.” Non-randomized trials, observational studies, case reports, and other non-randomized comparisons were excluded to ensure a high level of evidence and homogeneity within the analyzed data. The objective of this study is to conduct a meta-analysis to examine the effectiveness and safety of combining THM with adjuvant chemotherapy for the treatment of gastric cancer. The study was carried out in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). The study protocol has been registered on PROSPERO with the registration number CRD 42022354133. Our approach has included a quantitative synthesis of data through meta-analysis from studies that have met our pre-defined eligibility criteria, specifically those providing sufficient quantitative data and having comparable outcome measures. We have performed meta-analysis when we have found at least 2 studies that are sufficiently homogenous in terms of participants, interventions, comparators, and outcomes, as per our predefined protocol thresholds. The literature search was conducted by utilizing international web databases including KMBASE, KISS, OASIS, RISS, ScienceON, EMBASE, Pubmed, CNKI, Cochrane Library, and CiNii. To ensure a comprehensive coverage of the literature, articles published in English, Chinese, Japanese, and Korean were included. The search period for all databases extended through June 2022. The searching strategy of the PubMed database, which was searched in the corresponding database, detailed search strategy is provided in Table S1 ( Supplemental Material ). This research solely focused on the electronic searches. The search results were collected using the reference management program Endnote version 20 and duplicates were removed. Two independent researchers (SDK and JHK) evaluated the titles and abstracts of the studies; and excluded those that did not meet the criteria. The full texts were then reviewed and studies meeting the criteria were included, after consultation between the 2 researchers. If there was a disagreement, the issue was resolved by a third researcher (DHK). Excluded studies were recorded with reasons. The data were extracted by 2 independent researchers (SDK and JHK). The extracted data included the first author, study period, year of publication, participants, comparison, interventions in treatment and control groups, outcomes, and adverse events. If any data were missing from the material, the group could discuss the issue among themselves and reach out to the first author by email. To assess the quality of the selected literature data, we utilized the Cochrane Risk of Bias (ROB) tool. Two researchers (SDK and JHK) used ROB to assess the risk of bias. The risk of trial bias was assessed through a total of 7 items: random sequence generation and allocation concealment, blinding of participants and personnel, blinding of outcome assessment, incomplete outcome data, selective reporting, and other biases. If there was a disagreement, the issue was resolved by a third researcher (DHK). Building upon this foundation, our quality of evidence assessment applied the GRADE system to categorize evidence levels as high, moderate, low, or very low. RCTs initially offer high-quality evidence, which was then downgraded based on the presence of serious limitations like risk of bias, inconsistency, indirectness, and imprecision, and evaluated for potential publication bias. Upgrading of evidence was considered for factors such as a large effect size and dose-response relationship. The quality of evidence for each outcome was then summarized in a GRADE evidence profile, providing a clear and systematic assessment of the evidence strength and areas needing further research. Review Manager Software 5.4 was used to generate data results. In this study, the risk ratio (RR) with 95% confidence interval (CI) was used to measure dichotomous data. For continuous data, 95% CI with mean differences (MD) were used when measuring treatment outcomes on the same scale. Heterogeneity was calculated using Higgins’ I 2 in this study. An I 2 value greater than 50% has been considered indicative of substantial heterogeneity. In cases of substantial heterogeneity, we have explored potential sources through subgroup analyses and sensitivity analyses, based on predefined study characteristics such as population demographics, different types of herbal medicines, and chemotherapy regimens. Should substantial heterogeneity have remained unexplained, findings have been cautiously interpreted, and we have provided a narrative synthesis discussing the plausible reasons for these discrepancies. The meta-analysis, if conducted, has utilized a random-effects model to account for any identified heterogeneity among the included studies. To identify the predominant herbal interventions in the treatment of gastric cancer, a frequency analysis was conducted on the included studies. The frequency analysis aimed to determine the most commonly used herbs in the studies included in the meta-analysis. This analysis was carried out by tallying the occurrences of various herbs across the studies. Study Selection An initial search of 10 electronic databases yielded 3709 studies. After removing 483 duplicates, the remaining 3226 studies were evaluated based on their abstracts. This resulted in the further exclusion of 3129 studies, leaving 97 articles for full-text review. The eligibility criteria were then applied, a total of 61 studies were excluded for the following reasons: 14 studies included patients without a history of surgery or those who did not undergo surgery for gastric cancer, 4 studies had participants other than gastric cancer patients, 28 studies were not randomized controlled trials, and 15 studies had different study designs from the intended outcome. Consequently, a total of 36 randomized controlled trials were included in this analysis . Study Characteristics This meta-analysis includes 36 randomized controlled trials published up until 2021, with a total of 2176 participants diagnosed at different stages of gastric cancer, ranging from TNM stage Ⅰ to stage Ⅲ. - The experimental groups received chemotherapy combined with different TCM formulas as an adjuvant treatment, while the control groups received standard chemotherapy regimens, such as FOLFOX, XELOX, SOX, OLF, DC, and others. The treatment duration varied across the studies, ranging from 2 weeks to 12 months . The primary outcomes evaluated in these studies included disease control rate (DCR), overall response rate (ORR). DCR is defined as the sum of complete response, partial response, and stable disease. This measure provides a comprehensive assessment of the control over the disease progression. ORR is calculated as the sum of complete response and partial response, offering a focused view on the effectiveness of the treatment in reducing tumor size and progression. The secondary outcomes were set as Karnofsky Performance Status (KPS), ADR, Tumor Marker Levels, and Survival rate. Each of these outcomes offers a unique perspective on the overall impact of the treatment, encompassing both the efficacy and safety profile of combining Traditional Herbal Medicine with chemotherapy in Gastric Cancer treatment. Risk of Bias in Included Trials Based on the Cochrane risk of bias tool, the trials included in this review exhibited a moderate to high risk of bias due to insufficient methodological reporting . Most studies employed proper random sequence generation, while one study was considered to have a high risk of bias in this regard. Allocation concealment information was not provided in 18 trials, - ,25-27,29,31,32,34,40,42,46,47,49,50,52 leading to an unclear risk of bias assessment. Blinding participants or personnel was unfeasible since THM was administered solely to the experimental group. Detection bias was deemed to be at high risk in 20 studies, - , , , - , , , , , , , , , , , and no relevant information was reported in 4 studies. , , , Reporting bias was assessed as unclear risk in all studies due to the inability to locate the registered protocols. All studies were assessed to have a low risk of attrition and other biases . Primary Outcome Tumor response assessment A comprehensive review of 12 studies demonstrated a statistically significant increase in both DCR and ORR for those patients receiving a combination of THM and chemotherapy, , , , , , , - , , , compared to those receiving chemotherapy alone (RR 1.25, 95% CI [1.09, 1.45], I 2 = 79%, n = 835) and (RR 1.54, 95% CI [1.31, 1.81], I 2 = 0%, n = 835, ). To address the substantial heterogeneity in the DCR outcomes ( I 2 = 79%), 3 studies were excluded due to their notably high risk ratios and wide confidence intervals, which were identified as potential outliers. , , The recalculated data from the remaining studies still demonstrated an enhancement in DCR due to THM (RR 1.18, 95% CI [1.07, 1.29], I 2 = 42%, n = 646, ). However, the decrease in the I 2 value to 42% suggests the initial heterogeneity may have been driven by reporting bias. Secondary Outcomes Quality of life assessment In line with the established criteria for evaluating KPS score enhancement, 14 studies measured QoL through KPS score improvement. A combined analysis of these 14 studies revealed a significantly greater rate of KPS score enhancement in the group receiving both THM and chemotherapy compared to the group undergoing chemotherapy alone. This difference was statistically significant (MD of 6.84, 95% CI [5.77, 7.92], I 2 = 49%, n = 922, ). , , , , , , , , , , , - Adverse drug reactions assessment The findings from the meta-analysis revealed that the group receiving both THM and chemotherapy exhibited a reduced likelihood of experiencing adverse drug reactions compared to the chemotherapy-only group. This included lower risks of leukocyte reduction (RR 0.62, 95% CI [0.50, 0.76], I 2 = 49%, n = 1284, 16 trials), , , , , , , , , , , - , , , platelet reduction (RR 0.86, 95% CI [0.72, 1.01], I 2 = 10%, n = 1198, 16 trials), , , , , - , , , , - , , , liver injury (RR 0.59, 95% CI [0.42, 0.82], I 2 = 29%, n = 951, 13 trials), , , , , , , , , , , , renal injury (RR 0.46, 95% CI [0.28, 0.75], I 2 = 0%, n = 589, 7 trials), , , , , , , nausea and vomiting (RR 0.68, 95% CI [0.56, 0.84], I 2 = 51%, n = 1038, 14 trials), , , , , , , , - , , , diarrhea (RR 0.74, 95% CI [0.61, 0.88], I 2 = 4%, n = 900, 12 trials), , , , , , , , , , , , and neurotoxicity (RR 0.63, 95% CI [0.48, 0.84], I 2 = 0%, n = 651, 9 trials). , , , , , , , , Except for platelet reduction ( P = .07), all differences were statistically significant, with further details presented in . Tumor marker level In the THM plus chemotherapy group, there was a greater decrease in CEA compared to the chemotherapy-alone group, as demonstrated by the pooled results of 16 trials (MD −1.55, 95% CI [−2.23, −0.87], I 2 = 51%, n = 967). , , - , , , - , , - , , Moreover, the pooled results of 11 trials revealed a higher CA19-9 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −2.50, 95% CI [−3.91, −1.09], I 2 = 18%, n = 748). , , , , , , , , , , Conversely, the pooled results of 8 trials indicated no significant change in CA125 between the THM plus chemotherapy group and chemotherapy group (MD 0.12, 95%, CI [−1.47, 1.70], I 2 = 59%, n = 596). , , , , , , , Additionally, the pooled results of 6 trials showed a greater CA72-4 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −1.80, 95% CI [−2.59, −1.01], I 2 = 43%, n = 393). , , , , , The detailed information has been presented in . One-year survival rate The meta-analysis of 10 studies assessed the 1-year survival rate and found that THM plus chemotherapy group demonstrated a statistically significant improvement in the 1-year survival rate (RR 1.08, 95% CI [1.02, 1.14], I 2 = 0%, n = 868, 10 trials, ). , , , , , , , , , These findings provide strong evidence that the combination of THM and chemotherapy has a positive effect on the 1-year survival rate. Two-year survival rate The meta-analysis of 8 studies indicated that the combination of THM and chemotherapy was effective in improving the 2-year survival rate (RR 1.32, 95% CI [1.19, 1.47], I 2 = 0%, n = 712, 8 trials, ), providing robust evidence for the effectiveness of the THM and chemotherapy combination in enhancing the 2-year survival rate. Three-year survival rate The statistically significant improvement observed in the 3-year survival rate (RR 1.42, 95% CI [1.12, 1.79], I 2 = 0%, n = 343, 3 trials, ) further reinforces the efficacy of combining THM and chemotherapy as a promising approach. These findings provide valuable insights into the potential long-term benefits of this treatment strategy in improving patient outcomes. Publication Bias To investigate publication bias, funnel plots were created using RR, MD, and 1/(standard error: SE) values extracted from trials that measured ORR, KPS score, and 1-year survival rate. The funnel plot analysis for the ORR study exhibited a symmetrical shape , indicating a balanced distribution of studies across a range of effect sizes and their corresponding precision measures. This symmetry suggests the absence of significant publication bias or small study effects, reinforcing the reliability and robustness of the study’s findings regarding ORR. However, the funnel plots generated based on the KPS score and 1-year survival rate displayed an asymmetrical distribution ( and ), suggesting the potential presence of publication bias in these analyses. Quality of Evidence Assessment In the assessment of tumor response, the DCR and ORR were analyzed from 9 and 12 RCTs respectively, showing evidence of “Low” and “Moderate” certainty. Concerning the QoL, the KPC indicator from 14 RCTs demonstrated “Moderate” certainty. Adverse drug reactions showed varied results: leukocyte decrease, liver injury, nausea & vomiting, diarrhea, tumor marker levels (CEA, CA19-9, CA125, CA72-4) were all assessed with “Low” certainty, while platelet decrease, renal injury, and neurotoxicity held a “Moderate” certainty. For survival rate evaluations, the 1-, 2-, and 3-year survival rates all were categorized with “Moderate” certainty. The reasons for downgrading the evidence quality across these evaluations primarily included risk of bias and inconsistencies. Various outcomes also suffered from imprecision, often attributed to limited sample sizes. Detailed outcomes, effects, and absolute effects can be cross-referenced in the provided . Frequency Analysis The frequency analysis of the most commonly used herbs in the included studies unveiled significant trends in herbal prescriptions for post-surgical gastric cancer therapy. Among the 122 herbs observed over 36 prescriptions, the top 10 herbs are presented in . Atractylodes macrocephala exhibited the highest frequency, present in 75.0% of the analyzed prescriptions. Subsequently, Poria cocos showed a frequency of 63.9%, while Glycyrrhiza uralensis , Citrus reticulata , Astragalus membranaceus , and Hedyotis diffusa all shared a frequency of 61.1% across the prescriptions. Codonopsis pilosula and Pinellia ternata were also frequently prescribed, each appearing in 58.3% of the prescriptions. Angelicae Sinensis Radix and Coix lacryma-jobi were similarly common, noted in 38.9% of the prescriptions. These findings provide insight into the prevalent herbal interventions employed in the prescriptions analyzed, showcasing a selection of herbs frequently prescribed in post-surgical gastric cancer therapy involving THM. An initial search of 10 electronic databases yielded 3709 studies. After removing 483 duplicates, the remaining 3226 studies were evaluated based on their abstracts. This resulted in the further exclusion of 3129 studies, leaving 97 articles for full-text review. The eligibility criteria were then applied, a total of 61 studies were excluded for the following reasons: 14 studies included patients without a history of surgery or those who did not undergo surgery for gastric cancer, 4 studies had participants other than gastric cancer patients, 28 studies were not randomized controlled trials, and 15 studies had different study designs from the intended outcome. Consequently, a total of 36 randomized controlled trials were included in this analysis . This meta-analysis includes 36 randomized controlled trials published up until 2021, with a total of 2176 participants diagnosed at different stages of gastric cancer, ranging from TNM stage Ⅰ to stage Ⅲ. - The experimental groups received chemotherapy combined with different TCM formulas as an adjuvant treatment, while the control groups received standard chemotherapy regimens, such as FOLFOX, XELOX, SOX, OLF, DC, and others. The treatment duration varied across the studies, ranging from 2 weeks to 12 months . The primary outcomes evaluated in these studies included disease control rate (DCR), overall response rate (ORR). DCR is defined as the sum of complete response, partial response, and stable disease. This measure provides a comprehensive assessment of the control over the disease progression. ORR is calculated as the sum of complete response and partial response, offering a focused view on the effectiveness of the treatment in reducing tumor size and progression. The secondary outcomes were set as Karnofsky Performance Status (KPS), ADR, Tumor Marker Levels, and Survival rate. Each of these outcomes offers a unique perspective on the overall impact of the treatment, encompassing both the efficacy and safety profile of combining Traditional Herbal Medicine with chemotherapy in Gastric Cancer treatment. Based on the Cochrane risk of bias tool, the trials included in this review exhibited a moderate to high risk of bias due to insufficient methodological reporting . Most studies employed proper random sequence generation, while one study was considered to have a high risk of bias in this regard. Allocation concealment information was not provided in 18 trials, - ,25-27,29,31,32,34,40,42,46,47,49,50,52 leading to an unclear risk of bias assessment. Blinding participants or personnel was unfeasible since THM was administered solely to the experimental group. Detection bias was deemed to be at high risk in 20 studies, - , , , - , , , , , , , , , , , and no relevant information was reported in 4 studies. , , , Reporting bias was assessed as unclear risk in all studies due to the inability to locate the registered protocols. All studies were assessed to have a low risk of attrition and other biases . Tumor response assessment A comprehensive review of 12 studies demonstrated a statistically significant increase in both DCR and ORR for those patients receiving a combination of THM and chemotherapy, , , , , , , - , , , compared to those receiving chemotherapy alone (RR 1.25, 95% CI [1.09, 1.45], I 2 = 79%, n = 835) and (RR 1.54, 95% CI [1.31, 1.81], I 2 = 0%, n = 835, ). To address the substantial heterogeneity in the DCR outcomes ( I 2 = 79%), 3 studies were excluded due to their notably high risk ratios and wide confidence intervals, which were identified as potential outliers. , , The recalculated data from the remaining studies still demonstrated an enhancement in DCR due to THM (RR 1.18, 95% CI [1.07, 1.29], I 2 = 42%, n = 646, ). However, the decrease in the I 2 value to 42% suggests the initial heterogeneity may have been driven by reporting bias. A comprehensive review of 12 studies demonstrated a statistically significant increase in both DCR and ORR for those patients receiving a combination of THM and chemotherapy, , , , , , , - , , , compared to those receiving chemotherapy alone (RR 1.25, 95% CI [1.09, 1.45], I 2 = 79%, n = 835) and (RR 1.54, 95% CI [1.31, 1.81], I 2 = 0%, n = 835, ). To address the substantial heterogeneity in the DCR outcomes ( I 2 = 79%), 3 studies were excluded due to their notably high risk ratios and wide confidence intervals, which were identified as potential outliers. , , The recalculated data from the remaining studies still demonstrated an enhancement in DCR due to THM (RR 1.18, 95% CI [1.07, 1.29], I 2 = 42%, n = 646, ). However, the decrease in the I 2 value to 42% suggests the initial heterogeneity may have been driven by reporting bias. Quality of life assessment In line with the established criteria for evaluating KPS score enhancement, 14 studies measured QoL through KPS score improvement. A combined analysis of these 14 studies revealed a significantly greater rate of KPS score enhancement in the group receiving both THM and chemotherapy compared to the group undergoing chemotherapy alone. This difference was statistically significant (MD of 6.84, 95% CI [5.77, 7.92], I 2 = 49%, n = 922, ). , , , , , , , , , , , - Adverse drug reactions assessment The findings from the meta-analysis revealed that the group receiving both THM and chemotherapy exhibited a reduced likelihood of experiencing adverse drug reactions compared to the chemotherapy-only group. This included lower risks of leukocyte reduction (RR 0.62, 95% CI [0.50, 0.76], I 2 = 49%, n = 1284, 16 trials), , , , , , , , , , , - , , , platelet reduction (RR 0.86, 95% CI [0.72, 1.01], I 2 = 10%, n = 1198, 16 trials), , , , , - , , , , - , , , liver injury (RR 0.59, 95% CI [0.42, 0.82], I 2 = 29%, n = 951, 13 trials), , , , , , , , , , , , renal injury (RR 0.46, 95% CI [0.28, 0.75], I 2 = 0%, n = 589, 7 trials), , , , , , , nausea and vomiting (RR 0.68, 95% CI [0.56, 0.84], I 2 = 51%, n = 1038, 14 trials), , , , , , , , - , , , diarrhea (RR 0.74, 95% CI [0.61, 0.88], I 2 = 4%, n = 900, 12 trials), , , , , , , , , , , , and neurotoxicity (RR 0.63, 95% CI [0.48, 0.84], I 2 = 0%, n = 651, 9 trials). , , , , , , , , Except for platelet reduction ( P = .07), all differences were statistically significant, with further details presented in . Tumor marker level In the THM plus chemotherapy group, there was a greater decrease in CEA compared to the chemotherapy-alone group, as demonstrated by the pooled results of 16 trials (MD −1.55, 95% CI [−2.23, −0.87], I 2 = 51%, n = 967). , , - , , , - , , - , , Moreover, the pooled results of 11 trials revealed a higher CA19-9 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −2.50, 95% CI [−3.91, −1.09], I 2 = 18%, n = 748). , , , , , , , , , , Conversely, the pooled results of 8 trials indicated no significant change in CA125 between the THM plus chemotherapy group and chemotherapy group (MD 0.12, 95%, CI [−1.47, 1.70], I 2 = 59%, n = 596). , , , , , , , Additionally, the pooled results of 6 trials showed a greater CA72-4 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −1.80, 95% CI [−2.59, −1.01], I 2 = 43%, n = 393). , , , , , The detailed information has been presented in . One-year survival rate The meta-analysis of 10 studies assessed the 1-year survival rate and found that THM plus chemotherapy group demonstrated a statistically significant improvement in the 1-year survival rate (RR 1.08, 95% CI [1.02, 1.14], I 2 = 0%, n = 868, 10 trials, ). , , , , , , , , , These findings provide strong evidence that the combination of THM and chemotherapy has a positive effect on the 1-year survival rate. Two-year survival rate The meta-analysis of 8 studies indicated that the combination of THM and chemotherapy was effective in improving the 2-year survival rate (RR 1.32, 95% CI [1.19, 1.47], I 2 = 0%, n = 712, 8 trials, ), providing robust evidence for the effectiveness of the THM and chemotherapy combination in enhancing the 2-year survival rate. Three-year survival rate The statistically significant improvement observed in the 3-year survival rate (RR 1.42, 95% CI [1.12, 1.79], I 2 = 0%, n = 343, 3 trials, ) further reinforces the efficacy of combining THM and chemotherapy as a promising approach. These findings provide valuable insights into the potential long-term benefits of this treatment strategy in improving patient outcomes. In line with the established criteria for evaluating KPS score enhancement, 14 studies measured QoL through KPS score improvement. A combined analysis of these 14 studies revealed a significantly greater rate of KPS score enhancement in the group receiving both THM and chemotherapy compared to the group undergoing chemotherapy alone. This difference was statistically significant (MD of 6.84, 95% CI [5.77, 7.92], I 2 = 49%, n = 922, ). , , , , , , , , , , , - The findings from the meta-analysis revealed that the group receiving both THM and chemotherapy exhibited a reduced likelihood of experiencing adverse drug reactions compared to the chemotherapy-only group. This included lower risks of leukocyte reduction (RR 0.62, 95% CI [0.50, 0.76], I 2 = 49%, n = 1284, 16 trials), , , , , , , , , , , - , , , platelet reduction (RR 0.86, 95% CI [0.72, 1.01], I 2 = 10%, n = 1198, 16 trials), , , , , - , , , , - , , , liver injury (RR 0.59, 95% CI [0.42, 0.82], I 2 = 29%, n = 951, 13 trials), , , , , , , , , , , , renal injury (RR 0.46, 95% CI [0.28, 0.75], I 2 = 0%, n = 589, 7 trials), , , , , , , nausea and vomiting (RR 0.68, 95% CI [0.56, 0.84], I 2 = 51%, n = 1038, 14 trials), , , , , , , , - , , , diarrhea (RR 0.74, 95% CI [0.61, 0.88], I 2 = 4%, n = 900, 12 trials), , , , , , , , , , , , and neurotoxicity (RR 0.63, 95% CI [0.48, 0.84], I 2 = 0%, n = 651, 9 trials). , , , , , , , , Except for platelet reduction ( P = .07), all differences were statistically significant, with further details presented in . In the THM plus chemotherapy group, there was a greater decrease in CEA compared to the chemotherapy-alone group, as demonstrated by the pooled results of 16 trials (MD −1.55, 95% CI [−2.23, −0.87], I 2 = 51%, n = 967). , , - , , , - , , - , , Moreover, the pooled results of 11 trials revealed a higher CA19-9 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −2.50, 95% CI [−3.91, −1.09], I 2 = 18%, n = 748). , , , , , , , , , , Conversely, the pooled results of 8 trials indicated no significant change in CA125 between the THM plus chemotherapy group and chemotherapy group (MD 0.12, 95%, CI [−1.47, 1.70], I 2 = 59%, n = 596). , , , , , , , Additionally, the pooled results of 6 trials showed a greater CA72-4 decrease in the THM plus chemotherapy group than in the chemotherapy group (MD −1.80, 95% CI [−2.59, −1.01], I 2 = 43%, n = 393). , , , , , The detailed information has been presented in . The meta-analysis of 10 studies assessed the 1-year survival rate and found that THM plus chemotherapy group demonstrated a statistically significant improvement in the 1-year survival rate (RR 1.08, 95% CI [1.02, 1.14], I 2 = 0%, n = 868, 10 trials, ). , , , , , , , , , These findings provide strong evidence that the combination of THM and chemotherapy has a positive effect on the 1-year survival rate. The meta-analysis of 8 studies indicated that the combination of THM and chemotherapy was effective in improving the 2-year survival rate (RR 1.32, 95% CI [1.19, 1.47], I 2 = 0%, n = 712, 8 trials, ), providing robust evidence for the effectiveness of the THM and chemotherapy combination in enhancing the 2-year survival rate. The statistically significant improvement observed in the 3-year survival rate (RR 1.42, 95% CI [1.12, 1.79], I 2 = 0%, n = 343, 3 trials, ) further reinforces the efficacy of combining THM and chemotherapy as a promising approach. These findings provide valuable insights into the potential long-term benefits of this treatment strategy in improving patient outcomes. To investigate publication bias, funnel plots were created using RR, MD, and 1/(standard error: SE) values extracted from trials that measured ORR, KPS score, and 1-year survival rate. The funnel plot analysis for the ORR study exhibited a symmetrical shape , indicating a balanced distribution of studies across a range of effect sizes and their corresponding precision measures. This symmetry suggests the absence of significant publication bias or small study effects, reinforcing the reliability and robustness of the study’s findings regarding ORR. However, the funnel plots generated based on the KPS score and 1-year survival rate displayed an asymmetrical distribution ( and ), suggesting the potential presence of publication bias in these analyses. In the assessment of tumor response, the DCR and ORR were analyzed from 9 and 12 RCTs respectively, showing evidence of “Low” and “Moderate” certainty. Concerning the QoL, the KPC indicator from 14 RCTs demonstrated “Moderate” certainty. Adverse drug reactions showed varied results: leukocyte decrease, liver injury, nausea & vomiting, diarrhea, tumor marker levels (CEA, CA19-9, CA125, CA72-4) were all assessed with “Low” certainty, while platelet decrease, renal injury, and neurotoxicity held a “Moderate” certainty. For survival rate evaluations, the 1-, 2-, and 3-year survival rates all were categorized with “Moderate” certainty. The reasons for downgrading the evidence quality across these evaluations primarily included risk of bias and inconsistencies. Various outcomes also suffered from imprecision, often attributed to limited sample sizes. Detailed outcomes, effects, and absolute effects can be cross-referenced in the provided . The frequency analysis of the most commonly used herbs in the included studies unveiled significant trends in herbal prescriptions for post-surgical gastric cancer therapy. Among the 122 herbs observed over 36 prescriptions, the top 10 herbs are presented in . Atractylodes macrocephala exhibited the highest frequency, present in 75.0% of the analyzed prescriptions. Subsequently, Poria cocos showed a frequency of 63.9%, while Glycyrrhiza uralensis , Citrus reticulata , Astragalus membranaceus , and Hedyotis diffusa all shared a frequency of 61.1% across the prescriptions. Codonopsis pilosula and Pinellia ternata were also frequently prescribed, each appearing in 58.3% of the prescriptions. Angelicae Sinensis Radix and Coix lacryma-jobi were similarly common, noted in 38.9% of the prescriptions. These findings provide insight into the prevalent herbal interventions employed in the prescriptions analyzed, showcasing a selection of herbs frequently prescribed in post-surgical gastric cancer therapy involving THM. This meta-analysis and systematic review investigated the efficacy and safety of combining THM with chemotherapy, specifically for GC, across 36 randomized controlled trials involving 2176 patients. A critical evaluation of the included studies was performed using the RoB tool, which helped in assessing the potential biases in these trials. We identified that the trials exhibited a moderate to high risk of bias due to gaps in methodological reporting. Notably, a significant number lacked details on allocation concealment, and blinding of participants was challenging due to the nature of the intervention with THM. Additionally, a high risk of detection bias was observed in many studies, and the reporting bias remained uncertain for all included trials due to the absence of registered protocols. Primary outcomes revealed a positive association between THM and tumor response, with both DCR and ORR indicating statistically significant improvements. The RoB assessments underscore the need for further rigorously designed RCTs in this domain to strengthen the evidence base. A re-examination, after excluding 3 studies, resolved initial heterogeneity in DCR findings. Regarding the outcomes reported in this study, it is important to note that the outcome of “cancer recurrence prevention rate” was included in our initial registration record but was not reported in the final manuscript. This decision was due to the observed inconsistencies in how this outcome was measured across various studies, which could potentially compromise the reliability and comparability of our findings. Conversely, the outcome of “tumor markers,” while not initially included in the registration record, was later reported in our study. This inclusion was driven by a deeper analysis and understanding of the review’s objectives, where we recognized the significance of tumor markers in providing valuable insights into treatment efficacy and cancer progression. Secondary outcomes demonstrated that patients receiving both THM and chemotherapy had improved QoL scores and fewer adverse drug reactions compared to those on chemotherapy alone. Notable reductions were observed in specific reactions such as leukocyte and platelet reduction, liver and renal injuries, nausea, vomiting, diarrhea, and neurotoxicity, though platelet reduction was an exception with no significant statistical difference. One aspect that demands further discussion is the reporting of tumor markers. Our analysis underscores the importance of these biomarkers as they often serve as critical indicators of tumor burden and treatment response. Tumor markers can provide valuable insights into the pathophysiology of cancer, guide therapy decisions, and help in monitoring disease progression or response to treatment. In this review, we observed that combining THM with chemotherapy led to significant reductions in certain markers, except CA125. Detailed and consistent reporting of tumor markers would contribute substantially to the understanding of the mechanisms through which THM may potentiate the efficacy of chemotherapy. Additionally, survival rate analyses showcased remarkable enhancements in 1-, 2-, and 3-year survival rates with the THM and chemotherapy combination. To further validate our findings, particularly in light of the wide range of treatment durations in the included studies, we conducted a sensitivity analysis. This analysis confirmed that the duration of herbal medication, whether short-term or long-term, did not significantly influence the overall effectiveness of the treatments. This consistent finding across various studies indicates that differences in treatment durations did not introduce notable biases or affect the results, thus affirming the robustness and reliability of our conclusions, despite the diversity in treatment lengths. However, research is needed to determine whether the size of the effect varies depending on the composition and administration period of herbal medicine. Given these promising results, our research may offer new insights into the global challenge of treating gastric cancer, a disease fraught with complex risk factors and significant impacts on patients’ quality of life as detailed above. Gastric cancer is one of the commonly diagnosed diseases worldwide and is physically and psychologically distressing. Risk factors for gastric cancer include unavoidable variables such as race, age, and gender, as well as other risk factors such as infection with Helicobacter pylori bacteria, diets high in nitrates and nitrites, and smoking. Additionally, factors like genetics, medical history, and pernicious anemia also contribute to the onset of stomach cancer. Gastric cancer is commonly treated through surgery, radiation, and chemotherapy, but recurrence and side effects often occur. , Furthermore, patients can suffer from psychological symptoms related to tumor diagnosis, postoperative complications, and the side effects of treatment. Chemotherapeutic drugs commonly used in gastric cancer treatment, such as Oxaliplatin, typically cause side effects like nausea, vomiting, diarrhea, and loss of appetite. Also, adjuvant chemotherapy does not unequivocally improve survival rates. The treatment of gastric cancer, including surgery and chemotherapy, still involves high costs, is painful, imposes a significant burden on society, and most importantly, greatly reduces the quality of life for patients. Therefore, the treatment of gastric cancer remains an important challenge. One of the most researched options for gastric cancer is herbal medicine. THM can help in cancer treatment by reducing the side effects of conventional treatments or improving the quality of life. According to a meta-analysis of several RCTs, THM has been shown to improve cancer-related symptoms, enhance quality of life, and reduce the side effects of chemotherapy. This demonstrates the effectiveness of combining anticancer drugs with THM in the treatment of gastric cancer. According to the tumor response results of this paper, in the RCTs used for DCR and ORR research, medicines such as Astragalus membranaceus, Atractylodes macrocephala koidz, Radix angelica sinensis, Poria cocos, and Panax ginseng were widely used. Among them, Buzhong Yiqi decoction was used as an intervention in 3 studies. In the study by Xu, it was found that Buzhong Yiqi decoction promotes the proliferation of T lymphocytes, strengthens immunity, and prevents cancer cells from evading immune responses by reducing the expression of PD-1 and PD-L1. It also inhibits the expression of PD-L1 by suppressing the PI3K/AKT pathway. In the study that combined Buzhong Yiqi decoction with adjuvant chemotherapy, the OFL anticancer regimen used was a combination of oxaliplatin, leucovorin, and fluorouracil. Oxaliplatin in this anticancer drug promotes apoptosis of cancer cells, and fluorouracil interferes with the synthesis components of DNA and RNA in cancer cells. Leucovorin is used to enhance the effect of fluorouracil. Although the roles of OFL and Buzhong Yiqi decoction are different, from this perspective, it is considered that some anticancer drugs interact with the mechanism of THM and show better results than single adjuvant chemotherapy. Not only Buzhong Yiqi decoction, but also other THM with similar properties are being studied. In a study by Li, Yiqi Jianpi Huaji decoction, which includes herbal ingredients such as Astragalus membranaceus, Atractylodes macrocephala koidz, Radix angelica sinensis andCodonopsis pilosula, was found to inhibit cancer cell proliferation and help overcome multiple drug resistance. Chemotherapeutic drugs like fluorouracil inhibit the proliferation of cancer cells, promote cell death, interfere with the synthesis of DNA and RNA, and suppress the tumor microenvironment. Therefore, the use of THMs may help to prevent the progression of cancer and enhance the therapeutic effects in patients with gastric cancer. Similar results are being demonstrated in other types of cancer that use similar chemotherapeutic agents. It has been reported that combining THM with chemotherapy (combined with cisplatin and paclitaxel, gemcitabine, vinorelbine, etoposide) in the treatment of non-small cell lung cancer (NSCLC) is more effective. Compared to chemotherapy treatment alone, combining THM with chemotherapy improved QoL, clinical efficacy, KPS scores, and reduced toxicity (such as leukopenia, hemoglobin reduction, thrombocytopenia, myelosuppression, diarrhea, nausea and vomiting, and liver damage). The findings from this systematic review align with the findings from a previously presented study. , , A frequency analysis conducted within this review highlighted prevalent herbal medicines used in GC treatment, offering a deeper understanding of herbal medicine’s role in GC therapeutic strategies. Despite the evident benefits of combining THM with adjuvant chemotherapy for GC, there remain knowledge gaps in THM’s specifics, like discerning essential components and probing potential interactions between herbal medicines and standard treatments. With the varied nature of THM prescriptions, a meticulous evaluation of individual prescriptions is crucial. The absence of serious side effects from the combination therapy underscores the potential of this synergistic treatment. This study underscores the need for continued research to fathom the complex interactions between adjuvant chemotherapy and THM, aiming to identify specific elements boosting THM’s therapeutic advantages. In summation, the combined approach of THM and chemotherapy emerges as a promising, effective, and safe therapeutic pathway, potentially redefining future cancer treatments. Limitations This study has some limitations. Firstly, blinding participants or personnel was unfeasible since THM was administered solely to the experimental group. The absence of blinding icould introduce placebo effects, especially in subjective areas like quality of life. Secondly, there were many types of chemotherapy, making it difficult to perform subgroup analysis with the same chemotherapeutic agent. Thirdly, the THM of the studies was diverse, which may lead to variation. Finally, the funnel plot showed unbalanced studies, indicating the possibility of publication bias. Furthermore, all studies included in our analysis were published in China; there is a possibility of geographical publication bias which could affect the outcomes, as findings or perspectives from other regions or languages might vary. However, the meta-analysis results indicate that THM is an effective treatment method for gastric cancer, as it improves patients’ QoL, reduces side effects, and helps prevent the progression of GC. Overall, integrating THM into gastric cancer treatment regimens provides a promising strategy to enhance treatment outcomes and improve patient quality of life. However, more comprehensive and standardized research is needed for broader clinical application. This study has some limitations. Firstly, blinding participants or personnel was unfeasible since THM was administered solely to the experimental group. The absence of blinding icould introduce placebo effects, especially in subjective areas like quality of life. Secondly, there were many types of chemotherapy, making it difficult to perform subgroup analysis with the same chemotherapeutic agent. Thirdly, the THM of the studies was diverse, which may lead to variation. Finally, the funnel plot showed unbalanced studies, indicating the possibility of publication bias. Furthermore, all studies included in our analysis were published in China; there is a possibility of geographical publication bias which could affect the outcomes, as findings or perspectives from other regions or languages might vary. However, the meta-analysis results indicate that THM is an effective treatment method for gastric cancer, as it improves patients’ QoL, reduces side effects, and helps prevent the progression of GC. Overall, integrating THM into gastric cancer treatment regimens provides a promising strategy to enhance treatment outcomes and improve patient quality of life. However, more comprehensive and standardized research is needed for broader clinical application. The systematic review of studies on the combined use of adjuvant chemotherapy and THM in gastric cancer yields promising results. The combined therapy has shown significant improvements in tumor response, quality of life, and survival rates of patients. Furthermore, THM seems to synergize with adjuvant chemotherapy drugs to enhance their anticancer effects while also mitigating the side effects such as leukocyte reduction, liver and renal injuries, nausea, vomiting, diarrhea, and neurotoxicity. Tumor markers CEA, CA19-9, and CA72-4 displayed statistically significant results in the improvement of the quality of life and survival rates. However, the tumor marker CA125 and platelet reduction were not found to be statistically significant. Overall, the review suggests that the combination of adjuvant chemotherapy and THM could potentially be an effective treatment method for gastric cancer. However, considering the methodological limitations in the included studies, further well-designed research, particularly with placebo controls and more rigorous blinding and allocation concealment, is crucial to confirm these findings. sj-docx-1-ict-10.1177_15347354231226256 – Supplemental material for Comprehensive Evaluation of Traditional Herbal Medicine Combined With Adjuvant Chemotherapy on Post-Surgical Gastric Cancer: A Systematic Review and Meta-Analysis Click here for additional data file. Supplemental material, sj-docx-1-ict-10.1177_15347354231226256 for Comprehensive Evaluation of Traditional Herbal Medicine Combined With Adjuvant Chemotherapy on Post-Surgical Gastric Cancer: A Systematic Review and Meta-Analysis by Soo-Dam Kim, Jong-Hee Kim, Dong-Hyun Kim, Ji-Hye Park, Yabin Gong, Chengbing Sun, Hwa-Seung Yoo and So-Jung Park in Integrative Cancer Therapies |
Results from a cross-specialty consensus on optimal management of patients with chronic kidney disease (CKD): from screening to complications | 108257b9-f811-4c19-a38c-9c1eb3342862 | 10921537 | Internal Medicine[mh] | Chronic kidney disease (CKD) is defined as abnormalities of kidney structure or function, which are present for >3 months. This is indicated by a glomerular filtration rate (GFR) below 60 mL/min/1.73 m² or the presence of one or more markers of kidney damage. CKD is classified based on cause, GFR category and albuminuria category. Diabetes is the leading cause of CKD, followed by hypertension, suggesting a close relationship between the cardio-renal-metabolic (CRM) systems. The interplay between CKD, cardiovascular disease (CVD) and metabolic diseases such as type 2 diabetes mellitus (T2DM) is significant and has been termed ‘CRM’ disease. It has been suggested that CKD affects around 50% of people with T2DM, around 50% of heart failure (HF) patients, and up to 38% of those with hypertension, suggesting the need for a holistic approach to management of patients with CKD. Between 1990 and 2017, the prevalence of CKD was estimated to rise to 9.1% of the global population, with associated mortality increasing by 41.5%. This is closely associated with an increase in populations with risk factors such as diabetes, hypertension and pre-diabetes. As a consequence of these factors, 1.2 million people died in 2017 as a consequence of CKD. Early diagnosis and referral of CKD are key to reducing or avoiding progression to kidney failure, and reducing morbidity and mortality. The absence of symptoms in early stages of CKD requires that clinicians maintain suspicion in all patients, especially those with risk factors. Accurate screening, diagnosis and risk stratification need to be in place to support the aim of ‘earliest diagnosis’. A diagnosis of CKD is determined by laboratory confirmation of proteinuria or haematuria, and/or a reduction in the GFR, for more than 3 months duration. Two key markers of CKD include albuminuria (defined as a urinary albumin-to-creatine ratio (uACR) of >30 mg/g) and reduction in estimated GFR (eGFR), as calculated by the CKD-EPI based on creatinine alone (eGFR cr ) or on creatinine and cystatin C (eGFR cr-cys ). In practice, these methods may not be used effectively, an analysis of over 123 000 patient records reported a low frequency of uACR testing in patients with CKD, despite strong epidemiological evidence linking increased albuminuria with disease progression, kidney failure, cardiovascular events and premature mortality. Besides lifestyle modifications, disease-modifying therapies (DMTs) for CKD including renin–angiotensin–aldosterone system inhibitors (RAASi) and sodium-glucose co-transporter-2 inhibitors (SGLT2i), as recommended by Kidney Disease Improving Global Outcomes (KDIGO) for use in patients with CKD with hypertension or diabetes, and non-steroidal mineralocorticoid antagonists as add-on therapy in patients with T2DM and residual albuminuria. Despite the availability of these therapies, analysis of two large US healthcare systems showed that even though nearly two-thirds of the adults with CKD had diabetes, hypertension or pre-diabetes, rates of prescribing RAASi were low. Not only was DMT use below guideline-directed levels, but potentially nephrotoxic agents (such as non-steroidal anti-inflammatory drugs and proton pump inhibitors) were used more commonly than RAASi. There is clearly some disconnect between guideline recommendations and real-world practice. Guidelines for the management of CKD may not be clear or made known to primary care practitioners (PCPs). The objective of this project is to use a modified Delphi technique to examine the opinions of healthcare professionals (HCPs) towards aspects of CKD management across 11 countries, report these findings and develop practical recommendations for diagnosis and management of CKD. A steering group of experts (see author list) from 11 different countries convened in September 2022 to discuss current challenges in CKD management. The experts were defined as specialists in nephrology, endocrinology/diabetology, internal medicine and primary care medicine who had achieved an appropriate level of seniority within their field (eg, professors, clinical directors), or had published papers related to the management of CKD/HF/Diabetes, or had been involved in guidelines development. The steering group members were selected to provide representation across a range of development indicator levels external to Europe and North America (to avoid replication of previous published work ). Steering group members were recruited to represent countries outside of North America and Europe. Central and South America, Southeast Asia, Middle East and Africa were initially targeted. The process of recruitment involved identification of potential group members from each of these regions using desk research, followed by a snowball method until all target regions had at least one representative on the steering group. Although a wide representation was aimed, it is a limitation that there were not enough members from low-income countries. The Delphi technique used in this study was guided by Guidance on Conducting and REporting DElphi Studies. While guidelines exist, outcomes for CKD vary between countries, a modified Delphi approach was employed to understand where common care processes differ in local use, and how the attitudes of HCPs to elements of CKD management differ between countries. The overall process is outlined in . Six themes for focus for statement development were agreed , these were discussed further, and statements developed by the steering group working collaboratively. The statements were then collated, and the steering group independently rated the statements as either ‘accept’, ‘remove’ or ‘reword’ with suggested changes (as determined by a simple majority) with the potential for further group consultation for any significant differences of opinion on the fundamental principles of any statement. Once finalised, the steering group was agreed the final set of statements for testing. This constituted the initial round of consensus. The resulting 42 statements were developed into a Likert survey, which was then distributed by a third party (Sermo) in round 2 of the process. Panel members were recruited based on the following criteria: Employed within 1 of the 11 target countries. 25 respondents from each country in a broadly 2:1:1:1 ratio of primary care physician, consultant endocrinologist, consultant cardiologist, consultant nephrologist (or local equivalent). The identity of respondents was not known to the steering group or the independent facilitator. For each statement, respondents were offered a 4-point scale (‘strongly disagree’, ‘tend to disagree’, ‘tend to agree’ and ‘strongly agree’) to indicate their level of agreement with each statement. The survey also captured country, specialty, length of time in role and average number of patients with CKD managed over 3 months. Stopping criteria for data collection were defined as a target of 25 responses from each country (N=275) and a 4-week survey period. The target countries were chosen to reflect the steering group demographic, this would enable each steering group member to provide insight into results from their respective country. Panel members were recruited from the following countries: Argentina (UMI), Australia (HI), Brazil (UMI), Egypt (LMI), Guatemala (UMI), Mexico (UMI), Singapore (HI), South Korea (HI), Taiwan (HI), Thailand (UMI) and Turkey (UMI) (HI—high income, LMI—lower-middle income, UMI—upper-middle income, according to World Bank Classification.) The closing criteria for the study were defined a priori in line with best practice principles as: 90% of the final statement set achieving consensus threshold (defined at 75%, a widely accepted threshold). If these criteria were not met, statements would be modified, and the survey reissued as necessary for a maximum of three rounds. Consensus was further categorised as ‘high’ at ≥75% and ‘very high’ at ≥90%. Completed surveys were analysed using Microsoft Excel software. The responses were aggregated to provide an overall agreement level (ie, the number of responded expressing agreement as a percentage of the overall number of responses for each statement). Patient and public involvement None, the stated objective was to examine the opinions of HCPs towards aspects of CKD management across 11 countries. None, the stated objective was to examine the opinions of HCPs towards aspects of CKD management across 11 countries. Of the 45 initial statements created by the steering group, 32 were retained, 8 were modified, 5 were removed and 3 new statements were added. The final set was then sent out to the group via email for acceptance prior to progressing to round 2. In round 2, completed surveys from a panel of 274 were analysed to define the total level of agreement with each of the 42 statements. Respondents were either PCPs or consultants in cardiology, nephrology and endocrinology (including diabetology) . Most responders had more than 10 years experience in role (159/274) and only 13% (n=35) had less then 5 years in role. When asked to estimate the number of patients seen in a 3-month period, 177 responders (65%) stated that they saw more than 50 patients (either as inpatients or outpatients). This suggests the respondent cohort was sufficiently experienced to provide insight into aspects of CKD. Consensus was achieved for all statements, with very high agreement (>90%) in 37 (88%) statements, and high agreement (≥75 and≤89%) in 5 (12%) statements. Overall consensus agreement by statement is shown in and , detailed results showing percentages of agreement at each level can be found in and . 10.1136/bmjopen-2023-080891.supp2 Supplementary data 10.1136/bmjopen-2023-080891.supp1 Supplementary data As none of the statements failed to reach the predetermined threshold of 75%, only one round of survey was required. The results of the survey represent current opinions of the respondents and are not intended to contradict the established evidence base. Anonymised round 2 results data are available in . 10.1136/bmjopen-2023-080891.supp3 Supplementary data Note, in the discussion below, ‘S’ is used to denote ‘statement’ Earlier identification & screening of CKD (S1-9) Very high agreement (>90%) was observed for all statements in this topic, underscoring the key principle that early diagnosis of CKD is key to implementing strategies to slow disease progression. Responses suggest a strong support for the need for national kidney health screening and diagnostic programmes. Universal screening of the general population has been found not to be cost-effective, but systematic review has shown screening to be cost-effective in patients with hypertension or diabetes. Indeed, a KDIGO Controversies Conference concluded that targeted groups, such as those with hypertension, diabetes or CVD should be screened for CKD, and that an individualised approach should be taken to screen others based on a range of factors. Respondents agree that patients with risk factors should be screened for CKD at least annually by using eGFR and uACR where available, but identification of CKD is also challenging where awareness among healthcare staff and health literacy in the general population is poor, it is, therefore, recommended that national initiatives to improve these issues should support any screening programme. A minimal-resource prescreening tool has been developed and globally validated for CKD in people with T2DM. This demonstrates that age, gender, body mass index, duration of diabetes and blood pressure information can be used to identify those at an increased risk of CKD. This model does not require sophisticated diagnostics and can be used to guide cost-effective screening for CKD where resources are limited. Risk factors for CKD in CRM patients (S10–15) All statements in this topic achieved >90% consensus apart from statement 14 (85%). This is interesting and could be due to the wording of the statement and the specific use of COVID-19 as an example. Reference to COVID-19 was included in the statement as it was considered relevant by the steering group at the time of statement generation and evidence was beginning to emerge of potential kidney injury associated with COVID-19. Analysis by country found that Taiwan agreement was at 68% for this statement, noticeably lower than for other countries. Although 93% of respondents agree that acute kidney injury (AKI) is a risk factor for CKD and HF, it is interesting that 7% do not agree given the evidence base. Given the established interplay between the cardiac, renal and endocrine systems, it is heartening to see that 99% of respondents agree that a holistic approach is needed to provide personalised care for individuals with CKD. Where practical, models of care should be developed to deliver integrated multidisciplinary care to patients with CRM disease, as described by the CardioMetabolic Care Alliance, to use comprehensive, patient-centred, team-based approaches for aggressive secondary prevention. Implementing combined clinics to deliver medical care for patients with kidney disease and diabetes or CVD may reduce outpatient healthcare costs without compromising health outcomes. Combined multidisciplinary clinics for diabetes, CKD and CVD are also associated with a slower decline in GFR than usual care, and a significant reduction in the risk for all-cause mortality. However, global variation exists regarding resource availability and access to specialist care, and the use of multidisciplinary teams (MDTs) is not universal. In these regions, HCPs are encouraged to develop local contacts/networks to allow for interdisciplinary discussions of CKD cases where needed. Holistic management of CKD in CRM patients (S16–27) The broad objective of this consensus is to promote the earlier detection and management of CKD, and this principle applies regardless of resource limitations. As CKD-associated healthcare costs increase with disease progression, the economic argument supports this approach, and in patients with limited access to specialist care (including DMT), the role of patient education is paramount in improving health literacy and promoting lifestyle changes to reduce CKD risk. Coupled with this, the health system should look at how interventions such as DMTs can be used to slow progression of CKD and the need for dialysis (and associated costs), cost savings from slower progression to dialysis dependence might be reinvested in detection and diagnosis/national screening of high-risk patient groups. Very high agreement was observed for 10 of the statements in this topic, with 2 statements at lower levels of agreement (S23 and S25, 82% and 75%, respectively). Response to statement 23 is interesting, given the current debate regarding the benefits of stopping RAASi in advanced CKD. Results from a 52-week open-label UK study in patients with CKD stages 4–5 concluded ‘The STOP-ACEi trial did not find any benefit by stopping RAASi in advanced CKD’, and that ‘stopping RAASi in advanced CKD at an arbitrary GFR threshold is not the optimal approach’. Large observational studies have confirmed the cardioprotective benefits of RAASi in advanced CKD (including patients with concomitant T2DM). Linked to this is statement 21, respondents agree that a small decrease in eGFR is to be expected on initiation of DMT for CKD. During initiation and uptitration of RAASi treatment, a decline in kidney function of up to 30% within 4 weeks can be acceptable and it is important to avoid a knee-jerk response and reduce or stop DMT, and consultation with a nephrologist or MDT is recommended. Hyperkalaemia (HK) is potentially life-threatening; it may be acute or chronic and individuals with CKD are at an increased risk, which increases with the later stages of CKD. HK treatment is often stratified by serum potassium level, with borderline levels warranting modification of dietary potassium intake, followed by pharmaceutical management for larger or more sustained increases. Evidence to support recommending low potassium diets to patients with advanced CKD or ESRD is weak. Observational studies report weak associations between dietary potassium intake and potassium concentration and this approach may deprive patients of the beneficial cardiovascular effects associated with potassium-rich diets. When HK occurs, RAASi dose reduction or discontinuation are the most common used therapeutic options, but this approach is associated with worse cardiorenal outcomes and increased mortality. Once stopped, RAASi treatment is rarely reinitiated. In cases of mild-to-moderate HK, DMT should be maintained where possible, and an option for achieving this is using a potassium-lowering therapy such as patiromer or sodium zirconium cyclosilicate (S24, 91%). While the threshold for intervention in HK may vary by country and even individual physician, we can conclude that action may be considered when K + concentration reaches 5.0 mmol/L (S25, 75%) and certainly when at 5.5 mmol/L (S26, 93%) but with a degree of personalisation (in consultation with a nephrologist). Statements 25 and 26 were included to try and understand where the weight of opinion fell regarding interventions to manage HK, both statements achieved consensus, but the strongest agreement was for statement 26. On reflection, these statements could have been worded better to understand what would constitute appropriate action in these circumstances. Respondents very strongly agree that SGLT2i and RAASi therapies have a complementary cardiorenal protective action (S18, 98%) and that in patients with T2DM and HF, early SGLT2i use can prevent the development and progression of CKD (S19, 96%), in fact, SGLT2i may slow progression of CKD in patients without T2DM (S20, 91%). It is, therefore, recommended that SGLT2i and RAASi therapies are initiated as early as possible to both delay CKD progression and to therapeutically address the full CRM continuum—including T2DM and HF. The interplay between CKD, diabetes and CVD has been discussed, but CKD is associated with a number of comorbidities. A prospective UK cohort study of 1741 people with CKD stage 3 found that isolated CKD (without comorbidities) was present in only 4% of patients, and that common comorbidities include ‘painful condition’ (30%), anaemia (24%), thyroid disorders (12%), cerebrovascular disease (12%) and respiratory conditions (10%). These comorbidities should be identified and managed as part of a multidisciplinary approach. Guidelines (S28–35) While all statements in this topic achieved consensus threshold, the steering group were surprised that 23% of respondents did not agree that implementation of CKD guidelines is suboptimal. The lowest agreement levels were observed in South Korea (68%) and Guatemala (72%). This could be either genuine strength in implementation of guidelines in these countries (which should be investigated and replicated elsewhere), or the need for continuing professional development even among experienced physicians. Due to the vital role of primary care must play in identifying and referring suspected patients with CKD, respondents agree that clear guidelines for referral of patients are considered essential (S32, 98%). PCPs should be provided with clear criteria for referral and followed through with continuing education from an appropriate specialist (ideally a nephrologist). The focus on prevention can only be achieved through a whole-system approach with appropriate investment in awareness, screening and diagnosis programmes (S35, 96%). Cross-specialty alignment (Cardiology, Nephrology, Endocrinology, Primary Care & Policy Makers) (S35 - 39) Very high levels of agreement suggest strong support for multidisciplinary involvement in CKD management, and that PCPs should form part of the MDT. Given the prominent status of CKD as an emerging global health threat, there is a need to ensure that plans and funding are in place to enable optimal care. Stakeholders should engage with both local and national policy-makers to ensure that CKD is appropriately prioritised. Education of clinicians and patients (S40–42) Most responders agree on the need for up-to-date education for all HCP roles involved in CKD management, and that PCPs require structured education. Education of patients is a low-cost intervention, to encourage kidney healthy lifestyles and improve awareness of CKD among high-risk populations. Strengths and limitations 274 responses received with good representation from experienced physicians across 11 different countries is a strong basis for consensus, the study was designed to ensure that each country was represented by an equal number of responders. The majority of responders reported more than 10 years experience in role (159/274), suggesting that the results represent the views of experienced physicians. This study had some key limitations. Very high levels of agreement were achieved across the statements, this which may suggest that the statements were constructed as to achieve agreement (confirmation bias), or that perhaps they represent established good practice, a possible improvement to the process might be to provide survey responders (or a subset) with the opportunity to add or amend the statements prior to the full survey. It must also be acknowledged that associations with pharmaceutical companies may have led to unconscious bias the development of the consensus statements, but the study was designed to minimise the impact of this bias on results: the identity of steering group members was not disclosed to survey responders, and the identity of responders was not known to either the facilitator or the steering group. The lack of representation on the steering group (and subsequently the wider panel) of members from low-income and middle-income countries (LMICs) was a significant limitation. While the survey covered 11 countries of varying economic status, the range was from lower-middle income to high, a clear gap exists, namely those countries classified as ‘low income’ (eg, sub-Saharan Africa). Consequently, the findings of this study may not be generalisable to LMICs. In order to make such recommendations, a further Delphi process would be required specifically to engage with this demographic, this would provide insight into the specific challenges and considerations and allow for comparison with the current dataset. Aspects of patient choice and empowerment and consideration of the patient experience (outside of treatment outcomes) have not been discussed, this can be considered a limitation as the patient perspective may have significant bearing on the practicability and implementation of CKD management. Recommendations Early screening for CKD in high-risk groups is cost-effective for the health system (where resources are in place to support intervention). GFR (estimated by the CKD-EPI Creatinine Equation) and uACR (using albumin-to creatinine ratio) should be the screening method of choice for CKD. The CKD MDT should include the primary care physician to improve early intervention and decision-making. Intervene early in patients with CKD with proven therapies (ie, SGLT2i, RAASi, DMTs) to delay/prevent progression to kidney failure. RAASi therapies should be optimised in all patients with CKD and HF. Patients with CKD should have their HK managed appropriately when serum potassium level rises above >5.0 mmol/L. Chronic HK should be treated with a potassium binder (eg, patorimer, sodium zirconium cyclosilicate) to allow for the maintenance of DMTs. Guidelines should be practical with an executive summary/checklist to assist implementation by non-specialist HCPs. Guidelines should include clear criteria for when and how to refer to other specialists/MDT. Nephrologists should work together to deliver consistent and clear education regarding CKD management. Clinicians, professional associations, academic institutions and patient representative organisations need to engage with policy makers to ensure appropriate plans and funding are in place to deliver optimal CKD care. National kidney health programmes should be implemented to drive improvements to screening and diagnosis. Very high agreement (>90%) was observed for all statements in this topic, underscoring the key principle that early diagnosis of CKD is key to implementing strategies to slow disease progression. Responses suggest a strong support for the need for national kidney health screening and diagnostic programmes. Universal screening of the general population has been found not to be cost-effective, but systematic review has shown screening to be cost-effective in patients with hypertension or diabetes. Indeed, a KDIGO Controversies Conference concluded that targeted groups, such as those with hypertension, diabetes or CVD should be screened for CKD, and that an individualised approach should be taken to screen others based on a range of factors. Respondents agree that patients with risk factors should be screened for CKD at least annually by using eGFR and uACR where available, but identification of CKD is also challenging where awareness among healthcare staff and health literacy in the general population is poor, it is, therefore, recommended that national initiatives to improve these issues should support any screening programme. A minimal-resource prescreening tool has been developed and globally validated for CKD in people with T2DM. This demonstrates that age, gender, body mass index, duration of diabetes and blood pressure information can be used to identify those at an increased risk of CKD. This model does not require sophisticated diagnostics and can be used to guide cost-effective screening for CKD where resources are limited. All statements in this topic achieved >90% consensus apart from statement 14 (85%). This is interesting and could be due to the wording of the statement and the specific use of COVID-19 as an example. Reference to COVID-19 was included in the statement as it was considered relevant by the steering group at the time of statement generation and evidence was beginning to emerge of potential kidney injury associated with COVID-19. Analysis by country found that Taiwan agreement was at 68% for this statement, noticeably lower than for other countries. Although 93% of respondents agree that acute kidney injury (AKI) is a risk factor for CKD and HF, it is interesting that 7% do not agree given the evidence base. Given the established interplay between the cardiac, renal and endocrine systems, it is heartening to see that 99% of respondents agree that a holistic approach is needed to provide personalised care for individuals with CKD. Where practical, models of care should be developed to deliver integrated multidisciplinary care to patients with CRM disease, as described by the CardioMetabolic Care Alliance, to use comprehensive, patient-centred, team-based approaches for aggressive secondary prevention. Implementing combined clinics to deliver medical care for patients with kidney disease and diabetes or CVD may reduce outpatient healthcare costs without compromising health outcomes. Combined multidisciplinary clinics for diabetes, CKD and CVD are also associated with a slower decline in GFR than usual care, and a significant reduction in the risk for all-cause mortality. However, global variation exists regarding resource availability and access to specialist care, and the use of multidisciplinary teams (MDTs) is not universal. In these regions, HCPs are encouraged to develop local contacts/networks to allow for interdisciplinary discussions of CKD cases where needed. The broad objective of this consensus is to promote the earlier detection and management of CKD, and this principle applies regardless of resource limitations. As CKD-associated healthcare costs increase with disease progression, the economic argument supports this approach, and in patients with limited access to specialist care (including DMT), the role of patient education is paramount in improving health literacy and promoting lifestyle changes to reduce CKD risk. Coupled with this, the health system should look at how interventions such as DMTs can be used to slow progression of CKD and the need for dialysis (and associated costs), cost savings from slower progression to dialysis dependence might be reinvested in detection and diagnosis/national screening of high-risk patient groups. Very high agreement was observed for 10 of the statements in this topic, with 2 statements at lower levels of agreement (S23 and S25, 82% and 75%, respectively). Response to statement 23 is interesting, given the current debate regarding the benefits of stopping RAASi in advanced CKD. Results from a 52-week open-label UK study in patients with CKD stages 4–5 concluded ‘The STOP-ACEi trial did not find any benefit by stopping RAASi in advanced CKD’, and that ‘stopping RAASi in advanced CKD at an arbitrary GFR threshold is not the optimal approach’. Large observational studies have confirmed the cardioprotective benefits of RAASi in advanced CKD (including patients with concomitant T2DM). Linked to this is statement 21, respondents agree that a small decrease in eGFR is to be expected on initiation of DMT for CKD. During initiation and uptitration of RAASi treatment, a decline in kidney function of up to 30% within 4 weeks can be acceptable and it is important to avoid a knee-jerk response and reduce or stop DMT, and consultation with a nephrologist or MDT is recommended. Hyperkalaemia (HK) is potentially life-threatening; it may be acute or chronic and individuals with CKD are at an increased risk, which increases with the later stages of CKD. HK treatment is often stratified by serum potassium level, with borderline levels warranting modification of dietary potassium intake, followed by pharmaceutical management for larger or more sustained increases. Evidence to support recommending low potassium diets to patients with advanced CKD or ESRD is weak. Observational studies report weak associations between dietary potassium intake and potassium concentration and this approach may deprive patients of the beneficial cardiovascular effects associated with potassium-rich diets. When HK occurs, RAASi dose reduction or discontinuation are the most common used therapeutic options, but this approach is associated with worse cardiorenal outcomes and increased mortality. Once stopped, RAASi treatment is rarely reinitiated. In cases of mild-to-moderate HK, DMT should be maintained where possible, and an option for achieving this is using a potassium-lowering therapy such as patiromer or sodium zirconium cyclosilicate (S24, 91%). While the threshold for intervention in HK may vary by country and even individual physician, we can conclude that action may be considered when K + concentration reaches 5.0 mmol/L (S25, 75%) and certainly when at 5.5 mmol/L (S26, 93%) but with a degree of personalisation (in consultation with a nephrologist). Statements 25 and 26 were included to try and understand where the weight of opinion fell regarding interventions to manage HK, both statements achieved consensus, but the strongest agreement was for statement 26. On reflection, these statements could have been worded better to understand what would constitute appropriate action in these circumstances. Respondents very strongly agree that SGLT2i and RAASi therapies have a complementary cardiorenal protective action (S18, 98%) and that in patients with T2DM and HF, early SGLT2i use can prevent the development and progression of CKD (S19, 96%), in fact, SGLT2i may slow progression of CKD in patients without T2DM (S20, 91%). It is, therefore, recommended that SGLT2i and RAASi therapies are initiated as early as possible to both delay CKD progression and to therapeutically address the full CRM continuum—including T2DM and HF. The interplay between CKD, diabetes and CVD has been discussed, but CKD is associated with a number of comorbidities. A prospective UK cohort study of 1741 people with CKD stage 3 found that isolated CKD (without comorbidities) was present in only 4% of patients, and that common comorbidities include ‘painful condition’ (30%), anaemia (24%), thyroid disorders (12%), cerebrovascular disease (12%) and respiratory conditions (10%). These comorbidities should be identified and managed as part of a multidisciplinary approach. While all statements in this topic achieved consensus threshold, the steering group were surprised that 23% of respondents did not agree that implementation of CKD guidelines is suboptimal. The lowest agreement levels were observed in South Korea (68%) and Guatemala (72%). This could be either genuine strength in implementation of guidelines in these countries (which should be investigated and replicated elsewhere), or the need for continuing professional development even among experienced physicians. Due to the vital role of primary care must play in identifying and referring suspected patients with CKD, respondents agree that clear guidelines for referral of patients are considered essential (S32, 98%). PCPs should be provided with clear criteria for referral and followed through with continuing education from an appropriate specialist (ideally a nephrologist). The focus on prevention can only be achieved through a whole-system approach with appropriate investment in awareness, screening and diagnosis programmes (S35, 96%). Very high levels of agreement suggest strong support for multidisciplinary involvement in CKD management, and that PCPs should form part of the MDT. Given the prominent status of CKD as an emerging global health threat, there is a need to ensure that plans and funding are in place to enable optimal care. Stakeholders should engage with both local and national policy-makers to ensure that CKD is appropriately prioritised. Most responders agree on the need for up-to-date education for all HCP roles involved in CKD management, and that PCPs require structured education. Education of patients is a low-cost intervention, to encourage kidney healthy lifestyles and improve awareness of CKD among high-risk populations. 274 responses received with good representation from experienced physicians across 11 different countries is a strong basis for consensus, the study was designed to ensure that each country was represented by an equal number of responders. The majority of responders reported more than 10 years experience in role (159/274), suggesting that the results represent the views of experienced physicians. This study had some key limitations. Very high levels of agreement were achieved across the statements, this which may suggest that the statements were constructed as to achieve agreement (confirmation bias), or that perhaps they represent established good practice, a possible improvement to the process might be to provide survey responders (or a subset) with the opportunity to add or amend the statements prior to the full survey. It must also be acknowledged that associations with pharmaceutical companies may have led to unconscious bias the development of the consensus statements, but the study was designed to minimise the impact of this bias on results: the identity of steering group members was not disclosed to survey responders, and the identity of responders was not known to either the facilitator or the steering group. The lack of representation on the steering group (and subsequently the wider panel) of members from low-income and middle-income countries (LMICs) was a significant limitation. While the survey covered 11 countries of varying economic status, the range was from lower-middle income to high, a clear gap exists, namely those countries classified as ‘low income’ (eg, sub-Saharan Africa). Consequently, the findings of this study may not be generalisable to LMICs. In order to make such recommendations, a further Delphi process would be required specifically to engage with this demographic, this would provide insight into the specific challenges and considerations and allow for comparison with the current dataset. Aspects of patient choice and empowerment and consideration of the patient experience (outside of treatment outcomes) have not been discussed, this can be considered a limitation as the patient perspective may have significant bearing on the practicability and implementation of CKD management. Early screening for CKD in high-risk groups is cost-effective for the health system (where resources are in place to support intervention). GFR (estimated by the CKD-EPI Creatinine Equation) and uACR (using albumin-to creatinine ratio) should be the screening method of choice for CKD. The CKD MDT should include the primary care physician to improve early intervention and decision-making. Intervene early in patients with CKD with proven therapies (ie, SGLT2i, RAASi, DMTs) to delay/prevent progression to kidney failure. RAASi therapies should be optimised in all patients with CKD and HF. Patients with CKD should have their HK managed appropriately when serum potassium level rises above >5.0 mmol/L. Chronic HK should be treated with a potassium binder (eg, patorimer, sodium zirconium cyclosilicate) to allow for the maintenance of DMTs. Guidelines should be practical with an executive summary/checklist to assist implementation by non-specialist HCPs. Guidelines should include clear criteria for when and how to refer to other specialists/MDT. Nephrologists should work together to deliver consistent and clear education regarding CKD management. Clinicians, professional associations, academic institutions and patient representative organisations need to engage with policy makers to ensure appropriate plans and funding are in place to deliver optimal CKD care. National kidney health programmes should be implemented to drive improvements to screening and diagnosis. The steering group was able to form a set of recommendations specific to the 11 participant countries ranging from lower-middle income to high income and relevant to other countries with a similar demographic. Implementation of these recommendations has the potential to improve detection of CKD at an earlier stage in patients with risk factors. Earlier diagnosis provides the opportunity for early intervention with DMTs that can slow or halt the progression of CKD, in addition to reducing associated morbidity and mortality. Reviewer comments Author's manuscript |
Endometrial Carcinosarcomas are Almost Exclusively of p53abn Molecular Subtype After Exclusion of Mimics | 2940ac82-9edd-4dc3-94a0-50a4e6a5800a | 11771344 | Anatomy[mh] | Case Selection All cases diagnosed as carcinosarcoma based on histopathological examination of a hysterectomy specimen in a national cohort of endometrial carcinomas diagnosed/treated in a single calendar year (2016) from the institutional archives of 29 participating Canadian centers , were identified for this study. Clinicopathologic and outcomes data were collected as described previously , . A second set of cases consisted of all carcinosarcomas diagnosed after 2016 at the authors’ institution that were reported as being of NSMP molecular subtype. This study has been approved by the University of British Columbia institutional research ethics board. Histomorphologic Classification and Review The diagnosis of carcinosarcoma was based on the final diagnosis recorded in the pathology reports , and all these cases were subjected to histopathological review by 2 of the authors (J.H. and C.B.G.). One slide chosen by the participating site as being representative of the tumor was available for review, with all slides reviewed in a subset of cases. For the NSMP carcinosarcoma cases diagnosed after 2016 at the authors’ institutions, all slides were available for review. Molecular Subtype Diagnosis Proactive Molecular Risk Classifier for Endometrial Cancer associated immunomarkers (p53, MMR proteins) and POLE mutation testing by targeted next-generation sequencing was done as previously described , . POLE mut assignment was based on a list of 11 agreed-upon pathogenic mutations . Other genes assessed on the next-generation sequencing panel were as described previously and included TP53 . Cases with more than 1 molecular feature were classified in accordance with the segregation order and rationale described by León-Castillo et al . All cases diagnosed as carcinosarcoma based on histopathological examination of a hysterectomy specimen in a national cohort of endometrial carcinomas diagnosed/treated in a single calendar year (2016) from the institutional archives of 29 participating Canadian centers , were identified for this study. Clinicopathologic and outcomes data were collected as described previously , . A second set of cases consisted of all carcinosarcomas diagnosed after 2016 at the authors’ institution that were reported as being of NSMP molecular subtype. This study has been approved by the University of British Columbia institutional research ethics board. The diagnosis of carcinosarcoma was based on the final diagnosis recorded in the pathology reports , and all these cases were subjected to histopathological review by 2 of the authors (J.H. and C.B.G.). One slide chosen by the participating site as being representative of the tumor was available for review, with all slides reviewed in a subset of cases. For the NSMP carcinosarcoma cases diagnosed after 2016 at the authors’ institutions, all slides were available for review. Proactive Molecular Risk Classifier for Endometrial Cancer associated immunomarkers (p53, MMR proteins) and POLE mutation testing by targeted next-generation sequencing was done as previously described , . POLE mut assignment was based on a list of 11 agreed-upon pathogenic mutations . Other genes assessed on the next-generation sequencing panel were as described previously and included TP53 . Cases with more than 1 molecular feature were classified in accordance with the segregation order and rationale described by León-Castillo et al . There were 41 carcinosarcomas among the 973 hysterectomy specimens (4.2%) in the cohort of cases from 2016; 37 (91.2%) were p53abn, 2 (4.9%) were NSMP, and 1 each (2.4%) were POLE mut and MMRd, respectively. All 37 p53abn carcinosarcomas (as diagnosed based on the presence of mutant pattern p53 immunostaining together with wild-type POLE and expression of MMR proteins, that is, MMR proficient) had a TP53 mutation confirmed by sequencing. Of the 4 non-p53abn tumors (Fig. ), on review both NSMP tumors were corded and hyalinized (CHEC) pattern endometrioid EC (EEC). The MMRd tumor was a grade 1 EEC with reactive stromal proliferation, and the POLE mut tumor was grade 3 EEC with spindle cell growth, that is, none were confirmed to be carcinosarcoma. Eleven additional cases among the 37 p53abn tumors were not confirmed to be carcinosarcoma on review; 3 undifferentiated or dedifferentiated morphology, 5 endometrioid carcinomas with CHEC features, 2 carcinomas with prominent reactive spindle cell stroma rather than a sarcomatous component, and 1 adenosarcoma (Fig. – representative images). In the review of institutional cases diagnosed as NSMP carcinosarcoma after 2016, 3 cases were identified. On pathology review, one was reclassified as an adenosarcoma, and 2 as mesonephric-like adenocarcinoma with spindled cells grown and without heterologous elements. (Fig. – mesonephric-like cases). We show that all pathologically confirmed endometrial carcinosarcomas in a multicenter study are p53abn molecular subtypes. In the literature, the distribution of molecular subtypes of carcinosarcomas is more variable; p53abn account for 50% to 91%, MMRd for 2.4% to 26.1%, NSMP for 3% to 19%, and the POLE mut from 0% to 10.9% – , (summarized in Table ). There are a number of possible explanations for this variability. One significant problem identified in this study, however, was tumors originally diagnosed as carcinosarcoma that, based on diagnostic criteria and practice in 2023, would be diagnosed as something different. We identified diagnostic difficulties with known mimics of carcinosarcoma, such as CHEC pattern endometrioid carcinoma , , dedifferentiated/undifferentiated carcinoma , , and mesonephric-like carcinoma with spindle cell growth , , . All of these are recently described entities, and although they are included in the fifth edition of the WHO Classification of Female Genital Tumors , awareness of the range of appearances and diagnostic criteria for the distinction of these tumor types from carcinosarcoma remains work in progress. The subsequent institutional follow-up series of cases seen after 2016 identified 3 NSMP carcinosarcomas, of which 2 on review were mesonephric-like adenocarcinomas. These more recent cases add further evidence that carcinosarcomas of molecular subtypes other than p53abn are rare. Thus, although significant numbers of carcinosarcomas have been reported to be molecular subtypes other than p53abn (Table ), we believe that our findings demonstrate that almost all carcinosarcomas, as diagnosed based on current diagnostic criteria , are p53abn. This has significance in practice, as carcinosarcoma is an “aggressive histologic type” according to the FIGO 2023 staging of endometrial cancer, and p53abn is an aggressive molecular subtype ; as such, a diagnosis of either carcinosarcoma or p53abn can result in a change of tumor stage (upstaging). Based on our results, though, the molecular subtype of carcinosarcomas will consistently be p53abn, and situations where the stage of a given tumor will differ based on histotype (carcinosarcoma) versus molecular subtype will be rare. It is well established that most MMRd and POLE mut endometrial carcinomas are of endometrioid histotype, although POLE mut endometrial carcinomas have been known to be difficult to histotype reproducibly due to significant intratumoral heterogeneity of the tumor morphology , . POLE mut carcinosarcomas have been described in the literature, but all reports were published before the definition of pathogenic POLE mutations in early 2020 by Leon-Castillo et al . In a recent POLE mut individual patient meta-analysis of the literature, restricted to only those tumors with pathogenic POLE mutations, there were no carcinosarcomas among the 297 cases . Our carcinosarcoma cohort included 1 MMRd and 1 POLE mut tumor, of which the former showed reactive stromal proliferation and the latter spindled cell growth, and neither was confirmed to be a carcinosarcoma on pathologic review. A carcinosarcoma that is MMRd or POLE mut would create challenges in treatment planning (Should treatment be based on histotype or molecular subtype?). Gotoh et al showed that POLE mut and MSI-high carcinosarcomas had more favorable outcomes compared with NSMP carcinosarcomas, which had a similar prognosis to p53abn . Similar findings were also reported in a subsequent meta-analysis , but the diagnoses of both carcinosarcoma and molecular subtypes are not consistently based on current diagnostic criteria for these previous studies. In undertaking this study, 1 question we wished to address was whether NSMP carcinosarcoma existed, and if so, was the prognosis similar to or better than that of the more common p53abn carcinosarcoma. We identified no such cases, allowing us to conclude that NSMP carcinosarcoma will be encountered infrequently in practice and making studies of NSMP carcinosarcoma technically challenging or impossible because of their rarity. If such a case were encountered, it would be classified as an aggressive endometrial carcinoma histotype according to FIGO 2023 and treated as such according to the European Society of Gynaecological Oncology-The European SocieTy for Radiotherapy & Oncology- The European Society of Pathology and European Society of Medical Oncology guidelines, with adjuvant chemotherapy +/- radiation recommended in most cases; the absence of data notwithstanding, this approach seems reasonable. An unexpected finding in this study was the number of cases from the 2016 cohort where the diagnosis of carcinosarcoma was changed on pathology review. Although the focus of this study was not on diagnostic criteria and differential diagnosis, several observations can be made based on our reviews. In the past, it was possible to diagnose as carcinosarcoma any high-grade carcinoma with biphasic growth that included areas of spindle cell growth or loss of overt epithelial differentiation. There is now an emphasis on the presence of bona fide sarcomatous growth, with the cytologically high-grade epithelial and sarcomatous components “sharply juxtaposed,” that is, an abrupt transition between the components , a significant refinement of the diagnostic criteria. CHEC pattern endometrioid carcinomas can be high-grade and often have osteoid or chondroid matrix but should lack either overt sarcomatous differentiation or this abrupt transition. Dedifferentiated carcinoma is only recently characterized, and many or most such tumors would have been diagnosed as carcinosarcoma in the past based on their biphasic growth, but the undifferentiated component consists of more uniform cells without the pleomorphism and spindle cell morphology of the sarcomatous component of carcinosarcoma . Reactive spindle cell stroma has perhaps been underappreciated as a mimic of carcinosarcoma. As almost all carcinosarcomas show mutant pattern p53 staining, the absence of such staining in the spindle cell area, as well as the more uniform nuclear features of the spindle cells compared with true sarcomatous growth, can be used to support a diagnosis of reactive stroma rather than carcinosarcoma. Does the distinction between these other diagnostic entities and carcinosarcoma matter in a tumor that is p53abn, that is, is it clinically relevant? The larger question becomes whether there are meaningful subcategories within p53abn endometrial carcinoma that can be recognized based on histopathological features. In 1 meta-analysis, p53abn carcinosarcomas had an adverse progression-free survival compared with p53abn endometrioid and serous carcinomas ; however, in recent studies, no difference in progression-free or overall survival was found between serous carcinoma, endometrioid carcinoma and carcinosarcoma of p53abn molecular subtype , providing evidence of histotype being prognostically irrelevant in the context of p53abn molecular subtype, when the latter is known. Although there are opportunities for future studies of p53abn endometrial carcinoma to determine whether morphologic variants such as endometrioid carcinoma with CHEC pattern are of prognostic significance within this high-risk molecular subtype, it will be important that such studies address not only prognosis but provide reliable diagnostic criteria that allow for reproducible diagnosis; histotype diagnoses for high-grade endometrial carcinomas have historically suffered from suboptimal reproducibility , . It is telling that the ESGO/ESTRO/ECP, European Society of Medical Oncology, and FIGO 2023 risk assessment guidelines/staging guideline base risk category (and thus treatment) primarily on molecular subtype rather than histotype when molecular subtype is known , , . CHEC pattern endometrioid carcinoma is often low-grade and associated with loss of MMR proteins or of NSMP subtype, although high-grade CHEC pattern tumors associated with p53 abnormalities have been described , . CHEC pattern is rare and if associated with low-grade carcinoma and in the absence of mutant pattern, p53 immunostaining is associated with a favorable prognosis , , so separation from carcinosarcoma is important in that scenario. If, however, the carcinoma is high-grade and/or there is a mutant pattern p53 immunostaining in a CHEC pattern endometrioid carcinoma, such a distinction may not be clinically relevant, as noted above. Dedifferentiated/undifferentiated carcinoma is a very aggressive histotype of endometrial carcinoma, but the molecular abnormalities most commonly are mutations in genes encoding proteins in the SWI/SNF chromatin remodeling complex and frequent MLH1 promoter methylation, with wild-type TP53 . The underlying molecular pathology of dedifferentiated/undifferentiated carcinoma does not impact treatment at this time, but there are research efforts directed at developing more targeted therapies against cells with mutations in the SWI/SNF chromatin remodeling genes, and correct identification of these tumors may determine treatment in the future. Mesonephric-like carcinomas are mostly of NSMP molecular subtype , ; the diagnostic histopathological features and molecular pathology are being elucidated, but this is an important subset of endometrial carcinoma even though uncommon, as it is typically architecturally low-grade but is associated with aggressive behavior. In a recent study, Mirkovic et al reported observations on mesonephric-like carcinosarcomas and recommended that a diagnosis of mesonephric-like carcinosarcoma should be reserved for neoplasms with heterologous mesenchymal elements as mesonephric-like carcinomas often have spindle cell elements. As with the dedifferentiated/undifferentiated carcinomas, the diagnosis of mesonephric-like adenocarcinoma/carcinosarcoma does not result in specific treatment differences at this time, but accurate diagnosis of this histotype is an important starting point in the process of developing more personalized treatments based on the molecular pathology. Disagreement in the past about the classification of carcinosarcomas, that is, sarcoma versus carcinoma, has resulted in it being significantly understudied, and carcinosarcomas were routinely excluded from study cohorts, including clinical trials. A recent endometrial carcinosarcoma consensus statement has put a call to action to design ad hoc endometrial carcinosarcoma-oriented studies to develop new practice changing targeted therapies and to generate data to provide specific guidelines for the management of endometrial carcinosarcoma . Accurate diagnosis of carcinosarcoma to generate this data will be crucial. Our study identified several diagnostic difficulties with known mimics of carcinosarcoma in modern pathology practice, as the diagnosis of “carcinosarcoma” has evolved from a purely descriptive diagnosis to being based on specific criteria (a trend that has happened with a number of gynecologic cancers, such as clear cell carcinoma or small cell carcinoma, where the name reflects a prominent histologic feature that led to them being identified as a distinct tumor type, but the current diagnostic criteria incorporate other features, including immunohistochemical or other molecular pathology markers, and are not based solely on the notable feature after which they are named). More than a third of the carcinosarcomas diagnosed in 2016 were reclassified as other histotypes when following the 2020 WHO fifth edition diagnostic criteria. We recommend that all non-p53abn carcinosarcomas undergo expert gynecopathologic review before being enrolled in a clinical trial, and that special attention be paid to mimics of carcinosarcoma such as dedifferentiated/undifferentiated carcinoma, CHEC pattern endometrioid carcinoma, mesonephric-like carcinosarcoma/carcinosarcoma, and carcinoma with reactive spindle cell stroma. |
Not too sick, not too well: reducing the diagnostic void in pediatric emergency medicine | 77af2e30-fd88-4c50-9dbd-3ff2dae1602a | 11624129 | Pediatrics[mh] | There is a considerable emphasis in pediatric emergency medicine on the task of recognising the unwell child. This focus tends to be based on decision making around individual patients and fails to acknowledge that emergency physicians are usually responsible for dealing with multiple patients at any one time. As such, the rapid recognition of the child with no emergent medical condition – the well child – is important in terms of mitigating iatrogenia, utilization of resources, and surge capacity for acutely ill and injured children. An additional benefit to the rapid discharge of children with non-emergent conditions is to the treating clinical team, in terms of offloading cognitive burden and workflow bottlenecks. We explore models for recognising wellness in order to improve the ability of clinicians to focus on the significantly unwell children in their care. Over 30 million emergency department visits are made by children less than 18 years old in the United States yearly. Over 96% of children are treated and sent home. In pre-pandemic 2018, there were 100 visits per 100 persons aged 1 year and younger and 58 visits per 100 persons aged 1–4 years. During the same year, an estimated 34% of primary care visits made by children under 1 were for a “new problem”. Due to an array of systemic, demographic, and financial reasons the emergency department is increasingly becoming a destination of first choice for care, independent of acuity. , Traditionally the emphasis in emergency medicine has been the recognition of the seriously unwell child with a secondary emphasis on recognising frank wellness. Attempts to give structure to the assessment of unwell children typically result in poor sensitivity and specificity. For example one UK primary care based study found that only 6% of children assessed using the traffic light system of signs and symptoms were categorised as “green”, that is, not requiring urgent intervention. This poor specificity likely results from the normal physiology of unwell younger children who have a tendency to extreme signs and symptoms during uncomplicated lower risk infections. The speedy recognition of children who are not requiring admission has clear benefits including improving the ability of emergency departments to reduce crowding and therefore focus more clinical time to those remaining. This in turn may improve the ability of teams to recognise and treat the seriously unwell children in their care. Large patient volumes may saturate or overwhelm the capacity of clinicians, auxiliary staff, and space. The stressed environment may impact the triage and initial management of children presenting to the emergency department. A particular challenge is the distinction of an emergency medical condition (i.e. requiring immediate or urgent attention) from a medical complaint or concern that may be addressed outside of the emergency department. A good example of this is in self-resolving minor illness or injury. Most previously healthy children with viral syndrome do not require medical evaluation or professional treatment. When a family brings a child with a simple viral syndrome to the emergency department, there is an inherent discordance involving parental fear, parental convenience, disease severity, expectations of the visit, and the clinician’s assessment. Ever-increasing emergency department utilization amplifies the effect of every decision made by emergency clinicians, affecting patients, clinicians, and the system. It is imperative we reduce the decision-making load on acute and emergency clinicians. The standard understanding is that the layperson determines the emergency. That may be true in terms of the decision to go to the emergency department but often has little to do with the presence of an emergency medical condition. Data both before and after COVID highlight parental perception of illness is often far higher than the child’s actual illness. However passing judgement on parental decision making risks paternalism, if it can be challenging for clinicians to recognise serious illness why not so parents? And this creates a dichotomy, on the one hand, asking the family to go the ED purely for emergencies may risk the ill child staying at home and worsening. On the other hand, the current use of the ED as an entry point to the medical system is not sustainable and may put emergent patients at risk. Take for example young Anya (case study: Part A) Anya is 18 months old. She has had a cold for a couple of days. This morning her mother finds her to be pale, clingy, and not her usual self. She vomited once, and now refuses food. Anya is brought to the emergency department . She is seen on arrival and noted to be flushed and quiet. She is aware of her surroundings and reaches for objects. She has a raised respiratory rate (42 breaths per minute) and a high heart rate (165 beats per minute). There is no rash but her hands and feet feel cold. Her temperature is 40.1 degrees centigrade. She had spat out the acetaminophen (paracetamol) her mother had tried to give her in the morning . What now? Does Anya need to be prioritized, or is she safe to wait? Is Anya going to require investigations or admission? Given that febrile illness is the second most common presentation to Emergency Departments (after breathing difficulty) it is easy to see why this type of case poses a challenge. Fever in the young child can markedly affect their appearance, a cardinal feature in the initial assessment of children. Compounding the initial assessment are the parent or caregiver’s preconceived notions of the significance of fever. , On the one hand, clinicians aim to ally with parents to take their concerns seriously. On the other hand, the dissonance between perception of illness and safety risk of fever in children between parents and clinicians is often marked. , In the presented scenario, historically, protocols would flag this child is at risk of sepsis. The clinician may elect to observe or even undertake initial investigations. If parental concern was strong and the treating clinician felt that Anya’s overall appearance was not in keeping with a virus they may also elect to treat with antibiotics awaiting the results of various cultures. Regardless of the path chosen, the clinician wrestles with a feared retrospective criticism if the child were to return more unwell with a confirmed bacterial illness. Sepsis is a dynamic disease process. Clinicians see the individual patient at various stages of illness. A healthy child’s physiologic response to both benign viral syndrome and early bacteremia may be identical, especially early in the course. Viral infections often cause prime conditions for a subsequent bacterial superinfection, also with varying degrees of acuity. Progression to sepsis is rare and unpredictable; screening every child with invasive testing is not warranted. , Uncertainty, patient volumes, medicolegal risk, established practice patterns, and increasing reliance of the healthcare system on emergency medical services run in contradistinction to the above ideal practice pattern. The highly sensitive and non-specific systemic inflammatory response syndrome (SIRS) schema has been used with mixed results. SIRS became protocolized in many institutions, resulting in “SIRS alerts” and mandatory, albeit low-yield testing. Even in the high-risk population of children under three months of age, the presence of SIRS was not predictive of invasive bacterial illness. The Phoenix pediatric sepsis criteria were developed as a response to over-testing and over-utilization of inpatient resources. The paradigm has shifted from physiological response to assessment of end-organ risk (Table ). These definitions are far more specific than the previous screening criteria that were associated with a systemic inflammatory response syndrome (SIRS); the previous method of determining which patients needed intervention. The vast majority of children who present to Emergency Departments will not have sepsis. , Even at current vaccination rates, children over 3 months of age (presenting to a Children’s Emergency Department) display a rate of serious bacterial infection of less than 7%. The majority of bacterial infections follow an uncomplicated course with sepsis being a rare outcome. Finding the ‘sepsis’ needle in the ’emergency department’ haystack is a daily task. This is particularly true for the children aged between 6 months and 6 years old due to their tendency to have more exaggerated changes in physiological parameters when unwell with an uncomplicated infection. A previously healthy 2-year-old who initially appears miserable with volume depletion and tachycardia has reasonable potential to improve rapidly with hydration. Serial examinations during an observation period are important to track his recovery or to make the decision to intervene further. The need for sequential review in an overcrowded department creates a high-risk, decision-rich environment which is a significant cognitive and emotional load. There is no standardized definition of wellness available to health care professionals. To recognise the well child requires training, time, and thought. Parental descriptions and use of terminology concerning the child’s condition may not match that of the clinician. Age, duration of symptoms, and access to care outside of the emergency department all influence how quick an emergency physician may be to deem a child “well” (read: safe for discharge home). From a systems lens, emergency departments could not function if every child with normal vital signs and common, benign, self-limiting condition received an in-depth invasive investigation. From an interpersonal lens, there may be a dissonance between the wellness that the emergency physician sees and the miserable symptomatology the parent experiences. Common scenarios include presentations for fever, cough, rhinorrhea, vomiting, or refusal to eat. The effect of fever in particular on decision making is complex. Fever is a common feature of uncomplicated low risk illness and is non-specific. Due to a weak association between higher fever and serious bacterial infection many guidelines include a fever threshold as a discriminating feature in decision making. Unfortunately fever is too non-specific to have any clinical usefulness, regardless of its degree. In practice fever does influence the decision-making process due to the features commonly associated with a raised temperature: typically tachycardia and altered peripheral perfusion, alongside a reduced activity level and changes in behaviour. While fever itself is non-specific the overall appearance and behaviour of a child that is febrile at the time of assessment is often one that impairs the decision making of the clinician. For this reason well timed analgesia can be an extremely effective strategy in the emergency department, allowing a clinician to see a child who had a high fever an hour ago with all the associated features of sepsis yet now looks and behaves like a well child. The Phoenix score, with its focus on evidence of organ dysfunction is an important progression in our approach to recognising true sepsis. However to achieve this the Phoenix score requires extensive blood work. In pediatrics the ideal is to avoid the trauma of venepunture wherever possible. We therefore need a way of deciding when organ dysfunction is very unlikely. One model that works well for recognition of the well child is to focus on activity and behaviour. In paediatric practice these features give valuable clinical information about the current metabolic status and end organ function of the child being assessed. While non-specific features such as cough and fever support a process of differential diagnosis (upper or lower respiratory tract infection) the behaviour and actions of the child give important information about the clinical effects of the infection regardless of what the diagnosis is. A child with fever and a cough who is seen to be running around, eating, drinking, playing and chatting is demonstrating metabolic and cardiorespiratory sufficiency. Arguably this supersedes the distinction between upper and lower respiratory infection as a lower respiratory tract infection with minimal clinical effect can be managed conservatively in a similar way to upper respiratory tract infection. This model of predominantly using activity and behaviour for recognition of wellness essentially relies on the sensitive nature of higher cerebral function. The brain has a high metabolic requirement and function is much more quickly affected by any lack in perfusion, hypoxia or hypoglycemia than other vital organs. This is particularly true for higher cerebral function which is affected first while hindbrain function is preserved for longer when the brain is under stress. This physiological model explains why it is valid in clinical practice to allow an interactive smile to be an extremely valid examination finding. There are two relevant factors to an approach that focuses on spotting wellness. The first is that some ‘well appearing’ children need to be in an emergency department (think safeguarding or oncology) and the second is that rapid recognition of the well child may be challenging in children who are neurologically atypical at baseline. , It has been increasingly recognised that children with neurodivergence or neurodisability are at significant risk for poor outcomes when they become ill. Clinicians may misjudge wellness for an abnormality, and vice versa. For health care professionals assessing children who are not neurotypical, it is important to be aware of the specific difficulty and risk in the emergency department setting. A parent or carer who knows the child well and is able to verbally compare current state to the child’s baseline activity and behaviour can be an important substitute for a direct comparison of observation to a presumed normal. This requires trust by the health care professional of the caregiver’s judgement and experience. Decision making can be straightforward at the extremes of the well to unwell spectrum. Non-specific signs or symptoms may sway the clinician to investigate further. If after treatment and reassessment the child “proves” his wellness (i.e., normalized vital signs, improved appearance and activity), then intervention can be de-escalated. Conversely, concerning signs such as severe tachycardia, cold peripheries, poor responsiveness, and inactivity flag the child as unwell; he is at high-risk for decompensation even after initial partial improvement with resuscitative measures. What commonly creates a dilemma in paediatric emergency medicine is the child with a mixed picture, the “in-betweener”. Typically this is a child who is alert and active but tachycardic and symptomatic. As there is no single and universally accepted strategy for decision making in such children a variety of approaches exist. These include: Treat and re-evaluate Risk stratify with biomarkers Treat and admit This strategy is commonly used in emergency departments. The validity of this approach stems from the dilemma that, especially in young children, physiological response to uncomplicated illness is often extreme and indistinguishable from physiological compensation or decompensation due to sepsis. Given no high-risk medical comorbidities, the previously well child with a self-limiting viral illness may respond well to this strategy. In this approach, administration of an antipyretic or analgesic is being used more as an investigation based on the presumption that the physiological changes due to the illness will return to baseline while dysregulated parameters and signs of organ dysfunction will remain deranged. This approach works well when any abnormal parameters make a significant positive change and this correlates with a healthy activity level. The pitfalls of this approach are many including a lack of clear parameters for success. Does the heart rate need to return to normal or is a degree of normalisation sufficient? This is particularly problematic when the reassessment is handed over to a different person (for example following a change of shift) as this model of analgesia and review works best when the same person sees the effect of analgesia directly. The use of biomarkers (e.g. CRP or procalcitonin) in decision-making is often used in patients with some risk of decompensation or invasive illness, such as young infants. Their use in older, previously well children is debated. Given the range of presentations and medical complexity of patients who present to the ED, it is tempting to shed cognitive load and depend on an objective test. Applicability of the test depends on its performance, or accuracy. Accuracy is a function of prevalence of the disease as well as the sensitivity and specificity of the test itself. While biomarkers do have some correlation with the significance of infection there are no clear thresholds which allow for these tests to reliably rule out or rule in serious bacterial infection. Biomarkers also give no information about the clinical effect of an illness. If there were any test or formula that had good sensitivity and specificity it would be the gold standard approach. Since no such test exists biomarkers tend to be used in those cases who are neither well enough to immediately decide to discharge nor unwell enough for an immediate decision to treat. The very decision to use a biomarker is a critical intervention in itself and dependant on the experience of the clinician, the clinical context of the decision and the prevalence of disease. We acknowledge the role of biomarkers in validated algorithms, where the biomarkers allow determination of an action as a critical decision making node when a disease process has been identified. However, using biomarkers in a defensive fashion for a well, vaccinated child with likely viral source only causes harm to the patient, increases costs, and delays the care of other, sicker children. Therefore while biomarkers appear to have inherent face validity they are not always necessary. While little work has been done that could claim to compare the effectiveness of the different approaches there is interesting proxy evidence that suggests clinical decision making alone is not just valid but as safe and more efficient than adding biomarkers to the decision making process. The Petechiae in Children (PIC) study used local guidelines for children with fever and petechial rash and applied these guidelines to a large dataset of children with fever and non-blanching rash. The original paper demonstrated that all of the local guidelines used had better specificity than the current national guideline (which essentially used a blanket treat and decide approach) without losing sensitivity. All of the guidelines using biomarkers in decision making achieved a specificity of up to 36%. Examination of a different guideline which relied wholly on clinical decision making without the use of biomarkers improved specificity to 69% without the loss of any sensitivity. This highlights a need to do similar research comparing different approaches to decision making in all febrile children and not just those with non-blanching rash. Ultimately given the PIC study demonstrated petechiae are no longer a risk factor for sepsis in a population vaccinated against meningococcus, it is arguable that this publication has shown that clinical decision making without the use of biomarkers is both safe and perhaps more effective in some populations. This approach involves an early decision to admit the child with treatment ongoing. The benefit to the child would be to allow further treatment and testing and to assure a safe disposition from the emergency department. In an overburdened system, routing obviously ill children to other available clinicians may be a shrewd strategy. Early anchoring of the not-so-sick child (or one who could benefit from the above treat and reassess or risk-stratify strategies) may conversely lead to overtreatment and could overwhelm inpatient capacity. A default treat-and-admit approach is most valid in high-risk groups where the specificity will be greatest. For example, a well appearing, febrile, and tachycardic 3 day old is very high-risk for serious bacterial infection whereas a well appearing, febrile, and tachycardic 3-year-old is very low-risk. This is because there is an incidence of invasive bacterial illness in a well appearing 3 day old just on the basis of having a fever, whereas the well appearing 3 year old with a fever has a negligible (but not non-existent) risk of invasive bacterial illness. Both cases demonstrate an innate vigorous physiological reaction to fever. The 3-day-old, however, is at high-risk for decompensation and serious sequelae; the three-year-old has proven his robustness to recover. If high fever and vital sign abnormality were the deciding (read: anchoring) factor to admit regardless of emergency department course, harm may be done to those who would not benefit from admission. Traditionally the approach has been to sift out the sickest children through senior staff, triage and/or Early Warning Systems and then work through the remaining equivocal cases. Early Warning Systems have become synonymous with Early Warning Scores i.e. individual numerical scores based on physiology which correspond to various levels of escalation however Early Warning Systems are much broader strategies to recognise unwell children. They incorporate not only physiological measurements through the use of scores but require the use of subjective judgements of staff, and increasingly incorporate the views of the caregivers. Furthermore they are predicated on healthcare cultures which are not beholden to hierarchy and utilise communication processes aligned with human factors theory. A combination of effective decision-making approaches which increase the number of well children discharged early in the patient journey, allow prompt treatment of the most unwell and maximise focus on the group in the middle (the void) is needed. The delivery of this is complex given it requires multiple staff members and a patient group who may have evolving disease process. Figure brings together an early warning system decision tree approach to decision making in the Emergency Department. At its heart is the use of clinical expertise to determine the disposition of patients, but in a tiered approach, so that not all decisions need to be made by the most senior personal. Early Warning Scores, biomarkers and caregiver concern all have a role to play but are adjuncts within a decision tree which aims to remove the most well and unwell from the patient load and thereby reducing the cognitive strain by working in the void area. The application of the decision tree may result in the following conclusion to the case study Case Study Part B (Evaluating Risk) Anya has no high risk criteria (Box A). The initial clinical evaluation, performed by a junior doctor finds that Anya has an upper respiratory tract infection. Due to the abnormal physiological parameters (Box B) the junior consults a senior doctor who reviews Anya. The senior’s opinion is that Anya’s peripheral coldness, raised heart rate and respiratory rate are explained by a combination of fever and discomfort. They recommend a period of observation to re-evaluate after the analgesia has had an effect . A fundamental aim of pediatric care is to seek out sick children and prioritize them. This culture ensures the delivery of education, and of support, departments must audit and evaluate near-miss cases. This leads to a predisposition that children are presumed ill and that perhaps an overemphasis on that fact that every discharge home carries some degree of risk. Because acute care presentations often occur at the beginning of a disease process, clinicians vary in their comfort with uncertainty or risk tolerance. This experience is often difficult to teach and so communities of practice and other ongoing professional development strategies may best help clinicians understand the practice patterns of their peers. Opportunities to discuss, adjust or update practices are multiple, regardless of locale. The careful clinician can only make the best decision with the information and resources at the time of presentation. The risk of decompensation of a discharged patient may only removed by admitting all children, negating the value of a primary assessment service. For the risk to be acceptable we must avoid criticism of the decision to discharge where a valid clinical assessment and decision process has taken place. Instead it is key to accept that rarely a child who appears well will later become unwell. “Sick Children look Sick” wrote Green et al. nearly a decade ago. While the medico-legal implications of discharging a child for them to return more unwell are significant this occurrence does not mean the initial discharge decision was incorrect. In order to protect patients and staff the discharging clinician must provide excellent verbal and written safety netting advice. , By doing so we employ the parent or carer in an ongoing decision-making process that is as dynamic as the illnesses with which children present. The well-child visit to the emergency department may also be a teachable moment to engage parents in what clinical setting best matches common complaints (e.g., clinic, urgent care, or emergency department). Case Study part C (Resolution) Anya received acetaminophen per rectum and the emergency physician confirmed with her parents that she had no high-risk features. Shared decision-making included a short observation time and reassessment. Forty-five minutes later, Anya had defervesced; she was taking fluids and hungrily eating a cookie. Her repeat vital signs showed a heart rate of 135 beats per minute and a respiratory rate of 32 breaths per minute. The emergency physician was transparent in his logic to the parents and cautioned that although there is still a risk of bacterial infection now or in the near future, home monitoring was a safe first step. Careful precautionary advice was given. The family was discharged home, and the emergency physician was immediately called to the bedside of an apneic infant . The emergency care system is overloaded and above capacity. The mission of emergency departments to care for acutely ill and injured children is in conflict with the present-day clearinghouse phenomenon in which the spectrum of presentations is widely expanded to include many well children. In terms of preservation of mission, resources, and reducing harm, spotting the well child will become increasingly valuable. Clinician awareness of societal needs, patients’ access to healthcare, and a culture of satisfaction are substantial barriers to re-routing patients to the best venue for medical attention. A cultural shift is needed to enable prompt decision making regarding children at lowest risk of serious illness. In addition to institutional support, this will depend on adequately experienced clinicians to make decisions based on clinical judgement rather than biomarker output. |
Influence of High-Frequency Repetitive Transcranial Magnetic Stimulation on Neurobehavioral and Electrophysiology in Patients with Disorders of Consciousness | 80892084-bd77-4c1e-80cb-359552c07789 | 9699789 | Physiology[mh] | More and more patients with disorders of consciousness (DOC) are surviving from brain injury due to the ongoing improvements in intensive care and emergency medicine . As soon as these patients are medically stabilized, the attention of clinicians and families rapidly turns to planning for the needs to the recovery of consciousness . DOC is a highly challenging condition, and although multiple efforts have been made to facilitate recovery , only rare treatment schemes have been recommended by authoritative institutions . Currently, effective clinical protocols for managing patients with DOC are still lacking . In recent years, significant attention has been paid to repetitive transcranial magnetic stimulation (rTMS), a noninvasive and painless technique that has produced many inspiring beneficial results in the research of neurological diseases, such as stroke, Parkinson's disease, and Alzheimer's disease . Some published studies have also shown the potential therapeutic effects of rTMS in therapeutic interventions for DOC . Jang and Kwon reported that rTMS induced cognitive and neurophysiological modifications in one patient in a persistent vegetative state. Ge et al. showed that 10 Hz rTMS of the dorsolateral prefrontal cortex could improve the state of awareness of DOC patients in a nonrandomized controlled trial. However, other studies have failed to provide evidence of any obvious therapeutic effects of such treatment when compared with the control groups. Cincotta et al. assessed the effects of rTMS in 11 patients with DOC in a randomized sham-controlled study with a crossover design. In their study, significant differences were not observed in the JFK Coma Recovery Scale-Revised (CRS-R) and Clinical Global Impression Improvement (CGI-I) scale scores between the real or sham stimulation conditions. Naro et al. examined the feasibility of a single session of 10 Hz rTMS over the DLPFC in patients with DOC and did not find any clinical improvement or neural connectivity changes at the group level . On the other hand, there is still no consensus as to how best to measure the degree of consciousness impairment in noncommunicating patients and assess the modulation effects of the interventions on DOC . These results have significant ethical and practical implications for the caregivers and clinicians of DOC patients regarding outcome prognostication, medical care, rehabilitation services, and resource allocation . The current gold standard for assessing consciousness states used in previous studies is based on standardized clinical rating scales that are critically reliant on behavior observation . The results of such assessments are often confounded by underlying sensorimotor impairment and unrecognized cognitive and language deficits. Perhaps more important is the fact that patients' behavioral abilities may fluctuate across time, thus causing misdiagnosis . It is well known that consciousness is regulated by the activation of neural pathways. Connectivity is an important feature of neural pathways , and the disruption of pathway connectivity is related to the degree of consciousness breakdown , with a significant relationship to prognosis . Recent findings have suggested that the response to rTMS in DOC patients is mediated by the neural networks preserved after insult . With recent advances in computer instrumentation and signal processing over the past several years, the introduction of evoked potential (Ep) technologies has enabled the evaluation of the integrity of neural functional connectivity in a live human brain. EPs show increasing promise as powerful tools for assessing the severity of impairment and predicting the prognosis in patients with DOC , which are associated with a series of sensory events induced by the presence of specific sensory stimuli without being confounded by sedating medications and sleep . This process can be used to avoid misjudgments caused by sensorimotor, verbal, and cognitive deficits . More importantly, lengthy clinical practice has demonstrated that EPs can provide a reliable assessment of the connectivity of neural pathways . In particular, the N20 and P25 responses to median nerve stimulation by somatosensory-evoked potential (SEP) have been shown in many studies to be the predictor of the responsiveness prognosis in DOC patients . In other words, patients with a bilateral presence of the wave N20 and P25 responses to median nerve stimulation by SEP may be more likely to benefit from the treatment . Based on the principle of neural plasticity, rTMS can strengthen the connectivity of neural pathways through a long-term potentiation-like mechanism , stimulating arousability and functional integration within neural networks to facilitate the emergence of consciousness . Although numerous previous studies have suggested the potential role of neural pathways in behavioral modifications caused by HF-rTMS, whether HF-rTMS influences neural connectivity levels has not yet been directly investigated. Herein, we propose a new method for selecting patients according to their SEP before study enrollment, presenting results from a sham-controlled trial examining whether rTMS over the DLPFC affects neural connectivity levels while improving the level of consciousness in patients experiencing DOC. 2.1. Participants In our study, we included 50 patients with DOC who were consecutively admitted to the Department of Rehabilitation Medicine of The First Affiliated Hospital of Fujian Medical University from February 2020 to January 2022. The study was approved by the Ethics Committee of The First Affiliated Hospital of Fujian Medical University (approval number [2020]031). The entire study design and all procedures were performed in accordance with the Declaration of Helsinki. Written informed consent to participate in the study was obtained from the legal guardian of each patient, as patients were not deemed capable of giving consent. The http://chictr.org identifier is ChiCTR2000030419 ( http://www.chictr.org.cn/showproj.aspx?proj=50162 ). All patients enrolled in this study were 18–75 years of age, with an onset duration of 1–3 months, and met the diagnostic criteria for the vegetative state (VS) or minimally conscious state (MCS) when assessed with the CRS-R scale widely used to define levels of consciousness and monitor neurobehavioral recovery in patients . Brain lesions were confirmed, and communicating hydrocephalus was ruled out by magnetic resonance imaging or computerized tomography scans. The exclusion criteria were the unilateral or bilateral absence of N20; unstable vital signs; epileptic history or EEG epileptiform activity; implanted pacemakers and severe dysfunction of heart, liver, or kidney; previous neurological or psychiatric disorders; acute pneumonia and other extreme complications; craniotomy or metallic implantation on the right side of the head; and any other safety contraindications to TMS. The first SEP was administered to patients before being enrolled in the study to ensure the bilateral presence of the N20 and P25 components. Participants were randomly divided into an active group and a sham group using a random number table. All participants received a similar routine medication (amantadine, antiepileptic, anti-inflammatory, etc.) and a rehabilitation course (hyperbaric oxygen, passive exercises, electrical nerve stimulation, etc.) during the trial. On this basis, participants in the active group were treated with real rTMS, whereas those in the sham group were treated with sham stimulation . 2.2. Stimulation Protocol The rTMS was administered over five consecutive working days (from Monday to Friday) for six weeks. Stimulation intensity varied across this experiment was determined relative to the resting motor threshold (RMT) by stimulation of the M1 region corresponding to the right first dorsal interosseous (FDI) muscle representation (approximately position C3 of the 10/20 international electroencephalography system) and was recorded with an electromyogram amplifier module and surface electrodes. The patients were seated in a comfortable reclining chair and fitted with earplugs. The figure-8-shaped coil was placed at a tangent to the scalp, with the handle pointing backward and laterally at a 45° angle away from the midline. According to the International Federation of Clinical Neurophysiology Committee recommendations , the RMT intensity was defined as the minimum stimulus intensity that induces MEP greater than 50 μ V in at least five of 10 consecutive trials during muscle relaxation. The earplugs were inserted into the ears of patients, which continuously played a masking noise to prevent the interference of auditory potentials with TMS discharge during RMT measurement . The rTMS procedure consisted of a session of 1,000 pulses delivered in 10 trains of 10 Hz at an intensity of 90% RMT. Each train lasted 10 s with an intertrain pause of 60 s between each one. The coil was placed tangentially toward the scalp over the left DLPFC (position F3 of the 10/20 international electroencephalography system) for active stimulation. The junction region of the coil pointed backward and laterally at a 45°angle away from the midline . The placement of the coil is shown in . The sham rTMS was delivered using the same protocol except that the coil was held at an angle of 90° to the scalp . The protocol of stimulation was administered according to safety guidelines . The rTMS was performed by a physical therapist who was blinded to the assessments (NNZ). A registered nurse or physician was required to be present at every rTMS session, ensuring that if a seizure occurred during or after rTMS, the patient would be treated in time . The EEGs recorded biweekly were compared with the baseline EEG to identify possible patterns indicating an impending seizure. Structural MRIs were also completed postintervention; the MRIs were monitored by a neuroradiologist for changes from baseline, including hemorrhage and edema/toxic tissue. 2.3. Outcome Measures The assessments were performed pre- and postintervention in a quiet room, with patients lying on a comfortable bed. The complete clinical examinations were performed by a trained clinician (ZYW). The electrophysiological parameters were recorded using a NeMus 2 evoked potential system (EB Neuro S.p.A., Florence, Italy) by rehabilitation physicians (YJC and JMC). All the assessments were blinded to this experimental design. 2.3.1. Clinical Assessments The clinical assessments in this study included the CRS-R and Glasgow Coma Scale (GCS). CRS-R is a standardized tool consisting of 23 organized items divided into six subscales addressing auditory, visual, motor, verbal, communication, and arousal processes. The subscales are comprised of hierarchically arranged items associated with the brainstem, subcortical, and cortical processes. The score in each CRS-R subscale is determined according to the presence or absence of specific responses to a sensory stimulus, with a higher total score indicating a greater level of consciousness . GCS is a behavioral measure universally accepted as a gold standard for assessing the severity of a brain injury and level of consciousness in terms of a patient's ability to respond to stimuli; eye opening (maximum 4 points), best motor response (maximum 6 points), and verbal response (maximum 4 points) are all measured. Each level of response is assigned a number and added together to provide a total score between 3 and 15 ; the worse the response, the lower the number . Individual patients are best described by the three components of the Glasgow Coma Scale, whereas the derived total coma score can be used to characterize groups . 2.3.2. Electrophysiological Evaluations The SEP was recorded through Ag-AgCl surface electrodes that were placed over the bilateral supraclavicular fossae (Erb's point), spinous process of the sixth cervical vertebrae (Cv6), frontal pole (Fpz), and each somatosensory cortex contralateral to stimulation (C3′, C4′) according to the International 10–20 system. The SSEPs were recorded after median nerve stimulation of the wrist (duration: 0.2 ms; stimulus rate: 4.0 Hz). The impedance was kept below 3 k Ω , and SSEP was amplified with a bandpass of 20–1000 Hz. At least 300 responses were averaged into each waveform and obtained three times (a total of 900 responses). Next, the absolute latencies of N20 and the amplitudes of N20-P25 peak-peak (N20-P25 amplitudes) were measured. N20 was defined as the major negative peak with a latency of about 20 ms after stimulations, and P25 was defined as the major positive peak following the N20 . If the parameters were asymmetrical, the parameters on the more impaired side were recorded and used for analysis . Acoustic stimuli for BAEP were delivered through earphones. A masking white noise of 40 dB intensity was used on the contralateral side. Clicks of 100 us and 90–110 dB intensity was used at a rate of 10.7 Hz. At least three runs of 1,500 stimuli were averaged, and reproducibility was assessed by superimposing the traces. Recording electrodes were placed on bilateral mastoids (A1, A2), and the reference electrode was placed at the Fpz . The identification of waves for BAEP grading utilized Hall's classification as follows : grade 1, normal; grade 2, mild abnormality, moderate waveform differentiation with the following possible problems: prolonged I, III, or (and) V wave peak latency, prolonged interpeak latency of the I-III, III-V, or (and) I-V waves, peak-to-peak latency ratio of III − V/I − III > 1, and V/I wave amplitude ratio < 0.15; grade 3, moderate abnormality, poor waveform differentiation, and poor repeatability with the following possible problems: prolonged peak latency of III or V waves and the disappearance of V waves; and grade 4, severe abnormality, presence of I waves only, or disappearance of all waveforms. 2.4. Statistical Analysis Qualitative data were presented as numbers. The distribution of quantitative data was tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene's test. Normally distributed variables were presented as the mean (standard deviation) and nonnormally distributed variables as the median (interquartile range). The Mann–Whitney U test, the independent samples t -test, and the Chi-squared test were used for comparisons of data between the two groups with the baseline. Intragroup differences in pre- and postintervention were tested using a two-tailed unpaired Student's t -test and paired Wilcoxon rank-sum test. The Spearman rank correlation was used to test for a significant association between the total GCS and CRS-R scores, total scale scores, and electrophysiological parameters. The effects of the experimental intervention (changes) were calculated by subtracting the baseline data from the data obtained from postintervention (6 weeks) between the groups and were compared using the Mann–Whitney U test and the independent samples t -test. A p value of 0.05 or less was considered statistically significant. All statistics were performed using the SPSS software (version 23.0, IBM Corporation, Armonk, NY, USA). In our study, we included 50 patients with DOC who were consecutively admitted to the Department of Rehabilitation Medicine of The First Affiliated Hospital of Fujian Medical University from February 2020 to January 2022. The study was approved by the Ethics Committee of The First Affiliated Hospital of Fujian Medical University (approval number [2020]031). The entire study design and all procedures were performed in accordance with the Declaration of Helsinki. Written informed consent to participate in the study was obtained from the legal guardian of each patient, as patients were not deemed capable of giving consent. The http://chictr.org identifier is ChiCTR2000030419 ( http://www.chictr.org.cn/showproj.aspx?proj=50162 ). All patients enrolled in this study were 18–75 years of age, with an onset duration of 1–3 months, and met the diagnostic criteria for the vegetative state (VS) or minimally conscious state (MCS) when assessed with the CRS-R scale widely used to define levels of consciousness and monitor neurobehavioral recovery in patients . Brain lesions were confirmed, and communicating hydrocephalus was ruled out by magnetic resonance imaging or computerized tomography scans. The exclusion criteria were the unilateral or bilateral absence of N20; unstable vital signs; epileptic history or EEG epileptiform activity; implanted pacemakers and severe dysfunction of heart, liver, or kidney; previous neurological or psychiatric disorders; acute pneumonia and other extreme complications; craniotomy or metallic implantation on the right side of the head; and any other safety contraindications to TMS. The first SEP was administered to patients before being enrolled in the study to ensure the bilateral presence of the N20 and P25 components. Participants were randomly divided into an active group and a sham group using a random number table. All participants received a similar routine medication (amantadine, antiepileptic, anti-inflammatory, etc.) and a rehabilitation course (hyperbaric oxygen, passive exercises, electrical nerve stimulation, etc.) during the trial. On this basis, participants in the active group were treated with real rTMS, whereas those in the sham group were treated with sham stimulation . The rTMS was administered over five consecutive working days (from Monday to Friday) for six weeks. Stimulation intensity varied across this experiment was determined relative to the resting motor threshold (RMT) by stimulation of the M1 region corresponding to the right first dorsal interosseous (FDI) muscle representation (approximately position C3 of the 10/20 international electroencephalography system) and was recorded with an electromyogram amplifier module and surface electrodes. The patients were seated in a comfortable reclining chair and fitted with earplugs. The figure-8-shaped coil was placed at a tangent to the scalp, with the handle pointing backward and laterally at a 45° angle away from the midline. According to the International Federation of Clinical Neurophysiology Committee recommendations , the RMT intensity was defined as the minimum stimulus intensity that induces MEP greater than 50 μ V in at least five of 10 consecutive trials during muscle relaxation. The earplugs were inserted into the ears of patients, which continuously played a masking noise to prevent the interference of auditory potentials with TMS discharge during RMT measurement . The rTMS procedure consisted of a session of 1,000 pulses delivered in 10 trains of 10 Hz at an intensity of 90% RMT. Each train lasted 10 s with an intertrain pause of 60 s between each one. The coil was placed tangentially toward the scalp over the left DLPFC (position F3 of the 10/20 international electroencephalography system) for active stimulation. The junction region of the coil pointed backward and laterally at a 45°angle away from the midline . The placement of the coil is shown in . The sham rTMS was delivered using the same protocol except that the coil was held at an angle of 90° to the scalp . The protocol of stimulation was administered according to safety guidelines . The rTMS was performed by a physical therapist who was blinded to the assessments (NNZ). A registered nurse or physician was required to be present at every rTMS session, ensuring that if a seizure occurred during or after rTMS, the patient would be treated in time . The EEGs recorded biweekly were compared with the baseline EEG to identify possible patterns indicating an impending seizure. Structural MRIs were also completed postintervention; the MRIs were monitored by a neuroradiologist for changes from baseline, including hemorrhage and edema/toxic tissue. The assessments were performed pre- and postintervention in a quiet room, with patients lying on a comfortable bed. The complete clinical examinations were performed by a trained clinician (ZYW). The electrophysiological parameters were recorded using a NeMus 2 evoked potential system (EB Neuro S.p.A., Florence, Italy) by rehabilitation physicians (YJC and JMC). All the assessments were blinded to this experimental design. 2.3.1. Clinical Assessments The clinical assessments in this study included the CRS-R and Glasgow Coma Scale (GCS). CRS-R is a standardized tool consisting of 23 organized items divided into six subscales addressing auditory, visual, motor, verbal, communication, and arousal processes. The subscales are comprised of hierarchically arranged items associated with the brainstem, subcortical, and cortical processes. The score in each CRS-R subscale is determined according to the presence or absence of specific responses to a sensory stimulus, with a higher total score indicating a greater level of consciousness . GCS is a behavioral measure universally accepted as a gold standard for assessing the severity of a brain injury and level of consciousness in terms of a patient's ability to respond to stimuli; eye opening (maximum 4 points), best motor response (maximum 6 points), and verbal response (maximum 4 points) are all measured. Each level of response is assigned a number and added together to provide a total score between 3 and 15 ; the worse the response, the lower the number . Individual patients are best described by the three components of the Glasgow Coma Scale, whereas the derived total coma score can be used to characterize groups . 2.3.2. Electrophysiological Evaluations The SEP was recorded through Ag-AgCl surface electrodes that were placed over the bilateral supraclavicular fossae (Erb's point), spinous process of the sixth cervical vertebrae (Cv6), frontal pole (Fpz), and each somatosensory cortex contralateral to stimulation (C3′, C4′) according to the International 10–20 system. The SSEPs were recorded after median nerve stimulation of the wrist (duration: 0.2 ms; stimulus rate: 4.0 Hz). The impedance was kept below 3 k Ω , and SSEP was amplified with a bandpass of 20–1000 Hz. At least 300 responses were averaged into each waveform and obtained three times (a total of 900 responses). Next, the absolute latencies of N20 and the amplitudes of N20-P25 peak-peak (N20-P25 amplitudes) were measured. N20 was defined as the major negative peak with a latency of about 20 ms after stimulations, and P25 was defined as the major positive peak following the N20 . If the parameters were asymmetrical, the parameters on the more impaired side were recorded and used for analysis . Acoustic stimuli for BAEP were delivered through earphones. A masking white noise of 40 dB intensity was used on the contralateral side. Clicks of 100 us and 90–110 dB intensity was used at a rate of 10.7 Hz. At least three runs of 1,500 stimuli were averaged, and reproducibility was assessed by superimposing the traces. Recording electrodes were placed on bilateral mastoids (A1, A2), and the reference electrode was placed at the Fpz . The identification of waves for BAEP grading utilized Hall's classification as follows : grade 1, normal; grade 2, mild abnormality, moderate waveform differentiation with the following possible problems: prolonged I, III, or (and) V wave peak latency, prolonged interpeak latency of the I-III, III-V, or (and) I-V waves, peak-to-peak latency ratio of III − V/I − III > 1, and V/I wave amplitude ratio < 0.15; grade 3, moderate abnormality, poor waveform differentiation, and poor repeatability with the following possible problems: prolonged peak latency of III or V waves and the disappearance of V waves; and grade 4, severe abnormality, presence of I waves only, or disappearance of all waveforms. The clinical assessments in this study included the CRS-R and Glasgow Coma Scale (GCS). CRS-R is a standardized tool consisting of 23 organized items divided into six subscales addressing auditory, visual, motor, verbal, communication, and arousal processes. The subscales are comprised of hierarchically arranged items associated with the brainstem, subcortical, and cortical processes. The score in each CRS-R subscale is determined according to the presence or absence of specific responses to a sensory stimulus, with a higher total score indicating a greater level of consciousness . GCS is a behavioral measure universally accepted as a gold standard for assessing the severity of a brain injury and level of consciousness in terms of a patient's ability to respond to stimuli; eye opening (maximum 4 points), best motor response (maximum 6 points), and verbal response (maximum 4 points) are all measured. Each level of response is assigned a number and added together to provide a total score between 3 and 15 ; the worse the response, the lower the number . Individual patients are best described by the three components of the Glasgow Coma Scale, whereas the derived total coma score can be used to characterize groups . The SEP was recorded through Ag-AgCl surface electrodes that were placed over the bilateral supraclavicular fossae (Erb's point), spinous process of the sixth cervical vertebrae (Cv6), frontal pole (Fpz), and each somatosensory cortex contralateral to stimulation (C3′, C4′) according to the International 10–20 system. The SSEPs were recorded after median nerve stimulation of the wrist (duration: 0.2 ms; stimulus rate: 4.0 Hz). The impedance was kept below 3 k Ω , and SSEP was amplified with a bandpass of 20–1000 Hz. At least 300 responses were averaged into each waveform and obtained three times (a total of 900 responses). Next, the absolute latencies of N20 and the amplitudes of N20-P25 peak-peak (N20-P25 amplitudes) were measured. N20 was defined as the major negative peak with a latency of about 20 ms after stimulations, and P25 was defined as the major positive peak following the N20 . If the parameters were asymmetrical, the parameters on the more impaired side were recorded and used for analysis . Acoustic stimuli for BAEP were delivered through earphones. A masking white noise of 40 dB intensity was used on the contralateral side. Clicks of 100 us and 90–110 dB intensity was used at a rate of 10.7 Hz. At least three runs of 1,500 stimuli were averaged, and reproducibility was assessed by superimposing the traces. Recording electrodes were placed on bilateral mastoids (A1, A2), and the reference electrode was placed at the Fpz . The identification of waves for BAEP grading utilized Hall's classification as follows : grade 1, normal; grade 2, mild abnormality, moderate waveform differentiation with the following possible problems: prolonged I, III, or (and) V wave peak latency, prolonged interpeak latency of the I-III, III-V, or (and) I-V waves, peak-to-peak latency ratio of III − V/I − III > 1, and V/I wave amplitude ratio < 0.15; grade 3, moderate abnormality, poor waveform differentiation, and poor repeatability with the following possible problems: prolonged peak latency of III or V waves and the disappearance of V waves; and grade 4, severe abnormality, presence of I waves only, or disappearance of all waveforms. Qualitative data were presented as numbers. The distribution of quantitative data was tested for normality using the Shapiro–Wilk test and for homogeneity of variances using Levene's test. Normally distributed variables were presented as the mean (standard deviation) and nonnormally distributed variables as the median (interquartile range). The Mann–Whitney U test, the independent samples t -test, and the Chi-squared test were used for comparisons of data between the two groups with the baseline. Intragroup differences in pre- and postintervention were tested using a two-tailed unpaired Student's t -test and paired Wilcoxon rank-sum test. The Spearman rank correlation was used to test for a significant association between the total GCS and CRS-R scores, total scale scores, and electrophysiological parameters. The effects of the experimental intervention (changes) were calculated by subtracting the baseline data from the data obtained from postintervention (6 weeks) between the groups and were compared using the Mann–Whitney U test and the independent samples t -test. A p value of 0.05 or less was considered statistically significant. All statistics were performed using the SPSS software (version 23.0, IBM Corporation, Armonk, NY, USA). summarizes the demographic and clinical characteristics of all patients. Both groups were homogeneous for age, time since injury, etiology, total CRS-R, and GCS scale scores. Latencies of N20, N20-P25 amplitudes, and BAEP grade were also homogeneous at baseline for the active and sham groups (all p values > 0.05). All patients tolerated the study without complications, and no adverse effects were reported. The sample plots of SEP and BAEP in the two groups before and after interventions were provided in the Supplementary Materials (available ). 3.1. The Effects of rTMS on Clinical Assessment The total CRS-R score improved significantly at the end of the 6-week interventions compared to baseline in the active group and the sham group ( p value < 0.001 for both conditions). The improvements in the total GCS score were also considered with both the real ( p value < 0.001) and sham stimulation ( p value = 0.007) ( and ). The changes in score in total CRS-R score ( p value = 0.001) and GCS score ( p value = 0.014) were significantly higher in the active group than in the sham group postintervention ( and ). The scores for components of GCS and CRS-R scale in each group were provided in the Supplementary Materials (available ). 3.2. The Effects of rTMS on Electrophysiological Assessment N20-P25 amplitudes (all p value < 0.001) and BAEP grade ( p value = 0.022 vs. p value = 0.013) showed significant improvement in patients who received active rTMS at postintervention in comparison to baseline. Latencies of N20 improved significantly at postintervention compared to baseline in the active group ( p value < 0.001), but not in the sham group ( p value = 0.113) ( and ). The changes in latencies of N20, N20-P25 amplitudes, and BAEP grade were significantly different between the active and sham stimulation conditions ( p value = 0.018, p value = 0.011, and p value = 0.013, respectively). The details are summarized in and . 3.3. The Relationship between Clinical Assessments and Electrophysiological Parameters A strong and significant positive correlation was found between the total CRS-R score and the total GCS score postintervention in all patients ( r = 0.552, p value < 0.001). The latency of N20 at postintervention in all patients exhibited a significant negative correlation with the total CRS-R score ( r = −0.346, p value = 0.014). The grade of BAEP after interventions was related to the total CRS-R score ( r = −0.339, p value = 0.016). The N20-P25 amplitude after interventions was related to the total CRS-R score ( r = 0.0291, p value = 0.041). The changes in N20-P25 amplitude before and after interventions were related to the changes in total CRS-R score ( r = 0.370, p value = 0.008) and latency of N20 ( r = 0.453, p value = 0.001). The details are summarized in . The total CRS-R score improved significantly at the end of the 6-week interventions compared to baseline in the active group and the sham group ( p value < 0.001 for both conditions). The improvements in the total GCS score were also considered with both the real ( p value < 0.001) and sham stimulation ( p value = 0.007) ( and ). The changes in score in total CRS-R score ( p value = 0.001) and GCS score ( p value = 0.014) were significantly higher in the active group than in the sham group postintervention ( and ). The scores for components of GCS and CRS-R scale in each group were provided in the Supplementary Materials (available ). N20-P25 amplitudes (all p value < 0.001) and BAEP grade ( p value = 0.022 vs. p value = 0.013) showed significant improvement in patients who received active rTMS at postintervention in comparison to baseline. Latencies of N20 improved significantly at postintervention compared to baseline in the active group ( p value < 0.001), but not in the sham group ( p value = 0.113) ( and ). The changes in latencies of N20, N20-P25 amplitudes, and BAEP grade were significantly different between the active and sham stimulation conditions ( p value = 0.018, p value = 0.011, and p value = 0.013, respectively). The details are summarized in and . A strong and significant positive correlation was found between the total CRS-R score and the total GCS score postintervention in all patients ( r = 0.552, p value < 0.001). The latency of N20 at postintervention in all patients exhibited a significant negative correlation with the total CRS-R score ( r = −0.346, p value = 0.014). The grade of BAEP after interventions was related to the total CRS-R score ( r = −0.339, p value = 0.016). The N20-P25 amplitude after interventions was related to the total CRS-R score ( r = 0.0291, p value = 0.041). The changes in N20-P25 amplitude before and after interventions were related to the changes in total CRS-R score ( r = 0.370, p value = 0.008) and latency of N20 ( r = 0.453, p value = 0.001). The details are summarized in . As a representative of noninvasive brain stimulation (NIBS) techniques, transcranial magnetic stimulation (TMS) has been viewed as a potential experimental approach to DOC treatment, attracting increasing attention . Despite neurobehavioral gains in some research and clinical settings, there is a paucity of evidence regarding the effects of its application on neural activity . Therefore, the present randomized controlled clinical study was performed using electrophysiological and neurobehavioral assessments to explore clinical neurophysiological evidence in consciousness recovery during therapy according to an HF-rTMS protocol in patients with DOC. The results show that HF-rTMS can produce detectable electrophysiological modifications in DOC patients. There was also improvement in the CRS-R and GCS scores following six weeks of HF-rTMS to the DLPFC. More importantly, the findings of the electrophysiological assessments were, to some extent, compatible with the scores of clinical neurobehavior. The response to rTMS is mediated by the brain network that is preserved after insult. When neural connectivity is preserved, the thalamocortical system should respond to TMS with a complex activation pattern, involving various cortical areas; on the contrary, after losing connectivity, TMS pulses only produce a simple activation localized to the stimulation site . It is worth noting that the N20 and P25 components in SSEP are the primary cerebral responses to electrical stimulation applied to median nerves . The presence of the bilateral N20 and P25 components at baseline, especially the amplitude from N20 to P25, may be a strong predictor of return to consciousness in DOC patients , showing preservation of neural pathway connectivity . Bagnato et al. showed that N20-P25 amplitudes are related to consciousness recovery . In the report by Naro et al., the effect of a single session of rTMS is only shown in DOC patients with bilateral N20 . The residual neural pathway is capable of reacting as an efficient substrate for rTMS . Therefore, the SSEP may make it possible to select patients eligible for rTMS. In this study, the presence of bilateral N20 and P25 was determined by SEP in all patients before enrollment in the study. The presence of bilateral N20 and P25 in these patients could suggest that they may have a greater likelihood of recovery at baseline. In the present study, we also observed the improvements in clinical behavior scales assessed by the CRS-R and GCS in both groups at postintervention when compared to baseline. A disruption of interregional neural connectivity is associated with a breakdown in consciousness . Neural functional connectivity is an important characteristic to consider when describing consciousness levels . The generation and regulation of consciousness are heavily dependent on specific sensory input through thalamocortical pathways , and the connectivity of the pathways can be evaluated by the parameters of SSEP . Keren et al. reported that dynamic changes of N20 in amplitudes and latencies can be related to the changes in consciousness conditions in unaware patients . On the other hand, actions of the ascending reticular activating system (ARAS) which is predominately located in the midbrain and pons also play a significant role in the maintenance of consciousness. The connectivity of the brainstem network could reflect its capability to propagate ARAS signals throughout the cortex , which could then be assessed by BAEP . The patterns of five consecutive neurogenic waves in BAEP are closely related to specific neuroanatomical structures in the auditory pathway, including the cochlear nerve, cochlear nucleus, olivary complex, lateral lemniscus, and inferior colliculus . The presence or absence of these waves, their bilateral symmetry for parallel construction, and their characteristics are also often used to evaluate the severity and prognosis of DOC . In our study, SEP and BAEP were used to evaluate the connectivity of neural pathways and the severity of DOC. Along with the gains in clinical neurobehaviors, we also observed improvements in electrophysiological parameters for these patients at postintervention compared to baseline, particularly in the amplitudes of the N20-P25 and BAEP grades. These results indicate significant normalization of functional neural connectivity after stimulation treatment. Interestingly, Pisani et al. showed that the degree of neural functional connectivity is proportionally related to the consciousness level in patients suffering from DOC. In the present study, significant relationships were also observed between higher behavioral performance (CRS-R scores) and better levels of neural pathway connectivity including the latency of N20 and BAEP grade. The obvious positive relationship between the CRS-R score and the amplitude of N20-P25 was also observed. The induced effects of rTMS depend, in part, on the parameters of stimulation used. As such stimulation at high frequencies (>5 Hz) can induce neural excitation, the frequency commonly utilized in previous studies ranged from 5 to 20 Hz . Moreover, repetitive TMS may induce more significant perturbations in contrast to single TMS, with deep and sustained effects on subcortical regions that can be maintained long after completing rTMS sessions . Given the risk for seizure induction, the effective stimulation frequency in the rTMS design used for this study is repetitive stimulation at 10 Hz with 90% RMT. In addition, Louise-Bender et al. have highlighted the therapeutic effect of 10 Hz rTMS, concluding that in DOC patients, 30 applications may promote clinically significant neurobehavioral recovery . Therefore, 30 sessions of rTMS were performed in the present study, and active rTMS produced a greater elevation of changes in total CRS-R and GCS scores compared to sham stimulation. Notably, no side effects were observed for any of our patients either during or after the entire experiment. The negative results in the improvement of clinical assessment in the study by Naro et al. may be due to the use of only a single session of 10 Hz rTMS in DOC patients . The results from the present study suggest that the underlying mechanisms for behavioral gains could be attributed to the improved connectivity efficiency of a neural pathway. It has been proposed by Pisani et al. that rTMS is capable of modulating the efficient functional connectivity for the neural networks through long-term potentiation like synaptic plasticity mechanisms . Jane et al. observed that the volume of the neural tract of the right prefrontal cortex increased in concert with the provision of comprehensive rehabilitation including rTMS for months by using serial diffusion tensor tractography in a DOC patient in a clinical setting. Several basic studies have also shown that rTMS can remodel dendritic spines by promoting neuronal plasticity related to genes and protein expression . Hence, the improved neural connectivity could be related to the additional recruitment of dendritic (presynaptic or postsynaptic) plasticity by rTMS . The reconstruction of neural connectivity depends not only on local nerve regeneration, but also on effective stimulation of remaining nerve fibers in the damaged area to maximize their use . Our results in this study show that active rTMS significantly decreased the latencies of N20 and elevated the N20-P25 amplitudes compared to the sham stimulation; the latencies of N20 also improved significantly at postintervention compared to the baseline in the active group but not the sham group. Many studies have found that neurophysiological changes after sessions of rTMS in patients with prolonged DOC are related to clinical improvements . In this study, our results are consistent with previous observations that 30 sessions of rTMS altered neural functional connectivity and result in improved behavioral performance and that a positive correlation was observed between the change in CRS-R score and N20-P25 amplitude. The present sham-controlled study of 50 patients with bilateral N20 receiving real or sham rTMS stimulation for 30 sessions revealed higher behavioral gains (total CRS-R and GCS scores), as well as more significant improvement in the electrophysiological parameters (latencies of N20 and N20-P25 amplitudes and BAEP grades) of patients following real rTMS stimulation compared to those receiving sham stimulation. These findings indicate that preserved neural connectivity may be a key point of consciousness recovery in severe DOC patients. The residual plasticity potentiality can be properly triggered by rTMS to elevate neural connectivity and improve the level of consciousness for DOC patients. Future studies with larger sample sizes and the stratification of patients should be carried out to explore whether rTMS might also induce effects in patients with one or without the N20 component by other quantitative assessment means. In addition, this study also has certain limitations. First, the small sample size was largely due to the difficulty of finding eligible patients for such a long study. Second, the present study was a monocentric study. Third, the study did not investigate how long the rTMS-induced effects could last or the long-term prognosis for patients. The prognosis may related to many factors including family, economy, transfer, length of hospital stays, and subsequent treatment levels. Finally, the patient population was heterogeneous, representing patients with different kinds of lesions and diagnoses. In conclusion, rTMS could be a promising treatment strategy for DOC. The 10 Hz rTMS over the right DLPFC can effectively modulate neural functional connectivity and increase behavioral performance in DOC patients with the presence of bilateral N20 in a short term. Our preliminary results indicate that Eps might be useful for the assessment of the effects of rTMS, and an elevation in the connectivity of neural pathways may be one important potential mechanism of rTMS on DOC. However, this is a preliminary study in DOC patients with bilateral N20. Larger studies are needed to confirm the long-term effects and determine the safety in other DOC populations. |
Implementation and evaluation of a mentorship program in clinical master in family medicine during the COVID-19 pandemic at the Arabian Gulf University: a longitudinal study | b99b30df-ac94-4ef5-997b-652ef5d4e9bf | 11241932 | Family Medicine[mh] | Mentorship is an insightful process in which guidance is ensured from an experienced and trusted advisor in a supportive relationship, that requires active participation from both the mentor and the mentee . The mentor role was recognized historically to be responsible for the mentees’ education, shaping their character, and supporting the overall growth and development of the individual at a critical point . Mentoring is identified to be an important asset in academic medicine, which impacts and helps shape the careers of the future generation of healthcare providers . Mentoring programs are crucial in fostering a learner-centered environment for promoting professionalism and humanistic values while maintaining a work-life balance . Mentors are role models who can support in tracking and supporting the individual academic and personal progress, making links over time, and helping the mentee identify areas of improvement in a safe and non-judgmental relationship . Mentorship in academic medicine has also been recognized to have an important impact on personal growth and development, increased academic productivity, career guidance, job satisfaction, networking in the field of interest, and research productivity and publication . In postgraduate medical education, formal mentorship programs were distinguished to provide an effective teaching–learning strategy that is strongly associated with passing board exams and career preparation and satisfaction . Mentored medical residents were nearly twice as likely to describe excellent career preparation and highlighted the importance of mentoring to career advancement and identity formation . Early academic mentoring impacted positively career development in cardiothoracic surgery specialization as 24% of the mentored students and trainees have completed or are enrolled in higher research degrees, 18.9% were enrolled or have completed doctoral degrees, and 81% of participants have published at least one journal article . The Mentoring programs established in several medical schools worldwide vary in their goals and objectives . The process of implementation of these programs is adapted to fit specific institutional or target group needs . While some mentoring programs are designed for medical students in all years, or at specific stages of training, others are tailored to postgraduate medical training . Medical school mentoring programs are usually based on successful initiatives at other organizations and adapted to the context and feedback from different stakeholders. Needs analysis and program piloting are rarely conducted to ensure adequate design and effectiveness before implementation . Mentoring programs in different contexts vary in how mentors are assigned mentees, the mentor’s role, frequency, and format of meetings. While some programs allow mentees to choose their mentors, others are allocated randomly . Interestingly, students’ peer mentoring is integrated into some initiatives to support physician mentoring . Mentoring meetings are often held in person, but other modes of communication, such as email and phone, are increasingly being employed . The frequency of meetings varies according to the aims of the specific program and meetings might take place in a clinical setting, university, or outside the workplace . Mentoring activities in various programs tend to take place over a considerable period to enable the cultivation of successful mentor relationships . Finally, the topics covered during mentoring meetings may include diverse areas of discussion such as motivation, clinical supervision, discussion of specific mentee-selected topics, feedback, ethics, personal development plans, and career planning . Mentoring benefits are of value to mentees, mentors, medical programs, and institutions . Mentoring has been identified as fundamental to the retention and recruitment of trainees in different specialties, advancement in clinical care, as well as enhancement of research outputs and academia . Mentoring has been reported to support the personal and professional development of students and junior doctors through constructive feedback and observing positive role models as well as helping in developing insight into subspecialty training and career guidance, enhancing self-esteem, satisfaction, and stress management . Despite their benefits, mentoring programs might face several challenges. This is especially recognized when mentoring is informal and lacks structures and standards for consistency. Such challenges might arise when mentors are not trained and prepared for this role and lack a protected time for this function . Furthermore, mentee engagement with mentoring can be a challenge, when students’ engagement and perceived benefits are low . The literature review about mentorship programs revealed that they rarely rely on mixed methods to grasp a comprehensive understanding of the needs of trainees and the impact of the program. Furthermore, most of these programs are not based on standardized guidelines before their implementation. The longitudinal follow-up of the effectiveness of the program and multi-level control of the smooth running is lacking. This study builds on these previous experiences and attempts to address these gaps. It highlights the specific features of the mentoring program in The Clinical Master in Family Medicine (CMFM), at Arabian Gulf University and evaluates its effectiveness, especially during the critical period of the Coronavirus disease 2019 (COVID-19) pandemic. Study objectives This study aims to: i) highlight the innovative features of the mentorship program of the Clinical Master in Family Medicine (CMFM) at the Arabian Gulf University (AGU); ii) derive the major challenges faced by trainees and related corrective actions; iii) Evaluate the impact of this mentorship program (short term represented in trainees’ performance). Study design and population We conducted a longitudinal study design using a mixed-method approach for data collection. The study population includes two cohorts of 80 CMFM trainees enrolled and graduated between 2020 and 2023. Study settings and process of the mentorship program The CMFM program is a two-year clinically oriented postgraduate program at AGU which was established in April 2020 during the COVID-19 pandemic. The program combines different modalities of training, mainly clinical training in primary and secondary care, theoretical group activities, and quality improvement research projects. During this period, trainees are evaluated longitudinally through formative workplace-based assessments and summative end-of-each-year written (Multiple Choice Questions) and Objective Structured Clinical Examinations (OSCE). The program's intensity, implementation within the COVID-19 period, and the diversity of learning modalities within a short period required a contextualized mentorship program. Therefore, the academic committee conceived a mentoring program after an extensive literature review based on the program's needs and desired outcomes. The program was further adapted based on program piloting and stakeholders' feedback. Through this program, we aimed to ensure real-time monitoring of professional growth and optimal academic progress and well-being of trainees during each rotation. In addition, to identify struggling trainees timely who require personalized interventions. Recruitment of mentors, training, and mentoring meetings process Mentors were recruited based on their clinical experience in training in family medicine (more than five years), dedication, and motivation for the mentor role. All trainees enrolled in the CMFM program were enrolled in the mentoring program, and every five trainees were assigned a mentor by the program coordinator, however, there was flexibility in assigning mentees to specific mentors based on their request. A mentoring guide developed by the program committee was shared with mentors and mentees to provide an orientation and details about the process and outcomes of the program. Furthermore, induction workshops for both mentors and mentees were conducted to discuss details related to the mentoring program and the mentor's role. One-to-one meetings between the mentor and each trainee take place every twelve weeks but can be requested according to the mentee’s needs. For convenience and sustainability, the mentors and mentees had the option to conduct the meetings either face-to-face, virtual, or through phone calls. As part of the continuous monitoring and evaluation of the mentorship program, continuous meetings were conducted after each phase of training with both mentors and mentees separately, to obtain feedback and highlight areas for improvement. During mentoring meetings, mentors and mentees were encouraged to reflect on and discuss achievements, feedback, and potential problems (physical, mental, or social) that could affect training. Mentors also have access to the trainees' electronic portfolio (E-portfolio) documentation summarized in a dashboard for each mentee for each rotation. The academic committee considers five Key performance indicators (KPIs) in the dashboard of high priority. They are monitored in an electronic dashboard extracted from the learning platform “Moodle”. They include attendance, daily cases encountered (coded according to the international classification of primary care), skills, and procedures performed or observed by the trainee, and participation in educational activities. In addition, the mentor is encouraged to review and discuss with the trainee detailed documentation and reflection on selected submitted clinical cases to ensure deep learning and self-confidence. By the end of the meeting, the mentor and the mentee identify the gaps and agree on a plan for the coming period. The mentor can decide if the mentee needs a meeting with the academic committee in case of a serious issue that could hinder the trainee’s personal well-being and academic progress. To ensure a standardized process of information, the program’s committee conceived a structured electronic mentoring report form on the Moodle platform to guide discussion and probe areas that need follow-up and specific interventions by the program’s committee. High-risk trainees who were identified to have sub-optimal performance or health/well-being issues are subject to more extensive mentoring by the program’s committee until resolving the identified problems. Figure illustrates the multi-level framework and the cycle of the mentorship program during its implementation. The mentoring domains and related challenges are derived during every rotation at three levels: the mentors, the mentorship program coordinator, and the academic committee. At each level, specific tasks and tools are utilized to ensure a deep, comprehensive, and real-time assessment of the trainees’ progress and identify the threats. A personalized intervention plan is timely deployed and monitored at all levels until resolved. Data collection All trainees are enrolled in the mentorship program and included in this study (80 trainees, 40 for each cohort). All submitted mentoring reports by all the assigned mentors between September 2020 to April 2023 were included. In addition, to findings of academic committee meetings with the trainees. We extracted the data from, the electronic mentoring report form submitted by the mentors, and academic committee reports of meetings with trainees. Information collected from the mentoring reports includes the type, frequency of meetings, duration of meetings in minutes, and dashboard KPIs generated from the E-portfolio. The latter includes attendance (a percentage), daily cases encountered (a number with access to a Word file for every case), detailed reflective cases (a number with access to a Word file for every case), skills and procedures practiced (a number with a word file for every procedure), and educational activities (a number with word file for every activity). The type of challenges (available as a list of categories: health, psychological, social, time management, others, and the recommended action plan (available as a list of categories: specific topic reading/online course, adapt learning approaches, time management, engaging in team learning activities, others). The qualitative data collected from the mentors' documentation is provided in the open-text sections and academic committee meetings’ reports. It includes details related to training progress in primary and secondary care courses, progress in quality improvement projects, challenges faced by trainees, interventions/ recommendations agreed between mentors and mentees, and corrective actions implemented by the academic committee. The thematic analysis was conducted manually following the steps of the one sheet of paper (OSOP) technique of Ziebland. We integrated the transcripts from the mentorship text file, the academic committee file, and the review report file for every trainee. Two members of the academic committee read all the transcripts for every trainee and merged them into one transcript and then we extracted themes that emerged from the data. A second iteration of reading the transcripts permitted to linking of subthemes and quotes to the most likely related theme and subthemes as illustrated in Fig. . Data related to mentees’ GPAs were provided by the program’s officials and the university registration unit. Case definitions Cohort 1 refers to the first 40 trainees enrolled in the program in April 2020 and Cohort 2 refers to the group of 40 trainees who started the program in May 2021. Graduation with a master's degree requires the completion of the two-year curriculum and obtaining a cumulative GPA of a minimum of 3 out of 4. Trainees who had an accumulative GPA between 2 and 3 were entitled to a Diploma degree unless they wished to repeat a certain number of courses based on the university regulations to improve their GPA to obtain the master’s degree. A trainee is considered high risk if having one of the following situations: Not able to accomplish the required level of achievement in the KPIs (related to attendance, number of documented daily cases encountered, skills and procedures, continuous medical education activities, and quality of documentation in the reflective detailed clinical cases of the E-portfolio. If exposed to any health or psychosocial threat that prevents normal progress in the training. Having low academic performance in formative and/or summative assessments. Statistical analysis plan Statistical Analysis relies on a mixed method approach to calculate proportions and means for continuous quantitative variables, as well as thematic analysis for the qualitative textual information. The cohort and time effect were assessed using the T-test or the Mann–Whitney U test to account for the lack of normality (based on the significance of Kolmogorov–Smirnov). All quantitative analyses are performed using SPSS V28. The thematic analysis was conducted manually following the one sheet of paper (OSOP) technique of Ziebland . This study aims to: i) highlight the innovative features of the mentorship program of the Clinical Master in Family Medicine (CMFM) at the Arabian Gulf University (AGU); ii) derive the major challenges faced by trainees and related corrective actions; iii) Evaluate the impact of this mentorship program (short term represented in trainees’ performance). We conducted a longitudinal study design using a mixed-method approach for data collection. The study population includes two cohorts of 80 CMFM trainees enrolled and graduated between 2020 and 2023. The CMFM program is a two-year clinically oriented postgraduate program at AGU which was established in April 2020 during the COVID-19 pandemic. The program combines different modalities of training, mainly clinical training in primary and secondary care, theoretical group activities, and quality improvement research projects. During this period, trainees are evaluated longitudinally through formative workplace-based assessments and summative end-of-each-year written (Multiple Choice Questions) and Objective Structured Clinical Examinations (OSCE). The program's intensity, implementation within the COVID-19 period, and the diversity of learning modalities within a short period required a contextualized mentorship program. Therefore, the academic committee conceived a mentoring program after an extensive literature review based on the program's needs and desired outcomes. The program was further adapted based on program piloting and stakeholders' feedback. Through this program, we aimed to ensure real-time monitoring of professional growth and optimal academic progress and well-being of trainees during each rotation. In addition, to identify struggling trainees timely who require personalized interventions. Mentors were recruited based on their clinical experience in training in family medicine (more than five years), dedication, and motivation for the mentor role. All trainees enrolled in the CMFM program were enrolled in the mentoring program, and every five trainees were assigned a mentor by the program coordinator, however, there was flexibility in assigning mentees to specific mentors based on their request. A mentoring guide developed by the program committee was shared with mentors and mentees to provide an orientation and details about the process and outcomes of the program. Furthermore, induction workshops for both mentors and mentees were conducted to discuss details related to the mentoring program and the mentor's role. One-to-one meetings between the mentor and each trainee take place every twelve weeks but can be requested according to the mentee’s needs. For convenience and sustainability, the mentors and mentees had the option to conduct the meetings either face-to-face, virtual, or through phone calls. As part of the continuous monitoring and evaluation of the mentorship program, continuous meetings were conducted after each phase of training with both mentors and mentees separately, to obtain feedback and highlight areas for improvement. During mentoring meetings, mentors and mentees were encouraged to reflect on and discuss achievements, feedback, and potential problems (physical, mental, or social) that could affect training. Mentors also have access to the trainees' electronic portfolio (E-portfolio) documentation summarized in a dashboard for each mentee for each rotation. The academic committee considers five Key performance indicators (KPIs) in the dashboard of high priority. They are monitored in an electronic dashboard extracted from the learning platform “Moodle”. They include attendance, daily cases encountered (coded according to the international classification of primary care), skills, and procedures performed or observed by the trainee, and participation in educational activities. In addition, the mentor is encouraged to review and discuss with the trainee detailed documentation and reflection on selected submitted clinical cases to ensure deep learning and self-confidence. By the end of the meeting, the mentor and the mentee identify the gaps and agree on a plan for the coming period. The mentor can decide if the mentee needs a meeting with the academic committee in case of a serious issue that could hinder the trainee’s personal well-being and academic progress. To ensure a standardized process of information, the program’s committee conceived a structured electronic mentoring report form on the Moodle platform to guide discussion and probe areas that need follow-up and specific interventions by the program’s committee. High-risk trainees who were identified to have sub-optimal performance or health/well-being issues are subject to more extensive mentoring by the program’s committee until resolving the identified problems. Figure illustrates the multi-level framework and the cycle of the mentorship program during its implementation. The mentoring domains and related challenges are derived during every rotation at three levels: the mentors, the mentorship program coordinator, and the academic committee. At each level, specific tasks and tools are utilized to ensure a deep, comprehensive, and real-time assessment of the trainees’ progress and identify the threats. A personalized intervention plan is timely deployed and monitored at all levels until resolved. All trainees are enrolled in the mentorship program and included in this study (80 trainees, 40 for each cohort). All submitted mentoring reports by all the assigned mentors between September 2020 to April 2023 were included. In addition, to findings of academic committee meetings with the trainees. We extracted the data from, the electronic mentoring report form submitted by the mentors, and academic committee reports of meetings with trainees. Information collected from the mentoring reports includes the type, frequency of meetings, duration of meetings in minutes, and dashboard KPIs generated from the E-portfolio. The latter includes attendance (a percentage), daily cases encountered (a number with access to a Word file for every case), detailed reflective cases (a number with access to a Word file for every case), skills and procedures practiced (a number with a word file for every procedure), and educational activities (a number with word file for every activity). The type of challenges (available as a list of categories: health, psychological, social, time management, others, and the recommended action plan (available as a list of categories: specific topic reading/online course, adapt learning approaches, time management, engaging in team learning activities, others). The qualitative data collected from the mentors' documentation is provided in the open-text sections and academic committee meetings’ reports. It includes details related to training progress in primary and secondary care courses, progress in quality improvement projects, challenges faced by trainees, interventions/ recommendations agreed between mentors and mentees, and corrective actions implemented by the academic committee. The thematic analysis was conducted manually following the steps of the one sheet of paper (OSOP) technique of Ziebland. We integrated the transcripts from the mentorship text file, the academic committee file, and the review report file for every trainee. Two members of the academic committee read all the transcripts for every trainee and merged them into one transcript and then we extracted themes that emerged from the data. A second iteration of reading the transcripts permitted to linking of subthemes and quotes to the most likely related theme and subthemes as illustrated in Fig. . Data related to mentees’ GPAs were provided by the program’s officials and the university registration unit. Cohort 1 refers to the first 40 trainees enrolled in the program in April 2020 and Cohort 2 refers to the group of 40 trainees who started the program in May 2021. Graduation with a master's degree requires the completion of the two-year curriculum and obtaining a cumulative GPA of a minimum of 3 out of 4. Trainees who had an accumulative GPA between 2 and 3 were entitled to a Diploma degree unless they wished to repeat a certain number of courses based on the university regulations to improve their GPA to obtain the master’s degree. A trainee is considered high risk if having one of the following situations: Not able to accomplish the required level of achievement in the KPIs (related to attendance, number of documented daily cases encountered, skills and procedures, continuous medical education activities, and quality of documentation in the reflective detailed clinical cases of the E-portfolio. If exposed to any health or psychosocial threat that prevents normal progress in the training. Having low academic performance in formative and/or summative assessments. Statistical Analysis relies on a mixed method approach to calculate proportions and means for continuous quantitative variables, as well as thematic analysis for the qualitative textual information. The cohort and time effect were assessed using the T-test or the Mann–Whitney U test to account for the lack of normality (based on the significance of Kolmogorov–Smirnov). All quantitative analyses are performed using SPSS V28. The thematic analysis was conducted manually following the one sheet of paper (OSOP) technique of Ziebland . Participants’ characteristics All trainees are enrolled in the mentorship program and included in this study (80 trainees, 40 for each cohort). Most of the trainees were female (93.75%) and the mean age was 30.00 ± 2.19 years. This reflects the national statistics of primary care physicians in The Kingdom of Bahrain where most family physicians are female (77.7%). A total of 16 mentors were involved with a ratio of 5 trainees per mentor. The monitoring meetings were conducted either through phone calls (62%), virtually (29.7%), or face-to-face (8.3%). The mean number of meetings during the evaluation period (20 months) was 3.88 ± 2.31 and the mean duration for the meetings was 20.08 min ± 9.50. Data related to the mentorship program indicators are presented in Table . Challenges identified from the mentorship program and related interventions The analysis of the quantitative data related to the challenges reported by mentors revealed that time management was the most reported issue affecting the progress of the trainees (41.3%), followed by health-related (7.6%), social (4.6%), and psychological issues (3%). Interestingly, 43.6% of other types of challenges were reported, and they are detailed in the qualitative part of the study. Figure shows the presentation of different challenges as reported by mentors. The qualitative part of the study permitted to obtain very important information from struggling trainees, particularly sensitive information such as health and mental or psychosocial related issues. We extracted four main themes, such as challenges, and related subthemes data from the mentoring meeting as well as the academic committee face-to-face meeting reports. The main themes are related to trainees, training setting, E-portfolio, and COVID-19 challenges and are detailed below. Trainees’ related challenges Trainee-related challenges included five main sub-themes: health-related, psychological, social, learning style, and time management. Some of the trainees had chronic medical problems such as multiple sclerosis, systemic lupus erythematosus, sickle cell disease, and migraine as expressed by other trainees “ My migraine attacks are occurring more frequently and it affects my study” and “ “ I am afraid that my admission in the hospital will affect my training and need guidance on how to catch up”. In addition, others suffered from pre-existing mental health problems such as depression and anxiety as expressed “. Being diagnosed with medical and mental health problems and exposed to the additional stress caused by the intensive nature of the curriculum in the COVID-19 pandemic context, some trainees had low self-confidence and psychological challenges in their capacity to pursue the program as expressed by one of the trainees: “ I am not sure if I can cover all the training requirements, and study, and feel lost”. Some other trainees were facing some social challenges such as the death of a close relative, the birth of a new child, and being a caregiver of young children, as expressed by a trainee “ I feel that my study progress is slow since I need to manage between my study in the program and taking care of my two little daughters and their requirements”. The intensive character of the program generated serious challenges to some trainees related to time management and a fair balance between the program requirements and their other life aspects “ I am not sure if I am studying correctly….it is difficult to manage my time”. A group of trainees struggled to adapt their learning style to the primary care approach and setting which favors a learning based on clinical presentations. The latter challenge was very difficult to overcome particularly for freshly graduated trainees who are still influenced by the undergraduate learning mindsets. Trainees facing such challenges were considered by the academic committee at higher risk and required intensive mentoring. They were entitled to close monitoring to overcome a stressful period. Training setting challenges The training takes place in the regular primary care setting in which the trainers are primary care physicians assigned to train besides managing their scheduled patients’ lists. This situation permitted to immerse the trainees in a real context of family practice but created the challenge of trainers' dedication toward training and facing the problem of unavailability of training rooms in some health centers on some occasions busy trainers who were challenged to manage their role as trainers and other duties as stated by trainees sometimes: “There are no vacant consultation rooms and we alternate with the trainers in conducting the consultation…” and “The trainer is not available all the time to provide detailed feedback on my performance since she is busy as well with other tasks”. The study also permitted, through probing, “deviant cases” such as students suffering from mental health or chronic diseases that required special care such as a placement in a more psychologically- safe training environment (a more compassionate clinical trainer). Some other unexpected findings emerged from the probing such as the rejection of an experienced trainer because of a perceived “autocratic” vertical approach in training as well as loading the trainees by contents rather than best approaches in learning as expressed “My trainer is treating us in a very rigid way which makes me feel uncomfortable in my training and doubt if I’m not doing well”. Surprisingly, the same trainer was highly appreciated by other trainees. These conflicting patterns discovered through in-depth interviews might reflect different personality traits and cultural frameworks in the study group. E-portfolio-related challenges E-portfolio identified challenges were the suboptimal entry of cases and procedures encountered by some trainees and poor quality of documentation. The trainees did not consider electronic documentation of the daily activities as a high priority. It was usually left for a later time in the week, which increased the recall bias and incompleteness of information as expressed by a trainee “ Sometimes, I do not have the time to document in the E-portfolio and I have a lot of pending work related to my E-portfolio….I try to do it in the weakened”. This difficulty was reduced through feedback meetings and the improvement of E-portfolio forms in the Moodle platform. COVID-19 related challenges The program started during the first period of the COVID-19 pandemic when the social distancing precautions and regulations that included primary health care centers were strictly enforced. This resulted in a limitation in terms of the number and variety of clinical cases encountered especially in preventive and non-communicable diseases as expressed by a trainee “ There is a very low flow of patients and I am concerned that it will affect my learning”. In addition, we faced a gap in training for minor surgical procedures including those in primary care settings as expressed by a trainee “ I was not trained in any minor surgical procedures during this rotation….all minor non-urgent procedures were withheld due to the COVID-19 regulations”. Figure illustrates themes and sub-themes for these challenges. Triangulating the information from the mixed method approach permitted the CMFM program academic committee to obtain a comprehensive situation analysis of the progress of every trainee. Consequently, it allowed the timely implementation of relevant personalized corrective measures by the academic committee, to support the trainees and pursue their normal academic progress. These interventions are described in Table . Evaluation of the performance of the mentorship program Out of the 80 trainees, 12 (15%) were identified as high-risk trainees, 6 of the 12 (50% of the high-risk) graduated on time while the remaining had to repeat some courses to pass the exit assessment and obtain the master's degree. When we consider the mean global GPA of high-risk trainees ( n = 12) during year one, it was 3.22 (SE = 0.16) versus 3.41 (SE = 0.05) for low-risk trainees ( n = 68), ( P = 0.33). During year two the mean GPA for the low-risk trainees was 3.29 (SE = 0.07) versus 3.06 (SE = 0.03) for high-risk trainees, ( P = 0.04). Interestingly, the overall mean cumulative GPA was 3.35 (SE = 0.03) for the low-risk trainees, versus 3.14 (SE = 0.08) for the high-risk trainees, ( P = 0.043). These findings suggest that trainees are mainly challenged in the second year, but the discrepancy between the high-risk and low-risk trainees significantly reduced at the final cumulative GPA implying the effectiveness of the corrective action plans resulting from the mentorship program. The academic committee considered the GPA of trainees as one of the main outcomes reflecting the effect of monitoring, including the mentorship program, of progress of trainees, and the implementation of timely corrective actions. When we consider the mean GPA trends over time and cohorts, we realize that the mean GPA in year 1 (GPA 1) for Cohort 1 (40 trainees) was 3.43 (SE = 0.06) versus 3.45 (SE = 0.07) for Cohort 2 (40 trainees) ( P = 0.022). On the other hand, the mean GPA in year 2 (GPA2) for cohort 1 was 3.18 (SE = 0.04) versus 3.33 (SE = 0.04) for cohort 2, ( P = 0.02). Despite this slight significant difference, the two cohorts achieved equivalent successful GPAs in both years when we consider the cut-off of a minimum of 3 out of 4 required for the master’s degree. This was corroborated by the final cumulative mean GPA of 3.30 (SE = 0.03), versus 3.34 (SE = 0.05) for cohorts 1 and 2 respectively, ( P = 0.40). This reflects the stability of the performance of the program over time and cohorts. All trainees are enrolled in the mentorship program and included in this study (80 trainees, 40 for each cohort). Most of the trainees were female (93.75%) and the mean age was 30.00 ± 2.19 years. This reflects the national statistics of primary care physicians in The Kingdom of Bahrain where most family physicians are female (77.7%). A total of 16 mentors were involved with a ratio of 5 trainees per mentor. The monitoring meetings were conducted either through phone calls (62%), virtually (29.7%), or face-to-face (8.3%). The mean number of meetings during the evaluation period (20 months) was 3.88 ± 2.31 and the mean duration for the meetings was 20.08 min ± 9.50. Data related to the mentorship program indicators are presented in Table . The analysis of the quantitative data related to the challenges reported by mentors revealed that time management was the most reported issue affecting the progress of the trainees (41.3%), followed by health-related (7.6%), social (4.6%), and psychological issues (3%). Interestingly, 43.6% of other types of challenges were reported, and they are detailed in the qualitative part of the study. Figure shows the presentation of different challenges as reported by mentors. The qualitative part of the study permitted to obtain very important information from struggling trainees, particularly sensitive information such as health and mental or psychosocial related issues. We extracted four main themes, such as challenges, and related subthemes data from the mentoring meeting as well as the academic committee face-to-face meeting reports. The main themes are related to trainees, training setting, E-portfolio, and COVID-19 challenges and are detailed below. Trainee-related challenges included five main sub-themes: health-related, psychological, social, learning style, and time management. Some of the trainees had chronic medical problems such as multiple sclerosis, systemic lupus erythematosus, sickle cell disease, and migraine as expressed by other trainees “ My migraine attacks are occurring more frequently and it affects my study” and “ “ I am afraid that my admission in the hospital will affect my training and need guidance on how to catch up”. In addition, others suffered from pre-existing mental health problems such as depression and anxiety as expressed “. Being diagnosed with medical and mental health problems and exposed to the additional stress caused by the intensive nature of the curriculum in the COVID-19 pandemic context, some trainees had low self-confidence and psychological challenges in their capacity to pursue the program as expressed by one of the trainees: “ I am not sure if I can cover all the training requirements, and study, and feel lost”. Some other trainees were facing some social challenges such as the death of a close relative, the birth of a new child, and being a caregiver of young children, as expressed by a trainee “ I feel that my study progress is slow since I need to manage between my study in the program and taking care of my two little daughters and their requirements”. The intensive character of the program generated serious challenges to some trainees related to time management and a fair balance between the program requirements and their other life aspects “ I am not sure if I am studying correctly….it is difficult to manage my time”. A group of trainees struggled to adapt their learning style to the primary care approach and setting which favors a learning based on clinical presentations. The latter challenge was very difficult to overcome particularly for freshly graduated trainees who are still influenced by the undergraduate learning mindsets. Trainees facing such challenges were considered by the academic committee at higher risk and required intensive mentoring. They were entitled to close monitoring to overcome a stressful period. The training takes place in the regular primary care setting in which the trainers are primary care physicians assigned to train besides managing their scheduled patients’ lists. This situation permitted to immerse the trainees in a real context of family practice but created the challenge of trainers' dedication toward training and facing the problem of unavailability of training rooms in some health centers on some occasions busy trainers who were challenged to manage their role as trainers and other duties as stated by trainees sometimes: “There are no vacant consultation rooms and we alternate with the trainers in conducting the consultation…” and “The trainer is not available all the time to provide detailed feedback on my performance since she is busy as well with other tasks”. The study also permitted, through probing, “deviant cases” such as students suffering from mental health or chronic diseases that required special care such as a placement in a more psychologically- safe training environment (a more compassionate clinical trainer). Some other unexpected findings emerged from the probing such as the rejection of an experienced trainer because of a perceived “autocratic” vertical approach in training as well as loading the trainees by contents rather than best approaches in learning as expressed “My trainer is treating us in a very rigid way which makes me feel uncomfortable in my training and doubt if I’m not doing well”. Surprisingly, the same trainer was highly appreciated by other trainees. These conflicting patterns discovered through in-depth interviews might reflect different personality traits and cultural frameworks in the study group. E-portfolio identified challenges were the suboptimal entry of cases and procedures encountered by some trainees and poor quality of documentation. The trainees did not consider electronic documentation of the daily activities as a high priority. It was usually left for a later time in the week, which increased the recall bias and incompleteness of information as expressed by a trainee “ Sometimes, I do not have the time to document in the E-portfolio and I have a lot of pending work related to my E-portfolio….I try to do it in the weakened”. This difficulty was reduced through feedback meetings and the improvement of E-portfolio forms in the Moodle platform. The program started during the first period of the COVID-19 pandemic when the social distancing precautions and regulations that included primary health care centers were strictly enforced. This resulted in a limitation in terms of the number and variety of clinical cases encountered especially in preventive and non-communicable diseases as expressed by a trainee “ There is a very low flow of patients and I am concerned that it will affect my learning”. In addition, we faced a gap in training for minor surgical procedures including those in primary care settings as expressed by a trainee “ I was not trained in any minor surgical procedures during this rotation….all minor non-urgent procedures were withheld due to the COVID-19 regulations”. Figure illustrates themes and sub-themes for these challenges. Triangulating the information from the mixed method approach permitted the CMFM program academic committee to obtain a comprehensive situation analysis of the progress of every trainee. Consequently, it allowed the timely implementation of relevant personalized corrective measures by the academic committee, to support the trainees and pursue their normal academic progress. These interventions are described in Table . Out of the 80 trainees, 12 (15%) were identified as high-risk trainees, 6 of the 12 (50% of the high-risk) graduated on time while the remaining had to repeat some courses to pass the exit assessment and obtain the master's degree. When we consider the mean global GPA of high-risk trainees ( n = 12) during year one, it was 3.22 (SE = 0.16) versus 3.41 (SE = 0.05) for low-risk trainees ( n = 68), ( P = 0.33). During year two the mean GPA for the low-risk trainees was 3.29 (SE = 0.07) versus 3.06 (SE = 0.03) for high-risk trainees, ( P = 0.04). Interestingly, the overall mean cumulative GPA was 3.35 (SE = 0.03) for the low-risk trainees, versus 3.14 (SE = 0.08) for the high-risk trainees, ( P = 0.043). These findings suggest that trainees are mainly challenged in the second year, but the discrepancy between the high-risk and low-risk trainees significantly reduced at the final cumulative GPA implying the effectiveness of the corrective action plans resulting from the mentorship program. The academic committee considered the GPA of trainees as one of the main outcomes reflecting the effect of monitoring, including the mentorship program, of progress of trainees, and the implementation of timely corrective actions. When we consider the mean GPA trends over time and cohorts, we realize that the mean GPA in year 1 (GPA 1) for Cohort 1 (40 trainees) was 3.43 (SE = 0.06) versus 3.45 (SE = 0.07) for Cohort 2 (40 trainees) ( P = 0.022). On the other hand, the mean GPA in year 2 (GPA2) for cohort 1 was 3.18 (SE = 0.04) versus 3.33 (SE = 0.04) for cohort 2, ( P = 0.02). Despite this slight significant difference, the two cohorts achieved equivalent successful GPAs in both years when we consider the cut-off of a minimum of 3 out of 4 required for the master’s degree. This was corroborated by the final cumulative mean GPA of 3.30 (SE = 0.03), versus 3.34 (SE = 0.05) for cohorts 1 and 2 respectively, ( P = 0.40). This reflects the stability of the performance of the program over time and cohorts. We conducted a longitudinal study using mixed methods to describe the implementation of a mentorship program and evaluate its effectiveness in the context of an intensive two-year CMFM curriculum that started during the COVID-19 pandemic. We obtained a real-time comprehensive evaluation of the progress of trainees through parsimonious quantitative indicators and qualitative analysis of challenges they are facing, which was instrumental in designing real-time, personalized corrective actions. The mentorship program proved to be effective for the smooth academic progress of trainees and reduced the risk of failure in graduation. It supported trainees’ overall well-being while maintaining work-life balance and minimizing burnout. The CMFM mentorship program helped to ensure that the trainees’ progress was meeting curriculum standards and certain key performance indicators related to the training. Mentors provided a longitudinal and holistic evaluation of training that helped to bridge the gap between theoretical learning and clinical practice and suggested recommendations to ensure progress and attainment of appropriate skills and program standards on time. Our study confirmed that the COVID-19 pandemic threatened postgraduate medical trainees’ academic advancement by constraining opportunities for knowledge and skill acquisition, scholar productivity, and networking. On the other hand, the pandemic has created new opportunities. The exerted challenges of the pandemic-era circumstances required extra efforts and innovative solutions aimed at enhancing trainees’ academic progress while supporting work-life balance . In agreement with others, the consistent approach to mentoring, oversight continuous monitoring, and feedback facilitated oversight and regulation of the mentoring processes . Findings in the literature highlight that several health science faculties could avoid mentoring due to numerous factors including the lack of knowledge about the mentoring process, lack of confidence, and the fear of managing ‘challenging situations’, including problems of a personal nature . Indeed, to standardize the mentorship program and facilitate its implementation, the CMFM program committee provided a mentoring program guide and workshops targeting both mentors and mentees before implementation. Our findings confirm the importance of these preparatory aspects before launching the mentorship program. In addition, mentors attended several longitudinal workshops while the program was running to build their capacities as mentors, receive their feedback, and provide them with guidance and support. Similarly, we conducted periodic meetings with the trainees to identify any issue affecting the mentoring process and relationship that needs timely interventions. Trainees who were unsatisfied or faced any challenges related to the mentor–mentee relationship were assigned to other mentors to ensure the accomplishment of outcomes through academic and personal support. One of the innovative and strong aspects related to the CMFM mentorship program is structuring an electronic mentoring meeting report with a mixed structure in data collection that helped to harmonize mentor–mentee discussions during the meetings without compromising the specific needs of trainees. Since mentors are composed of family physician consultants and some of them are involved as trainers, we expected the possibility for mentors to focus more on trainee-related issues compared to other factors. Indeed, mentors with integrated physician and mentor identities can embrace liminality and develop as mentors, this was addressed through guidance and support . This tendency is reflected in the frequency of challenges listed in the quantitative study. However, this was fixed by the findings of the qualitative section in the form that permitted to grasp a richer understanding of the trainees' global progress and constraints for pertinent and customized corrective actions as detailed in the findings. This mixed format provided a standardized structure without compromising flexibility in areas of discussion, rapid interventions, and follow-up . Another strength related to the CMFM mentorship program is the formal one-to-one mentoring providing a safe and non-judgmental environment for discussions and personalized advice and guidance. This contrasts with other mentorship programs where the mentor conducts group meetings with mentees . The various options, face-to-face, virtual, and phone calls, for mentoring meetings, eased continuity in meetings, especially during the period of COVID-19. Another strength of our mentorship program is a cascade of checkpoints and interventions at the level of mentors, students’ feedback meetings, and academic committee oversight particularly for trainees at high risk. This permitted a large consensus about corrective plans and strong governance of a complex and intensive program. The identified challenges through the CMFM mentoring program are consistent with those reported in other postgraduate clinical training programs. The major reduction in the volume of inpatients and outpatients encountered during the pandemic affected the number and diversity of clinical exposure and mitigated drastically the opportunities for trainees to perform physical examinations and essential procedures, which can be mastered mostly during clinical training . It was reported similarly in other studies, that the ongoing pandemic has added new stressors while aggravating the existing ones for students and trainees . On the contrary, the pandemic has created new opportunities for the CMFM program academic committee, trainers, and mentors, to sustain and enhance training outcomes. Trainers had more time dedicated to interactions and discussions around selected clinical cases leading to deep clinical learning and high self-confidence. The role of teleconsultation, underutilized in the pre-COVID era, was integrated to ensure continuity of healthcare delivery during the current pandemic by the healthcare system, which offered an opportunity for integrating training on telemedicine and teleconsultation. These skills are nowadays necessary to continue with safe, high-quality delivery of services and increase this modality of care integration in healthcare systems . Another innovative aspect of the CMFM program was the utilization of trainees' E-portfolio entries related to the clinical cases encountered, skills and procedures that they were exposed to, and mentoring meeting reports to identify gaps related to clinical exposure, mainly during the COVID-19 pandemic. This allowed the implementation of pioneering interventions such as engaging learners in experiences that simulate reality and compensate for cases of fewer encounters. Trainers with the help of program administrators also integrated simulated scenarios followed by constructive feedback discussions during clinical training and on weekly educational activities. We also highlight the big added value of on-campus skills and procedures training in the Medical Skills and Simulation Center using high-fidelity mannequins. Simulation is a useful modality to supplement training in real clinical situations because it allows control over the sequence of tasks offered to learners, provides opportunities to offer support and guidance to learners, prevents unsafe and dangerous situations, and creates tasks that rarely occur in the real world. It is also an excellent form that supports inter-professional and communication skills education . We also integrated team-based learning to promote active learning and enhance inter-professional skills development . In addition, trainees whose training was disrupted for any reason (birth of a new child, contact with COVID-19 cases, health-related), and whose situation allowed distance learning were provided with a distance learning toolkit containing clinical scenarios followed by smart questions and recommended online courses related to the ongoing clinical rotation. They received more intense mentorship and administrative support to overcome their challenges. The monitoring of smart few KPIs during the mentorship program permitted, in our context, the early detection of struggling trainees before the summative exam, allowing timely corrective actions to be implemented. The CMFM program is system-centered and integrated into primary health care, which increased the ownership; by health authorities and preparedness to facilitate any action needed to better prepare the training environment for an optimized outcome and increased the recruitment of graduated trainees. The provision of a protected time for the trainers to discussions around clinical cases of high educational value and the availability of independent consultation lists and rooms under the supervision of the trainers was particularly instrumental in facilitating deep learning and enhancing the level of self-confidence, safety of trainees and their immersion in a real context of practice. The learning environment, an important dimension in our mentorship program, was adapted to promote changes in students' thinking strategies as well as their development as flexible, reflective learners . These endeavors require support from mentors and program administrators with rigorous expectations and good facilitation skills. The mentorship program was successful and effective, particularly when coupled with longitudinal meetings and feedback from trainees, trainers, and mentors to get the best comprehensive analysis of the situation and to implement the most appropriate intervention plans. Students’ voices and perspectives as important stakeholders in the process of learning are essential to providing emotional and cognitive support that enhances learning and prevents burnout . In addition to the mentioned benefits for all trainees, the mentorship program identified 12 trainees (5%) at high risk for failure. Six of them (50%) achieved high scores and obtained their degrees at the end of the program due to early identification, extensive follow-up, and support by the program’s committee. Five out of the remaining six trainees obtained their degrees after repeating a few courses to ensure the needed level of safety and competency. Only one trainee out of the two cohorts (80 trainees) graduated with a diploma because the final GPA was less than the threshold of 3/4 as required by AGU regulations. On the other hand, when we consider the overall GPA of high-risk trainees and the rest of the cohort, the difference is not significant which reflects the effectiveness of corrective plans. In addition, analysis of GPA through years and cohorts demonstrates the stability of the performance of the CMFM program over time, partly due to the mentorship program. This study highlights the importance of a mentorship program in supporting and monitoring postgraduate training in family medicine practice. The lessons learned here lay the foundation for the design of formal and contextualized mentorship programs that align with the training context and curriculum, and the importance of engaging both mentors and mentees in the mentoring process through several aspects including training, guidance, and longitudinal monitoring. All of these aspects, in addition to setting specific key performance indicators, are essential for sustainability, robustness, and meeting intended outcomes. Our recommendations are in alignment with the literature findings regarding the value of designing a customized, holistic, longitudinal mentoring assessment tool in facilitating mentoring and providing timely and specific support to the evolving needs of mentees as they develop their clinical competencies . In addition to the importance of institutional support, adapting programs to local needs and resources, and mentors’ engagement and training for sustainability and performance . Mentorship programs can be instrumental, as we found in this study, in identifying challenges associated with postgraduate clinical training and executing promptly corrective measures. These challenges can be trainee-related (time management, study style, and physical, mental, and social well-being issues), training-setting-related, and implementation phase-related (COVID-19 pandemic in our situation). Mentorship was reported to be positively associated with specific academic performance, attitudes, and minimizing psychological stress . Addressing these challenges and facilitating identification by triangulating findings from different stakeholders supported the timely implementation of appropriate interventions and the optimization of results. The mentorship program has proven to be beneficial in ensuring trainees' smooth academic development and lowering the risks of failure to graduate. It improved trainees' overall well-being while also promoting work-life balance and reducing burnout. The CMFM program was a success story, in our hands, due to the inclusiveness of all stakeholders and the robustness of design, process, and monitoring despite the constraints of the COVID-19 pandemic. To the best of our knowledge, the format of the contextualized formal mentoring program and the mixed-method approach as well as the multiple levels of oversight are novel in this study. Despite its originality and the significance of its findings, this study has some limitations. The mentors’ reports might be prone to subjectivity. The COVID-19 pandemic and other confounders might affect trainees’ performance represented in GPA. However, this is out of the scope of this study. As a future perspective, more detailed qualitative studies targeting mentees and mentors probing their experience are highly recommended, particularly after the COVID-19 pandemic. Evaluating the level of satisfaction and the mentoring experience from their perspectives will provide another insight that we have not formally measured in the current study. A mentorship program implemented in the CMFM program of Arabian Gulf University integrated key performance indicators extracted from a parsimonious e-portfolio and mixed data from mentorship forms as well as periodic face-to-face meetings with different stakeholders. Triangulating longitudinal information using mixed methods design and analyzing at multiple levels permitted timely personalized pertinent interventions. |
Update of Robotic Surgery in Benign Gynecological Pathology: Systematic Review | 06c0ed9c-e271-4641-96b6-9d5b6dcadba7 | 9024779 | Gynaecology[mh] | In the last decades, minimally invasive surgery (MIS) was widespread both in benign and malignant pathologies . Furthermore, since the Food and Drug Administration (FDA) approval in 2005, the application of robotic surgery (RS) in gynecology was adopted all over the world . MIS is associated with a minor length of hospital stay, less blood loss, a reduction in postoperative pain, and superior long-term quality of life compared to the open approach . Furthermore, the MIS approach is also encouraged by Enhanced Recovery After Surgery (ERAS) recommendations as a tool to improve fast recovery after surgery . However, laparoscopy (LPS) and RS require a fair number of procedures for one to become confident with the surgical gestures, with a slow learning curve. LPS is characterized by two-dimensional visualization, a limited range of motions, difficulty with hand-eye coordination, and enhanced physiologic tremors . Therefore, the introduction of RS provided the same LPS advantages with additional improvements. Moreover, RS with the 3D visualization, wristed instrumentation, and improved ergonomics can facilitate the surgical gestures of inexperienced surgeons . However, due to the emerging technology and specific equipment, RS has higher costs and longer operative times compared to open and LPS approaches. In light of these data, the RS application presents known advantages for patients and surgeons but not always strong scientific evidence to support its use in clinical practice. The present study aimed to provide an update on RS in benign gynecological pathology by reporting the scientific recommendations and the high-value scientific literature available to date. For this purpose, only randomized clinical trials (RCT) and large retrospective cohort studies are discussed in the present review. A systematic review of the literature was performed in double-blind by two authors (VAC and ES). The analysis was conducted from September 2021 to January 2022. A third author (SC) checked the selected articles. Research on Pubmed, Web of Science, and Scopus was carried out using the following keywords: “robotic surgery” and “gynecology”, “robotic surgery” and “myomectomy”, “robotic surgery” and “hysterectomy”, “robotic surgery” and “endometriosis”, “robotic surgery” and “pelvic organ prolapse”, “robotic surgery” and “benign gynecological disease”. The agreement about potential relevance was reached by consensus of the researchers and according to PRISMA statement guidelines . After the first selection, the authors evaluated the full-text copies of selected papers and separately extracted relevant data regarding study characteristics and outcomes. All bibliographies were analyzed to evaluate additional eligible studies. Only RCT and retrospective cohort trials were included in the present review in order to synthesize the relevant evidence about the current role of RS. Studies considered not in line with the purpose of the study, prospective non-randomized trials, case reports, analysis with a small number of patients (<20 cases), redundant studies, and articles not in the English language were excluded. Since no RCTs comparing robotic myomectomy to other surgical techniques have been published yet, only retrospective cohort studies were included in this case. The electronic database search provided a total of 2130 studies published between 2005 and 2021. Of these, 258 duplicates, 781 case reports, 63 studies not in the English language, and 1009 works not fitting the review scope were excluded from the analysis. The study selection flowchart is shown in . Twenty-two studies were considered eligible for the study, eight studies regarding robotic myomectomy , five studies on robotic hysterectomy , five studies about RS in endometriosis treatment , and four studies on robotic pelvic organ prolapse (POP) treatment . Overall, 12 RCT and 10 retrospective studies were included in the analysis. The total of patients enrolled was 269,728, 1721 in the myomectomy group, 265,100 in the hysterectomy group, 1527 in the endometriosis surgical treatment group, and 1380 patients who received treatment for POP. To better illustrate the results of the research and describe scientific evidence about different gynecological procedures, the main findings are reported in chapters: robotic myomectomy, robotic hysterectomy, robotic endometriosis eradication, and robotic pelvic organ prolapse treatment. 3.1. Robotic Myomectomy Leiomyoma is the most common benign gynecologic tumor diagnosed in women during reproductive age. The true incidence in the general population is unknown because fibromas are often asymptomatic. However, almost 60% of women in reproductive age have fibroids . The most common clinical presentation is abnormal uterine bleeding, bulk symptoms, and infertility. Fibroids can be classified depending on their uterine localizations according to the International Federation of Gynecology and Obstetrics (FIGO) . Myomectomy is a safe treatment in symptomatic patients who desire to preserve their fertility. The fertility preserving surgical approach includes abdominal myomectomy (AM), laparoscopic myomectomy (LM), and robotic-assisted myomectomy (RAM). The appropriate surgical treatment should be individualized depending on myoma dimensions, number, localization, and surgeon skills. In a large prospective randomized trial published in 2000 comparing LM and AM, LPS was associated with a minor length of hospital stay, less blood loss, smaller scars, faster recovery, and a non-inferiority pregnancy rate . However, LPS is also characterized by some limitations. In the absence of a wide range of motion and limited visualization as in the case of a large uterus, laparoscopic dissection may be challenging. Besides, experts’ opinions suggest that LPS is contraindicated for fibroids greater than 10–12 cm and in the presence of more than three lesions requiring multiple uterine incisions. RM has gained wide acceptance because robotic endowrist instruments offer better maneuverability and facilitated sutures. Moreover, RS is comparable to LPS in terms of enhanced recovery, perioperative outcomes, and cosmetic results. Limitations may derive from the lack of haptic feedback, in particular in controlling strength in suturing, in case of need for closure of cavity defect after myomectomy, and the individuation and location of small myomas. Furthermore, the removal of large myomas could be laborious due to the reduction of the surgical field visibility. To date, no randomized clinical trials comparing RM to open or laparoscopic approaches are available in the literature. However, retrospective non-inferiority trials support RM feasibility. In 2007, Advincula et al. published a retrospective case-matched study including 58 patients with symptomatic leiomyomas undergoing AM or RAM. The results showed higher postoperative complications, greater blood loss, and longer hospital stays in the AM group. Nevertheless, higher costs and longer operative times were reported in the RAM group . In a retrospective analysis of 81 LM and RAM cases, Bedient et al. reported comparable short-term outcomes for both approaches, while long-term outcomes were not assessed . Along with these results, Nezhat et al., in a retrospective case-matched study of 50 patients (35 LM and 15 RAM), reported longer operative times in the RS group and comparable post-operative short-term outcomes when compared to LM. Furthermore, the same authors reported that the main RAM advantage was the flattened learning curve that could allow less experienced endoscopic surgeons to perform MIS . In 2012, with a large retrospective trial (115 LMs and 174 RAMs), Gargiulo et al. reported that LM and RAM have comparable short-term surgical outcomes . RAM had longer operative times and larger estimated blood loss; however, during LM, a higher rate of the barbed suture were performed (67.9% vs. 5%), with significant impact on suturing time and blood loss. Subsequently, Barakat et al. published a large retrospective study on 575 myomectomies, comparing AM, LA, and RAM. The authors found that RAM was associated with decreased blood loss and less length of hospital stay compared with traditional LPS and AM. Furthermore, RM and LM shared comparable advantages compared to open surgery in terms of perioperative outcomes. However, myoma diameters were significantly higher in the robotic and open surgery arms compared to the laparoscopic group . In line with these authors, Gobern et al. reported shorter hospital stays and decreased blood loss in the MIS group in a retrospective analysis of 308 procedures (169 AM, 73 LM, and 66 RAM) . In a recent large retrospective trial conducted by Özbaşlı et al., the authors reported the safety and feasibility of a robotic-assisted approach in patients with large uterine size and myomas. Moreover, RAM patients experienced significantly reduced post-operative pain compared to AM and LM patients . Long-term surgical outcomes were investigated in a retrospective study conducted by Flyckt et al. analyzing 133 myomectomies (80 AMs, 28 LM, and 25 RAM). After a median follow-up of eight years, women wishing for pregnancy showed a 55% pregnancy rate without a statistically significant difference in the three groups. Moreover, the bleeding symptom control was similar regardless of the surgical approach used . Furthermore, no cases of uterine rupture were reported in the MIS groups . In line with these authors, in a recent retrospective case series, Goldberg et al. reported a 70% pregnancy rate in 123 patients undergoing RAM . In conclusion, in the absence of randomized prospective trials, several noninferiority studies are now available to indicate that RAM is as effective and safe as conventional LM. Moreover, the value of RS could offer a minimally invasive approach to patients that otherwise would be treated with open surgery. The limitations are related to the higher costs and longer operating time. 3.2. Robotic Hysterectomy Hysterectomy is one of the most performed surgical procedures worldwide. In 90% of cases, benign pathologies are the main indication for the surgical procedure . Surgical approaches to benign hysterectomy include laparotomy, LPS, vaginal and robotic techniques . Over time, both open and vaginal approaches are decreasing in popularity, while the widespread adoption of robotic-assisted hysterectomy gave access to a larger number of patients to minimally invasive techniques, even in cases of severe obesity . First, in 2009 and subsequently in 2021, the American College of Obstetricians and Gynecologists (ACOG) recommended the MIS approach as the gold standard for hysterectomy. Furthermore, among minimally invasive techniques, the vaginal route should be the primary choice whenever feasible . Nevertheless, concern for malignancy, large uterine size, a fixed uterus, or the lack of confidence of the surgeon may preclude the vaginal approach. When the vaginal route is not indicated or feasible, LPS is mentioned as the preferred alternative to open surgery . Advantages of laparoscopic hysterectomy (LH) over open abdominal hysterectomy (AH) include decreased postoperative pain, shorter hospital stay, and quicker return to daily activities . However, the steep learning curve, counter-intuitive hand movement, as well as limited instruments movement and two-dimensional visualization are limitations of the technique . On the other hand, robotic hysterectomy (RH) requires a lower level of technical skill with more intuitive surgical gestures compared to LH . As a consequence, RH gained great popularity thanks to the easy adoption of the technique, even in the absence of strong evidence supporting RH over LH . Despite lacking a strict indication for use of robotic-assisted hysterectomy, the RH may represent a suitable minimally invasive option in less optimal candidates for LPS. In particular, RS may offer a favorable alternative in severely obese patients . In a population-based retrospective study conducted by Wright et al. on 264,758 women undergoing hysterectomy for benign disease, the authors found that LH and RH share comparable postoperative outcomes, although RS was associated with higher costs . RH advantages and disadvantages were also assessed in randomized clinical trials. In a blinded, prospective randomized controlled trial conducted by Paraiso et al., 53 patients were randomized to LH (n = 27) and RH (n = 26). The authors reported a low complication rate for both approaches without statistically significant differences between the two groups. No intraoperative lesions or need for transfusions was registered. Furthermore, RH was associated with longer operative time, good postoperative pain control, and a fast return to daily activities . In line with these results, Sarlos et al., in an RCT enrolling 95 patients who underwent LH or RH reported higher operating times in the robotic group and similar surgical and postoperative outcomes. Furthermore, patients enrolled in the RH arm reported a higher level of short term postoperative quality of life . Lonnerfors et al. in an RCT with 122 patients (61 LH vs. 61 RH), also reported better short-term outcomes and a lower rate of postoperative complications in RH compared to the LH group. Concerning operative times, there were no differences between LH and RH. This may suggest that, where RS is well implemented, operating room time is not affected . In agreement with this observation, Deimling et al. found no significant difference in operating time between LH and RH within the 144 cases analyzed. The mean operative time in the RH group was 73.9 min and 74.9 min in the LH group. The Authors concluded that RS when performed by experienced surgeons is not inferior to LPS in terms of operative time . In summary, to date, there are no clear indications for RH over other minimally invasive techniques. At present, the main indications include patient obesity, uterine size, and surgeons’ expertise. For benign pathologies, RS appears non-inferior to LPS in the hands of expert surgeons, but with increased costs. The main advantage provided by RH adoption is a greater number of patients who gained access to a minimally invasive approach. 3.3. Robotic Endometriosis Treatment Endometriosis is a chronic inflammatory condition that affects women during reproductive age. Endometriosis is associated with pelvic pain and infertility, but the severity of symptoms is not predictive of the stage of the disease. Endometriosis eradication is one of the most complex laparoscopic surgeries due to the distortion of the normal anatomy, adhesions, and hypomobility of the pelvic organs . Surgical treatment depends on symptoms, response to medications, and women’s fertility status. Currently, although MIS is the approach of choice, no indication as to which MIS approach to prefer is present in the literature . LPS is accepted as the preferred technique because of comparable outcomes to open surgery with the known advantages of MIS . To date, scientific evidence about RS in endometriosis cases is limited. Many studies report that RS in endometriosis is a feasible and safe option . However, most of these studies are retrospective in nature or with a limited number of cases reported. On the other hand, RS uses are reported in complex cases of deep infiltrating endometriosis with urinary and bowel involvement. The LAROSE trial is a prospective randomized clinical trial comparing LPS to RS in terms of operative times and perioperative outcomes in endometriotic disease. In the 53 patients enrolled (38 LPS vs. 35 RS), RS was shown to be non-inferior to LPS for both aspects. Even after adjustment for the stage of disease, operative times and quality of life after a six-month follow-up were similar. Further evidence comes from retrospective clinical trials comparing robotic and laparoscopic approaches. These retrospective studies showed minor operative times for LPS and superimposable complication rates compared to RS. However, due to their retrospective nature, comparison between LPS and RS is limited by the lack of randomization and the heterogeneity of stage of disease between the two approaches. Furthermore, surgeon experience and the need for other specialists in advanced stages should also be investigated. In conclusion, MIS is the gold standard for endometriosis surgical treatment. Currently, both robotic and LPS are acceptable techniques for endometriosis surgical treatment. RS offers enhanced visualization and higher dexterity that can overcome some LPS limitations. Furthermore, according to the current evidence, RS could be the best option in complex cases with deep infiltrating endometriosis. 3.4. Robotic Pelvic Organ Prolapse Treatment Pelvic organ prolapse (POP) is a common cause of morbidity in women with a remarkable impact on quality of life . Surgery can offer a wide range of options to restore pelvic anatomy and function. The surgical approach depends on the surgeon’s experience, patient’s performance status, age, and patient comorbidity. The gold standard surgical treatment for grade 2–4 vaginal vault prolapse is sacrocolpopexy. Sacrocolpopexy superiority compared to other techniques, such as sacrospinous vaginal apex suspension, has been confirmed in a randomized clinical trial . Furthermore, open abdominal sacrocolpopexy (ASC) is associated with optimal long-term outcomes. However, the advent of MIS and its known advantages compared to open surgery made its application in POP surgery an issue of interest. In a randomized clinical trial by Freeman in 2013, non-inferiority of LPS vs. ASC in terms of perioperative outcomes and anatomic restoration according to the Pelvic Organ Prolapse Quantification System (POP-Q) were equivalent . In addition, the robotic approach offers better visualization during dissection and easier suturing compared to LPS. As a consequence, RS may represent a feasible option for providing greater access to patients and surgeons to minimally invasive techniques due to a flatter learning curve . Robotic and laparoscopic sacrocolpopexy has been compared in randomized clinical trials. Paraiso et al. enrolled 78 patients with 2–4 stage POP, 38 in the laparoscopic group, and 40 in the robotic group. Robotic sacrocolpopexy (RSC) was associated with longer operative time, increased postoperative pain, higher costs, and no benefits in terms of the anatomic and functional success of the technique after a one-year follow-up compared to laparoscopic sacrocolpopexy (LSC) . Anger et al. randomized 78 patients with symptomatic POP to LSC and RSC. The primary outcome was to evaluate costs over the six weeks after surgery. Secondary outcomes were perioperative complications, postoperative pain, and clinical long-term outcomes after six months of follow-up. The results showed higher costs, longer operative time, and increased postoperative pain for the robotic approach with overlapping long-term outcomes compared to LSC. The author hypothesized that the lack of tactile feedback may hinder the surgeon’s control of pressure exerted on ports with a slight temporary increase in postoperative pain . Furthermore, a high success rate for minimally invasive sacrocolpopexy was confirmed in an ancillary analysis after a one-year follow-up without differences between the two groups . Illiano et al. in 2019 published a non-inferiority RCT comparing RSC to LSC for POP repair in patients with symptomatic POP-Q stage III-IV. Both arms showed excellent results with a 100% cure rate of apical compartment defect. RSC also showed a higher restoration rate of the anterior and posterior compartment compared to LSC but without statistical significance . In a large retrospective trial published by Nosti et al. on 1124 patients (589 ASC vs. 273 LSC vs. 262 RSC), the open approach was associated with a higher rate of intraoperative and postoperative complications compared to minimally invasive sacrocolpopexy (MISC). MISC was associated with less blood loss, minor length of hospital stay, and longer operative time compared to ASC, especially in the robotic group. Furthermore, RSC patients experienced a minor rate of postoperative complications compared to LSC . According to the available evidence, RSC may be considered a non-inferior alternative compared to LSC. The advantage provided by a flatter learning curve in the robotic approach may have value for surgeons with no experience in LPS. On the other hand, RS is associated with higher costs and longer operative times compared to LSC. Leiomyoma is the most common benign gynecologic tumor diagnosed in women during reproductive age. The true incidence in the general population is unknown because fibromas are often asymptomatic. However, almost 60% of women in reproductive age have fibroids . The most common clinical presentation is abnormal uterine bleeding, bulk symptoms, and infertility. Fibroids can be classified depending on their uterine localizations according to the International Federation of Gynecology and Obstetrics (FIGO) . Myomectomy is a safe treatment in symptomatic patients who desire to preserve their fertility. The fertility preserving surgical approach includes abdominal myomectomy (AM), laparoscopic myomectomy (LM), and robotic-assisted myomectomy (RAM). The appropriate surgical treatment should be individualized depending on myoma dimensions, number, localization, and surgeon skills. In a large prospective randomized trial published in 2000 comparing LM and AM, LPS was associated with a minor length of hospital stay, less blood loss, smaller scars, faster recovery, and a non-inferiority pregnancy rate . However, LPS is also characterized by some limitations. In the absence of a wide range of motion and limited visualization as in the case of a large uterus, laparoscopic dissection may be challenging. Besides, experts’ opinions suggest that LPS is contraindicated for fibroids greater than 10–12 cm and in the presence of more than three lesions requiring multiple uterine incisions. RM has gained wide acceptance because robotic endowrist instruments offer better maneuverability and facilitated sutures. Moreover, RS is comparable to LPS in terms of enhanced recovery, perioperative outcomes, and cosmetic results. Limitations may derive from the lack of haptic feedback, in particular in controlling strength in suturing, in case of need for closure of cavity defect after myomectomy, and the individuation and location of small myomas. Furthermore, the removal of large myomas could be laborious due to the reduction of the surgical field visibility. To date, no randomized clinical trials comparing RM to open or laparoscopic approaches are available in the literature. However, retrospective non-inferiority trials support RM feasibility. In 2007, Advincula et al. published a retrospective case-matched study including 58 patients with symptomatic leiomyomas undergoing AM or RAM. The results showed higher postoperative complications, greater blood loss, and longer hospital stays in the AM group. Nevertheless, higher costs and longer operative times were reported in the RAM group . In a retrospective analysis of 81 LM and RAM cases, Bedient et al. reported comparable short-term outcomes for both approaches, while long-term outcomes were not assessed . Along with these results, Nezhat et al., in a retrospective case-matched study of 50 patients (35 LM and 15 RAM), reported longer operative times in the RS group and comparable post-operative short-term outcomes when compared to LM. Furthermore, the same authors reported that the main RAM advantage was the flattened learning curve that could allow less experienced endoscopic surgeons to perform MIS . In 2012, with a large retrospective trial (115 LMs and 174 RAMs), Gargiulo et al. reported that LM and RAM have comparable short-term surgical outcomes . RAM had longer operative times and larger estimated blood loss; however, during LM, a higher rate of the barbed suture were performed (67.9% vs. 5%), with significant impact on suturing time and blood loss. Subsequently, Barakat et al. published a large retrospective study on 575 myomectomies, comparing AM, LA, and RAM. The authors found that RAM was associated with decreased blood loss and less length of hospital stay compared with traditional LPS and AM. Furthermore, RM and LM shared comparable advantages compared to open surgery in terms of perioperative outcomes. However, myoma diameters were significantly higher in the robotic and open surgery arms compared to the laparoscopic group . In line with these authors, Gobern et al. reported shorter hospital stays and decreased blood loss in the MIS group in a retrospective analysis of 308 procedures (169 AM, 73 LM, and 66 RAM) . In a recent large retrospective trial conducted by Özbaşlı et al., the authors reported the safety and feasibility of a robotic-assisted approach in patients with large uterine size and myomas. Moreover, RAM patients experienced significantly reduced post-operative pain compared to AM and LM patients . Long-term surgical outcomes were investigated in a retrospective study conducted by Flyckt et al. analyzing 133 myomectomies (80 AMs, 28 LM, and 25 RAM). After a median follow-up of eight years, women wishing for pregnancy showed a 55% pregnancy rate without a statistically significant difference in the three groups. Moreover, the bleeding symptom control was similar regardless of the surgical approach used . Furthermore, no cases of uterine rupture were reported in the MIS groups . In line with these authors, in a recent retrospective case series, Goldberg et al. reported a 70% pregnancy rate in 123 patients undergoing RAM . In conclusion, in the absence of randomized prospective trials, several noninferiority studies are now available to indicate that RAM is as effective and safe as conventional LM. Moreover, the value of RS could offer a minimally invasive approach to patients that otherwise would be treated with open surgery. The limitations are related to the higher costs and longer operating time. Hysterectomy is one of the most performed surgical procedures worldwide. In 90% of cases, benign pathologies are the main indication for the surgical procedure . Surgical approaches to benign hysterectomy include laparotomy, LPS, vaginal and robotic techniques . Over time, both open and vaginal approaches are decreasing in popularity, while the widespread adoption of robotic-assisted hysterectomy gave access to a larger number of patients to minimally invasive techniques, even in cases of severe obesity . First, in 2009 and subsequently in 2021, the American College of Obstetricians and Gynecologists (ACOG) recommended the MIS approach as the gold standard for hysterectomy. Furthermore, among minimally invasive techniques, the vaginal route should be the primary choice whenever feasible . Nevertheless, concern for malignancy, large uterine size, a fixed uterus, or the lack of confidence of the surgeon may preclude the vaginal approach. When the vaginal route is not indicated or feasible, LPS is mentioned as the preferred alternative to open surgery . Advantages of laparoscopic hysterectomy (LH) over open abdominal hysterectomy (AH) include decreased postoperative pain, shorter hospital stay, and quicker return to daily activities . However, the steep learning curve, counter-intuitive hand movement, as well as limited instruments movement and two-dimensional visualization are limitations of the technique . On the other hand, robotic hysterectomy (RH) requires a lower level of technical skill with more intuitive surgical gestures compared to LH . As a consequence, RH gained great popularity thanks to the easy adoption of the technique, even in the absence of strong evidence supporting RH over LH . Despite lacking a strict indication for use of robotic-assisted hysterectomy, the RH may represent a suitable minimally invasive option in less optimal candidates for LPS. In particular, RS may offer a favorable alternative in severely obese patients . In a population-based retrospective study conducted by Wright et al. on 264,758 women undergoing hysterectomy for benign disease, the authors found that LH and RH share comparable postoperative outcomes, although RS was associated with higher costs . RH advantages and disadvantages were also assessed in randomized clinical trials. In a blinded, prospective randomized controlled trial conducted by Paraiso et al., 53 patients were randomized to LH (n = 27) and RH (n = 26). The authors reported a low complication rate for both approaches without statistically significant differences between the two groups. No intraoperative lesions or need for transfusions was registered. Furthermore, RH was associated with longer operative time, good postoperative pain control, and a fast return to daily activities . In line with these results, Sarlos et al., in an RCT enrolling 95 patients who underwent LH or RH reported higher operating times in the robotic group and similar surgical and postoperative outcomes. Furthermore, patients enrolled in the RH arm reported a higher level of short term postoperative quality of life . Lonnerfors et al. in an RCT with 122 patients (61 LH vs. 61 RH), also reported better short-term outcomes and a lower rate of postoperative complications in RH compared to the LH group. Concerning operative times, there were no differences between LH and RH. This may suggest that, where RS is well implemented, operating room time is not affected . In agreement with this observation, Deimling et al. found no significant difference in operating time between LH and RH within the 144 cases analyzed. The mean operative time in the RH group was 73.9 min and 74.9 min in the LH group. The Authors concluded that RS when performed by experienced surgeons is not inferior to LPS in terms of operative time . In summary, to date, there are no clear indications for RH over other minimally invasive techniques. At present, the main indications include patient obesity, uterine size, and surgeons’ expertise. For benign pathologies, RS appears non-inferior to LPS in the hands of expert surgeons, but with increased costs. The main advantage provided by RH adoption is a greater number of patients who gained access to a minimally invasive approach. Endometriosis is a chronic inflammatory condition that affects women during reproductive age. Endometriosis is associated with pelvic pain and infertility, but the severity of symptoms is not predictive of the stage of the disease. Endometriosis eradication is one of the most complex laparoscopic surgeries due to the distortion of the normal anatomy, adhesions, and hypomobility of the pelvic organs . Surgical treatment depends on symptoms, response to medications, and women’s fertility status. Currently, although MIS is the approach of choice, no indication as to which MIS approach to prefer is present in the literature . LPS is accepted as the preferred technique because of comparable outcomes to open surgery with the known advantages of MIS . To date, scientific evidence about RS in endometriosis cases is limited. Many studies report that RS in endometriosis is a feasible and safe option . However, most of these studies are retrospective in nature or with a limited number of cases reported. On the other hand, RS uses are reported in complex cases of deep infiltrating endometriosis with urinary and bowel involvement. The LAROSE trial is a prospective randomized clinical trial comparing LPS to RS in terms of operative times and perioperative outcomes in endometriotic disease. In the 53 patients enrolled (38 LPS vs. 35 RS), RS was shown to be non-inferior to LPS for both aspects. Even after adjustment for the stage of disease, operative times and quality of life after a six-month follow-up were similar. Further evidence comes from retrospective clinical trials comparing robotic and laparoscopic approaches. These retrospective studies showed minor operative times for LPS and superimposable complication rates compared to RS. However, due to their retrospective nature, comparison between LPS and RS is limited by the lack of randomization and the heterogeneity of stage of disease between the two approaches. Furthermore, surgeon experience and the need for other specialists in advanced stages should also be investigated. In conclusion, MIS is the gold standard for endometriosis surgical treatment. Currently, both robotic and LPS are acceptable techniques for endometriosis surgical treatment. RS offers enhanced visualization and higher dexterity that can overcome some LPS limitations. Furthermore, according to the current evidence, RS could be the best option in complex cases with deep infiltrating endometriosis. Pelvic organ prolapse (POP) is a common cause of morbidity in women with a remarkable impact on quality of life . Surgery can offer a wide range of options to restore pelvic anatomy and function. The surgical approach depends on the surgeon’s experience, patient’s performance status, age, and patient comorbidity. The gold standard surgical treatment for grade 2–4 vaginal vault prolapse is sacrocolpopexy. Sacrocolpopexy superiority compared to other techniques, such as sacrospinous vaginal apex suspension, has been confirmed in a randomized clinical trial . Furthermore, open abdominal sacrocolpopexy (ASC) is associated with optimal long-term outcomes. However, the advent of MIS and its known advantages compared to open surgery made its application in POP surgery an issue of interest. In a randomized clinical trial by Freeman in 2013, non-inferiority of LPS vs. ASC in terms of perioperative outcomes and anatomic restoration according to the Pelvic Organ Prolapse Quantification System (POP-Q) were equivalent . In addition, the robotic approach offers better visualization during dissection and easier suturing compared to LPS. As a consequence, RS may represent a feasible option for providing greater access to patients and surgeons to minimally invasive techniques due to a flatter learning curve . Robotic and laparoscopic sacrocolpopexy has been compared in randomized clinical trials. Paraiso et al. enrolled 78 patients with 2–4 stage POP, 38 in the laparoscopic group, and 40 in the robotic group. Robotic sacrocolpopexy (RSC) was associated with longer operative time, increased postoperative pain, higher costs, and no benefits in terms of the anatomic and functional success of the technique after a one-year follow-up compared to laparoscopic sacrocolpopexy (LSC) . Anger et al. randomized 78 patients with symptomatic POP to LSC and RSC. The primary outcome was to evaluate costs over the six weeks after surgery. Secondary outcomes were perioperative complications, postoperative pain, and clinical long-term outcomes after six months of follow-up. The results showed higher costs, longer operative time, and increased postoperative pain for the robotic approach with overlapping long-term outcomes compared to LSC. The author hypothesized that the lack of tactile feedback may hinder the surgeon’s control of pressure exerted on ports with a slight temporary increase in postoperative pain . Furthermore, a high success rate for minimally invasive sacrocolpopexy was confirmed in an ancillary analysis after a one-year follow-up without differences between the two groups . Illiano et al. in 2019 published a non-inferiority RCT comparing RSC to LSC for POP repair in patients with symptomatic POP-Q stage III-IV. Both arms showed excellent results with a 100% cure rate of apical compartment defect. RSC also showed a higher restoration rate of the anterior and posterior compartment compared to LSC but without statistical significance . In a large retrospective trial published by Nosti et al. on 1124 patients (589 ASC vs. 273 LSC vs. 262 RSC), the open approach was associated with a higher rate of intraoperative and postoperative complications compared to minimally invasive sacrocolpopexy (MISC). MISC was associated with less blood loss, minor length of hospital stay, and longer operative time compared to ASC, especially in the robotic group. Furthermore, RSC patients experienced a minor rate of postoperative complications compared to LSC . According to the available evidence, RSC may be considered a non-inferior alternative compared to LSC. The advantage provided by a flatter learning curve in the robotic approach may have value for surgeons with no experience in LPS. On the other hand, RS is associated with higher costs and longer operative times compared to LSC. Currently, a minimally invasive approach is suggested in benign gynecological pathologies. According to the available evidence, RS has comparable clinical outcomes compared to LPS, but at the expense of higher costs and longer operating times. On the other hand, the introduction of RS has allowed a growing number of patients to gain access to MIS and benefit from a minimally invasive treatment due to a flattened learning curve and enhanced dexterity and visualization. |
Improving healthcare delivery at a district hospital through teaching interns – A short report | 47b9e0c1-5f59-4a48-bcb7-006aeaf6bc78 | 11079332 | Family Medicine[mh] | Currently, in the Malawi healthcare system, clinical officers (COs) and medical assistants (MAs) remain the main providers of primary healthcare. , Family medicine is relatively new to Malawi’s healthcare system. One of the aims of family medicine is to strengthen and improve the primary health care provision though collaborating, mentoring and teaching other members of the healthcare team. , The leadership role of family physician is also one of the main competencies. While this report focuses on teaching and mentoring roles, it should be noted that these roles are made possible and anchored by the leadership role. Every district in Malawi has at least two doctors managing the social and healthcare needs of the local population. The medical doctors in the district are involved in administrative work and have minimal time for clinical practice. As such, in most district hospitals COs form the backbone of patient care provision. These are cadres that have a 3-year training in clinical medicine; they work side by side with MAs and nurses. The MAs have 2-year training in clinical medicine and are awarded with a certificate in clinical medicine after graduating. Clinical officers learn the science of medicine and put it into clinical practice in much the same way doctors do in medical schools although CO study involves less detail because of the shorter training schedule. They are awarded a diploma in clinical medicine when they graduate. After graduating, they must complete a year of clinical practice in a district hospital under the supervision of qualified COs or available medical doctors; this is called a CO internship. Medical Assistants work under the supervision of COs in managing non-complicated medical cases. They may run rural primary care clinics, with or without a CO on site, and they refer complicated conditions to the district hospitals. Intern medical assistants (IMOs), like intern clinical officers (ICOs), do their internship at the district hospitals. Apart from MoH workforce, the Department of Family Medicine (FM) of Kamuzu University of Health Sciences (KUHeS) has its district base at Mangochi District Hospital (MDH). Family physicians and residents from FM department assist in provision of mentorship and teaching to other cadres. One must understand that work-based learning takes various strategies and approaches. Mangochi District Hospital interns (ICOs or IMAs) are the front-liners for the 24 h on-call team. However, observations from patient handover meetings between on-call and day shift team revealed a gap in medical knowledge among interns. Thus, the clinical lecture series (CLS) specifically focused on management of common primary care conditions. Clinical lecture series are sessions organised by the family medicine department that are done every 2 weeks with the aim of improving the medical knowledge and skills of the IMOs and ICOs. This report focuses on deliberate mentorship and support to address the gaps identified in the medical knowledge and skills of IMOs and ICOs. Family medicine residents also strengthen their teaching skills through facilitation of CLS session as future consultants and leaders. In brief, the following gaps were identified before starting CLS sessions: (1) inadequate knowledge in making diagnosis and management of the common conditions presenting to MDH, (2) lack of practical skills in the common procedures, such as thoracentesis and others and (3) lack of use of evidence-based medicine and its application to the local clinical setting. In 2016, the FM department joined with MoH, taking an active role in the training of the intern COs and intern MAs through CLS. Later, interns from laboratory and pharmacy also joined the classes. To make the teaching more appropriate and useful, the interns themselves choose the topics to be covered and who among them will prepare the presentation. The job of a family physician or resident is to facilitate the class, adding more knowledge to the interns’ presentations and making corrections. To avoid disrupting daily work schedules, these classes take place during lunch hour from noon to 13:30. In the beginning, the turnout was not very good, with perhaps half of the interns attending, that is, less than 15. At very period, MDH tends to have about 30 interns. However, these lectures have grown into one of the most attended classes. Currently, more than 90% of the interns at MDH attend, that is, 28–30 interns. The teaching focuses on basic medical science, evidence-based medicine, best clinical practice as well as clinical skills; common primary health care patient conditions are included. The practical sessions include common emergency room interventions such as cardiopulmonary resuscitation (CPR), advanced cardiac life support (ACLS), common procedures such as thoracentesis and suturing techniques. Manikins are used in some of these sessions. The sessions are relevant to the interns’ clinical practice. The knowledge gained from these lectures includes how to apply the skills to the clinical scenarios that interns come across quite often in their hospital work. Practical sessions, special physical examination or practical sessions are included. Scientific evidence and management of diseases are adapted to the resource-limited setting of the district hospital. Through interviews with Intern COs (ICOs) and Intern MAs (IMAs) who have been part of this CLS, it was revealed that the teaching sessions had been very important in their professional development and healthcare provision. They liked the fact that they were preparing the topics and presentations. The classes take a form of a flipped class, in which learners are given material and prepare before the class is delivered; the active involvement in their own teaching was the most cited positive thing in the feedback interviews. Some other important points were change of clinical practice: on learning new information such as the management of a particular condition, participants reported that they changed their clinical practice according to the newly gained knowledge. They reported: improved confidence in their ability to effectively manage difficult cases. improved teamwork and collaboration among the interns. We conducted two focus group discussions (FGDs). One group consisted of 15 senior interns or qualified COs or MAs, the other one consisted of 15 junior internees at MDH who were regular attendees of CLS. Both groups expressed that CLS was important to their career development. They overall agreed it was one feature that attracted them to come learn at MDH and it improved their ability to care for patients. summaries themes and comments on outcomes unveiled from FGDS on CLS sessions. Here are couple samples of the extracts from the FGDs showing influence on career development and perceived improved clinical practice: ‘I decided to do my internship at MDH after my friends had told me it was a good place for learning; I was told, with the presence of Family Medicine doctors, there is a very good intern-mentorship program.’ (male, junior intern clinical officer, 26 years old) ‘CLS has assisted me to always look beyond the guidelines and find scientifically sound alternatives to guidelines when I am stuck.’ (male, clinical officer, 29 years old) Over the years the CLS have become one of the unique features of MDH in terms of interns’ mentorship. The feedback shows that CLS capacitated lower cadres (ICOs and IMAs) of Mangochi District Hospital in healthcare provision. Clinical lecture series have proved as effective learning tool during internship training. The classes have also improved the relationship between the MoH team and the FM department members. The improvement in patient management cannot be over-emphasised. We have seen a general change in the perception by the MoH team of our FM presence at MDH; while in the beginning, we could be perceived as a parallel structure, now the CLS is recognised as part of the formal teaching programme for internees at MDH. The CLS are very important especially to the residents of FM; their active participation and facilitation of the CLS prepare them for the lifetime roles of a teacher, a consultant and a leader in primary health care. Following internship, the relation continues for those clinicians who remain to work at MDH. Our ability to provide meals during the sessions is supported by the NGO Seed Global Health, which has sustained the presentation of these lectures for years. However, this may not be feasible in settings with financial constraints and without external support. So, a different arrangement or time may be needed. The CLS programme has proved to be a good tool to facilitate mentorship of junior cadres in the primary health care at district hospital. The presence of family medicine at Mangochi District Hospital has contributed to teaching and improved skills of the COs and MA internees. The authors would like to acknowledge the contribution of SEEDS Global Health for funding the continuous lecture series (CLS), through the Department of medicine; we also acknowledge the contribution by the Department of Family Medicine in delivering the lectures; we acknowledge all current and previous interns at Mangichi District Hospital for their very important feedback on CLS. |
Was ist neu im Management der peripheren arteriellen Verschlusskrankheit und der aortalen Erkrankungen? | 998f4f02-c054-417a-be30-b7b92f041e5c | 11772412 | Internal Medicine[mh] | Die neuen Leitlinien der European Society of Cardiology (ESC) zum Management der peripheren arteriellen Krankheit und von Aortenerkrankungen („peripheral arterial and aortic diseases“, PAAD) beinhalten einige Neuerungen, die im Folgenden zusammengefasst werden sollen . Bemerkenswert ist die Zusammenlegung von 2 zuvor separat geführten Leitliniendokumenten (ESC-Leitlinie zu Aortenerkrankungen von 2014 und ESC-Leitlinie zur peripheren arteriellen Verschlusskrankheit [pAVK] von 2017) zu einem Dokument . Im aktuellen ESC-Leitliniendokument wird auf die zunehmende Komplexität von Patienten mit pAVK und Aortenerkrankungen hingewiesen, und es werden ein ganzheitlicher Zugang sowie Multidisziplinarität im Patientenmanagement gefordert . Eine weitere Neuerung zeigt sich bei der Nomenklatur. Im 2017 publizierten ESC-Leitliniendokument zur pAVK wurde die Bezeichnung „periphere arterielle Erkrankung“ („peripheral arterial disease“, PAD) als übergeordneter Begriff für sämtliche peripheren, nichtkoronaren Gefäßerkrankungen definiert . Unter diesem Überbegriff wurden atherosklerotische Erkrankungen der Karotiden und Vertebralarterien, der Arterien der oberen Extremitäten, der Mesenterialarterien, der Nierenarterien sowie der Arterien der unteren Extremitäten („lower extremity artery disease“, LEAD) zusammengefasst. In der aktuellen ESC-Leitlinie 2024 wird der 2017 eingeführte Begriff LEAD nicht mehr verwendet, und die pAVK der unteren Extremität wird, wie schon vor der früheren Leitlinie, als PAD bezeichnet . Wie schon in der ESC-Leitlinie zur pAVK von 2017 wird im aktuellen Leitliniendokument die bei pAVK vorkommende Koprävalenz von koronarer und zerebrovaskulärer Atherosklerose unterstrichen, was das hohe Risiko koronarer und zerebrovaskulärer Ereignisse bei Patienten mit pAVK erklärt. Für das Management der pAVK wurden folglich als Therapieziele die Reduktion des Risikos von MACE („major adverse cardiac events“), die Reduktion des Risikos von MALE („major adverse limb events“) und die Verbesserung der Lebensqualität festgelegt . Bei Aortenerkrankungen stellen die Prävention, die Screeningmaßnahmen und die rechtzeitige Initiierung der Therapie wesentliche Maßnahmen zur Reduktion der Morbidität und Mortalität dar. In der aktuellen ESC-Leitlinie wird empfohlen, bei Patienten ab 65 Jahre, insbesondere bei Vorliegen von kardiovaskulären Risikofaktoren, ein pAVK-Screening in Erwägung zu ziehen . Bei Patienten mit 2 oder mehr kardiovaskulären Risikofaktoren kann ein pAVK-Screening unabhängig vom Alter erwogen werden . Als Screeningmethode wird die Ermittlung des Köchel-Arm-Index („ankle-brachial index“, ABI) empfohlen. Ein ABI von 0,9 oder weniger kann dabei als diagnostisches Kriterium für das Vorliegen einer pAVK gewertet werden . Bei Patienten mit Diabetes mellitus oder chronischer Nierenfunktionseinschränkung kann es infolge einer Mediasklerose zur Messung falsch-hoher Knöcheldruckwerte und folglich zur Ermittlung falsch-hoher ABI-Werte (> 1,4) kommen. In diesem Fall besteht eine I/B-Empfehlung, alternativ zum ABI die Messung des Zehendrucks, die Ermittlung des Zehen-Arm-Index („toe-brachial index“) oder Dopplerkurvenanalysen einzusetzen (; Abb. ). Je nach klinischer Präsentation werden unterschiedliche pAVK-Stadien unterschieden (Fontaine- oder Rutherford-Klassifikation). Im aktuellen ESC-Leitliniendokument wird eine Gruppierung in „asymptomatische pAVK“ (entspricht dem Fontaine-Stadium I), „symptomatische (belastungsabhängige) pAVK“ (entspricht den Fontaine-Stadien IIa und IIb) und „chronische beinbedrohende Ischämie“ („chronic limb-threatening ischemia“ [CLTI]; entspricht Fontaine-Stadium III und IV) vorgeschlagen (Tab. ). Um der trotz vereinfachter Klassifikation für pAVK-Patienten bestehenden Vielfalt von klinischen Erscheinungsbildern nachzukommen, führt das aktuelle ESC-Leitliniendokument den Begriff der pAVK-Syndrome ein. Der Begriff „pAVK-Syndrom“ beschreibt das mögliche Vorliegen chronischer Wunden in unterschiedlichen klinischen Stadien der pAVK . Ziel dieser Begrifflichkeit ist, das Vorhandensein einer chronischen Wunde bei pAVK-Patienten vom Vorliegen einer Minderperfusion, wie bei Vorliegen einer CLTI, zu entkoppeln. Um Wunden standardisiert zu charakterisieren und den Einfluss von Minderperfusion, Infektion und Wundausmaß zu graduieren, wird die Verwendung der WIfI(„wound, ischemia and foot infection“)-Klassifikation empfohlen . Im Behandlungsalgorithmus für pAVK-Syndrome wird empfohlen, ein Gefäßteam in die Entscheidungsfindung bezüglich der besten Therapieoptionen einzubeziehen. Für Patienten mit symptomatischer (belastungsabhängiger) pAVK empfehlen die aktuellen ESC-Leitlinien ein supervidiertes Gehtraining (Klasse I, Evidenzgrad A; ). Das Gehtraining sollte bevorzugt an einer Gesundheitseinrichtung (3-mal pro Woche, 30 min pro Einheit, über eine Periode von ≥ 3 Monaten) stattfinden. Sollte das Gehtraining an einer Gesundheitseinrichtung nicht verfügbar sein, wird ein strukturiertes heimbasiertes Gehtraining mit Supervision (Kontrollvisiten oder Telefonate und Trainingsdokumentation mit Logbüchern) empfohlen . Einen weiteren Schwerpunkt setzt die aktuelle ESC-Leitlinie auf die Nachuntersuchung der Patienten mit pAVK nach Therapieinitiierung. Im Rahmen der klinischen Evaluation soll neben dem kardiovaskulären Risikoprofil und dem klinischen Stadium der pAVK die Lebensqualität der Patienten standardisiert erhoben und dokumentiert werden . Bei symptomatischer (belastungsabhängiger) pAVK besteht bei fehlender Besserung der Lebensqualität eine IIb/B-Empfehlung für die Planung eines revaskularisierenden Eingriffs . Bei endovaskulären Eingriffen wird femoropopliteal der Einsatz medikamentenbeschichteter Ballone oder Stents zur Reduktion des Rezidivrisikos nach erfolgreicher Revaskularisation empfohlen . Bei langstreckigen femoropoplitealen Verschlüssen sollte bei guter Venenverfügbarkeit und niedrigem operativen Risiko eine chirurgische Venenbypassanlage in Erwägung gezogen werden . Bei Vorliegen einer CLTI (Tab. ) besteht in der ESC-Leitlinie eine klare Empfehlung für eine zeitnahe Revaskularisation zum Extremitätenerhalt . In diesem Zusammenhang wird bei CLTI eine Bildgebung der gesamten betroffenen Extremität empfohlen, um den Eingriff planen zu können . Zur Reduktion des Risikos ischämischer Ereignisse und zur Vermeidung eines erhöhten Blutungsrisikos wird ein individuelles Vorgehen in der antithrombotischen Therapie empfohlen. In diesem Zusammenhang sollten die klinische Präsentation („limb presentation“), das jeweilige Blutungsrisiko und die ggf. bestehende Notwendigkeit einer Antikoagulation aufgrund einer Komorbidität (z. B. Vorhofflimmern) berücksichtigt werden. Hervorgehoben wird in der aktuellen ESC-Leitlinie bei symptomatischer pAVK mit niedrigem Blutungsrisiko die „dual-pathway inhibition“ mit Acetylsalicylsäure (ASS) und niedrig dosiertem Rivaroxaban (1-mal täglich ASS 100 mg plus 2‑mal täglich Rivaroxaban 2,5 mg; ). Das Management von Patienten mit pAVK und aortalen Erkrankungen ist charakterisiert durch nichtmedikamentöse und medikamentöse Maßnahmen, sowie – bei ausgewählten Patienten – durch endovaskuläre oder chirurgische Interventionen. Hinsichtlich Lebensstilmodifikation wird im aktuellen ESC-Leitliniendokument erstmals eine Ernährungsempfehlung für Patienten mit pAVK und Aortenerkrankungen abgegeben (ausgewogene mediterrane ballaststoff- und flavonoidreiche Ernährung mit hohem Anteil an Hülsenfrüchten; ). Zusätzlich werden die Patientenaufklärung und verhaltensmodifizierende Interventionen mit dem Ziel der Nikotinkarenz empfohlen, wobei unterstützend vorübergehend E‑Zigaretten zum Einsatz gebracht werden können . Bei der medikamentösen Therapie spielen neben der oben genannten antithrombotischen Medikation die antihypertensive und die lipidsenkende Therapie sowie die Behandlung des Diabetes eine wesentliche Rolle, um das kardiovaskuläre Risiko zu senken. Bei pAVK besteht eine Klasse-I/Evidenzgrad-A-Empfehlung zu einer Statintherapie . Das LDL(„low-density lipoprotein“)-Cholesterin(LDL-C)-Ziel liegt bei weniger als 55 mg/dl, und das Ausgangs-LDL‑C sollte um mehr als 50 % im Vergleich zum Ausgangswert gesenkt werden . Wird das LDL-C-Ziel mit einer maximal tolerierten Statindosis nicht erreicht, sollte Ezitimib ergänzt werden . Reicht diese Kombination weiterhin nicht aus, um das LDL-C-Ziel zu erreichen, findet sich im aktuellen ESC-Leitliniendokument eine I/A-Empfehlung für den Einsatz eines PCSK9(Proproteinkonvertase Subtilisin/Kexin Typ 9)-Inhibitors . Bei Statinunverträglichkeit und fehlendem Erreichen des LDL-C-Ziels mit Ezitimib sollte Bempedoinsäure oder eine Kombination aus Bempedoinsäure mit einem PCSK9-Inhibitor zum Einsatz kommen . Bei der Blutdrucktherapie besteht im aktuellen Leitliniendokument eine I/A-Empfehlung für ein systolisches Blutdruckziel von 120–129 mm Hg, wobei primär ACE(„angiotensin-converting enzyme“)-Hemmer oder Angiotensinrezeptorblocker zum Einsatz kommen sollten . Es wird in 2 separaten Empfehlungen ein etwas liberaleres Blutdruckziel von weniger als 140/90 mm Hg für fragile Patienten mit pAVK und Aortenerkrankungen empfohlen (Alter ≥ 85 Jahre, Pflegebedürftigkeit, orthostatische Hypotension, Lebenserwartung < 3 Jahre; ). Die Empfehlungen für die antihypertensive Medikation wurden mit den zeitgleich veröffentlichten ESC-Leitlinien zum Management erhöhten Blutdrucks in Einklang gebracht . Beim Management von Diabetes mellitus besteht für Patienten mit pAVK und Aortenerkrankungen ein HbA 1c (Hämoglobin A 1c )-Ziel von weniger als 7 % (Empfehlungsgrad I, Evidenzgrad A), wobei auch hier eine Individualisierung des HbA 1c -Ziels in Abhängigkeit von den Komorbiditäten und der Lebenserwartung als Konsensusempfehlung ergänzt wurde . In Analogie zu der vor 1 Jahr publizierten ESC-Leitlinie zu kardiovaskulären Erkrankungen bei Diabetes empfiehlt auch das aktuelle Leitliniendokument unabhängig vom HbA 1c -Wert SGLT(„sodium-glucose linked transporter 2“)-Hemmer oder GLP(„glucagon-like peptide“)-1-Rezeptor-Agonisten mit nachgewiesenem kardiovaskulärem Nutzen . Karotis- und Subklaviastenosen Das ESC-Leitliniendokument beinhaltet für Patienten mit 2 oder mehr kardiovaskulären Risikofaktoren eine IIb/C-Empfehlung für ein sonographisches Screening hinsichtlich einer Karotisstenose . Für Patienten mit diagnostizierter Karotisstenose werden – in Abhängigkeit vom Fehlen oder Vorliegen einer zugehörigen neurologischen Symptomatik – 2 separate Behandlungsalgorithmen vorgeschlagen. Die zugrunde liegende Quantifizierung des Stenosegrades erfolgt primär sonographisch (PSV [„peak systolic velocity“]: 125–230 cm/s oder ≥ 180 cm/s oder ≥ 125 cm/s, gemeinsam mit dem Vorliegen einer Ratio der PSV aus der A. carotis interna und der PSV aus der A. carotis communis ≥ 2 als Kriterien für einen Stenosegrad von 50–69 %; PSV > 230 cm/s als Kriterium für einen Stenosegrad ≥ 70 %), wobei bei symptomatischer Karotisstenose eine weiterführende Bildgebung mittels Computertomographie-Angiographie (CTA) oder Magnetresonanzangiographie (MRA) durchgeführt werden sollte . Bei asymptomatischer wie auch bei symptomatischer Karotisstenose wurde in den aktuellen Empfehlungen die optimale medikamentöse Therapie (OMT) zu Beginn der jeweiligen Behandlungsalgorithmen hervorgehoben . Eine klare I/A-Empfehlung für eine zeitnahe Revaskularisation besteht lediglich bei der symptomatischen Karotisstenose mit einem Stenosegrad von 70–99 % . Bei 50–69 % einer symptomatischen Karotisstenose kann eine Revaskularisation in Erwägung gezogen werden (Klasse-IIa/A-Empfehlung). Bei asymptomatischer 60–99 %iger Karotisstenose wird empfohlen, eine Revaskularisation individuell, d. h. in Abhängigkeit vom jeweiligen Risikoprofil bzw. vom Eingriffsrisiko, in Erwägung zu ziehen (Klasse-IIb/B-Empfehlung; ). Die Revaskularisationsmethode (Karotisdesobliteration vs. Karotisstent) soll ebenso in Abhängigkeit von klinischen und anatomischen Gegebenheiten gewählt werden: klinische Risikofaktoren: Herzinsuffizienz (NYHA [New York Heart Association] III/IV), instabile Angina pectoris (CCS [Canadian Cardiovascular Society] III/IV), koronare Herzkrankheit mit Hauptstammläsion oder > Einkoronargefäßerkrankung mit 70 %iger Stenose, rezenter Myokardinfarkt (< 30 Tage), geplante Herzoperation (< 30 Tage), linksventrikuläre Ejektionsfraktion (LVEF) < 30 %, schwere Lungenerkrankung, schwere Nierenfunktionseinschränkung; anatomische Risikofaktoren: chirurgisch schwierig/nicht zugängliche Läsionen (Höhe Wirbelkörper C2 oder darüber, unter dem Schlüsselbein), ipsilaterale Strahlentherapie, immobilisierte Halswirbelsäule, kontralateraler Karotisverschluss (erhöhtes Schlaganfallsrisiko), kontralaterale Rekurrensparese, Tracheostoma. Zum nicht-invasiven Screening hinsichtlich Subklaviastenosen werden bei Patienten mit pAVK oder aortalen Erkrankungen beidseitige Blutdruckmessungen empfohlen (Blutdruckdifferenz > 10–15 mm Hg hinweisgebend für eine Subklaviastenose; ). Bei Vorliegen einer symptomatischen Subklaviastenose besteht im ESC-Leitliniendokument eine IIa/B-Empfehlung für eine Revaskularisation, wobei ein endovaskulärer Zugang einem offen-chirurgischen Vorgehen vorgezogen werden sollte . Eine routinemäßige Revaskularisation von asymptomatischen Subklaviastenosen wird nicht empfohlen . Nierenarterienstenose Bei Verdacht auf das Vorliegen einer Nierenarterienstenose empfehlen die ESC-Leitlinien auch primär eine duplexsonographische Diagnostik (Kriterien: PSV ≥ 200 cm/s entspricht > 50 %iger Stenose, renoaortale Ratio der PSV ≥ 3,5 entspricht ≥ 60 %iger Stenose bzw. Seitendifferenz des intrarenalen Widerstandsindex ≥ 0,5; ). Bei inkonklusivem duplexsonographischen Befund oder Verdacht auf das Vorliegen einer Nierenarterienstenose besteht eine I/B-Indikation für eine weiterführende Bildgebung mittels CTA oder MRA. Bei Vorliegen von Risikomarkern (rasch progredienter therapierefraktärer Hypertonus, rasche Verschlechterung der Nierenfunktion, hypertensives Lungenödem, Vorliegen einer Einzelniere) und erhaltener Nierenvitalität (Nierengröße > 8 cm, Nierenkortex > 0,5 cm, Albumin-Kreatinin-Ratio < 20 mg/mmol, Widerstandsindex < 0,8) wird nach OMT eine invasive katheterbasierte Abklärung mit ggf. Stentimplantation empfohlen . Ähnliches gilt für bilaterale Nierenarterienstenosen und für Patienten mit fibromuskulärer Dysplasie, wobei bei Letzterer eine primäre Ballondilatation gegenüber einer primären Stentimplantation bevorzugt werden sollte. Von einer routinemäßigen Revaskularisation einer einseitigen Nierenarterienstenose ohne Vorliegen oben genannter Risikomarker wird abgeraten, ebenso bei Fehlen der Nierenvitalität (Empfehlungsgrad III, Evidenzgrad A; ). Stenosen der Mesenterialarterien Bei Verdacht auf chronische atherosklerotische Stenosen der Mesenterialarterien empfiehlt das ESC-Leitliniendokument zur bildgebenden Diagnostik eine CTA . Je nach bildgebendem Befund werden atherosklerotische Stenosen von der nichtokklusiven mesenterialen Ischämie (NOMI) und vom Ligamentum-arcuatum-Syndrom unterschieden. Unter dem Begriff NOMI wird eine mesenteriale Ischämie verstanden, welche sich nicht auf Verschlüsse großer Mesenterialarterien zurückführen lässt, sondern auf eine Minderperfusion in der mesenterialen Endstrombahn, häufig infolge einer Vasokonstriktion in diesem Bereich, wie z. B. bei schwerer Herzinsuffizienz. Das Ligamentum-arcuatum-Syndrom benennt ein Kompressionssyndrom des Truncus coeliacus durch das Ligamentum arcuatum des Zwerchfells, was folglich zu einer symptomatischen Perfusionsstörung führen kann. Während eine Revaskularisation von asymptomatischen Stenosen abgelehnt wird (III/C), empfiehlt das aktuelle ESC-Leitliniendokument bei symptomatischen Stenosen die Einbindung eines Gefäßteams in den Entscheidungsprozess hinsichtlich der Revaskularisationsindikation und -methode. Das ESC-Leitliniendokument beinhaltet für Patienten mit 2 oder mehr kardiovaskulären Risikofaktoren eine IIb/C-Empfehlung für ein sonographisches Screening hinsichtlich einer Karotisstenose . Für Patienten mit diagnostizierter Karotisstenose werden – in Abhängigkeit vom Fehlen oder Vorliegen einer zugehörigen neurologischen Symptomatik – 2 separate Behandlungsalgorithmen vorgeschlagen. Die zugrunde liegende Quantifizierung des Stenosegrades erfolgt primär sonographisch (PSV [„peak systolic velocity“]: 125–230 cm/s oder ≥ 180 cm/s oder ≥ 125 cm/s, gemeinsam mit dem Vorliegen einer Ratio der PSV aus der A. carotis interna und der PSV aus der A. carotis communis ≥ 2 als Kriterien für einen Stenosegrad von 50–69 %; PSV > 230 cm/s als Kriterium für einen Stenosegrad ≥ 70 %), wobei bei symptomatischer Karotisstenose eine weiterführende Bildgebung mittels Computertomographie-Angiographie (CTA) oder Magnetresonanzangiographie (MRA) durchgeführt werden sollte . Bei asymptomatischer wie auch bei symptomatischer Karotisstenose wurde in den aktuellen Empfehlungen die optimale medikamentöse Therapie (OMT) zu Beginn der jeweiligen Behandlungsalgorithmen hervorgehoben . Eine klare I/A-Empfehlung für eine zeitnahe Revaskularisation besteht lediglich bei der symptomatischen Karotisstenose mit einem Stenosegrad von 70–99 % . Bei 50–69 % einer symptomatischen Karotisstenose kann eine Revaskularisation in Erwägung gezogen werden (Klasse-IIa/A-Empfehlung). Bei asymptomatischer 60–99 %iger Karotisstenose wird empfohlen, eine Revaskularisation individuell, d. h. in Abhängigkeit vom jeweiligen Risikoprofil bzw. vom Eingriffsrisiko, in Erwägung zu ziehen (Klasse-IIb/B-Empfehlung; ). Die Revaskularisationsmethode (Karotisdesobliteration vs. Karotisstent) soll ebenso in Abhängigkeit von klinischen und anatomischen Gegebenheiten gewählt werden: klinische Risikofaktoren: Herzinsuffizienz (NYHA [New York Heart Association] III/IV), instabile Angina pectoris (CCS [Canadian Cardiovascular Society] III/IV), koronare Herzkrankheit mit Hauptstammläsion oder > Einkoronargefäßerkrankung mit 70 %iger Stenose, rezenter Myokardinfarkt (< 30 Tage), geplante Herzoperation (< 30 Tage), linksventrikuläre Ejektionsfraktion (LVEF) < 30 %, schwere Lungenerkrankung, schwere Nierenfunktionseinschränkung; anatomische Risikofaktoren: chirurgisch schwierig/nicht zugängliche Läsionen (Höhe Wirbelkörper C2 oder darüber, unter dem Schlüsselbein), ipsilaterale Strahlentherapie, immobilisierte Halswirbelsäule, kontralateraler Karotisverschluss (erhöhtes Schlaganfallsrisiko), kontralaterale Rekurrensparese, Tracheostoma. Zum nicht-invasiven Screening hinsichtlich Subklaviastenosen werden bei Patienten mit pAVK oder aortalen Erkrankungen beidseitige Blutdruckmessungen empfohlen (Blutdruckdifferenz > 10–15 mm Hg hinweisgebend für eine Subklaviastenose; ). Bei Vorliegen einer symptomatischen Subklaviastenose besteht im ESC-Leitliniendokument eine IIa/B-Empfehlung für eine Revaskularisation, wobei ein endovaskulärer Zugang einem offen-chirurgischen Vorgehen vorgezogen werden sollte . Eine routinemäßige Revaskularisation von asymptomatischen Subklaviastenosen wird nicht empfohlen . Bei Verdacht auf das Vorliegen einer Nierenarterienstenose empfehlen die ESC-Leitlinien auch primär eine duplexsonographische Diagnostik (Kriterien: PSV ≥ 200 cm/s entspricht > 50 %iger Stenose, renoaortale Ratio der PSV ≥ 3,5 entspricht ≥ 60 %iger Stenose bzw. Seitendifferenz des intrarenalen Widerstandsindex ≥ 0,5; ). Bei inkonklusivem duplexsonographischen Befund oder Verdacht auf das Vorliegen einer Nierenarterienstenose besteht eine I/B-Indikation für eine weiterführende Bildgebung mittels CTA oder MRA. Bei Vorliegen von Risikomarkern (rasch progredienter therapierefraktärer Hypertonus, rasche Verschlechterung der Nierenfunktion, hypertensives Lungenödem, Vorliegen einer Einzelniere) und erhaltener Nierenvitalität (Nierengröße > 8 cm, Nierenkortex > 0,5 cm, Albumin-Kreatinin-Ratio < 20 mg/mmol, Widerstandsindex < 0,8) wird nach OMT eine invasive katheterbasierte Abklärung mit ggf. Stentimplantation empfohlen . Ähnliches gilt für bilaterale Nierenarterienstenosen und für Patienten mit fibromuskulärer Dysplasie, wobei bei Letzterer eine primäre Ballondilatation gegenüber einer primären Stentimplantation bevorzugt werden sollte. Von einer routinemäßigen Revaskularisation einer einseitigen Nierenarterienstenose ohne Vorliegen oben genannter Risikomarker wird abgeraten, ebenso bei Fehlen der Nierenvitalität (Empfehlungsgrad III, Evidenzgrad A; ). Bei Verdacht auf chronische atherosklerotische Stenosen der Mesenterialarterien empfiehlt das ESC-Leitliniendokument zur bildgebenden Diagnostik eine CTA . Je nach bildgebendem Befund werden atherosklerotische Stenosen von der nichtokklusiven mesenterialen Ischämie (NOMI) und vom Ligamentum-arcuatum-Syndrom unterschieden. Unter dem Begriff NOMI wird eine mesenteriale Ischämie verstanden, welche sich nicht auf Verschlüsse großer Mesenterialarterien zurückführen lässt, sondern auf eine Minderperfusion in der mesenterialen Endstrombahn, häufig infolge einer Vasokonstriktion in diesem Bereich, wie z. B. bei schwerer Herzinsuffizienz. Das Ligamentum-arcuatum-Syndrom benennt ein Kompressionssyndrom des Truncus coeliacus durch das Ligamentum arcuatum des Zwerchfells, was folglich zu einer symptomatischen Perfusionsstörung führen kann. Während eine Revaskularisation von asymptomatischen Stenosen abgelehnt wird (III/C), empfiehlt das aktuelle ESC-Leitliniendokument bei symptomatischen Stenosen die Einbindung eines Gefäßteams in den Entscheidungsprozess hinsichtlich der Revaskularisationsindikation und -methode. Bei Vorliegen atheromatöser Plaques infolge aortaler Atherosklerose werden von der ESC-Leitlinie – unabhängig von der Anamnese früherer embolischer Ereignisse – eine 1‑fache plättchenfunktionshemmende Therapie und eine lipidsenkende Therapie mit einem LDL-C-Ziel von weniger als 55 mg/dl empfohlen . Hinsichtlich eines Screenings auf das Vorliegen eines abdominellen Aortenaneurysmas (AAA) empfiehlt die ESC-Leitlinie 2024 die Ultraschalldiagnostik bei Männern ab 65 Jahre mit Raucheranamnese und bei Männern ab 75 Jahre unabhängig vom Raucherstatus. Bei Frauen ab 75 Jahre wird ein AAA-Screening bei Vorliegen einer Raucher- oder Hypertonieanamnese empfohlen . Für Familienmitglieder von Patienten mit AAA besteht in der Leitlinie eine Empfehlung für ein AAA-Screening ab einem Alter von 50 Jahren (Empfehlungsgrad I, Evidenzgrad C; ). Nach Diagnose eines AAA sollte ein duplexsonographisches Screening hinsichtlich femoropoplitealer Aneurysmen in Erwägung gezogen werden. Bei Männern besteht ab einem AAA-Durchmesser von mehr als 55 mm und bei Frauen ab einem AAA-Durchmesser von 50 mm eine Klasse-I/Evidenzgrad-A-Empfehlung für eine Sanierung eines AAA . Wie im letzten Aortenleitliniendokument wird auch im aktuellen Papier bei einem AAA-Wachstum von 5 mm oder mehr in 6 Monaten oder von mehr als 10 mm in 1 Jahr empfohlen, eine AAA-Sanierung in Erwägung zu ziehen . Bei sakkulären AAA wird empfohlen, eine AAA-Sanierung schon ab einem maximalen Querdurchmesser von 45 mm in Erwägung zu ziehen . Wichtig ist der Zusatz, dass die AAA-Sanierung bei einer voraussichtlichen Lebenserwartung von unter 2 Jahren kritisch hinterfragt werden sollte. Außerdem besteht keine Indikation (III/C-Empfehlung), vor einer AAA-Sanierung routinemäßig eine koronarangiographische Abklärung oder routinemäßig eine koronare Revaskularisation durchzuführen . Bei Diagnose einer thorakalen Aortendilatation wird eine echokardiographische Untersuchung der Aortenklappe empfohlen (I/B-Empfehlung; ). Zur Dokumentation des Baseline-Durchmessers sowie zur Beurteilung der aortalen Symmetrie sollte jedoch eine weiterführende Bildgebung mit kardialer Magnetresonanztomographie oder kardialer CT folgen . Bei einer Dilatation der Aorta ascendens und trikuspider Aortenklappe wird eine operative Sanierung bei einem Aortendurchmesser oder bei einem Durchmesser der Aortenwurzel von 55 mm oder mehr empfohlen . Neben den genannten Durchmesserangaben werden zusätzliche morphologische Kriterien in der Indikationsstellung zur aortalen Sanierung in Erwägung gezogen (Tab. ; ). Eine operative Sanierung wird bei bikuspider Aortopathie bei einem Durchmesser von 55 mm oder mehr empfohlen. Bei bikuspider Aortopathie und Root-Phänotyp (aortale Dilatation mit tubulärem Durchmesser > Sinusdurchmesser) wird eine Sanierung bei einem Durchmesser von 50 mm oder mehr empfohlen . Bei Aortenbogenaneurysma wird bei thorakalen Beschwerden und niedrigem oder intermediärem operativen Risiko eine offen-chirurgische Sanierung empfohlen (Klasse-I/Evidenzgrad-B-Empfehlung; ). Bei asymptomatischen Patienten sollte bei einem Durchmesser von 55 mm oder mehr eine chirurgische Sanierung erfolgen (Klasse-IIa/Evidenzgrad-B-Empfehlung; ). Bei Aneurysma der deszendierenden thorakalen Aorta wird bei einem Durchmesser von 55 mm oder mehr eine Sanierung empfohlen (Klasse-I/Evidenzgrad-B-Empfehlung; ). Präsentieren sich Patienten mit Verdacht auf ein akutes Aortensyndrom, wird ein multiparametrischer diagnostischer Algorithmus hervorgehoben . Hierbei werden Hochrisikobedingungen (Marfan-Syndrom, Familienanamnese, bekannte Aortenklappenerkrankung, rezente aortale Manipulation, bekanntes Aortenaneurysma), Hochrisikoschmerzsymptome (Brust- oder abdomineller „reißender“ starker Schmerz mit plötzlichem Beginn) und Hochrisikountersuchungsergebnisse (hämodynamische Instabilität, peripheres Perfusionsdefizit, neurologisches Defizit, neues Strömungsgeräusch) evaluiert und entsprechend Punkte vergeben. Je nach Punktzahl in diesem Algorithmus kann eine Risikoeinstufung vorgenommen werden und die weitere diagnostische Abklärung erfolgen. Bei der Bildgebung mittels CT sollte eine EKG(Elektrokardiogramm)-getriggerte kardiale CT mit zusätzlicher Darstellung der gesamten Aorta erfolgen. Die medikamentöse Therapie orientiert sich an der Blutdruck‑, Herzfrequenz- und Schmerzkontrolle (Abb. ); die Sanierung sollte an einem erfahrenen Aortengefäßzentrum erfolgen. Bei penetrierenden Aortenulzera (PAU) wird bei Typ-A-PAU (Lokalisation in der Aorta ascendens) eine operative Sanierung empfohlen, während bei Typ-B-PAU (Lokalisation in der mittleren oder deszendierenden Aorta thoracalis) ein initial konservatives Vorgehen mit Imaging-Kontrollen empfohlen wird (Klasse-I/Evidenzgrad-C-Empfehlung; ). Bei komplizierten Typ-B-PAU oder unkompliziertem Typ-B-PAU mit Risikokonstellation wird eine endovaskuläre Versorgung empfohlen. Bei ausgewählten Patienten mit unkompliziertem Typ-A-PAU ohne Risikokonstellation und mit hohem operativen Risiko kann auch ein abwartendes Vorgehen gewählt werden . Beim Management genetischer Erkrankungen mit Beteiligung der Aorta wird ein individualisiertes Vorgehen an einem Aortengefäßzentrum empfohlen. Die ESC-Leitlinie empfiehlt bei Patienten mit Aortenwurzel‑/Ascendensaneurysma oder thorakaler Aortendissektion das Einholen der Familienanamnese über zumindest 3 Generationen hinsichtlich thorakaler Aortenerkrankungen, plötzlichen Herztodes und peripherer oder intrakranieller Aneurysmen (Empfehlungsgrad I, Evidenzgrad B; ). Bei Vorliegen eines Aortenwurzel‑/Ascendensaneurysmas oder einer thorakalen Aortendissektion mit Risikofaktoren für hereditäre Aortenerkrankungen werden eine genetische Beratung und ggf. Testung an einem Zentrum empfohlen. Bei Patienten mit Marfan-Syndrom wird eine medikamentöse Therapie mit Betablockern oder Angiotensinrezeptorblockern in maximal tolerierter Dosierung empfohlen, um die Entwicklung einer Aortendilatation zu bremsen (Empfehlungsgrad I, Evidenzgrad A; ). Bei Patienten mit Loeys-Dietz-Syndrom werden im aktuellen ESC-Leitliniendokument Aortenwurzeldurchmessergrenzwerte (> 45 mm) für ein operatives Vorgehen angeführt . Der Schwerpunkt der ESC(European Society of Cardiology)-Leitlinien 2024 zur peripheren arteriellen Verschlusskrankheit (pPAVK) und zu Aortenerkrankungen liegt auf einem ganzheitlichen, gefäßmedizinischen Ansatz. Dieser Ansatz hebt die Prävention, die frühe Diagnose, ein individuelles patientenzentriertes Management und eine regelmäßige Nachsorge bei diesen Krankheitsbildern hervor und fordert die dafür erforderliche Multidisziplinarität im Patientenmanagement. |
British South Asian ancestry participants views of pharmacogenomics clinical implementation and research: a thematic analysis | e9d89066-4a91-4907-80dd-29e129b358ae | 10661738 | Pharmacology[mh] | The South Asian ancestry population is a rapidly growing demographic in the United Kingdom (UK), now representing 10% of the national population . South Asian ancestry populations are under-represented in both genomics studies and clinical trials which provide the data that underpin therapeutic licensure by regulators . The UK South Asian ancestry population suffers from high rates of multi-morbidity and will therefore be exposed to polypharmacy. This means they are more likely to experience adverse drug events and drug-drug interactions as compared with other populations due to exposure to higher numbers of medications. Pharmacogenomics (PGx) uses genetic information to predict some of the interindividual variability in response to therapeutics and can help to personalise medication choice to get the right drug to the right patient at the right dose and the right time. PGx can therefore increase efficacy, decrease adverse drug reactions (ADRs), and mitigate drug-drug interactions. The potential benefits of PGx for the UK South Asian ancestry population are substantial, so it is vital engage the community in discussions about PGx clinical implementation and use of generated clinical data for future research. PGx has potential to address some inequalities by nature of ancestral variation in polymorphism prevalence. For example, it would personalise therapy for those who are poor CYP2C19 metabolizers (higher prevalence in Asian and Oceanic ancestry populations) or ultra-rapid CYP2D6 metabolizers (more likely in those of Oceanic, Ashkenazi Jewish and middle eastern populations) . However, PGx implementation could make inequalities worse if historically under-represented ancestral groups, such as the South Asian ancestry population, do not engage with the PGx research that will flow from clinical implementation . This is because unless there is research engagement from diverse ancestral groups, PGx polymorphisms cannot be validated in diverse populations, and polymorphisms specific to non-European ancestral groups may be missed. The UK National Health Service (NHS) has committed to examining the evidence for PGx implementation in the next one to three years as part of the national genomic medicine strategy .The benefit of patient and public engagement (PPI) in clinical service development is well established. Systematic review shows that care process outcomes emerged from high-level enagagement . Furthermore, engagement can improve the relevance and credibility of research, aligning the research community and research population, and improve accountability to the research population . PPI is critical to shaping and driving PGx implementation. Enhanced research participation from historically underrepresented communities is vital to the goal of using PGx to address health inequality. This is particularly important when there might be disproportionate benefit to historically under-represented communities and potential trust barriers to be overcome. The objective of this qualitative study is to understand UK South Asian ancestry participants attitudes toward PGx clinical implementation and potential barriers and facilitators in relation to PGx data sharing for research. Due to the lack of any prior PGx public acceptability work in this cohort demographic, focus groups were chosen as a methodology to canvas public input with minimal assumptions. We recruited to focus groups from existing participants in the Genes & Health cohort study . Genes & Health participants were originally recruited 2015-present in community and healthcare settings . Inclusion criteria were age 16 or older and self-identified as Bangladeshi or Pakistani ancestry. 150 participants who had recently engaged with follow-up studies locally were sent an SMS inviting them to join the focus groups. This was supplemented with invites extended directly to recent or future participants by telephone. Demographic information was collected from participants in a brief survey administered prior to the discussion. Four focus groups were conducted with 9–12 participants per group. Two groups were mixed gender, one was male only and one was female only. Simultaneous interpretation was available to participants in Urdu and Bengali. A brief introduction was given on PGx and then PGx clinical implementation and use of clinically generated PGx information for research were discussed. The focus groups took a semi-structured approach using a topic guide which asked questions about PGx implementation, concerns about taking a PGx test, and sharing clinical PGx data with third parties for research (the topic guide is provided in the appendix). A literature review was undertaken to inform the topic guide development. Though information regarding public and patient perspectives of PGx is scant and high level there are common themes in the literature which served as a starting point for the semi-structured topic guide used (supplementary table ) . A clinician investigator led the focus groups, enabling participants to ask questions about the topic (i.e., how genetic testing samples could be collected). Focus groups were recorded and abridged transcription was performed. The data was analysed thematically, using an inductive approach, to describe perceived utility of and barriers to clinical PGX implementation and subsequent PGx research . This study was approved by the Queen Mary University of London Research Ethics Committee (QMERC22.353). Written informed consent was obtained for participation in the study. Participants were given a 50 GBP voucher to thank them for their time and participation. A member checking session was held to discuss the results of the thematic analysis. Focus group demographics There were 42 participants across the four groups, 64% female. 26% were born in the UK or Europe. 52% were born in Bangladesh and 17% in Pakistan. 36% reported university level education. More detailed information to characterise each focus group is shown in Table . Thematic analysis Main themes that emerged are shown in Table . For PGx clinical implementation these were: benefits, communication, timing of testing in the clinical care pathway, custodianship of data, cost, trust, education and outreach. Themes that emerged from discussion of sharing clinical PGx data for research purposes were: benefits, trust, education, data sharing facilitators, barriers to data sharing and safeguards. Themes were consistent across all groups, and all groups emphasised trust as primary and interlaced with all other themes. The relationships between these themes are illustrated in Fig. (Fig. - mind map). These key themes are expanded on below, with sub-themes, to illustrate participant insights (Table ). PGx clinical implementation Benefits Benefits of clinical PGx implementation: which medicine ‘suits’ me PGx was perceived to be beneficial to individuals, by making medication choice more tailored, with less trial and error: “ which medicine suits me, I think that would be a good idea ”. There was particular interest in implementing PGx for gene-drug pairs where there are known to be high prevalence of polymorphisms in South Asian ancestry groups, and therefore a higher risk of inefficacy or toxicity in this community. Risk of ADRs were perceived to be a big concern in taking medications, and to impact on compliance. There were concerns that ADRs can be worse or more long-lasting that the original treatment indication, and that if participants knew of someone who reacted badly to a medication, they would not want to take it: “For example, I take a medicine and I react really badly to it. Everyone in this room might sit there and think, wow, she’s taking that medication and she’s had a really bad reaction. Maybe I shouldn’t take that medicine.” Participants felt that PGx had the potential to mitigate this reaction by reassuring people that genetic risk of ADRs had been checked. The potential to avoid broad contraindications with a more targeted approach was raised by several participant anecdotes. For example, one participant suggested that with more precise PGx stratification we might better understand which asthmatics are likely to have a bad reaction to ibuprofen and not withhold it from those who are not likely to have an ADR. Communication Communication to support clinical PGx implementation Participants felt that limited information was desired for clinical PGx use at the point of care. There was a strong preference for use of simple language to communicate PGx. Participants thought that the easiest way of conveying the utility of PGx was to identify which medicines “ suit ” you/your body. Participants generally agreed that for clinical indication a minimal explanation of PGx testing to inform medication choice (similar to a routine blood test) was sufficient. Many participants didn’t think it was necessary or helpful to include the fact that DNA/genetics are being tested. For example, as one participant reflected elderly people might not understand what genes are in comparison to younger people. Given this, they suggested presenting PGx as something that would help clinicians make sure that the medicines they prescribe are “ more suitable for you ” would result in an explanation that would make sense to a wider range of people. This sentiment of offering PGx clinically for medicine optimisation without detailed discussion of genetics was echoed by the majority of participants across all focus groups. There was a strong preference that communication around PGx be led by General practitioners (GPs). GPs were described as trusted sources of information and having the skill and resources to support communication where language and literacy barriers are present: “ GP can explain very well” . Timing Timing of testing in the clinical pathway: who would benefit the most and how should testing eligibility reflect that? PGx was viewed as particularly helpful to those who suffer from polypharmacy. People taking many medications were perceived as most likely to benefit from PGx testing, by decreasing risk of side effects and drug-drug interactions. In addition to identifying polypharmacy as increasing risk of ADRs, participants felt that enhancing efficacy from medication for those with the most morbidity was important, regardless of age. In the words of one participant, which provoked broad agreement “ you could have someone that is like half the age and has already been using so many different medicines. They aren’t working for them and they wanna know why it’s not working .” Due to the shared view amongst participants that the greatest beneficiaries of PGx implementation would be those with the most morbidity, they proposed the idea of a secondary prevention speciality clinic. They felt that this would mean that people at high risk would be able to benefit from PGx innovations “ as soon as possible ” rather than having to wait potentially many years for pre-emptive PGx to be rolled out across everyday clinical practice for all people via NHS primary care. While benefits of more personalised medicine were thought to be particularly promising for multimorbid patients, if resources allowed participants liked the idea of PGx panel testing for all at birth, so that the information would be there pre-emptively to optimise medication choice throughout life. Several participants suggested they would welcome PGx testing as a part of routine neonatal testing: “ you know the kids are born and then they offer the next day the hearing test …in the hospital without leaving? You can offer [PGx] at the same time .” Participants had no concerns about doing PGx testing on babies, provided sample extraction was not painful or harmful. Parents were much more concerned with the risks of a perceived trial and error prescribing approach that did not consider genetic data which could indicate high ADR risk. Testing in primary care . Participants felt strongly that pre-emptive PGx testing via the GP was preferable to point of care testing in hospital at the time of indication for therapeutic (ie in the example of CYP2C19 testing to help guide anti-platelet choice after a myocardial infarction). The reasons were multifactorial; the GP was first point of contact, had all patient information, provided continuity of care, and was perceived to communicate well. Participants liked the idea of having PGx testing before there was a treatment indication, and felt primary care was the right place for this kind of anticipatory testing. There was also a concern that anything viewed as not essential may not reliably happen in acute care settings. Furthermore, participants thought of primary care as a less threatening and more personal setting where there was a higher likelihood of receiving information about test results and being in a state to understand that information, as compared with hospitals. “ Going to the GP… it’s a lot more personal than going to a hospital… if you’re at a hospital it just kind of feels alien ”. They also felt that need for acute care was associated with fear: “ people go in hospital when [they are] in danger…I can call the GP and book an appointment… when you go to hospital [there’s] always danger there ”. Participants felt that due to the acute nature of secondary care communication was limited, and patients were often unaware of investigations ordered. As one participant surmised: “ We don’t even know probably half of the things they do. No one questions about the medicine or why they’re taking the blood test. There’s no choice ”. Custodian of data Maximising benefits of clinical PGx testing: transfer of information across care settings Benefits of PGx were thought to be greatest if PGx results could be effectively shared across care settings, particularly primary and secondary care but also community pharmacy settings. Some participants felt that integration across care settings of existing analogous data is not good. One parent illustrated this with an anecdote: “I have an example: One of my sons [is] allergic [to] ibuprofen. So, this information I can see …the GP shared with me…but always I have to tell [them] in a hospital, don’t give ibuprofen to him, because he has a reaction with that” . However, another participant gave examples of successful programmes where important medical information travels with patients, suggesting the same could be done with PGx: “ Shouldn’t be a problem because you already have medical bracelets and tags for people with different…conditions… so they could be identified if some something was to happen to them in public you can see that necklace or bracelet .” Participants liked the idea of an NHS app having PGx information that could travel with the patient and allow self-advocacy. For example, in the words of one participant: “It should be the GP as well as the patient who has that information because… sometimes… the GP don’t really listen properly… if she knows what her needs are… she can show it and say this is what it is. This is my genetic result” (translated from Bengali). Some participants saw community pharmacists as care providers that could give more personalised advice if they had access to PGx results. This could take some strain off primary care. However, others perceived sharing clinical PGx data with private chemists as a risk that could lead to inflated prices. Cost Balancing benefits against costs The benefits of clinical PGx implementation, particularly as pre-emptive testing for all nationally, were weighed against the risk of overburdening resource limited NHS services and clinicians, which participants felt protective of. There was trust in the NHS and NHS clinicians and a perception that the benefit of PGx implementation would need to outweigh added financial strain and time constraints on these institutions and professionals. There was a feeling that any preventive endeavour would be lower priority, as compared with testing which responded to clinical need. In the words of one participant, which the other participants expressed agreement with: “you know they’re suggesting a GP visit should cost people money…what about the cost of the test… would it cause too much pressure? …In advance you are doing a testing… Maybe you need it in the future or not…still you are doing it … they’re asking for less pressure on the NHS then you’re putting so much more pressure on the NHS.” Many participants felt that streamlined logistics of PGx implementation were crucial to ensure the inconvenience of participation wasn’t perceived to outweigh the benefits. A further concern to the integrity of existing services and professionals was any added threat of litigation. This concern further highlighted the protective feeling participants had toward the NHS, and the requirement that the benefits outweigh the all-inclusive costs: “Could this open up the NHS or the GP to liable action, i.e., being sued? Because they have the genetic markers there. You gave the medicine, but now obviously they got it wrong…Patient then sues the GP/brings action against the NHS because you’ve given me a wrong medicine, even though you’ve had my genetic markers.” Trust Trust was a central theme in discussion of clinical PGx implementation and was impacted by and impacted on communication and education. The role of trust in clinical PGx implementation: ‘GP they trust’ There were strong feelings of trust in the NHS and health care providers, particularly primary care practitioners. Examples of broadly shared articulated trust in GP were common. Despite this trust, participants commonly cited concerns about side effects leading to medication non-compliance. “ Some people are quite scared of taking any medication because of all the side effects. Even if they get the medication from the GP… they’re going to ask how many side effects [and then] don’t take it” . Participants thought that a more personalised approach to prescribing using genetic information would enhance trust in prescribers and prescriptions, because people would have more confidence in the selection of therapeutics knowing it was aligned with their personal test results. “ After genetic test when doctor will prescribe medicine obviously there’re going to involve more trust on this ”. Participants thought that this enhanced trust would improve medication compliance, as demonstrated by one participant: “For example, if I go doctor then they just prescribe me paracetamol? Yeah. If they tell me. OK have 100 [dose]. Maybe I’m gonna have 20 or 30. But after the blood test or whatever test done. If he give me 100 then I’m gonna say yeah I’m gonna finish the 100 because it’s been done by test… In the first time, he gave me 100, I’m not gonna take it.” Ancestry specific representation in research generated evidence for therapeutics was noted to build trust in a clinical setting: “ If you get a medication out and say we tested it on these kind of… people… and that was beneficial… This drug was good for Asian community… So it’s better to take that.” The implication was that participants know that ancestry is sometimes linked with response to medication. Therefore, proportionate ancestry representation in evidence base assessing efficacy and ADRs builds trust in clinical practice by demonstrating that a specific medicine has a favourable risk-benefit profile in their community. Due to trans-ancestry variation in pharmacogene polymorphisms and historically non-diverse clinical trial cohorts this is an important point in how clinical PGx implementation interfaces with trust. Interestingly, there were no concerns from participants around misuse of data within clinical care pathways. Participants unanimously felt that their clinical data was secure through standard NHS data protection pathways and that PGx data would be no different. In the words of two participants: “ I think the GDPR legislation makes me comfortable with sharing my information with the GP and the NHS, so I don’t see any hindrance…sharing my information ”; “ the current GDPR is quite broad ”. However, participants felt that any sharing of personal data with private entities such as chemists could result in price gouging if, for example, pharmacies discovered they were serving a population who were much more likely to respond well to one specific medication. This was a widely shared concern. Education and outreach Education to support clinical PGx implementation There was consensus that national roll out of pharmacogenomic testing should be accompanied by public health level education, with outreach, and clear communication to facilitate trust. It was clear from the focus group discussions that it is important to differentiate diagnostic genetic testing or genetic testing to predict disease risk from PGx testing. There was a general concern from participants that the level of genetic literacy in the UK South Asian community is low. There was a feeling that people with more lived experience of disease and medication use were more likely to understand and be interested in PGx. Outreach and engagement Participants universally acknowledged that GPs would not be able to discuss PGx with each person. This was an impetus for support for a broader public health and outreach awareness campaign proposed by participants. Forums such as local mosques, Islamic centres, schools, fairs, shopping centres and GP surgeries were suggested to disseminate information about PGx. “ The mosque …some Islamic mosques have community services [centres] as well… the kids there are learning…the elders are coming there…women are coming there…mosques have a community system…the ladies are very much involved in that .” Multi language leaflets and videos were enthusiastically suggested, as was propagation of information via social media. The importance of leadership in the community, and community and family links, were paramount. Therefore, secular and faith leaders and heads of family were perceived to play a key role in propagating information. There was also a suggestion from participants in every group that information can be disseminated in families by incorporating education about genetics generally and PGx specifically in schools. “ Getting children to understand…maybe they can go home to their parents…if you come to schools and talk about it ”. Education and misinformation – lessons learnt from the COVID-19 pandemic Participants framed their experience with dissemination of new medical information through experiences with COVID-19 vaccines. There was a broadly shared view that misinformation around the COVID-19 pandemic and vaccines had eroded trust between the community and health care. There was perceived to be a new reluctance to engage in any non-essential clinical tests: “You know covid changed everything. Do you think that people will go for the blood test or genetic test that don’t know why you are using this, why we need this? So you need to educate them what is the importance for them. Otherwise, it’s very hard for the Asian community to come.” The pandemic highlighted a need for high quality accessible information regarding new developments in therapeutics related clinical care (“ to spread information and minimise misinformation ” in the words of one participant), and ability to understand which demographics different forms of information was reaching. Misinformation was a concern, particularly via social media platforms, where it can be widely disseminated: “ There’s so much data on the internet, and so much information it can be false ”. Education with outreach was seen as a solution to the problem of misinformation. Social media was seen as an effective tool to combat misinformation and democratise knowledge via accessible multi-media campaigns. Sharing clinical PGx data for research purposes Themes that emerged from discussion of sharing clinical PGx data for research purposes were: benefits, trust, education, data sharing facilitators, barriers to data sharing and safeguards. Benefits Benefits of research using PGx clinical data: ‘whatever is necessary to help the community’ . Research that could be generated from use of PGx clinical data was felt to be beneficial to the community with some risks to the individual’s privacy. Participants felt favourably about contributing data to support research which would benefit the community, and the good of the community had a central role in discussion. As one participant said, and others echoed: “ What’s the point in just having the blood test done and not going for research. I think that goes hand in hand…I would take it… Whatever is necessary to help the community ”. However, there were strong feelings about privacy and concerns that any data sharing may breach privacy and open potential for misuse: “I think data protection is very important in our lives…Yeah like how we said it should be between …researchers and GP…I don’t think I would like everyone to know… what benefits me… I would like to have privacy ourselves as well” . These privacy concerns were counterbalanced by the benefits of community representation in research to develop community specific knowledge. A participant highlighted concern that research on medicine is only done in some people, but then the medicine is used in all people, and participants agreed broadly that it is reassuring to be treated with medicine when the evidence base for medicine use included their community. “ When scientists do research there is one portion of the population but how [do] they apply that information onto the big portion of the population? ” (translated from Urdu). Participants felt more favourably about taking medication that had been trialled in their ancestry group and felt that ancestry specific research could drive changes in medication or supplement taking behaviour. For example, a participant gave an example of impact on behaviour driven by community specific research: most people in the community didn’t take vitamin D supplements, and then research that South Asians often lack vitamin D was disseminated. This research specific to the South Asian community then convinced many people in the community to take vitamin D: “ They got some information Asian people lack vitamin D. Apparently it’s in the genes or something…majority of the Asian people, my family members, all of them, they take vitamin D” . Benefits of data sharing to generate further research specific to the South Asian community was perceived as outweighing the potential risks of data misuse generally, particularly with appropriate safeguards: “ if it benefits the community by sharing the data… with their permission, with their consent, if this is shared in the research team that’s fine also… keeping data secure, confidential with her permission .” Trust Trust in PGx research: protective and harmful factors Willingness to share clinical data for research purposes revolved around trust. Participants felt that more personalised therapy through PGx clinical implementation would enhance trust and therefore contribute to increased willingness to engage with and share data for research. Trust was engendered by institutional affiliations (i.e., NHS, medical practitioners, national regulatory bodies such as the Medicines Healthcare Regulatory Association (MHRA)). Trust was supported by safeguards in data protection and de-identification of data used for research. Participants also found the non-diagnostic nature of PGx testing reassuring, and keeping the scope of PGx testing to non-disease diagnostic genes was a factor that enhanced trust: “ I feel if like it’s really narrowed down in front of you it would be safer …” . Trust in the individual recruiting to research was also a factor. Trust leading to research engagement could be gained by endorsement of a family member, faith leader, or community leader: “ If my relative did it, I might [do it]. Some people trust in relatives…People trust more family ”. Trust was harmed by insecure data, a history of data breach or association with individuals or institutions that were not trusted. Lack of consistency in information and profit as a motivating factor were other factors which harmed trust. Lack of trust leads to concerns about data misuse Misuse of data by non-trusted entities was a concern. This was a central disincentive to research participation: “ People really don’t want to share their information. They might have doubt on the people using to do research. That’s why they don’t want to share ” (Translated from Urdu). Concerns regarding the specific nature of potential data misuse ranged from breaches of privacy and financial exploitation to the potential for malicious actors to use genomics data for racially motivated genocide. “In theory… if someone wants to target a …specific group of people like South Asians… if I target that gene it could set off a virus that could only affect these people…I think I’ve seen it in a film, when they target a specific gene … they set this gas off but it will only effect people with this gene…South Asian genes” . This latter was perceived by some participants to be hyperbolic, and the level of time, knowledge and resources needed to misuse data in such a nefarious way were cited as protective: “ to get to the point of …killing hundreds of thousands… is far-fetched. We’d need to dissect …an entire genome, which would take a very long time, and a lot of work .” Lack of trust in profit driven research Participants across all groups expressed concern that pharmaceutical industry was not trustworthy due to profit as a motivating factor. “ Medicine is about helping people and saving lives…They’ve developed the drugs but they’re big businesses as well …”. Some extended this logic to private chemists and pharmacists working at chemists, as profit was felt to be the bottom line. The perceived conflict of interest created by profit as a primary goal was felt to lead to risk of information misuse. There were concerns about benefit hoarding for profit. Many participants across all groups worried that if industry were to get PGx data for research they would find a way to profit at the expense of the community and withhold benefits from the community. Several participants felt there was a risk of price gouging if a therapeutic was found to be particularly beneficial to their community: “but when the makers know that then they will increase the prices. And you know we are very careful about our health so we will spend money.” Another participant in a different group expressed the same sentiment: “If this information is being delivered to industry, will it affect the cost of the medicine?… if we’re getting a tablet for 1 pound we might then get it for 3 pounds” (translated from Urdu). There was a negative view toward proprietary patents as tools to restrict availability of therapeutics. There was a concern that if lifesaving medications were discovered from genetic data, patents would mean that the medicine would not be affordable or accessible to the participant communities that had contributed data to the research. However, trust in national regulators was seen by some participants to counterbalance the risk of unrestrained industry: “ business is business at the end of the day. Some businessmen are OK. If the regulator doesn’t allow, then they won’t get it [the medication]. They need to allow it first .” Feeding back research results facilitates trust Feeding back research results was crucial to ongoing research engagement through building trust: “ if someone sees a result then they will become more involved ”. Participants agreed that if research results were fed back it would support education and engagement and facilitate trust via grassroots community communication. As one participant said of receiving feedback on how she contributed to a study: “ And then you would speak to, like, your friends and family…They would open up. They would be like, wow, that’s so cool… It would build trust between communities .” Some participants felt that personalised feedback on an individual level had an even more powerful impact, and there were suggestions that researchers could build trust further by contacting individual participants to make them aware of how their data had contributed to a study. Trust in therapeutics research through the lens of the COVID-19 pandemic Participants expressed their experience with trust, and mistrust, in therapeutics and research through the lens of the COVID-19 pandemic. Participants reported a change in context and trust toward therapeutics research due to the pandemic. There was broad agreement that lack of trust had manifested in strongly divided opinions on the safety and efficacy of the COVID-19 vaccine: “for example…, covid injection, half of the people …didn’t have it…a lot of people I know, they didn’t go for that injection…it’s their choice end of the day…but there’s a reason why they didn’t have it…because they don’t trust maybe, they didn’t believe” . Lack of trust toward the COVID-19 vaccine within the South Asian community was widely felt to be prompted by the perceived pace of research and social media reports of trusted health care practitioners refusing COVID-19 vaccination. Because of the nature of the pandemic, some participants saw COVID-19 vaccines and treatments as initially experimental or offered without a full understanding of possible effects. However, it was felt that this mistrust would not extend to PGx research focused on optimising personal risk/benefit profile for existing medications. Education Education to facilitate PGx research In contrast to the skeletal information desired for clinical PGx use at the point of care, participants felt that a lot of information and education was needed to responsibly organise sharing of the generated clinical data for research. “ for research: you have to make sure you understand it perfectly and it has to be accurate information given to you. Clinical, that’s something you just do…easier to do…research you have to be really accurate .” Participants highlighted lack of awareness of research, and lack of scientific and genetic literacy as significant barriers to research engagement. “this is the reason there is less data from these groups: because the lack of education and they don’t participate if they don’t understand anything.” Language barriers were also cited as key hurdles to engagement of this community with research. However, participants also perceived a lot of interest in advancing health and medication related knowledge in the community and suggested that community ties offered vehicles to public engagement. The public health engagement campaign suggestions outlined above around national PGx roll out were echoed strongly in the discussion of education to facilitate PGx data sharing for research. Engaging with local community members for grass-roots education was advised by participants. But some participants perceived a lack of scientific literacy to be a significant barrier to community exchange of information: “How to educate those people? Like when you speak to other people they don’t know, like when she will leave from here, what she would say to her neighbour … what is that genetic information to do with the medication? We take medication everyday” (translated from Urdu). Many participants expressed interest in being trained to be community champions and volunteered to disseminate PGx information to facilitate research engagement: “ in East London mosque they have events and things… I’m here today. I understand. I will go and spread to my friends and family. So, it’s like word of mouth will get spread .” Data sharing facilitators Data sharing was the key concept on which research from a hypothetical clinical PGx service hinged. Participants required prompting to distinguishing PGx testing for clinical use from sharing clinical PGx data for research. Factors supporting PGx data sharing for research Data sharing was desirable if the researchers did not have a financial stake, and benefits would be shared. There was a common perception that without research, the use of medicines will not improve, but an understanding that the risk is to the participating individuals while the benefit would be for future individuals: “If you don’t share it, you don’t advance really. So, you have to come to some sort of compromise where you are sharing the results they need, or do you want to just not share it and be stuck and not give two hoots about what happens in the future. You have to draw that line somewhere.” . The perceived “good” of the research purpose was a key motif: “ So the point is how it works when we share for the good purpose, not for the bad purpose. So, it can help, so definitely [we should] share ”. There was broad consensus across individuals and groups that the idea of good as compared with bad purpose had an association with the trustworthiness of the researchers: “ we have a concern, so we can only share these things [PGx data] with trusty [trustworthy] ones and [make ourselves aware ].” The perceived trustworthiness of both individual researchers and associated institutions were determining factors in weighing willingness to share clinical PGx data for research purposes. Health care professionals, academic institutions, and the regulatory body (the MHRA) were considered trustworthy and therefore participants were happy to share PGx data with these groups for research with the protection of standard data de-identification and data protection. Barriers to data sharing and safeguards Barriers to sharing clinical PGx data for research and potential safeguards Participants across all groups broadly acknowledged that some people would not like to share data as a rule, due to privacy concerns: “There are people with those [privacy] concerns and those concerns are very real” . Data ownership was an important topic linked with privacy, and many participants wanted to maintain control over access to their data “It’s my information. That’s mine, do you know what I mean, it’s an invasion of privacy where you don’t have control over who gets to see your information.” As compared with healthcare practitioners and academics, there were very different perspectives on sharing PGx data for research with industry. Concerns revolved around trust, as outlined above. Most participants felt that industry has an inherent conflict of interest as a profit driven private enterprise and therefore could not be trusted to prioritise benefit sharing/the health of the community over potentially exploitive options: “ pharmaceutical companies are only thinking about their profits then it’s not good to share our information with them ”. Others felt that there is an inherent risk to not doing research: “ without research there will be always risk, there’s no cure ”. Some perceived the benefits of data sharing with industry to outweigh potential harms: “It improves the medicine, so it improves the patient care ”. Confidentiality and anonymization of data were important safeguards to protect privacy. “ It’s anonymous isn’t it…so the people who are doing it, they don’t know who it is. They have to have that barrier that [data] is confidential and not to be leaked to anyone .” Well-articulated policies around data protection and management of any breach of data protection were perceived by many participants to be crucial safeguards: “It’s important what they’re going to do with the data but also if there is a breach of data, what happens… if they find that information was leaked to the public…what they do ”. Transparency about potential conflicts of interest and opportunities to opt out of data sharing with non-trusted research partners were desirable. “I think everyone should be given the option to opt in and opt out, so I think that’s potentially a way of going forward … so you [can] opt in for pharmaceuticals or universities and … and so on…You can label them as non-profit organisation and for-profit organisations and so. That would build confidence in the person that is being involved in the research.” Safeguards against financial exploitation due to knowledge of individual or community PGx data would be protective. There were 42 participants across the four groups, 64% female. 26% were born in the UK or Europe. 52% were born in Bangladesh and 17% in Pakistan. 36% reported university level education. More detailed information to characterise each focus group is shown in Table . Main themes that emerged are shown in Table . For PGx clinical implementation these were: benefits, communication, timing of testing in the clinical care pathway, custodianship of data, cost, trust, education and outreach. Themes that emerged from discussion of sharing clinical PGx data for research purposes were: benefits, trust, education, data sharing facilitators, barriers to data sharing and safeguards. Themes were consistent across all groups, and all groups emphasised trust as primary and interlaced with all other themes. The relationships between these themes are illustrated in Fig. (Fig. - mind map). These key themes are expanded on below, with sub-themes, to illustrate participant insights (Table ). PGx clinical implementation Benefits Benefits of clinical PGx implementation: which medicine ‘suits’ me PGx was perceived to be beneficial to individuals, by making medication choice more tailored, with less trial and error: “ which medicine suits me, I think that would be a good idea ”. There was particular interest in implementing PGx for gene-drug pairs where there are known to be high prevalence of polymorphisms in South Asian ancestry groups, and therefore a higher risk of inefficacy or toxicity in this community. Risk of ADRs were perceived to be a big concern in taking medications, and to impact on compliance. There were concerns that ADRs can be worse or more long-lasting that the original treatment indication, and that if participants knew of someone who reacted badly to a medication, they would not want to take it: “For example, I take a medicine and I react really badly to it. Everyone in this room might sit there and think, wow, she’s taking that medication and she’s had a really bad reaction. Maybe I shouldn’t take that medicine.” Participants felt that PGx had the potential to mitigate this reaction by reassuring people that genetic risk of ADRs had been checked. The potential to avoid broad contraindications with a more targeted approach was raised by several participant anecdotes. For example, one participant suggested that with more precise PGx stratification we might better understand which asthmatics are likely to have a bad reaction to ibuprofen and not withhold it from those who are not likely to have an ADR. Communication Communication to support clinical PGx implementation Participants felt that limited information was desired for clinical PGx use at the point of care. There was a strong preference for use of simple language to communicate PGx. Participants thought that the easiest way of conveying the utility of PGx was to identify which medicines “ suit ” you/your body. Participants generally agreed that for clinical indication a minimal explanation of PGx testing to inform medication choice (similar to a routine blood test) was sufficient. Many participants didn’t think it was necessary or helpful to include the fact that DNA/genetics are being tested. For example, as one participant reflected elderly people might not understand what genes are in comparison to younger people. Given this, they suggested presenting PGx as something that would help clinicians make sure that the medicines they prescribe are “ more suitable for you ” would result in an explanation that would make sense to a wider range of people. This sentiment of offering PGx clinically for medicine optimisation without detailed discussion of genetics was echoed by the majority of participants across all focus groups. There was a strong preference that communication around PGx be led by General practitioners (GPs). GPs were described as trusted sources of information and having the skill and resources to support communication where language and literacy barriers are present: “ GP can explain very well” . Timing Timing of testing in the clinical pathway: who would benefit the most and how should testing eligibility reflect that? PGx was viewed as particularly helpful to those who suffer from polypharmacy. People taking many medications were perceived as most likely to benefit from PGx testing, by decreasing risk of side effects and drug-drug interactions. In addition to identifying polypharmacy as increasing risk of ADRs, participants felt that enhancing efficacy from medication for those with the most morbidity was important, regardless of age. In the words of one participant, which provoked broad agreement “ you could have someone that is like half the age and has already been using so many different medicines. They aren’t working for them and they wanna know why it’s not working .” Due to the shared view amongst participants that the greatest beneficiaries of PGx implementation would be those with the most morbidity, they proposed the idea of a secondary prevention speciality clinic. They felt that this would mean that people at high risk would be able to benefit from PGx innovations “ as soon as possible ” rather than having to wait potentially many years for pre-emptive PGx to be rolled out across everyday clinical practice for all people via NHS primary care. While benefits of more personalised medicine were thought to be particularly promising for multimorbid patients, if resources allowed participants liked the idea of PGx panel testing for all at birth, so that the information would be there pre-emptively to optimise medication choice throughout life. Several participants suggested they would welcome PGx testing as a part of routine neonatal testing: “ you know the kids are born and then they offer the next day the hearing test …in the hospital without leaving? You can offer [PGx] at the same time .” Participants had no concerns about doing PGx testing on babies, provided sample extraction was not painful or harmful. Parents were much more concerned with the risks of a perceived trial and error prescribing approach that did not consider genetic data which could indicate high ADR risk. Testing in primary care . Participants felt strongly that pre-emptive PGx testing via the GP was preferable to point of care testing in hospital at the time of indication for therapeutic (ie in the example of CYP2C19 testing to help guide anti-platelet choice after a myocardial infarction). The reasons were multifactorial; the GP was first point of contact, had all patient information, provided continuity of care, and was perceived to communicate well. Participants liked the idea of having PGx testing before there was a treatment indication, and felt primary care was the right place for this kind of anticipatory testing. There was also a concern that anything viewed as not essential may not reliably happen in acute care settings. Furthermore, participants thought of primary care as a less threatening and more personal setting where there was a higher likelihood of receiving information about test results and being in a state to understand that information, as compared with hospitals. “ Going to the GP… it’s a lot more personal than going to a hospital… if you’re at a hospital it just kind of feels alien ”. They also felt that need for acute care was associated with fear: “ people go in hospital when [they are] in danger…I can call the GP and book an appointment… when you go to hospital [there’s] always danger there ”. Participants felt that due to the acute nature of secondary care communication was limited, and patients were often unaware of investigations ordered. As one participant surmised: “ We don’t even know probably half of the things they do. No one questions about the medicine or why they’re taking the blood test. There’s no choice ”. Custodian of data Maximising benefits of clinical PGx testing: transfer of information across care settings Benefits of PGx were thought to be greatest if PGx results could be effectively shared across care settings, particularly primary and secondary care but also community pharmacy settings. Some participants felt that integration across care settings of existing analogous data is not good. One parent illustrated this with an anecdote: “I have an example: One of my sons [is] allergic [to] ibuprofen. So, this information I can see …the GP shared with me…but always I have to tell [them] in a hospital, don’t give ibuprofen to him, because he has a reaction with that” . However, another participant gave examples of successful programmes where important medical information travels with patients, suggesting the same could be done with PGx: “ Shouldn’t be a problem because you already have medical bracelets and tags for people with different…conditions… so they could be identified if some something was to happen to them in public you can see that necklace or bracelet .” Participants liked the idea of an NHS app having PGx information that could travel with the patient and allow self-advocacy. For example, in the words of one participant: “It should be the GP as well as the patient who has that information because… sometimes… the GP don’t really listen properly… if she knows what her needs are… she can show it and say this is what it is. This is my genetic result” (translated from Bengali). Some participants saw community pharmacists as care providers that could give more personalised advice if they had access to PGx results. This could take some strain off primary care. However, others perceived sharing clinical PGx data with private chemists as a risk that could lead to inflated prices. Cost Balancing benefits against costs The benefits of clinical PGx implementation, particularly as pre-emptive testing for all nationally, were weighed against the risk of overburdening resource limited NHS services and clinicians, which participants felt protective of. There was trust in the NHS and NHS clinicians and a perception that the benefit of PGx implementation would need to outweigh added financial strain and time constraints on these institutions and professionals. There was a feeling that any preventive endeavour would be lower priority, as compared with testing which responded to clinical need. In the words of one participant, which the other participants expressed agreement with: “you know they’re suggesting a GP visit should cost people money…what about the cost of the test… would it cause too much pressure? …In advance you are doing a testing… Maybe you need it in the future or not…still you are doing it … they’re asking for less pressure on the NHS then you’re putting so much more pressure on the NHS.” Many participants felt that streamlined logistics of PGx implementation were crucial to ensure the inconvenience of participation wasn’t perceived to outweigh the benefits. A further concern to the integrity of existing services and professionals was any added threat of litigation. This concern further highlighted the protective feeling participants had toward the NHS, and the requirement that the benefits outweigh the all-inclusive costs: “Could this open up the NHS or the GP to liable action, i.e., being sued? Because they have the genetic markers there. You gave the medicine, but now obviously they got it wrong…Patient then sues the GP/brings action against the NHS because you’ve given me a wrong medicine, even though you’ve had my genetic markers.” Trust Trust was a central theme in discussion of clinical PGx implementation and was impacted by and impacted on communication and education. The role of trust in clinical PGx implementation: ‘GP they trust’ There were strong feelings of trust in the NHS and health care providers, particularly primary care practitioners. Examples of broadly shared articulated trust in GP were common. Despite this trust, participants commonly cited concerns about side effects leading to medication non-compliance. “ Some people are quite scared of taking any medication because of all the side effects. Even if they get the medication from the GP… they’re going to ask how many side effects [and then] don’t take it” . Participants thought that a more personalised approach to prescribing using genetic information would enhance trust in prescribers and prescriptions, because people would have more confidence in the selection of therapeutics knowing it was aligned with their personal test results. “ After genetic test when doctor will prescribe medicine obviously there’re going to involve more trust on this ”. Participants thought that this enhanced trust would improve medication compliance, as demonstrated by one participant: “For example, if I go doctor then they just prescribe me paracetamol? Yeah. If they tell me. OK have 100 [dose]. Maybe I’m gonna have 20 or 30. But after the blood test or whatever test done. If he give me 100 then I’m gonna say yeah I’m gonna finish the 100 because it’s been done by test… In the first time, he gave me 100, I’m not gonna take it.” Ancestry specific representation in research generated evidence for therapeutics was noted to build trust in a clinical setting: “ If you get a medication out and say we tested it on these kind of… people… and that was beneficial… This drug was good for Asian community… So it’s better to take that.” The implication was that participants know that ancestry is sometimes linked with response to medication. Therefore, proportionate ancestry representation in evidence base assessing efficacy and ADRs builds trust in clinical practice by demonstrating that a specific medicine has a favourable risk-benefit profile in their community. Due to trans-ancestry variation in pharmacogene polymorphisms and historically non-diverse clinical trial cohorts this is an important point in how clinical PGx implementation interfaces with trust. Interestingly, there were no concerns from participants around misuse of data within clinical care pathways. Participants unanimously felt that their clinical data was secure through standard NHS data protection pathways and that PGx data would be no different. In the words of two participants: “ I think the GDPR legislation makes me comfortable with sharing my information with the GP and the NHS, so I don’t see any hindrance…sharing my information ”; “ the current GDPR is quite broad ”. However, participants felt that any sharing of personal data with private entities such as chemists could result in price gouging if, for example, pharmacies discovered they were serving a population who were much more likely to respond well to one specific medication. This was a widely shared concern. Education and outreach Education to support clinical PGx implementation There was consensus that national roll out of pharmacogenomic testing should be accompanied by public health level education, with outreach, and clear communication to facilitate trust. It was clear from the focus group discussions that it is important to differentiate diagnostic genetic testing or genetic testing to predict disease risk from PGx testing. There was a general concern from participants that the level of genetic literacy in the UK South Asian community is low. There was a feeling that people with more lived experience of disease and medication use were more likely to understand and be interested in PGx. Outreach and engagement Participants universally acknowledged that GPs would not be able to discuss PGx with each person. This was an impetus for support for a broader public health and outreach awareness campaign proposed by participants. Forums such as local mosques, Islamic centres, schools, fairs, shopping centres and GP surgeries were suggested to disseminate information about PGx. “ The mosque …some Islamic mosques have community services [centres] as well… the kids there are learning…the elders are coming there…women are coming there…mosques have a community system…the ladies are very much involved in that .” Multi language leaflets and videos were enthusiastically suggested, as was propagation of information via social media. The importance of leadership in the community, and community and family links, were paramount. Therefore, secular and faith leaders and heads of family were perceived to play a key role in propagating information. There was also a suggestion from participants in every group that information can be disseminated in families by incorporating education about genetics generally and PGx specifically in schools. “ Getting children to understand…maybe they can go home to their parents…if you come to schools and talk about it ”. Education and misinformation – lessons learnt from the COVID-19 pandemic Participants framed their experience with dissemination of new medical information through experiences with COVID-19 vaccines. There was a broadly shared view that misinformation around the COVID-19 pandemic and vaccines had eroded trust between the community and health care. There was perceived to be a new reluctance to engage in any non-essential clinical tests: “You know covid changed everything. Do you think that people will go for the blood test or genetic test that don’t know why you are using this, why we need this? So you need to educate them what is the importance for them. Otherwise, it’s very hard for the Asian community to come.” The pandemic highlighted a need for high quality accessible information regarding new developments in therapeutics related clinical care (“ to spread information and minimise misinformation ” in the words of one participant), and ability to understand which demographics different forms of information was reaching. Misinformation was a concern, particularly via social media platforms, where it can be widely disseminated: “ There’s so much data on the internet, and so much information it can be false ”. Education with outreach was seen as a solution to the problem of misinformation. Social media was seen as an effective tool to combat misinformation and democratise knowledge via accessible multi-media campaigns. Sharing clinical PGx data for research purposes Themes that emerged from discussion of sharing clinical PGx data for research purposes were: benefits, trust, education, data sharing facilitators, barriers to data sharing and safeguards. Benefits Benefits of research using PGx clinical data: ‘whatever is necessary to help the community’ . Research that could be generated from use of PGx clinical data was felt to be beneficial to the community with some risks to the individual’s privacy. Participants felt favourably about contributing data to support research which would benefit the community, and the good of the community had a central role in discussion. As one participant said, and others echoed: “ What’s the point in just having the blood test done and not going for research. I think that goes hand in hand…I would take it… Whatever is necessary to help the community ”. However, there were strong feelings about privacy and concerns that any data sharing may breach privacy and open potential for misuse: “I think data protection is very important in our lives…Yeah like how we said it should be between …researchers and GP…I don’t think I would like everyone to know… what benefits me… I would like to have privacy ourselves as well” . These privacy concerns were counterbalanced by the benefits of community representation in research to develop community specific knowledge. A participant highlighted concern that research on medicine is only done in some people, but then the medicine is used in all people, and participants agreed broadly that it is reassuring to be treated with medicine when the evidence base for medicine use included their community. “ When scientists do research there is one portion of the population but how [do] they apply that information onto the big portion of the population? ” (translated from Urdu). Participants felt more favourably about taking medication that had been trialled in their ancestry group and felt that ancestry specific research could drive changes in medication or supplement taking behaviour. For example, a participant gave an example of impact on behaviour driven by community specific research: most people in the community didn’t take vitamin D supplements, and then research that South Asians often lack vitamin D was disseminated. This research specific to the South Asian community then convinced many people in the community to take vitamin D: “ They got some information Asian people lack vitamin D. Apparently it’s in the genes or something…majority of the Asian people, my family members, all of them, they take vitamin D” . Benefits of data sharing to generate further research specific to the South Asian community was perceived as outweighing the potential risks of data misuse generally, particularly with appropriate safeguards: “ if it benefits the community by sharing the data… with their permission, with their consent, if this is shared in the research team that’s fine also… keeping data secure, confidential with her permission .” Trust Trust in PGx research: protective and harmful factors Willingness to share clinical data for research purposes revolved around trust. Participants felt that more personalised therapy through PGx clinical implementation would enhance trust and therefore contribute to increased willingness to engage with and share data for research. Trust was engendered by institutional affiliations (i.e., NHS, medical practitioners, national regulatory bodies such as the Medicines Healthcare Regulatory Association (MHRA)). Trust was supported by safeguards in data protection and de-identification of data used for research. Participants also found the non-diagnostic nature of PGx testing reassuring, and keeping the scope of PGx testing to non-disease diagnostic genes was a factor that enhanced trust: “ I feel if like it’s really narrowed down in front of you it would be safer …” . Trust in the individual recruiting to research was also a factor. Trust leading to research engagement could be gained by endorsement of a family member, faith leader, or community leader: “ If my relative did it, I might [do it]. Some people trust in relatives…People trust more family ”. Trust was harmed by insecure data, a history of data breach or association with individuals or institutions that were not trusted. Lack of consistency in information and profit as a motivating factor were other factors which harmed trust. Lack of trust leads to concerns about data misuse Misuse of data by non-trusted entities was a concern. This was a central disincentive to research participation: “ People really don’t want to share their information. They might have doubt on the people using to do research. That’s why they don’t want to share ” (Translated from Urdu). Concerns regarding the specific nature of potential data misuse ranged from breaches of privacy and financial exploitation to the potential for malicious actors to use genomics data for racially motivated genocide. “In theory… if someone wants to target a …specific group of people like South Asians… if I target that gene it could set off a virus that could only affect these people…I think I’ve seen it in a film, when they target a specific gene … they set this gas off but it will only effect people with this gene…South Asian genes” . This latter was perceived by some participants to be hyperbolic, and the level of time, knowledge and resources needed to misuse data in such a nefarious way were cited as protective: “ to get to the point of …killing hundreds of thousands… is far-fetched. We’d need to dissect …an entire genome, which would take a very long time, and a lot of work .” Lack of trust in profit driven research Participants across all groups expressed concern that pharmaceutical industry was not trustworthy due to profit as a motivating factor. “ Medicine is about helping people and saving lives…They’ve developed the drugs but they’re big businesses as well …”. Some extended this logic to private chemists and pharmacists working at chemists, as profit was felt to be the bottom line. The perceived conflict of interest created by profit as a primary goal was felt to lead to risk of information misuse. There were concerns about benefit hoarding for profit. Many participants across all groups worried that if industry were to get PGx data for research they would find a way to profit at the expense of the community and withhold benefits from the community. Several participants felt there was a risk of price gouging if a therapeutic was found to be particularly beneficial to their community: “but when the makers know that then they will increase the prices. And you know we are very careful about our health so we will spend money.” Another participant in a different group expressed the same sentiment: “If this information is being delivered to industry, will it affect the cost of the medicine?… if we’re getting a tablet for 1 pound we might then get it for 3 pounds” (translated from Urdu). There was a negative view toward proprietary patents as tools to restrict availability of therapeutics. There was a concern that if lifesaving medications were discovered from genetic data, patents would mean that the medicine would not be affordable or accessible to the participant communities that had contributed data to the research. However, trust in national regulators was seen by some participants to counterbalance the risk of unrestrained industry: “ business is business at the end of the day. Some businessmen are OK. If the regulator doesn’t allow, then they won’t get it [the medication]. They need to allow it first .” Feeding back research results facilitates trust Feeding back research results was crucial to ongoing research engagement through building trust: “ if someone sees a result then they will become more involved ”. Participants agreed that if research results were fed back it would support education and engagement and facilitate trust via grassroots community communication. As one participant said of receiving feedback on how she contributed to a study: “ And then you would speak to, like, your friends and family…They would open up. They would be like, wow, that’s so cool… It would build trust between communities .” Some participants felt that personalised feedback on an individual level had an even more powerful impact, and there were suggestions that researchers could build trust further by contacting individual participants to make them aware of how their data had contributed to a study. Trust in therapeutics research through the lens of the COVID-19 pandemic Participants expressed their experience with trust, and mistrust, in therapeutics and research through the lens of the COVID-19 pandemic. Participants reported a change in context and trust toward therapeutics research due to the pandemic. There was broad agreement that lack of trust had manifested in strongly divided opinions on the safety and efficacy of the COVID-19 vaccine: “for example…, covid injection, half of the people …didn’t have it…a lot of people I know, they didn’t go for that injection…it’s their choice end of the day…but there’s a reason why they didn’t have it…because they don’t trust maybe, they didn’t believe” . Lack of trust toward the COVID-19 vaccine within the South Asian community was widely felt to be prompted by the perceived pace of research and social media reports of trusted health care practitioners refusing COVID-19 vaccination. Because of the nature of the pandemic, some participants saw COVID-19 vaccines and treatments as initially experimental or offered without a full understanding of possible effects. However, it was felt that this mistrust would not extend to PGx research focused on optimising personal risk/benefit profile for existing medications. Education Education to facilitate PGx research In contrast to the skeletal information desired for clinical PGx use at the point of care, participants felt that a lot of information and education was needed to responsibly organise sharing of the generated clinical data for research. “ for research: you have to make sure you understand it perfectly and it has to be accurate information given to you. Clinical, that’s something you just do…easier to do…research you have to be really accurate .” Participants highlighted lack of awareness of research, and lack of scientific and genetic literacy as significant barriers to research engagement. “this is the reason there is less data from these groups: because the lack of education and they don’t participate if they don’t understand anything.” Language barriers were also cited as key hurdles to engagement of this community with research. However, participants also perceived a lot of interest in advancing health and medication related knowledge in the community and suggested that community ties offered vehicles to public engagement. The public health engagement campaign suggestions outlined above around national PGx roll out were echoed strongly in the discussion of education to facilitate PGx data sharing for research. Engaging with local community members for grass-roots education was advised by participants. But some participants perceived a lack of scientific literacy to be a significant barrier to community exchange of information: “How to educate those people? Like when you speak to other people they don’t know, like when she will leave from here, what she would say to her neighbour … what is that genetic information to do with the medication? We take medication everyday” (translated from Urdu). Many participants expressed interest in being trained to be community champions and volunteered to disseminate PGx information to facilitate research engagement: “ in East London mosque they have events and things… I’m here today. I understand. I will go and spread to my friends and family. So, it’s like word of mouth will get spread .” Data sharing facilitators Data sharing was the key concept on which research from a hypothetical clinical PGx service hinged. Participants required prompting to distinguishing PGx testing for clinical use from sharing clinical PGx data for research. Factors supporting PGx data sharing for research Data sharing was desirable if the researchers did not have a financial stake, and benefits would be shared. There was a common perception that without research, the use of medicines will not improve, but an understanding that the risk is to the participating individuals while the benefit would be for future individuals: “If you don’t share it, you don’t advance really. So, you have to come to some sort of compromise where you are sharing the results they need, or do you want to just not share it and be stuck and not give two hoots about what happens in the future. You have to draw that line somewhere.” . The perceived “good” of the research purpose was a key motif: “ So the point is how it works when we share for the good purpose, not for the bad purpose. So, it can help, so definitely [we should] share ”. There was broad consensus across individuals and groups that the idea of good as compared with bad purpose had an association with the trustworthiness of the researchers: “ we have a concern, so we can only share these things [PGx data] with trusty [trustworthy] ones and [make ourselves aware ].” The perceived trustworthiness of both individual researchers and associated institutions were determining factors in weighing willingness to share clinical PGx data for research purposes. Health care professionals, academic institutions, and the regulatory body (the MHRA) were considered trustworthy and therefore participants were happy to share PGx data with these groups for research with the protection of standard data de-identification and data protection. Barriers to data sharing and safeguards Barriers to sharing clinical PGx data for research and potential safeguards Participants across all groups broadly acknowledged that some people would not like to share data as a rule, due to privacy concerns: “There are people with those [privacy] concerns and those concerns are very real” . Data ownership was an important topic linked with privacy, and many participants wanted to maintain control over access to their data “It’s my information. That’s mine, do you know what I mean, it’s an invasion of privacy where you don’t have control over who gets to see your information.” As compared with healthcare practitioners and academics, there were very different perspectives on sharing PGx data for research with industry. Concerns revolved around trust, as outlined above. Most participants felt that industry has an inherent conflict of interest as a profit driven private enterprise and therefore could not be trusted to prioritise benefit sharing/the health of the community over potentially exploitive options: “ pharmaceutical companies are only thinking about their profits then it’s not good to share our information with them ”. Others felt that there is an inherent risk to not doing research: “ without research there will be always risk, there’s no cure ”. Some perceived the benefits of data sharing with industry to outweigh potential harms: “It improves the medicine, so it improves the patient care ”. Confidentiality and anonymization of data were important safeguards to protect privacy. “ It’s anonymous isn’t it…so the people who are doing it, they don’t know who it is. They have to have that barrier that [data] is confidential and not to be leaked to anyone .” Well-articulated policies around data protection and management of any breach of data protection were perceived by many participants to be crucial safeguards: “It’s important what they’re going to do with the data but also if there is a breach of data, what happens… if they find that information was leaked to the public…what they do ”. Transparency about potential conflicts of interest and opportunities to opt out of data sharing with non-trusted research partners were desirable. “I think everyone should be given the option to opt in and opt out, so I think that’s potentially a way of going forward … so you [can] opt in for pharmaceuticals or universities and … and so on…You can label them as non-profit organisation and for-profit organisations and so. That would build confidence in the person that is being involved in the research.” Safeguards against financial exploitation due to knowledge of individual or community PGx data would be protective. Benefits Benefits of clinical PGx implementation: which medicine ‘suits’ me PGx was perceived to be beneficial to individuals, by making medication choice more tailored, with less trial and error: “ which medicine suits me, I think that would be a good idea ”. There was particular interest in implementing PGx for gene-drug pairs where there are known to be high prevalence of polymorphisms in South Asian ancestry groups, and therefore a higher risk of inefficacy or toxicity in this community. Risk of ADRs were perceived to be a big concern in taking medications, and to impact on compliance. There were concerns that ADRs can be worse or more long-lasting that the original treatment indication, and that if participants knew of someone who reacted badly to a medication, they would not want to take it: “For example, I take a medicine and I react really badly to it. Everyone in this room might sit there and think, wow, she’s taking that medication and she’s had a really bad reaction. Maybe I shouldn’t take that medicine.” Participants felt that PGx had the potential to mitigate this reaction by reassuring people that genetic risk of ADRs had been checked. The potential to avoid broad contraindications with a more targeted approach was raised by several participant anecdotes. For example, one participant suggested that with more precise PGx stratification we might better understand which asthmatics are likely to have a bad reaction to ibuprofen and not withhold it from those who are not likely to have an ADR. Communication Communication to support clinical PGx implementation Participants felt that limited information was desired for clinical PGx use at the point of care. There was a strong preference for use of simple language to communicate PGx. Participants thought that the easiest way of conveying the utility of PGx was to identify which medicines “ suit ” you/your body. Participants generally agreed that for clinical indication a minimal explanation of PGx testing to inform medication choice (similar to a routine blood test) was sufficient. Many participants didn’t think it was necessary or helpful to include the fact that DNA/genetics are being tested. For example, as one participant reflected elderly people might not understand what genes are in comparison to younger people. Given this, they suggested presenting PGx as something that would help clinicians make sure that the medicines they prescribe are “ more suitable for you ” would result in an explanation that would make sense to a wider range of people. This sentiment of offering PGx clinically for medicine optimisation without detailed discussion of genetics was echoed by the majority of participants across all focus groups. There was a strong preference that communication around PGx be led by General practitioners (GPs). GPs were described as trusted sources of information and having the skill and resources to support communication where language and literacy barriers are present: “ GP can explain very well” . Timing Timing of testing in the clinical pathway: who would benefit the most and how should testing eligibility reflect that? PGx was viewed as particularly helpful to those who suffer from polypharmacy. People taking many medications were perceived as most likely to benefit from PGx testing, by decreasing risk of side effects and drug-drug interactions. In addition to identifying polypharmacy as increasing risk of ADRs, participants felt that enhancing efficacy from medication for those with the most morbidity was important, regardless of age. In the words of one participant, which provoked broad agreement “ you could have someone that is like half the age and has already been using so many different medicines. They aren’t working for them and they wanna know why it’s not working .” Due to the shared view amongst participants that the greatest beneficiaries of PGx implementation would be those with the most morbidity, they proposed the idea of a secondary prevention speciality clinic. They felt that this would mean that people at high risk would be able to benefit from PGx innovations “ as soon as possible ” rather than having to wait potentially many years for pre-emptive PGx to be rolled out across everyday clinical practice for all people via NHS primary care. While benefits of more personalised medicine were thought to be particularly promising for multimorbid patients, if resources allowed participants liked the idea of PGx panel testing for all at birth, so that the information would be there pre-emptively to optimise medication choice throughout life. Several participants suggested they would welcome PGx testing as a part of routine neonatal testing: “ you know the kids are born and then they offer the next day the hearing test …in the hospital without leaving? You can offer [PGx] at the same time .” Participants had no concerns about doing PGx testing on babies, provided sample extraction was not painful or harmful. Parents were much more concerned with the risks of a perceived trial and error prescribing approach that did not consider genetic data which could indicate high ADR risk. Testing in primary care . Participants felt strongly that pre-emptive PGx testing via the GP was preferable to point of care testing in hospital at the time of indication for therapeutic (ie in the example of CYP2C19 testing to help guide anti-platelet choice after a myocardial infarction). The reasons were multifactorial; the GP was first point of contact, had all patient information, provided continuity of care, and was perceived to communicate well. Participants liked the idea of having PGx testing before there was a treatment indication, and felt primary care was the right place for this kind of anticipatory testing. There was also a concern that anything viewed as not essential may not reliably happen in acute care settings. Furthermore, participants thought of primary care as a less threatening and more personal setting where there was a higher likelihood of receiving information about test results and being in a state to understand that information, as compared with hospitals. “ Going to the GP… it’s a lot more personal than going to a hospital… if you’re at a hospital it just kind of feels alien ”. They also felt that need for acute care was associated with fear: “ people go in hospital when [they are] in danger…I can call the GP and book an appointment… when you go to hospital [there’s] always danger there ”. Participants felt that due to the acute nature of secondary care communication was limited, and patients were often unaware of investigations ordered. As one participant surmised: “ We don’t even know probably half of the things they do. No one questions about the medicine or why they’re taking the blood test. There’s no choice ”. Custodian of data Maximising benefits of clinical PGx testing: transfer of information across care settings Benefits of PGx were thought to be greatest if PGx results could be effectively shared across care settings, particularly primary and secondary care but also community pharmacy settings. Some participants felt that integration across care settings of existing analogous data is not good. One parent illustrated this with an anecdote: “I have an example: One of my sons [is] allergic [to] ibuprofen. So, this information I can see …the GP shared with me…but always I have to tell [them] in a hospital, don’t give ibuprofen to him, because he has a reaction with that” . However, another participant gave examples of successful programmes where important medical information travels with patients, suggesting the same could be done with PGx: “ Shouldn’t be a problem because you already have medical bracelets and tags for people with different…conditions… so they could be identified if some something was to happen to them in public you can see that necklace or bracelet .” Participants liked the idea of an NHS app having PGx information that could travel with the patient and allow self-advocacy. For example, in the words of one participant: “It should be the GP as well as the patient who has that information because… sometimes… the GP don’t really listen properly… if she knows what her needs are… she can show it and say this is what it is. This is my genetic result” (translated from Bengali). Some participants saw community pharmacists as care providers that could give more personalised advice if they had access to PGx results. This could take some strain off primary care. However, others perceived sharing clinical PGx data with private chemists as a risk that could lead to inflated prices. Cost Balancing benefits against costs The benefits of clinical PGx implementation, particularly as pre-emptive testing for all nationally, were weighed against the risk of overburdening resource limited NHS services and clinicians, which participants felt protective of. There was trust in the NHS and NHS clinicians and a perception that the benefit of PGx implementation would need to outweigh added financial strain and time constraints on these institutions and professionals. There was a feeling that any preventive endeavour would be lower priority, as compared with testing which responded to clinical need. In the words of one participant, which the other participants expressed agreement with: “you know they’re suggesting a GP visit should cost people money…what about the cost of the test… would it cause too much pressure? …In advance you are doing a testing… Maybe you need it in the future or not…still you are doing it … they’re asking for less pressure on the NHS then you’re putting so much more pressure on the NHS.” Many participants felt that streamlined logistics of PGx implementation were crucial to ensure the inconvenience of participation wasn’t perceived to outweigh the benefits. A further concern to the integrity of existing services and professionals was any added threat of litigation. This concern further highlighted the protective feeling participants had toward the NHS, and the requirement that the benefits outweigh the all-inclusive costs: “Could this open up the NHS or the GP to liable action, i.e., being sued? Because they have the genetic markers there. You gave the medicine, but now obviously they got it wrong…Patient then sues the GP/brings action against the NHS because you’ve given me a wrong medicine, even though you’ve had my genetic markers.” Trust Trust was a central theme in discussion of clinical PGx implementation and was impacted by and impacted on communication and education. The role of trust in clinical PGx implementation: ‘GP they trust’ There were strong feelings of trust in the NHS and health care providers, particularly primary care practitioners. Examples of broadly shared articulated trust in GP were common. Despite this trust, participants commonly cited concerns about side effects leading to medication non-compliance. “ Some people are quite scared of taking any medication because of all the side effects. Even if they get the medication from the GP… they’re going to ask how many side effects [and then] don’t take it” . Participants thought that a more personalised approach to prescribing using genetic information would enhance trust in prescribers and prescriptions, because people would have more confidence in the selection of therapeutics knowing it was aligned with their personal test results. “ After genetic test when doctor will prescribe medicine obviously there’re going to involve more trust on this ”. Participants thought that this enhanced trust would improve medication compliance, as demonstrated by one participant: “For example, if I go doctor then they just prescribe me paracetamol? Yeah. If they tell me. OK have 100 [dose]. Maybe I’m gonna have 20 or 30. But after the blood test or whatever test done. If he give me 100 then I’m gonna say yeah I’m gonna finish the 100 because it’s been done by test… In the first time, he gave me 100, I’m not gonna take it.” Ancestry specific representation in research generated evidence for therapeutics was noted to build trust in a clinical setting: “ If you get a medication out and say we tested it on these kind of… people… and that was beneficial… This drug was good for Asian community… So it’s better to take that.” The implication was that participants know that ancestry is sometimes linked with response to medication. Therefore, proportionate ancestry representation in evidence base assessing efficacy and ADRs builds trust in clinical practice by demonstrating that a specific medicine has a favourable risk-benefit profile in their community. Due to trans-ancestry variation in pharmacogene polymorphisms and historically non-diverse clinical trial cohorts this is an important point in how clinical PGx implementation interfaces with trust. Interestingly, there were no concerns from participants around misuse of data within clinical care pathways. Participants unanimously felt that their clinical data was secure through standard NHS data protection pathways and that PGx data would be no different. In the words of two participants: “ I think the GDPR legislation makes me comfortable with sharing my information with the GP and the NHS, so I don’t see any hindrance…sharing my information ”; “ the current GDPR is quite broad ”. However, participants felt that any sharing of personal data with private entities such as chemists could result in price gouging if, for example, pharmacies discovered they were serving a population who were much more likely to respond well to one specific medication. This was a widely shared concern. Education and outreach Education to support clinical PGx implementation There was consensus that national roll out of pharmacogenomic testing should be accompanied by public health level education, with outreach, and clear communication to facilitate trust. It was clear from the focus group discussions that it is important to differentiate diagnostic genetic testing or genetic testing to predict disease risk from PGx testing. There was a general concern from participants that the level of genetic literacy in the UK South Asian community is low. There was a feeling that people with more lived experience of disease and medication use were more likely to understand and be interested in PGx. Outreach and engagement Participants universally acknowledged that GPs would not be able to discuss PGx with each person. This was an impetus for support for a broader public health and outreach awareness campaign proposed by participants. Forums such as local mosques, Islamic centres, schools, fairs, shopping centres and GP surgeries were suggested to disseminate information about PGx. “ The mosque …some Islamic mosques have community services [centres] as well… the kids there are learning…the elders are coming there…women are coming there…mosques have a community system…the ladies are very much involved in that .” Multi language leaflets and videos were enthusiastically suggested, as was propagation of information via social media. The importance of leadership in the community, and community and family links, were paramount. Therefore, secular and faith leaders and heads of family were perceived to play a key role in propagating information. There was also a suggestion from participants in every group that information can be disseminated in families by incorporating education about genetics generally and PGx specifically in schools. “ Getting children to understand…maybe they can go home to their parents…if you come to schools and talk about it ”. Education and misinformation – lessons learnt from the COVID-19 pandemic Participants framed their experience with dissemination of new medical information through experiences with COVID-19 vaccines. There was a broadly shared view that misinformation around the COVID-19 pandemic and vaccines had eroded trust between the community and health care. There was perceived to be a new reluctance to engage in any non-essential clinical tests: “You know covid changed everything. Do you think that people will go for the blood test or genetic test that don’t know why you are using this, why we need this? So you need to educate them what is the importance for them. Otherwise, it’s very hard for the Asian community to come.” The pandemic highlighted a need for high quality accessible information regarding new developments in therapeutics related clinical care (“ to spread information and minimise misinformation ” in the words of one participant), and ability to understand which demographics different forms of information was reaching. Misinformation was a concern, particularly via social media platforms, where it can be widely disseminated: “ There’s so much data on the internet, and so much information it can be false ”. Education with outreach was seen as a solution to the problem of misinformation. Social media was seen as an effective tool to combat misinformation and democratise knowledge via accessible multi-media campaigns. Benefits of clinical PGx implementation: which medicine ‘suits’ me PGx was perceived to be beneficial to individuals, by making medication choice more tailored, with less trial and error: “ which medicine suits me, I think that would be a good idea ”. There was particular interest in implementing PGx for gene-drug pairs where there are known to be high prevalence of polymorphisms in South Asian ancestry groups, and therefore a higher risk of inefficacy or toxicity in this community. Risk of ADRs were perceived to be a big concern in taking medications, and to impact on compliance. There were concerns that ADRs can be worse or more long-lasting that the original treatment indication, and that if participants knew of someone who reacted badly to a medication, they would not want to take it: “For example, I take a medicine and I react really badly to it. Everyone in this room might sit there and think, wow, she’s taking that medication and she’s had a really bad reaction. Maybe I shouldn’t take that medicine.” Participants felt that PGx had the potential to mitigate this reaction by reassuring people that genetic risk of ADRs had been checked. The potential to avoid broad contraindications with a more targeted approach was raised by several participant anecdotes. For example, one participant suggested that with more precise PGx stratification we might better understand which asthmatics are likely to have a bad reaction to ibuprofen and not withhold it from those who are not likely to have an ADR. Communication to support clinical PGx implementation Participants felt that limited information was desired for clinical PGx use at the point of care. There was a strong preference for use of simple language to communicate PGx. Participants thought that the easiest way of conveying the utility of PGx was to identify which medicines “ suit ” you/your body. Participants generally agreed that for clinical indication a minimal explanation of PGx testing to inform medication choice (similar to a routine blood test) was sufficient. Many participants didn’t think it was necessary or helpful to include the fact that DNA/genetics are being tested. For example, as one participant reflected elderly people might not understand what genes are in comparison to younger people. Given this, they suggested presenting PGx as something that would help clinicians make sure that the medicines they prescribe are “ more suitable for you ” would result in an explanation that would make sense to a wider range of people. This sentiment of offering PGx clinically for medicine optimisation without detailed discussion of genetics was echoed by the majority of participants across all focus groups. There was a strong preference that communication around PGx be led by General practitioners (GPs). GPs were described as trusted sources of information and having the skill and resources to support communication where language and literacy barriers are present: “ GP can explain very well” . Timing of testing in the clinical pathway: who would benefit the most and how should testing eligibility reflect that? PGx was viewed as particularly helpful to those who suffer from polypharmacy. People taking many medications were perceived as most likely to benefit from PGx testing, by decreasing risk of side effects and drug-drug interactions. In addition to identifying polypharmacy as increasing risk of ADRs, participants felt that enhancing efficacy from medication for those with the most morbidity was important, regardless of age. In the words of one participant, which provoked broad agreement “ you could have someone that is like half the age and has already been using so many different medicines. They aren’t working for them and they wanna know why it’s not working .” Due to the shared view amongst participants that the greatest beneficiaries of PGx implementation would be those with the most morbidity, they proposed the idea of a secondary prevention speciality clinic. They felt that this would mean that people at high risk would be able to benefit from PGx innovations “ as soon as possible ” rather than having to wait potentially many years for pre-emptive PGx to be rolled out across everyday clinical practice for all people via NHS primary care. While benefits of more personalised medicine were thought to be particularly promising for multimorbid patients, if resources allowed participants liked the idea of PGx panel testing for all at birth, so that the information would be there pre-emptively to optimise medication choice throughout life. Several participants suggested they would welcome PGx testing as a part of routine neonatal testing: “ you know the kids are born and then they offer the next day the hearing test …in the hospital without leaving? You can offer [PGx] at the same time .” Participants had no concerns about doing PGx testing on babies, provided sample extraction was not painful or harmful. Parents were much more concerned with the risks of a perceived trial and error prescribing approach that did not consider genetic data which could indicate high ADR risk. Testing in primary care . Participants felt strongly that pre-emptive PGx testing via the GP was preferable to point of care testing in hospital at the time of indication for therapeutic (ie in the example of CYP2C19 testing to help guide anti-platelet choice after a myocardial infarction). The reasons were multifactorial; the GP was first point of contact, had all patient information, provided continuity of care, and was perceived to communicate well. Participants liked the idea of having PGx testing before there was a treatment indication, and felt primary care was the right place for this kind of anticipatory testing. There was also a concern that anything viewed as not essential may not reliably happen in acute care settings. Furthermore, participants thought of primary care as a less threatening and more personal setting where there was a higher likelihood of receiving information about test results and being in a state to understand that information, as compared with hospitals. “ Going to the GP… it’s a lot more personal than going to a hospital… if you’re at a hospital it just kind of feels alien ”. They also felt that need for acute care was associated with fear: “ people go in hospital when [they are] in danger…I can call the GP and book an appointment… when you go to hospital [there’s] always danger there ”. Participants felt that due to the acute nature of secondary care communication was limited, and patients were often unaware of investigations ordered. As one participant surmised: “ We don’t even know probably half of the things they do. No one questions about the medicine or why they’re taking the blood test. There’s no choice ”. Maximising benefits of clinical PGx testing: transfer of information across care settings Benefits of PGx were thought to be greatest if PGx results could be effectively shared across care settings, particularly primary and secondary care but also community pharmacy settings. Some participants felt that integration across care settings of existing analogous data is not good. One parent illustrated this with an anecdote: “I have an example: One of my sons [is] allergic [to] ibuprofen. So, this information I can see …the GP shared with me…but always I have to tell [them] in a hospital, don’t give ibuprofen to him, because he has a reaction with that” . However, another participant gave examples of successful programmes where important medical information travels with patients, suggesting the same could be done with PGx: “ Shouldn’t be a problem because you already have medical bracelets and tags for people with different…conditions… so they could be identified if some something was to happen to them in public you can see that necklace or bracelet .” Participants liked the idea of an NHS app having PGx information that could travel with the patient and allow self-advocacy. For example, in the words of one participant: “It should be the GP as well as the patient who has that information because… sometimes… the GP don’t really listen properly… if she knows what her needs are… she can show it and say this is what it is. This is my genetic result” (translated from Bengali). Some participants saw community pharmacists as care providers that could give more personalised advice if they had access to PGx results. This could take some strain off primary care. However, others perceived sharing clinical PGx data with private chemists as a risk that could lead to inflated prices. Balancing benefits against costs The benefits of clinical PGx implementation, particularly as pre-emptive testing for all nationally, were weighed against the risk of overburdening resource limited NHS services and clinicians, which participants felt protective of. There was trust in the NHS and NHS clinicians and a perception that the benefit of PGx implementation would need to outweigh added financial strain and time constraints on these institutions and professionals. There was a feeling that any preventive endeavour would be lower priority, as compared with testing which responded to clinical need. In the words of one participant, which the other participants expressed agreement with: “you know they’re suggesting a GP visit should cost people money…what about the cost of the test… would it cause too much pressure? …In advance you are doing a testing… Maybe you need it in the future or not…still you are doing it … they’re asking for less pressure on the NHS then you’re putting so much more pressure on the NHS.” Many participants felt that streamlined logistics of PGx implementation were crucial to ensure the inconvenience of participation wasn’t perceived to outweigh the benefits. A further concern to the integrity of existing services and professionals was any added threat of litigation. This concern further highlighted the protective feeling participants had toward the NHS, and the requirement that the benefits outweigh the all-inclusive costs: “Could this open up the NHS or the GP to liable action, i.e., being sued? Because they have the genetic markers there. You gave the medicine, but now obviously they got it wrong…Patient then sues the GP/brings action against the NHS because you’ve given me a wrong medicine, even though you’ve had my genetic markers.” Trust was a central theme in discussion of clinical PGx implementation and was impacted by and impacted on communication and education. The role of trust in clinical PGx implementation: ‘GP they trust’ There were strong feelings of trust in the NHS and health care providers, particularly primary care practitioners. Examples of broadly shared articulated trust in GP were common. Despite this trust, participants commonly cited concerns about side effects leading to medication non-compliance. “ Some people are quite scared of taking any medication because of all the side effects. Even if they get the medication from the GP… they’re going to ask how many side effects [and then] don’t take it” . Participants thought that a more personalised approach to prescribing using genetic information would enhance trust in prescribers and prescriptions, because people would have more confidence in the selection of therapeutics knowing it was aligned with their personal test results. “ After genetic test when doctor will prescribe medicine obviously there’re going to involve more trust on this ”. Participants thought that this enhanced trust would improve medication compliance, as demonstrated by one participant: “For example, if I go doctor then they just prescribe me paracetamol? Yeah. If they tell me. OK have 100 [dose]. Maybe I’m gonna have 20 or 30. But after the blood test or whatever test done. If he give me 100 then I’m gonna say yeah I’m gonna finish the 100 because it’s been done by test… In the first time, he gave me 100, I’m not gonna take it.” Ancestry specific representation in research generated evidence for therapeutics was noted to build trust in a clinical setting: “ If you get a medication out and say we tested it on these kind of… people… and that was beneficial… This drug was good for Asian community… So it’s better to take that.” The implication was that participants know that ancestry is sometimes linked with response to medication. Therefore, proportionate ancestry representation in evidence base assessing efficacy and ADRs builds trust in clinical practice by demonstrating that a specific medicine has a favourable risk-benefit profile in their community. Due to trans-ancestry variation in pharmacogene polymorphisms and historically non-diverse clinical trial cohorts this is an important point in how clinical PGx implementation interfaces with trust. Interestingly, there were no concerns from participants around misuse of data within clinical care pathways. Participants unanimously felt that their clinical data was secure through standard NHS data protection pathways and that PGx data would be no different. In the words of two participants: “ I think the GDPR legislation makes me comfortable with sharing my information with the GP and the NHS, so I don’t see any hindrance…sharing my information ”; “ the current GDPR is quite broad ”. However, participants felt that any sharing of personal data with private entities such as chemists could result in price gouging if, for example, pharmacies discovered they were serving a population who were much more likely to respond well to one specific medication. This was a widely shared concern. Education to support clinical PGx implementation There was consensus that national roll out of pharmacogenomic testing should be accompanied by public health level education, with outreach, and clear communication to facilitate trust. It was clear from the focus group discussions that it is important to differentiate diagnostic genetic testing or genetic testing to predict disease risk from PGx testing. There was a general concern from participants that the level of genetic literacy in the UK South Asian community is low. There was a feeling that people with more lived experience of disease and medication use were more likely to understand and be interested in PGx. Outreach and engagement Participants universally acknowledged that GPs would not be able to discuss PGx with each person. This was an impetus for support for a broader public health and outreach awareness campaign proposed by participants. Forums such as local mosques, Islamic centres, schools, fairs, shopping centres and GP surgeries were suggested to disseminate information about PGx. “ The mosque …some Islamic mosques have community services [centres] as well… the kids there are learning…the elders are coming there…women are coming there…mosques have a community system…the ladies are very much involved in that .” Multi language leaflets and videos were enthusiastically suggested, as was propagation of information via social media. The importance of leadership in the community, and community and family links, were paramount. Therefore, secular and faith leaders and heads of family were perceived to play a key role in propagating information. There was also a suggestion from participants in every group that information can be disseminated in families by incorporating education about genetics generally and PGx specifically in schools. “ Getting children to understand…maybe they can go home to their parents…if you come to schools and talk about it ”. Education and misinformation – lessons learnt from the COVID-19 pandemic Participants framed their experience with dissemination of new medical information through experiences with COVID-19 vaccines. There was a broadly shared view that misinformation around the COVID-19 pandemic and vaccines had eroded trust between the community and health care. There was perceived to be a new reluctance to engage in any non-essential clinical tests: “You know covid changed everything. Do you think that people will go for the blood test or genetic test that don’t know why you are using this, why we need this? So you need to educate them what is the importance for them. Otherwise, it’s very hard for the Asian community to come.” The pandemic highlighted a need for high quality accessible information regarding new developments in therapeutics related clinical care (“ to spread information and minimise misinformation ” in the words of one participant), and ability to understand which demographics different forms of information was reaching. Misinformation was a concern, particularly via social media platforms, where it can be widely disseminated: “ There’s so much data on the internet, and so much information it can be false ”. Education with outreach was seen as a solution to the problem of misinformation. Social media was seen as an effective tool to combat misinformation and democratise knowledge via accessible multi-media campaigns. Themes that emerged from discussion of sharing clinical PGx data for research purposes were: benefits, trust, education, data sharing facilitators, barriers to data sharing and safeguards. Benefits Benefits of research using PGx clinical data: ‘whatever is necessary to help the community’ . Research that could be generated from use of PGx clinical data was felt to be beneficial to the community with some risks to the individual’s privacy. Participants felt favourably about contributing data to support research which would benefit the community, and the good of the community had a central role in discussion. As one participant said, and others echoed: “ What’s the point in just having the blood test done and not going for research. I think that goes hand in hand…I would take it… Whatever is necessary to help the community ”. However, there were strong feelings about privacy and concerns that any data sharing may breach privacy and open potential for misuse: “I think data protection is very important in our lives…Yeah like how we said it should be between …researchers and GP…I don’t think I would like everyone to know… what benefits me… I would like to have privacy ourselves as well” . These privacy concerns were counterbalanced by the benefits of community representation in research to develop community specific knowledge. A participant highlighted concern that research on medicine is only done in some people, but then the medicine is used in all people, and participants agreed broadly that it is reassuring to be treated with medicine when the evidence base for medicine use included their community. “ When scientists do research there is one portion of the population but how [do] they apply that information onto the big portion of the population? ” (translated from Urdu). Participants felt more favourably about taking medication that had been trialled in their ancestry group and felt that ancestry specific research could drive changes in medication or supplement taking behaviour. For example, a participant gave an example of impact on behaviour driven by community specific research: most people in the community didn’t take vitamin D supplements, and then research that South Asians often lack vitamin D was disseminated. This research specific to the South Asian community then convinced many people in the community to take vitamin D: “ They got some information Asian people lack vitamin D. Apparently it’s in the genes or something…majority of the Asian people, my family members, all of them, they take vitamin D” . Benefits of data sharing to generate further research specific to the South Asian community was perceived as outweighing the potential risks of data misuse generally, particularly with appropriate safeguards: “ if it benefits the community by sharing the data… with their permission, with their consent, if this is shared in the research team that’s fine also… keeping data secure, confidential with her permission .” Trust Trust in PGx research: protective and harmful factors Willingness to share clinical data for research purposes revolved around trust. Participants felt that more personalised therapy through PGx clinical implementation would enhance trust and therefore contribute to increased willingness to engage with and share data for research. Trust was engendered by institutional affiliations (i.e., NHS, medical practitioners, national regulatory bodies such as the Medicines Healthcare Regulatory Association (MHRA)). Trust was supported by safeguards in data protection and de-identification of data used for research. Participants also found the non-diagnostic nature of PGx testing reassuring, and keeping the scope of PGx testing to non-disease diagnostic genes was a factor that enhanced trust: “ I feel if like it’s really narrowed down in front of you it would be safer …” . Trust in the individual recruiting to research was also a factor. Trust leading to research engagement could be gained by endorsement of a family member, faith leader, or community leader: “ If my relative did it, I might [do it]. Some people trust in relatives…People trust more family ”. Trust was harmed by insecure data, a history of data breach or association with individuals or institutions that were not trusted. Lack of consistency in information and profit as a motivating factor were other factors which harmed trust. Lack of trust leads to concerns about data misuse Misuse of data by non-trusted entities was a concern. This was a central disincentive to research participation: “ People really don’t want to share their information. They might have doubt on the people using to do research. That’s why they don’t want to share ” (Translated from Urdu). Concerns regarding the specific nature of potential data misuse ranged from breaches of privacy and financial exploitation to the potential for malicious actors to use genomics data for racially motivated genocide. “In theory… if someone wants to target a …specific group of people like South Asians… if I target that gene it could set off a virus that could only affect these people…I think I’ve seen it in a film, when they target a specific gene … they set this gas off but it will only effect people with this gene…South Asian genes” . This latter was perceived by some participants to be hyperbolic, and the level of time, knowledge and resources needed to misuse data in such a nefarious way were cited as protective: “ to get to the point of …killing hundreds of thousands… is far-fetched. We’d need to dissect …an entire genome, which would take a very long time, and a lot of work .” Lack of trust in profit driven research Participants across all groups expressed concern that pharmaceutical industry was not trustworthy due to profit as a motivating factor. “ Medicine is about helping people and saving lives…They’ve developed the drugs but they’re big businesses as well …”. Some extended this logic to private chemists and pharmacists working at chemists, as profit was felt to be the bottom line. The perceived conflict of interest created by profit as a primary goal was felt to lead to risk of information misuse. There were concerns about benefit hoarding for profit. Many participants across all groups worried that if industry were to get PGx data for research they would find a way to profit at the expense of the community and withhold benefits from the community. Several participants felt there was a risk of price gouging if a therapeutic was found to be particularly beneficial to their community: “but when the makers know that then they will increase the prices. And you know we are very careful about our health so we will spend money.” Another participant in a different group expressed the same sentiment: “If this information is being delivered to industry, will it affect the cost of the medicine?… if we’re getting a tablet for 1 pound we might then get it for 3 pounds” (translated from Urdu). There was a negative view toward proprietary patents as tools to restrict availability of therapeutics. There was a concern that if lifesaving medications were discovered from genetic data, patents would mean that the medicine would not be affordable or accessible to the participant communities that had contributed data to the research. However, trust in national regulators was seen by some participants to counterbalance the risk of unrestrained industry: “ business is business at the end of the day. Some businessmen are OK. If the regulator doesn’t allow, then they won’t get it [the medication]. They need to allow it first .” Feeding back research results facilitates trust Feeding back research results was crucial to ongoing research engagement through building trust: “ if someone sees a result then they will become more involved ”. Participants agreed that if research results were fed back it would support education and engagement and facilitate trust via grassroots community communication. As one participant said of receiving feedback on how she contributed to a study: “ And then you would speak to, like, your friends and family…They would open up. They would be like, wow, that’s so cool… It would build trust between communities .” Some participants felt that personalised feedback on an individual level had an even more powerful impact, and there were suggestions that researchers could build trust further by contacting individual participants to make them aware of how their data had contributed to a study. Trust in therapeutics research through the lens of the COVID-19 pandemic Participants expressed their experience with trust, and mistrust, in therapeutics and research through the lens of the COVID-19 pandemic. Participants reported a change in context and trust toward therapeutics research due to the pandemic. There was broad agreement that lack of trust had manifested in strongly divided opinions on the safety and efficacy of the COVID-19 vaccine: “for example…, covid injection, half of the people …didn’t have it…a lot of people I know, they didn’t go for that injection…it’s their choice end of the day…but there’s a reason why they didn’t have it…because they don’t trust maybe, they didn’t believe” . Lack of trust toward the COVID-19 vaccine within the South Asian community was widely felt to be prompted by the perceived pace of research and social media reports of trusted health care practitioners refusing COVID-19 vaccination. Because of the nature of the pandemic, some participants saw COVID-19 vaccines and treatments as initially experimental or offered without a full understanding of possible effects. However, it was felt that this mistrust would not extend to PGx research focused on optimising personal risk/benefit profile for existing medications. Education Education to facilitate PGx research In contrast to the skeletal information desired for clinical PGx use at the point of care, participants felt that a lot of information and education was needed to responsibly organise sharing of the generated clinical data for research. “ for research: you have to make sure you understand it perfectly and it has to be accurate information given to you. Clinical, that’s something you just do…easier to do…research you have to be really accurate .” Participants highlighted lack of awareness of research, and lack of scientific and genetic literacy as significant barriers to research engagement. “this is the reason there is less data from these groups: because the lack of education and they don’t participate if they don’t understand anything.” Language barriers were also cited as key hurdles to engagement of this community with research. However, participants also perceived a lot of interest in advancing health and medication related knowledge in the community and suggested that community ties offered vehicles to public engagement. The public health engagement campaign suggestions outlined above around national PGx roll out were echoed strongly in the discussion of education to facilitate PGx data sharing for research. Engaging with local community members for grass-roots education was advised by participants. But some participants perceived a lack of scientific literacy to be a significant barrier to community exchange of information: “How to educate those people? Like when you speak to other people they don’t know, like when she will leave from here, what she would say to her neighbour … what is that genetic information to do with the medication? We take medication everyday” (translated from Urdu). Many participants expressed interest in being trained to be community champions and volunteered to disseminate PGx information to facilitate research engagement: “ in East London mosque they have events and things… I’m here today. I understand. I will go and spread to my friends and family. So, it’s like word of mouth will get spread .” Data sharing facilitators Data sharing was the key concept on which research from a hypothetical clinical PGx service hinged. Participants required prompting to distinguishing PGx testing for clinical use from sharing clinical PGx data for research. Factors supporting PGx data sharing for research Data sharing was desirable if the researchers did not have a financial stake, and benefits would be shared. There was a common perception that without research, the use of medicines will not improve, but an understanding that the risk is to the participating individuals while the benefit would be for future individuals: “If you don’t share it, you don’t advance really. So, you have to come to some sort of compromise where you are sharing the results they need, or do you want to just not share it and be stuck and not give two hoots about what happens in the future. You have to draw that line somewhere.” . The perceived “good” of the research purpose was a key motif: “ So the point is how it works when we share for the good purpose, not for the bad purpose. So, it can help, so definitely [we should] share ”. There was broad consensus across individuals and groups that the idea of good as compared with bad purpose had an association with the trustworthiness of the researchers: “ we have a concern, so we can only share these things [PGx data] with trusty [trustworthy] ones and [make ourselves aware ].” The perceived trustworthiness of both individual researchers and associated institutions were determining factors in weighing willingness to share clinical PGx data for research purposes. Health care professionals, academic institutions, and the regulatory body (the MHRA) were considered trustworthy and therefore participants were happy to share PGx data with these groups for research with the protection of standard data de-identification and data protection. Barriers to data sharing and safeguards Barriers to sharing clinical PGx data for research and potential safeguards Participants across all groups broadly acknowledged that some people would not like to share data as a rule, due to privacy concerns: “There are people with those [privacy] concerns and those concerns are very real” . Data ownership was an important topic linked with privacy, and many participants wanted to maintain control over access to their data “It’s my information. That’s mine, do you know what I mean, it’s an invasion of privacy where you don’t have control over who gets to see your information.” As compared with healthcare practitioners and academics, there were very different perspectives on sharing PGx data for research with industry. Concerns revolved around trust, as outlined above. Most participants felt that industry has an inherent conflict of interest as a profit driven private enterprise and therefore could not be trusted to prioritise benefit sharing/the health of the community over potentially exploitive options: “ pharmaceutical companies are only thinking about their profits then it’s not good to share our information with them ”. Others felt that there is an inherent risk to not doing research: “ without research there will be always risk, there’s no cure ”. Some perceived the benefits of data sharing with industry to outweigh potential harms: “It improves the medicine, so it improves the patient care ”. Confidentiality and anonymization of data were important safeguards to protect privacy. “ It’s anonymous isn’t it…so the people who are doing it, they don’t know who it is. They have to have that barrier that [data] is confidential and not to be leaked to anyone .” Well-articulated policies around data protection and management of any breach of data protection were perceived by many participants to be crucial safeguards: “It’s important what they’re going to do with the data but also if there is a breach of data, what happens… if they find that information was leaked to the public…what they do ”. Transparency about potential conflicts of interest and opportunities to opt out of data sharing with non-trusted research partners were desirable. “I think everyone should be given the option to opt in and opt out, so I think that’s potentially a way of going forward … so you [can] opt in for pharmaceuticals or universities and … and so on…You can label them as non-profit organisation and for-profit organisations and so. That would build confidence in the person that is being involved in the research.” Safeguards against financial exploitation due to knowledge of individual or community PGx data would be protective. Benefits of research using PGx clinical data: ‘whatever is necessary to help the community’ . Research that could be generated from use of PGx clinical data was felt to be beneficial to the community with some risks to the individual’s privacy. Participants felt favourably about contributing data to support research which would benefit the community, and the good of the community had a central role in discussion. As one participant said, and others echoed: “ What’s the point in just having the blood test done and not going for research. I think that goes hand in hand…I would take it… Whatever is necessary to help the community ”. However, there were strong feelings about privacy and concerns that any data sharing may breach privacy and open potential for misuse: “I think data protection is very important in our lives…Yeah like how we said it should be between …researchers and GP…I don’t think I would like everyone to know… what benefits me… I would like to have privacy ourselves as well” . These privacy concerns were counterbalanced by the benefits of community representation in research to develop community specific knowledge. A participant highlighted concern that research on medicine is only done in some people, but then the medicine is used in all people, and participants agreed broadly that it is reassuring to be treated with medicine when the evidence base for medicine use included their community. “ When scientists do research there is one portion of the population but how [do] they apply that information onto the big portion of the population? ” (translated from Urdu). Participants felt more favourably about taking medication that had been trialled in their ancestry group and felt that ancestry specific research could drive changes in medication or supplement taking behaviour. For example, a participant gave an example of impact on behaviour driven by community specific research: most people in the community didn’t take vitamin D supplements, and then research that South Asians often lack vitamin D was disseminated. This research specific to the South Asian community then convinced many people in the community to take vitamin D: “ They got some information Asian people lack vitamin D. Apparently it’s in the genes or something…majority of the Asian people, my family members, all of them, they take vitamin D” . Benefits of data sharing to generate further research specific to the South Asian community was perceived as outweighing the potential risks of data misuse generally, particularly with appropriate safeguards: “ if it benefits the community by sharing the data… with their permission, with their consent, if this is shared in the research team that’s fine also… keeping data secure, confidential with her permission .” Trust in PGx research: protective and harmful factors Willingness to share clinical data for research purposes revolved around trust. Participants felt that more personalised therapy through PGx clinical implementation would enhance trust and therefore contribute to increased willingness to engage with and share data for research. Trust was engendered by institutional affiliations (i.e., NHS, medical practitioners, national regulatory bodies such as the Medicines Healthcare Regulatory Association (MHRA)). Trust was supported by safeguards in data protection and de-identification of data used for research. Participants also found the non-diagnostic nature of PGx testing reassuring, and keeping the scope of PGx testing to non-disease diagnostic genes was a factor that enhanced trust: “ I feel if like it’s really narrowed down in front of you it would be safer …” . Trust in the individual recruiting to research was also a factor. Trust leading to research engagement could be gained by endorsement of a family member, faith leader, or community leader: “ If my relative did it, I might [do it]. Some people trust in relatives…People trust more family ”. Trust was harmed by insecure data, a history of data breach or association with individuals or institutions that were not trusted. Lack of consistency in information and profit as a motivating factor were other factors which harmed trust. Lack of trust leads to concerns about data misuse Misuse of data by non-trusted entities was a concern. This was a central disincentive to research participation: “ People really don’t want to share their information. They might have doubt on the people using to do research. That’s why they don’t want to share ” (Translated from Urdu). Concerns regarding the specific nature of potential data misuse ranged from breaches of privacy and financial exploitation to the potential for malicious actors to use genomics data for racially motivated genocide. “In theory… if someone wants to target a …specific group of people like South Asians… if I target that gene it could set off a virus that could only affect these people…I think I’ve seen it in a film, when they target a specific gene … they set this gas off but it will only effect people with this gene…South Asian genes” . This latter was perceived by some participants to be hyperbolic, and the level of time, knowledge and resources needed to misuse data in such a nefarious way were cited as protective: “ to get to the point of …killing hundreds of thousands… is far-fetched. We’d need to dissect …an entire genome, which would take a very long time, and a lot of work .” Lack of trust in profit driven research Participants across all groups expressed concern that pharmaceutical industry was not trustworthy due to profit as a motivating factor. “ Medicine is about helping people and saving lives…They’ve developed the drugs but they’re big businesses as well …”. Some extended this logic to private chemists and pharmacists working at chemists, as profit was felt to be the bottom line. The perceived conflict of interest created by profit as a primary goal was felt to lead to risk of information misuse. There were concerns about benefit hoarding for profit. Many participants across all groups worried that if industry were to get PGx data for research they would find a way to profit at the expense of the community and withhold benefits from the community. Several participants felt there was a risk of price gouging if a therapeutic was found to be particularly beneficial to their community: “but when the makers know that then they will increase the prices. And you know we are very careful about our health so we will spend money.” Another participant in a different group expressed the same sentiment: “If this information is being delivered to industry, will it affect the cost of the medicine?… if we’re getting a tablet for 1 pound we might then get it for 3 pounds” (translated from Urdu). There was a negative view toward proprietary patents as tools to restrict availability of therapeutics. There was a concern that if lifesaving medications were discovered from genetic data, patents would mean that the medicine would not be affordable or accessible to the participant communities that had contributed data to the research. However, trust in national regulators was seen by some participants to counterbalance the risk of unrestrained industry: “ business is business at the end of the day. Some businessmen are OK. If the regulator doesn’t allow, then they won’t get it [the medication]. They need to allow it first .” Feeding back research results facilitates trust Feeding back research results was crucial to ongoing research engagement through building trust: “ if someone sees a result then they will become more involved ”. Participants agreed that if research results were fed back it would support education and engagement and facilitate trust via grassroots community communication. As one participant said of receiving feedback on how she contributed to a study: “ And then you would speak to, like, your friends and family…They would open up. They would be like, wow, that’s so cool… It would build trust between communities .” Some participants felt that personalised feedback on an individual level had an even more powerful impact, and there were suggestions that researchers could build trust further by contacting individual participants to make them aware of how their data had contributed to a study. Trust in therapeutics research through the lens of the COVID-19 pandemic Participants expressed their experience with trust, and mistrust, in therapeutics and research through the lens of the COVID-19 pandemic. Participants reported a change in context and trust toward therapeutics research due to the pandemic. There was broad agreement that lack of trust had manifested in strongly divided opinions on the safety and efficacy of the COVID-19 vaccine: “for example…, covid injection, half of the people …didn’t have it…a lot of people I know, they didn’t go for that injection…it’s their choice end of the day…but there’s a reason why they didn’t have it…because they don’t trust maybe, they didn’t believe” . Lack of trust toward the COVID-19 vaccine within the South Asian community was widely felt to be prompted by the perceived pace of research and social media reports of trusted health care practitioners refusing COVID-19 vaccination. Because of the nature of the pandemic, some participants saw COVID-19 vaccines and treatments as initially experimental or offered without a full understanding of possible effects. However, it was felt that this mistrust would not extend to PGx research focused on optimising personal risk/benefit profile for existing medications. Education to facilitate PGx research In contrast to the skeletal information desired for clinical PGx use at the point of care, participants felt that a lot of information and education was needed to responsibly organise sharing of the generated clinical data for research. “ for research: you have to make sure you understand it perfectly and it has to be accurate information given to you. Clinical, that’s something you just do…easier to do…research you have to be really accurate .” Participants highlighted lack of awareness of research, and lack of scientific and genetic literacy as significant barriers to research engagement. “this is the reason there is less data from these groups: because the lack of education and they don’t participate if they don’t understand anything.” Language barriers were also cited as key hurdles to engagement of this community with research. However, participants also perceived a lot of interest in advancing health and medication related knowledge in the community and suggested that community ties offered vehicles to public engagement. The public health engagement campaign suggestions outlined above around national PGx roll out were echoed strongly in the discussion of education to facilitate PGx data sharing for research. Engaging with local community members for grass-roots education was advised by participants. But some participants perceived a lack of scientific literacy to be a significant barrier to community exchange of information: “How to educate those people? Like when you speak to other people they don’t know, like when she will leave from here, what she would say to her neighbour … what is that genetic information to do with the medication? We take medication everyday” (translated from Urdu). Many participants expressed interest in being trained to be community champions and volunteered to disseminate PGx information to facilitate research engagement: “ in East London mosque they have events and things… I’m here today. I understand. I will go and spread to my friends and family. So, it’s like word of mouth will get spread .” Data sharing was the key concept on which research from a hypothetical clinical PGx service hinged. Participants required prompting to distinguishing PGx testing for clinical use from sharing clinical PGx data for research. Factors supporting PGx data sharing for research Data sharing was desirable if the researchers did not have a financial stake, and benefits would be shared. There was a common perception that without research, the use of medicines will not improve, but an understanding that the risk is to the participating individuals while the benefit would be for future individuals: “If you don’t share it, you don’t advance really. So, you have to come to some sort of compromise where you are sharing the results they need, or do you want to just not share it and be stuck and not give two hoots about what happens in the future. You have to draw that line somewhere.” . The perceived “good” of the research purpose was a key motif: “ So the point is how it works when we share for the good purpose, not for the bad purpose. So, it can help, so definitely [we should] share ”. There was broad consensus across individuals and groups that the idea of good as compared with bad purpose had an association with the trustworthiness of the researchers: “ we have a concern, so we can only share these things [PGx data] with trusty [trustworthy] ones and [make ourselves aware ].” The perceived trustworthiness of both individual researchers and associated institutions were determining factors in weighing willingness to share clinical PGx data for research purposes. Health care professionals, academic institutions, and the regulatory body (the MHRA) were considered trustworthy and therefore participants were happy to share PGx data with these groups for research with the protection of standard data de-identification and data protection. Barriers to sharing clinical PGx data for research and potential safeguards Participants across all groups broadly acknowledged that some people would not like to share data as a rule, due to privacy concerns: “There are people with those [privacy] concerns and those concerns are very real” . Data ownership was an important topic linked with privacy, and many participants wanted to maintain control over access to their data “It’s my information. That’s mine, do you know what I mean, it’s an invasion of privacy where you don’t have control over who gets to see your information.” As compared with healthcare practitioners and academics, there were very different perspectives on sharing PGx data for research with industry. Concerns revolved around trust, as outlined above. Most participants felt that industry has an inherent conflict of interest as a profit driven private enterprise and therefore could not be trusted to prioritise benefit sharing/the health of the community over potentially exploitive options: “ pharmaceutical companies are only thinking about their profits then it’s not good to share our information with them ”. Others felt that there is an inherent risk to not doing research: “ without research there will be always risk, there’s no cure ”. Some perceived the benefits of data sharing with industry to outweigh potential harms: “It improves the medicine, so it improves the patient care ”. Confidentiality and anonymization of data were important safeguards to protect privacy. “ It’s anonymous isn’t it…so the people who are doing it, they don’t know who it is. They have to have that barrier that [data] is confidential and not to be leaked to anyone .” Well-articulated policies around data protection and management of any breach of data protection were perceived by many participants to be crucial safeguards: “It’s important what they’re going to do with the data but also if there is a breach of data, what happens… if they find that information was leaked to the public…what they do ”. Transparency about potential conflicts of interest and opportunities to opt out of data sharing with non-trusted research partners were desirable. “I think everyone should be given the option to opt in and opt out, so I think that’s potentially a way of going forward … so you [can] opt in for pharmaceuticals or universities and … and so on…You can label them as non-profit organisation and for-profit organisations and so. That would build confidence in the person that is being involved in the research.” Safeguards against financial exploitation due to knowledge of individual or community PGx data would be protective. There were key cross-cutting themes common to discussion of both clinical implementation and use of clinical data for research. These included: benefits, the central role of trust, concerns about baseline education and desire for public heath level campaign to address this perceived need, and data sharing/custodianship. These echoed existing themes in the literature around the central importance of public awareness, education, trust, and data custodianship (Supplementary table ). However, the interaction between the key themes across clinical application and research domains was rich, particularly around trust, and adds some novel detailed insight around building trust within this population. PGx implementation with appropriate population wide education and clinician communication was perceived to have the potential to enhance trust in clinical care systems by personalising therapy to individuals, particularly those from under-represented ancestorial groups. This increased trust was thought likely to contribute to increased medication compliance. Trust drives willingness to share data and engage with research, and participants linked increased trust in clinical prescribing with increased willingness to share data toward advancing PGx because they could see PGx benefits in action (i.e., there is clinical value proven from PGx research). Representation of the South Asian ancestry group in therapeutic evidence base through research increases trust in the evidence base for medicine use and may increase compliance with therapeutics. Therefore, participants constructed a circular trust building and benefit model that could see a well implemented PGx roll out promote increased medication compliance via trust in clinical systems and increased research representation, which would then feed information back into clinical practice, further supporting trust (Fig. ). The relationship between participants and GPs were key to promoting this model of trust, as was feeding back utility of research to those who choose to participate, public health level education campaigns, and stakeholder guided data gating. Therefore, if the NHS decides to adopt panel PGx testing nationwide, educational and engagement initiatives should proceed the roll out, with accessible materials in multiple languages that can be disseminated either by championing individuals or via multi-media/social media. Engaging with community leaders to disseminate information is a valuable approach, as well as optimising intergenerational information sharing by educating those in school. Success of a national PGx programme is likely to hinge on the level of trust built into the rollout. Some of that trust is engendered already by trusted individuals, professionals, and associations, but some must be earned by education and engagement initiatives with the public. The COVID-19 pandemic demonstrated how easily misinformation can be disseminated and erode trust. The unanimous emphasise on mistrust kindled by the COVID-19 pandemic have implications for PGx, particularly in Black, Asian and minority ethnic groups, not prior discussed to our knowledge. These findings highlight the importance of building from existing trusted relationships with GPs and carefully considering stakeholder suggested safeguards to preserve trust. Trust can be supported by robust and transparent policies around protection of data, management of data security breaches, and stakeholder input on proposed data sharing. Sharing any data which could be used by private entities for fiscal gain is likely to be a particular source of contention and therefore should be continually informed by stakeholder consultation. Policies that would protect against price gouging as a result of proprietary gains from clinical PGx data sharing for research should be considered. This study suggests that pre-emptive PGx roll out via primary care is the preferred approach in the long term, but participants highlighted secondary care prevention clinics as a high benefit population in which to pilot panel PGx testing. Strengths and limitations of this study This is the first study to engage UK participants of South Asian ancestry in discussion of facilitators and barriers to PGx implementation and secondary research. Further research should be done quantitatively to canvas large scale public awareness and attitudes to PGx clinical implementation, utility, and sharing PGx data for research in this community. The study was made possible by collaboration with the Genes & Health research team and their links and pre-existing trust building with the community. However, participants recruited from a cohort who have chosen to participate in a large-scale genetic research study may not be representative in their attitudes toward PGx. Clinical implications This participant data from an under-characterised and disproportionately morbid population within the UK is valuable to influence policy on PGx implementation. Inclusive engagement studies can increase the likelihood that PGx implementation would become a tool to improve the health of this group at high risk of polypharmacy and support underpinnings for data sharing to generate PGx research specific to this under-represented population. Such a stakeholder informed approach will support PGx to be a tool which reduces instead of exacerbating health inequality. This is the first study to engage UK participants of South Asian ancestry in discussion of facilitators and barriers to PGx implementation and secondary research. Further research should be done quantitatively to canvas large scale public awareness and attitudes to PGx clinical implementation, utility, and sharing PGx data for research in this community. The study was made possible by collaboration with the Genes & Health research team and their links and pre-existing trust building with the community. However, participants recruited from a cohort who have chosen to participate in a large-scale genetic research study may not be representative in their attitudes toward PGx. This participant data from an under-characterised and disproportionately morbid population within the UK is valuable to influence policy on PGx implementation. Inclusive engagement studies can increase the likelihood that PGx implementation would become a tool to improve the health of this group at high risk of polypharmacy and support underpinnings for data sharing to generate PGx research specific to this under-represented population. Such a stakeholder informed approach will support PGx to be a tool which reduces instead of exacerbating health inequality. Supplemental Material |
Usability of an artificially intelligence-powered triage platform for adult ophthalmic emergencies: a mixed methods study | 944002e4-1434-4f72-acf9-d4f1a5c75ad7 | 10728059 | Ophthalmology[mh] | In the UK, ophthalmology is currently the busiest outpatient speciality within the NHS accounting for almost 8 million attendances in 2021/22 . In the hospital eye service itself, there is growing demand for emergency-based eye services – . With the wide variation in the severity of conditions seen and the high attendance in ophthalmic casualty units, most units have introduced a triage system that allocates a patient to the appropriate category of urgency . It has been found that up to 80% of those attending eye casualty (EC) do not require urgent ophthalmic management following triage and up to 60% were seen and discharged at their first visit , – . The Royal College of Ophthalmologists have recommended upskilling and supporting of allied health professionals (AHP) in order to meet this increasing demand for current eye service, where this will enable safe and appropriate management of patients . Machine learning algorithms could support AHP on improving their accuracy and speed of the triage process. Machine learning algorithms used in the general emergency department have shown that they are better than clinicians at predicting the need for admission , . Adoption of medical devices for observation and treatment of patients is becoming more common. User error caused by inadequate medical device usability have become an increasing cause for concern. Many medical devices developed without applying a usability engineering process have found their device to be difficult to learn and impractical by users and may limit its feasibility for use in future studies and clinical practice . A novel online platform interface with an integrated machine learning algorithm known as the DemDx Ophthalmology Triage System (DOTS) has recently been developed. DOTS has been designed to guide and support AHP clinical decision-making when triaging patients who present in an ophthalmic emergency-based setting. As far as the authors are aware, there are no studies that have evaluated the usability of any potential artificial intelligence (AI) powered triage platform for ophthalmology. The objective of this study was to evaluate its interface, usability, safety and gain insight on the acceptability of DOTS by AHP before clinical implementation. Platform development The data input form for DOTS (Fig. ) was developed using the triage form used in Accident and Emergency (A&E) for adults at Moorfields Eye Hospital (MEH) that reflected demographics, presentation, red flags and triage outcomes. The development of ophthalmic signs, symptoms and relevant patient history to select by users were developed using clinical guidelines – and by experienced MEH clinicians working in eye casualty. Data was then captured from 12,584 attendances at MEH A&E from 11,733 patients by trained ophthalmic nurses working in triage from 9th August 2021 to April 30th 2022. UK ophthalmic nurses are healthcare practitioners who have a Batchelor’s degree or degree apprenticeship in nursing. They are registered with the Nursing and Midwifery Council and attain ophthalmic subspecialisation through exposure to patients under supervision and completion of competency-based signoffs through internal protocols within their workplace that enable them to work in their designated role. Ophthalmic nurses also have to demonstrate continuing professional development in their field to maintain registration. The AI algorithm was developed using Python 3.8.10.13 ( https://www.python.org ) and was trained on the captured data. Four architectures were evaluated for each model, chosen to include simple models as baseline (Logistic Regression and Decision Tree) as well as the ones considered the state-of-the-art for tabular data, including tree bagging (Random Forest) and tree boosting (XGBoost). The model with the highest weighted average of specificities for emergency and urgency was selected as best-performing and was tested using internal and external data sets (1,269 and 761 patients, respectively). This model showed higher specificity and similar sensitivity regarding triage outcomes when compared to ophthalmic nurses, which was then utilised for this study; further details of the development of the algorithm and its’ testing has been described by Brandao-de-Resende et al. . After the user has inputted the data into DOTS, the results page (Fig. ) displayed the AI suggested triage outcome within a coloured background using a traffic light system that reflects urgency; red: see in A&E same day, walk to specialty clinics same day; yellow: referred to urgent care clinic within the week; green; treated/advice given at triage or see general practitioner/optometrist within a fortnight. For diagnosis prediction, two models were built: (1) the most probable emergency/urgency differentials, and (2) the most probable elective differentials. When the triage model predicts the case as emergency/urgency, a list of the most probable emergency/urgency differentials as “most probable diagnoses” and the list of the most probable elective differentials as ‘consider less serious diagnoses’ were displayed. When the triage model predicts the case as elective, a list of the most probable emergency/urgency differentials as “serious diagnoses to be considered” and the list of the most probable elective differentials as “most probable diagnoses” was shown to the user. With each list of three diagnoses, adjacent probability bars indicating the likelihood of each diagnosis and where appropriate, a red flag indicating the condition would require same day attendance in ophthalmic A&E was displayed. A refine results feature was also incorporated to support history taking and data entry that was based on the algorithm outputs, which could refine the triage and diagnosis as well as a copy to clipboard feature that would enable users to transfer the output for the patient and/or practitioner documentation. Both layout of DOTS, interface and features were developed iteratively based on suggestions and feedback from a multi-disciplinary team of experts and users that included; clinicians working in ophthalmic A&E, an independent research steering committee (comprised of academics and clinicians), patient public involvement panel, a user interface experience designer and digital support from informatics and developers involved in electronic patient records for ophthalmology ( https://openeyes.apperta.org ) and a Digital Health Accelerator company. Usability testing A prospective mixed methods cohort usability study for the newly developed platform was conducted at MEH NHS Foundation Trust, City Road, UK between October and December 2022. Participating clinicians UK registered ophthalmic professionals based at MEH, City Road, UK were recruited for the study via an email invitation to employed ophthalmic nurses, optometrists and ophthalmologists for their expressions of interest in evaluating the usability of a new artificial intelligence integrated triage platform for use in an ocular emergency setting. Inclusion criteria included qualified clinicians (doctors, nurses, optometrists, other AHP) who actively triage patients who present with ocular emergencies within primary care or an eye casualty setting. Written and informed consent was obtained for all participants, all personal information was pseudonymised and a study number was generated. The study was approved by the MEH Research Ethics Committee and complied with the tenets of the Declaration of Helsinki. IRAS number 290843. ethics approval number 21/LO/0294, approved on the 4th August 2022. After the participant had consented, an initial online survey was completed asking information on professional status, gender, days worked in primary/secondary care, years of registration, work experience in eye casualty and experience with digital clinical applications and thoughts on the use of AI to support triage (Supplementary ). Think aloud interview On completion of the initial survey, participants were booked into an individual single 1-h appointment slot with the researcher either face to face or virtually to test and provide feedback regarding the online triage platform. Participants were provided access to the online platform and were provided instructions on the processes involved at the interview prior commencement. The first stage involved participants interfacing with the platform using an ocular emergency case based on their own experience and ‘thinking aloud.’ The ‘thinking aloud’ is a method used to gather data in usability testing in product design and development, in psychology and a range of social sciences (e.g., reading, writing, translation research, decision making, and process tracing). Think aloud interviews' are a type of qualitative interview where you ask participants to look at/use/engage with the intervention and to say out loud any thoughts that come to mind as they work through it. From a usability testing context, observers can take notes of what participants say and do, without attempting to interpret or influence their actions and words, and especially noting places where they encounter difficulty during the interview as this would represent real-world interaction. Participants were asked to think aloud regarding the usability and features of the newly developed platform for the data input page and the results page. The researcher observed each participant and followed the method described above. No participant had any prior exposure or usage of the platform. The interview was recorded using audio only via MSTeams and transcripts were autogenerated for subsequent analysis. Questionnaire and interview After the think aloud interview, participants completed an online questionnaire on the presentation, usability, safety, navigation and accessibility of the platform that was divided into 3 sections; patient information, results and overall impression. Answers were rated using either a 5-point Likert scale i.e. 5 as very easy, 1 very difficult) or binary choice (Yes or No). There was also an additional free text boxes where users could elaborate on their feedback about the platform (Supplementary ). After the questionnaire, the interviewer conducted a semi-structured interview where participants were asked questions to do an in-depth exploration of their feedback on the platform that was recorded. the researcher expanded on the topics that the participant has described during their Think Aloud session as well as asking them open-ended questions about their platform experience that included what they liked, improvements, how easy or difficult they found navigating the platform challenges and features as well as the platforms’ potential and how likely they were to use it. Before recruitment the study was piloted to refine the interview questions and think aloud task. No data was used from the pilot in the analysis. Sample size Recommendations by the US Food and Drugs Administration for the development of medical devices recommend utilisation of a multidisciplinary team of at least 15 users . Statistical analysis The data captured from the questionnaires were analysed using SPSS Statistics software version 29.0 ( http://www.ibm.com/SPSS_statistics ). Interview transcripts were analysed using framework analysis, applying the code tree model by Van Waes . This method of analysis starts deductively with a priori codes from the study’s aims and objectives and the subsequent analysis is inductive . DS read and reread the transcripts for familiarisation and data immersion. Transcripts were then coded to create a coding/thematic framework based on the code tree model to identify themes. The mapping was discussed to ensure codes that could be interpreted as being more than one function were allocated to the most appropriate code tree domain. The code tree analysis model followed two main dimensions: usability and perceived usefulness. The usability was coded from different perspectives as suggested by Van Waes : (1) navigation strategy (i.e., which navigation platforms did the participant use and (2) navigation problems, what were the navigation barriers the participant came across). Three categories were subsequently identified related to the navigation strategies and problems: (1) use of navigational elements, (2) layout, and (3) instructions. Regarding the perceived usefulness of the platform, the negative and positive remarks regarding the content presented on the website with three subcodes: (1) satisfaction with information modality, (2) information preferences, and (3) satisfaction with comprehensibility. Finally, regarding the intention to use the platform were coded in terms of whether and why participants indicated they would or would not use it in the ED setting. The code tree is presented in Fig. . The data input form for DOTS (Fig. ) was developed using the triage form used in Accident and Emergency (A&E) for adults at Moorfields Eye Hospital (MEH) that reflected demographics, presentation, red flags and triage outcomes. The development of ophthalmic signs, symptoms and relevant patient history to select by users were developed using clinical guidelines – and by experienced MEH clinicians working in eye casualty. Data was then captured from 12,584 attendances at MEH A&E from 11,733 patients by trained ophthalmic nurses working in triage from 9th August 2021 to April 30th 2022. UK ophthalmic nurses are healthcare practitioners who have a Batchelor’s degree or degree apprenticeship in nursing. They are registered with the Nursing and Midwifery Council and attain ophthalmic subspecialisation through exposure to patients under supervision and completion of competency-based signoffs through internal protocols within their workplace that enable them to work in their designated role. Ophthalmic nurses also have to demonstrate continuing professional development in their field to maintain registration. The AI algorithm was developed using Python 3.8.10.13 ( https://www.python.org ) and was trained on the captured data. Four architectures were evaluated for each model, chosen to include simple models as baseline (Logistic Regression and Decision Tree) as well as the ones considered the state-of-the-art for tabular data, including tree bagging (Random Forest) and tree boosting (XGBoost). The model with the highest weighted average of specificities for emergency and urgency was selected as best-performing and was tested using internal and external data sets (1,269 and 761 patients, respectively). This model showed higher specificity and similar sensitivity regarding triage outcomes when compared to ophthalmic nurses, which was then utilised for this study; further details of the development of the algorithm and its’ testing has been described by Brandao-de-Resende et al. . After the user has inputted the data into DOTS, the results page (Fig. ) displayed the AI suggested triage outcome within a coloured background using a traffic light system that reflects urgency; red: see in A&E same day, walk to specialty clinics same day; yellow: referred to urgent care clinic within the week; green; treated/advice given at triage or see general practitioner/optometrist within a fortnight. For diagnosis prediction, two models were built: (1) the most probable emergency/urgency differentials, and (2) the most probable elective differentials. When the triage model predicts the case as emergency/urgency, a list of the most probable emergency/urgency differentials as “most probable diagnoses” and the list of the most probable elective differentials as ‘consider less serious diagnoses’ were displayed. When the triage model predicts the case as elective, a list of the most probable emergency/urgency differentials as “serious diagnoses to be considered” and the list of the most probable elective differentials as “most probable diagnoses” was shown to the user. With each list of three diagnoses, adjacent probability bars indicating the likelihood of each diagnosis and where appropriate, a red flag indicating the condition would require same day attendance in ophthalmic A&E was displayed. A refine results feature was also incorporated to support history taking and data entry that was based on the algorithm outputs, which could refine the triage and diagnosis as well as a copy to clipboard feature that would enable users to transfer the output for the patient and/or practitioner documentation. Both layout of DOTS, interface and features were developed iteratively based on suggestions and feedback from a multi-disciplinary team of experts and users that included; clinicians working in ophthalmic A&E, an independent research steering committee (comprised of academics and clinicians), patient public involvement panel, a user interface experience designer and digital support from informatics and developers involved in electronic patient records for ophthalmology ( https://openeyes.apperta.org ) and a Digital Health Accelerator company. A prospective mixed methods cohort usability study for the newly developed platform was conducted at MEH NHS Foundation Trust, City Road, UK between October and December 2022. UK registered ophthalmic professionals based at MEH, City Road, UK were recruited for the study via an email invitation to employed ophthalmic nurses, optometrists and ophthalmologists for their expressions of interest in evaluating the usability of a new artificial intelligence integrated triage platform for use in an ocular emergency setting. Inclusion criteria included qualified clinicians (doctors, nurses, optometrists, other AHP) who actively triage patients who present with ocular emergencies within primary care or an eye casualty setting. Written and informed consent was obtained for all participants, all personal information was pseudonymised and a study number was generated. The study was approved by the MEH Research Ethics Committee and complied with the tenets of the Declaration of Helsinki. IRAS number 290843. ethics approval number 21/LO/0294, approved on the 4th August 2022. After the participant had consented, an initial online survey was completed asking information on professional status, gender, days worked in primary/secondary care, years of registration, work experience in eye casualty and experience with digital clinical applications and thoughts on the use of AI to support triage (Supplementary ). On completion of the initial survey, participants were booked into an individual single 1-h appointment slot with the researcher either face to face or virtually to test and provide feedback regarding the online triage platform. Participants were provided access to the online platform and were provided instructions on the processes involved at the interview prior commencement. The first stage involved participants interfacing with the platform using an ocular emergency case based on their own experience and ‘thinking aloud.’ The ‘thinking aloud’ is a method used to gather data in usability testing in product design and development, in psychology and a range of social sciences (e.g., reading, writing, translation research, decision making, and process tracing). Think aloud interviews' are a type of qualitative interview where you ask participants to look at/use/engage with the intervention and to say out loud any thoughts that come to mind as they work through it. From a usability testing context, observers can take notes of what participants say and do, without attempting to interpret or influence their actions and words, and especially noting places where they encounter difficulty during the interview as this would represent real-world interaction. Participants were asked to think aloud regarding the usability and features of the newly developed platform for the data input page and the results page. The researcher observed each participant and followed the method described above. No participant had any prior exposure or usage of the platform. The interview was recorded using audio only via MSTeams and transcripts were autogenerated for subsequent analysis. After the think aloud interview, participants completed an online questionnaire on the presentation, usability, safety, navigation and accessibility of the platform that was divided into 3 sections; patient information, results and overall impression. Answers were rated using either a 5-point Likert scale i.e. 5 as very easy, 1 very difficult) or binary choice (Yes or No). There was also an additional free text boxes where users could elaborate on their feedback about the platform (Supplementary ). After the questionnaire, the interviewer conducted a semi-structured interview where participants were asked questions to do an in-depth exploration of their feedback on the platform that was recorded. the researcher expanded on the topics that the participant has described during their Think Aloud session as well as asking them open-ended questions about their platform experience that included what they liked, improvements, how easy or difficult they found navigating the platform challenges and features as well as the platforms’ potential and how likely they were to use it. Before recruitment the study was piloted to refine the interview questions and think aloud task. No data was used from the pilot in the analysis. Recommendations by the US Food and Drugs Administration for the development of medical devices recommend utilisation of a multidisciplinary team of at least 15 users . The data captured from the questionnaires were analysed using SPSS Statistics software version 29.0 ( http://www.ibm.com/SPSS_statistics ). Interview transcripts were analysed using framework analysis, applying the code tree model by Van Waes . This method of analysis starts deductively with a priori codes from the study’s aims and objectives and the subsequent analysis is inductive . DS read and reread the transcripts for familiarisation and data immersion. Transcripts were then coded to create a coding/thematic framework based on the code tree model to identify themes. The mapping was discussed to ensure codes that could be interpreted as being more than one function were allocated to the most appropriate code tree domain. The code tree analysis model followed two main dimensions: usability and perceived usefulness. The usability was coded from different perspectives as suggested by Van Waes : (1) navigation strategy (i.e., which navigation platforms did the participant use and (2) navigation problems, what were the navigation barriers the participant came across). Three categories were subsequently identified related to the navigation strategies and problems: (1) use of navigational elements, (2) layout, and (3) instructions. Regarding the perceived usefulness of the platform, the negative and positive remarks regarding the content presented on the website with three subcodes: (1) satisfaction with information modality, (2) information preferences, and (3) satisfaction with comprehensibility. Finally, regarding the intention to use the platform were coded in terms of whether and why participants indicated they would or would not use it in the ED setting. The code tree is presented in Fig. . Participant demographics Twenty study participants were included in the study. There were 8 ophthalmic nurses, 7 optometrists and 5 consultant ophthalmologists recruited. The majority of participants (85%) worked in a dedicated eye casualty environment or urgent care clinic in the hospital, further details of the study participants are in Table . Quantitative results Participants experience of digital applications, triage and artificial intelligence In the initial questionnaire prior DOTS interaction, eight of the participants reported they have used a digital application as a clinical reference guide, where they referred to ease of access and clear instructions as a positive feature for its utilisation and regular use but none had used a digital application to support their clinical decision making. From the 17 participants that have experience in ophthalmic triage in EC, 6 (30%) reported concerns in their current triage process, where a quarter of all respondents specifically mentioned they have experienced uncertainty in triage and had to ‘sometimes seek medical advice’ or ‘refer to protocols.’ 15 (75%) participants were positively receptive to using AI to support triage decisions, four were not sure and one was cautious of triage decisions being dependent on patient verbal input. DOTS usability: quantitative In the subsequent questionnaire that surveyed the participants experience with DOTS revealed that they all found the colours and text were suitable and legible, and they could identify all the information that was inputted before submitting for AI analysis. 90% or more of participants found; they could identify all the input fields; satisfacation with the number of options available for data input selection regarding signs and symptoms; platform order reflected their current triage workflow and were confident in entering the data into all the fields. 10 participants suggested that the signs and symptoms section could be expanded; three participants mentioned one additional symptom each; two participants reported missing symptoms but were already present in the platform; two participants said there should be more symptoms but did not specify which ones; two suggested quantification of symptoms and one suggested free text in the data input. 95% of participants felt the processing time for the algorithm to present an output to be fast, 90% of participants found it was easy to identify the suggested triage output from DOTS and it was clear that the tool’s suggestions were to aid their clinical decision making. 65% of participants did not identify the edit function that could change the inputted data if required. 85% of participants identified a suggested diagnosis was a red flag and they could identify the patient details. 80% were able to interpret the probability bars that were displayed alongside the diagnosis. Nineteen participants found the triage outcome matched their individual simulated ocular emergency case and one didn’t report this. Ten participants found the diagnosis outcome matched their cases; 5 didn’t discuss this, 4 didn't match and 1 was not sure. Overall, 85% of participants felt the platform was safe to use in managing patients and the summary report that was generated was acceptable. 90% or more of participants found the platform was easy to navigate through the different sections and easy to use, in addition nearly all participants (95%) were willing to use this tool in their clinical workflow. DOTS usability: qualitative Navigational strategies and problems We identified three categories of navigation strategies that led to problems in optimally navigating DOTS: (1) use of navigational elements, (2) layout, and (3) instructions. Participants frequently commented that it was easy for them to navigate through the different sections of the platform. However, some participants noted navigational issues. Use of navigational elements on data entry and results pages Most participants commented that the platform was easy to use, and they could easily identify the navigational icons and understand their purpose. Ophthalmologists found the data entry process straightforward. One suggestion was made to improve the navigational experience related to the addition of a progress bar so users could see their progress to complete the triage, which could be particularly pertinent in an EC setting: “ It would be kind of good if it gives you an idea that ‘don't worry, you don't have another 10 pages to go’ " (Participant 20, consultant ophthalmologist). Regarding the ‘Edit’ function in the patient’s medical information box on the results page, participants noted that it would be useful to be able to close the box with a “cross added at the top” as well as “greying the ‘refine results’ button at the bottom of the box when no further information has been to be entered” (Participant 20, consultant ophthalmologist) would improve the navigation flow through the page. Layout of the results page Some participants commented that more space should be made to contrast the triage outcome with the report of the most probable diagnosis: “ Maybe there should be a couple of spaces in between so that it differentiates ” (Participant 18, nurse). Most participants appreciated the intuitive and logical layout of the platform, though some did not notice the ‘Edit’ function at the top of the page. In addition, participants found the presentation of the traffic colours (red, yellow, green) useful to highlight the emergency of the triage outcome: “It's a good way of doing it because there's something highlighted in red and it’s immediately grabbing your attention. If it's green, you got something that's nationwide. If it’s red, it is an important message.” (Participant 10, optometrist). Despite finding the layout of the platform clear, participants acknowledged that receiving further training prior would help to familiarise themselves with the platform more easily: “I think that once you're taught what the sections are and the purpose of them, then it would become quite easy to use ” (Participant 12, optometrist). Instructions for data entry and results interpretation Participants were provided with limited instructions prior to the task in order to test the intuitiveness of the platform. Participants appreciated that it was a simple tool to use, nevertheless, they wanted further instructions on how to navigate through the platform. Two participants highlighted that the summarised instructions needed to be displayed in the data inputting stage without referring to the user guide tab. For instance, information could be added on the first page explaining the data collection process, the different options on the results page including, how to use the ‘Refine results’ tool and copying to clipboard. Perceived usefulness Perceived usefulness was measured in terms of satisfaction with the content of DOTS and intention to use it. Regarding satisfaction with the content, we identified three categories: (1) satisfaction with information modality, (2) information preferences, and (3) satisfaction with information comprehensibility. Content on the data entry and results pages Satisfaction with information modality Regarding the modality of how information was presented on both the data entry and results pages, participants appreciated the speed of the outcome delivery and the simplicity of the display of the triage outcome: “ It was quick, it didn’t take me to 60 million different screens to get to an answer ” (Participant 10, optometrist). In addition, all participants acknowledged the effectiveness of the implemented strategies in reducing input errors, such as the utilisation of a validation entry for patients’ hospital number, the copy and paste function, and the edit function. Participants saw the red flags on the results page as a clear way of catching their attention on the importance of the information. However, several participants commented that because of this, the triage decision should be displayed in a clearer way, so it is not overstepped by the diagnosis section, which is a less prioritised outcome from the triage platform: “ My only concern is the red flag confusion [in the diagnosis section].I think what you're alluding to here is the triage output which has come up because it's a triage platform. It needs to be made a lot bigger, bolder and clearer that this is the [triage] outcome ” (Participant 13, nurse). The probability bars displayed in the results output were intended to demonstrate the likelihood of a particular diagnosis. Nevertheless, many found them confusing and were unable to interpret the information that the bars represented. This issue is further discussed in the theme of 'Satisfaction with comprehensibility’'. Information preferences Participants were satisfied with the triage decisions and the level of information on the platform on both pages. They found the refine results prompts particularly useful as “ this would tease out some other questions you may not have asked the patient and it would help you to add to it” (Participant 13, nurse) or "if you've forgotten to ask the question” (Participant 16, nurse). In the data entry page, participants often stated that information related to demographics was comprehensive but needed to be more specific about gender and be defined as ‘gender at birth’. Furthermore, they often mentioned preferring entering patients’ date of birth rather than an age since that is what is entered in patients’ medical records. Most participants commented they were satisfied with the number of options when inputting signs and symptoms, however a participant mentioned the significance of asymptomatic patients that was missing from the platform. “Yes, we have a lot of patients who come to EC who are asymptomatic and then in letter sent by the opticians, it says they need to be seen in EC” (Participant 5, nurse). Participants noted “pain” was missing in the ‘red flag’ checkbox section and in general the platform’s red flags should be expanded to allow adding more red flags options: “The triage people need a few more options for red flags. Because I know red eye is red eye but that is actually a red flag as well because it could be a post operation red eye, so I think it has to reflect the ophthalmology side of things as well a bit more” (Participant 3, consultant ophthalmologist). Satisfaction with comprehensibility of the results Participants generally indicated that the red flags and probability bars were useful. However, many were uncertain about interpreting the bars and found it challenging to understand the diagnosis results due to the red flags. Hence, clearer instructions on how to interpret the red flags and probability bars were needed: “ It wasn't easy to understand because there's so much information. There are two sets of information, as in the these [the ones with red flags] are the ones to consider, and these [without red flags].” (Participant 4, optometrist) “ Not sure these bars mean, maybe that is the likelihood? ” (Participant 19, consultant ophthalmologist). Intention to use Most participants felt that DOTS is a valuable triage platform and intended to use it in the future, although consultant ophthalmologists were less likely to find the platform useful for themselves than other healthcare professionals potentially due to their extensive training and experience working in the EC setting. Participants often cited the platform as particularly helpful to AHP or those who are in training or are less experienced in acute ophthalmology and can support their decision making in triage: “ This is a very good platform for healthcare professionals. The area of triage is a bit alarming for some of us, who are new and having a backup system of this nature helps. It will be a supportive tool for whoever's coming to do triage so they are not alone in that room trying to make decisions all by themselves. ” (Participant 13, nurse). Practitioners found the 'most probable diagnoses' feature helpful. They viewed it as an additional source of information and support for clinical decision making or referring patients in the eye care pathway: “ Well, it [the most probable diagnosis feature] will help in terms of shortening, the time that they're in charge, and you'll be able to decide if the patients need to stay in or they could be redirected somewhere else straight away in terms of the referral .” (Participant 20, nurse). Twenty study participants were included in the study. There were 8 ophthalmic nurses, 7 optometrists and 5 consultant ophthalmologists recruited. The majority of participants (85%) worked in a dedicated eye casualty environment or urgent care clinic in the hospital, further details of the study participants are in Table . Participants experience of digital applications, triage and artificial intelligence In the initial questionnaire prior DOTS interaction, eight of the participants reported they have used a digital application as a clinical reference guide, where they referred to ease of access and clear instructions as a positive feature for its utilisation and regular use but none had used a digital application to support their clinical decision making. From the 17 participants that have experience in ophthalmic triage in EC, 6 (30%) reported concerns in their current triage process, where a quarter of all respondents specifically mentioned they have experienced uncertainty in triage and had to ‘sometimes seek medical advice’ or ‘refer to protocols.’ 15 (75%) participants were positively receptive to using AI to support triage decisions, four were not sure and one was cautious of triage decisions being dependent on patient verbal input. In the initial questionnaire prior DOTS interaction, eight of the participants reported they have used a digital application as a clinical reference guide, where they referred to ease of access and clear instructions as a positive feature for its utilisation and regular use but none had used a digital application to support their clinical decision making. From the 17 participants that have experience in ophthalmic triage in EC, 6 (30%) reported concerns in their current triage process, where a quarter of all respondents specifically mentioned they have experienced uncertainty in triage and had to ‘sometimes seek medical advice’ or ‘refer to protocols.’ 15 (75%) participants were positively receptive to using AI to support triage decisions, four were not sure and one was cautious of triage decisions being dependent on patient verbal input. In the subsequent questionnaire that surveyed the participants experience with DOTS revealed that they all found the colours and text were suitable and legible, and they could identify all the information that was inputted before submitting for AI analysis. 90% or more of participants found; they could identify all the input fields; satisfacation with the number of options available for data input selection regarding signs and symptoms; platform order reflected their current triage workflow and were confident in entering the data into all the fields. 10 participants suggested that the signs and symptoms section could be expanded; three participants mentioned one additional symptom each; two participants reported missing symptoms but were already present in the platform; two participants said there should be more symptoms but did not specify which ones; two suggested quantification of symptoms and one suggested free text in the data input. 95% of participants felt the processing time for the algorithm to present an output to be fast, 90% of participants found it was easy to identify the suggested triage output from DOTS and it was clear that the tool’s suggestions were to aid their clinical decision making. 65% of participants did not identify the edit function that could change the inputted data if required. 85% of participants identified a suggested diagnosis was a red flag and they could identify the patient details. 80% were able to interpret the probability bars that were displayed alongside the diagnosis. Nineteen participants found the triage outcome matched their individual simulated ocular emergency case and one didn’t report this. Ten participants found the diagnosis outcome matched their cases; 5 didn’t discuss this, 4 didn't match and 1 was not sure. Overall, 85% of participants felt the platform was safe to use in managing patients and the summary report that was generated was acceptable. 90% or more of participants found the platform was easy to navigate through the different sections and easy to use, in addition nearly all participants (95%) were willing to use this tool in their clinical workflow. Navigational strategies and problems We identified three categories of navigation strategies that led to problems in optimally navigating DOTS: (1) use of navigational elements, (2) layout, and (3) instructions. Participants frequently commented that it was easy for them to navigate through the different sections of the platform. However, some participants noted navigational issues. Use of navigational elements on data entry and results pages Most participants commented that the platform was easy to use, and they could easily identify the navigational icons and understand their purpose. Ophthalmologists found the data entry process straightforward. One suggestion was made to improve the navigational experience related to the addition of a progress bar so users could see their progress to complete the triage, which could be particularly pertinent in an EC setting: “ It would be kind of good if it gives you an idea that ‘don't worry, you don't have another 10 pages to go’ " (Participant 20, consultant ophthalmologist). Regarding the ‘Edit’ function in the patient’s medical information box on the results page, participants noted that it would be useful to be able to close the box with a “cross added at the top” as well as “greying the ‘refine results’ button at the bottom of the box when no further information has been to be entered” (Participant 20, consultant ophthalmologist) would improve the navigation flow through the page. Layout of the results page Some participants commented that more space should be made to contrast the triage outcome with the report of the most probable diagnosis: “ Maybe there should be a couple of spaces in between so that it differentiates ” (Participant 18, nurse). Most participants appreciated the intuitive and logical layout of the platform, though some did not notice the ‘Edit’ function at the top of the page. In addition, participants found the presentation of the traffic colours (red, yellow, green) useful to highlight the emergency of the triage outcome: “It's a good way of doing it because there's something highlighted in red and it’s immediately grabbing your attention. If it's green, you got something that's nationwide. If it’s red, it is an important message.” (Participant 10, optometrist). Despite finding the layout of the platform clear, participants acknowledged that receiving further training prior would help to familiarise themselves with the platform more easily: “I think that once you're taught what the sections are and the purpose of them, then it would become quite easy to use ” (Participant 12, optometrist). Instructions for data entry and results interpretation Participants were provided with limited instructions prior to the task in order to test the intuitiveness of the platform. Participants appreciated that it was a simple tool to use, nevertheless, they wanted further instructions on how to navigate through the platform. Two participants highlighted that the summarised instructions needed to be displayed in the data inputting stage without referring to the user guide tab. For instance, information could be added on the first page explaining the data collection process, the different options on the results page including, how to use the ‘Refine results’ tool and copying to clipboard. Perceived usefulness Perceived usefulness was measured in terms of satisfaction with the content of DOTS and intention to use it. Regarding satisfaction with the content, we identified three categories: (1) satisfaction with information modality, (2) information preferences, and (3) satisfaction with information comprehensibility. Content on the data entry and results pages Satisfaction with information modality Regarding the modality of how information was presented on both the data entry and results pages, participants appreciated the speed of the outcome delivery and the simplicity of the display of the triage outcome: “ It was quick, it didn’t take me to 60 million different screens to get to an answer ” (Participant 10, optometrist). In addition, all participants acknowledged the effectiveness of the implemented strategies in reducing input errors, such as the utilisation of a validation entry for patients’ hospital number, the copy and paste function, and the edit function. Participants saw the red flags on the results page as a clear way of catching their attention on the importance of the information. However, several participants commented that because of this, the triage decision should be displayed in a clearer way, so it is not overstepped by the diagnosis section, which is a less prioritised outcome from the triage platform: “ My only concern is the red flag confusion [in the diagnosis section].I think what you're alluding to here is the triage output which has come up because it's a triage platform. It needs to be made a lot bigger, bolder and clearer that this is the [triage] outcome ” (Participant 13, nurse). The probability bars displayed in the results output were intended to demonstrate the likelihood of a particular diagnosis. Nevertheless, many found them confusing and were unable to interpret the information that the bars represented. This issue is further discussed in the theme of 'Satisfaction with comprehensibility’'. Information preferences Participants were satisfied with the triage decisions and the level of information on the platform on both pages. They found the refine results prompts particularly useful as “ this would tease out some other questions you may not have asked the patient and it would help you to add to it” (Participant 13, nurse) or "if you've forgotten to ask the question” (Participant 16, nurse). In the data entry page, participants often stated that information related to demographics was comprehensive but needed to be more specific about gender and be defined as ‘gender at birth’. Furthermore, they often mentioned preferring entering patients’ date of birth rather than an age since that is what is entered in patients’ medical records. Most participants commented they were satisfied with the number of options when inputting signs and symptoms, however a participant mentioned the significance of asymptomatic patients that was missing from the platform. “Yes, we have a lot of patients who come to EC who are asymptomatic and then in letter sent by the opticians, it says they need to be seen in EC” (Participant 5, nurse). Participants noted “pain” was missing in the ‘red flag’ checkbox section and in general the platform’s red flags should be expanded to allow adding more red flags options: “The triage people need a few more options for red flags. Because I know red eye is red eye but that is actually a red flag as well because it could be a post operation red eye, so I think it has to reflect the ophthalmology side of things as well a bit more” (Participant 3, consultant ophthalmologist). Satisfaction with comprehensibility of the results Participants generally indicated that the red flags and probability bars were useful. However, many were uncertain about interpreting the bars and found it challenging to understand the diagnosis results due to the red flags. Hence, clearer instructions on how to interpret the red flags and probability bars were needed: “ It wasn't easy to understand because there's so much information. There are two sets of information, as in the these [the ones with red flags] are the ones to consider, and these [without red flags].” (Participant 4, optometrist) “ Not sure these bars mean, maybe that is the likelihood? ” (Participant 19, consultant ophthalmologist). Intention to use Most participants felt that DOTS is a valuable triage platform and intended to use it in the future, although consultant ophthalmologists were less likely to find the platform useful for themselves than other healthcare professionals potentially due to their extensive training and experience working in the EC setting. Participants often cited the platform as particularly helpful to AHP or those who are in training or are less experienced in acute ophthalmology and can support their decision making in triage: “ This is a very good platform for healthcare professionals. The area of triage is a bit alarming for some of us, who are new and having a backup system of this nature helps. It will be a supportive tool for whoever's coming to do triage so they are not alone in that room trying to make decisions all by themselves. ” (Participant 13, nurse). Practitioners found the 'most probable diagnoses' feature helpful. They viewed it as an additional source of information and support for clinical decision making or referring patients in the eye care pathway: “ Well, it [the most probable diagnosis feature] will help in terms of shortening, the time that they're in charge, and you'll be able to decide if the patients need to stay in or they could be redirected somewhere else straight away in terms of the referral .” (Participant 20, nurse). We identified three categories of navigation strategies that led to problems in optimally navigating DOTS: (1) use of navigational elements, (2) layout, and (3) instructions. Participants frequently commented that it was easy for them to navigate through the different sections of the platform. However, some participants noted navigational issues. Use of navigational elements on data entry and results pages Most participants commented that the platform was easy to use, and they could easily identify the navigational icons and understand their purpose. Ophthalmologists found the data entry process straightforward. One suggestion was made to improve the navigational experience related to the addition of a progress bar so users could see their progress to complete the triage, which could be particularly pertinent in an EC setting: “ It would be kind of good if it gives you an idea that ‘don't worry, you don't have another 10 pages to go’ " (Participant 20, consultant ophthalmologist). Regarding the ‘Edit’ function in the patient’s medical information box on the results page, participants noted that it would be useful to be able to close the box with a “cross added at the top” as well as “greying the ‘refine results’ button at the bottom of the box when no further information has been to be entered” (Participant 20, consultant ophthalmologist) would improve the navigation flow through the page. Layout of the results page Some participants commented that more space should be made to contrast the triage outcome with the report of the most probable diagnosis: “ Maybe there should be a couple of spaces in between so that it differentiates ” (Participant 18, nurse). Most participants appreciated the intuitive and logical layout of the platform, though some did not notice the ‘Edit’ function at the top of the page. In addition, participants found the presentation of the traffic colours (red, yellow, green) useful to highlight the emergency of the triage outcome: “It's a good way of doing it because there's something highlighted in red and it’s immediately grabbing your attention. If it's green, you got something that's nationwide. If it’s red, it is an important message.” (Participant 10, optometrist). Despite finding the layout of the platform clear, participants acknowledged that receiving further training prior would help to familiarise themselves with the platform more easily: “I think that once you're taught what the sections are and the purpose of them, then it would become quite easy to use ” (Participant 12, optometrist). Instructions for data entry and results interpretation Participants were provided with limited instructions prior to the task in order to test the intuitiveness of the platform. Participants appreciated that it was a simple tool to use, nevertheless, they wanted further instructions on how to navigate through the platform. Two participants highlighted that the summarised instructions needed to be displayed in the data inputting stage without referring to the user guide tab. For instance, information could be added on the first page explaining the data collection process, the different options on the results page including, how to use the ‘Refine results’ tool and copying to clipboard. Most participants commented that the platform was easy to use, and they could easily identify the navigational icons and understand their purpose. Ophthalmologists found the data entry process straightforward. One suggestion was made to improve the navigational experience related to the addition of a progress bar so users could see their progress to complete the triage, which could be particularly pertinent in an EC setting: “ It would be kind of good if it gives you an idea that ‘don't worry, you don't have another 10 pages to go’ " (Participant 20, consultant ophthalmologist). Regarding the ‘Edit’ function in the patient’s medical information box on the results page, participants noted that it would be useful to be able to close the box with a “cross added at the top” as well as “greying the ‘refine results’ button at the bottom of the box when no further information has been to be entered” (Participant 20, consultant ophthalmologist) would improve the navigation flow through the page. Some participants commented that more space should be made to contrast the triage outcome with the report of the most probable diagnosis: “ Maybe there should be a couple of spaces in between so that it differentiates ” (Participant 18, nurse). Most participants appreciated the intuitive and logical layout of the platform, though some did not notice the ‘Edit’ function at the top of the page. In addition, participants found the presentation of the traffic colours (red, yellow, green) useful to highlight the emergency of the triage outcome: “It's a good way of doing it because there's something highlighted in red and it’s immediately grabbing your attention. If it's green, you got something that's nationwide. If it’s red, it is an important message.” (Participant 10, optometrist). Despite finding the layout of the platform clear, participants acknowledged that receiving further training prior would help to familiarise themselves with the platform more easily: “I think that once you're taught what the sections are and the purpose of them, then it would become quite easy to use ” (Participant 12, optometrist). Participants were provided with limited instructions prior to the task in order to test the intuitiveness of the platform. Participants appreciated that it was a simple tool to use, nevertheless, they wanted further instructions on how to navigate through the platform. Two participants highlighted that the summarised instructions needed to be displayed in the data inputting stage without referring to the user guide tab. For instance, information could be added on the first page explaining the data collection process, the different options on the results page including, how to use the ‘Refine results’ tool and copying to clipboard. Perceived usefulness was measured in terms of satisfaction with the content of DOTS and intention to use it. Regarding satisfaction with the content, we identified three categories: (1) satisfaction with information modality, (2) information preferences, and (3) satisfaction with information comprehensibility. Content on the data entry and results pages Satisfaction with information modality Regarding the modality of how information was presented on both the data entry and results pages, participants appreciated the speed of the outcome delivery and the simplicity of the display of the triage outcome: “ It was quick, it didn’t take me to 60 million different screens to get to an answer ” (Participant 10, optometrist). In addition, all participants acknowledged the effectiveness of the implemented strategies in reducing input errors, such as the utilisation of a validation entry for patients’ hospital number, the copy and paste function, and the edit function. Participants saw the red flags on the results page as a clear way of catching their attention on the importance of the information. However, several participants commented that because of this, the triage decision should be displayed in a clearer way, so it is not overstepped by the diagnosis section, which is a less prioritised outcome from the triage platform: “ My only concern is the red flag confusion [in the diagnosis section].I think what you're alluding to here is the triage output which has come up because it's a triage platform. It needs to be made a lot bigger, bolder and clearer that this is the [triage] outcome ” (Participant 13, nurse). The probability bars displayed in the results output were intended to demonstrate the likelihood of a particular diagnosis. Nevertheless, many found them confusing and were unable to interpret the information that the bars represented. This issue is further discussed in the theme of 'Satisfaction with comprehensibility’'. Information preferences Participants were satisfied with the triage decisions and the level of information on the platform on both pages. They found the refine results prompts particularly useful as “ this would tease out some other questions you may not have asked the patient and it would help you to add to it” (Participant 13, nurse) or "if you've forgotten to ask the question” (Participant 16, nurse). In the data entry page, participants often stated that information related to demographics was comprehensive but needed to be more specific about gender and be defined as ‘gender at birth’. Furthermore, they often mentioned preferring entering patients’ date of birth rather than an age since that is what is entered in patients’ medical records. Most participants commented they were satisfied with the number of options when inputting signs and symptoms, however a participant mentioned the significance of asymptomatic patients that was missing from the platform. “Yes, we have a lot of patients who come to EC who are asymptomatic and then in letter sent by the opticians, it says they need to be seen in EC” (Participant 5, nurse). Participants noted “pain” was missing in the ‘red flag’ checkbox section and in general the platform’s red flags should be expanded to allow adding more red flags options: “The triage people need a few more options for red flags. Because I know red eye is red eye but that is actually a red flag as well because it could be a post operation red eye, so I think it has to reflect the ophthalmology side of things as well a bit more” (Participant 3, consultant ophthalmologist). Satisfaction with comprehensibility of the results Participants generally indicated that the red flags and probability bars were useful. However, many were uncertain about interpreting the bars and found it challenging to understand the diagnosis results due to the red flags. Hence, clearer instructions on how to interpret the red flags and probability bars were needed: “ It wasn't easy to understand because there's so much information. There are two sets of information, as in the these [the ones with red flags] are the ones to consider, and these [without red flags].” (Participant 4, optometrist) “ Not sure these bars mean, maybe that is the likelihood? ” (Participant 19, consultant ophthalmologist). Intention to use Most participants felt that DOTS is a valuable triage platform and intended to use it in the future, although consultant ophthalmologists were less likely to find the platform useful for themselves than other healthcare professionals potentially due to their extensive training and experience working in the EC setting. Participants often cited the platform as particularly helpful to AHP or those who are in training or are less experienced in acute ophthalmology and can support their decision making in triage: “ This is a very good platform for healthcare professionals. The area of triage is a bit alarming for some of us, who are new and having a backup system of this nature helps. It will be a supportive tool for whoever's coming to do triage so they are not alone in that room trying to make decisions all by themselves. ” (Participant 13, nurse). Practitioners found the 'most probable diagnoses' feature helpful. They viewed it as an additional source of information and support for clinical decision making or referring patients in the eye care pathway: “ Well, it [the most probable diagnosis feature] will help in terms of shortening, the time that they're in charge, and you'll be able to decide if the patients need to stay in or they could be redirected somewhere else straight away in terms of the referral .” (Participant 20, nurse). Satisfaction with information modality Regarding the modality of how information was presented on both the data entry and results pages, participants appreciated the speed of the outcome delivery and the simplicity of the display of the triage outcome: “ It was quick, it didn’t take me to 60 million different screens to get to an answer ” (Participant 10, optometrist). In addition, all participants acknowledged the effectiveness of the implemented strategies in reducing input errors, such as the utilisation of a validation entry for patients’ hospital number, the copy and paste function, and the edit function. Participants saw the red flags on the results page as a clear way of catching their attention on the importance of the information. However, several participants commented that because of this, the triage decision should be displayed in a clearer way, so it is not overstepped by the diagnosis section, which is a less prioritised outcome from the triage platform: “ My only concern is the red flag confusion [in the diagnosis section].I think what you're alluding to here is the triage output which has come up because it's a triage platform. It needs to be made a lot bigger, bolder and clearer that this is the [triage] outcome ” (Participant 13, nurse). The probability bars displayed in the results output were intended to demonstrate the likelihood of a particular diagnosis. Nevertheless, many found them confusing and were unable to interpret the information that the bars represented. This issue is further discussed in the theme of 'Satisfaction with comprehensibility’'. Participants were satisfied with the triage decisions and the level of information on the platform on both pages. They found the refine results prompts particularly useful as “ this would tease out some other questions you may not have asked the patient and it would help you to add to it” (Participant 13, nurse) or "if you've forgotten to ask the question” (Participant 16, nurse). In the data entry page, participants often stated that information related to demographics was comprehensive but needed to be more specific about gender and be defined as ‘gender at birth’. Furthermore, they often mentioned preferring entering patients’ date of birth rather than an age since that is what is entered in patients’ medical records. Most participants commented they were satisfied with the number of options when inputting signs and symptoms, however a participant mentioned the significance of asymptomatic patients that was missing from the platform. “Yes, we have a lot of patients who come to EC who are asymptomatic and then in letter sent by the opticians, it says they need to be seen in EC” (Participant 5, nurse). Participants noted “pain” was missing in the ‘red flag’ checkbox section and in general the platform’s red flags should be expanded to allow adding more red flags options: “The triage people need a few more options for red flags. Because I know red eye is red eye but that is actually a red flag as well because it could be a post operation red eye, so I think it has to reflect the ophthalmology side of things as well a bit more” (Participant 3, consultant ophthalmologist). Participants generally indicated that the red flags and probability bars were useful. However, many were uncertain about interpreting the bars and found it challenging to understand the diagnosis results due to the red flags. Hence, clearer instructions on how to interpret the red flags and probability bars were needed: “ It wasn't easy to understand because there's so much information. There are two sets of information, as in the these [the ones with red flags] are the ones to consider, and these [without red flags].” (Participant 4, optometrist) “ Not sure these bars mean, maybe that is the likelihood? ” (Participant 19, consultant ophthalmologist). Most participants felt that DOTS is a valuable triage platform and intended to use it in the future, although consultant ophthalmologists were less likely to find the platform useful for themselves than other healthcare professionals potentially due to their extensive training and experience working in the EC setting. Participants often cited the platform as particularly helpful to AHP or those who are in training or are less experienced in acute ophthalmology and can support their decision making in triage: “ This is a very good platform for healthcare professionals. The area of triage is a bit alarming for some of us, who are new and having a backup system of this nature helps. It will be a supportive tool for whoever's coming to do triage so they are not alone in that room trying to make decisions all by themselves. ” (Participant 13, nurse). Practitioners found the 'most probable diagnoses' feature helpful. They viewed it as an additional source of information and support for clinical decision making or referring patients in the eye care pathway: “ Well, it [the most probable diagnosis feature] will help in terms of shortening, the time that they're in charge, and you'll be able to decide if the patients need to stay in or they could be redirected somewhere else straight away in terms of the referral .” (Participant 20, nurse). Device safety has been found to be correlated to the usability of a product , , where usability has been identified as a key component of good practice in the development of digital applications . In this study, most clinicians found the tool safe to use, easy to navigate, rapid in providing an output and were willing to use the platform in their workflow that would support these themes for clinical integration. A quarter of clinicians had reported feeling uncertainty when undertaking ophthalmic triage in their career, which was surprising considering most were experienced clinicians where four out the five had least 18 years of clinical experience and it is often the assumption that those with more experience, would have greater confidence in clinical decision making . With the DOTS tool having high concordance with the participants expected triage outcomes (95%) and the positive reception regarding users being able to refine the symptoms, this may have impacted clinician’s acceptability and perception of patient safety; where there was an increase of twenty percent willingness to use an AI assisted tool pre and post study. These findings are supportive of other studies that have found clinicians’ acceptance and attitudes towards electronic health record systems are related closely to system usability – . Putting user experience in the context of Web-based health information tools, evaluation of usability and perceived usefulness in terms of content and intention to use the tool is important . Where usability is related to the ease of use of a system, perceived usefulness addresses “the degree to which a person believes that using a particular system would enhance his or her job performance” . These themes were explored in the think-aloud section of the study, where participants found that the data fields were comprehensive with the red flags and probability bars providing useful information. However, there was uncertainty in the interpretation of the latter by twenty percent of the cohort and a third of participants missed the edit function within the platform. Whilst participants were provided with instructions prior commencement of the study with an explanation regarding the platform’s features, this could have led to information overload as this was the first time they had interacted with the platform that may have resulted in misidentification and misinterpretation for some users. With medical devices being an integral part of clinical practice in healthcare settings, nurses are facing an increase in the number of these with varying degrees of complexity , , it’s therefore vital that the provision of both initial and ongoing training on device management and use complement good device designs to foster positive attitudes , which can also minimise the risk of misinterpretation. Other aspects of usability are the extent to which the tool meets the user’s needs and abilities in terms of navigation strategy and problems . Navigation was acceptable in DOTS where participants appreciated the intuitive and logical layout of the platform that reflected their triage workflow and use of the traffic light system to guide interpretation. However, some of the navigational problems were attributable to the layout of the platform, for instance, the ability to close the refine results box and the need for more space between the triage outcome and the most probable diagnosis for ease of distinction; as the triage outcome is the primary function of the platform where nearly all the participants found the triage outcome matched their simulated ocular emergency. This re-enforces the iterative process of medical devices that usability studies are conducted to identify any problems that need addressing before clinical use where the number of participants for this study was sufficient . The US Food and Drug Administration for the development of medical devices recommended a multidisciplinary team approach, with at least an inclusion of 15 users . One of the major strengths of this study was the number of stakeholders across several disciplines that were involved in the development of the triage tool; usability was then evaluated by the relevant number of clinicians who actively work in eye casualty, signifying the applicability of testing for the target users of the device. Taking a user-centred approach to development increases the likelihood of a device being used regularly, correctly and users being satisfied . Think-aloud observations are a classic method to assess user experience of Web-based interfaces , an additional strength of this study was to use this in conjunction with the quantitative survey, where this combination can support validity . Whilst the tool’s usability was evaluated by experienced clinicians working in its intended setting; some clinicians recommended that this tool would be useful for those in training or who were less experienced but these groups were not evaluated, which may limit the generalisability of the study’s findings. Furthermore, this study may have induced a Hawthorne effect for the evaluated healthcare practitioners particularly if their performance is being measured. In conclusion, the DOTS tool was found to be easy to use, reflected users current data entry at triage and was easy to navigate as well as being fast and matching their expected triage output amongst the majority of clinicians who examine ocular emergencies. Nearly all practitioners indicated that they would use the platform following formal device validation when triaging patients and recognised that the tool’s purpose was to aid their decision-making. The study identified features in the platform that required improvements to the interface to increase usability and the need for further clarification to users, re-enforcing the importance of clear instructions being provided for all its’ functions prior use. After validating the tool in terms of usability, further research is required to evaluate DOTS in a real-world clinical trial that is currently underway to determine its performance and usability with clinicians working in ophthalmic triage with a range of expertise, where continuing feedback will support the iterative process of platform development. Supplementary Information 1. Supplementary Information 2. |
The creation of a pediatric surgical checklist for adult providers | 0a40b2d0-0c11-4147-968d-632cf5dd73d2 | 11375845 | Pediatrics[mh] | Surgery is a vital element of healthcare with the potential to cause serious harm when performed in an unsafe manner. A recent World Health Organization (WHO) survey estimates complications occur in about a quarter of surgical patients . A large portion of cases in which those serious complications occur are preventable and are related to non-technical skills . To reduce adverse events such as these, the WHO developed a Surgical Safety Checklist (SSC) in 2008. The checklist comprises three phases and 19 items addressing a variety of perioperative safety measures. The mechanism for improving surgical safety is two-fold: through direct action it standardizes what the team does for every procedure and indirectly it promotes a culture of safety in the operating room . This checklist and others inspired by it have been implemented worldwide with a variety of results. There is heterogeneity in terms of outcomes studied, however, overall multiple papers suggest that checklists are beneficial: decreasing cost, complications and mortality while improving teamwork and communication. The current literature also highlights the importance of staff perception of SSC with staff attitudes towards SSC affecting how often it is utilized and how it is altered to better adapt to their context . As the focus of research on surgical checklists has increasingly shifted to include more tailored checklists, their application in pediatric surgery remains largely unexplored. This gap in the literature is of particular importance as it could assist adult surgeons who often must operate on children in emergency circumstances. This is especially true in rural settings and in low- and middle-income countries (LMIC) like Uganda where general surgeons perform the majority of general pediatric surgeries . In the USA as many as 40% of all pediatric inpatient surgical procedures are performed in adult hospitals . Furthermore, children are far more complex than just smaller adults yet the WHO SSC does not consider and fully address the intricacies of pediatric surgery. Given the potential worldwide impact of a pediatric surgery checklist for adult general surgeons, we reviewed existing literature on surgical checklists and created a fundamental checklist that surgeons in a variety of resource settings can utilize. Resuscitation, consent, pain control and postoperative care for pediatric patients all require special consideration when the adult surgeon is called to operate on a child. Low and high resource settings may contract or expand this checklist based on their resources and needs. This essential checklist of considerations serves as a guide for adult surgeons needing to operate on children. The literature review was conducted using PubMed and the University of Illinois library. Papers with text words and subject headings including “surgical checklist” were identified and reviewed. Reference lists from papers identified in the PubMed search were also reviewed and included when appropriate. We used “surgical checklist” as the keyword search due to the limited availability of pediatric specific checklists and our desire to evaluate all existing papers evaluating checklists’ outcomes to learn the process of creating an effective checklist from them. Two studies out of 42 explicitly mentioned pediatric surgery cases, multiple papers did include patients of all ages however did not provide exact breakdowns. D.R. and E.N. performed independent review of the existing literature for qualifying studies which were then discussed with G.K. to ensure they fit inclusion criteria. We included all papers Jan 2008-October 2023 on the topic. All authors reviewed the list of included papers. Pediatric surgeons at the University of Illinois at Chicago (UIC) Division of Pediatric Surgery and the Paediatric Surgical Foundation of Uganda (PSFU) identified checklist items that they felt were both vital and specific to pediatric general surgery. Dr. Phyllis Kisa from Mulago National Referral Hospital and Dr. Martin Situma from Mbarara Regional Referral Hospital in Uganda participated in the creation of this checklist and provided valuable insight into its potential real world application in LMICs from their own clinical experience. Dr. Lobe, Dr. Sims, and Dr. Rojnica from the UIC Division of Pediatric Surgery also helped create checklist items they deemed essential for adult surgeons performing pediatric surgery in their setting. We then integrated checklist items from UIC and PSFU with key findings from our comprehensive literature review to create three age appropriate, contextually adaptable checklists for pediatric surgery. The majority of papers reviewed employed the WHO SSC and its specific adaptations. (Table ) . No existing pediatric surgery checklists were identified in our review of the literature. Checklist effect on complications and mortality Checklists have been shown to reduce postoperative complications, including SSI and mortality. The WHO SSC specifically targets mortality , SSI , pneumonia , return to the operating room , urinary tract infection, intubation, and sepsis . The WHO SSC has shown positive changes in regards to all of these targets . Thromboembolism (DVT), however, was not a target. Investigations have shown that although the WHO SSC does affect measures like mortality and SSI it does not affect postoperative measures of safety and quality that are not targeted, like DVT . Maternal sepsis rates were also found to be reduced with the use of the WHO SSC with adherence negatively correlating with sepsis rates . Further, there is evidence that intraoperative blood loss and incidence of postoperative intestinal fistula formation was lower with the SSC . Impact on mortality and SSI has been suggested to be more significant in emergency settings in low and middle income countries . Checklist effect on teamwork, communication, and culture of safety The impact of SSC implementation on teamwork and communication was almost unanimously positive across all the studies. After SSC intervention, Molina et al. reported improvements in team discussions, physician receptiveness to quality improvements, and overall communication by 15%, 9%, and 11.9%, respectively . Zingiryan et al. reported improved communication in 76.4% of participants . White et al. (2018) reported improved teamwork and communication in 91% and 89% of participants . Tan et al. (2021) reported improved communication in 85% of participants . One study, however, stood out as an exception; it demonstrated that while nurses and anesthesiologists experienced significantly fewer communication failures, surgeons found no difference in communication with SSC use . Despite this outlier, other studies note that although nursing staff involvement is especially important for compliance, support from surgeons is also critical . Notably, safety culture also improved and was likely correlated with fidelity to a checklist . However, that fidelity appeared to be compromised when staff perceived the checklists as “add ons” . Checklist financial impact Few studies investigated the financial impact of SSC; however, those that did noted SSC implementation was a cost effective health intervention. Checklist implementation costs, length/cost of hospital stay, blood transfusion, antibiotics used in the OR, the cost of OR time, and the economic gain from additional years of life expectancy were considered in studies that did evaluate the financial impact of SSC. In their single-center assessment, Healey et al. (2020) determined that for every 100 admissions the SSC cost $900 to implement but saved $55,899 overall . Yu et al. (2020) discovered significantly lower hospitalization costs while Haugen et al. witnessed a 40% reduction in blood transfusion costs with implementation of the SSC . The SSC incremental cost-effectiveness ratio (ICER) for one year of life loss averted was $31–118 and for every $1 spent on checklist implementation $9–62 was saved . Checklist creation Research indicates that checklists perform best when they are targeted, simple, and contextually appropriate . Almeida et al. (2021) analyzed all surgeries performed at their hospital or in their country to gain a more comprehensive view of SSC impact . Their findings highlighted the need for a tailored checklist . Others found that involving hospital staff in checklist creation helps create a contextually appropriate checklist . Although contextually appropriate checklists are best, this of course has its limits. A checklist made for just one setting has more limited utility. With this in mind, using findings from our literature review, and receiving input from pediatric surgeons in HICs and LMICs we created three age specific, adaptable, general pediatric surgery checklists: Neonatal, Infant, and Toddler/Child. These checklists have room for contextually-appropriate modifications depending on the operation and resources available. Below is the Neonatal checklist as an example. All three checklists are also located in the appendix. We also determined that there are important points on neonatal, infant, and child physiology that the provider should be aware of prior to following the checklist, administering resuscitation, and delivering anesthesia (Appendix 2). This information complements the checklists and should serve as a reference for providers who care for the sick surgical child. Broselow Tape is an additional reference that can be used to estimate appropriate tube sizes, medication doses, and defibrillator shock doses but its accuracy has been shown to be limited in recent studies . Checklists have been shown to reduce postoperative complications, including SSI and mortality. The WHO SSC specifically targets mortality , SSI , pneumonia , return to the operating room , urinary tract infection, intubation, and sepsis . The WHO SSC has shown positive changes in regards to all of these targets . Thromboembolism (DVT), however, was not a target. Investigations have shown that although the WHO SSC does affect measures like mortality and SSI it does not affect postoperative measures of safety and quality that are not targeted, like DVT . Maternal sepsis rates were also found to be reduced with the use of the WHO SSC with adherence negatively correlating with sepsis rates . Further, there is evidence that intraoperative blood loss and incidence of postoperative intestinal fistula formation was lower with the SSC . Impact on mortality and SSI has been suggested to be more significant in emergency settings in low and middle income countries . The impact of SSC implementation on teamwork and communication was almost unanimously positive across all the studies. After SSC intervention, Molina et al. reported improvements in team discussions, physician receptiveness to quality improvements, and overall communication by 15%, 9%, and 11.9%, respectively . Zingiryan et al. reported improved communication in 76.4% of participants . White et al. (2018) reported improved teamwork and communication in 91% and 89% of participants . Tan et al. (2021) reported improved communication in 85% of participants . One study, however, stood out as an exception; it demonstrated that while nurses and anesthesiologists experienced significantly fewer communication failures, surgeons found no difference in communication with SSC use . Despite this outlier, other studies note that although nursing staff involvement is especially important for compliance, support from surgeons is also critical . Notably, safety culture also improved and was likely correlated with fidelity to a checklist . However, that fidelity appeared to be compromised when staff perceived the checklists as “add ons” . Few studies investigated the financial impact of SSC; however, those that did noted SSC implementation was a cost effective health intervention. Checklist implementation costs, length/cost of hospital stay, blood transfusion, antibiotics used in the OR, the cost of OR time, and the economic gain from additional years of life expectancy were considered in studies that did evaluate the financial impact of SSC. In their single-center assessment, Healey et al. (2020) determined that for every 100 admissions the SSC cost $900 to implement but saved $55,899 overall . Yu et al. (2020) discovered significantly lower hospitalization costs while Haugen et al. witnessed a 40% reduction in blood transfusion costs with implementation of the SSC . The SSC incremental cost-effectiveness ratio (ICER) for one year of life loss averted was $31–118 and for every $1 spent on checklist implementation $9–62 was saved . Research indicates that checklists perform best when they are targeted, simple, and contextually appropriate . Almeida et al. (2021) analyzed all surgeries performed at their hospital or in their country to gain a more comprehensive view of SSC impact . Their findings highlighted the need for a tailored checklist . Others found that involving hospital staff in checklist creation helps create a contextually appropriate checklist . Although contextually appropriate checklists are best, this of course has its limits. A checklist made for just one setting has more limited utility. With this in mind, using findings from our literature review, and receiving input from pediatric surgeons in HICs and LMICs we created three age specific, adaptable, general pediatric surgery checklists: Neonatal, Infant, and Toddler/Child. These checklists have room for contextually-appropriate modifications depending on the operation and resources available. Below is the Neonatal checklist as an example. All three checklists are also located in the appendix. We also determined that there are important points on neonatal, infant, and child physiology that the provider should be aware of prior to following the checklist, administering resuscitation, and delivering anesthesia (Appendix 2). This information complements the checklists and should serve as a reference for providers who care for the sick surgical child. Broselow Tape is an additional reference that can be used to estimate appropriate tube sizes, medication doses, and defibrillator shock doses but its accuracy has been shown to be limited in recent studies . Resuscitation Access as large bore as able to place: 24 gauge for neonates, 22 gauge for infants, Weigh neonate Initial bolus resuscitation with crystalloid fluids: 10–20 cc/kg (0.9% NaCl or LR) Maintenance fluid rate by weight using the 4–2-1 rule Urine output: < 1 year: 2–3 cc/kg/hr 1–3 years: 1.5–2 cc/kg/hr > 3 years: 1–1.5 cc/kg/hr Lab value targets Potassium > 3.7 Bicarb > 28 HGB > 10 If bowel is resected it will lower HBG and more transfusion may be required Achieve normothermia—use skin to skin contact, heating blankets, and warm saline to maintain the neonate’s temperature between 36.5 and 37.5 degrees celsius Abdominal concerns Perform a digital rectal exam Decompress the neonate with an NG tube Concern for sepsis? If yes then IV antibiotics such as penicillin/ampicillin and gentamicin Weight-based dosing according to institution protocols Pain Control Weight-based per institution protocols and available medications (paracetamol, morphine etc.) Do NOT use NSAIDS for patients with age < 6 months, asthma, systemic steroids and bleeding disorders Do NOT use Aspirin for patients with age < 12 years Consent obtained from legal guardian Preop and Anesthesia Parents educated on patient’s condition, procedure, and expectations in culturally appropriate and sensitive manner Size appropriate pediatric monitoring equipment available and functioning Size appropriate pediatric respiratory equipment available and functioning Avoid Halothane if possible Breast Milk up to 4 h before scheduled procedure Clear Liquids up to 2 h before scheduled procedure Formula and Solids up to 6 h before scheduled procedure Endotracheal tube size Use little finger as a measure Perioperative Anxiety (most prevalent ages 1–5 y/o) Mother present if possible, to soothe child—even during induction if necessary Oral midazolam administered if necessary Surgery Formulate plan for maintaining child’s temperature during the operation Adjust electrocautery and laparoscopic insufflation settings for patient size, weight and age, place grounding away from site of surgery or potential spillage Laparoscopic insufflation settings for patient size, weight and age Weight based dosing of prophylactic antibiotics Post-operative Antiemetics available Parent/guardian present to help differentiate pain from anxiety. Avoid overdistention of stomach if mask ventilation necessary Post-operative fluid status assessed Research focusing on a variety of surgical subspecialties including general surgery, neurosurgery, plastic surgery, otolaryngology, orthopedics have shown the positive impact of checklists on clinical outcomes . The evidence for checklist impact overall, however, is quite heterogeneous in terms of outcomes studied and the estimated magnitude of the impact of the checklist. Table attached in the appendix displays the current literature on checklists and shows this variation in existing literature. Nevertheless, the consensus impact of SSC remains generally positive. One challenge in evaluating checklist implementation is that different research groups have investigated different post-surgical outcomes. Studies have focused on surgical site infections, in-hospital mortality, overall mortality, blood loss, reoperation, embolism and other adverse outcomes. Although this complicates the overall picture when comparing studies and some types of post-surgical outcome have limited evidence, it also provides a more complete description of the many elements that might be improved through the use of the SSC. Another critical element of SSC use explored throughout the literature is the variability in adherence and attitudes towards SSC and their impact on clinical outcomes. Overall, staff attitudes are critical for utilization compliance . This perhaps suggests that regular training and education on the purpose of SSC are important for engagement of the team. Training should specifically target collaboration with the surgical team since their cooperation is the most commonly cited obstacle to successful implementation . These trainings should also have implementation procedures which consider previous experiences and feedback in order to most effectively create a culture of safety. When implementing a SSC it is also important to consider the burden on a workforce in under-resourced settings that is often stretched too thin. Ultimately, SSC’s should not create more work but rather decrease workload through improved patient outcomes. Although a majority of providers have positive opinions of surgical checklists, there remains a gap in knowledge about their use. In order to bridge this gap there is some evidence that digital SSC displayed on OR monitors increases engagement and accessibility. Many settings, however, do not have an OR computer monitor and efforts to bridge this gap must be made elsewhere . As with the consideration of not creating more work it is vital to adapt these findings to the local resources as the goal of the SSC is to standardize surgical care and provide guidance. Towards the goal of providing standardized guidance for pediatric patients, Ugandan pediatric surgeons also developed the Pediatric Emergency Surgery Course (PESC). It is a three day course targeted at rural general surgeons and healthcare providers . Similar to this checklist the course aims to improve resuscitation and referral patterns for complex surgical conditions such as high anorectal malformations. It also aims to increase provider confidence treating less complex conditions such as pyloric stenosis. The course has been reviewed favorably, demonstrating statistically significant improvements in provider knowledge . In the future, checklist implementation could coincide with educational interventions such as the PESC. Not only should future work coincide with contextually appropriate training but also investigations and feedback should be gathered from providers who use the checklist so improvements can be made. As stakeholders improve surgical outcomes and safety locally and globally special consideration should be given to pediatric surgery checklists. Surgical disease represents roughly 28% of the world’s burden of disease . This burden disproportionately affects children in LMICs; up to 85% of children in LMICs have a surgically-treatable condition by age 15 . The lack of a pediatric surgery checklist for any setting further demonstrates the need and potential benefits of a pediatric surgery checklist that can be adapted for different resource levels. Further research on the topic is necessary especially regarding the differences between implementing such a checklist in HIC and LMIC hospitals. As the first checklist seeking to inform surgical care on children for providers without significant specialized training and in urgent, and often resource limited settings, it is important to evaluate its implementation and effectiveness for adult general surgeons. Although this checklist had input from pediatric surgeons in HICs and LMICs, UIC, Mulago and Mbarara were the only institutions represented in its creation. Our pediatric checklist seeks to integrate as much knowledge from the pediatric surgeons involved in its creation, however it is limited to their experiences and the resources available in their institutions. We acknowledge that other checklists exist already and some may argue against the utility of this checklist and its specificity to pediatric general surgery. It has however been shown throughout this paper that specific checklists have a role to play in different surgical subspecialties, thus supporting our work in the creation of this framework for pediatric general surgery . We have sought to create a comprehensive checklist with input from multiple pediatric surgeons. from both high and low resource settings. We acknowledge that SCC implementation is a process in itself requiring multidisciplinary review and feedback. Effective implementation requires adaptation to specific context in order to achieve local buy-in. We have had extensive conversations with surgeons in high and low resource settings to determine the best way of making this checklist easily adaptable regardless of resource availability. Our cross cultural checklist based on a comprehensive literature review illustrates the importance of adapting checklists to local practices to enhance them instead of implementing a generic checklist. The aim of the pediatric SCC is ultimately to enhance current practice and fill in gaps which exist in pediatric surgery by creating a framework that is both standardized and flexible to the context. This iteration was based on a panel of pediatric surgeons from different resource levels. A next step would be to obtain multidisciplinary feedback from other providers such as nurses, anesthesiologists during the implementation portion of this checklist. Although there exist books with pediatric surgery considerations, a concise checklist indicating clear actions that are important for providers is necessary for settings with limited resources. Countries such as Uganda with few pediatric surgeons, general surgeons are required to fill the gaps and provide care to children without a clear standard of care. The next step for standardizing pediatric surgical care in resource limited settings would be evaluating the effectiveness of our pediatric surgery checklist in practice by adult general surgeons in a variety of settings in HICs and LMICs. The benefits of surgical checklists are far reaching: improved teamwork, communication, clinical outcomes, and patient safety all while saving hospitals and patients money. Keeping in mind that checklists are most effective when they are tailored to the context and the patient, we created three general pediatric surgery checklists that can be adapted to different settings based on resource availability and specific needs. This is the first set of checklists developed specifically for pediatric surgery and providers should carefully weigh their benefits as they consider how to appropriately use them in their practice. This peer reviewed checklist steeped in robust literature review is a critical first step in further standardization of pediatric surgical care and highlights the most important considerations in pediatric surgery in a way that is accessible and concise for general surgeons to use in their practice. Supplementary Material 1. Supplementary Material 2. |
Assessing ChatGPT’s capacity for clinical decision support in pediatrics: A comparative study with pediatricians using KIDMAP of Rasch analysis | 8768a457-0b16-48f3-b6d8-825c39ed17d5 | 10289633 | Pediatrics[mh] | ChatGPT, which stands for Chat Generative Pretrained Transformer, is a cutting-edge language model created by OpenAI. Its purpose is to generate text that resembles human language when given a prompt or context. ChatGPT has been trained on a vast amount of Internet text data, which allows it to comprehend and generate a wide range of language styles and topics. ChatGPT is a versatile language model developed by OpenAI with numerous applications. One major use of ChatGPT is for text generation, which has the potential to transform how we create content, including academic publications. With the increasing sophistication of language models such as ChatGPT, distinguishing between text generated by humans and that produced by artificial intelligence (AI) will become increasingly challenging. It is able to respond to user prompts by answering questions and writing essays, poems, love letters, computer code, or business plans. ChatGPT can solve problems, including math or physics. 1.1. Assessing ChatGPT’s capacity for clinical decision support According to Gilat and Cole, there are significant concerns regarding ChatGPT’s reliability and fairness, echoing reports from the popular press that have highlighted the chatbot’s issues with misinformation. The authors warn that ChatGPT may not always provide accurate information, and there are worries that ChatGPT could be manipulated to disseminate false information or used to generate “deepfakes.” Prior research in medical question answering has often focused on more specific tasks to evaluate the model performance of ChatGPT. For example, Jin et al achieved 68.1% accuracy with their model for answering yes-or-no questions that can be found in PubMed-available abstracts; ChatGPT was performed with accuracy on 4 datasets on the United States Medical Licensing Examination: 64.4% (56/87) on NBME-Free-Step1, and 57.8% (59/102) on NBME-Free-Step2 ; a ChatGPT score of 1.83 (out of 2), a select all that apply (SATA) average percentage correct rate of 88.9% for breast cancer screening prompts, a SATA average percentage correct rate of 58.3% for breast pain prompts, and an average Open-ended (OE) score of 1.125 (out of 2) was achieved by ChatGPT ; answers to prompts about Kawasaki disease in ChatGPT can be graded by A− ; Ha and Yaneva reported only a 29% accuracy on 454 USMLE Step 1 and Step 2 questions on automatic question answering for medical multiple choice questions (MCQs). 1.2. ChatGPT for patients in pediatrics Pediatrics is a specialized medical field that deals with the healthcare needs of infants, children, and adolescents. Clinical decision-making in pediatrics requires a thorough understanding of age-specific developmental patterns, growth milestones, and unique clinical presentations. Assessing ChatGPT’s capacity for clinical decision support (CDS) in pediatrics is particularly relevant as it addresses the specific challenges and considerations in this field. Effective clinical decision-making in pediatrics can significantly impact patient outcomes, as early detection and appropriate management of pediatric conditions are crucial. By evaluating ChatGPT’s performance in pediatric CDS, this study aims to enhance the quality of care provided to pediatric patients and potentially improve health outcomes in this vulnerable population. While the use of large language models has gained attention in various domains, there is a scarcity of research specifically evaluating their application in pediatrics. It is required to fill a gap in the existing literature by focusing on pediatrics and assessing the capabilities of ChatGPT in this specialized medical field. Furthermore, pediatric medicine involves unique considerations such as growth and development, age-specific clinical presentations, and the need for age-appropriate communication. By evaluating ChatGPT’s performance in pediatrics, we can understand how well the ChatGPT can adapt to the specific needs of pediatric patients and provide accurate and contextually appropriate CDS. In this study, the first research question is what is ChatGPT’s grade (e.g., A, B, C, D, or E) about questions in the field of pediatrics. 1.3. Subjective judgments of ChatGPT in an OE format In ChatGPT, there are 2 types of prompts: MCQs and OEs. The OE format of ChatGPT is more subjective than the MCQs. Examining the correlation coefficient and differences in measures of ChatGPT scores between 2 pediatricians assessing ChatGPT answers in an OE format would be useful and necessary. In the second research question, we examine whether Rasch analysis based on the rating scale model (RSM) can be used to examine differences in the assessment of ChatGPT performance evaluated by pediatricians. A study using Rasch analysis to examine the performance of ChatGPT has not yet been published in the literature. It is thus necessary to compare the assessment differences in ChatGPT responses between judges. 1.4. Features of Rasch analysis Rasch analysis is a statistical technique used to analyze the performance of individuals on tests or assessments. When applied to examine the performance of ChatGPT, Rasch analysis can help researchers evaluate the quality of its responses and identify any areas where improvements may be needed. Some features of Rasch analysis applied to examine ChatGPT performance are listed below: Item difficulty : Rasch analysis can help to determine the level of difficulty of each question or prompt that ChatGPT is presented with. This information can be used to identify areas where ChatGPT may be struggling (e.g., performance is lower when the questions are harder) or excelling (e.g., performance is higher when the questions are easier). Person ability : Rasch analysis can also help to measure the ability level of ChatGPT based on its responses to the questions or prompts. This can provide insight into ChatGPT’s overall performance and help to identify areas where improvements may be needed. Item fit statistics : Rasch analysis produces item fit statistics, which measure the degree to which each question or prompt fits the overall model. This information can be used to identify items that may need to be revised or removed from the assessment. Differential item functioning (DIF ) : Rasch analysis can detect DIF, which occurs when different groups of people (such as males and females or people from different cultural backgrounds) respond differently to the same item. This information can be used to identify potentially biased items and improve the fairness of the assessment. Three visualizations are frequently applied to present item features and person measures, including Wright Map with groups, DIF using forest plots, and KIDMAP. 1.5. Study aims This study aimed to determine the ChatGPT grade (e.g., A, B, C, or D) compared to a normal sample and the difference in the assessment of ChatGPT between pediatricians. According to Gilat and Cole, there are significant concerns regarding ChatGPT’s reliability and fairness, echoing reports from the popular press that have highlighted the chatbot’s issues with misinformation. The authors warn that ChatGPT may not always provide accurate information, and there are worries that ChatGPT could be manipulated to disseminate false information or used to generate “deepfakes.” Prior research in medical question answering has often focused on more specific tasks to evaluate the model performance of ChatGPT. For example, Jin et al achieved 68.1% accuracy with their model for answering yes-or-no questions that can be found in PubMed-available abstracts; ChatGPT was performed with accuracy on 4 datasets on the United States Medical Licensing Examination: 64.4% (56/87) on NBME-Free-Step1, and 57.8% (59/102) on NBME-Free-Step2 ; a ChatGPT score of 1.83 (out of 2), a select all that apply (SATA) average percentage correct rate of 88.9% for breast cancer screening prompts, a SATA average percentage correct rate of 58.3% for breast pain prompts, and an average Open-ended (OE) score of 1.125 (out of 2) was achieved by ChatGPT ; answers to prompts about Kawasaki disease in ChatGPT can be graded by A− ; Ha and Yaneva reported only a 29% accuracy on 454 USMLE Step 1 and Step 2 questions on automatic question answering for medical multiple choice questions (MCQs). Pediatrics is a specialized medical field that deals with the healthcare needs of infants, children, and adolescents. Clinical decision-making in pediatrics requires a thorough understanding of age-specific developmental patterns, growth milestones, and unique clinical presentations. Assessing ChatGPT’s capacity for clinical decision support (CDS) in pediatrics is particularly relevant as it addresses the specific challenges and considerations in this field. Effective clinical decision-making in pediatrics can significantly impact patient outcomes, as early detection and appropriate management of pediatric conditions are crucial. By evaluating ChatGPT’s performance in pediatric CDS, this study aims to enhance the quality of care provided to pediatric patients and potentially improve health outcomes in this vulnerable population. While the use of large language models has gained attention in various domains, there is a scarcity of research specifically evaluating their application in pediatrics. It is required to fill a gap in the existing literature by focusing on pediatrics and assessing the capabilities of ChatGPT in this specialized medical field. Furthermore, pediatric medicine involves unique considerations such as growth and development, age-specific clinical presentations, and the need for age-appropriate communication. By evaluating ChatGPT’s performance in pediatrics, we can understand how well the ChatGPT can adapt to the specific needs of pediatric patients and provide accurate and contextually appropriate CDS. In this study, the first research question is what is ChatGPT’s grade (e.g., A, B, C, D, or E) about questions in the field of pediatrics. In ChatGPT, there are 2 types of prompts: MCQs and OEs. The OE format of ChatGPT is more subjective than the MCQs. Examining the correlation coefficient and differences in measures of ChatGPT scores between 2 pediatricians assessing ChatGPT answers in an OE format would be useful and necessary. In the second research question, we examine whether Rasch analysis based on the rating scale model (RSM) can be used to examine differences in the assessment of ChatGPT performance evaluated by pediatricians. A study using Rasch analysis to examine the performance of ChatGPT has not yet been published in the literature. It is thus necessary to compare the assessment differences in ChatGPT responses between judges. Rasch analysis is a statistical technique used to analyze the performance of individuals on tests or assessments. When applied to examine the performance of ChatGPT, Rasch analysis can help researchers evaluate the quality of its responses and identify any areas where improvements may be needed. Some features of Rasch analysis applied to examine ChatGPT performance are listed below: Item difficulty : Rasch analysis can help to determine the level of difficulty of each question or prompt that ChatGPT is presented with. This information can be used to identify areas where ChatGPT may be struggling (e.g., performance is lower when the questions are harder) or excelling (e.g., performance is higher when the questions are easier). Person ability : Rasch analysis can also help to measure the ability level of ChatGPT based on its responses to the questions or prompts. This can provide insight into ChatGPT’s overall performance and help to identify areas where improvements may be needed. Item fit statistics : Rasch analysis produces item fit statistics, which measure the degree to which each question or prompt fits the overall model. This information can be used to identify items that may need to be revised or removed from the assessment. Differential item functioning (DIF ) : Rasch analysis can detect DIF, which occurs when different groups of people (such as males and females or people from different cultural backgrounds) respond differently to the same item. This information can be used to identify potentially biased items and improve the fairness of the assessment. Three visualizations are frequently applied to present item features and person measures, including Wright Map with groups, DIF using forest plots, and KIDMAP. This study aimed to determine the ChatGPT grade (e.g., A, B, C, or D) compared to a normal sample and the difference in the assessment of ChatGPT between pediatricians. 2.1. Data source In the study, 300 virtual participants responded to 8 symptom-related items (Table ) with 5 ordinal categories and were analyzed according to item difficulty (with logit unit, i.e., log odds: [−2, −1.33, −0.66, 0.00,0.66, 1.33, 1.99, and 2.5]) in the Rasch model in accordance with normal distribution of person measures. Each item in Table was prompted. Answers from ChatGPT were gathered and scored on a scale of 0 to 100 by 2 pediatricians; see Supplemental Digital Content 1, http://links.lww.com/MD/J151 (responses by CHatGPT), Table (comments by pediatricians), and the MP4 video (demonstrations of prompts and answers from ChatGPT). The 302 virtual participants were randomly divided into 2 groups based on gender. There were 5 grades (from A to E) assigned based on the person measures (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits). By using the formula round (Score − minimum) ÷ (maximum − minimum) × 5, 0), the scores from the 2 sets of assessments by the 2 pediatricians were transformed into 5-category scores from 0 to 4. As a final step, the 302 individuals (including the 2 assessed by the 2 pediatricians) were analyzed using Raschonline software. This study used simulated data shown in Supplemental Digital Content 2, http://links.lww.com/MD/J152 . According does not require ethical approval due to its design. 2.2. R person responses using RaschOnline RaschOnline based on Rasch RSM was used to analyze the data. The multitomous data can therefore be analyzed using RaschOnline. In the Rasch model, the probability and standardized error (SE) of the person estimate can be expressed as Equations 1 and 2: f ( θ n − δ i ) = P r o ( X n i | θ ) = e ( θ n − δ i ) 1 + e ( θ n − δ i ) (1) S E ( θ ) ^ = 1 ∑ i = 1 L ( p ′ i ( θ ) ) 2 P i ( θ ) Q i ( θ ) (2) where θ and δ are defined as person ability and item difficulty, respectively. L is the item length. p ′ i ( θ ) is the first-order derivative for person n with ability ′ on item i in Equation 1; P i ( θ ) is identical to Equation 1; Q i ( θ ) refers to Equation 3, as shown below: Q i ( θ ) = 1 − P i ( θ ) = 1 − e ( θ n − δ i ) 1 + e ( θ n − δ i ) = 1 + e ( θ n − δ i ) − e ( θ n − δ i ) 1 + e ( θ n − δ i ) = 1 1 + e ( θ n − δ i ) (3) The processes of the first-order derivative for S E ( θ ) ^ in Equation 2 are described below: P ′ i ( θ ) = e ( θ n − δ i ) ( 1 + e ( θ n − δ i ) ) − e ( θ n − δ i ) ( 0 + e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 = e ( θ n − δ i ) ( 1 + e ( θ n − δ i ) − e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 = e ( θ n − δ i ) × 1 ( 1 + e ( θ n − δ i ) ) 2 = P i ( θ ) × Q i ( θ ) (4) Equation 2 can then be extended to Equation 5, indicating that person SE is associated with the inverse of its total variances across all items. S E ( θ ) ^ = 1 ∑ i = 1 L ( P i ′ ( θ ) ) 2 P i ( θ ) Q i ( θ ) = 1 ∑ i = 1 L ( P i ( θ ) × Q i ( θ ) ) 2 P i ( θ ) Q i ( θ ) = 1 ∑ i = 1 L P i ( θ ) × Q i ( θ ) (5) The processes of the first-order derivative for variance (denoted by V a r n i ) on ( θ n − δ i ) can also be described based on Equation 4 and are shown below: V a r n i = f ′ ( θ n − δ i ) = ( e ( θ n − δ i ) 1 + e ( θ n − δ i ) ) ′ = ( e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 (6) If ( e ( θ n − δ i ) ) is replaced with e x p ∑ x k = 0 ( θ n − ( δ i + τ k ) ) , the variance for person n on item i adaptive to the RSM equals the result in Equation 6. Through the Newton–Raphson iteration method and the person estimate and S E ( θ ) ^ in Equations 1 and 5, RaschOnline was programed and developed. To visualize item features and individual measures, several visualizations are commonly used, including Wright Map with groups, DIF using forest plots, and KIDMAP. The method of drawing these visualizations refers to the manual of RaschOnline and Supplemental Digital Content 3, http://links.lww.com/MD/J153 (how to conduct this study). 2.3. Two tasks required to achieve the study goals 2.3.1. Determining the grade of ChatGPT performance (Task 1). Rasch analysis was employed to observe item features and person responses (e.g., the determination of grade in ChatGPT performance), and some significant terms in Rasch analysis are defined below: Wright Map with groups was used to display sample distributions of groups compared to the overall sample of item difficulties and person performance abilities with a log-odds(=logit) unit on a common equal-interval continuum. Analysis of variance (ANOVA) was performed to examine differences in measures between groups (e.g., Female and Male). DIF analysis was performed to examine whether there are items in favor of a specific group (e.g., Female or Male). For example, a specific item might be in favor of female (or male) to be easy in response. Details about DIF are in Supplemental Digital Content 3, http://links.lww.com/MD/J153 . In the KIDMAP, individual person performance is assessed using the Z score (observed × expected ÷ SD) across items. The Z scores of items outside the upper limit (> 2.0) indicate that the observed responses are significantly higher than those expected or Z scores (<-2.0) with unexpected responses based on the individual’s ability. In Task 1, the first study goal of the determination of ChatGPT’s grade (e.g., A, B, C, D, or E) compared to a normal sample would be achieved, based on the criteria of grades from A to E (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits). and (2) the difference in the assessment of ChatGPT between pediatricians by Rasch measures and their corresponding individual standard errors (SE). 2.3.2. The difference in assessment of ChatGPT between pediatricians (Task 2). Using Rasch analysis, the 2 participants assessed by the 2 pediatricians had their own individual measures and standard errors (SEs). If the 95% confidence intervals (CIs) of the 2 measures are overlaid, no difference is apparent between them. In Task 2, the second study goal of the difference in assessment of ChatGPT between pediatricians would be determined based on their 95% CIs. 2.4. Statistical tools and data analysis To perform descriptive statistics and frequency distributions, as well as to calculate the correlation coefficient of scores assessed by the 2 pediatricians and the difference in person measures between groups, IBM SPSS Statistics 22.0 for Windows (SPSS Inc., Chicago, IL) and MedCalc 9.5.0.0 for Windows (MedCalc Software, Ostend, Belgium) were used. Type I errors were set at a significance level of 0.05. In addition, we calculated the intraclass correlation coefficient (ICC) to evaluate interrater reliability. ICC estimates and 95% CIs were reported based on a 2-way mixed-effects model (mean of k raters type and consistency definition). For ICC estimates, below 0.5 represents low reliability; 0.5 to 0.74 represents moderate reliability; 0.75 to 0.90 represents good reliability; and greater than 0.9 represents excellent reliability ; see Supplemental Digital Content 4, http://links.lww.com/MD/J154 . The 2 tables contain 8 items and comments by pediatricians, as shown in Section 2.1. Six visualizations include distribution of item difficulties, DIF or DIF-free in items between groups, scatter plot for assessments by the 2 pediatricians, Wright map to observe item fit statistics (e.g., Infit mean square error, MNSQ < 1.5), KIDMAP for ChatGPT A, and KIDMAP for ChatGPT B. Details about how to conduct this study can be found in the link (MP4) provided in reference and in the Supplementary Digital Contents. In the study, 300 virtual participants responded to 8 symptom-related items (Table ) with 5 ordinal categories and were analyzed according to item difficulty (with logit unit, i.e., log odds: [−2, −1.33, −0.66, 0.00,0.66, 1.33, 1.99, and 2.5]) in the Rasch model in accordance with normal distribution of person measures. Each item in Table was prompted. Answers from ChatGPT were gathered and scored on a scale of 0 to 100 by 2 pediatricians; see Supplemental Digital Content 1, http://links.lww.com/MD/J151 (responses by CHatGPT), Table (comments by pediatricians), and the MP4 video (demonstrations of prompts and answers from ChatGPT). The 302 virtual participants were randomly divided into 2 groups based on gender. There were 5 grades (from A to E) assigned based on the person measures (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits). By using the formula round (Score − minimum) ÷ (maximum − minimum) × 5, 0), the scores from the 2 sets of assessments by the 2 pediatricians were transformed into 5-category scores from 0 to 4. As a final step, the 302 individuals (including the 2 assessed by the 2 pediatricians) were analyzed using Raschonline software. This study used simulated data shown in Supplemental Digital Content 2, http://links.lww.com/MD/J152 . According does not require ethical approval due to its design. RaschOnline based on Rasch RSM was used to analyze the data. The multitomous data can therefore be analyzed using RaschOnline. In the Rasch model, the probability and standardized error (SE) of the person estimate can be expressed as Equations 1 and 2: f ( θ n − δ i ) = P r o ( X n i | θ ) = e ( θ n − δ i ) 1 + e ( θ n − δ i ) (1) S E ( θ ) ^ = 1 ∑ i = 1 L ( p ′ i ( θ ) ) 2 P i ( θ ) Q i ( θ ) (2) where θ and δ are defined as person ability and item difficulty, respectively. L is the item length. p ′ i ( θ ) is the first-order derivative for person n with ability ′ on item i in Equation 1; P i ( θ ) is identical to Equation 1; Q i ( θ ) refers to Equation 3, as shown below: Q i ( θ ) = 1 − P i ( θ ) = 1 − e ( θ n − δ i ) 1 + e ( θ n − δ i ) = 1 + e ( θ n − δ i ) − e ( θ n − δ i ) 1 + e ( θ n − δ i ) = 1 1 + e ( θ n − δ i ) (3) The processes of the first-order derivative for S E ( θ ) ^ in Equation 2 are described below: P ′ i ( θ ) = e ( θ n − δ i ) ( 1 + e ( θ n − δ i ) ) − e ( θ n − δ i ) ( 0 + e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 = e ( θ n − δ i ) ( 1 + e ( θ n − δ i ) − e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 = e ( θ n − δ i ) × 1 ( 1 + e ( θ n − δ i ) ) 2 = P i ( θ ) × Q i ( θ ) (4) Equation 2 can then be extended to Equation 5, indicating that person SE is associated with the inverse of its total variances across all items. S E ( θ ) ^ = 1 ∑ i = 1 L ( P i ′ ( θ ) ) 2 P i ( θ ) Q i ( θ ) = 1 ∑ i = 1 L ( P i ( θ ) × Q i ( θ ) ) 2 P i ( θ ) Q i ( θ ) = 1 ∑ i = 1 L P i ( θ ) × Q i ( θ ) (5) The processes of the first-order derivative for variance (denoted by V a r n i ) on ( θ n − δ i ) can also be described based on Equation 4 and are shown below: V a r n i = f ′ ( θ n − δ i ) = ( e ( θ n − δ i ) 1 + e ( θ n − δ i ) ) ′ = ( e ( θ n − δ i ) ) ( 1 + e ( θ n − δ i ) ) 2 (6) If ( e ( θ n − δ i ) ) is replaced with e x p ∑ x k = 0 ( θ n − ( δ i + τ k ) ) , the variance for person n on item i adaptive to the RSM equals the result in Equation 6. Through the Newton–Raphson iteration method and the person estimate and S E ( θ ) ^ in Equations 1 and 5, RaschOnline was programed and developed. To visualize item features and individual measures, several visualizations are commonly used, including Wright Map with groups, DIF using forest plots, and KIDMAP. The method of drawing these visualizations refers to the manual of RaschOnline and Supplemental Digital Content 3, http://links.lww.com/MD/J153 (how to conduct this study). 2.3.1. Determining the grade of ChatGPT performance (Task 1). Rasch analysis was employed to observe item features and person responses (e.g., the determination of grade in ChatGPT performance), and some significant terms in Rasch analysis are defined below: Wright Map with groups was used to display sample distributions of groups compared to the overall sample of item difficulties and person performance abilities with a log-odds(=logit) unit on a common equal-interval continuum. Analysis of variance (ANOVA) was performed to examine differences in measures between groups (e.g., Female and Male). DIF analysis was performed to examine whether there are items in favor of a specific group (e.g., Female or Male). For example, a specific item might be in favor of female (or male) to be easy in response. Details about DIF are in Supplemental Digital Content 3, http://links.lww.com/MD/J153 . In the KIDMAP, individual person performance is assessed using the Z score (observed × expected ÷ SD) across items. The Z scores of items outside the upper limit (> 2.0) indicate that the observed responses are significantly higher than those expected or Z scores (<-2.0) with unexpected responses based on the individual’s ability. In Task 1, the first study goal of the determination of ChatGPT’s grade (e.g., A, B, C, D, or E) compared to a normal sample would be achieved, based on the criteria of grades from A to E (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits). and (2) the difference in the assessment of ChatGPT between pediatricians by Rasch measures and their corresponding individual standard errors (SE). 2.3.2. The difference in assessment of ChatGPT between pediatricians (Task 2). Using Rasch analysis, the 2 participants assessed by the 2 pediatricians had their own individual measures and standard errors (SEs). If the 95% confidence intervals (CIs) of the 2 measures are overlaid, no difference is apparent between them. In Task 2, the second study goal of the difference in assessment of ChatGPT between pediatricians would be determined based on their 95% CIs. Rasch analysis was employed to observe item features and person responses (e.g., the determination of grade in ChatGPT performance), and some significant terms in Rasch analysis are defined below: Wright Map with groups was used to display sample distributions of groups compared to the overall sample of item difficulties and person performance abilities with a log-odds(=logit) unit on a common equal-interval continuum. Analysis of variance (ANOVA) was performed to examine differences in measures between groups (e.g., Female and Male). DIF analysis was performed to examine whether there are items in favor of a specific group (e.g., Female or Male). For example, a specific item might be in favor of female (or male) to be easy in response. Details about DIF are in Supplemental Digital Content 3, http://links.lww.com/MD/J153 . In the KIDMAP, individual person performance is assessed using the Z score (observed × expected ÷ SD) across items. The Z scores of items outside the upper limit (> 2.0) indicate that the observed responses are significantly higher than those expected or Z scores (<-2.0) with unexpected responses based on the individual’s ability. In Task 1, the first study goal of the determination of ChatGPT’s grade (e.g., A, B, C, D, or E) compared to a normal sample would be achieved, based on the criteria of grades from A to E (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits). and (2) the difference in the assessment of ChatGPT between pediatricians by Rasch measures and their corresponding individual standard errors (SE). Using Rasch analysis, the 2 participants assessed by the 2 pediatricians had their own individual measures and standard errors (SEs). If the 95% confidence intervals (CIs) of the 2 measures are overlaid, no difference is apparent between them. In Task 2, the second study goal of the difference in assessment of ChatGPT between pediatricians would be determined based on their 95% CIs. To perform descriptive statistics and frequency distributions, as well as to calculate the correlation coefficient of scores assessed by the 2 pediatricians and the difference in person measures between groups, IBM SPSS Statistics 22.0 for Windows (SPSS Inc., Chicago, IL) and MedCalc 9.5.0.0 for Windows (MedCalc Software, Ostend, Belgium) were used. Type I errors were set at a significance level of 0.05. In addition, we calculated the intraclass correlation coefficient (ICC) to evaluate interrater reliability. ICC estimates and 95% CIs were reported based on a 2-way mixed-effects model (mean of k raters type and consistency definition). For ICC estimates, below 0.5 represents low reliability; 0.5 to 0.74 represents moderate reliability; 0.75 to 0.90 represents good reliability; and greater than 0.9 represents excellent reliability ; see Supplemental Digital Content 4, http://links.lww.com/MD/J154 . The 2 tables contain 8 items and comments by pediatricians, as shown in Section 2.1. Six visualizations include distribution of item difficulties, DIF or DIF-free in items between groups, scatter plot for assessments by the 2 pediatricians, Wright map to observe item fit statistics (e.g., Infit mean square error, MNSQ < 1.5), KIDMAP for ChatGPT A, and KIDMAP for ChatGPT B. Details about how to conduct this study can be found in the link (MP4) provided in reference and in the Supplementary Digital Contents. 3.1. Determining the grade of ChatGPT performance The distribution of item difficulties is shown in Figure . All 8 items fit to Rasch rather well (i.e., MNSQs of Infit and Outfit less than 1.5). The reason is that all data were simulated under the Rasch RSM model. Two easier items are #1 (What is about that pediatric patients are considered to have a fever above body temperature?) and # 8 (o you think it is possible for ChatGPT AI to replace medical doctors at this moment or in the future?). Several items are more difficult than these two; see the total summation scores in the last column of Fig. ). A higher score corresponds to an easier item. The items in Figure are all DIF-free. The reason for this is that all responses are generated using a Rasch RSM model, and the gender groups are randomly assigned to each virtual participant. Figure illustrates the scatter plot of the 2 pediatricians’ scores. The original scores evaluated by pediatricians A and B are depicted on axes y and x, respectively. There is a correlation coefficient of 0.66 with t = 2.16 and df = 6 ( P < .01). The ICC (=0.61) is identical to the 2-way ANOVA shown in Supplemental Digital Content 2, http://links.lww.com/MD/J152 , implying that the moderate reliability (within 0.5–0.74) was observed between the 2 pediatricians in terms of assessment of consistency. The Wright map in Figure illustrates several findings. First, item difficulties are arranged from harder to easier on the right panel. Second, the middle panel displays person measures distributed from high to low abilities. Third, the left panel shows the display of person measures in groups. Fourth, the bottom panel indicates that there is no significant difference in measures between the 2 groups of males and females ( P = .853). Finally, based on the grade criteria of person measures (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits), GPT301 and GPG302, with measures of −0.83 and 0.90 logits, are classified into grade C, indicating their medium performance in answering questions about symptoms in pediatrics when compared to the normal sample generated by responses under the Rasch rating scale model. The first study goal of ChatGPT’s ability as medium performance (grade C) has been determined. 3.2. The difference in the assessment of ChatGPT between pediatricians According to Figures and , the responses of GPT301 and GPT302 are expected within the upper and lower limits (i.e., Zscore in item i = (observed − expected)/(standard deviation) of item i < 2.0). The Outfit MNSQs are smaller than 2.0, which indicates that no aberrant responses exist in items (i.e., person responses are consistent with Rasch’s expectations). In Figures and , we observed that the abilities of GPT301 and GPT 302 are located in the middle of the person-measure distribution, with grade C. According to 2 pediatricians’ assessments, ChatGPT had an average score of −0.89 logits and 0.90 logits, with SEs of 0.37 and 0.41, respectively; see the bottom panel of Figure . A significant difference in performance assessments between pediatricians was found ( P < .05). Accordingly, this study confirms the second goal of finding differences in pediatrician bias of ChatGPT between the 2 pediatricians. 3.3. Online dashboards shown on Google Maps For readers who wish to manipulate dashboards independently, those QR codes are provided in Figures (or at links ). The distribution of item difficulties is shown in Figure . All 8 items fit to Rasch rather well (i.e., MNSQs of Infit and Outfit less than 1.5). The reason is that all data were simulated under the Rasch RSM model. Two easier items are #1 (What is about that pediatric patients are considered to have a fever above body temperature?) and # 8 (o you think it is possible for ChatGPT AI to replace medical doctors at this moment or in the future?). Several items are more difficult than these two; see the total summation scores in the last column of Fig. ). A higher score corresponds to an easier item. The items in Figure are all DIF-free. The reason for this is that all responses are generated using a Rasch RSM model, and the gender groups are randomly assigned to each virtual participant. Figure illustrates the scatter plot of the 2 pediatricians’ scores. The original scores evaluated by pediatricians A and B are depicted on axes y and x, respectively. There is a correlation coefficient of 0.66 with t = 2.16 and df = 6 ( P < .01). The ICC (=0.61) is identical to the 2-way ANOVA shown in Supplemental Digital Content 2, http://links.lww.com/MD/J152 , implying that the moderate reliability (within 0.5–0.74) was observed between the 2 pediatricians in terms of assessment of consistency. The Wright map in Figure illustrates several findings. First, item difficulties are arranged from harder to easier on the right panel. Second, the middle panel displays person measures distributed from high to low abilities. Third, the left panel shows the display of person measures in groups. Fourth, the bottom panel indicates that there is no significant difference in measures between the 2 groups of males and females ( P = .853). Finally, based on the grade criteria of person measures (e.g., >3.0, >1.5, >−1.5, >−3.0, and <=−3.0 logits), GPT301 and GPG302, with measures of −0.83 and 0.90 logits, are classified into grade C, indicating their medium performance in answering questions about symptoms in pediatrics when compared to the normal sample generated by responses under the Rasch rating scale model. The first study goal of ChatGPT’s ability as medium performance (grade C) has been determined. According to Figures and , the responses of GPT301 and GPT302 are expected within the upper and lower limits (i.e., Zscore in item i = (observed − expected)/(standard deviation) of item i < 2.0). The Outfit MNSQs are smaller than 2.0, which indicates that no aberrant responses exist in items (i.e., person responses are consistent with Rasch’s expectations). In Figures and , we observed that the abilities of GPT301 and GPT 302 are located in the middle of the person-measure distribution, with grade C. According to 2 pediatricians’ assessments, ChatGPT had an average score of −0.89 logits and 0.90 logits, with SEs of 0.37 and 0.41, respectively; see the bottom panel of Figure . A significant difference in performance assessments between pediatricians was found ( P < .05). Accordingly, this study confirms the second goal of finding differences in pediatrician bias of ChatGPT between the 2 pediatricians. For readers who wish to manipulate dashboards independently, those QR codes are provided in Figures (or at links ). 4.1. Principal findings According to 2 pediatricians’ assessments, ChatGPT with a grade of C had an average score of −0.89 logits and 0.90 logits. There was a significant difference in in pediatrician bias of ChatGPT between pediatricians ( P < .05), with scores of −0.89 (SE = 0.37) and 0.90 (SE = 0.41) in a unit of log odds (logits in Rasch analysis), respectively. However, the ICC (=0.61) shows that the moderate reliability (within 0.5–0.74) was observed between the 2 pediatricians in terms of assessment of consistency. Accordingly, 2 objectives have been achieved: ChatGPT’s ability as medium performance (grade C) and differences in the assessment of ChatGPT between pediatricians. 4.2. What this knowledge adds to what we already knew The performance of a large language model called ChatGPT was evaluated on the United States Medical Licensing Exam (USMLE), which includes 3 exams: Step 1, Step 2CK, and Step 3. The results showed that ChatGPT performed at or near the passing threshold for all 3 exams, without any specialized training or reinforcement. In a study, a total of 36 AI-generated suggestions and 29 human-generated suggestions for 7 alerts were analyzed by 5 clinicians. Among the top 20 suggestions based on the survey scores, 9 were generated by ChatGPT. The AI-generated suggestions were noted for providing unique perspectives and were evaluated as highly understandable and relevant. However, their usefulness was deemed moderate, with low acceptance due to concerns regarding bias, inversion, and redundancy, similar to the finding in our study. Prior research on medical question answering has focused on evaluating model performance on specific tasks. ChatGPT has achieved accuracy on various datasets, including answering yes-or-no questions from PubMed-available abstracts and questions on the US Medical Licensing Examination. ChatGPT has also demonstrated high accuracy rates for breast cancer screening prompts and SATA prompts. Additionally, ChatGPT has been graded as a minor for answering prompts about Kawasaki disease. In comparison, a study reported only 29% accuracy on medical Multiple Choice Questions (MCQs). This study seeks to determine ChatGPT grade C for questions in the field of pediatrics, similar results to previous studies. The potential for ChatGPT and future virtual assistants become important tools for patients and healthcare providers based on assessments ranging from basic fact-based questions to complex clinical questions in a study. Although ChatGPT was able to provide interpretable responses that reduced the likelihood of causing alarm compared to Google’s feature snippet, there is an urgent need for regulators and healthcare professionals to be involved in developing standards for minimum quality and to educate patients about the limitations of emerging AI assistants. To raise awareness about the paradigm shift in medical education and research, the future of medical education and research is a blessing or a blight in disguise and needs to be given careful consideration. Gilat and Cole have raised concerns about ChatGPT’s reliability and fairness, in line with reports in the popular press regarding misinformation issues. The authors caution that ChatGPT may not always provide accurate information, and there are fears that it could be manipulated to spread false information or produce “deepfakes. ” Based on the results of this study, ChatGPT is capable of providing answers to pediatric-related questions with an average level (with grade C) of accuracy and consistency across the 8 prompts provided. According to the study, ChatGPT can serve as a useful tool for healthcare professionals in diagnosing and treating pediatric patients, but caution should be taken into consideration. As prompts to ChatGPT with item 8 (Do you think it is possible for ChatGPT AI to replace medical doctors at this moment or in the future?), ChatGPT does not have such personal beliefs or opinions about the question: while ChatGPT and other AI technologies have shown promise in aiding medical decision-making, they are not capable of replacing medical doctors at this moment or in the foreseeable future. This is because the field of medicine requires a human touch that includes empathy, ethical judgment, and communication skills, which are not yet replicable by AI or ChatGPT. Additionally, healthcare providers have ethical and legal responsibilities to provide the best possible care to their patients, which cannot be replaced by a machine or, said, ChatGPT. It should be noted that the study objective was not to prove that ChatGPT suggestions are superior to human suggestions, but rather to demonstrate how ChatGPT suggestions can enhance traditional techniques for CDS maintenance and optimization. This approach aligns with the fundamental theorem of medical informatics, which emphasizes the creation of computer systems that augment human intelligence, leading to improved performance when humans and computers work together compared to humans alone. 4.3. The strengths and features of this study In this study, the capacity of ChatGPT was evaluated by 2 pediatricians for CDS in pediatrics. The study compared ChatGPT’s responses to 8 items on clinical symptoms commonly encountered by pediatricians, and 2 pediatricians assessed its answers using the Wright Map and KIDMAP to compare ChatGPT ability with other virtual participants in Rasch analysis. The study found that ChatGPT has the potential to improve the clinical workflow and the responsible use of clinical decision-making, and it demonstrated the feasibility of using ChatGPT for patients with symptoms commonly encountered in pediatrics. According to this study, ChatGPT has an average level (with grade C) of ability to answer pediatric questions, and the effectiveness of ChatGPT is determined by the judgers’ leniency and severity as pediatricians B and A with 0.90 and −0.83 logits, respectively. We suggest that the methods and visualizations used in this study can be replicated in future research uising RaschOnline. The distinct features of this study include the following: RaschOnline, which is based on Rasch RSM, was used to analyze the data. This allowed us to use 3 visualizations to display item features and person responses online: Wright Map with groups, DIF using forest plots, and KIDMAP. These visualizations had not been previously demonstrated in the literature and can be accessed on RaschOnline for further details and demonstrations. ChatGPT has an average level of ability in answering pediatric questions using objective measurement of Rasch analysis. The effectiveness of ChatGPT is determined by the judges’ leniency and severity, and caution should be taken in future ChatGPT assessments. 4.4. Limitations and directions for future study This study has certain limitations that may motivate further research. The first concern is that the data were generated using Rasch simulation responses based on the Rasch RSM model. The real and virtual responses to the 8 pediatric items were compared. Second, RashOnline has clearly been shown to be applicable in use rather than traditional professional statistical software (e.g., WINSTEPS, Quest, ConQuest, RUMM2030, WINMIRA, LPCM-Win, and R-language Rasch software ), and further research should be conducted to determine whether the visualizations generated using Google Maps in RaschOnline are more straightforward and easier to use for general researchers. Third, on the basis of the study sample size (n = 300 in this study), it is not possible to draw reliable and valid conclusions. For a reliable and accurate assessment, there is a need for a larger sample size in future research. Fourth, in this study, only 8 items were used. A test or assessment that contains more items will be more reliable. To assess ChatGPT ability, more items will be needed in the future. Fifth, in the case of an open-ended (OE) assessment, ChatGPT’s ability is dependent upon the judge’s leniency and severity. The results of our study revealed that the 2 pediatricians had distinctly different attitudes toward the responses provided by the ChatGPT. The conditions of leniency and severity in the assessment of ChatGPT should be stricter in the future. Sixth, in future studies, it is possible to recruit medical students in a classroom or pediatricians at an international conference to evaluate their perceptions of ChatGPT’s responses to the 8 items using Rasch analysis, in addition to the simulated data based on the Rasch model as did in this study. While this approach may involve higher costs and require more manpower compared to the current study, it holds potential for validating the results obtained. It is anticipated that the findings from such studies could yield similar results. Further validating the effectiveness of ChatGPT in the evaluated context based on their ratings on ChatGPT for acceptance, relevance, understanding, and usefulness is required in the future. Finally, while AI technologies such as ChatGPT have shown promise in aiding medical decision-making in specific areas, such as diagnosing certain conditions or interpreting medical images, they are not yet advanced enough to replace medical doctors in complex diagnoses or treatment planning. However, as technology continues to evolve, it is possible that AI could play an increasingly significant role in healthcare decision-making in the future. 4.5. Conclusions The results showed that ChatGPT had an average score of −0.89 logits and 0.90 logits with a grade of C, and there was a significant difference in performance assessments between pediatricians. This study has demonstrated the potential of ChatGPT as a valuable tool for CDS in pediatrics. The findings have shed light on ChatGPT’s performance in responding to clinical symptoms commonly encountered in pediatric practice, and its ability to provide accurate and contextually relevant information. By utilizing ChatGPT, healthcare professionals can benefit from improved clinical workflows and decision-making processes in the future. Furthermore, the use of RaschOnline is recommended for visalizations in assessments based on Rasch analysis because it has an easy copy and paste interface. According to 2 pediatricians’ assessments, ChatGPT with a grade of C had an average score of −0.89 logits and 0.90 logits. There was a significant difference in in pediatrician bias of ChatGPT between pediatricians ( P < .05), with scores of −0.89 (SE = 0.37) and 0.90 (SE = 0.41) in a unit of log odds (logits in Rasch analysis), respectively. However, the ICC (=0.61) shows that the moderate reliability (within 0.5–0.74) was observed between the 2 pediatricians in terms of assessment of consistency. Accordingly, 2 objectives have been achieved: ChatGPT’s ability as medium performance (grade C) and differences in the assessment of ChatGPT between pediatricians. The performance of a large language model called ChatGPT was evaluated on the United States Medical Licensing Exam (USMLE), which includes 3 exams: Step 1, Step 2CK, and Step 3. The results showed that ChatGPT performed at or near the passing threshold for all 3 exams, without any specialized training or reinforcement. In a study, a total of 36 AI-generated suggestions and 29 human-generated suggestions for 7 alerts were analyzed by 5 clinicians. Among the top 20 suggestions based on the survey scores, 9 were generated by ChatGPT. The AI-generated suggestions were noted for providing unique perspectives and were evaluated as highly understandable and relevant. However, their usefulness was deemed moderate, with low acceptance due to concerns regarding bias, inversion, and redundancy, similar to the finding in our study. Prior research on medical question answering has focused on evaluating model performance on specific tasks. ChatGPT has achieved accuracy on various datasets, including answering yes-or-no questions from PubMed-available abstracts and questions on the US Medical Licensing Examination. ChatGPT has also demonstrated high accuracy rates for breast cancer screening prompts and SATA prompts. Additionally, ChatGPT has been graded as a minor for answering prompts about Kawasaki disease. In comparison, a study reported only 29% accuracy on medical Multiple Choice Questions (MCQs). This study seeks to determine ChatGPT grade C for questions in the field of pediatrics, similar results to previous studies. The potential for ChatGPT and future virtual assistants become important tools for patients and healthcare providers based on assessments ranging from basic fact-based questions to complex clinical questions in a study. Although ChatGPT was able to provide interpretable responses that reduced the likelihood of causing alarm compared to Google’s feature snippet, there is an urgent need for regulators and healthcare professionals to be involved in developing standards for minimum quality and to educate patients about the limitations of emerging AI assistants. To raise awareness about the paradigm shift in medical education and research, the future of medical education and research is a blessing or a blight in disguise and needs to be given careful consideration. Gilat and Cole have raised concerns about ChatGPT’s reliability and fairness, in line with reports in the popular press regarding misinformation issues. The authors caution that ChatGPT may not always provide accurate information, and there are fears that it could be manipulated to spread false information or produce “deepfakes. ” Based on the results of this study, ChatGPT is capable of providing answers to pediatric-related questions with an average level (with grade C) of accuracy and consistency across the 8 prompts provided. According to the study, ChatGPT can serve as a useful tool for healthcare professionals in diagnosing and treating pediatric patients, but caution should be taken into consideration. As prompts to ChatGPT with item 8 (Do you think it is possible for ChatGPT AI to replace medical doctors at this moment or in the future?), ChatGPT does not have such personal beliefs or opinions about the question: while ChatGPT and other AI technologies have shown promise in aiding medical decision-making, they are not capable of replacing medical doctors at this moment or in the foreseeable future. This is because the field of medicine requires a human touch that includes empathy, ethical judgment, and communication skills, which are not yet replicable by AI or ChatGPT. Additionally, healthcare providers have ethical and legal responsibilities to provide the best possible care to their patients, which cannot be replaced by a machine or, said, ChatGPT. It should be noted that the study objective was not to prove that ChatGPT suggestions are superior to human suggestions, but rather to demonstrate how ChatGPT suggestions can enhance traditional techniques for CDS maintenance and optimization. This approach aligns with the fundamental theorem of medical informatics, which emphasizes the creation of computer systems that augment human intelligence, leading to improved performance when humans and computers work together compared to humans alone. In this study, the capacity of ChatGPT was evaluated by 2 pediatricians for CDS in pediatrics. The study compared ChatGPT’s responses to 8 items on clinical symptoms commonly encountered by pediatricians, and 2 pediatricians assessed its answers using the Wright Map and KIDMAP to compare ChatGPT ability with other virtual participants in Rasch analysis. The study found that ChatGPT has the potential to improve the clinical workflow and the responsible use of clinical decision-making, and it demonstrated the feasibility of using ChatGPT for patients with symptoms commonly encountered in pediatrics. According to this study, ChatGPT has an average level (with grade C) of ability to answer pediatric questions, and the effectiveness of ChatGPT is determined by the judgers’ leniency and severity as pediatricians B and A with 0.90 and −0.83 logits, respectively. We suggest that the methods and visualizations used in this study can be replicated in future research uising RaschOnline. The distinct features of this study include the following: RaschOnline, which is based on Rasch RSM, was used to analyze the data. This allowed us to use 3 visualizations to display item features and person responses online: Wright Map with groups, DIF using forest plots, and KIDMAP. These visualizations had not been previously demonstrated in the literature and can be accessed on RaschOnline for further details and demonstrations. ChatGPT has an average level of ability in answering pediatric questions using objective measurement of Rasch analysis. The effectiveness of ChatGPT is determined by the judges’ leniency and severity, and caution should be taken in future ChatGPT assessments. This study has certain limitations that may motivate further research. The first concern is that the data were generated using Rasch simulation responses based on the Rasch RSM model. The real and virtual responses to the 8 pediatric items were compared. Second, RashOnline has clearly been shown to be applicable in use rather than traditional professional statistical software (e.g., WINSTEPS, Quest, ConQuest, RUMM2030, WINMIRA, LPCM-Win, and R-language Rasch software ), and further research should be conducted to determine whether the visualizations generated using Google Maps in RaschOnline are more straightforward and easier to use for general researchers. Third, on the basis of the study sample size (n = 300 in this study), it is not possible to draw reliable and valid conclusions. For a reliable and accurate assessment, there is a need for a larger sample size in future research. Fourth, in this study, only 8 items were used. A test or assessment that contains more items will be more reliable. To assess ChatGPT ability, more items will be needed in the future. Fifth, in the case of an open-ended (OE) assessment, ChatGPT’s ability is dependent upon the judge’s leniency and severity. The results of our study revealed that the 2 pediatricians had distinctly different attitudes toward the responses provided by the ChatGPT. The conditions of leniency and severity in the assessment of ChatGPT should be stricter in the future. Sixth, in future studies, it is possible to recruit medical students in a classroom or pediatricians at an international conference to evaluate their perceptions of ChatGPT’s responses to the 8 items using Rasch analysis, in addition to the simulated data based on the Rasch model as did in this study. While this approach may involve higher costs and require more manpower compared to the current study, it holds potential for validating the results obtained. It is anticipated that the findings from such studies could yield similar results. Further validating the effectiveness of ChatGPT in the evaluated context based on their ratings on ChatGPT for acceptance, relevance, understanding, and usefulness is required in the future. Finally, while AI technologies such as ChatGPT have shown promise in aiding medical decision-making in specific areas, such as diagnosing certain conditions or interpreting medical images, they are not yet advanced enough to replace medical doctors in complex diagnoses or treatment planning. However, as technology continues to evolve, it is possible that AI could play an increasingly significant role in healthcare decision-making in the future. The results showed that ChatGPT had an average score of −0.89 logits and 0.90 logits with a grade of C, and there was a significant difference in performance assessments between pediatricians. This study has demonstrated the potential of ChatGPT as a valuable tool for CDS in pediatrics. The findings have shed light on ChatGPT’s performance in responding to clinical symptoms commonly encountered in pediatric practice, and its ability to provide accurate and contextually relevant information. By utilizing ChatGPT, healthcare professionals can benefit from improved clinical workflows and decision-making processes in the future. Furthermore, the use of RaschOnline is recommended for visalizations in assessments based on Rasch analysis because it has an easy copy and paste interface. We thank AJE (American Journal Experts at https://www.aje.com/ ) for the English language review of this manuscript. All authors declare no conflicts of interest. Conceptualization: Hsu-Ju Kao, Wen-Chung Wang, Julie Chi Chow. Investigation: Willy Chou. Methodology: Tsair-wei Chien. |
Desert Endemic Plants in Algeria: A Review on Traditional Uses, Phytochemistry, Polyphenolic Compounds and Pharmacological Activities | 7bd2f06a-b05a-456d-9aa3-9d230c3ab9cc | 9959599 | Pharmacology[mh] | Recently, a great deal of research has been completed on traditional medicine and its applications. It has been determined that conventional medicine is a body of knowledge, abilities, and procedures based on concepts, beliefs, and experiences that are particular to various cultures and are used to maintain health as well as prevent, identify, treat, and improve a variety of physical and mental illnesses . On the other hand, the study of medicinal plants is a method for examining the links and interactions between the biological and cultural components of the environment . Nowadays, ethnobotanical studies are seen to be the best method for finding new medicinal plants or concentrating on those that have already been recognized as having bioactive components , and only a few studies have been completed in the Hot Arid Regions in terms of assessing chemical contents of medicinal plants, especially in terms of identifying the structure of bioactive elements of traditional medicinal plants . The Sahara Desert encompasses the majority of the northern part of the African continent, stretching from the Atlantic to the Red Sea, and because of the extremely low and irregular precipitation, high temperatures, wide temperature range, and protracted droughts that characterize this eco-region’s climate, many living organisms find it difficult to survive . Furthermore, this eco-region’s geomorphological characteristics are primarily related to the growth of sporadic vegetation . Although there is relatively little rainfall each year, it is enough to support plant life on practically all northern Sahara Desert landscapes. Thus, a landscape-specific vegetation has an extremely unbalanced density and structure that are influenced by the habitat’s environmental factors. In depressions such as Wadi beds, the flora is thicker, but on desert plateaus, pavements, and sand dunes, there is scattered vegetation with limited canopy cover . Under these harsh climatic conditions and a variety of pressures in these desert areas, however, many Saharian plants have been discovered that are still in use today as they have developed thanks to ethnic medicine, including morphine, opium, and anesthetic alkaloids, these plants may act as storage for natural, safe, and effective macromolecules that can be used as antioxidants . The aim of this review was to discover plants that grow spontaneously in the Saharian and Algerian areas (Hot Arid Regions) that are utilized in traditional medicines by indigenous people. This is the first attempt at a comprehensive study of the therapeutic qualities of such medicinal plants, which are likely to attract pharmacologists and biological control researchers for further critical and scientific confirmation. 2.1. Collection of Information Published literature between 2000 and 2021 in the form of books and articles downloaded from databases: PubMed, Scopus, Science Direct, Wiley Online Library, and Google Scholar using keywords such as medicine practitioner, traditional medicine, and traditional medicinal plant were used as sources of information. 2.2. Identification of Medicinal Plant A comprehensive ethnobotanical survey of medicinal plants in Saharian Algerian areas was conducted to gather information on the most significant families of ethnomedicinal plant species utilized by the inhabitants of the research area. Plant materials were identified with the assistance of humans and healers . Published literature between 2000 and 2021 in the form of books and articles downloaded from databases: PubMed, Scopus, Science Direct, Wiley Online Library, and Google Scholar using keywords such as medicine practitioner, traditional medicine, and traditional medicinal plant were used as sources of information. A comprehensive ethnobotanical survey of medicinal plants in Saharian Algerian areas was conducted to gather information on the most significant families of ethnomedicinal plant species utilized by the inhabitants of the research area. Plant materials were identified with the assistance of humans and healers . According to the World Health Organization (WHO), traditional medicine is used by 80% of the people in the developing world. In recent decades, the industrialized world has seen an increase in the use of complementary and alternative medicine (CAM), notably herbal treatments . Herbal medications are made up of herbs, herbal materials, herbal preparations, and completed herbal products that have active components that are plant parts or other plant materials . While herbal medicines are used by 90% of the population in Ethiopia for basic healthcare, studies in affluent nations such as Germany and Canada reveal that at least 70% of the population has tried medicine (CAM) at least once . It’s possible that ancient civilizations’ extensive knowledge of herbal medicines, established through trial and error over many years, as well as the most significant cures, were carefully passed down from generation to generation . Indeed, contemporary allopathic medicine has its roots in this old medicine, and many significant novel treatments are expected to be created and marketed in the future from African biodiversity, just as they have in the past by following the leads offered by traditional knowledge and experiences . The polyphenol profile of alcoholic or aqueous extracts of medicinal plants has recently been the subject of various investigations. For instance, the paper of Binello et al. demonstrated microwave-assisted extraction (MAE) and ultrasound procedures for the selective green extraction of polyphenols from lemon balm, where it was found that the compound rosmarinic acid is the main constituent of the phenolic fractions, and it was also determined that ethanol is an excellent solvent for both MAE ultrasonography methods. Reactive oxygen species are involved in a variety of illnesses; hence, plant extracts containing low molecular mass chemicals have been utilized in phytotherapy since ancient times. Many naturally occurring substances have been shown to have significant action as radical scavengers and lipid oxidation inhibitors . Plant antioxidants such as phenolic compounds (see ) (tannins, flavonoids, anthrocyanins, chalcones, xanthones, liganans, depsides, and depsidones), terpenes (sesquterpens and diterpines), alkaloids, and organic sulfur compounds, in addition to alpha-tocopherol and beta-carotene, are useful as antioxidants . A significant number of studies on the antioxidant activity of various plant extracts and powders have been conducted. The findings of these tests show that many secondary metabolites, particularly phenolic compounds such as flavonoids and tannins, etc. . 4.1. Polyphenols Numerous plants contain polyphenols, which are vital nutrients in the diets of both people and animals. The finest polypharmacy against the onset of chronic illness is found in fruits and vegetables, which include a wide range of antioxidant components, such as polyphenols . Polyphenols play an important role in the scavenging of free radicals. In several herbs, vegetables, and fruits, there was a clear link between antioxidant activity and total phenolic concentration . Polyphenols are a sizable class of physiologically useful chemicals that are found in nature; they may be broken down into four categories . Bioflavonoids are the first group. Anthocyanins and proanthocyanidins are two families of bioflavonoids that are closely related (OPCs). The xanthones are the final group. 4.2. Flavonoids Flavonoids work as antioxidants by neutralizing oxidizing radicals such as superoxide and hydroxyl radicals, as well as through chelation . Due to the electron-donating capability of their phenolic groups, flavonoids can also serve as a strong chain-breaking antioxidant. Flavonoids have powerful antioxidant activity; their capacity to scavenge hydroxyl radicals may be their most significant function, and it underpins many of their activities in the body . Flavonoids have been shown to protect against ischemia tissue damage by serving as free radical scavengers, and by functioning as antioxidants, they have numerous positive benefits such as anti-inflammatory, antiallergic, antiviral, and anticancer properties. They’ve also been linked to a reduction in the risk of liver disease, cataracts, and cardiovascular disease . They have direct antioxidant action as well as protective effects on other antioxidants such as vitamins C and E . In addition, their ability to influence membrane-dependent processes such as free radical-induced membrane lipid peroxidation is linked not only to structural features but also to their ability to interact with and permeate lipid bilayers . Polyphenolic compounds are especially susceptible to peroxynitrite-dependent reactions and are powerful inhibitors of nitrous acid-dependent nitration and DNA deamination in vitro, and the role can be exerted in vivo; thus, flavonoids may provide gastro-protective effects when high levels of RNS are produced, in addition to their effects on ROS . Anthocyanins are a kind of flavonoid, which is a family of antioxidant chemicals. Anthocyanins are the pigments that give red, purple, and blue plants their vibrant colors. They may be found in a variety of foods. Anthocyanins may have anti-inflammatory, antiviral, and anti-cancer properties in addition to serving as antioxidants and combating free radicals . Anthocyanin-rich compounds have long been utilized in herbal medicine to treat a variety of blood vessel-related diseases, including chronic venous insufficiency, high blood pressure, and diabetic retinopathy. They have also been used to treat a variety of other illnesses, such as colds and urinary tract infections. Anthocyanins may also help protect against major health issues such as heart disease and cancer . 4.3. Alkaloids A diverse set of naturally occurring compounds known as alkaloids perform a wide range of biological functions. Many of these have important medical uses. The most well-known alkaloids, such as morphine, quinine, strychnine, and cocaine, are made from plants, which are also the first source of alkaloids . The identification of novel alkaloids in these less complex microbial species has benefited from the quick advances in molecular biology and genome sequencing, and a wealth of biosynthetic knowledge about these substances has been acquired in a number of recent mechanistic studies. There are certainly still a lot more microbe-derived alkaloids to be discovered . Metabolic engineering initiatives are a significant biosynthetic research application since many alkaloids have medicinal utility. Alkaloid production has been increased by metabolic engineering, and the biosynthetic enzyme engineering of alkaloids has been used to logically alter the structure of alkaloids in simpler hosts. Recently, there has been substantial advancement in the study of alkaloids, biosynthetic pathways, and metabolic engineering . Numerous plants contain polyphenols, which are vital nutrients in the diets of both people and animals. The finest polypharmacy against the onset of chronic illness is found in fruits and vegetables, which include a wide range of antioxidant components, such as polyphenols . Polyphenols play an important role in the scavenging of free radicals. In several herbs, vegetables, and fruits, there was a clear link between antioxidant activity and total phenolic concentration . Polyphenols are a sizable class of physiologically useful chemicals that are found in nature; they may be broken down into four categories . Bioflavonoids are the first group. Anthocyanins and proanthocyanidins are two families of bioflavonoids that are closely related (OPCs). The xanthones are the final group. Flavonoids work as antioxidants by neutralizing oxidizing radicals such as superoxide and hydroxyl radicals, as well as through chelation . Due to the electron-donating capability of their phenolic groups, flavonoids can also serve as a strong chain-breaking antioxidant. Flavonoids have powerful antioxidant activity; their capacity to scavenge hydroxyl radicals may be their most significant function, and it underpins many of their activities in the body . Flavonoids have been shown to protect against ischemia tissue damage by serving as free radical scavengers, and by functioning as antioxidants, they have numerous positive benefits such as anti-inflammatory, antiallergic, antiviral, and anticancer properties. They’ve also been linked to a reduction in the risk of liver disease, cataracts, and cardiovascular disease . They have direct antioxidant action as well as protective effects on other antioxidants such as vitamins C and E . In addition, their ability to influence membrane-dependent processes such as free radical-induced membrane lipid peroxidation is linked not only to structural features but also to their ability to interact with and permeate lipid bilayers . Polyphenolic compounds are especially susceptible to peroxynitrite-dependent reactions and are powerful inhibitors of nitrous acid-dependent nitration and DNA deamination in vitro, and the role can be exerted in vivo; thus, flavonoids may provide gastro-protective effects when high levels of RNS are produced, in addition to their effects on ROS . Anthocyanins are a kind of flavonoid, which is a family of antioxidant chemicals. Anthocyanins are the pigments that give red, purple, and blue plants their vibrant colors. They may be found in a variety of foods. Anthocyanins may have anti-inflammatory, antiviral, and anti-cancer properties in addition to serving as antioxidants and combating free radicals . Anthocyanin-rich compounds have long been utilized in herbal medicine to treat a variety of blood vessel-related diseases, including chronic venous insufficiency, high blood pressure, and diabetic retinopathy. They have also been used to treat a variety of other illnesses, such as colds and urinary tract infections. Anthocyanins may also help protect against major health issues such as heart disease and cancer . A diverse set of naturally occurring compounds known as alkaloids perform a wide range of biological functions. Many of these have important medical uses. The most well-known alkaloids, such as morphine, quinine, strychnine, and cocaine, are made from plants, which are also the first source of alkaloids . The identification of novel alkaloids in these less complex microbial species has benefited from the quick advances in molecular biology and genome sequencing, and a wealth of biosynthetic knowledge about these substances has been acquired in a number of recent mechanistic studies. There are certainly still a lot more microbe-derived alkaloids to be discovered . Metabolic engineering initiatives are a significant biosynthetic research application since many alkaloids have medicinal utility. Alkaloid production has been increased by metabolic engineering, and the biosynthetic enzyme engineering of alkaloids has been used to logically alter the structure of alkaloids in simpler hosts. Recently, there has been substantial advancement in the study of alkaloids, biosynthetic pathways, and metabolic engineering . 5.1. Search Results The main emphasis in the current review was on the most important types of plants used . From the search above, more than 150 ethnobotanical articles and phytochemistry and pharmacology papers were retained and have been approved by this review. 5.2. Ethnobotanical Studies shows a list of medicinal plants used in Saharan regions for traditional treatment, as well as their habits, portions utilized, route of administration, nations where they are used, toxicity, and ethnopharmacology. Using websites, the proper names and authorities were listed according to the International Convention of Botanical Nomenclature (( www.theplantlist.org , accessed on 2 January 2023), JSTOR ( http://plants.jstor.org , accessed on 2 January 2023), and Tropicos ( www.tropicos.org , accessed on 2 January 2023)) 5.3. Pharmacological Studies Several plants have demonstrated promising results in studies. In vitro, in vivo, and in clinical trials, it possesses healing effects. We provide here pharmacological research, summarized in , that has looked into Saharan medicinal plants that are used to cure a variety of illnesses directly or indirectly. Some of these plants were not found in ethnobotanical studies, and the author is the only one who can confirm their usage in traditional medicine. 5.4. Ethnopharmacology and Toxicological Evidence Due to the lack of dose recommendations in herbal medicine, additional drug discovery methods such as toxicological, clinical, and lab studies are required to scientifically confirm folkloric use and unravel the possible toxicity of the implicated plants . Through this study, not enough information has been found about the toxicity of these plants. This implies that more work still needs to be completed on medicinal plants before they are recommended to patients to prevent adverse side effects. 5.5. List of Multi-Purpose Plants and Their Uses in the Saharan Regions 5.5.1. Retama retam Webb. (Family: Fabaceae; Vernacular Name: Rtem) Useful parts : Aerial part (Infusion, powder, compressed herbal). Investigation: Analgesic, antiseptic, and anti-inflammatory. The stem of this plant is utilized in cauterization in traditional medicine. It can also help with rheumatism, scorpion stings, and injuries. Despite their obvious relevance in traditional medicine, the number of studies on chemical components with biological activities (therapeutic virtues) on the Retama retam Webb. , particularly in Algeria, is sparse compared with other herbs . Selaimia A et al., (2020) assessed antioxidant activity using the DPPH technique. The findings of the DPPH activities indicated that the extracts of the Retama had the most antioxidant activity, with the ethyl acetate extract of the leaves having the highest antioxidant activity. According to the antimicrobial activity research, the extracts have a varied activity that varies depending on the strain examined. It has been found to be ineffective against meticillin-resistant Staphylococcus aureus, although it is extremely sensitive to Candida albican . Another investigation found that the extracts of these plants, dried in various ways, exhibit antibacterial activity suppression for both bacterial strains ( Escherichia coli and Pseudomonas aeruginosa ). Retama retam , on the other hand, was shown to have limited antibacterial action against Pseudomonas aeruginosa and high antibacterial activity against other bacteria . These findings suggest that the herb might be useful in the treatment of viral infections. Their ability to distinguish between different microbes. 5.5.2. Astragalus cruciatus Link (Family: Fabaceae; Vernacular Name: Akifa) Astragalus cruciatus Link. belongs to the genus Astragalus L. of the family Fabaceae. Ithas four synonyms “ A. aristidis Coss .”, “ A. radiatus Ehrenb. ”, “ A. trabutianus Batt. ”, and A. corrugates Bertol . Species of the Astragalus genus are used as therapeutic herbs in traditional medicine all throughout the world to treat stomach ulcers, cough, chronic bronchitis, hypertension, gynecological problems, diabetes, and scorpion poisonous stings . Immunostimulant, cardiovascular, and antiviral properties have been found in plants from the same genus . Saponin, phenolic, and polysaccharide chemicals are the physiologically active ingredients of Astragalus species, while thenitro-toxins, imidazoline alkaloids, and selenium derivatives are poisonous components . The Astragalus genus is well-known for its abundance of bioactive secondary metabolites. Saponins and flavonoids have been isolated and characterized in previous phytochemical studies of several Astragalus species. However, there has never been any constitutional or pharmacological research on Astragalus cruciatus Link. 5.5.3. Genista saharae (Coss. & Dur.) (Family: Fabaceae; Vernacular Name: Marekh) Investigation: Cold, influenza, respiratory system problems. It contains flavonoids compounds . Genista saharae is a leafless, spontaneous fabaceae that is colloquially called as “Tellegit” in Algeria. For the reason that it is known as a source of chemical components with antioxidant properties, this medicinal plant is used in traditional pharmacopeia. Sofiane Guettaf’s research focuses on examining the phytochemical content and assessing the antioxidant activity of aerial portions of an aqueous extract of Genista saharae (AEG) under settings similar to its traditional use. The qualitative chemical composition of AEG was assessed via phytochemical screening utilizing precipitation and coloring reactions. Furthermore, spectrophotometric techniques were used to determine the total phenolics, flavonoids, tannins, and carotene contents. Finally, the antioxidant properties of AEG were determined using three different methods: DPPH, reducing power, and β carotene bleaching tests. Phénolic compounds, flavonoids, alkaloids, tannins, terpenoids, glycosides, steroids, and saponins are among the biomolecules found in AEG, according to the findings. Furthermore, the quantitative examination reveals a significant amount of total phenolics, tannins, and β carotene. In sum, EAG has a strong antioxidant activity against β carotene bleaching, which validates its usage by traditional healers. As a result, Genista saharae is an excellent antioxidant source. The presence of tannins or other phenolic chemicals such as terpenoids, β carotene, and saponins may explain its high antioxidant activity . The chemical composition and antioxidant activity of this genus’ alcoholic extracts have been studied before . Unfortunately, there is presently no phytochemical or biological information regarding this shrub. There is no information in the literature on the antioxidant effects of its aqueous extract. As a result, all of the Genista saharae findings are consistent with its traditional use. 5.5.4. Astragalus gyzensis Bunge (Family: Fabaceae; Vernacular Name: Foul Alibil) Investigation: Scorpion stings and snake bites. According to ref. , this plant is used to treat snake bites and is a highly hardy plant in the Saharan climate, which is characterized by a variety of stresses. Animals find this plant to be moderately appealing, especially when it is in blossom. Volatiles, which are constantly released into the atmosphere, can account for this. Many studies involving the essential oils of plants have been conducted in recent years for the purpose of discovering natural products and anti-disease actives. Essential oils contain a variety of biological activities, including larvicidal efficacy against mosquitos . The essential oil may be utilized to produce natural pesticides and help reduce the negative effects of synthetic goods, including buildup, resistance, and contamination. 5.5.5. Euphorbia guyoniana Boiss. & Reut (Family: Euphorbiaceae; Vernacular Name: Lebina) Investigation: Diarrhea, skin diseases, scorpion stings and snake bites. This plant, similar to many euphorbias, is extremely poisonous due to the presence of toxic white latex. The nomads, on the other hand, utilize it to protect themselves from snake bites . Euphorbia guyoniana Boiss. and Reut. , a member of the Euphorbiaceae family, was the plant studied in this study. This species may be found across Algeria’s desert and pre-desert areas . It’s called “Lebbina” in the area and grows 30–100 cm tall with upright and branching stems that produce very poisonous white latex. More than 2000 species of Euphorbia have been used in traditional medicine to treat skin problems, including dermatitis, gastrointestinal issues, bacterial or fungal infections, and even some forms of cancer. Algerian Sahara nomads also utilize E. guyoniana to protect themselves against snake bites . It has significant antioxidant capabilities, according to a recent study . The high amount of secondary chemicals in E. guyoniana , such as terpenoids, alkaloids, and flavonoids, contributes to its positive benefits. 5.5.6. Ephedra alata DC. (Family: Ephedraceae; Vernacular Name: Alanda) Parts of use: Leaves and boughs (Maceration, inhalation, herb tea). Investigation: This plant is used to cure a variety of ailments, including colds, influenza, respiratory issues, hypertension, body aches, whooping cough, and cancer . 5.5.7. Heliathemum lipii (L.) Pers . (Family: Cistaceae; Vernacular Name: Samhari) The genus Helianthemum belongs to the family Cistaceae , which are widespread in the Mediterranean region. Across the world, this genus includes 70 species . In Algeria and Pakistan, this genus has a single species Helianthemum lippii (L.) Pers. The vernacular name of this species is different in different regions and continents, for examplef the name of Al Samhari (in the region of Oued Souf: Southeast of Algeria) ; Reguig (in the region of Ouargla: South of Algeria) Tahsowat and Alrjik (South-West of Algeria); Alrkaroq (in Kuwait); Umm Souika (the Arabian Peninsula) ; and Sun Flower (Northeast of Jordan) . This plant is highly intriguing from an ecological and economic standpoint . It belongs to the pastoral plants , plays a key role in the battle against desertification, and contributes to the stability of desertification-prone areas. The species’ powder is used to treat skin rashes . It’s also used in Libya to treat rot and rashes, as well as to prevent illness . In Morocco, this plant with the Bedouins can cause lameness in camels in the name of GAF or Kraft (a type of arthritis). But in reality, the toxicity of this plant has not yet been proven . 5.5.8. Cyperus conglomeratus (Family: Cyperaceae; Vernacular Name: Saad) Cyperus rotundus is one of the best known species of the genus as a medicinal plant, featuring in Indian, Chinese, and Japanese traditional medicines for spasms, stomach and intestinal problems, and menstrual irregularities . Several writers have studied C. rotundus extensively; the most notable essential oils isolated from it are α-pinene, β-pinene, α-copaene, cyperene, cyperotundone, α-cyperone, and caryophyllene oxide . For the reason that the southern coast, eastern south, and central parts of Iran have dry and very hot air for eight months of the year, this species can withstand harsh environmental conditions . This species has been recorded to be used as a pectoral, emollient, diuretic, stimulant, analgesic, and anthelmintic therapy in traditional medicine. 5.5.9. Calligonum comosum L’herit (Family: Polygonaceae; Vernacular Name: Larta) Parts of use: Leaves, roots, boughs (infusion, decoction). Investigation: This plant can be used against scorpion stingsand snake bites , vermifuge. The antibacterial activity of Calligonum comosum L. Her. , “Arta,” a member of the family Polygonaceae, was investigated. It is a tropical and subtropical plant that is widely distributed in the United Arab Emirates and Saudi Arabia. Previous research found that an ethanolic extract of Calligonum comosum aerial parts greatly decreased the rise in carrageenan-induced hind paw oedema in rats. Furthermore, a pre-treatment with the extract inhibited the acute stomach ulcers caused by phenylbutazone and indomethacin in a dose-dependent manner . Recently, researchers discovered that extracts from several Calligonum comosum plant sections have significant antibacterial activity against four harmful bacteria . Anthraquinones and flanovonids are the most frequent chemical components of Arta, according to prior investigations. In addition, when Calligonum comosum was treated with organic solvents, dehydrodicatechin was extracted, which showed cytotoxic and antioxidant activities . 5.5.10. Plantago albicans L. (Family: Plantaginaceae; Vernacular Name: Linem) Is an herbaceous plant that grows wild in subtropical and temperate climates and may be grown readily in Tunisia, Algeria, and Libya. The extracts of Plantago species are often used in traditional medicine due to their hepatoprotective , analgesic, anti-inflammatory, and antipyretic properties. Furthermore, P. albicans caused structural and functional changes in liver and heart tissue . In fact, this genus contains a high amount of primary and secondary metabolites . 5.5.11. Limoniastrum guyonianum Coss & Dur. (Family: Plumbaginaceae; Vernacular Name: Zita) Investigation: This plant can be used as a treatment for several diseases, including: diabetes, scorpion stings and snake bites, headaches, constipation, hypertension, renal illness, anemia, antiseptics, burns, leprosy, wounds, ulcers, diabetes, anemia, cough, constipation, gas, obesity, tonsillitis, and flu . 5.5.12. Tamarix boveana (Family: Tamaricaceae; Vernacular Name: Tarfa) Investigation: This plant can be used as a treatment for several diseases, including: cough, hemorrhage, diuretic, antiseptic, leprosy, injuries and ulcers, scorpions and insect bites, renal diseases, diarrhea, anemia, gum and mouth inflammation, gastric ulcer, cephalalgia, hypertension, diabetes, joint disease, and pancreatic inflammation . 5.5.13. Traganum nudatum Del. (Family: Chenopodiaceae; Vernacular Name: Damran) Parts of use: Leaves (compressed maceration, powder, and ointment). Investigation: Rheumatism, skin diseases, diarrhea, rheumatism wound dermatoses . This plant is well renowned for its tasty fruit as well as its wood for burning . 5.5.14. Bassia muricata L. Asch. (Family: Chenopodiaceae; Vernacular Name: Ghabitha) Investigation: Analgesic, antiseptic, and anti-inflammatory. It is a rich plant with triterpenoids and saponins . It grows in sandy environments in North Africa, the East Mediterranean region, Sinai, Saudi Arabia, and Iran as an annual plant . It is a significant plant in traditional medicine, where it is used as an analgesic, antipyretic, and nephrotic. It also contains various biological properties, including antioxidants . The extract of B. muricata has been shown to lower white blood cell counts, enhance prothrombin time, reduce blood pressure, and have antibacterial properties . Flavonoid glycosides and other phytochemical components of B. muricata have been isolated and identified . 5.5.15. Atriplex halimus L. (Family: Chenopodiaceae; Vernacular Name: Gatef) Investigation: this plant can be used as a treatment for several diseases, including: cysts in the uterus, diabetes, stomach pain, constipation, diarrhea, gas, hypertension, antiseptic, burns, fever, anemia, otitis, rheumatism, diuretic, vermifuge, vomiting, wounds and ulcers, tonsillitis, gallbladder disease, fortify the gums, infertility, prostate, fall of placenta, nephrolithiasis, hypercholesterolemia . Streptozotocin-induced diabetic rats showed both long-term and short-term antidiabetic effects. The aqueous extract was given orally at a dose of 200 mg/kg BW for 30 days to rats divided into four groups: normal rats, diabetic rats (50 mg/kg BW of streptozotocin), and diabetic and normal rats treated with 200 mg/kg BW in the short-term study after administration of a glucose dose of 3 g/kg . When compared with the results before treatment, the aqueous extract resulted in a substantial reduction (54%) in blood glucose in the treated diabetic group at the end of the trial. The plant extract also has an antihyperglycemic effect, lowering diabetic rats’ fasting blood glucose levels by 23 and 41% after 2 and 3 h, respectively . The aqueous extract is high in tannins and flavonoids, which the researchers believe may have an effect on the pancreas via increasing beta cell insulin production. 5.5.16. Zygophyllum album L. (Family: Zygophyllaceae; Vernacular Name: Agga) Parts of use: Leaves, stems, and fruits (Decoction, powder, ointment). Investigation : Diabetes, purgatives and laxatives, anti-viruses and fungi, indigestion. This plant can be used to treat diabetes, indigestion, skin problems, as an analgesic, and as a disinfectant, according to . This herb is utilized in Tunisian traditional medicine as a rheumatism, gout, and asthma treatment . It’s also a diuretic, a local anesthetic, an antihistaminic, and an antidiabetic . 5.5.17. Matricaria pubescens (Desf) (Family: Asteraceae; Vernacular Name: Guartoufa) Parts of use: Leaves (Powder). Investigation: It is used as a treatment for: scorpion stings and snake bites, colds and respiratory difficulties, bleeding, diuretics, fever, astringents and stimulants, stomach and stomach aches, and constipation. This herb is used to treat scorpion stings and snake bites in the Oued Righ area (El Oued, Algeria) . 5.5.18. Launaea resedifolia O. K. (Family: Asteraceae; Vernacular Name: Athid) The Launaea resedifolia O. K family is one of the biggest angiosperm families, with around 340 genera and 3350 species divided into 11 tribes. They are found all over the world, primarily in temperate parts of the northern hemisphere . The Irano-Turanian, Mediterranean, and Saharao-Sindian areas are the family’s primary distribution hubs. 5.5.19. Solanum nigrum L. (Family: Solanaceae; Vernacular Name: Anb Aldib) Parts of use: Leaf, stem, and fruits are the parts of the plant that are used. It is a poisonous plant that is classified as active and hazardous in the pharmacopoeia. It is for external use . Investigation: It is used as a treatment for: diuretic, chronic liver enlargement, diarrhea, and piles; also effective against skin illness and anthrax. Fruits are used to treat a variety of ailments, including heart disease, hiccups, asthma, fever, bronchitis, and diarrhea. Ringworm can be treated using green fruit pastes. Fruit juice is an expectorant and cooling drink that can be used to treat fevers, gonorrhea, giddiness, and inflammations . 5.5.20. Erodium glaucophyllum L. Her. (Family: Geraniaceae; Vernacular Name: Tommir) Parts of use: All its parts are useful. Investigation: It is used as a treatment for: diarrhea, colds, influenza, and respiratory system disorders are investigated. It’s a medicinal herb that’s good for diarrhea, an astringent, allergies, and oxytocin . Erodium glaucophyllum L. is a common herb in the Nile valley, the western Mediterranean coastal region, and the deserts, and goes by the Arabic names Kahkul, Lesan Hamad, Kabshia, Ragma, Dahma, Murrar, and Tamir. 5.5.21. Cleome arabica L. (Family: Capparidaceae; Vernacular Name: Nettin) Parts of use: Leaves (Infusion, maceration). Investigation: Rheumatism, urinary tract. It is a plant that is high in flavone chemicals, particularly flavonoids, and is diuretic. It can also help with rhumatism, arthritis, and diarrhea . 5.5.22. Neurada procumbens L. (Family: Rosaceae; Vernacular Name: Anfal/Saadan) Parts of use : Leaves, seeds and fruits. Investigation: Analgesic, antiseptic, anti-inflammatory, astringent, and stimulant. The Bedouin have always regarded them as edible medical plants . The Bedouins see it as a sort of camel meal that is both safe and edible. The herb has also been used to cure diarrhea and dysentery in traditional Arabic medicine. It’s also been utilized to boost heart and respiratory processes as a stimulant . Its water extract made the mice’s blood pressure rise. Only one study examined the chemical constituents of this plant, which revealed that it contains alkaloids, flavonoids, saponins, sterols and terpenes, volatile oils, and tannins, as well as a hypertensive effect of its ethanol extract, indicating that cardiovascular patients should exercise caution when using this plant . It was also used to cure stroke in people and animals in Pakistan, and its dried star-shaped fruits may be mixed with rose water in the summer as a cooling agent and with dry nuts in the winter to stimulate nerves . In addition to dihydroflavonol glycosides, prior findings in Egypt suggest that this plant contains polysaccharides (gum, mucilage) . On the other hand, mineral monitoring, particularly for toxic elements, is one of the most important aspects of ensuring the quality of medicinal plants because elements such as Fe, Ca, and Zn play an important role in many processes of human metabolism and thus contribute significantly to human health , as well as plant growth processes, even in small amounts. The main emphasis in the current review was on the most important types of plants used . From the search above, more than 150 ethnobotanical articles and phytochemistry and pharmacology papers were retained and have been approved by this review. shows a list of medicinal plants used in Saharan regions for traditional treatment, as well as their habits, portions utilized, route of administration, nations where they are used, toxicity, and ethnopharmacology. Using websites, the proper names and authorities were listed according to the International Convention of Botanical Nomenclature (( www.theplantlist.org , accessed on 2 January 2023), JSTOR ( http://plants.jstor.org , accessed on 2 January 2023), and Tropicos ( www.tropicos.org , accessed on 2 January 2023)) Several plants have demonstrated promising results in studies. In vitro, in vivo, and in clinical trials, it possesses healing effects. We provide here pharmacological research, summarized in , that has looked into Saharan medicinal plants that are used to cure a variety of illnesses directly or indirectly. Some of these plants were not found in ethnobotanical studies, and the author is the only one who can confirm their usage in traditional medicine. Due to the lack of dose recommendations in herbal medicine, additional drug discovery methods such as toxicological, clinical, and lab studies are required to scientifically confirm folkloric use and unravel the possible toxicity of the implicated plants . Through this study, not enough information has been found about the toxicity of these plants. This implies that more work still needs to be completed on medicinal plants before they are recommended to patients to prevent adverse side effects. 5.5.1. Retama retam Webb. (Family: Fabaceae; Vernacular Name: Rtem) Useful parts : Aerial part (Infusion, powder, compressed herbal). Investigation: Analgesic, antiseptic, and anti-inflammatory. The stem of this plant is utilized in cauterization in traditional medicine. It can also help with rheumatism, scorpion stings, and injuries. Despite their obvious relevance in traditional medicine, the number of studies on chemical components with biological activities (therapeutic virtues) on the Retama retam Webb. , particularly in Algeria, is sparse compared with other herbs . Selaimia A et al., (2020) assessed antioxidant activity using the DPPH technique. The findings of the DPPH activities indicated that the extracts of the Retama had the most antioxidant activity, with the ethyl acetate extract of the leaves having the highest antioxidant activity. According to the antimicrobial activity research, the extracts have a varied activity that varies depending on the strain examined. It has been found to be ineffective against meticillin-resistant Staphylococcus aureus, although it is extremely sensitive to Candida albican . Another investigation found that the extracts of these plants, dried in various ways, exhibit antibacterial activity suppression for both bacterial strains ( Escherichia coli and Pseudomonas aeruginosa ). Retama retam , on the other hand, was shown to have limited antibacterial action against Pseudomonas aeruginosa and high antibacterial activity against other bacteria . These findings suggest that the herb might be useful in the treatment of viral infections. Their ability to distinguish between different microbes. 5.5.2. Astragalus cruciatus Link (Family: Fabaceae; Vernacular Name: Akifa) Astragalus cruciatus Link. belongs to the genus Astragalus L. of the family Fabaceae. Ithas four synonyms “ A. aristidis Coss .”, “ A. radiatus Ehrenb. ”, “ A. trabutianus Batt. ”, and A. corrugates Bertol . Species of the Astragalus genus are used as therapeutic herbs in traditional medicine all throughout the world to treat stomach ulcers, cough, chronic bronchitis, hypertension, gynecological problems, diabetes, and scorpion poisonous stings . Immunostimulant, cardiovascular, and antiviral properties have been found in plants from the same genus . Saponin, phenolic, and polysaccharide chemicals are the physiologically active ingredients of Astragalus species, while thenitro-toxins, imidazoline alkaloids, and selenium derivatives are poisonous components . The Astragalus genus is well-known for its abundance of bioactive secondary metabolites. Saponins and flavonoids have been isolated and characterized in previous phytochemical studies of several Astragalus species. However, there has never been any constitutional or pharmacological research on Astragalus cruciatus Link. 5.5.3. Genista saharae (Coss. & Dur.) (Family: Fabaceae; Vernacular Name: Marekh) Investigation: Cold, influenza, respiratory system problems. It contains flavonoids compounds . Genista saharae is a leafless, spontaneous fabaceae that is colloquially called as “Tellegit” in Algeria. For the reason that it is known as a source of chemical components with antioxidant properties, this medicinal plant is used in traditional pharmacopeia. Sofiane Guettaf’s research focuses on examining the phytochemical content and assessing the antioxidant activity of aerial portions of an aqueous extract of Genista saharae (AEG) under settings similar to its traditional use. The qualitative chemical composition of AEG was assessed via phytochemical screening utilizing precipitation and coloring reactions. Furthermore, spectrophotometric techniques were used to determine the total phenolics, flavonoids, tannins, and carotene contents. Finally, the antioxidant properties of AEG were determined using three different methods: DPPH, reducing power, and β carotene bleaching tests. Phénolic compounds, flavonoids, alkaloids, tannins, terpenoids, glycosides, steroids, and saponins are among the biomolecules found in AEG, according to the findings. Furthermore, the quantitative examination reveals a significant amount of total phenolics, tannins, and β carotene. In sum, EAG has a strong antioxidant activity against β carotene bleaching, which validates its usage by traditional healers. As a result, Genista saharae is an excellent antioxidant source. The presence of tannins or other phenolic chemicals such as terpenoids, β carotene, and saponins may explain its high antioxidant activity . The chemical composition and antioxidant activity of this genus’ alcoholic extracts have been studied before . Unfortunately, there is presently no phytochemical or biological information regarding this shrub. There is no information in the literature on the antioxidant effects of its aqueous extract. As a result, all of the Genista saharae findings are consistent with its traditional use. 5.5.4. Astragalus gyzensis Bunge (Family: Fabaceae; Vernacular Name: Foul Alibil) Investigation: Scorpion stings and snake bites. According to ref. , this plant is used to treat snake bites and is a highly hardy plant in the Saharan climate, which is characterized by a variety of stresses. Animals find this plant to be moderately appealing, especially when it is in blossom. Volatiles, which are constantly released into the atmosphere, can account for this. Many studies involving the essential oils of plants have been conducted in recent years for the purpose of discovering natural products and anti-disease actives. Essential oils contain a variety of biological activities, including larvicidal efficacy against mosquitos . The essential oil may be utilized to produce natural pesticides and help reduce the negative effects of synthetic goods, including buildup, resistance, and contamination. 5.5.5. Euphorbia guyoniana Boiss. & Reut (Family: Euphorbiaceae; Vernacular Name: Lebina) Investigation: Diarrhea, skin diseases, scorpion stings and snake bites. This plant, similar to many euphorbias, is extremely poisonous due to the presence of toxic white latex. The nomads, on the other hand, utilize it to protect themselves from snake bites . Euphorbia guyoniana Boiss. and Reut. , a member of the Euphorbiaceae family, was the plant studied in this study. This species may be found across Algeria’s desert and pre-desert areas . It’s called “Lebbina” in the area and grows 30–100 cm tall with upright and branching stems that produce very poisonous white latex. More than 2000 species of Euphorbia have been used in traditional medicine to treat skin problems, including dermatitis, gastrointestinal issues, bacterial or fungal infections, and even some forms of cancer. Algerian Sahara nomads also utilize E. guyoniana to protect themselves against snake bites . It has significant antioxidant capabilities, according to a recent study . The high amount of secondary chemicals in E. guyoniana , such as terpenoids, alkaloids, and flavonoids, contributes to its positive benefits. 5.5.6. Ephedra alata DC. (Family: Ephedraceae; Vernacular Name: Alanda) Parts of use: Leaves and boughs (Maceration, inhalation, herb tea). Investigation: This plant is used to cure a variety of ailments, including colds, influenza, respiratory issues, hypertension, body aches, whooping cough, and cancer . 5.5.7. Heliathemum lipii (L.) Pers . (Family: Cistaceae; Vernacular Name: Samhari) The genus Helianthemum belongs to the family Cistaceae , which are widespread in the Mediterranean region. Across the world, this genus includes 70 species . In Algeria and Pakistan, this genus has a single species Helianthemum lippii (L.) Pers. The vernacular name of this species is different in different regions and continents, for examplef the name of Al Samhari (in the region of Oued Souf: Southeast of Algeria) ; Reguig (in the region of Ouargla: South of Algeria) Tahsowat and Alrjik (South-West of Algeria); Alrkaroq (in Kuwait); Umm Souika (the Arabian Peninsula) ; and Sun Flower (Northeast of Jordan) . This plant is highly intriguing from an ecological and economic standpoint . It belongs to the pastoral plants , plays a key role in the battle against desertification, and contributes to the stability of desertification-prone areas. The species’ powder is used to treat skin rashes . It’s also used in Libya to treat rot and rashes, as well as to prevent illness . In Morocco, this plant with the Bedouins can cause lameness in camels in the name of GAF or Kraft (a type of arthritis). But in reality, the toxicity of this plant has not yet been proven . 5.5.8. Cyperus conglomeratus (Family: Cyperaceae; Vernacular Name: Saad) Cyperus rotundus is one of the best known species of the genus as a medicinal plant, featuring in Indian, Chinese, and Japanese traditional medicines for spasms, stomach and intestinal problems, and menstrual irregularities . Several writers have studied C. rotundus extensively; the most notable essential oils isolated from it are α-pinene, β-pinene, α-copaene, cyperene, cyperotundone, α-cyperone, and caryophyllene oxide . For the reason that the southern coast, eastern south, and central parts of Iran have dry and very hot air for eight months of the year, this species can withstand harsh environmental conditions . This species has been recorded to be used as a pectoral, emollient, diuretic, stimulant, analgesic, and anthelmintic therapy in traditional medicine. 5.5.9. Calligonum comosum L’herit (Family: Polygonaceae; Vernacular Name: Larta) Parts of use: Leaves, roots, boughs (infusion, decoction). Investigation: This plant can be used against scorpion stingsand snake bites , vermifuge. The antibacterial activity of Calligonum comosum L. Her. , “Arta,” a member of the family Polygonaceae, was investigated. It is a tropical and subtropical plant that is widely distributed in the United Arab Emirates and Saudi Arabia. Previous research found that an ethanolic extract of Calligonum comosum aerial parts greatly decreased the rise in carrageenan-induced hind paw oedema in rats. Furthermore, a pre-treatment with the extract inhibited the acute stomach ulcers caused by phenylbutazone and indomethacin in a dose-dependent manner . Recently, researchers discovered that extracts from several Calligonum comosum plant sections have significant antibacterial activity against four harmful bacteria . Anthraquinones and flanovonids are the most frequent chemical components of Arta, according to prior investigations. In addition, when Calligonum comosum was treated with organic solvents, dehydrodicatechin was extracted, which showed cytotoxic and antioxidant activities . 5.5.10. Plantago albicans L. (Family: Plantaginaceae; Vernacular Name: Linem) Is an herbaceous plant that grows wild in subtropical and temperate climates and may be grown readily in Tunisia, Algeria, and Libya. The extracts of Plantago species are often used in traditional medicine due to their hepatoprotective , analgesic, anti-inflammatory, and antipyretic properties. Furthermore, P. albicans caused structural and functional changes in liver and heart tissue . In fact, this genus contains a high amount of primary and secondary metabolites . 5.5.11. Limoniastrum guyonianum Coss & Dur. (Family: Plumbaginaceae; Vernacular Name: Zita) Investigation: This plant can be used as a treatment for several diseases, including: diabetes, scorpion stings and snake bites, headaches, constipation, hypertension, renal illness, anemia, antiseptics, burns, leprosy, wounds, ulcers, diabetes, anemia, cough, constipation, gas, obesity, tonsillitis, and flu . 5.5.12. Tamarix boveana (Family: Tamaricaceae; Vernacular Name: Tarfa) Investigation: This plant can be used as a treatment for several diseases, including: cough, hemorrhage, diuretic, antiseptic, leprosy, injuries and ulcers, scorpions and insect bites, renal diseases, diarrhea, anemia, gum and mouth inflammation, gastric ulcer, cephalalgia, hypertension, diabetes, joint disease, and pancreatic inflammation . 5.5.13. Traganum nudatum Del. (Family: Chenopodiaceae; Vernacular Name: Damran) Parts of use: Leaves (compressed maceration, powder, and ointment). Investigation: Rheumatism, skin diseases, diarrhea, rheumatism wound dermatoses . This plant is well renowned for its tasty fruit as well as its wood for burning . 5.5.14. Bassia muricata L. Asch. (Family: Chenopodiaceae; Vernacular Name: Ghabitha) Investigation: Analgesic, antiseptic, and anti-inflammatory. It is a rich plant with triterpenoids and saponins . It grows in sandy environments in North Africa, the East Mediterranean region, Sinai, Saudi Arabia, and Iran as an annual plant . It is a significant plant in traditional medicine, where it is used as an analgesic, antipyretic, and nephrotic. It also contains various biological properties, including antioxidants . The extract of B. muricata has been shown to lower white blood cell counts, enhance prothrombin time, reduce blood pressure, and have antibacterial properties . Flavonoid glycosides and other phytochemical components of B. muricata have been isolated and identified . 5.5.15. Atriplex halimus L. (Family: Chenopodiaceae; Vernacular Name: Gatef) Investigation: this plant can be used as a treatment for several diseases, including: cysts in the uterus, diabetes, stomach pain, constipation, diarrhea, gas, hypertension, antiseptic, burns, fever, anemia, otitis, rheumatism, diuretic, vermifuge, vomiting, wounds and ulcers, tonsillitis, gallbladder disease, fortify the gums, infertility, prostate, fall of placenta, nephrolithiasis, hypercholesterolemia . Streptozotocin-induced diabetic rats showed both long-term and short-term antidiabetic effects. The aqueous extract was given orally at a dose of 200 mg/kg BW for 30 days to rats divided into four groups: normal rats, diabetic rats (50 mg/kg BW of streptozotocin), and diabetic and normal rats treated with 200 mg/kg BW in the short-term study after administration of a glucose dose of 3 g/kg . When compared with the results before treatment, the aqueous extract resulted in a substantial reduction (54%) in blood glucose in the treated diabetic group at the end of the trial. The plant extract also has an antihyperglycemic effect, lowering diabetic rats’ fasting blood glucose levels by 23 and 41% after 2 and 3 h, respectively . The aqueous extract is high in tannins and flavonoids, which the researchers believe may have an effect on the pancreas via increasing beta cell insulin production. 5.5.16. Zygophyllum album L. (Family: Zygophyllaceae; Vernacular Name: Agga) Parts of use: Leaves, stems, and fruits (Decoction, powder, ointment). Investigation : Diabetes, purgatives and laxatives, anti-viruses and fungi, indigestion. This plant can be used to treat diabetes, indigestion, skin problems, as an analgesic, and as a disinfectant, according to . This herb is utilized in Tunisian traditional medicine as a rheumatism, gout, and asthma treatment . It’s also a diuretic, a local anesthetic, an antihistaminic, and an antidiabetic . 5.5.17. Matricaria pubescens (Desf) (Family: Asteraceae; Vernacular Name: Guartoufa) Parts of use: Leaves (Powder). Investigation: It is used as a treatment for: scorpion stings and snake bites, colds and respiratory difficulties, bleeding, diuretics, fever, astringents and stimulants, stomach and stomach aches, and constipation. This herb is used to treat scorpion stings and snake bites in the Oued Righ area (El Oued, Algeria) . 5.5.18. Launaea resedifolia O. K. (Family: Asteraceae; Vernacular Name: Athid) The Launaea resedifolia O. K family is one of the biggest angiosperm families, with around 340 genera and 3350 species divided into 11 tribes. They are found all over the world, primarily in temperate parts of the northern hemisphere . The Irano-Turanian, Mediterranean, and Saharao-Sindian areas are the family’s primary distribution hubs. 5.5.19. Solanum nigrum L. (Family: Solanaceae; Vernacular Name: Anb Aldib) Parts of use: Leaf, stem, and fruits are the parts of the plant that are used. It is a poisonous plant that is classified as active and hazardous in the pharmacopoeia. It is for external use . Investigation: It is used as a treatment for: diuretic, chronic liver enlargement, diarrhea, and piles; also effective against skin illness and anthrax. Fruits are used to treat a variety of ailments, including heart disease, hiccups, asthma, fever, bronchitis, and diarrhea. Ringworm can be treated using green fruit pastes. Fruit juice is an expectorant and cooling drink that can be used to treat fevers, gonorrhea, giddiness, and inflammations . 5.5.20. Erodium glaucophyllum L. Her. (Family: Geraniaceae; Vernacular Name: Tommir) Parts of use: All its parts are useful. Investigation: It is used as a treatment for: diarrhea, colds, influenza, and respiratory system disorders are investigated. It’s a medicinal herb that’s good for diarrhea, an astringent, allergies, and oxytocin . Erodium glaucophyllum L. is a common herb in the Nile valley, the western Mediterranean coastal region, and the deserts, and goes by the Arabic names Kahkul, Lesan Hamad, Kabshia, Ragma, Dahma, Murrar, and Tamir. 5.5.21. Cleome arabica L. (Family: Capparidaceae; Vernacular Name: Nettin) Parts of use: Leaves (Infusion, maceration). Investigation: Rheumatism, urinary tract. It is a plant that is high in flavone chemicals, particularly flavonoids, and is diuretic. It can also help with rhumatism, arthritis, and diarrhea . 5.5.22. Neurada procumbens L. (Family: Rosaceae; Vernacular Name: Anfal/Saadan) Parts of use : Leaves, seeds and fruits. Investigation: Analgesic, antiseptic, anti-inflammatory, astringent, and stimulant. The Bedouin have always regarded them as edible medical plants . The Bedouins see it as a sort of camel meal that is both safe and edible. The herb has also been used to cure diarrhea and dysentery in traditional Arabic medicine. It’s also been utilized to boost heart and respiratory processes as a stimulant . Its water extract made the mice’s blood pressure rise. Only one study examined the chemical constituents of this plant, which revealed that it contains alkaloids, flavonoids, saponins, sterols and terpenes, volatile oils, and tannins, as well as a hypertensive effect of its ethanol extract, indicating that cardiovascular patients should exercise caution when using this plant . It was also used to cure stroke in people and animals in Pakistan, and its dried star-shaped fruits may be mixed with rose water in the summer as a cooling agent and with dry nuts in the winter to stimulate nerves . In addition to dihydroflavonol glycosides, prior findings in Egypt suggest that this plant contains polysaccharides (gum, mucilage) . On the other hand, mineral monitoring, particularly for toxic elements, is one of the most important aspects of ensuring the quality of medicinal plants because elements such as Fe, Ca, and Zn play an important role in many processes of human metabolism and thus contribute significantly to human health , as well as plant growth processes, even in small amounts. Retama retam Webb. (Family: Fabaceae; Vernacular Name: Rtem) Useful parts : Aerial part (Infusion, powder, compressed herbal). Investigation: Analgesic, antiseptic, and anti-inflammatory. The stem of this plant is utilized in cauterization in traditional medicine. It can also help with rheumatism, scorpion stings, and injuries. Despite their obvious relevance in traditional medicine, the number of studies on chemical components with biological activities (therapeutic virtues) on the Retama retam Webb. , particularly in Algeria, is sparse compared with other herbs . Selaimia A et al., (2020) assessed antioxidant activity using the DPPH technique. The findings of the DPPH activities indicated that the extracts of the Retama had the most antioxidant activity, with the ethyl acetate extract of the leaves having the highest antioxidant activity. According to the antimicrobial activity research, the extracts have a varied activity that varies depending on the strain examined. It has been found to be ineffective against meticillin-resistant Staphylococcus aureus, although it is extremely sensitive to Candida albican . Another investigation found that the extracts of these plants, dried in various ways, exhibit antibacterial activity suppression for both bacterial strains ( Escherichia coli and Pseudomonas aeruginosa ). Retama retam , on the other hand, was shown to have limited antibacterial action against Pseudomonas aeruginosa and high antibacterial activity against other bacteria . These findings suggest that the herb might be useful in the treatment of viral infections. Their ability to distinguish between different microbes. Astragalus cruciatus Link (Family: Fabaceae; Vernacular Name: Akifa) Astragalus cruciatus Link. belongs to the genus Astragalus L. of the family Fabaceae. Ithas four synonyms “ A. aristidis Coss .”, “ A. radiatus Ehrenb. ”, “ A. trabutianus Batt. ”, and A. corrugates Bertol . Species of the Astragalus genus are used as therapeutic herbs in traditional medicine all throughout the world to treat stomach ulcers, cough, chronic bronchitis, hypertension, gynecological problems, diabetes, and scorpion poisonous stings . Immunostimulant, cardiovascular, and antiviral properties have been found in plants from the same genus . Saponin, phenolic, and polysaccharide chemicals are the physiologically active ingredients of Astragalus species, while thenitro-toxins, imidazoline alkaloids, and selenium derivatives are poisonous components . The Astragalus genus is well-known for its abundance of bioactive secondary metabolites. Saponins and flavonoids have been isolated and characterized in previous phytochemical studies of several Astragalus species. However, there has never been any constitutional or pharmacological research on Astragalus cruciatus Link. Genista saharae (Coss. & Dur.) (Family: Fabaceae; Vernacular Name: Marekh) Investigation: Cold, influenza, respiratory system problems. It contains flavonoids compounds . Genista saharae is a leafless, spontaneous fabaceae that is colloquially called as “Tellegit” in Algeria. For the reason that it is known as a source of chemical components with antioxidant properties, this medicinal plant is used in traditional pharmacopeia. Sofiane Guettaf’s research focuses on examining the phytochemical content and assessing the antioxidant activity of aerial portions of an aqueous extract of Genista saharae (AEG) under settings similar to its traditional use. The qualitative chemical composition of AEG was assessed via phytochemical screening utilizing precipitation and coloring reactions. Furthermore, spectrophotometric techniques were used to determine the total phenolics, flavonoids, tannins, and carotene contents. Finally, the antioxidant properties of AEG were determined using three different methods: DPPH, reducing power, and β carotene bleaching tests. Phénolic compounds, flavonoids, alkaloids, tannins, terpenoids, glycosides, steroids, and saponins are among the biomolecules found in AEG, according to the findings. Furthermore, the quantitative examination reveals a significant amount of total phenolics, tannins, and β carotene. In sum, EAG has a strong antioxidant activity against β carotene bleaching, which validates its usage by traditional healers. As a result, Genista saharae is an excellent antioxidant source. The presence of tannins or other phenolic chemicals such as terpenoids, β carotene, and saponins may explain its high antioxidant activity . The chemical composition and antioxidant activity of this genus’ alcoholic extracts have been studied before . Unfortunately, there is presently no phytochemical or biological information regarding this shrub. There is no information in the literature on the antioxidant effects of its aqueous extract. As a result, all of the Genista saharae findings are consistent with its traditional use. Astragalus gyzensis Bunge (Family: Fabaceae; Vernacular Name: Foul Alibil) Investigation: Scorpion stings and snake bites. According to ref. , this plant is used to treat snake bites and is a highly hardy plant in the Saharan climate, which is characterized by a variety of stresses. Animals find this plant to be moderately appealing, especially when it is in blossom. Volatiles, which are constantly released into the atmosphere, can account for this. Many studies involving the essential oils of plants have been conducted in recent years for the purpose of discovering natural products and anti-disease actives. Essential oils contain a variety of biological activities, including larvicidal efficacy against mosquitos . The essential oil may be utilized to produce natural pesticides and help reduce the negative effects of synthetic goods, including buildup, resistance, and contamination. Euphorbia guyoniana Boiss. & Reut (Family: Euphorbiaceae; Vernacular Name: Lebina) Investigation: Diarrhea, skin diseases, scorpion stings and snake bites. This plant, similar to many euphorbias, is extremely poisonous due to the presence of toxic white latex. The nomads, on the other hand, utilize it to protect themselves from snake bites . Euphorbia guyoniana Boiss. and Reut. , a member of the Euphorbiaceae family, was the plant studied in this study. This species may be found across Algeria’s desert and pre-desert areas . It’s called “Lebbina” in the area and grows 30–100 cm tall with upright and branching stems that produce very poisonous white latex. More than 2000 species of Euphorbia have been used in traditional medicine to treat skin problems, including dermatitis, gastrointestinal issues, bacterial or fungal infections, and even some forms of cancer. Algerian Sahara nomads also utilize E. guyoniana to protect themselves against snake bites . It has significant antioxidant capabilities, according to a recent study . The high amount of secondary chemicals in E. guyoniana , such as terpenoids, alkaloids, and flavonoids, contributes to its positive benefits. Ephedra alata DC. (Family: Ephedraceae; Vernacular Name: Alanda) Parts of use: Leaves and boughs (Maceration, inhalation, herb tea). Investigation: This plant is used to cure a variety of ailments, including colds, influenza, respiratory issues, hypertension, body aches, whooping cough, and cancer . Heliathemum lipii (L.) Pers . (Family: Cistaceae; Vernacular Name: Samhari) The genus Helianthemum belongs to the family Cistaceae , which are widespread in the Mediterranean region. Across the world, this genus includes 70 species . In Algeria and Pakistan, this genus has a single species Helianthemum lippii (L.) Pers. The vernacular name of this species is different in different regions and continents, for examplef the name of Al Samhari (in the region of Oued Souf: Southeast of Algeria) ; Reguig (in the region of Ouargla: South of Algeria) Tahsowat and Alrjik (South-West of Algeria); Alrkaroq (in Kuwait); Umm Souika (the Arabian Peninsula) ; and Sun Flower (Northeast of Jordan) . This plant is highly intriguing from an ecological and economic standpoint . It belongs to the pastoral plants , plays a key role in the battle against desertification, and contributes to the stability of desertification-prone areas. The species’ powder is used to treat skin rashes . It’s also used in Libya to treat rot and rashes, as well as to prevent illness . In Morocco, this plant with the Bedouins can cause lameness in camels in the name of GAF or Kraft (a type of arthritis). But in reality, the toxicity of this plant has not yet been proven . Cyperus conglomeratus (Family: Cyperaceae; Vernacular Name: Saad) Cyperus rotundus is one of the best known species of the genus as a medicinal plant, featuring in Indian, Chinese, and Japanese traditional medicines for spasms, stomach and intestinal problems, and menstrual irregularities . Several writers have studied C. rotundus extensively; the most notable essential oils isolated from it are α-pinene, β-pinene, α-copaene, cyperene, cyperotundone, α-cyperone, and caryophyllene oxide . For the reason that the southern coast, eastern south, and central parts of Iran have dry and very hot air for eight months of the year, this species can withstand harsh environmental conditions . This species has been recorded to be used as a pectoral, emollient, diuretic, stimulant, analgesic, and anthelmintic therapy in traditional medicine. Calligonum comosum L’herit (Family: Polygonaceae; Vernacular Name: Larta) Parts of use: Leaves, roots, boughs (infusion, decoction). Investigation: This plant can be used against scorpion stingsand snake bites , vermifuge. The antibacterial activity of Calligonum comosum L. Her. , “Arta,” a member of the family Polygonaceae, was investigated. It is a tropical and subtropical plant that is widely distributed in the United Arab Emirates and Saudi Arabia. Previous research found that an ethanolic extract of Calligonum comosum aerial parts greatly decreased the rise in carrageenan-induced hind paw oedema in rats. Furthermore, a pre-treatment with the extract inhibited the acute stomach ulcers caused by phenylbutazone and indomethacin in a dose-dependent manner . Recently, researchers discovered that extracts from several Calligonum comosum plant sections have significant antibacterial activity against four harmful bacteria . Anthraquinones and flanovonids are the most frequent chemical components of Arta, according to prior investigations. In addition, when Calligonum comosum was treated with organic solvents, dehydrodicatechin was extracted, which showed cytotoxic and antioxidant activities . Plantago albicans L. (Family: Plantaginaceae; Vernacular Name: Linem) Is an herbaceous plant that grows wild in subtropical and temperate climates and may be grown readily in Tunisia, Algeria, and Libya. The extracts of Plantago species are often used in traditional medicine due to their hepatoprotective , analgesic, anti-inflammatory, and antipyretic properties. Furthermore, P. albicans caused structural and functional changes in liver and heart tissue . In fact, this genus contains a high amount of primary and secondary metabolites . Limoniastrum guyonianum Coss & Dur. (Family: Plumbaginaceae; Vernacular Name: Zita) Investigation: This plant can be used as a treatment for several diseases, including: diabetes, scorpion stings and snake bites, headaches, constipation, hypertension, renal illness, anemia, antiseptics, burns, leprosy, wounds, ulcers, diabetes, anemia, cough, constipation, gas, obesity, tonsillitis, and flu . Tamarix boveana (Family: Tamaricaceae; Vernacular Name: Tarfa) Investigation: This plant can be used as a treatment for several diseases, including: cough, hemorrhage, diuretic, antiseptic, leprosy, injuries and ulcers, scorpions and insect bites, renal diseases, diarrhea, anemia, gum and mouth inflammation, gastric ulcer, cephalalgia, hypertension, diabetes, joint disease, and pancreatic inflammation . Traganum nudatum Del. (Family: Chenopodiaceae; Vernacular Name: Damran) Parts of use: Leaves (compressed maceration, powder, and ointment). Investigation: Rheumatism, skin diseases, diarrhea, rheumatism wound dermatoses . This plant is well renowned for its tasty fruit as well as its wood for burning . Bassia muricata L. Asch. (Family: Chenopodiaceae; Vernacular Name: Ghabitha) Investigation: Analgesic, antiseptic, and anti-inflammatory. It is a rich plant with triterpenoids and saponins . It grows in sandy environments in North Africa, the East Mediterranean region, Sinai, Saudi Arabia, and Iran as an annual plant . It is a significant plant in traditional medicine, where it is used as an analgesic, antipyretic, and nephrotic. It also contains various biological properties, including antioxidants . The extract of B. muricata has been shown to lower white blood cell counts, enhance prothrombin time, reduce blood pressure, and have antibacterial properties . Flavonoid glycosides and other phytochemical components of B. muricata have been isolated and identified . Atriplex halimus L. (Family: Chenopodiaceae; Vernacular Name: Gatef) Investigation: this plant can be used as a treatment for several diseases, including: cysts in the uterus, diabetes, stomach pain, constipation, diarrhea, gas, hypertension, antiseptic, burns, fever, anemia, otitis, rheumatism, diuretic, vermifuge, vomiting, wounds and ulcers, tonsillitis, gallbladder disease, fortify the gums, infertility, prostate, fall of placenta, nephrolithiasis, hypercholesterolemia . Streptozotocin-induced diabetic rats showed both long-term and short-term antidiabetic effects. The aqueous extract was given orally at a dose of 200 mg/kg BW for 30 days to rats divided into four groups: normal rats, diabetic rats (50 mg/kg BW of streptozotocin), and diabetic and normal rats treated with 200 mg/kg BW in the short-term study after administration of a glucose dose of 3 g/kg . When compared with the results before treatment, the aqueous extract resulted in a substantial reduction (54%) in blood glucose in the treated diabetic group at the end of the trial. The plant extract also has an antihyperglycemic effect, lowering diabetic rats’ fasting blood glucose levels by 23 and 41% after 2 and 3 h, respectively . The aqueous extract is high in tannins and flavonoids, which the researchers believe may have an effect on the pancreas via increasing beta cell insulin production. Zygophyllum album L. (Family: Zygophyllaceae; Vernacular Name: Agga) Parts of use: Leaves, stems, and fruits (Decoction, powder, ointment). Investigation : Diabetes, purgatives and laxatives, anti-viruses and fungi, indigestion. This plant can be used to treat diabetes, indigestion, skin problems, as an analgesic, and as a disinfectant, according to . This herb is utilized in Tunisian traditional medicine as a rheumatism, gout, and asthma treatment . It’s also a diuretic, a local anesthetic, an antihistaminic, and an antidiabetic . Matricaria pubescens (Desf) (Family: Asteraceae; Vernacular Name: Guartoufa) Parts of use: Leaves (Powder). Investigation: It is used as a treatment for: scorpion stings and snake bites, colds and respiratory difficulties, bleeding, diuretics, fever, astringents and stimulants, stomach and stomach aches, and constipation. This herb is used to treat scorpion stings and snake bites in the Oued Righ area (El Oued, Algeria) . Launaea resedifolia O. K. (Family: Asteraceae; Vernacular Name: Athid) The Launaea resedifolia O. K family is one of the biggest angiosperm families, with around 340 genera and 3350 species divided into 11 tribes. They are found all over the world, primarily in temperate parts of the northern hemisphere . The Irano-Turanian, Mediterranean, and Saharao-Sindian areas are the family’s primary distribution hubs. Solanum nigrum L. (Family: Solanaceae; Vernacular Name: Anb Aldib) Parts of use: Leaf, stem, and fruits are the parts of the plant that are used. It is a poisonous plant that is classified as active and hazardous in the pharmacopoeia. It is for external use . Investigation: It is used as a treatment for: diuretic, chronic liver enlargement, diarrhea, and piles; also effective against skin illness and anthrax. Fruits are used to treat a variety of ailments, including heart disease, hiccups, asthma, fever, bronchitis, and diarrhea. Ringworm can be treated using green fruit pastes. Fruit juice is an expectorant and cooling drink that can be used to treat fevers, gonorrhea, giddiness, and inflammations . Erodium glaucophyllum L. Her. (Family: Geraniaceae; Vernacular Name: Tommir) Parts of use: All its parts are useful. Investigation: It is used as a treatment for: diarrhea, colds, influenza, and respiratory system disorders are investigated. It’s a medicinal herb that’s good for diarrhea, an astringent, allergies, and oxytocin . Erodium glaucophyllum L. is a common herb in the Nile valley, the western Mediterranean coastal region, and the deserts, and goes by the Arabic names Kahkul, Lesan Hamad, Kabshia, Ragma, Dahma, Murrar, and Tamir. Cleome arabica L. (Family: Capparidaceae; Vernacular Name: Nettin) Parts of use: Leaves (Infusion, maceration). Investigation: Rheumatism, urinary tract. It is a plant that is high in flavone chemicals, particularly flavonoids, and is diuretic. It can also help with rhumatism, arthritis, and diarrhea . Neurada procumbens L. (Family: Rosaceae; Vernacular Name: Anfal/Saadan) Parts of use : Leaves, seeds and fruits. Investigation: Analgesic, antiseptic, anti-inflammatory, astringent, and stimulant. The Bedouin have always regarded them as edible medical plants . The Bedouins see it as a sort of camel meal that is both safe and edible. The herb has also been used to cure diarrhea and dysentery in traditional Arabic medicine. It’s also been utilized to boost heart and respiratory processes as a stimulant . Its water extract made the mice’s blood pressure rise. Only one study examined the chemical constituents of this plant, which revealed that it contains alkaloids, flavonoids, saponins, sterols and terpenes, volatile oils, and tannins, as well as a hypertensive effect of its ethanol extract, indicating that cardiovascular patients should exercise caution when using this plant . It was also used to cure stroke in people and animals in Pakistan, and its dried star-shaped fruits may be mixed with rose water in the summer as a cooling agent and with dry nuts in the winter to stimulate nerves . In addition to dihydroflavonol glycosides, prior findings in Egypt suggest that this plant contains polysaccharides (gum, mucilage) . On the other hand, mineral monitoring, particularly for toxic elements, is one of the most important aspects of ensuring the quality of medicinal plants because elements such as Fe, Ca, and Zn play an important role in many processes of human metabolism and thus contribute significantly to human health , as well as plant growth processes, even in small amounts. There are many plants with therapeutic potential, as has become evident over the past few decades. It is also commonly recognised that traditional medicinal plants may be able to provide prospective template molecules for the development of new drugs. Numerous of the plants included here show very potential therapeutic properties, suggesting that more clinical research should be completed on them. However, only a few of them have compelling scientific and clinical support . Considerable work should be put into finding and describing the bioactive components present in these plants, given the state of scientific understanding . It is a challenging and potentially sophisticated medical process to identify therapeutic components in traditional plant-based remedies . There is still a dearth of literature resulting from the last decade’s investigations addressing procedures to be adopted for quality assurance, authentication, and standardization of crude plant products, despite ongoing thorough and mechanism-oriented evaluations of medicinal plants from the Saharan region’s flora. The formulation of the finished product, extraction techniques, and proper raw material management may all contribute to achieving the desired consistency . In fact, it has been acknowledged that one of the main obstacles to the formation of a modern phytomedicine business in the Saharan region is the lack of proper validation of traditional knowledge, as well as technological needs and quality control standards. For buyers, both domestic and foreign, this makes assessing the efficacy and safety of plants and extracts, as well as contrasting batches of commodities from different locations or years, extremely challenging . Potential safety issues, such as the contamination of medicinal plant products with heavy metals from traditional medical supplies from the Saharan region, must also be addressed, and regulatory regulations must be properly developed and put into place. By using controlled settings for growth (under GACP) and processing, contamination of medicinal plant material must be kept to a minimum (under Good Manufacturing Practice). For the reason that it is simpler to regulate the supply chain and contamination is limited, cultivated plant material is chosen in the medicinal plant sector . On the other hand, correct identification of medicinal plant material is critical to the quality control process; the source of the plant material must be established unambiguously. Following that, during the material’s processing phases, microbiological contamination (fungal, bacterial, and any possible human diseases) must be verified. Chemical, pharmacological, and toxicological assessments, completed in accordance with Good Laboratory Practices (GLPs) principles, will validate the bioactive characteristics of the material during processing . These tests are routinely used to predict the safety of newly produced products, where clinical safety and efficacy must be established in lengthy, in-depth investigations during the early stages of a medicinal agent’s development. The unit dosage forms generated after that will be regarded as safe as long as the standard operating procedures are followed. Regardless, quality assurance processes must be implemented to ensure that the factory’s goods are of excellent quality, safety, and efficacy . Alkaloids and flavonoids have well-known antimicrobial and spasmolytic properties . In contrast to alkaloids, catechic and gallic tannins, flavonoids, and saponins are abundant in organic extracts of P. lentiscus leaves, according to research by Barbouchi . The A. herba-alba is abundant in compound phenolics, flavonoids, tannins, and anthocyanins, as shown by Khlifi’s study . According to Najafi et al. , the ethanolic extract of C. colocynth seeds includes tannins, alkaloids, flavonoids, and saponins, whereas Benariba et al. found that catechic tannins and flavonoids are plentiful in hydromethanol extracts. These variations in outcomes may be related to variations in the harvesting region, soil composition, climate, harvest season, solvents, and experimental extraction settings. The phenolic acid derivatives and polyphenolic components, primarily flavonoid glycosides, are the primary biological components of most of the studied therapeutic plants and herbs . Flavonoids are so-called secondary plant chemicals that have a variety of physiological and pharmacological effects (see ) . They possess diverse biological properties such as antioxidant, antiageing, anti-carcinogen, anti-inflammatory, anti-atherosclerosis, cardioprotective and improved endothelial function . The majority of these biological effects are thought to be due to their inherent decreasing capacities. They may also provide indirect protection by triggering endogenous defense mechanisms and altering physiological processes . Phytosterols are another category of chemicals found in these plants and herbs. The most notable of their bioactivities is their ability to decrease blood cholesterol by partially inhibiting intestinal cholesterol absorption . Possible antiatherogenic action is one of the claimed benefits of phytosterols, as well as the immune boosting and anti-inflammatory activities carried out mostly by beta-sitosterol, which are all claimed advantages of phytosterols. Furthermore, there is growing evidence that certain of these plants and herbs’ sterols may have especially protective benefits against the development of various malignancies, including colorectal, prostate, and breast cancers . It’s unclear if mechanisms other than phytosterols’ well-known cholesterol-lowering effect may also play a role in these possible health advantages . 7.1. Antioxidant and Detoxicating Activity A molecule known as an antioxidant is one that can slow down or prevent the oxidation of other molecules. In the chemical process of oxidation, electrons are moved from a substance to an oxidizing agent . Oxidation activities generate free radicals, which trigger a cascade of detrimental cell-damaging events. Antioxidants can work as free radical scavengers, helping to eliminate dangerous free radicals and their byproducts while also inhibiting further oxidation reactions by being oxidized. Several factors, some of which are depicted in , influence how effective antioxidants are. Antioxidants such as thiols or polyphenols are therefore commonly utilized as reducing agents . According to the radical hypothesis of human physiology, active free radicals are involved in almost every cellular breakdown process that results in cell death. Oxidative stress is thought to play a role in a number of chronic and degenerative diseases, including cancer, autoimmune disorders, aging, cataracts, rheumatoid arthritis, cardiovascular diseases, and neurological disorders . In the majority of the plants analyzed, phenolic components have been linked in several studies to a significant degree of antioxidant bioactivity. The relationship between these two characteristics, however, is not entirely evident . 7.2. Anti-Inflammatory Activity Inflammation is a physiological response to physical or biological substances causing harm to tissues or cells, comprising a variety of responses aimed at removing the source and repairing the damage. Polyphenols are one of the most common types of phytochemicals with anti-inflammatory characteristics, and many plants with polyphenols as secondary metabolites have been shown to have potential anti-inflammatory capabilities . 7.3. Benign Prostatic Hyperplasia A burning sensation, a strong and frequent urge to urinate, discomfort in the lower back or abdomen, and difficulty urinating can all be symptoms of prostatitis or prostate inflammation. The most important use of plants or herbs in medicine is for the preventative and curative treatment of prostate disorders. An enlarged prostate is known as benign prostatic hyperplasia (BPH). BPH is caused by non-cancerous growths inside the prostate. Chronic prostatitis is particularly frequent in senior men, possibly due to hormonal changes and aging. It is not surprising that more people are turning to phytotherapy and other alternative treatments as opposed to pharmaceutical therapies because they have fewer side effects than antibiotics. 7.4. Antidiabetic Activity Aqueous extracts of leaves from numerous plants have been found to help reduce blood glucose levels. Additionally, it exhibited a sizable hypolipidemic effect, bringing down blood triglyceride and total cholesterol levels. The plant extracts outperformed the synthetic drug metformin significantly in terms of antihyperglycemia and antihypertriacyl-glycerolaemia. The results also suggest that the plant extract could be used to treat type 2 diabetes and the associated dyslipidemia. These polyphenolic compounds act as monomers or oligomers in epididymal fat cells, enhancing insulin activity in vitro and demonstrating insulin-like action as well as an antioxidant impact in vitro . 7.5. Antiviral Effect For the reason that viruses have defied prevention or therapy longer than any other form of life, infectious viral illnesses continue to be a serious danger to public health. Traditional folk medicine utilizes medicinal plants to treat a variety of illnesses, including infectious infections. At 1000 g/mL with Rf 104, hydro-alcoholic extracts of several medicinal plants were shown to have virucidal action against the herpes simplex-1 virus (HSV). The capacity of plant extract dilutions to suppress the generated cytopathogenic effect (CPE) is represented as a reduction factor (Rf) of the virus titer in an antiviral bioassay. The study was carried out by the University of Bristol in the UK using a technique called end point titration technique . 7.6. Antineoplastic Properties Only evaluating the effect on cell viability, as some of the reports included in our review did, is not a valuable report because cell cultures are highly susceptible to environmental factors such as temperature and osmotic pressure. Another reason for the low reliability of in vitro antineoplastic studies is that in a living organism, the plant extract is exposed to several other cells besides the target tissue; thus, the extract may be toxic to normal tissues as well, which is one of the main reasons why several agents have been barred from clinical use as anticancer agents. Another issue is that malignant cells generally acquire resistance to anticancer medicines after prolonged exposure, which is one of the main reasons why researchers are continuously looking for novel anticancer treatments. As a result, in vitro antineoplastic activity does not imply anticancer activity, and more data, such as antitumor characteristics in tumor-bearing animals, is needed to determine whether a plant is worthy of further investigation as a source of anticancer drugs . A molecule known as an antioxidant is one that can slow down or prevent the oxidation of other molecules. In the chemical process of oxidation, electrons are moved from a substance to an oxidizing agent . Oxidation activities generate free radicals, which trigger a cascade of detrimental cell-damaging events. Antioxidants can work as free radical scavengers, helping to eliminate dangerous free radicals and their byproducts while also inhibiting further oxidation reactions by being oxidized. Several factors, some of which are depicted in , influence how effective antioxidants are. Antioxidants such as thiols or polyphenols are therefore commonly utilized as reducing agents . According to the radical hypothesis of human physiology, active free radicals are involved in almost every cellular breakdown process that results in cell death. Oxidative stress is thought to play a role in a number of chronic and degenerative diseases, including cancer, autoimmune disorders, aging, cataracts, rheumatoid arthritis, cardiovascular diseases, and neurological disorders . In the majority of the plants analyzed, phenolic components have been linked in several studies to a significant degree of antioxidant bioactivity. The relationship between these two characteristics, however, is not entirely evident . Inflammation is a physiological response to physical or biological substances causing harm to tissues or cells, comprising a variety of responses aimed at removing the source and repairing the damage. Polyphenols are one of the most common types of phytochemicals with anti-inflammatory characteristics, and many plants with polyphenols as secondary metabolites have been shown to have potential anti-inflammatory capabilities . A burning sensation, a strong and frequent urge to urinate, discomfort in the lower back or abdomen, and difficulty urinating can all be symptoms of prostatitis or prostate inflammation. The most important use of plants or herbs in medicine is for the preventative and curative treatment of prostate disorders. An enlarged prostate is known as benign prostatic hyperplasia (BPH). BPH is caused by non-cancerous growths inside the prostate. Chronic prostatitis is particularly frequent in senior men, possibly due to hormonal changes and aging. It is not surprising that more people are turning to phytotherapy and other alternative treatments as opposed to pharmaceutical therapies because they have fewer side effects than antibiotics. Aqueous extracts of leaves from numerous plants have been found to help reduce blood glucose levels. Additionally, it exhibited a sizable hypolipidemic effect, bringing down blood triglyceride and total cholesterol levels. The plant extracts outperformed the synthetic drug metformin significantly in terms of antihyperglycemia and antihypertriacyl-glycerolaemia. The results also suggest that the plant extract could be used to treat type 2 diabetes and the associated dyslipidemia. These polyphenolic compounds act as monomers or oligomers in epididymal fat cells, enhancing insulin activity in vitro and demonstrating insulin-like action as well as an antioxidant impact in vitro . For the reason that viruses have defied prevention or therapy longer than any other form of life, infectious viral illnesses continue to be a serious danger to public health. Traditional folk medicine utilizes medicinal plants to treat a variety of illnesses, including infectious infections. At 1000 g/mL with Rf 104, hydro-alcoholic extracts of several medicinal plants were shown to have virucidal action against the herpes simplex-1 virus (HSV). The capacity of plant extract dilutions to suppress the generated cytopathogenic effect (CPE) is represented as a reduction factor (Rf) of the virus titer in an antiviral bioassay. The study was carried out by the University of Bristol in the UK using a technique called end point titration technique . Only evaluating the effect on cell viability, as some of the reports included in our review did, is not a valuable report because cell cultures are highly susceptible to environmental factors such as temperature and osmotic pressure. Another reason for the low reliability of in vitro antineoplastic studies is that in a living organism, the plant extract is exposed to several other cells besides the target tissue; thus, the extract may be toxic to normal tissues as well, which is one of the main reasons why several agents have been barred from clinical use as anticancer agents. Another issue is that malignant cells generally acquire resistance to anticancer medicines after prolonged exposure, which is one of the main reasons why researchers are continuously looking for novel anticancer treatments. As a result, in vitro antineoplastic activity does not imply anticancer activity, and more data, such as antitumor characteristics in tumor-bearing animals, is needed to determine whether a plant is worthy of further investigation as a source of anticancer drugs . Medicinal plants used to cure humans have bestowed a plethora of herbal remedies on the Sahara, which local people acquire, maintain, and pass on to the next generation . The widespread usage of traditional medicine in Hot Arid Regions, which is mostly comprised of medicinal plants, has been connected to cultural and economic factors. As a result, the World Health Organization (WHO) urges member desert nations to promote and incorporate traditional medicinal practices into their health systems . Plants generally include a variety of phytochemicals, also known as secondary metabolites, that can benefit health singly, additively, or synergistically . Indeed, unlike pharmacological medicines, medicinal plants frequently have multiple compounds acting together catalytically and synergistically to generate a combined impact that is greater than the sum of the individual components’ activities . By speeding up or slowing down the absorption of the primary therapeutic component, the combined activities of these drugs tend to boost its activity. Secondary metabolites derived from plants may improve the stability of active compounds or phytochemicals, reduce the occurrence of unwanted side effects, and have an additive, potentiating, or antagonistic impact . It has been proposed that the enormous diversity of chemical structures found in these plants are specialized secondary metabolites involved in the organism’s relationship with the environment, such as pollinator attractants, signal products, defensive substances against predators and parasites, or pest and disease resistance . Bitter substances that aid in the removal of waste products and pollutants, anti-inflammatory compounds that reduce swelling and pain, phenolic compounds that act as antioxidants and venotonics, antibacterial and antifungal tannins that act as natural antibiotics, and diuretic substances that aid in the removal of waste products and pollutants, as well as alkaloids that improve mood and provide a sense of well-being, are just a few examples . Although some may believe that isolating phytochemicals and using them as single chemical entities is a better option, which has led to the replacement of plant extracts, a growing body of evidence suggests that using crude and/or standardized extracts rather than isolated single compounds may have some medical benefits . Concerns about natural resources, particularly plant species, continue to be a global problem . Unfortunately, the rate of plant biodiversity loss is expected to accelerate . According to available data, the severity of the situation is strongly pronounced in hot areas—the arid desert, as shown by the high number of plant species now under threat (critically endangered, endangered, or vulnerable). Multiple causes contribute to the loss of plant diversity, including habitat degradation, alien species competition, death from imported diseases, pollution, and overexploitation for a variety of purposes, including food, shelter, and medicine . Due to the large number of species, the medicinal use of plants is one of the most important uses of natural resources . Because of their substantial contributions to healthcare, financial income, cultural identity, and livelihood security, the value of medicinal plants is basically unlimited . The Desert African medicinal plant business has recently been under increased attention as a result of rising concerns about the sustainability of wild plants, which are traditionally the primary source of traditional medicines . The first stage in selecting species with conservation or resource management priorities is to gather information on the species that are sold, their prices, and the marketed volumes . However, throughout Africa, a lack of scientific data on these critical components of the medicinal trade is prevalent and continues to be a serious issue . Recent quantitative studies analyzing the quantities of medicinal plants traded have been reported for a few African nations, including South Africa , Ghana , Gabon , Tanzania , and Sierra Leone , as the relevance and utility of such data have grown. The information gained from this research will undoubtedly aid in the assessment of the conservation status of frequently used medicinal species. Previous research has found that wild populations of medicinal plants are preferentially targeted, posing particular challenges for conservationists . Their viability is determined by the kind of vegetation collected, relative abundance, and growth rates . The vulnerability of medicinal plant species to overharvesting is also impacted by the life forms and plant parts utilized. Woody plants (shrubs and trees) make up the majority of the 51 most significant medicinal plants (65%). Sierra Leone , Gabon , South Africa , and Ghana have all documented significant usage of non-sustainable organ harvesting. The removal of wood, bark, bulbs/corms, roots, or complete plants from any medicinal plant usually results in the death of an individual species . On the other hand, harvesting leaves, fruits, or seeds, on the other hand, is typically seen as less harmful to plant survival. Intensive pruning, on the other hand, may have an effect on the reproductive success of such plants. These concerns can be divided into four categories from an ecological standpoint: (1) growth, survival, and reproduction rates; (2) population structure and dynamics; (3) community structure and composition, plant-animal and plant-plant interactions; and (4) nutrient and organic matter dynamics, energy exchange . As a result, while creating and assessing ways to minimize the negative impacts of medicinal plant overharvesting, researchers must consider the aforementioned processes (which correspond to distinct ecological levels). The medicinal plant industry is extensive, comprising collectors/local sellers, and consumers, as well as heavily reliant sectors such as nutraceutical and pharmaceutical firms that require enormous volumes of raw material . As a result, the existing conservation techniques have been impacted by the diversity of interested parties and the success of any future approaches/solutions. To alleviate the constant strain on medicinal plant populations in the wild, it is necessary to take rigorous steps to ensure the continuing availability of these precious natural resources . Increased regulation and the adoption of sustainable wild gathering methods can provide enough protection for some species; however, a more feasible long-term solution may be an increase in desert medicinal plant cultivation . Medicinal plant cultivation usually needs a lot of care and attention. Depending on the grade of medicinal plant components required, the circumstances and time of cultivation are different . In comparison to Europe and Asia, medicinal plant production in Africa’s Saharan areas is still in its infancy. Strong attempts toward commercial medicinal plant production has recently been recorded in the northern (Egypt, Libya, Morocco, Tunisia), eastern (Uganda, Kenya, and Tanzania), western (Nigeria, Ghana and Sierra Leone), and southern Africa (South Africa and Madagascar) . Cultivation initiatives for some of the most significant African medicinal plants are a priority among academics and policymakers. Furthermore, growing evidence from countries across the continents, such as South Africa and Sierra Leone , strongly suggests that other equally valuable medicinal plants can be grown on a small and large scale with the right approach. Despite rare reports of medicinal plant growth, there is a distinct dearth of recorded information on the scale of such operations. As a result, additional research in this field is necessary. Based on extensive recent research in ethnopharmacology, green chemistry, and pharmaceutical chemistry, it demonstrates a renaissance in interest in plant medicines, including desert plants, for the treatment and prevention of many ailments. In hot, dry climates, medicinal plants continue to play an essential role in the rural healthcare system. Until now, the successful treatment of illnesses using plant products has not yet been thoroughly proven using rigorous scientific criteria to compete with the current conventional treatments; thus, a number of significant challenges must be overcome and addressed before their full potential can be realized. The current review focused on the necessity for accurate recording of medicinal plants utilized by residents of the Saharan region to treat prevalent illnesses. The findings of this study revealed a rich diversity of medicinal plants used to treat various disease conditions as well as ethnomedicinal knowledge; as a result, if the trade of Saharan region herbal products is to increase, local laws must be TRIPS compliant, and issues of sustainable use and development of plant products must be addressed at the same time. Studies have indicated that all medicinal plants that have never been reported previously yet have been utilized for millennia to cure a variety of severe ailments should be investigated for their unknown potential uses. Communities would surely benefit from further research and promotion of medicine as they work to preserve knowledge and incorporate particular techniques into healthcare services. This may open the door to further study by pharmacologists and phytochemists. Two things should be mentioned. First, local knowledge might be turned into pharmaceuticals or other products for sale, and the locals who are the guardians of this knowledge should be thanked and fairly compensated. Second, overexploitation of medicinal plants will inevitably jeopardize their existence, necessitating conservation efforts. |
Exploring the ambiguity in the anatomical terminology among Dental professionals | 733ba03f-a55e-46c9-9bf6-6c86492ef652 | 11342683 | Anatomy[mh] | Anatomical terminology constitutes a specialized collection of terms employed to delineate the intricate structures within the human body, forming the foundational basis for communication across various healthcare disciplines, including dentistry. The vast majority of these terms are specific and discernible exclusively to professionals, making them uncommon in daily communication . The history of anatomical terms can be traced back over 2500 years. From ancient Greece to the present day, these terms have developed in various regions over time, driven by evolving usage needs. This diversification has led to an increase in the quantity of terms and a greater degree of complexity. Systematic efforts have been made to organize these terms, resulting in the development of the International Anatomical Terminology. Its first edition was recognized as Nomina Anatomica. Subsequent revisions and refinements have led to the current standard lexicon known as Terminologia Anatomica (TA) . Previous studies on discrepancies in anatomical terminology have shown that many anatomical terms also have synonyms. For example, the term ‘pineal gland’ has 16 synonyms, including pineal body, epiphysis, and parietal eye . Therefore, this might intensify the academic burden on students, compelling them to commit a greater extent of these terms to memory . Consequently, each newly emerging field endeavoured to formulate novel anatomical terminology tailored to its specific communication needs . New sets of terminology have emerged. Some of these terms were not found in TA, such as the ‘retromolar triangle’ whereas the other terms were synonyms of TA terms, or even terms identical to TA but with new meanings. For instance, the term ‘internal oblique line of mandible’ is synonymous with the TA term ‘mylohyoid line,’ which refers to the attachment of the mylohyoid muscle . Additionally, as a non-TA term, it denotes a bony ridge on the inner aspect of the mandibular ramus, extending from the coronoid process to the last molar, serving as the insertion point for the deep tendon of the temporalis muscle . Initially, anatomical terms were systematically compiled to facilitate communication within the healthcare community. However, with the creation of these terms, encompassing not only existing TA terms but also new terminologies, synonyms, and homonyms, there has been a notable increase in the overall volume of terms heightening the risk of confusion in both communication and interpretation . Undoubtedly, it imposes a substantial burden on learners in the field. Dentistry, similar to medical professions, constitutes a complex and ever-evolving domain requiring a profound understanding of human anatomy. Dental professionals rely extensively on anatomical terminology to communicate effectively, diagnose oral health conditions, and administer appropriate treatments. However, not only anatomical terms but also specific dental jargon, such as “centric occlusion,” lacks consensus among academics. This lack of agreement poses challenges for dental students in comprehending and applying inconsistent terminologies found across various learning resources, ultimately leading to confusion in knowledge acquisition . Furthermore, inconsistent terminology might complicate literature reviews and scientific discussions, potentially impeding advancements in dental research. Nevertheless, this matter has solely been addressed within academic literature and has not been thoroughly investigated concerning the confusion related to anatomical terms in the clinical setting . Therefore, this study aimed to explore and gather evidence to demonstrate the ambiguity surrounding the utilization of anatomical terms among dental practitioners, thereby enhancing awareness regarding the potential for miscommunication in clinical practice. This cross-sectional study was conducted among dental practitioners with a minimum of 1 year of clinical practice experience in Thailand including Bangkok and other provinces. A total of 78 participants were selected using convenience sampling. All participants provided written consent before the commencement of data collection. The data collection process involved interviews using a questionnaire designed to gather information on 13 selected anatomical terms based on their common usage in dental practice. These terms were determined by a literature review of English-written books, research papers in the field of dentistry, as well as medical dictionaries and dental glossaries. These 13 terms were categorized into two groups: Group 1, comprising 3 terms with clear and unambiguous meanings in scientific literatures, including Mental foramen, Condyle of mandible, and Greater palatine foramen; and Group 2, consisting of 10 terms with ambiguous meanings in scientific literatures, including Mandibular notch, Zygoma, Mylohyoid ridge, Incisive canal, Internal oblique ridge (line), Temporal crest of mandible, Coronoid notch, Sigmoid notch, Mandibular fossa, and External oblique ridge. During the interviews, participants were asked to identify the location of each anatomical term based on their understanding and usage on a provided artificial skull by interviewer (PT), and the responses were recorded on the paper containing skull figure (Fig. ) by data recorder (LM). There was no modification of interviewer’s behaviour and participants’ responses. In the first part, to ensure the authenticity of responses, the Group 1 terms were taken to verify that participants’ answers were not influenced by ignorance or arbitrary guessing. Eligible participants who mislabelled or incorrectly indicated any of these terms were excluded from the study. In the second part of the interview, participants were required to identify the Group 2 terms on an artificial skull and provide explanations for each term such as the importance of this location or a landmark for some operations. All data were recorded in the record form (Supplementary ), and the findings were reported as descriptive statistics by researchers (KC, TA, PG). This study received approval from the Mahidol Ethical Approval Committee (MU-DT/PY-IRB 2011/059.1209). Among the 78 participants, all participating dentists were eligible for the study. The average ages were 43.8 years old (ranging from 28 to 60 years old) with working experiences in dentistry ranging from 5 to 35 years. The majority specialized in prosthetic dentistry ( N = 28), followed by advanced general dentistry ( N = 15), oral and maxillofacial surgery ( N = 10), operative dentistry and endodontics ( N = 10), oral medicine ( N = 5), oral and maxillofacial radiology ( N = 5), and paediatric dentistry ( N = 5). All participants have already completed their postgraduate studies. While they all graduated with a bachelor’s degree in Dentistry in Thailand, their postgraduate training varied, taking place both in Thailand and internationally in countries such as the United States, the United Kingdom, Germany and Japan. All participants correctly identified and labelled three anatomical terms with clear clinical significance: condyle of the mandible, mental foramen, and greater palatine foramen. Therefore, no participants were excluded from the study. Table shows the results for the 10 anatomical terms that have ambiguous meaning in clinical use. Mandibular notch was in two different areas on the skulls: the concave region of the superior border of the ramus (25%) and the concave region of the anterior margin of the ramus (25%), while 33% of participants were unable to define this term. Coronoid notch was frequently confused with mandibular notch and was located at the concave region of the superior border of the ramus (51.32%) and the concave region of the anterior border of the ramus (18.42%), while 13.16% of participants reported not knowing or being unable to define this term. More than three-quarters of participants (77.62%) reported being unable to define the term sigmoid notch, but 17.11% of dental surgeon experts indicated it as the concave region of the superior border of the ramus. Zygoma was located in two different areas, namely the zygomatic bone (50.5%) and the zygomatic bone with zygomatic arch (31.7%). Mylohyoid ridge was indicated at the bone crest, which lies on the medial aspect of the body of the mandible acting as the attachment of the mylohyoid muscle (84.5%), and in other areas or not known (6% and 9.5%, respectively). Incisive canal was labelled on the maxilla area, where the nasopalatine nerve passes through the canal and opens into the incisive foramen by 90.8% of participants. However, a few dentists located this canal in a different area, namely the ramus of the mandible, where the canal was the passing way for the inferior alveolar nerve. Internal oblique ridge (line) was located at the bone crest as the inner side of the coronoid process, which lies on the medial surface of the ramus to the area nearby the distal end of the mandibular third molar by 85.5% of participants. However, 6.5% of participants indicated this term at the medial surface of the ramus, which is the distal end of the mandibular third molar only. In addition, most participants (85.5%) were unable to define the term temporal crest of mandible. Sixty percent of participants were unable to locate the mandibular fossa, and this term was in different areas by about 34% of participants. Moreover, the mandibular fossa was replaced by other terms such as “temporomandibular fossa,” “articular fossa,” “glenoid fossa,” and “condylar fossa” in the interview. Finally, the external oblique ridge was located at the anterior aspect of the ramus until the end of the coronoid process (50%) or at the bone crest of the distal body of the mandible (47.4%). The usage of anatomical terms is essential for dental professionals to communicate with each other and to understand the location of various parts of the head and skull. The present study found that some anatomical terms used in the dental clinical setting have different meanings from their definition in the anatomical terminology textbooks. This discrepancy can lead to confusion and miscommunication among dental professionals, especially for those who are just starting their education. In this study, the terms “Mandibular fossa” or “fossa mandibularis” was defined in the TA as “A prominent depression in the inferior surface of the squamous part of the temporal bone at the base of the zygomatic process in which the condyloid process of the mandible rests” . Naming a fossa or depression based on its articulating structure is a common practice in anatomical terminology. Similar examples can be found in general anatomy, such as the olecranon fossa of the humerus, which accommodates the olecranon of the ulna, the vermian fossa of the occipital bone, which houses part of the inferior cerebellar vermis, and the digastric fossa of the mandible, which serves as the attachment site for the anterior belly of the digastric muscle. Moreover, in a local anaesthesia textbook for dentists , another meaning of the term “mandibular fossa” is employed to denote the foramen located on the medial surface of the mandibular ramus, a structure more commonly recognized as the “mandibular foramen”. Surprisingly, the result of this study indicated that very few dentists used “mandibular fossa” in either meaning according to the TA term (2.6%) or in the context of “mandibular foramen” (2.6%). The majority of dentists are unfamiliar with the term (60.5%). This lack of familiarity might be due to the occurrence of alternative terms in dentistry. For the first meaning, other terms like “articular fossa” or “glenoid fossa of the temporal bone” are more commonly used. Additionally, for the second meaning, dentists frequently use the term “mandibular foramen”, rendering the term “mandibular fossa” obsolete. Consequently, “mandibular fossa” is a term that most dentists are unfamiliar with, despite its fundamental relevance to the direct practice of dentistry. The terms “Mandibular notch,” “Coronoid notch,” and “Sigmoid notch” illustrated the complexity arising from homonyms and synonyms in dental terminology. According to the TA term, “Mandibular notch” is considered synonymous with “Sigmoid notch,” and “Mandibular incisure” or “Incisura Mandibulae” referring to “the concave area at the superior border of the mandibular ramus” . However, in anatomical textbooks designed for dental students, “Mandibular notch” was not only presented in the context of the TA term but also described as a synonym for “Coronoid notch” , a term that was not included in the TA. Furthermore, in various dental textbooks, “Mandibular notch” was homonymously defined as “the concave area at the anterior border of the mandibular ramus“ and was synonymous with “coronoid notch” in this context . This confusion between “Mandibular notch” and “Coronoid notch” held considerable significance within dentistry, emphasizing the variability in the use and interpretation of these terms within the same professional community. The present study revealed the considerable variability in the usage and understanding of the terms “Mandibular notch,” “Coronoid notch,” and “Sigmoid notch” among dentists, especially within specific dental subspecialties like oral and maxillofacial surgery. The research showed that even among dental professionals, there were discrepancies in the interpretation of these terms. Notably, the term “Sigmoid notch,” as per the TA term, was rarely used in dental literature and was unfamiliar to most dentists. Instead, dentists tended to use alternative terms such as “Coronoid notch” or “Mandibular notch,” leading to potential misunderstandings, especially in interdisciplinary communication within dentistry. This aligned with the present findings, which revealed that dentists described both terms in similar contexts. However, the term “Sigmoid notch,” which was an anatomical term, was rarely encountered in dental literature, making most dentists unfamiliar with its meaning, despite its relevance to dentistry-related contexts. Moreover, among the dentists who did specify the meaning of “Sigmoid notch” according to the TA term (17.1%), nearly all reported by participants who specialized in oral and maxillofacial surgery, reflecting that even within the dental profession, there were variations in the use of terminology among different subspecialties. The present study further demonstrated that the terms “Mandibular notch,” “Coronoid notch,” and “Sigmoid notch” were used interchangeably, not only in describing the “anterior” or “superior” border of the mandibular ramus but also in other meanings that were not consistently documented in written literatures. This inconsistency and interchangeability in the usage of these terms emphasized the need for standardized and precise anatomical terminology, especially in the field of dentistry, to ensure clear communication and understanding among professionals, regardless of their subspecialties. The emergence of broader meanings for specific terms over time is a common phenomenon in language evolution. This evolution is often driven by the necessity to describe specific anatomical regions more precisely due to the development of specialized knowledge within various fields. Consequently, new anatomical terms may be coincided to meet the demands of evolving academic disciplines. This process can lead to the creation of new meanings for existing terms, especially in response to the changing requirements of specific fields. Over time, as these new meanings become widely accepted within particular disciplines, they may not pose issues . For instance, the use of the term “sigmoid notch” in oral and maxillofacial surgery might not cause confusion within that specific subspecialty. However, challenges arise when interdisciplinary communication occurs, even among different subspecialties within dentistry. In these situations, professionals might use different terms, such as “coronoid notch” or “mandibular notch,” leading to misunderstandings and miscommunications. Therefore, it remains essential to establish precise and standardized anatomical terminology to facilitate effective communication and understanding across various dental subspecialties and, more broadly, within the field of healthcare. “The concave area at the anterior border of the mandibular ramus” was not only referred to as the Mandibular notch or Coronoid notch but the present study demonstrated that most dentists were also familiar with another term, the External oblique ridge. Interestingly, even though almost all dentists described this term in a similar location, they defined its boundaries differently. Half of them specified the inferior end of the external oblique ridge in the area distal to the last molar along the anterior border of the ramus. However, the other dentists (47.4%) extended the definition of the inferior end of the external oblique ridge to the area encompassing the second premolar. The latter group primarily consisted of oral and maxillofacial radiologists. Based on their experience with radiographic images, they observed the radiopaque nature of the external oblique ridge extending to the second premolar. This variation in descriptions supported the differences in interpretations of the term, which depend on the expertise and specialized focus of each dental subspecialty. The terms “Internal oblique ridge” or “internal oblique line” are another example of the creation of new terms to provide to the specific needs of dental contexts. These terms refer to the bony ridge on the medial surface of the mandibular ramus, extending from the coronoid process to the posterior area of the last molar tooth . This ridge serves as the distal attachment of the deep tendon of the temporalis muscle, which holds clinical significance in dentistry. Surprisingly, this specific region is not mentioned in the TA or general anatomical literature. According to Cunningham’s anatomy textbook , it is briefly described as a “blunt ridge”. Therefore, the term “Internal oblique ridge” was coincided to facilitate communication within the dental field and could only be found in the dental textbooks such as Jorgensen and Hayden’s textbook . This illustrated the necessity of creating specialized terms to address specific anatomical features significant in dental practice, even if they were not extensively covered in general anatomical literature. Additionally, in dental textbooks, the term “Internal oblique ridge” could also be found under such different names as the “temporal crest of the mandible” and “mandibular temporal crest” . However, the study revealed that the majority of dentists were more familiar with the term “Internal oblique ridge”, as opposed to terms like “temporal crest” when referring to the bony ridge serving as the distal attachment of the deep tendon of the temporalis muscle. This indicated a clearer preference for the term “Internal oblique ridge” among dentists, showcasing the importance of standardized terminology for effective communication within the dental field. The study demonstrated that even though specialized terms were coincided within specific fields and documented in written literatures, they might not gain popularity in clinical usage and could fade away over time. When these terms were introduced, they could lead to confusion in meaning, as evidenced by the study results. This underscored the importance of caution and precision in using terminologies, both in educational contexts and clinical communication, especially in interdisciplinary communication within specialized fields. The results were in line with the previous studies which terminologies need to be expanded to the terms that used by clinicians and should accommodate with both clinical and anatomical works . This highlights the importance of clear and consistent communication within and across medical community included dental specialties, as well as the need for standardized terminology in the field. However, there were limitations in this study to be further improved. First, the study was conducted with a small sample size and a specific sampling method. Although the interviewees had experience in training both in Thailand and internationally, the limited sample size raises concerns about the generalizability of the data. A multi-centre study should be warranted in the conclusion. However, the results of this study could help dental educators become aware of this issue. Second, the selection of anatomical landmarks was limited to those in previous reports; expanding the terminology specific to the dental field might improve the study by focusing on dental communication. Third, the study design limited opportunities for data analysis. Further studies should improve data collection and provide more thorough analysis. However, this field in dentistry lacks evidence, so this study highlights the issue of confusion among academics that needs to be addressed. Furthermore, the present study highlighted the emergence of new and broader meanings for existing anatomical terms over time. This phenomenon was common in language evolution, where terms expanded in meaning based on the specific needs and developments within different scientific disciplines. The new terminology has not been defined or approved by international committee or clarified the detail of terminology, while commonly using in dental field only. To our concern, the synonym and homonyms could make students confused due to the various terms. Moreover, dental textbooks had different terms to indicate various anatomical areas. This problem could affect students who just start in dental study would be confused in learning, practicing, or using those term without awareness among students and professors or specialists . Overall, the findings of this study suggest that there is a need to systematically revise the usage of anatomical terms between anatomists and clinicians to reduce ambiguity and miscommunication in the dental clinical setting . Standardizing the terminology used in dental education and clinical practice would also benefit dental professionals and students by providing clear and consistent communication in the field . In conclusion, the study emphasizes the importance of clear and consistent terminology in the field of dentistry and the need for effective communication, especially when dealing with interdisciplinary or subspecialty interactions. This issue could lead to confusion and miscommunication among dental professionals and students, especially those who are just starting their dental education . We recommend that this problem be addressed by increasing awareness among dental professionals and students and by revising the usage of anatomical terms in a more systematic and standardized manner . While several terms were compiled in TA, they were still insufficient to keep pace with the rapid academic advancements occurring in various fields today. This would lead to better communication and collaboration among dental professionals and between dental and medical professionals, improving patient care and outcomes. In conclusion, our study highlights the philological ambiguity of anatomical terms among different specialties of dentistry. The usage of anatomical terms varies among specialties, with some terms having different meanings or being used to describe different locations than their original definitions. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Hard and soft tissue alterations after the application of different soft tissue grafting materials during immediate dental implant placement: a systematic review and Bayesian network meta-analysis | b1b7bcea-ad9f-41c9-886f-bcc97b9c32d1 | 11789362 | Dentistry[mh] | The immediate dental implantation experienced an upstream in its related published research after 2017 . Reducing the edentulism duration and minimizing the number of surgical procedures are two main reasons for immediate implant placement popularity and application that make it a suitable treatment option for both dentists and patients . There are two main aspects to every successful dental implant placement: biological and aesthetic features. The quality and harmony of the soft tissue around implants not only impact the aesthetic outcomes of dental implants but also specify health-related outcomes such as bleeding on probing . Soft tissue thickness can influence the biological width around dental implants. An adequate amount of soft tissue acts as a barrier against microorganisms in the mouth, facilitates oral hygiene, and guarantees aesthetics by covering the greyish color of implants and abutments . Previously, in 2012, Hsu et al., in a decision tree and case report, suggested that soft tissue grafting can be considered in sites with less than 2 mm of keratinized mucosal width, less than 2 mm of mucosal thickness, and sites in which the implants are placed in a buccal position . Moreover, a study by Kadkhodazadeh et al. offered a protocol for the efficient timing of soft tissue grafting around dental implants . There are three major types of soft tissue grafts around dental implants: autogenous soft tissue grafts, such as subepithelial connective tissue graft (CTG), allogenous grafting materials, and xenogenic graft substitutes, like xenogenic collagen matrix material (XCM) or acellular dermal matrix (ADM). CTG is usually harvested from the tuberosity or hard palate; therefore, it is associated with a higher patient morbidity rate . The success of autogenous grafts usually depends on patient anatomy, requires more surgeries, and can cause the patient some discomfort, while in xenogenic grafts, fewer surgeries and a faster healing process are seen. XCM acts as a space-making scaffold for new peri-implant tissue formation and holds angiogenic properties . On the other hand, the effectiveness of xenogenic grafts is lower than autogenous grafts due to the tissue shrinkage and lower quality of attached tissues in grafts . Since the benefits of different adjunctive soft tissue grafting materials in immediate implant placement have not yet been fully discovered and there is no systematic review and network meta-analysis available in the literature to evaluate the clinical outcomes of different materials used simultaneously with immediate implant placement, the present systematic review and network meta-analysis aimed to comprehensively assess the effects of different types of soft tissue grafts on different aspects of immediate implant treatment success and rank the available soft tissue graft options in the each desired outcome. Protocol This systematic review has been conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension statement for network meta-analysis (PRISMA-NMA) guidance . The protocol of this review has been prospectively registered in the PROSPERO on July 23, 2024 (CRD42024568586). The available literature has been qualitatively and quantitatively analyzed concerning the following question: “In patients with non-restorable teeth, who receive immediate dental implants (P), what is the best adjunctive soft tissue grafting approach among different autogenous, allogenous, and xenogenous grafts (I), to achieve the desired hard and soft tissue structure (O), compared to sites without grafting (C)?”. Eligibility criteria PICO P (Population) Human patients who need immediate dental implant placement after tooth extraction. I (Intervention) Randomized clinical trials in which adjunctive soft tissue grafting using different autogenous, allogenous, xenogenous soft tissue grafts around immediately placed dental implants has been done. C (Comparison) Comparison between different autogenous, allogenous, xenogenous soft tissue grafts, and No Treatment (control group). O (Outcome) Primary outcomes: Pink Esthetic Score (PES), Marginal Interproximal Bone Level Changes (MIBL), Buccal Bone Thickness Changes (BBT), Keratinized Tissue Width Changes (KTW), Soft Tissue Thickness Changes (STT), Papilla Height Changes (PH), Midfacial Gingival Margin Level Changes (MGML). Secondary outcomes: Prosthetic and Surgical Complications Inclusion criteria Human original studies Randomized clinical trials (RCTs) Studies used soft tissue grafts around immediately placed dental implants at the time of surgery. Exclusion criteria Articles not written in English Observational, case report, case series. Studies did not compare different soft tissue grafts with each other or No Treatment; and instead, they compared soft tissue grafts with other treatment modalities for peri-implant soft tissue enhancement. Information sources and search strategy PubMed, Scopus, and ISI Web of Science databases have been searched electronically up to May 9, 2024 (Table 1/S1). The search was updated on September 13, 2024. All extracted records have been imported into Mendeley Reference Manager to remove the duplicates. For further details, see Appendix S1. Study selection and data collection process Study selection has been carried out according to the eligibility criteria. firstly, four authors (F.R, A.Y, K.H, and A.M) were calibrated by (A.A) in terms of study selection and data extraction. three authors (F.R, A.Y, and A.M), independently, screened all records based on their titles and abstracts. Afterward, three authors (F.R, A.Y, and K.H) screened the full-text of the articles and extracted the data into the pre-designed tables (by A.A), independently. All disagreements at any stage were resolved after a discussion by the final verdict of the first author. Data items The following data items have been extracted into the tables for each study: Treatment arms and groups Population characteristics Initial ridge condition Placed implants’ characteristics Follow-up period Implant loading details Missing tooth/teeth numbers Insertion torque Baseline measurements (If any were available) Clinical outcomes (PES, MIBL, BBT, KTW, STT, PH, and MGML) Prosthetic and surgical complications Other outcomes and observations (If any were available) Risk of bias assessment The Cochrane Collaboration’s tool for randomized clinical trials (RoB 2) has been used to assess the individual studies’ risk of bias. Firstly, three authors (F.R, A.Y, and K.H) were calibrated by (A.A) in terms of utilization of the risk of bias tool in the bias assessment. Three authors (F.R, A.Y, and K.H) independently appraised all studies concerning the following domains: random sequence generation, concealment of the allocation, blinding, incomplete outcome data report, and other sources of bias. All disagreements at any stage were resolved after a discussion by the final verdict of the first author. Statistical analysis Due to the limited number of studies available on different treatment arms, a random-effect Bayesian network meta-analysis, considering the No Treatment group as the reference, has been conducted to compare various available treatment arms. Moreover, a traditional pair-wise random-effect Bayesian meta-analysis was used, and at least two studies were available comparing two specific treatment arms. The effect measure for all outcomes was mean difference. For the sake of transitivity and making the results of the network meta-analysis clinically interpretable, for each specific outcome, a period of follow-ups that are most frequently reported for that outcome was chosen, and reports of the shorter and longer follow-up times were disregarded from the quantitative analysis. After the data had been explored, a 6 – 12-month period was chosen for PES, BBT, KTW, STT, PH, and MGML, and a 12 – 24-month period was chosen for MIBL. Of the studies on the same populations, only one that matched the other included studies from the follow-up time perspective was chosen for each specific outcome. In cases where a study reported two follow-up time points within the specified follow-up period of an outcome, the longer one was selected for the analysis. Eventually, the exact follow-up time of the studies included in the analyses was considered as a covariate to be tested for their significant effect and accounting for their effect if it enhanced the model fit. All studies had a parallel design and included single implants; hence, the transitivity assumption was applicable regarding these matters. Moreover, the initial conditions of the sockets (All were non-compromised or minimally compromised) were relatively similar allowing all of the treatment arms be potentially applicable for all of the patients. The treatment arms were evaluated to be similar in terms of the patient’s age and gender, whether they raised a flap or placed a flapless implant, immediate or delayed provisionalization, use of the bone graft materials, and inclusion of the defective sockets, qualitatively. By the way, it was planned to use network meta-regression to explore the effect of these covariates, if they were reported in all included studies in the analysis, to see if they have a significant effect. The deviance information criterion (DIC) was used for model selection, as a model with lower DIC is considered better-fitted and more parsimonious. The between-study heterogeneity was evaluated using I 2 . To assess the consistency assumption globally and locally, a comparison between the consistency and inconsistency models’ DIC and node-splitting has been used, respectively. The node-splitting model used was introduced by van Valkenhoef et al. , which only split potential inconsistent loops (loops with independent indirect estimation). If there was an evident inconsistency, the source was investigated by accounting for various predictive and confounding pre-defined covariates using network meta-regression. Furthermore, to find the best-fitted model and catch as much between-study heterogeneity as possible, the predictive covariates accounted for their effect on the model to see if they could reduce the DIC value. The sensitivity analyses in this study comprised fitting a model with a different between-study heterogeneity prior type. Moreover, if the final model accounted for the effect of a covariate, the plain model without regression was also fitted as a sensitivity analysis. The results of the analyses are presented in the league tables (comprising both network and traditional pair-wise meta-analysis results). The surface under cumulative ranking (SUCRA) was utilized to rank the treatment modalities. To assess the publication bias, the comparison-specific funnel plots were generated. The network geometry and funnel plots were produced using the STATA 15.0 software (StataCorp LP, Lakeway Drive, College Station, TX, USA). All analyses were synthesized using GeMTC and Bayesmeta R packages. A 95% credible interval (CrI) was used to determine the finding’s significance. For further details, see Appendix S1. Certainty of the meta-evidence assessment The Grades of Recommendations, Assessment, Development, and Evaluation (GRADE) approach customized for the network meta-analysis was used to assess the overall certainty and strength of the generated meta-evidence . First, the certainty of all direct network estimations was calculated. The final certainty of the network meta-analysis evidence has been achieved considering the certainty of the direct estimates and the weight of participation of both direct and indirect estimates in the overall network meta-analysis estimation. This systematic review has been conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension statement for network meta-analysis (PRISMA-NMA) guidance . The protocol of this review has been prospectively registered in the PROSPERO on July 23, 2024 (CRD42024568586). The available literature has been qualitatively and quantitatively analyzed concerning the following question: “In patients with non-restorable teeth, who receive immediate dental implants (P), what is the best adjunctive soft tissue grafting approach among different autogenous, allogenous, and xenogenous grafts (I), to achieve the desired hard and soft tissue structure (O), compared to sites without grafting (C)?”. PICO P (Population) Human patients who need immediate dental implant placement after tooth extraction. I (Intervention) Randomized clinical trials in which adjunctive soft tissue grafting using different autogenous, allogenous, xenogenous soft tissue grafts around immediately placed dental implants has been done. C (Comparison) Comparison between different autogenous, allogenous, xenogenous soft tissue grafts, and No Treatment (control group). O (Outcome) Primary outcomes: Pink Esthetic Score (PES), Marginal Interproximal Bone Level Changes (MIBL), Buccal Bone Thickness Changes (BBT), Keratinized Tissue Width Changes (KTW), Soft Tissue Thickness Changes (STT), Papilla Height Changes (PH), Midfacial Gingival Margin Level Changes (MGML). Secondary outcomes: Prosthetic and Surgical Complications Inclusion criteria Human original studies Randomized clinical trials (RCTs) Studies used soft tissue grafts around immediately placed dental implants at the time of surgery. Exclusion criteria Articles not written in English Observational, case report, case series. Studies did not compare different soft tissue grafts with each other or No Treatment; and instead, they compared soft tissue grafts with other treatment modalities for peri-implant soft tissue enhancement. P (Population) Human patients who need immediate dental implant placement after tooth extraction. I (Intervention) Randomized clinical trials in which adjunctive soft tissue grafting using different autogenous, allogenous, xenogenous soft tissue grafts around immediately placed dental implants has been done. C (Comparison) Comparison between different autogenous, allogenous, xenogenous soft tissue grafts, and No Treatment (control group). O (Outcome) Primary outcomes: Pink Esthetic Score (PES), Marginal Interproximal Bone Level Changes (MIBL), Buccal Bone Thickness Changes (BBT), Keratinized Tissue Width Changes (KTW), Soft Tissue Thickness Changes (STT), Papilla Height Changes (PH), Midfacial Gingival Margin Level Changes (MGML). Secondary outcomes: Prosthetic and Surgical Complications Human patients who need immediate dental implant placement after tooth extraction. Randomized clinical trials in which adjunctive soft tissue grafting using different autogenous, allogenous, xenogenous soft tissue grafts around immediately placed dental implants has been done. Comparison between different autogenous, allogenous, xenogenous soft tissue grafts, and No Treatment (control group). Primary outcomes: Pink Esthetic Score (PES), Marginal Interproximal Bone Level Changes (MIBL), Buccal Bone Thickness Changes (BBT), Keratinized Tissue Width Changes (KTW), Soft Tissue Thickness Changes (STT), Papilla Height Changes (PH), Midfacial Gingival Margin Level Changes (MGML). Secondary outcomes: Prosthetic and Surgical Complications Human original studies Randomized clinical trials (RCTs) Studies used soft tissue grafts around immediately placed dental implants at the time of surgery. Articles not written in English Observational, case report, case series. Studies did not compare different soft tissue grafts with each other or No Treatment; and instead, they compared soft tissue grafts with other treatment modalities for peri-implant soft tissue enhancement. PubMed, Scopus, and ISI Web of Science databases have been searched electronically up to May 9, 2024 (Table 1/S1). The search was updated on September 13, 2024. All extracted records have been imported into Mendeley Reference Manager to remove the duplicates. For further details, see Appendix S1. Study selection has been carried out according to the eligibility criteria. firstly, four authors (F.R, A.Y, K.H, and A.M) were calibrated by (A.A) in terms of study selection and data extraction. three authors (F.R, A.Y, and A.M), independently, screened all records based on their titles and abstracts. Afterward, three authors (F.R, A.Y, and K.H) screened the full-text of the articles and extracted the data into the pre-designed tables (by A.A), independently. All disagreements at any stage were resolved after a discussion by the final verdict of the first author. The following data items have been extracted into the tables for each study: Treatment arms and groups Population characteristics Initial ridge condition Placed implants’ characteristics Follow-up period Implant loading details Missing tooth/teeth numbers Insertion torque Baseline measurements (If any were available) Clinical outcomes (PES, MIBL, BBT, KTW, STT, PH, and MGML) Prosthetic and surgical complications Other outcomes and observations (If any were available) The Cochrane Collaboration’s tool for randomized clinical trials (RoB 2) has been used to assess the individual studies’ risk of bias. Firstly, three authors (F.R, A.Y, and K.H) were calibrated by (A.A) in terms of utilization of the risk of bias tool in the bias assessment. Three authors (F.R, A.Y, and K.H) independently appraised all studies concerning the following domains: random sequence generation, concealment of the allocation, blinding, incomplete outcome data report, and other sources of bias. All disagreements at any stage were resolved after a discussion by the final verdict of the first author. Due to the limited number of studies available on different treatment arms, a random-effect Bayesian network meta-analysis, considering the No Treatment group as the reference, has been conducted to compare various available treatment arms. Moreover, a traditional pair-wise random-effect Bayesian meta-analysis was used, and at least two studies were available comparing two specific treatment arms. The effect measure for all outcomes was mean difference. For the sake of transitivity and making the results of the network meta-analysis clinically interpretable, for each specific outcome, a period of follow-ups that are most frequently reported for that outcome was chosen, and reports of the shorter and longer follow-up times were disregarded from the quantitative analysis. After the data had been explored, a 6 – 12-month period was chosen for PES, BBT, KTW, STT, PH, and MGML, and a 12 – 24-month period was chosen for MIBL. Of the studies on the same populations, only one that matched the other included studies from the follow-up time perspective was chosen for each specific outcome. In cases where a study reported two follow-up time points within the specified follow-up period of an outcome, the longer one was selected for the analysis. Eventually, the exact follow-up time of the studies included in the analyses was considered as a covariate to be tested for their significant effect and accounting for their effect if it enhanced the model fit. All studies had a parallel design and included single implants; hence, the transitivity assumption was applicable regarding these matters. Moreover, the initial conditions of the sockets (All were non-compromised or minimally compromised) were relatively similar allowing all of the treatment arms be potentially applicable for all of the patients. The treatment arms were evaluated to be similar in terms of the patient’s age and gender, whether they raised a flap or placed a flapless implant, immediate or delayed provisionalization, use of the bone graft materials, and inclusion of the defective sockets, qualitatively. By the way, it was planned to use network meta-regression to explore the effect of these covariates, if they were reported in all included studies in the analysis, to see if they have a significant effect. The deviance information criterion (DIC) was used for model selection, as a model with lower DIC is considered better-fitted and more parsimonious. The between-study heterogeneity was evaluated using I 2 . To assess the consistency assumption globally and locally, a comparison between the consistency and inconsistency models’ DIC and node-splitting has been used, respectively. The node-splitting model used was introduced by van Valkenhoef et al. , which only split potential inconsistent loops (loops with independent indirect estimation). If there was an evident inconsistency, the source was investigated by accounting for various predictive and confounding pre-defined covariates using network meta-regression. Furthermore, to find the best-fitted model and catch as much between-study heterogeneity as possible, the predictive covariates accounted for their effect on the model to see if they could reduce the DIC value. The sensitivity analyses in this study comprised fitting a model with a different between-study heterogeneity prior type. Moreover, if the final model accounted for the effect of a covariate, the plain model without regression was also fitted as a sensitivity analysis. The results of the analyses are presented in the league tables (comprising both network and traditional pair-wise meta-analysis results). The surface under cumulative ranking (SUCRA) was utilized to rank the treatment modalities. To assess the publication bias, the comparison-specific funnel plots were generated. The network geometry and funnel plots were produced using the STATA 15.0 software (StataCorp LP, Lakeway Drive, College Station, TX, USA). All analyses were synthesized using GeMTC and Bayesmeta R packages. A 95% credible interval (CrI) was used to determine the finding’s significance. For further details, see Appendix S1. The Grades of Recommendations, Assessment, Development, and Evaluation (GRADE) approach customized for the network meta-analysis was used to assess the overall certainty and strength of the generated meta-evidence . First, the certainty of all direct network estimations was calculated. The final certainty of the network meta-analysis evidence has been achieved considering the certainty of the direct estimates and the weight of participation of both direct and indirect estimates in the overall network meta-analysis estimation. Study selection Figure shows the flow diagram of the study. A total of 1532 records were found after the primary search, and after removing duplicates, 909 studies remained. After the primary and final screening toward the inclusion and exclusion criteria, 26 articles were included in this review. Among all these studies, 21 articles were considered for the qualitative and quantitative analysis. Characteristics of the included studies Among all included studies, 19, 1, 4, 1, 2, and 16 treatment arms were available on CTG, a combination of CTG with platelet-rich fibrin (L-PRF), Mucoderm, Mucograft, AlloDerm, and No Treatment, respectively. Moreover, from the included studies, two of them and four of them were performed and reported on the same populations. The methodological characteristics of the included studies are summarized in Table . This systematic review pooled 530 immediate implants placed. Soft tissue grafts were applied in 320 implant placements; CTG was used in 238, CTG + L-PRF was used in 6, porcine-derived collagen matrices in 51 (43 Mucoderm and 8 Mucograft), and acellular dermal matrices (AlloDerm) in 25. Furthermore, bone graft was used in 434 immediate implant placements. It was mentioned in the included studies, except Abd El-Aziz et al. study , that implants replaced teeth ranging from 1 to 5, maxillary anterior or premolar regions. All implant types were bone level. Implants with different widths, ranging from 3.2 to 5 mm, and lengths, ranging from 9 to 18 mm, were employed, respectively. Nine studies did not mention the diameter width of implants and nine did not mention the length of implants . Sixteen studies mentioned the type of restorations used; all were non-splinted single crowns as in 11 studies were screw-retained and 11 studies cement-retained . Various follow-up periods with different onset times were observed in studies ranging from 2 weeks to 5 years. Thirteen studies reported on the insertion torque of the implants employing different methodologies for reporting ranging between 20 to 48.55 N-cm. Risk of bias assessment The overall quality assessment of the studies is summarized in Fig. . None of the studies exhibited a high risk of bias. Randomization process was the most problematic domain among all domains due to the lack of a clear allocation concealment process, and 10 studies raised some concerns regarding this issue . Five studies raised some concerns due to deviations from intended intervention (D2) . There was only one study showing some concerns missing outcome data (D3) and in measurement of the outcome (D4) . Selection of the reported results (D5) was the only domains that raised no concerns in the included studies. In summary, among the 21 included studies, 11 showed an overall some concerns regarding bias While the 10 others demonstrated an overall low risk of bias . Moreover, an outcome specific assessment of the risk of bias are presented in the Appendix S1 as Figs. 1/S1, 2/S1, 3/S1, 4/S1, 5/S1, 6/S1, 7/S1 correspond to assessment of the risk of bias concerning studies reporting PES, MIBL, BBT, KTW, STT, PH, and MGML. Qualitative synthesis Table summarizes the PES/WES, MIBL, BBT, KTW, STT, PH, MGML, and the complications experienced in the studies. Twelve studies analyzed mean PES in using grafts and no grafts groups, reporting all studies no significant difference between them. Two studies reported WES points with no significant difference but a slightly lower point in CTG groups. Ten studies reported MIBL . Notably, Zuiderveld et al. reported the highest median MIBL in a follow-up of 12 months gain at 0.9 mm (Interquartile Range (IQR): 0.40 – 1.2mm) in the mesial interproximal region across all of the studies, achieved through the CTG as the grafting material. In contrast, Abd El-Aziz et al. documented the lowest mean change in a follow-up of 6 months at −1.19 ± 1.48 mm in buccal crestal region without any grafting materials. Among the studies using grafting material, six studies presented a decrease in mean (or median) MIBL despite using CTG as grafting material, and Lee et al. reported mean MIBL of −0.69 ± 0.74 mm in mesial and −0.39 ± 2.24 mm in the distal region with AlloDerm as grafting material in a 12 months follow-up. Five studies reported BBT changes Jiang et al. mentioned the highest BBT of 2.72 ± 0.73 mm based on the 0 mm distance from the implant’s shoulder with no grafting material in a 6-month follow-up. Contrary, a decrease in BBT was documented in 2 studies , reporting the most decreasing change of −2.17 ± 2.39 mm based on the 0 mm distance from the implant’s shoulder with CTG as grafting material in a 24 months follow-up . KTW was presented by nine studies . Sharafuddin et al. reported the highest value change of 15.42 ± 18.60 mm without any grafting material in a 3-month follow-up. However, the lowest value change of −30.62 ± 35.88 mm without any grafting material was reported in Sharafuddin et al. study in a 6-month follow-up. Two studies reported decreased KTW in immediate implant placements with AlloDerm as grafting material in which the lowest rate was reported as −0.47 ± 1.06 mm . Seven studies reported implant loss due to failure in osseointegration . Quantitative synthesis The transitivity assessment has been qualitatively appraised as described in " " section. The potential covariates (the patient’s age and gender, whether a study raised a flap or flapless, immediate or delayed provisionalization, use of the bone graft materials, and inclusion of the defective sockets) were distributed similarly between different treatment arms qualitatively; however, all expect for patient’s age and gender, were considered to be tested as predictive covariates for their effects on the final estimation in the model. Individual outcome measures The detailed results of the network meta-analysis for the estimation of each PES, MIBL, BBT, KTW, STT, PH, and MGML outcomes are reported in appendices S3, S4, S5, S6, S7, S8, and S9, respectively. For further details, see Appendix S2. Pink esthetic score Figure A elicits the geometry of the overall network of the studies reporting PES included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S3), which is resolved by fixed-effect accounting for the effect of follow-up time in the final model (Table 4/S3). The network meta-regression showed that the follow-up time has a significant effect on the estimation of PES in both fixed- (−2.68 [95% CrI: −4.68, −0.65]) and random-effect (for CTG: −2.69 [95% CrI: −4.68, −0.60]). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: Mucoderm (0.81) – CTG (0.67) – Mucograft (0.52) – No Treatment (0.27) – CTG + L-PRF (0.24). Figure A shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Marginal interproximal bone level changes Figure B elicits the geometry of the overall network of the studies reporting MIBL included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S4). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 12 – 24 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.59) – AlloDerm (0.56) – Mucoderm (0.50) – No Treatment (0.35). Figure B shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Buccal bone thickness changes Figure C elicits the geometry of the overall network of the studies reporting BBT included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S5). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: No Treatment (0.57) – Mucograft (0.48) – CTG (0.45). Figure C shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Keratinized tissue width changes Figure D elicits the geometry of the overall network of the studies reporting KTW included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S6). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.67) – CTG + L-PRF (0.5) – Mucoderm (0.64) – AlloDerm (0.27) – No Treatment (0.27). Figure D shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Soft tissue thickness changes Figure E elicits the geometry of the overall network of the studies reporting STT included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S7), which is resolved by random-effect accounting for the effect of baseline soft tissue thickness value in the final model (Table 4/S7). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. Only the adjunctive application of CTG showed a significantly higher amount of STT after 6 – 12 months of follow-up compared to No Treatment (1.13 mm [95% CrI: 0.04, 2.34]). The other comparisons did not demonstrate any significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.69) – AlloDerm (0.63) – CTG + L-PRF (0.58) – Mucograft (0.42) – No Treatment (0.19). Figure E shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Papilla height changes Figure F elicits the geometry of the overall network of the studies reporting PH included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S8). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.73) – Mucograft (0.65) – No Treatment (0.43) – AlloDerm (0.19). Figure F shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Midfacial gingival margin level changes Figure G elicits the geometry of the overall network of the studies reporting MGML included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S9). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.91) – No Treatment (0.39) – AlloDerm (0.20). Figure G shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Certainty of the meta-evidence assessment A summary of the GRADE assessment regarding each outcome and each comparison is demonstrated in Table . The problematic domains were the within study bias and imprecision causing the certainty of the meta-evidence to be down-graded. Only meta-evidence receiving a high certainty in this study were CTG vs. No Treatment in the KTW and Mucoderm vs. No Treatment in the MIBL. Overall, in different outcomes, 18, 16, and 12 comparisons received a low, moderate, and very low certainty. Figure shows the flow diagram of the study. A total of 1532 records were found after the primary search, and after removing duplicates, 909 studies remained. After the primary and final screening toward the inclusion and exclusion criteria, 26 articles were included in this review. Among all these studies, 21 articles were considered for the qualitative and quantitative analysis. Among all included studies, 19, 1, 4, 1, 2, and 16 treatment arms were available on CTG, a combination of CTG with platelet-rich fibrin (L-PRF), Mucoderm, Mucograft, AlloDerm, and No Treatment, respectively. Moreover, from the included studies, two of them and four of them were performed and reported on the same populations. The methodological characteristics of the included studies are summarized in Table . This systematic review pooled 530 immediate implants placed. Soft tissue grafts were applied in 320 implant placements; CTG was used in 238, CTG + L-PRF was used in 6, porcine-derived collagen matrices in 51 (43 Mucoderm and 8 Mucograft), and acellular dermal matrices (AlloDerm) in 25. Furthermore, bone graft was used in 434 immediate implant placements. It was mentioned in the included studies, except Abd El-Aziz et al. study , that implants replaced teeth ranging from 1 to 5, maxillary anterior or premolar regions. All implant types were bone level. Implants with different widths, ranging from 3.2 to 5 mm, and lengths, ranging from 9 to 18 mm, were employed, respectively. Nine studies did not mention the diameter width of implants and nine did not mention the length of implants . Sixteen studies mentioned the type of restorations used; all were non-splinted single crowns as in 11 studies were screw-retained and 11 studies cement-retained . Various follow-up periods with different onset times were observed in studies ranging from 2 weeks to 5 years. Thirteen studies reported on the insertion torque of the implants employing different methodologies for reporting ranging between 20 to 48.55 N-cm. The overall quality assessment of the studies is summarized in Fig. . None of the studies exhibited a high risk of bias. Randomization process was the most problematic domain among all domains due to the lack of a clear allocation concealment process, and 10 studies raised some concerns regarding this issue . Five studies raised some concerns due to deviations from intended intervention (D2) . There was only one study showing some concerns missing outcome data (D3) and in measurement of the outcome (D4) . Selection of the reported results (D5) was the only domains that raised no concerns in the included studies. In summary, among the 21 included studies, 11 showed an overall some concerns regarding bias While the 10 others demonstrated an overall low risk of bias . Moreover, an outcome specific assessment of the risk of bias are presented in the Appendix S1 as Figs. 1/S1, 2/S1, 3/S1, 4/S1, 5/S1, 6/S1, 7/S1 correspond to assessment of the risk of bias concerning studies reporting PES, MIBL, BBT, KTW, STT, PH, and MGML. Table summarizes the PES/WES, MIBL, BBT, KTW, STT, PH, MGML, and the complications experienced in the studies. Twelve studies analyzed mean PES in using grafts and no grafts groups, reporting all studies no significant difference between them. Two studies reported WES points with no significant difference but a slightly lower point in CTG groups. Ten studies reported MIBL . Notably, Zuiderveld et al. reported the highest median MIBL in a follow-up of 12 months gain at 0.9 mm (Interquartile Range (IQR): 0.40 – 1.2mm) in the mesial interproximal region across all of the studies, achieved through the CTG as the grafting material. In contrast, Abd El-Aziz et al. documented the lowest mean change in a follow-up of 6 months at −1.19 ± 1.48 mm in buccal crestal region without any grafting materials. Among the studies using grafting material, six studies presented a decrease in mean (or median) MIBL despite using CTG as grafting material, and Lee et al. reported mean MIBL of −0.69 ± 0.74 mm in mesial and −0.39 ± 2.24 mm in the distal region with AlloDerm as grafting material in a 12 months follow-up. Five studies reported BBT changes Jiang et al. mentioned the highest BBT of 2.72 ± 0.73 mm based on the 0 mm distance from the implant’s shoulder with no grafting material in a 6-month follow-up. Contrary, a decrease in BBT was documented in 2 studies , reporting the most decreasing change of −2.17 ± 2.39 mm based on the 0 mm distance from the implant’s shoulder with CTG as grafting material in a 24 months follow-up . KTW was presented by nine studies . Sharafuddin et al. reported the highest value change of 15.42 ± 18.60 mm without any grafting material in a 3-month follow-up. However, the lowest value change of −30.62 ± 35.88 mm without any grafting material was reported in Sharafuddin et al. study in a 6-month follow-up. Two studies reported decreased KTW in immediate implant placements with AlloDerm as grafting material in which the lowest rate was reported as −0.47 ± 1.06 mm . Seven studies reported implant loss due to failure in osseointegration . The transitivity assessment has been qualitatively appraised as described in " " section. The potential covariates (the patient’s age and gender, whether a study raised a flap or flapless, immediate or delayed provisionalization, use of the bone graft materials, and inclusion of the defective sockets) were distributed similarly between different treatment arms qualitatively; however, all expect for patient’s age and gender, were considered to be tested as predictive covariates for their effects on the final estimation in the model. Individual outcome measures The detailed results of the network meta-analysis for the estimation of each PES, MIBL, BBT, KTW, STT, PH, and MGML outcomes are reported in appendices S3, S4, S5, S6, S7, S8, and S9, respectively. For further details, see Appendix S2. Pink esthetic score Figure A elicits the geometry of the overall network of the studies reporting PES included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S3), which is resolved by fixed-effect accounting for the effect of follow-up time in the final model (Table 4/S3). The network meta-regression showed that the follow-up time has a significant effect on the estimation of PES in both fixed- (−2.68 [95% CrI: −4.68, −0.65]) and random-effect (for CTG: −2.69 [95% CrI: −4.68, −0.60]). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: Mucoderm (0.81) – CTG (0.67) – Mucograft (0.52) – No Treatment (0.27) – CTG + L-PRF (0.24). Figure A shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Marginal interproximal bone level changes Figure B elicits the geometry of the overall network of the studies reporting MIBL included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S4). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 12 – 24 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.59) – AlloDerm (0.56) – Mucoderm (0.50) – No Treatment (0.35). Figure B shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Buccal bone thickness changes Figure C elicits the geometry of the overall network of the studies reporting BBT included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S5). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: No Treatment (0.57) – Mucograft (0.48) – CTG (0.45). Figure C shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Keratinized tissue width changes Figure D elicits the geometry of the overall network of the studies reporting KTW included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S6). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.67) – CTG + L-PRF (0.5) – Mucoderm (0.64) – AlloDerm (0.27) – No Treatment (0.27). Figure D shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Soft tissue thickness changes Figure E elicits the geometry of the overall network of the studies reporting STT included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S7), which is resolved by random-effect accounting for the effect of baseline soft tissue thickness value in the final model (Table 4/S7). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. Only the adjunctive application of CTG showed a significantly higher amount of STT after 6 – 12 months of follow-up compared to No Treatment (1.13 mm [95% CrI: 0.04, 2.34]). The other comparisons did not demonstrate any significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.69) – AlloDerm (0.63) – CTG + L-PRF (0.58) – Mucograft (0.42) – No Treatment (0.19). Figure E shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Papilla height changes Figure F elicits the geometry of the overall network of the studies reporting PH included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S8). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.73) – Mucograft (0.65) – No Treatment (0.43) – AlloDerm (0.19). Figure F shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Midfacial gingival margin level changes Figure G elicits the geometry of the overall network of the studies reporting MGML included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S9). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.91) – No Treatment (0.39) – AlloDerm (0.20). Figure G shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. The detailed results of the network meta-analysis for the estimation of each PES, MIBL, BBT, KTW, STT, PH, and MGML outcomes are reported in appendices S3, S4, S5, S6, S7, S8, and S9, respectively. For further details, see Appendix S2. Figure A elicits the geometry of the overall network of the studies reporting PES included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S3), which is resolved by fixed-effect accounting for the effect of follow-up time in the final model (Table 4/S3). The network meta-regression showed that the follow-up time has a significant effect on the estimation of PES in both fixed- (−2.68 [95% CrI: −4.68, −0.65]) and random-effect (for CTG: −2.69 [95% CrI: −4.68, −0.60]). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: Mucoderm (0.81) – CTG (0.67) – Mucograft (0.52) – No Treatment (0.27) – CTG + L-PRF (0.24). Figure A shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure B elicits the geometry of the overall network of the studies reporting MIBL included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S4). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 12 – 24 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.59) – AlloDerm (0.56) – Mucoderm (0.50) – No Treatment (0.35). Figure B shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure C elicits the geometry of the overall network of the studies reporting BBT included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S5). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: No Treatment (0.57) – Mucograft (0.48) – CTG (0.45). Figure C shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure D elicits the geometry of the overall network of the studies reporting KTW included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S6). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.67) – CTG + L-PRF (0.5) – Mucoderm (0.64) – AlloDerm (0.27) – No Treatment (0.27). Figure D shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure E elicits the geometry of the overall network of the studies reporting STT included in the network meta-analysis. A global inconsistency has been observed in the plain model (Table 1/S7), which is resolved by random-effect accounting for the effect of baseline soft tissue thickness value in the final model (Table 4/S7). No significant effect was observed among other predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. Only the adjunctive application of CTG showed a significantly higher amount of STT after 6 – 12 months of follow-up compared to No Treatment (1.13 mm [95% CrI: 0.04, 2.34]). The other comparisons did not demonstrate any significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.69) – AlloDerm (0.63) – CTG + L-PRF (0.58) – Mucograft (0.42) – No Treatment (0.19). Figure E shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure F elicits the geometry of the overall network of the studies reporting PH included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S8). No significant effect was observed among the predictive and confounder variables. There was not a significant sign of loop inconsistency according to the node-splitting. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.73) – Mucograft (0.65) – No Treatment (0.43) – AlloDerm (0.19). Figure F shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. Figure G elicits the geometry of the overall network of the studies reporting MGML included in the network meta-analysis. No global inconsistency has been observed in the plain model (Table 1/S9). No significant effect was observed among the predictive and confounder variables. There was no potential inconsistent loop to be investigated in the network geometry. The funnel plot did not show an observable sign of publication bias. There was no significant difference between treatment arms after 6 – 12 months of follow-up according to both network and traditional pair-wise meta-analysis (Table ). The treatment ranking according to the SUCRA value was as follows: CTG (0.91) – No Treatment (0.39) – AlloDerm (0.20). Figure G shows the forest plot of the comparison between each treatment arm and the No Treatment group. The sensitivity analysis results were not significantly different from the main results. A summary of the GRADE assessment regarding each outcome and each comparison is demonstrated in Table . The problematic domains were the within study bias and imprecision causing the certainty of the meta-evidence to be down-graded. Only meta-evidence receiving a high certainty in this study were CTG vs. No Treatment in the KTW and Mucoderm vs. No Treatment in the MIBL. Overall, in different outcomes, 18, 16, and 12 comparisons received a low, moderate, and very low certainty. Interpretation of the results This review aimed to evaluate the effect of different adjunctive soft tissue grafts on the clinical outcomes of immediately placed implants. Figure elicits the SUCRA values of each treatment modality regarding each of the outcomes. Despite the fact that there are some available decision trees in the literature regarding the application of soft tissue grafting around immediately placed dental implants, still there is no widely accepted guideline and consensus on the subject, especially for the sockets with minimally hard and soft tissue deficiencies in which it is not clear that soft tissue grafting with different materials are beneficial or have any differences regarding the implant-related clinical outcomes. Since most of the studies in the present review evaluated the effect using different soft tissue grafts on the aforementioned condition and compared them with not placing any grafts, the results of this study can be considered beneficial to the available literature. The esthetic outcomes of an immediately placed implant can be dependent on the location of the tooth, mucosal margin position, soft tissue phenotype, and spatial position of the placed implant in relation to the available hard and soft tissue . Moreover, planning for a provisional restoration showed a beneficial effect on the emergence profile of the soft tissue and, consequently, a superior esthetic outcome . In the present review, it was tried to investigate and account for the effect of these factors even though they were qualitatively similar in the included studies. Nonetheless, except for the follow-up time, other tested covariates did not show a significant effect on all outcomes. This result could be because of the relatively limited number of studies reporting each outcome, as the regression analyses could be heavily associated with the number of observations, in our case, studies. The long-term stability and health of the restorations supported by the implants are associated with the quantity of the peri-implant keratinized mucosal width and thickness. It has been shown that a keratinized tissue width higher than 2 mm can reduce the chance of soft tissue inflammation and recession, bacterial plaque aggregation, and hygiene maintenance. On the other hand, more than 2 mm of soft tissue thickness demonstrated a protective influence on the peri-implant marginal bone . According to the SUCRA values and rankings, our analyses illustrated that CTG, compared to other treatments, could be associated with a higher STT after 6 – 12 months and, consequently, a higher MIBL after 12 – 24 months. Mucograft, on the other hand, demonstrated a weak performance among graft materials regarding the STT, which might be attributed to its higher rate of thickness loss and maintained space, consequently . Interestingly, a higher BBT was achieved after 6 – 12 months in No Treatment had been applied. This outcome could be attributed to less soft tissue manipulation when the implants are placed without soft tissue grafting. Coherently, Kuebler and Noelken, in 2024, evaluated the effect of adjunctive CTG grafting alongside bone grafting in immediate implant placement and demonstrated that sockets grafted with CTG and bone grafts experienced higher PES, lower recession, but thinner buccal bone wall . The findings of the present study were in convergence with similar reviews. In 2024, Tommasato et al., in a systematic review, for non-immediately placed implants, compared the effect of autogenous and collagen matrices on the outcomes of peri-implant soft tissue augmentation . With an insistence on the CTG as superior material in most outcomes and free gingival graft (FGG) in increasing the keratinized tissue width after six months, their frequentist network meta-analysis revealed the efficacy of the autogenous grafts (FGG and CTG) is higher than that of xenogenous soft tissue grafts. However, except for the keratinized tissue width increasing after six months, they did not find any other significant differences between different treatment modalities in other outcomes. Coherently, our findings suggest that CTG possessed a higher efficacy in improving MIBL, KTW, STT, PH, and MGML than other treatments. In the Tommasato et al. study, all available xenogenic, as well as allogenic, substitutes were combined together; however, their various structure, porosity, number of layers, origin of collagens, and thickness, which are critical properties for the regeneration process, can cause inaccuracies in the conclusion. Torra-Moneny et al., in a systematic review and meta-analysis in 2024, assessed the impact of the application of CTG on the peri-implant soft tissue of the immediate implants compared to no grafting modality . In agreement with our findings, they suggest that there is no significant difference in the buccal gingival margin level between the application of CTG and No Treatment. Although CTG is highly effective, they come with several significant drawbacks. For instance, they often require longer surgical procedures and can cause complications at the donor site, potentially leading to additional surgeries. These issues can result in considerable post-operative pain and discomfort for patients , especially those concerned about surgical complications. Additionally, the availability of CTGs may be limited for larger surgical areas, which could discourage patients from starting or continuing treatments involving CTG. Despite the fact that the application of CTG showed superior efficacy in the improvement of the MIBL, KTW, STT, PH, and MGML, Mucoderm demonstrated higher, but relatively comparable to CTG, amounts of achieved PES, as a comprehensive outcome of an immediately placed implant, after 6 – 12 months. Moreover, based on the presence of no statistically significant difference between different types of grafts, a clinical recommendation needs to be supported by a richer literature, and therefore, more clinical investigations should be conducted on this subject. Hence, according to current evidence, the utilization of allogeneic and xenogeneic soft tissue grafts may be deemed appropriate for individuals exhibiting mild to moderate soft tissue deficiencies, those demonstrating limited compliance, or patients who exhibit apprehension regarding potential postoperative discomfort, adverse events, or morbidity. Eventually, the findings regarding CTG + L-PRF and Mucograft treatment modalities may not be completely relied on because there is only one study available on them. Moreover, a caution should be made regarding the evidence graded with low and very low certainty as they are limited by imprecision or bias and are more likely to be changed with emerging of new studies in the field. Limitations and recommendations for future studies Since to the authors knowledge there is no review comprehensively comparing different soft tissue grafting materials regarding the clinical outcomes of immediately placed implants, this study can be considered novel and can contribute the expansion of the available literature. However, the present systematic review has its limitations. First, the imprecision of some of the comparisons due to the lack of a reliable number of studies comparing those treatment arms affected the certainty and quality of the generated meta-evidence. Although most of the included studies have an overall low risk of bias, an unclear risk of bias affected the certainty of comparisons between AlloDerm and CTG or No Treatment in KTW, STT, and PH outcomes. Moreover, a comprehensive comparison between all available treatment modalities could not be achieved, as it is demonstrated in Table that many of the cells are not filled, because the included studies on different treatment arms are not distributed balanced. Furthermore, the reports on the different outcomes are mostly within a short-term follow-up time, which negatively influences the generalizability of the results. Eventually, despite the fact that the follow-up time was considered as a potential covariate in the regression models and also has been restricted to a limited period to satisfy the assumption of transitivity, still this network meta-analysis has this limitation that the included studies have different follow-up times which may violate the transitivity; thus, the results should be interpretated with cautions. It would be beneficial to the literature if future studies focus on the mentioned missing comparisons between available treatment arms in the different outcomes. Furthermore, higher follow-up time periods (longer than 12 months) will enable future evidence with more power and certainty. Finally, based on the current literature, allogenic and xenogenic soft tissue substitutes showed no significant difference compared to the autogenous grafts, and they are attributed to less post-operative pain and discomfort; hence, to recommend the CTG as a gold standard, further investigation seems to be necessary for a more comprehensive conclusion with higher certainty, and making clinical recommendation instead of suggestions. This review aimed to evaluate the effect of different adjunctive soft tissue grafts on the clinical outcomes of immediately placed implants. Figure elicits the SUCRA values of each treatment modality regarding each of the outcomes. Despite the fact that there are some available decision trees in the literature regarding the application of soft tissue grafting around immediately placed dental implants, still there is no widely accepted guideline and consensus on the subject, especially for the sockets with minimally hard and soft tissue deficiencies in which it is not clear that soft tissue grafting with different materials are beneficial or have any differences regarding the implant-related clinical outcomes. Since most of the studies in the present review evaluated the effect using different soft tissue grafts on the aforementioned condition and compared them with not placing any grafts, the results of this study can be considered beneficial to the available literature. The esthetic outcomes of an immediately placed implant can be dependent on the location of the tooth, mucosal margin position, soft tissue phenotype, and spatial position of the placed implant in relation to the available hard and soft tissue . Moreover, planning for a provisional restoration showed a beneficial effect on the emergence profile of the soft tissue and, consequently, a superior esthetic outcome . In the present review, it was tried to investigate and account for the effect of these factors even though they were qualitatively similar in the included studies. Nonetheless, except for the follow-up time, other tested covariates did not show a significant effect on all outcomes. This result could be because of the relatively limited number of studies reporting each outcome, as the regression analyses could be heavily associated with the number of observations, in our case, studies. The long-term stability and health of the restorations supported by the implants are associated with the quantity of the peri-implant keratinized mucosal width and thickness. It has been shown that a keratinized tissue width higher than 2 mm can reduce the chance of soft tissue inflammation and recession, bacterial plaque aggregation, and hygiene maintenance. On the other hand, more than 2 mm of soft tissue thickness demonstrated a protective influence on the peri-implant marginal bone . According to the SUCRA values and rankings, our analyses illustrated that CTG, compared to other treatments, could be associated with a higher STT after 6 – 12 months and, consequently, a higher MIBL after 12 – 24 months. Mucograft, on the other hand, demonstrated a weak performance among graft materials regarding the STT, which might be attributed to its higher rate of thickness loss and maintained space, consequently . Interestingly, a higher BBT was achieved after 6 – 12 months in No Treatment had been applied. This outcome could be attributed to less soft tissue manipulation when the implants are placed without soft tissue grafting. Coherently, Kuebler and Noelken, in 2024, evaluated the effect of adjunctive CTG grafting alongside bone grafting in immediate implant placement and demonstrated that sockets grafted with CTG and bone grafts experienced higher PES, lower recession, but thinner buccal bone wall . The findings of the present study were in convergence with similar reviews. In 2024, Tommasato et al., in a systematic review, for non-immediately placed implants, compared the effect of autogenous and collagen matrices on the outcomes of peri-implant soft tissue augmentation . With an insistence on the CTG as superior material in most outcomes and free gingival graft (FGG) in increasing the keratinized tissue width after six months, their frequentist network meta-analysis revealed the efficacy of the autogenous grafts (FGG and CTG) is higher than that of xenogenous soft tissue grafts. However, except for the keratinized tissue width increasing after six months, they did not find any other significant differences between different treatment modalities in other outcomes. Coherently, our findings suggest that CTG possessed a higher efficacy in improving MIBL, KTW, STT, PH, and MGML than other treatments. In the Tommasato et al. study, all available xenogenic, as well as allogenic, substitutes were combined together; however, their various structure, porosity, number of layers, origin of collagens, and thickness, which are critical properties for the regeneration process, can cause inaccuracies in the conclusion. Torra-Moneny et al., in a systematic review and meta-analysis in 2024, assessed the impact of the application of CTG on the peri-implant soft tissue of the immediate implants compared to no grafting modality . In agreement with our findings, they suggest that there is no significant difference in the buccal gingival margin level between the application of CTG and No Treatment. Although CTG is highly effective, they come with several significant drawbacks. For instance, they often require longer surgical procedures and can cause complications at the donor site, potentially leading to additional surgeries. These issues can result in considerable post-operative pain and discomfort for patients , especially those concerned about surgical complications. Additionally, the availability of CTGs may be limited for larger surgical areas, which could discourage patients from starting or continuing treatments involving CTG. Despite the fact that the application of CTG showed superior efficacy in the improvement of the MIBL, KTW, STT, PH, and MGML, Mucoderm demonstrated higher, but relatively comparable to CTG, amounts of achieved PES, as a comprehensive outcome of an immediately placed implant, after 6 – 12 months. Moreover, based on the presence of no statistically significant difference between different types of grafts, a clinical recommendation needs to be supported by a richer literature, and therefore, more clinical investigations should be conducted on this subject. Hence, according to current evidence, the utilization of allogeneic and xenogeneic soft tissue grafts may be deemed appropriate for individuals exhibiting mild to moderate soft tissue deficiencies, those demonstrating limited compliance, or patients who exhibit apprehension regarding potential postoperative discomfort, adverse events, or morbidity. Eventually, the findings regarding CTG + L-PRF and Mucograft treatment modalities may not be completely relied on because there is only one study available on them. Moreover, a caution should be made regarding the evidence graded with low and very low certainty as they are limited by imprecision or bias and are more likely to be changed with emerging of new studies in the field. Since to the authors knowledge there is no review comprehensively comparing different soft tissue grafting materials regarding the clinical outcomes of immediately placed implants, this study can be considered novel and can contribute the expansion of the available literature. However, the present systematic review has its limitations. First, the imprecision of some of the comparisons due to the lack of a reliable number of studies comparing those treatment arms affected the certainty and quality of the generated meta-evidence. Although most of the included studies have an overall low risk of bias, an unclear risk of bias affected the certainty of comparisons between AlloDerm and CTG or No Treatment in KTW, STT, and PH outcomes. Moreover, a comprehensive comparison between all available treatment modalities could not be achieved, as it is demonstrated in Table that many of the cells are not filled, because the included studies on different treatment arms are not distributed balanced. Furthermore, the reports on the different outcomes are mostly within a short-term follow-up time, which negatively influences the generalizability of the results. Eventually, despite the fact that the follow-up time was considered as a potential covariate in the regression models and also has been restricted to a limited period to satisfy the assumption of transitivity, still this network meta-analysis has this limitation that the included studies have different follow-up times which may violate the transitivity; thus, the results should be interpretated with cautions. It would be beneficial to the literature if future studies focus on the mentioned missing comparisons between available treatment arms in the different outcomes. Furthermore, higher follow-up time periods (longer than 12 months) will enable future evidence with more power and certainty. Finally, based on the current literature, allogenic and xenogenic soft tissue substitutes showed no significant difference compared to the autogenous grafts, and they are attributed to less post-operative pain and discomfort; hence, to recommend the CTG as a gold standard, further investigation seems to be necessary for a more comprehensive conclusion with higher certainty, and making clinical recommendation instead of suggestions. Considering the limitations of the present review, the following conclusions can be drawn: There was not any significant difference between different autogenous, allogenous, and xenogenous soft tissue grafts regarding any of the investigated implant-related clinical outcomes, However, based on the treatment rankings, although CTG illustrated higher efficacy in the improvement of most of the immediate implant clinical outcomes (MIBL, KTW, STT, PH, and MGML), Mucoderm superiorly, but relatively comparable to CTG, enhanced the most important outcome of an immediately placed implant (PES). Hence, no clinical recommendation can be drawn based on the current literature, and the use of CTG can be only a suggestion. Nonetheless, since the current evidence shows no significant difference between different graft types, allogeneic and xenogeneic soft tissue grafts, especially xenogenic collagen matrices, may be suitable for patients with mild soft tissue thickness loss, limited compliance, or major concerns about postoperative complications and morbidity. The application of CTG showed significantly higher amounts of soft tissue thickness gain compared to the not applying any soft tissue grafts after 6 – 12 months and can be suggested for the clinical scenario, especially in cases with a thin soft tissue thickness. However, based on the treatment rankings, adjunctive soft tissue grafting intervention, except for BBT, demonstrated higher efficacy in the improvement of clinical outcomes of the immediately placed implants compared to No Treatment in 6 – 12 months (12 – 24 months for MIBL). The PES score of the immediately placed implants decreases between 6 – 12 months of follow-up regardless of the grafting with different materials or non-grafting approach. Supplementary Material 1. Supplementary Material 2. Supplementary Material 3. Supplementary Material 4. Supplementary Material 5. Supplementary Material 6. Supplementary Material 7. Supplementary Material 8. Supplementary Material 9. |
Clinicopathological study and molecular subtyping of muscle-invasive bladder cancer (MIBC) using dual immunohistochemical (IHC) markers | 2f0e6f9a-6d06-4eb5-913a-55809d4c4f9d | 11759442 | Anatomy[mh] | Carcinoma of urinary bladder presents with wide range of clinical features and consists of diverse histological types that influences the disease progression, prognosis, and recurrence . It is a common malignancy occurring in the urinary tract, that accounted for 613,791 new cases and 220,349 cancer-related deaths in the world in 2022. In the same year, India reported 22,548 cases, constituting 10.5% of the total cases of bladder cancers in Asia, which occurred three to four times more commonly in males than females . Histologically, bladder carcinomas are of several types that include urothelial carcinomas, squamous cell carcinoma, adenocarcinoma, urachal carcinoma, diverticular carcinoma, tumours of Mullerian type etc . The presence or absence of detrusor muscle invasion is an important determinant of prognosis; hence they are classified into non-muscle invasive bladder cancer (NMIBC), which account for nearly 80% of cases and muscle-invasive bladder cancer (MIBC) making up the remaining cases . Genomic profiling and mutational studies in NMIBCs and MIBCs have identified several molecular subtypes; the primary molecular subtypes of MIBC being luminal and basal subtypes which express markers of urothelial and basal/squamous differentiation respectively . These groups are reported to be associated with distinct clinicopathological features, with basal subtype exhibiting worse prognosis with better response to chemotherapy, compared to the luminal subtype. A comprehensive analysis of these processes helps in accurate diagnosis and multimodality treatment approaches. Studies have demonstrated the utility of surrogate immunohistochemistry (IHC) markers to identify the molecular subtypes using GATA3, CK20 as luminal markers while CK5/6, CK14 as basal markers. A meta-analysis on three cohorts of bladder carcinoma (937 samples) to analyse the gene expression profiles using a panel of immunohistochemical markers, demonstrated that the two subtypes can be identified using only two markers - GATA3 and CK5/6 for luminal and basal tumours respectively, with an accuracy of more than 90% . This study aimed study the clinicopathological features of MIBCs and classify them into molecular subtypes using a limited panel of two antibodies-GATA3 and CK5/6 and analyse their correlation with the biological behaviour. Case selection A 5-year retrospective study (January 2017 to December 2021) of all diagnosed cases of carcinoma of the bladder with muscle invasion (MIBC) retrieved from the database of Department of Pathology was conducted, that included transurethral resection specimens of bladder tumour (TURBT) as well as radical cystectomy specimens. NMIBCs, mesenchymal tumours and lymphomas were excluded from the study. The study was approved by the institutional ethics committee (IEC2: 449/2022). The clinico-demographic data, cystoscopy findings and treatment details were obtained from the electronic medical records. Follow up details including the duration of follow-up, patient survival and recurrence data were obtained wherever available. Hematoxylin and Eosin (H & E) slides were reviewed for histomorphological features and classified according to the World Health Organization (WHO) 5th edition and pathological staging was done as per TNM and American Joint Committee on Cancer (AJCC) guidelines. Immunohistochemistry IHC was performed on all the cases using GATA3 (clone L50-823, mouse monoclonal antibody, Biocare Medical) and CK5/6 (clone CK5/6.007, mouse monoclonal antibody, Biocare Medical). The representative areas of muscle-invasive tumour were identified, marked on the slide and in the formalin-fixed paraffin-embedded blocks. Tissue microarray (TMA) was constructed by retrieving 5 mm tumour tissue cores, and each recipient block was embedded with seven cores. Manual IHC staining was performed on the tumour samples along with appropriate positive controls for both antibodies (breast cancer and prostate tissue for GATA3 and CK5/6 respectively), starting with heat-induced epitope retrieval. Primary antibody incubation was done for one hour followed by horse-radish peroxidase labelled MACH1 antibody with Betazoid diaminobenzidine as chromogen. Counterstain with hematoxylin was performed and the results were interpreted. The IHC slides were interpreted as positive if the tumour cells showed nuclear stain for GATA3 and cytoplasmic or membrane stain for CK5/6. Based on the staining percentage in the tumour cell population, they were declared positive if staining was more than 10% and negative if less than 10% similar to study by Bejrananda et al. IHC-based molecular subtypes Molecular subclassification using surrogate IHC markers – GATA3 and CK5/6 were done as follows. GATA3 positive, CK5/6 negative - Luminal subtype. GATA3 negative, CK5/6 positive - Basal subtype. GATA3 negative, CK5/6 negative - Double negative subtype. Tumours staining for both GATA3 and CK5/6 staining, with CK5/6 observed in < 10% tumour population were grouped under the luminal subtype as some luminal tumours are shown to possess scattered CK5/6 positive cells . Statistics Data was analysed using IBM-SPSS Statistics for Windows version 23.0 (Armonk, NY: IBM Corp). The categorical data was expressed in terms of proportions and percentages. Continuous variables were expressed using mean and standard deviation. T test was used to compare means, and Chi-square test was employed to study the association between the categorical variables. A p value of < 0.05 was considered statistically significant. A 5-year retrospective study (January 2017 to December 2021) of all diagnosed cases of carcinoma of the bladder with muscle invasion (MIBC) retrieved from the database of Department of Pathology was conducted, that included transurethral resection specimens of bladder tumour (TURBT) as well as radical cystectomy specimens. NMIBCs, mesenchymal tumours and lymphomas were excluded from the study. The study was approved by the institutional ethics committee (IEC2: 449/2022). The clinico-demographic data, cystoscopy findings and treatment details were obtained from the electronic medical records. Follow up details including the duration of follow-up, patient survival and recurrence data were obtained wherever available. Hematoxylin and Eosin (H & E) slides were reviewed for histomorphological features and classified according to the World Health Organization (WHO) 5th edition and pathological staging was done as per TNM and American Joint Committee on Cancer (AJCC) guidelines. IHC was performed on all the cases using GATA3 (clone L50-823, mouse monoclonal antibody, Biocare Medical) and CK5/6 (clone CK5/6.007, mouse monoclonal antibody, Biocare Medical). The representative areas of muscle-invasive tumour were identified, marked on the slide and in the formalin-fixed paraffin-embedded blocks. Tissue microarray (TMA) was constructed by retrieving 5 mm tumour tissue cores, and each recipient block was embedded with seven cores. Manual IHC staining was performed on the tumour samples along with appropriate positive controls for both antibodies (breast cancer and prostate tissue for GATA3 and CK5/6 respectively), starting with heat-induced epitope retrieval. Primary antibody incubation was done for one hour followed by horse-radish peroxidase labelled MACH1 antibody with Betazoid diaminobenzidine as chromogen. Counterstain with hematoxylin was performed and the results were interpreted. The IHC slides were interpreted as positive if the tumour cells showed nuclear stain for GATA3 and cytoplasmic or membrane stain for CK5/6. Based on the staining percentage in the tumour cell population, they were declared positive if staining was more than 10% and negative if less than 10% similar to study by Bejrananda et al. Molecular subclassification using surrogate IHC markers – GATA3 and CK5/6 were done as follows. GATA3 positive, CK5/6 negative - Luminal subtype. GATA3 negative, CK5/6 positive - Basal subtype. GATA3 negative, CK5/6 negative - Double negative subtype. Tumours staining for both GATA3 and CK5/6 staining, with CK5/6 observed in < 10% tumour population were grouped under the luminal subtype as some luminal tumours are shown to possess scattered CK5/6 positive cells . Data was analysed using IBM-SPSS Statistics for Windows version 23.0 (Armonk, NY: IBM Corp). The categorical data was expressed in terms of proportions and percentages. Continuous variables were expressed using mean and standard deviation. T test was used to compare means, and Chi-square test was employed to study the association between the categorical variables. A p value of < 0.05 was considered statistically significant. During the 5-year study period, 400 cases of bladder carcinomas were received, of which 66 (16.5%) cases were MIBC with detrusor muscle invasion. Majority were TURBT samples constituting 48 cases (72.7%) while 18 cases (27.3%) were radical cystectomy specimens. The clinicopathologic features of MIBCs are summarized in Table . The age of the patients ranged from 40 to 85 years with mean age at diagnosis being 65.91 years. Male to female ratio was 3.71:1. Most common clinical manifestation noted was painless haematuria in 86.4% of patients, followed by burning micturition in 40.9% patients. Cystoscopically, sites involved were lateral walls (45.5%), followed by dome (22.1%), neck of bladder (21.7%), anterior wall (19.7%), posterior wall (19.7%), trigone (16.7%), and ureteric orifice (3.0%). The lesions were solitary in most patients (75.8%) with a mean size of 2.98 cm. Histopathological examination showed that most of the cases were conventional urothelial carcinomas in 54.5% cases, and urothelial carcinoma with divergent differentiation in 36.4% of which squamous differentiation was the most common. Non-urothelial carcinomas -squamous cell carcinoma in 6.1% and neuroendocrine carcinoma in 3% cases noted. Pathological staging and lymph node involvement were available for 18 and 11 cystectomy specimens respectively, listed in Table . Based on the expression of GATA 3 and CK5/6 expression, 41 cases (62.1%) were classified as luminal subtype expressing GATA3 IHC (Fig. ). 20 cases (30.3%) demonstrating CK5/6 expression as basal subtype (Fig. ) while the remaining five (7.6%) cases did not express either of the 2 markers and were classified as the double-negative subtype (Fig. ). Twenty-one cases which demonstrated positive GATA3 expression with scattered or basal strips of CK5/6 staining in < 10% tumour population were included in the luminal subtype (Fig. ). The association of the clinicopathologic features with these subtypes are given in Table . IHC-basal subtype presented at a younger mean age ( p = 0.049). A male preponderance was noted among all subtypes. On cystoscopy, most cases showing multiple lesions belonged to the luminal subtype while larger lesions were associated with basal subtype. On microscopy, predominant papillary architecture was observed commonly in the luminal subtype (76.7%). Conventional urothelial carcinomas was identified to be predominantly of luminal subtype (88.9%) while urothelial carcinomas with squamous differentiation and subtypes and pure squamous cell carcinomas (SCC) belonged to the basal subtype on histopathological examination. Although pathological staging in 18 cases of MIBC in cystectomy specimens revealed most pT4 cases to be of basal subtype (66.7%), there was no statistically significant difference in pathological staging between the IHC molecular subtypes. Necrosis was a common feature seen in all the 3 subtypes, with all double-negative cases demonstrating it. Perineural invasion (PNI) was noted in both luminal and basal subtypes, with slight preponderance in luminal cases. In contrast, lymphovascular invasion (LVI) ( p < 0.05) and lymph node involvement ( p < 0.022) were noted more commonly with basal subtype. Patients were followed up for a period of three years which was available only for 17 patients, rest were lost to follow up. Two cases (one luminal, one basal type) showed recurrence. Luminal subtype of the high grade conventional urothelial carcinoma showed similar features on recurrence as the primary tumor, while the basal type of high-grade urothelial carcinoma showed divergent sarcomatoid differentiation in the recurrent tumor. Distant metastasis to bone, lung and liver was seen in three patients of luminal subtype and double negative subtype respectively. Post-radical cystectomy, eight patients died due to post-operative complications such as aspiration pneumonia, acute pulmonary embolism and sepsis. Bladder cancers are heterogeneous at the molecular level with distinct pathways involved in pathogenesis of NMIBCs and MIBCs. Various groups have studied the genomic landscape across bladder tumours, utilizing individual study cohorts that helped to stratify the patients for prognosis and to predict response to neoadjuvant chemotherapy. Several classifications have categorised both NMIBCs and MIBCs into various molecular subtypes using mRNA assays or IHC markers. Each system identified different clusters, starting with two subtypes (luminal-like and basal-like) and recently leading to the development of the consensus classification system identifying six molecular subtypes: luminal-papillary, luminal unstable, luminal non-specified, stroma-rich, basal/squamous neuroendocrine-like . Studies have indicated that the gene expression profiles of the two primary molecular subtypes: luminal and basal subtypes mirror the expression patterns of normal luminal and basal urothelial cells. Using surrogate IHC markers, bladder tumours can be classified as luminal when they express GATA3, and CK20, and basal when they show expression of CK5/6 and CK14 . To effectively incorporate the classification system into clinical practice, surrogate IHC analysis plays an important role, especially in resource-poor settings. MIBCs presented commonly in the elderly population in our study with mean age of 65.91 years, comparable to the findings of other studies . Most participants were males, and a similar male preponderance was noted by various authors . Data on smoking history revealed 63% (17/27) to be smokers. Other studies also reported smoking in 71.4% and 71% participants respectively, thus indicating a strong relationship between smoking and bladder carcinoma . Cystoscopy noted most lesions to be solitary (75.8%) with a mean size of 2.98 cm, like the findings of Satoh E et al. where 72.2% of MIBCs were found to be solitary measuring ≥ 1 cm . The commonest sites involved were the lateral walls followed by the dome, trigone similar to the study by Stephenson et al. who noted 37.1% occurring in lateral wall followed by posterior walls and trigone . Histopathological examination showed papillary architecture in 45.5% of all tumours, in contrast to only 25% noted by Jangir H et al. . Most tumours were conventional urothelial carcinomas followed by urothelial carcinomas with squamous differentiation, as also noted in various studies while SCC was seen in higher numbers in study by Naga NK et al. . Only small numbers of giant cell and sarcomatoid subtypes as well as neuroendocrine carcinomas were identified in our study, coinciding with other studies . Our study demonstrates that GATA3 and CK5/6 immunostaining was noted in 41 and 20 cases respectively. The luminal subtype constituted 62.1% of the study population. A meta-analysis on transcriptome expression identified 70% and 81.5% luminal tumours in the MD Anderson and Lund cohort, comprising of both NMIBCs and MIBCs while a lower 52% was noted in The Cancer Genome Atlas (TCGA) cohort including only MIBCs . Olkhov Mitsel et al. reported a higher (78.8%) number under the IHC-based luminal subtype which could be because the cases expressing both GATA3 and CK5/6 were included in this molecular subtype while lower numbers were seen in various other studies which identified mixed subtype as a separate group . The basal subtype was demonstrated in 30.3% MIBCs in our study which was comparable to the 26% of the MD Anderson cohort, while TCGA and the Lund cohort reported higher (44%) and lower (13.3%) basal tumours respectively . Other studies have also demonstrated similar results in MIBCs . The double negative subtype was observed in only 7.6% of our study population, coinciding with the observations in the MD Anderson, Lund and TCGA cohort (4%, 5% and 4% respectively) . The luminal subtype was associated with the following characteristics: presentation at an older age compared to other subtypes while other studies identified this group to present earlier . Of the 17 with a history of smoking, 11 were identified to be smokers coinciding with the findings of Sun X et al. . On microscopy, papillary morphology was appreciated predominantly in this subtype (76.7%), which was comparable to the 72.7% tumours of the luminal papillary, luminal non-specified and luminal unstable subtypes studied by Benitez et al. . Most cases of conventional urothelial carcinoma along with few showing divergent squamous differentiation were associated with luminal subtype, as was seen in numerous analyses . The basal subtype significantly occurred at an earlier age (mean − 62.70) than other subtypes, like other studies . The female patients in the study population were equally distributed between basal and luminal subtypes, in contrast to the female predominance demonstrated in other studies . Smoking was observed in 6/9 basal MIBCs, which is consistent with research demonstrating significant association between smoking and basal subtype . Histopathological examination revealed the basal subtype associated with non-papillary morphology as was demonstrated by other studies . Urothelial carcinoma with squamous differentiation and pure squamous cell carcinoma were of this basal subtype which was statistically significant. Similar findings were also observed by other researchers . All the cases of urothelial carcinoma - giant cell type, squamous cell carcinoma and neuroendocrine carcinoma were negative on IHC for GATA3, which agreed with the results of Naga NK et al. . LVI was noted in most cases, like other studies while Sanguedolce et al. showed lower LVI in the basal subtype . PNI was observed in more than half the cases, reinforcing the findings of Serag Eldien MM et al. that PNI was significantly associated with cases showing CK5/6 positivity and low GATA3 expression . Moderate to brisk stromal inflammatory infiltrate was identified, which was in concordance with studies showing high stromal infiltration in the basal/squamous subtype . In our study, the five cases of double-negative subtype were all males, aligning with other studies . No smokers were noted in this group. Histology showed three cases of conventional urothelial carcinoma, predominantly non-papillary type and two neuroendocrine carcinomas. This lack of expression of both markers in neuroendocrine carcinoma was also shown in other studies while some studies noted GATA3 expression . All cases demonstrated necrosis. Brisk stromal inflammatory infiltrate was not identified in this subtype, like the findings in other studies . Six cases of pT4 stage in cystectomy specimens were of the basal subtype (66.7%) similar to findings of other studies showing basal tumours mostly being pT3 and T4 cases . Lymph node involvement was also more commonly seen in basal subtype like the findings noted by other researchers . Limited followup data was available for only 17 patients. The two patients who developed recurrence belonged to the luminal and basal subtypes, while those with distant metastasis were seen in the double negative and luminal subtype. Bejrananda et al. noted the absence of CK5/6 or GATA3 expression to be associated with poor survival and strongly predicted a negative outcome . The basal subtype showed a poor outcome with one patient showing tumour recurrence while five patients expired during the follow-up period mainly due to post-operative morbidities. The adverse outcome in the basal subtype is usually attributed to GATA3 loss, promoting tumour suppressor gene downregulation and transition from epithelial to mesenchymal type leading to increased invasiveness and spread of the tumour . Helal DS and co-workers noted that most basal subtype patients in their study cohort presented with higher T stage with the presence of lymphovascular invasion and nodal involvement, contributing to the unfavorable outcome, similar to our study . The worst outcome was reported in the double negative group, with a higher risk of death . The limitations of this study are the retrospective design in a single-centre setting and the availability of limited follow-up data due to which survival studies could not be performed. TMA was utilized to perform GATA3 and CK5/6 IHC analysis. While this method is cost and time-effective, it utilizes only a small area of the tumour tissue for staining and may not reflect the tumour heterogeneity seen commonly in bladder carcinomas. Further workup using gene profiling in a larger cohort to validate the IHC-based molecular subtypes was not done. MIBCs have been documented to have genomic instability and multiple mutations, resulting in poor prognosis in these patients, despite intensive surgery and chemotherapy. Many researchers have demonstrated the prognostic and predictive beneficial information obtained by ascertaining the molecular subtypes and immunophenotypes as regards to patient management. To effectively incorporate the classification system into clinical practice, surrogate IHC analysis plays an important role in identifying the molecular subtypes. Accurate identification of histological variants, molecular subtypes, and immunophenotypes of urothelial carcinomas helps in risk stratification according to biological aggressiveness. This data will also provide important information about patient treatment and prognostication. |
Analysis of deaths due to gender-based violence: An autopsy-based cross-sectional study from Mumbai | 64fe0133-a719-4f56-bbe5-4cfbd987c6f2 | 11722706 | Forensic Medicine[mh] | Gender-based violence (GBV), particularly against women, remains a pervasive global public health issue, contributing to significant morbidity and mortality. GBV encompasses a wide range of harmful acts, including physical, sexual, and psychological violence, perpetrated by intimate partners or family members. In India, despite legislative efforts to protect women, GBV-related deaths continue to be underreported or misclassified as accidents or suicides, masking the true extent of the problem. Data from the National Family Health Survey-5 (published in December 2021) reveals that 29.3% of ever-married women in India have experienced spousal violence. However, many deaths, particularly those involving burns, poisoning, or hanging, remain misclassified due to sociocultural stigma, inadequate investigation, or pressure from relatives. As a result, the true burden of GBV-related deaths is difficult to ascertain, hindering effective policy implementation and interventions. The United Nations defines violence against women as “ any act of gender-based violence that results in, or is likely to result in, physical, sexual, or psychological harm or suffering to women, including threats of such acts, coercion, or arbitrary deprivation of liberty, whether occurring in public or private life .” This definition emphasizes the multifaceted nature of GBV, which often remains hidden within domestic settings, manifesting as intentional homicides or suicides that are the result of prolonged psychological abuse. In India, cultural practices such as dowry contribute to a significant number of unnatural deaths among women, often reported as accidental burns or suicides, despite evidence of foul play. Dowry-related violence, for instance, accounts for a substantial portion of GBV-related deaths, particularly in rural areas, where sociocultural pressures to conform to traditional gender roles are stronger. These deaths often go unrecognized as GBV due to a lack of investigative rigor, further complicating data collection and policy formulation. This study aimed to address this critical gap by examining the prevalence and characteristics of GBV-related deaths among women. By analyzing 5 years of autopsy data from a medical teaching institution in Mumbai, we seek to determine the proportion of deaths attributable to GBV, focusing on cases misclassified as accidents or suicides. This study further explores the challenges in reporting and investigating such deaths, advocating for enhanced data collection and policy reform to mitigate the impact of GBV on women’s lives. Study design and setting This retrospective cross-sectional study was conducted at a tertiary hospital in Mumbai. The study period spanned from May 2017 to April 2022. Study population and sampling The sampling frame included records of autopsies performed on women, girls, and non-binary individuals who died due to unnatural causes within the specified period. A convenience sampling technique was employed. Inclusion criteria were autopsies on individuals with a history suggestive of GBV, including deaths certified as unnatural, of undetermined intention, accidental exposure, or intoxication, and those with injuries indicative of GBV. Cases with discrepancies between victim statements, post-mortem examination findings, and contextual information were included to identify patterns suggestive of intentional harm, even if initially classified as accidental. Exclusion criteria included deaths from natural causes, chronic or mental illnesses, and other pathological conditions. Accidental deaths such as falls, train accidents, or road crashes were reviewed for indications of GBV and excluded if such indicators were absent. Data collection A data collection tool was developed to capture household and individual characteristics, details of the deceased, mechanism and manner of injury, and perpetrator information. Data were sourced from autopsy records, police statements, inquest reports, and chargesheets. Data extraction involved translating records from Marathi to English, and all relevant information was compiled and verified against available chargesheets or FIRs. Missing information was noted. Data were entered into an Excel spreadsheet for analysis. Descriptive statistical measures such as mean, median, and proportions were used for data analysis. Ethical considerations The study protocol was reviewed and approved by the institutional ethics committee. Due to the retrospective nature of the study and the use of anonymized data, informed consent from the relatives of the deceased was waived. Ethical guidelines adhered to include ICH-GCP, CDSCO-GCP, the Declaration of Helsinki, and ICMR 2006 guidelines. The study followed guidelines for structured abstracts, data management practices, and adherence to ethical standards to enhance data collection and reporting accuracy. This retrospective cross-sectional study was conducted at a tertiary hospital in Mumbai. The study period spanned from May 2017 to April 2022. The sampling frame included records of autopsies performed on women, girls, and non-binary individuals who died due to unnatural causes within the specified period. A convenience sampling technique was employed. Inclusion criteria were autopsies on individuals with a history suggestive of GBV, including deaths certified as unnatural, of undetermined intention, accidental exposure, or intoxication, and those with injuries indicative of GBV. Cases with discrepancies between victim statements, post-mortem examination findings, and contextual information were included to identify patterns suggestive of intentional harm, even if initially classified as accidental. Exclusion criteria included deaths from natural causes, chronic or mental illnesses, and other pathological conditions. Accidental deaths such as falls, train accidents, or road crashes were reviewed for indications of GBV and excluded if such indicators were absent. A data collection tool was developed to capture household and individual characteristics, details of the deceased, mechanism and manner of injury, and perpetrator information. Data were sourced from autopsy records, police statements, inquest reports, and chargesheets. Data extraction involved translating records from Marathi to English, and all relevant information was compiled and verified against available chargesheets or FIRs. Missing information was noted. Data were entered into an Excel spreadsheet for analysis. Descriptive statistical measures such as mean, median, and proportions were used for data analysis. The study protocol was reviewed and approved by the institutional ethics committee. Due to the retrospective nature of the study and the use of anonymized data, informed consent from the relatives of the deceased was waived. Ethical guidelines adhered to include ICH-GCP, CDSCO-GCP, the Declaration of Helsinki, and ICMR 2006 guidelines. The study followed guidelines for structured abstracts, data management practices, and adherence to ethical standards to enhance data collection and reporting accuracy. During the study period, a total of 6190 autopsies were conducted, of which 1467 were female cases. Of these female autopsy cases, 57.3% (840) were determined to have died from unnatural causes. GBV was identified in 12.3% (181 cases) of these unnatural deaths. Of the 181 cases with suspected GBV, 23% (42 cases) had a documented history of violence based on statements from victims or their relatives, while the remaining 77% (139 cases) exhibited signs of potential GBV, such as discrepancies in injury sites, patterns, circumstances, autopsy findings, and statements from relatives, despite lacking a clear history of violence. No cases involving non-binary individuals or persons with disabilities were identified. illustrates the year-wise distribution of deaths attributed to GBV. Of the 181 GBV cases, 48% (86 cases) were suicides, 47% (85 cases) were accidents, and 6% (10 cases) were homicides. In terms of residence, 77% (139 cases) of the victims lived in urban areas, while 23% (42 cases) resided in rural areas. Deaths occurred in home or private spaces (99%, 179 cases), with only 1% (2 cases) occurring outside the home. Among urban residents, 50% (69 cases) died by suicide, whereas in rural areas, 57% (24 cases) died due to accidents . shows the age distribution. Out of the 181 cases, 75% (136 cases) were aged 15–44 years, with the highest proportion (45%) being between 15 and 29 years. The mean age of the victims was 34.8 years, with a 95% confidence interval ranging from 32.30 to 37.32. The median age was 30.5 years (age range: 11 months–87 years). When considering with manner of death, the median age of victims who died by suicide was 28 years, accidental deaths was 32 years, and homicides was 24.5 years. Regarding marital and pregnancy status, 67% (121 cases) of victims were married, 29% (53) were unmarried, and 4% (7 cases) were either widowed or divorced. Among the married victims, 4% (5 cases) were pregnant at the time of death. illustrates the nature of injuries in GBV cases. Burns were the leading cause of death, accounting for 58% (105 cases) of the deaths, followed by hanging in 20% (36 cases) and poisoning in 16% (29 cases). In addition, 3% (5 cases) were reported as deaths due to fall from a height, and 3% (6) resulted from aggravated assaults. shows that fire or flames were the most prevalent method of killing in accidental cases (75% (64 cases)). For suicides, drugs and chemical substances were used in 51% (44 cases), while bodily force (suicidal jump and hanging) accounted for 48% (41 cases). Half of all homicide deaths 50% (5 cases) involved drugs and chemicals. The underlying reasons for acts of violence in 114 cases were studied. Of these, 58% (66 cases) were related to marital problems, 29% (33 cases) to family issues, and 13% (15 cases) to intimate relationships or affairs. Marital disputes and family issues were identified as primary factors accounting for 87% (99 cases) of all GBV-related deaths, while unsuccessful intimate relationships contributed to 13% (15 cases) of the deaths. In terms of perpetrators, husbands or intimate partners were responsible in 61% (69 cases) of incidents. Other family members were identified in 35% (40 cases), while both the husband and a family member were involved in 4% (5 cases) of cases. shows the post-incident survival duration of victims. Of the 181 cases, 21% (46 cases) were brought dead to the hospital. Victims of hanging and jumping typically survived less than a day. Assault victims survived for an average of 3 days (range: 0–16 days), poison victims survived 5 days (range: 0–43 days), and burn victims survived 6 days (range: 0–102 days). In 37% (67 cases), the reason for death remains unclear due to fragmented data. There were discrepancies in post-mortem examination findings (e.g. injury site or pattern) when compared to history provided by the relatives or the information accessed from the police reports. summarizes the sources of variables and missing data in the study. Significant gaps in sociodemographic data related to both victims and perpetrators were noted in the autopsy and police reports, highlighting the data gaps. The study examines the prevalence and characteristics of deaths resulting from GBV in females of all ages, including non-binary individuals, based on autopsy reports. This study underscores the alarming prevalence of 12.3% of GBV as a significant contributor to female mortality. Our findings highlight that many deaths, categorized as accidental or suicidal, are rooted in GBV, pointing to the hidden nature of this violence. Our analysis also revealed that most victims were young, between the ages of 15–29 years, highlighting the heightened vulnerability of this demographic to intimate partner violence and marital conflict. These deaths were often the result of prolonged psychological or physical abuse, further contributing to the underreporting of violence within domestic settings. This pattern is consistent with other research that identifies younger, married women as disproportionately affected by GBV, particularly in cases involving dowry disputes. In our study, the deaths occurred in home or private spaces (99%, 179 cases), with only 1% (2 cases) occurring outside the home, aligning with other research findings. When considering the manner of death, the median age of victims who died by suicide was 28 years, accidental deaths was 32 years, and homicides was 24.5 years. This aligns with other studies that have shown that most victims of unnatural deaths are typically 21–40 years old. Regarding marital and pregnancy status, 67% (121 cases) of victims were married, 29% (53) were unmarried, and 4% (7 cases) were either widowed or divorced, which is consistent with findings from another research. Burns were the leading cause of death, accounting for 58% (105 cases) of the deaths, consistent with other research findings. The high prevalence of burns, accounting for 58% of deaths, is particularly concerning as burns are frequently reported as accidental, particularly in dowry-related incidents. In the context of India, dowry-related deaths, often referred to as bride-burning, are a significant issue across all social classes. When dowry demands are not met, it can lead to severe marital conflict, and in some instances, women may choose suicide as a means of escaping such brutality. Unfortunately, burn injuries are frequently reported as accidents and are not thoroughly investigated, which obscures the underlying violence and the true nature of these deaths. This underscores the need for improved investigative protocols to differentiate between accidental and intentional burns. The prevalence of hanging (20%) and poisoning (16%) as methods of death also mirrors findings from other studies on unnatural deaths, where these methods are commonly misreported. Half of all homicide deaths (50% (5 cases)) involved drugs and chemicals, consistent with other studies showing the prevalence of accidents and suicides. While this finding aligns with previous research, especially in the context of dowry-related violence, our study also brings attention to the systemic underreporting and misclassification of such deaths, which obscures the true scope of GBV. In terms of perpetrators, husbands or intimate partners were responsible in 61% (69 cases) of incidents. Other family members were identified in 35% (40 cases), while both the husband and a family member were involved in 4% (5 cases) of cases, consistent with findings from other studies. A key finding of this study is the critical role played by intimate partners, who were responsible for 61% of the GBV-related deaths. The majority of incidents occurred in private homes, where violence remains hidden from public scrutiny. This reflects the broader issue of domestic violence, where victims often suffer in isolation, exacerbated by cultural norms that discourage reporting. The significant role of other family members (35%) as perpetrators further illustrates the embedded nature of violence within domestic and familial structures. This is consistent with other studies, which also emphasizes that marriage, marital discord, and domestic environments often exacerbate the risk of GBV-related deaths. One of the novel contributions of this study is its identification of the underlying violence in deaths often classified as accidents or suicides. Most unnatural deaths in women are often classified as accidents or suicides due to families refraining from reporting foul play or fear of social stigma. The fact that many victims survive for days after sustaining burn injuries, compared to more immediate fatalities from hanging, poisoning, fall from height, or assault, highlights a potential window of opportunity for intervention into the circumstances of death as many cases of GBV remain undetected due to superficial categorization. There were discrepancies in post-mortem examination findings (e.g., injury site or pattern) when compared to history provided by the relatives or the information accessed from the police reports, as highlighted by the UNODC report on gender-related killing of women and girls, 2021 where 4 out of 10 cases lack such information. The absence of contextual information in a substantial portion of cases is a limitation as it prevents a more nuanced understanding of the social and interpersonal dynamics leading to these deaths. This lack of data is not unique to this study; it reflects a broader, global issue in GBV research, where the social stigma associated with reporting violence and the fear of legal or societal repercussions result in underreporting. Compared to other research, this study strengthens the argument for improved investigation protocols, particularly in cases of female deaths labeled as accidental or suicidal. A key limitation of our study is the reliance on forensic autopsy data, which may not fully capture the scope of GBV-related deaths, particularly in cases where deaths are misclassified due to reporting biases. Furthermore, the absence of detailed contextual information in a significant number of cases limits the ability to fully explore the underlying causes of violence, which remains a challenge in many GBV studies worldwide. In addition, cultural, and societal biases play a role in both the reporting and investigation of these deaths, potentially skewing the data. Our findings have significant practical implications. There is an urgent need for forensic and law enforcement agencies to develop standardized protocols for investigating deaths suspected to involve GBV. Training in recognizing signs of underlying violence, particularly in cases initially reported as accidental or suicidal, is critical. Moreover, improving the quality and consistency of death reporting through enhanced data collection practices is vital. Institutional reforms aimed at strengthening ethical data collection and capacity building among stakeholders could lead to more reliable estimates of GBV-related mortality, thereby influencing policy and prevention strategies. In conclusion, the study highlights the pervasive nature of GBV, particularly in intimate partner relationships, and stresses the need for systemic changes in how these cases are identified, reported, and addressed. It further emphasizes the critical need for improved investigative protocols and comprehensive data collection to reveal the true extent of GBV-related deaths. Enhancing the quality of death reporting is essential for generating reliable estimates of the true impact of GBV. An integrated approach, involving both legal and public health frameworks, is essential to reducing GBV-related mortality. While our findings are specific to a particular region, they reflect a broader global trend of underreporting and misclassification of GBV-related deaths. Future research should focus on cross-national comparisons and longitudinal studies that investigate the long-term health impacts of GBV. Conflicts of interest There are no conflicts of interest. There are no conflicts of interest. |
Metabolomic characteristics of cord blood from neonates with hyperkalemia after antenatal exposure to ritodrine and magnesium sulfate | 6b830820-cef7-4a3d-bbf7-f12416e39772 | 11739404 | Biochemistry[mh] | Selective beta2-adrenergic receptor (β2-AR) agonists, such as terbutaline and ritodrine, have been historically used as uterine contraction inhibitors , . The U.S. Food and Drug Administration issued a warning against prolonged use (beyond 48–72 h) of selective β2-AR agonists in pregnant women for the prevention of preterm labor due to an increased risk of severe cardiovascular side effects for both the mother and the fetus in 2011. However, in Japan, the use of ritodrine (with caution with respect to its potential side effects) was widely applied as a treatment option for preterm labor threatened patients based on the guidelines published by the Japan Society of Obstetrics and Gynecology until 2017. The current Japanese guidelines published in 2020 and 2023 still include this treatment. Magnesium sulfate (MgSO 4 ) has been widely applied to prevent seizures in women with preeclampsia or eclampsia . MgSO 4 is recommended for use in women at risk of giving birth at less than 32 weeks of gestation for neuroprotection of their infants . In Japan, the use of MgSO 4 as a tocolytic agent has been included in the guidelines since 2008. Consequently, prolonged tocolytic therapy involving the combined use of ritodrine and MgSO 4 has been used in Japan when the control of uterine contractions is not achievable with either ritodrine or MgSO 4 alone , , . Recent reports have highlighted an increased risk of hyperkalemia in neonates born to mothers treated with ritodrine and MgSO 4 during pregnancy , . Potassium, the major intracellular cation, is essential for maintaining vital physiological functions . Neonates must maintain a net positive potassium balance to support their growth, primarily by minimizing renal potassium losses . Consequently, neonatal plasma potassium concentrations are higher than those observed in children and adults . Preterm infants, particularly those born earlier in gestation, are especially vulnerable to elevated extracellular potassium concentrations due to additional risk factors such as acute kidney injury, hypotension, acidosis, hemorrhagic disorders, and transfusion therapy, which can lead to life-threatening complications . The incidence of neonatal hyperkalemia is notably high in extremely low birth weight infants, with rates ranging from 25 to 50%, and nearly all infants born before 25 weeks of gestation develop hyperkalemia – . Although the incidence of hyperkalemia in neonates born between 32 and 36 weeks of gestation is relatively low (approximately 8%), exposure to both ritodrine and MgSO₄ significantly increases its incidence . Severe hyperkalemia, with potassium levels exceeding 9 mEq/L, was associated with arrhythmias in 60% of cases . Sychlowy et al. reported that only one of seven infants with hyperkalemia-induced arrhythmias survived . In addition, hyperkalemia can lead to intracranial complications such as intraventricular hemorrhage and periventricular leukomalacia . Given that ritodrine and MgSO 4 are administered to women at risk of preterm labor, these drugs may also be independent risk factors for hyperkalemia in preterm neonates. Recognizing the risk associated with prenatal exposure to both ritodrine and MgSO 4 is crucial for predicting extremely high potassium levels and the resulting fatal arrhythmias. The underlying mechanisms of neonatal hyperkalemia have scarcely been investigated due to the lack of concurrent administration of ritodrine and MgSO 4 to pregnant women in the United States and Europe. The identification of markers that enable an early and accurate diagnosis of neonatal hyperkalemia resulting from exposure to ritodrine and MgSO 4 could potentially assist in stratifying risk groups, thus aiding in the formulation of appropriate therapeutic strategies to improve patient management. Metabolomics provides information on all biochemical activities, including substrates, products, and cofactors, of all enzymatic reactions in a specific biological system at a single time point . Changes in the serum metabolome have been reported in neonatal conditions such as infections, perinatal asphyxia, cardiac diseases, and renal disorders, and metabolomics approaches are valuable for the early diagnosis and assessment of the severity of neonatal diseases . The metabolism of fetuses may be affected by the administration of ritodrine and MgSO 4 in pregnant women; hence, subtle changes in the serum metabolome of umbilical cord blood may contribute to elucidating the mechanisms of neonatal hyperkalemia that develops in association with the combined use of ritodrine and MgSO 4 . In this study, we conducted a metabolomics analysis of the serum in umbilical cord blood to elucidate metabolic changes in neonates with antenatal exposure ritodrine and MgSO 4 . Additionally, to identify predictors of neonatal hyperkalemia, we compared the clinical symptoms and serum metabolites in the umbilical cord blood of neonates born to pregnant women who received ritodrine and MgSO 4 with those of neonates born to mothers who did not receive such treatment. Perinatal characteristics In this study, we investigated the clinical characteristics of neonatal hyperkalemia caused by antenatal exposure to ritodrine or MgSO 4 . This investigation targeted preterm infants at 24–36 weeks of gestational age, a period when treatment for threatened preterm labor is administered. Throughout the designated research period, 563 infants were admitted to our hospital’s neonatal intensive care unit (NICU), among whom 241 were born at 24–36 weeks of gestation (Supplementary Figure ). To minimize the influence of confounding factors due to uncertain prior treatments, we excluded cases with uncertain details regarding the administration of specific drugs, such as tocolytic therapies, including their doses and duration. Consequently, 99 were excluded from the study for the following reasons: unknown maternal drug history (n = 71), presence of multiple malformation syndromes or chromosomal abnormalities (n = 8), hemolytic anemia or intracranial hemorrhage (n = 19), and a maternal history of anticancer drug use. Thus, a final cohort of 142 infants was included in the study. Among these infants, 68 mothers did not receive antenatal therapy, 36 mothers received antenatal ritodrine, 15 mothers received antenatal MgSO 4 , and 23 mothers received antenatal ritodrine and MgSO 4 before delivery. Data on the administration of ritodrine and MgSO 4 in the four groups (neither ritodrine nor MgSO 4 , ritodrine alone, MgSO 4 alone, and both ritodrine and MgSO 4 ) are shown in Supplementary Table . Regarding the administration of ritodrine to mothers, the both ritodrine and MgSO 4 group exhibited a longer administration duration and a higher total dosage than the ritodrine alone group. Among the cases of MgSO 4 administration to mothers, the both ritodrine and MgSO 4 group had a longer administration duration and a higher total dosage than the MgSO 4 alone group. Maternal serum magnesium concentrations were similar between the MgSO 4 alone group and the both ritodrine and MgSO 4 group (Table ). The perinatal characteristics of the four groups are shown in Table . Although no significant differences in serum K + levels were observed immediately after birth among the 4 groups, the highest serum K + level within 48 h after birth was significantly higher in the both ritodrine and MgSO 4 group compared to the neither ritodrine nor MgSO 4 group (Fig. ). Regarding the characteristics of the infants, significant differences were found among the 4 groups in sex, gestational age, birth weight, rate of twin pregnancies, incidence of premature rupture of membranes (PROM), Apgar score at 5 min, urine output within 48 h post-birth, and rate of insulin/glucose therapy. Blood tests conducted immediately after birth revealed significant intergroup differences in albumin (ALB), total bilirubin (T-Bil), alkaline phosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH), sodium (Na), chloride (Cl), calcium (Ca), phosphorus (P), hemoglobin (Hb), and platelet counts (PLT). Regarding maternal characteristics, the incidence of gestational hypertension was significantly different between the groups. Given that MgSO 4 is effective in managing preeclampsia and providing neuroprotection in extremely low birth weight infants, infants in the MgSO 4 alone group had significantly lower gestational ages and birth weights, and their mothers had a significantly higher frequency of gestational hypertension. To minimize the confounding effects of prematurity due to lower gestational age and to identify parameters strongly associated with exposure to ritodrine and MgSO 4 , we conducted an analysis focusing solely on infants born at 32–36 weeks of gestation (Supplementary Table ). This analysis was further confirmed by sensitivity analyses, which excluded groups with combined ritodrine and MgSO 4 treatment durations of < 3 days (n = 10) and < 14 days (n = 10) (Supplementary Table ). As a result, significant differences were consistently observed in peak serum K + levels within 48 h post-birth, the rate of twin pregnancies and PROM, urine output within 48 h post-birth, and serum levels of T-Bil, ALP, Na, Cl, P, Hb, and PLT immediately after birth. In twin pregnancies, uterine contractions were often poorly controlled, leading to a higher rate of combined use of ritodrine and MgSO 4 . Additionally, for cases without membrane rupture, long-term use of tocolytics up to 36 weeks resulted in a lower PROM rate in the both ritodrine and MgSO 4 group compared to those in the neither ritodrine nor MgSO 4 group or the ritodrine alone group. Factors associated with serum potassium levels in neonates A multiple regression analysis was conducted to explore factors that predict the severity of neonatal hyperkalemia in neonates exposed to ritodrine and MgSO 4 . The highest potassium level within 48 h after birth was used as the dependent variable, and clinical characteristics that were significantly different between the four groups were used as independent variables (Table ). The results of the analysis showed that the factor most related to the severity of neonatal hyperkalemia was the serum phosphorus level immediately after birth (β = 0.288, p < 0.001). Alterations in metabolism resulting from antenatal ritodrine and MgSO 4 as revealed by a multivariate analysis of metabolites To determine the mechanism underlying the induction of neonatal hyperkalemia in association with the maternal administration of antenatal ritodrine and MgSO 4 , serum metabolites in umbilical cord blood collected from newborns born at 24–36 weeks of gestation were analyzed using GC-MS/MS (Supplementary Figure and Supplementary Table ). As a result, 293 metabolites were detected in all samples (Supplementary Table ). The orthogonal partial least-squares discriminant analysis (OPLS-DA) model was applied for the multivariate analysis of metabolites. The resulting score scatter plot presented distinctive clusters among the 4 groups (neither ritodrine nor MgSO 4 , ritodrine alone, MgSO 4 alone, and both ritodrine and MgSO 4 ) (Fig. ). Pairwise comparisons were conducted to identify potential distinctive predictors associated with neonatal hyperkalemia triggered by exposure to both ritodrine and MgSO 4 within a multitude of metabolites identified in the serum of the umbilical cord blood. Specifically, comparisons were made between the neither ritodrine nor MgSO 4 in conjunction with the ritodrine alone group (Fig. A), as well as between the neither ritodrine nor MgSO 4 group in conjunction with the MgSO 4 alone group (Fig. B), and between the neither ritodrine nor MgSO 4 group in conjunction with the both ritodrine and MgSO 4 group (Fig. C). Additionally, pairwise comparisons were performed between the ritodrine alone group and the both ritodrine and MgSO 4 group (Supplementary Figure A), and between the MgSO 4 alone group and the both ritodrine and MgSO 4 group (Supplementary Figure B). A clustering analysis between these two sets of groups revealed substantial dissimilarities, implying significant metabolite diversity among these groups. An S-plot analysis (Fig. D–F and Supplementary Figure C and D) and variable importance in projection (VIP) values identified 16 significant metabolites in the ritodrine alone group, 4 in the MgSO 4 alone group, and 18 in the both ritodrine and MgSO 4 group (Table and Supplementary Table ). A more comprehensive analysis of the pathways and networks most closely associated with the metabolic changes induced by exposure to both ritodrine and MgSO 4 was performed using a pathway enrichment analysis in MetaboAnalyst 5.0. The metabolic pathways are presented in Fig. , Supplementary Figure , and Supplementary Table , based on the metabolites that showed an increase in the serum of umbilical cord blood. The pathways related to amino acid biosynthesis and carbohydrate metabolism were notably influenced in the ritodrine alone group. Beta-alanine metabolism involving spermine was affected in the MgSO 4 alone group. In the both ritodrine and MgSO 4 group, the pathways associated with amino acid biosynthesis, carbohydrate metabolism, and glycolysis/gluconeogenesis were primarily affected. This group exhibited a trend similar to that observed in the ritodrine alone group. To further elucidate the specific metabolic pathways arising from exposure to both ritodrine and MgSO 4 , we assessed the metabolite pathways with significant differences between the both ritodrine and MgSO 4 group and the ritodrine alone group. As a result, the citrate cycle emerged as the most affected metabolic pathway in the both ritodrine and MgSO 4 group in comparison to the ritodrine alone group (Supplementary Figure and Supplementary Table ). The citrate cycle did not exhibit significant activation in the magnesium sulfate monotherapy group, suggesting a synergistic effect resulting from the combined effect of ritodrine and MgSO 4 . Correlation between metabolite levels and postnatal peak potassium levels To identify potential predictors of the postnatal elevation of serum potassium levels in neonates born to mothers administered both ritodrine and MgSO 4 , we examined 18 specific metabolites showing distinctive increases in both ritodrine and MgSO 4 group compared to the neither ritodrine nor MgSO 4 group, along with their correlation with peak potassium values within 48 h after birth. Employing the peak potassium level within 48 h postnatally as the dependent variable, a multiple regression analysis was conducted with these specific metabolites as independent variables. The analysis revealed that citric acid was the factor most strongly associated with the severity of neonatal hyperkalemia (Supplementary Table ). Furthermore, we validated the correlation between peak potassium levels (which showed the most significant association in clinical tests) and phosphorus levels with citric acid. The relationships among peak potassium, phosphorus, and citric acid levels were quantified using Pearson’s correlation coefficients (Table ). Citric acid levels exhibited the highest positive correlation with peak potassium levels (r = 0.551, p < 0.01), indicating a positive correlation. In addition, citric acid levels were positively correlated with phosphorus levels (r = 0.491, P < 0.01). Based on these findings, serum citric acid and phosphorus levels immediately after birth are considered to be potential predictors of neonatal hyperkalemia. To further validate citric acid and phosphate as potential biomarkers of ritodrine-induced and MgSO 4 -induced hyperkalemia, we performed a receiver operating characteristic (ROC) curve analysis. Supplementary Figure shows the ROC curves for citric acid and phosphate in the prediction of hyperkalemia, defined as a maximum potassium level of 6 mEq/l postnatally. Citric acid demonstrated an area under the curve (AUC) of 0.782, indicating a good discriminatory capacity. A cutoff value for citric acid, set at 1.26 times the average peak area obtained from a GC/MS analysis, yielded 66.67% sensitivity and 95.83% specificity, with a PPV of 85.7 and an NPV of 88.5. Phosphate levels had an AUC of 0.755, suggesting a fair discriminatory capacity. Using a cut-off value of > 6.8 mg/dl, phosphate levels achieved 55.56% sensitivity and 91.67% specificity, with a PPV of 71.4, and an NPV of 84.6. These findings indicate that both citric acid and phosphate are promising biomarkers of hyperkalemia in the context of ritodrine and MgSO 4 , with citric acid having higher specificity and overall predictive accuracy. In this study, we investigated the clinical characteristics of neonatal hyperkalemia caused by antenatal exposure to ritodrine or MgSO 4 . This investigation targeted preterm infants at 24–36 weeks of gestational age, a period when treatment for threatened preterm labor is administered. Throughout the designated research period, 563 infants were admitted to our hospital’s neonatal intensive care unit (NICU), among whom 241 were born at 24–36 weeks of gestation (Supplementary Figure ). To minimize the influence of confounding factors due to uncertain prior treatments, we excluded cases with uncertain details regarding the administration of specific drugs, such as tocolytic therapies, including their doses and duration. Consequently, 99 were excluded from the study for the following reasons: unknown maternal drug history (n = 71), presence of multiple malformation syndromes or chromosomal abnormalities (n = 8), hemolytic anemia or intracranial hemorrhage (n = 19), and a maternal history of anticancer drug use. Thus, a final cohort of 142 infants was included in the study. Among these infants, 68 mothers did not receive antenatal therapy, 36 mothers received antenatal ritodrine, 15 mothers received antenatal MgSO 4 , and 23 mothers received antenatal ritodrine and MgSO 4 before delivery. Data on the administration of ritodrine and MgSO 4 in the four groups (neither ritodrine nor MgSO 4 , ritodrine alone, MgSO 4 alone, and both ritodrine and MgSO 4 ) are shown in Supplementary Table . Regarding the administration of ritodrine to mothers, the both ritodrine and MgSO 4 group exhibited a longer administration duration and a higher total dosage than the ritodrine alone group. Among the cases of MgSO 4 administration to mothers, the both ritodrine and MgSO 4 group had a longer administration duration and a higher total dosage than the MgSO 4 alone group. Maternal serum magnesium concentrations were similar between the MgSO 4 alone group and the both ritodrine and MgSO 4 group (Table ). The perinatal characteristics of the four groups are shown in Table . Although no significant differences in serum K + levels were observed immediately after birth among the 4 groups, the highest serum K + level within 48 h after birth was significantly higher in the both ritodrine and MgSO 4 group compared to the neither ritodrine nor MgSO 4 group (Fig. ). Regarding the characteristics of the infants, significant differences were found among the 4 groups in sex, gestational age, birth weight, rate of twin pregnancies, incidence of premature rupture of membranes (PROM), Apgar score at 5 min, urine output within 48 h post-birth, and rate of insulin/glucose therapy. Blood tests conducted immediately after birth revealed significant intergroup differences in albumin (ALB), total bilirubin (T-Bil), alkaline phosphatase (ALP), creatine kinase (CK), lactate dehydrogenase (LDH), sodium (Na), chloride (Cl), calcium (Ca), phosphorus (P), hemoglobin (Hb), and platelet counts (PLT). Regarding maternal characteristics, the incidence of gestational hypertension was significantly different between the groups. Given that MgSO 4 is effective in managing preeclampsia and providing neuroprotection in extremely low birth weight infants, infants in the MgSO 4 alone group had significantly lower gestational ages and birth weights, and their mothers had a significantly higher frequency of gestational hypertension. To minimize the confounding effects of prematurity due to lower gestational age and to identify parameters strongly associated with exposure to ritodrine and MgSO 4 , we conducted an analysis focusing solely on infants born at 32–36 weeks of gestation (Supplementary Table ). This analysis was further confirmed by sensitivity analyses, which excluded groups with combined ritodrine and MgSO 4 treatment durations of < 3 days (n = 10) and < 14 days (n = 10) (Supplementary Table ). As a result, significant differences were consistently observed in peak serum K + levels within 48 h post-birth, the rate of twin pregnancies and PROM, urine output within 48 h post-birth, and serum levels of T-Bil, ALP, Na, Cl, P, Hb, and PLT immediately after birth. In twin pregnancies, uterine contractions were often poorly controlled, leading to a higher rate of combined use of ritodrine and MgSO 4 . Additionally, for cases without membrane rupture, long-term use of tocolytics up to 36 weeks resulted in a lower PROM rate in the both ritodrine and MgSO 4 group compared to those in the neither ritodrine nor MgSO 4 group or the ritodrine alone group. A multiple regression analysis was conducted to explore factors that predict the severity of neonatal hyperkalemia in neonates exposed to ritodrine and MgSO 4 . The highest potassium level within 48 h after birth was used as the dependent variable, and clinical characteristics that were significantly different between the four groups were used as independent variables (Table ). The results of the analysis showed that the factor most related to the severity of neonatal hyperkalemia was the serum phosphorus level immediately after birth (β = 0.288, p < 0.001). 4 as revealed by a multivariate analysis of metabolites To determine the mechanism underlying the induction of neonatal hyperkalemia in association with the maternal administration of antenatal ritodrine and MgSO 4 , serum metabolites in umbilical cord blood collected from newborns born at 24–36 weeks of gestation were analyzed using GC-MS/MS (Supplementary Figure and Supplementary Table ). As a result, 293 metabolites were detected in all samples (Supplementary Table ). The orthogonal partial least-squares discriminant analysis (OPLS-DA) model was applied for the multivariate analysis of metabolites. The resulting score scatter plot presented distinctive clusters among the 4 groups (neither ritodrine nor MgSO 4 , ritodrine alone, MgSO 4 alone, and both ritodrine and MgSO 4 ) (Fig. ). Pairwise comparisons were conducted to identify potential distinctive predictors associated with neonatal hyperkalemia triggered by exposure to both ritodrine and MgSO 4 within a multitude of metabolites identified in the serum of the umbilical cord blood. Specifically, comparisons were made between the neither ritodrine nor MgSO 4 in conjunction with the ritodrine alone group (Fig. A), as well as between the neither ritodrine nor MgSO 4 group in conjunction with the MgSO 4 alone group (Fig. B), and between the neither ritodrine nor MgSO 4 group in conjunction with the both ritodrine and MgSO 4 group (Fig. C). Additionally, pairwise comparisons were performed between the ritodrine alone group and the both ritodrine and MgSO 4 group (Supplementary Figure A), and between the MgSO 4 alone group and the both ritodrine and MgSO 4 group (Supplementary Figure B). A clustering analysis between these two sets of groups revealed substantial dissimilarities, implying significant metabolite diversity among these groups. An S-plot analysis (Fig. D–F and Supplementary Figure C and D) and variable importance in projection (VIP) values identified 16 significant metabolites in the ritodrine alone group, 4 in the MgSO 4 alone group, and 18 in the both ritodrine and MgSO 4 group (Table and Supplementary Table ). A more comprehensive analysis of the pathways and networks most closely associated with the metabolic changes induced by exposure to both ritodrine and MgSO 4 was performed using a pathway enrichment analysis in MetaboAnalyst 5.0. The metabolic pathways are presented in Fig. , Supplementary Figure , and Supplementary Table , based on the metabolites that showed an increase in the serum of umbilical cord blood. The pathways related to amino acid biosynthesis and carbohydrate metabolism were notably influenced in the ritodrine alone group. Beta-alanine metabolism involving spermine was affected in the MgSO 4 alone group. In the both ritodrine and MgSO 4 group, the pathways associated with amino acid biosynthesis, carbohydrate metabolism, and glycolysis/gluconeogenesis were primarily affected. This group exhibited a trend similar to that observed in the ritodrine alone group. To further elucidate the specific metabolic pathways arising from exposure to both ritodrine and MgSO 4 , we assessed the metabolite pathways with significant differences between the both ritodrine and MgSO 4 group and the ritodrine alone group. As a result, the citrate cycle emerged as the most affected metabolic pathway in the both ritodrine and MgSO 4 group in comparison to the ritodrine alone group (Supplementary Figure and Supplementary Table ). The citrate cycle did not exhibit significant activation in the magnesium sulfate monotherapy group, suggesting a synergistic effect resulting from the combined effect of ritodrine and MgSO 4 . To identify potential predictors of the postnatal elevation of serum potassium levels in neonates born to mothers administered both ritodrine and MgSO 4 , we examined 18 specific metabolites showing distinctive increases in both ritodrine and MgSO 4 group compared to the neither ritodrine nor MgSO 4 group, along with their correlation with peak potassium values within 48 h after birth. Employing the peak potassium level within 48 h postnatally as the dependent variable, a multiple regression analysis was conducted with these specific metabolites as independent variables. The analysis revealed that citric acid was the factor most strongly associated with the severity of neonatal hyperkalemia (Supplementary Table ). Furthermore, we validated the correlation between peak potassium levels (which showed the most significant association in clinical tests) and phosphorus levels with citric acid. The relationships among peak potassium, phosphorus, and citric acid levels were quantified using Pearson’s correlation coefficients (Table ). Citric acid levels exhibited the highest positive correlation with peak potassium levels (r = 0.551, p < 0.01), indicating a positive correlation. In addition, citric acid levels were positively correlated with phosphorus levels (r = 0.491, P < 0.01). Based on these findings, serum citric acid and phosphorus levels immediately after birth are considered to be potential predictors of neonatal hyperkalemia. To further validate citric acid and phosphate as potential biomarkers of ritodrine-induced and MgSO 4 -induced hyperkalemia, we performed a receiver operating characteristic (ROC) curve analysis. Supplementary Figure shows the ROC curves for citric acid and phosphate in the prediction of hyperkalemia, defined as a maximum potassium level of 6 mEq/l postnatally. Citric acid demonstrated an area under the curve (AUC) of 0.782, indicating a good discriminatory capacity. A cutoff value for citric acid, set at 1.26 times the average peak area obtained from a GC/MS analysis, yielded 66.67% sensitivity and 95.83% specificity, with a PPV of 85.7 and an NPV of 88.5. Phosphate levels had an AUC of 0.755, suggesting a fair discriminatory capacity. Using a cut-off value of > 6.8 mg/dl, phosphate levels achieved 55.56% sensitivity and 91.67% specificity, with a PPV of 71.4, and an NPV of 84.6. These findings indicate that both citric acid and phosphate are promising biomarkers of hyperkalemia in the context of ritodrine and MgSO 4 , with citric acid having higher specificity and overall predictive accuracy. In this study, we demonstrated that the serum maximum potassium levels in newborns born to mothers administered antenatal ritodrine and MgSO 4 for preterm labor increased significantly within 48 h of birth, although their serum potassium levels were normal immediately after birth. Furthermore, we revealed that neonates exposed to both ritodrine and MgSO 4 exhibited enhanced metabolic pathways different from those observed in neonates exposed to ritodrine or MgSO 4 alone, using a comprehensive analysis of serum metabolites in umbilical cord blood. Despite changes in the fetal metabolism, unaltered potassium levels immediately after birth in infants exposed to ritodrine and MgSO 4 are presumably regulated by the abundance of potassium channels in the placenta . To support this finding, no correlation was found between pH and potassium levels in umbilical cord blood immediately after birth, despite acidosis increasing the concentration of potassium ions in blood in adults , . Recent research has reported that the combined use of ritodrine and MgSO 4 in mothers is a risk factor for hyperkalemia in preterm infants , . The findings of this study confirmed that the antenatal administration of ritodrine and MgSO 4 to mothers induced hyperkalemia in preterm infants. Furthermore, a metabolomics analysis revealed activation not only in amino acid biosynthesis, sugar metabolism, and glycolysis/gluconeogenesis, but also in a distinctive citrate cycle in the neonatal group exposed to both ritodrine and MgSO 4 . Therefore, this study suggests a synergistic effect of ritodrine and MgSO 4 , which can induce distinctive metabolic changes and trigger neonatal hyperkalemia. The significantly increased citric acid observed in the both ritodrine and MgSO 4 group was produced in the citrate cycle through aldol condensation of oxaloacetic acid and acetyl CoA, the final products of the preceding turn of the cycle . Acetyl-CoA originates from glucose through the glycolytic pathway . Ritodrine functions as a β2-AR agonist. The stimulation of hepatic β2 adrenergic receptors by ritodrine activates hepatic adenylate cyclase, promoting hepatic gluconeogenesis and glycogenesis, thereby inducing hyperglycemia , . Prolonged glucose stimulation induces citrate synthesis in the mitochondria via the glycolysis pathway, leading to increased cytoplasmic and extracellular citrate . The administration of MgSO 4 to pregnant women results in an increased concentration of magnesium within the fetal serum and intracellular spaces, which is facilitated by the ease with which MgSO 4 traverses the placenta . The elevation in intracellular magnesium levels facilitates the uptake of glucose into the cell via Glut4, a component of the glycolytic pathway . Additionally, magnesium serves as a cofactor for enzymes in the glycolysis pathway, including hexokinase, phosphofructokinase, phosphoglycerate kinase, and enolase , . In our study, neonates with antenatal exposure to both ritodrine and MgSO 4 exhibited significant activation of metabolic pathways, including the citrate cycle, branched-chain amino acids, alanine, and fatty acid synthesis, derived from metabolites generated during glycolysis. Therefore, the synergistic effect of both ritodrine and MgSO 4 suggests that they activate glycolysis and the derived metabolic routes. Glycolysis regulates cell membrane ion transporters (pumps, exchangers, and channels) and modulates the intracellular distribution of potassium ions . Adenosine triphosphate (ATP) is produced through three processes: glycolysis, the citrate cycle, and oxidative phosphorylation, starting from intracellular glucose . The cell membrane features Na–K ATPase and ATP-sensitive K channels, with an increase in intracellular ATP levels, leading to elevated intracellular potassium concentrations , . Generally, stimulation of β2 adrenergic receptors by ritodrine involves the activation of Na–K ATPase pumps on the cell membrane, facilitating the intracellular transition of potassium . The rebound following the cessation of ritodrine is known to cause the release of potassium from intracellular spaces into the serum – . As approximately 98% of potassium resides intracellularly, even a slight efflux of intracellular potassium results in a significant increase in serum potassium concentration . The rebound of potassium after the injection of insulin or thiopentone has also been reported to induce the uptake of potassium into cells, indicating that rapid changes in mechanisms regulating potassium distribution in the body are risk factors for hyperkalemia . In our study, neonates exposed to both ritodrine and MgSO 4 may have experienced an enhanced influx of potassium into cells due to the activation of Na–K ATPase pumps by ritodrine coupled with increased metabolic pathways related to glycolysis. The postulated etiology in our study is shown in Fig. . Therefore, neonates exposed to both ritodrine and MgSO 4 may have a heightened quantity of potassium redistribution from intracellular to serum compartments following the discontinuation of these medications, potentially increasing their susceptibility to postnatal hyperkalemia. The citric acid levels in umbilical cord blood serum were positively correlated with the highest potassium values within the first 48 h of life, suggesting its potential as a marker for the early detection of neonatal hyperkalemia (Table ). However, citric acid is not routinely analyzed in the blood of preterm infants immediately after birth. In our investigation, serum phosphorus levels immediately after birth demonstrated a positive correlation with citric acid levels in the serum of umbilical cord blood and the highest potassium values within the first 48 h after delivery (Table ). The presence of hypermagnesemia induced by MgSO 4 administration inhibits parathyroid hormone secretion and induces hyperphosphatemia in the fetus . Furthermore, ritodrine-induced insulin promotes the reabsorption of phosphate in the proximal tubules . Therefore, the elevation in serum levels of phosphorus and citric acid due to the influence of ritodrine and MgSO 4 can occur through different mechanisms. Consequently, serum levels of phosphorus and citric acid immediately after birth can independently serve as potential predictors of the onset of neonatal hyperkalemia. Neonatal hyperkalemia is generally recognized to occur in infants born at < 32 weeks of gestation and requires close monitoring of serum potassium levels over time . In contrast, babies born between 32 and 36 weeks of gestation have a lower incidence of hyperkalemia; however, exposure to ritodrine and MgSO 4 increases the risk of hyperkalemia . Due to the perceived low risk of hyperkalemia in infants born between 32 and 36 weeks of gestation, there may be a delay in recognizing hyperkalemia. Hyperkalemia can appear as early as 9 h after birth and peak around 26 h postpartum . In such cases, the evaluation of serum citric acid and phosphorus levels at birth could lead to more vigilant potassium monitoring, ensuring timely detection and treatment of hyperkalemia. Furthermore, predicting neonatal hyperkalemia could facilitate the early initiation of glucose-insulin therapy, a treatment for hyperkalemia, and assist in the decision making about the introduction of early caffeine therapy, which has recently been reported to have preventive effects against neonatal hyperkalemia . These early interventions may mitigate the risk of fatal arrhythmias due to hyperkalemia and potentially improve neonatal outcomes. The present study was associated with several limitations. First, it was a retrospective analysis conducted at a single institution, and was limited to the analysis of the serum of umbilical cord blood from a subset of cases. In this study, we limited our investigation to cases to minimize any bias due to treatment strategies for threatened preterm labor; however, treatment strategies that depended on individual physicians might affect the clinical and laboratory data. Additionally, pregnant women who received both ritodrine and MgSO 4 had longer durations of drug administration and higher dosages than those who received ritodrine or MgSO 4 alone. This discrepancy in the drug administration parameters may have influenced the outcomes of this study. Furthermore, the study did not assess neonatal blood magnesium concentrations or urinary potassium excretion rates. Additionally, the effects of diuretics and steroid hormones, which can influence the maternal, fetal, and neonatal serum potassium levels, were not considered in this study. Prospective randomized controlled trials are necessary to eliminate potential bias and elucidate the remaining issues. The present study revealed that neonates exposed to both ritodrine and MgSO 4 may experience synergistic effects of these drugs, leading to the activation of glycolysis and the derived metabolic routes, potentially inducing hyperkalemia. Citric acid levels in the serum of umbilical cord blood or serum phosphorus levels immediately after birth may serve as predictors of neonatal hyperkalemia. The early prediction of neonatal hyperkalemia would facilitate the timely introduction of preventive or therapeutic drugs for hyperkalemia. Furthermore, our findings hold the potential to contribute to future drug development aimed at preventing neonatal hyperkalemia by targeting molecules involved in glycolysis and the citrate cycle. Ethics This study was approved by the Institutional Review Board of Oita University Hospital, Japan (Oita University, 2413). It follows the tenets of the Declaration of Helsinki. The need to obtain informed consent from the study participants was waived by the Institutional Review Board of Oita University Hospital since this is a retrospective analysis. Patients who were eligible for this study had the opportunity to refuse to participate in the study by opting out. The research was conducted in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects established by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare. Study population This study included preterm infants born at 24–36 weeks of gestation at Oita University Hospital’s NICU between April 2017 and August 2022. Oita University Hospital is equipped to handle deliveries as early as 22 weeks of gestation. Infants with uncertain maternal drug use, multiple malformation syndrome, chromosomal abnormalities, hemolytic anemia, intracranial hemorrhage, maternal malignancy, or history of anticancer drug treatment were excluded from the study. In our study, we focused on preterm infants born at 24–36 weeks of gestation. The reasons were based on several considerations regarding gestational age-dependent management and health outcomes of preterm infants. Infants born at Oita University Hospital at ≥ 37 weeks of gestational age are managed by obstetricians after delivery. These infants do not routinely undergo electrolyte monitoring unless they present with clinical symptoms. Regardless of clinical symptoms, infants exposed to ritodrine within 24 h before birth were routinely measured for blood glucose levels after birth. Pediatricians evaluate and treat infants who develop hypoglycemia or other clinical problems. In particular, none of the term infants (born at ≥ 37 weeks of gestation) managed by pediatricians developed hyperkalemia prior to this study. Infants born at 22–23 weeks of gestation were excluded due to the variability in resuscitation practices, prognosis, and ethical considerations surrounding the initiation of intensive care, which always involves in-depth discussions with the parents. Consequently, we investigated preterm infants born at 24–36 weeks of gestation. The umbilical cord blood serum analysis used residual samples from a previous study approved by the ethics committee due to limited sample availability. Therefore, the study included cases of infants born between April 2020 and August 2022 with gestational ages ranging from 24 to 36 weeks. Protocol of tocolytic treatment During the study period, the protocol of our hospital for tocolytic treatment was as follows. For threatened preterm labor, we began with the administration of ritodrine hydrochloride, with an initial dose of 50 μg/min administered via continuous intravenous infusion. The dose was increased to 100 μg/min, 150 μg/min, and up to 200 μg/min depending on the severity of the uterine contractions. If uterine contractions were not adequately controlled with ritodrine hydrochloride at 200 μg/min, MgSO 4 was used either in combination with ritodrine or as a monotherapy if patients could not tolerate ritodrine due to side effects. The administration of MgSO 4 began with an initial dose of 2 g administered intravenously for 20 min, followed by a continuous intravenous infusion at 1 g/h. If uterine contractions remained uncontrolled, the dosage was increased by 0.5 g/h, up to a maximum of 2 g/h. Tocolytic drugs were adjusted according to the uterine contraction status and were typically discontinued at 36 weeks of gestation. If the membranes ruptured, tocolytic therapy was stopped at 34 weeks. In cases of clinical chorioamnionitis or non-reassuring fetal status, tocolytic drugs were immediately stopped and delivery was started. Regarding the order of drug selection and administration, ritodrine hydrochloride was administered first, with magnesium sulfate added if contractions were not controlled with ritodrine alone or if ritodrine could not be used due to side effects. Clinical observations We retrospectively collected comprehensive clinical data from medical records of the participants, encompassing the following aspects: (a) information on the administration of ritodrine and MgSO 4 , which included duration of injections, final infusion rate, total dose, and maternal serum magnesium levels; (b) perinatal status of infants, encompassing factors such as gestational age, sex, weight, height, route of delivery, singleton or multiplet, Apgar scores at 1 min and 5 min, post-birth urinary output, and insulin/glucose perfusion; (c) laboratory data at birth, comprising umbilical artery pH, white blood cell (WBC) count, Hb levels, PLT count, and serum levels of total protein (TP), ALB, T-Bil, aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALP, CK, LDH, blood urea nitrogen (BUN), creatinine (CRE), Na, K, Cl, Ca, P, and C-reactive protein (CRP); (d) highest serum K level in neonate within 48 h of birth; and (e) maternal data included age, parity, obstetric complications (gestational hypertension and gestational diabetes), and with or without complication of PROM during delivery. In this study, we focused on the laboratory data obtained from infants immediately after birth, because blood tests were of all newborns were performed immediately after delivery, ensuring a consistent set of data for each infant. On the contrary, the timing of blood sampling for mothers during pregnancy varied depending on their underlying conditions and no uniform data from mothers was available around the time of delivery. Therefore, we only evaluated the infant laboratory data to ensure the consistency and reliability of the statistical analysis. Neonatal blood tests were carried out at intervals of no more than 24 h from birth to 48 h of age. If potassium levels tended to increase, blood tests were performed repeatedly every 3–6 h. Sampling specimens and storage Umbilical cord blood samples were collected at delivery using plastic syringes. Samples were centrifuged for 10 min at 1200× g , the serum was decanted, and serum samples were stored at − 80 °C until use in an analysis. Metabolomic analyses The analysis of metabolites was performed by GC-MS/MS. The GC-MS/MS analysis was performed on a GCMS-TQ8040 system (Shimadzu Corporation, Kyoto, Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm inner diameter, film thickness 1 μm; Agilent, Santa Clara, CA, USA). Each 1-μm aliquot of the derivatized sample solution was automatically injected in splitless mode into a gas–liquid chromatography column using an auto-injector (AOC-20i; Shimadzu Corporation). During the GCMS-TQ8040 analysis, the injector temperature was maintained at 280 °C, and helium was used as the carrier gas at a constant flow rate of 39.0 cm/s. The GC column temperature was programmed to remain at 100 °C for 4 min, then to rise to 320 °C at a rate of 10 °C/min, holding at 320 °C for an additional 11 min. The ionization voltage was set to 70 eV. Argon was used for collision-induced dissociation. Metabolite detection was performed using the Smart Metabolite Database Ver. 3 software program (Shimadzu Corporation) using the method described in a previous study with some modifications . The 2-isopropylmalic acid contained in the extraction solution was also used to evaluate the stability of the GC-MS/MS analysis system. Peak identification was performed automatically and then confirmed manually based on the specific precursor and product ions as well as the retention time using the method described in our previous study . Statistical analyses Statistical analyses were performed using the SPSS software program version 29.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism software version 8 (GraphPad Software, Inc., San Diego, CA, USA). Perinatal characteristics among the four groups were assessed using Fisher’s exact probability test for nominal variables and the Kruskal–Wallis test for continuous variables. The highest serum potassium concentration was observed in the Shapiro–Wilk and Brown–Forsythe tests for normal distribution and homogeneity, respectively. Since the data exhibited a normal distribution, a one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparison test was employed for the analysis. We conducted a sensitivity analysis to address the potential variability in the duration of combined therapy with ritodrine and MgSO 4 . Specifically, we excluded any participants with a combined treatment duration of 3 days or less, as well as those with a duration of 14 days or less, to assess the robustness of our findings. A stepwise multiple regression analysis was performed to evaluate the relationship between potassium concentration and clinical characteristics. The correlation between serum metabolites in umbilical cord blood and serum potassium concentration of phosphorus was analyzed using the Pearson correlation coefficient. A significance threshold of p < 0.05 was employed. The specificity and sensitivity were assessed by the AUC of the ROC curves. Integral metabolomics datasets were imported into the SIMCA version 13.0.3.0 software program (Umetrics, Umea, Sweden) for multivariate statistical analyses. OPLS-DA with Pareto scaling was used to visualize the differences between the metabolomic datasets and extract significant metabolites. The primary distinctions in metabolites between each group were identified through an S-plot analysis, which visualizes both the covariance and correlation between metabolites and the modelled class designation. Significant metabolites were selected based on compounds with a p (corr) value of > 0.7 and VIP values of > 1.0, a commonly employed metric to summarize the significance of each variable in model construction . In the pathway analysis, to determine the pathways altered between metabolomics data sets, MetPA and MSEA with significant metabolites were performed using the MetaboAnalyst 5.0 software program ( https://www.metaboanalyst.ca/ , accessed on June 23, 2023), which is a free web-based tool that combines results from a potent pathway enrichment analysis pertaining to the conditions under study . This study was approved by the Institutional Review Board of Oita University Hospital, Japan (Oita University, 2413). It follows the tenets of the Declaration of Helsinki. The need to obtain informed consent from the study participants was waived by the Institutional Review Board of Oita University Hospital since this is a retrospective analysis. Patients who were eligible for this study had the opportunity to refuse to participate in the study by opting out. The research was conducted in accordance with the Ethical Guidelines for Medical and Health Research Involving Human Subjects established by the Ministry of Education, Culture, Sports, Science and Technology and the Ministry of Health, Labour and Welfare. This study included preterm infants born at 24–36 weeks of gestation at Oita University Hospital’s NICU between April 2017 and August 2022. Oita University Hospital is equipped to handle deliveries as early as 22 weeks of gestation. Infants with uncertain maternal drug use, multiple malformation syndrome, chromosomal abnormalities, hemolytic anemia, intracranial hemorrhage, maternal malignancy, or history of anticancer drug treatment were excluded from the study. In our study, we focused on preterm infants born at 24–36 weeks of gestation. The reasons were based on several considerations regarding gestational age-dependent management and health outcomes of preterm infants. Infants born at Oita University Hospital at ≥ 37 weeks of gestational age are managed by obstetricians after delivery. These infants do not routinely undergo electrolyte monitoring unless they present with clinical symptoms. Regardless of clinical symptoms, infants exposed to ritodrine within 24 h before birth were routinely measured for blood glucose levels after birth. Pediatricians evaluate and treat infants who develop hypoglycemia or other clinical problems. In particular, none of the term infants (born at ≥ 37 weeks of gestation) managed by pediatricians developed hyperkalemia prior to this study. Infants born at 22–23 weeks of gestation were excluded due to the variability in resuscitation practices, prognosis, and ethical considerations surrounding the initiation of intensive care, which always involves in-depth discussions with the parents. Consequently, we investigated preterm infants born at 24–36 weeks of gestation. The umbilical cord blood serum analysis used residual samples from a previous study approved by the ethics committee due to limited sample availability. Therefore, the study included cases of infants born between April 2020 and August 2022 with gestational ages ranging from 24 to 36 weeks. During the study period, the protocol of our hospital for tocolytic treatment was as follows. For threatened preterm labor, we began with the administration of ritodrine hydrochloride, with an initial dose of 50 μg/min administered via continuous intravenous infusion. The dose was increased to 100 μg/min, 150 μg/min, and up to 200 μg/min depending on the severity of the uterine contractions. If uterine contractions were not adequately controlled with ritodrine hydrochloride at 200 μg/min, MgSO 4 was used either in combination with ritodrine or as a monotherapy if patients could not tolerate ritodrine due to side effects. The administration of MgSO 4 began with an initial dose of 2 g administered intravenously for 20 min, followed by a continuous intravenous infusion at 1 g/h. If uterine contractions remained uncontrolled, the dosage was increased by 0.5 g/h, up to a maximum of 2 g/h. Tocolytic drugs were adjusted according to the uterine contraction status and were typically discontinued at 36 weeks of gestation. If the membranes ruptured, tocolytic therapy was stopped at 34 weeks. In cases of clinical chorioamnionitis or non-reassuring fetal status, tocolytic drugs were immediately stopped and delivery was started. Regarding the order of drug selection and administration, ritodrine hydrochloride was administered first, with magnesium sulfate added if contractions were not controlled with ritodrine alone or if ritodrine could not be used due to side effects. We retrospectively collected comprehensive clinical data from medical records of the participants, encompassing the following aspects: (a) information on the administration of ritodrine and MgSO 4 , which included duration of injections, final infusion rate, total dose, and maternal serum magnesium levels; (b) perinatal status of infants, encompassing factors such as gestational age, sex, weight, height, route of delivery, singleton or multiplet, Apgar scores at 1 min and 5 min, post-birth urinary output, and insulin/glucose perfusion; (c) laboratory data at birth, comprising umbilical artery pH, white blood cell (WBC) count, Hb levels, PLT count, and serum levels of total protein (TP), ALB, T-Bil, aspartate aminotransferase (AST), alanine aminotransferase (ALT), ALP, CK, LDH, blood urea nitrogen (BUN), creatinine (CRE), Na, K, Cl, Ca, P, and C-reactive protein (CRP); (d) highest serum K level in neonate within 48 h of birth; and (e) maternal data included age, parity, obstetric complications (gestational hypertension and gestational diabetes), and with or without complication of PROM during delivery. In this study, we focused on the laboratory data obtained from infants immediately after birth, because blood tests were of all newborns were performed immediately after delivery, ensuring a consistent set of data for each infant. On the contrary, the timing of blood sampling for mothers during pregnancy varied depending on their underlying conditions and no uniform data from mothers was available around the time of delivery. Therefore, we only evaluated the infant laboratory data to ensure the consistency and reliability of the statistical analysis. Neonatal blood tests were carried out at intervals of no more than 24 h from birth to 48 h of age. If potassium levels tended to increase, blood tests were performed repeatedly every 3–6 h. Umbilical cord blood samples were collected at delivery using plastic syringes. Samples were centrifuged for 10 min at 1200× g , the serum was decanted, and serum samples were stored at − 80 °C until use in an analysis. The analysis of metabolites was performed by GC-MS/MS. The GC-MS/MS analysis was performed on a GCMS-TQ8040 system (Shimadzu Corporation, Kyoto, Japan) equipped with a DB-5 capillary column (30 m × 0.25 mm inner diameter, film thickness 1 μm; Agilent, Santa Clara, CA, USA). Each 1-μm aliquot of the derivatized sample solution was automatically injected in splitless mode into a gas–liquid chromatography column using an auto-injector (AOC-20i; Shimadzu Corporation). During the GCMS-TQ8040 analysis, the injector temperature was maintained at 280 °C, and helium was used as the carrier gas at a constant flow rate of 39.0 cm/s. The GC column temperature was programmed to remain at 100 °C for 4 min, then to rise to 320 °C at a rate of 10 °C/min, holding at 320 °C for an additional 11 min. The ionization voltage was set to 70 eV. Argon was used for collision-induced dissociation. Metabolite detection was performed using the Smart Metabolite Database Ver. 3 software program (Shimadzu Corporation) using the method described in a previous study with some modifications . The 2-isopropylmalic acid contained in the extraction solution was also used to evaluate the stability of the GC-MS/MS analysis system. Peak identification was performed automatically and then confirmed manually based on the specific precursor and product ions as well as the retention time using the method described in our previous study . Statistical analyses were performed using the SPSS software program version 29.0 (IBM Corporation, Armonk, NY, USA) and GraphPad Prism software version 8 (GraphPad Software, Inc., San Diego, CA, USA). Perinatal characteristics among the four groups were assessed using Fisher’s exact probability test for nominal variables and the Kruskal–Wallis test for continuous variables. The highest serum potassium concentration was observed in the Shapiro–Wilk and Brown–Forsythe tests for normal distribution and homogeneity, respectively. Since the data exhibited a normal distribution, a one-way analysis of variance (ANOVA) followed by Tukey’s pairwise comparison test was employed for the analysis. We conducted a sensitivity analysis to address the potential variability in the duration of combined therapy with ritodrine and MgSO 4 . Specifically, we excluded any participants with a combined treatment duration of 3 days or less, as well as those with a duration of 14 days or less, to assess the robustness of our findings. A stepwise multiple regression analysis was performed to evaluate the relationship between potassium concentration and clinical characteristics. The correlation between serum metabolites in umbilical cord blood and serum potassium concentration of phosphorus was analyzed using the Pearson correlation coefficient. A significance threshold of p < 0.05 was employed. The specificity and sensitivity were assessed by the AUC of the ROC curves. Integral metabolomics datasets were imported into the SIMCA version 13.0.3.0 software program (Umetrics, Umea, Sweden) for multivariate statistical analyses. OPLS-DA with Pareto scaling was used to visualize the differences between the metabolomic datasets and extract significant metabolites. The primary distinctions in metabolites between each group were identified through an S-plot analysis, which visualizes both the covariance and correlation between metabolites and the modelled class designation. Significant metabolites were selected based on compounds with a p (corr) value of > 0.7 and VIP values of > 1.0, a commonly employed metric to summarize the significance of each variable in model construction . In the pathway analysis, to determine the pathways altered between metabolomics data sets, MetPA and MSEA with significant metabolites were performed using the MetaboAnalyst 5.0 software program ( https://www.metaboanalyst.ca/ , accessed on June 23, 2023), which is a free web-based tool that combines results from a potent pathway enrichment analysis pertaining to the conditions under study . Supplementary Information 1. Supplementary Information 2. |
Prevalence of HER3 Expression in Pancreatic Cancer Patients Treated With Systemic Chemotherapy | e1201710-b40b-4670-af1f-c152104877b5 | 11626478 | Anatomy[mh] | Introduction Human epidermal growth factor receptor 3 (HER3/ErbB3) is a receptor tyrosine kinase that belongs to the HER family and shows little or no intrinsic tyrosine kinase activity . In comparison with other epidermal growth factor receptor (EGFR) family members, HER3 shows differences at critical residues in its intracellular kinase domain, which is locked in an inactive‐like conformation, leading to 1000‐fold weaker kinase activity than that of EGFR . However, HER3 can form heterodimers with other EGFR family members, which is responsible for activating oncogenic signaling pathways . Furthermore, HER3 activates the PI3K/Akt/mTOR signaling pathway for cancer cell survival by directly binding to PI3K . HER3 also activates the MAPK and the JAK–STAT and proto‐oncogene c‐Src signaling pathways for cancer cell proliferation . HER3 expression is associated with disease progression and metastasis in various types of cancer . Two systematic analyses across multiple malignant tumor types, including pancreatic cancer, confirmed that HER3 expression was associated with worse overall survival and a 1.6‐fold higher risk of death than that in HER3‐negative patients . HER3 expression also serves as a bypass mechanism for various therapies, and elevated HER3 signaling confers resistance to multiple therapeutic agents . HER3 expression is reported to be potentially altered before and after systemic therapy. HER3 expression levels are high in NSCLC with EGFR mutations, and EGFR inhibition increases HER3 expression . HER3 is considered to be a target for anticancer treatment . The variation in HER3 expression before and after systemic therapy is also important from the perspective of drug development. Pancreatic cancer is one of the most aggressive tumors and the fourth‐leading cause of cancer‐related deaths in the United States and Japan . Patients with this disease have an extremely poor prognosis, and the US “Cancer Statistics, 2023” reported that the 5‐year relative survival rate for pancreatic cancer was 12% for all stages and only 3% for metastatic disease (the most common form) . Pancreatic cancer is expected to become the second‐leading cause of cancer‐related deaths by 2030 . Therefore, novel therapies with improved efficacy are urgently needed to improve the prognosis of patients with this disease. Only a few reports on HER3 expression in pancreatic cancer have been published to date, and little is known about the clinical importance of HER3 expression in pancreatic cancer patients. Given the hypothesis that HER3 expression after chemotherapy may be different from that previously reported at the time of diagnosis, we investigated the status of HER3 expression after chemotherapy for pancreatic cancer and evaluated the associations among HER3 expression, clinicopathological features, and patient clinical outcomes. Materials and Methods 2.1 Patients We retrospectively reviewed the medical records of patients with pancreatic cancer treated at the National Cancer Center Hospital between January 2010 and June 2020. Only patients who had previously undergone chemotherapy and for whom post‐chemotherapy tissue samples collected after or during the treatments were available were included in this study. Patients currently undergoing treatment or follow‐up at our institution were excluded from this study to avoid any clinical disadvantages of obtaining tissue specimens. Clinical and histopathological characteristics of the patients, including age, sex, ECOG performance status, histopathology type, primary site of the pancreatic tumor lesion (head, body, tail), disease stage at diagnosis (resectable, locally advanced, metastatic), tumor size, tissue collection method, chemotherapy regimen, and carcinoembryonic antigen and carbohydrate antigen 19‐9 levels at tissue collection, were collected and analyzed. Tumor response was analyzed using the Response Evaluation Criteria in Solid Tumors version 1.1 . Data were retrospectively collected from medical chart reviews and electronic records. This study was approved by the Institutional Review Board of the National Cancer Center, Tokyo, Japan (Approval Number: 2018‐149). Written informed consent had been obtained from all participants. 2.2 Immunohistochemical Staining and Evaluation HER3 membrane expression was assessed by immunohistochemistry (IHC) in pre‐ and post‐chemotherapy tissue samples using previously reported methods . Briefly, HER3 IHC was performed on 4‐μm‐thick sections prepared from paraffin blocks of 10% neutral buffered formalin‐fixed specimens. Sections of each sample were deparaffinized, and antigen retrieval was performed using a PT Link machine (Dako; Agilent Technologies Inc.) at high pH. Sections were stained with a rabbit monoclonal antibody against HER3/ErbB3 (1:59 dilution; clone D22C5; Cell Signaling Technology Inc.) using the Dako autostainer Link48 (Dako) and EnVision Flex Mini Kit (Dako) according to the manufacturer's instructions. The slides were counterstained with hematoxylin. HER3 expression was evaluated as IHC scores of 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). 2.3 Analysis of Genomic Alterations We analyzed genomic alterations in eight patients who had undergone testing for genomic alterations under normal medical care and analyzed the findings in relation to HER3 expression. In Japan, three types of next‐generation sequencing‐based cancer genome profiling (CGP) tests—OncoGuide NCC Oncopanel System, FoundationOne CDx, and FoundationOne Liquid CDx—are reimbursed by the national health insurance system and implemented in routine oncological practice. Only genetic mutations reported as “pathogenic” or “likely pathogenic” were included in the analysis. 2.4 Statistical Analysis Overall survival (OS) was defined as the period from the start of chemotherapy (neoadjuvant therapy or first‐line chemotherapy) to death or last follow‐up. OS was estimated using the Kaplan–Meier method and compared between independent groups using the log‐rank test. Statistical significance was set at p < 0.05. Statistical analysis was performed using EZR software version 1.38 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) . Patients We retrospectively reviewed the medical records of patients with pancreatic cancer treated at the National Cancer Center Hospital between January 2010 and June 2020. Only patients who had previously undergone chemotherapy and for whom post‐chemotherapy tissue samples collected after or during the treatments were available were included in this study. Patients currently undergoing treatment or follow‐up at our institution were excluded from this study to avoid any clinical disadvantages of obtaining tissue specimens. Clinical and histopathological characteristics of the patients, including age, sex, ECOG performance status, histopathology type, primary site of the pancreatic tumor lesion (head, body, tail), disease stage at diagnosis (resectable, locally advanced, metastatic), tumor size, tissue collection method, chemotherapy regimen, and carcinoembryonic antigen and carbohydrate antigen 19‐9 levels at tissue collection, were collected and analyzed. Tumor response was analyzed using the Response Evaluation Criteria in Solid Tumors version 1.1 . Data were retrospectively collected from medical chart reviews and electronic records. This study was approved by the Institutional Review Board of the National Cancer Center, Tokyo, Japan (Approval Number: 2018‐149). Written informed consent had been obtained from all participants. Immunohistochemical Staining and Evaluation HER3 membrane expression was assessed by immunohistochemistry (IHC) in pre‐ and post‐chemotherapy tissue samples using previously reported methods . Briefly, HER3 IHC was performed on 4‐μm‐thick sections prepared from paraffin blocks of 10% neutral buffered formalin‐fixed specimens. Sections of each sample were deparaffinized, and antigen retrieval was performed using a PT Link machine (Dako; Agilent Technologies Inc.) at high pH. Sections were stained with a rabbit monoclonal antibody against HER3/ErbB3 (1:59 dilution; clone D22C5; Cell Signaling Technology Inc.) using the Dako autostainer Link48 (Dako) and EnVision Flex Mini Kit (Dako) according to the manufacturer's instructions. The slides were counterstained with hematoxylin. HER3 expression was evaluated as IHC scores of 0 (no staining), 1+ (weak staining), 2+ (moderate staining), and 3+ (strong staining). Analysis of Genomic Alterations We analyzed genomic alterations in eight patients who had undergone testing for genomic alterations under normal medical care and analyzed the findings in relation to HER3 expression. In Japan, three types of next‐generation sequencing‐based cancer genome profiling (CGP) tests—OncoGuide NCC Oncopanel System, FoundationOne CDx, and FoundationOne Liquid CDx—are reimbursed by the national health insurance system and implemented in routine oncological practice. Only genetic mutations reported as “pathogenic” or “likely pathogenic” were included in the analysis. Statistical Analysis Overall survival (OS) was defined as the period from the start of chemotherapy (neoadjuvant therapy or first‐line chemotherapy) to death or last follow‐up. OS was estimated using the Kaplan–Meier method and compared between independent groups using the log‐rank test. Statistical significance was set at p < 0.05. Statistical analysis was performed using EZR software version 1.38 (Saitama Medical Center, Jichi Medical University, Saitama, Japan) . Results 3.1 Patient Characteristics A Total of 41 patients who were eligible for HER3 expression analysis after chemotherapy were included in this study (Figure and Table ). In addition, for five patients, pre‐chemotherapy tissue samples were available in sufficient amounts for HER3 expression analysis. Of 41 post‐chemotherapy tissue samples, HER3 expression IHC scores ≥ 1+ were observed in 40 patients (98%), while HER3 scores ≥ 2+ were observed in 26 patients (63%) (Figures and ). Clinicopathological features of 41 patients are summarized in Table , where patients were divided, on the basis of their HER3 expression status, into two groups: 2+/3+ and 0/1+. Overall, the primary site was the pancreatic head in 68% of the patients, and 93% of the patients were diagnosed as showing adenocarcinoma on histopathological assessments. Specimens were mainly obtained by surgery (81%), while a few were obtained by biopsy (19%). There was no major difference in patient background between the HER3 (2+/3+) group and the HER3 (0/1+) group, but the median tumor size of the primary lesion was 30 mm in the HER3 (2+/3+) group and 35 mm in the HER3 (0/1+) group. Approximately 40% of the patients showed a locally advanced stage at diagnosis in both groups; 46% of the tumors were resectable and 12% were metastatic in the HER3 (2+/3+) group, and 20% were resectable and 33% were metastatic in the HER3 (0/1+) group. Chemotherapy administered prior to specimen collection was often gemcitabine plus S‐1 for patients with resectable disease and gemcitabine plus nab‐paclitaxel (GnP) or FOLFIRINOX (oxaliplatin, irinotecan, leucovorin, and fluorouracil) for patients with locally advanced disease or distant metastases. With respect to differences in HER3 expression by chemotherapy regimen, GnP showed HER3 (2+/3+) in all 5 patients, followed by GEM+S‐1 and FOLFIRINOX, which showed HER3 (2+/3+) in 73.3% and 50% of patients, respectively (Table ). 3.2 Association of HER3 Expression After Chemotherapy With Overall Survival In 38 cases of adenocarcinoma, excluding three cases of rare subtypes, the median OS was 21.0 months (95% CI: 15.6–32.2) in the HER3 (2+/3+) group and 17.1 months (95% CI: 8.9–27.8) in the HER3 (0/1 +) group. No significant difference in OS ( p = 0.602) was observed between the HER3 (2+/3+) and HER3 (0/1+) groups (Figure ). Subgroup analysis by disease stage also showed no significant difference in OS by HER3 expression (Figure ). 3.3 Differences in HER3 Expression Pre‐ and Post‐Chemotherapy Pre‐chemotherapy HER3 status was analyzed in five cases, allowing the comparison of HER3 expression pre‐ and post‐chemotherapy (Figure ). Only one case showed an increase in the IHC score for HER3 expression after chemotherapy, while the remaining cases showed no change in the IHC score (one case) or a reduction in the score (three cases; Table ). When HER3 expression scores were categorized as 2+/3+ or 0/1+, the scores changed from 2+/3+ to 0/1+ in one case, from 0/1+ to 2+/3+ in another case, and remained at 2+/3+ in three cases. Assessments of the efficacy of chemotherapy among these five patients revealed that a partial response according to RECIST 1.1 was observed in three patients, and the carbohydrate antigen 19‐9 (CA19‐9) level was lower than the pretreatment level in four patients. Among the three cases showing partial response, two showed altered HER3 IHC expression (one showed an increase in the score and the other showed a decrease). 3.4 HER3 Expression and Genomic Alterations Using the results of the CGP test in eight cases, we examined the association between HER3 expression by IHC and genomic alterations (Figure ). HER3 expression was observed in all eight cases, with an IHC score of 3+ in three cases, 2+ in one case, and 1+ in four cases. A tissue‐based CGP test was performed in seven patients, five of whom underwent testing with FoundationOne CDx and two with the OncoGuide NCC Oncopanel System, which were performed on the same samples tested for HER3 expression. The remaining patient was tested using the liquid‐based FoundationOne Liquid CDx, which was performed at a different timepoint from tissue collection. No cases of HER3 amplification or mutation were identified. No amplifications or mutations were observed in HER family members EGFR and HER2. Seven of the eight cases were adenocarcinomas, and both KRAS and TP53 mutations were observed in these seven cases. The remaining case was an acinar cell carcinoma that showed a RAF1 fusion, no KRAS or TP53 mutations, and an IHC score of 2+. All patients had a low tumor mutation burden and showed a microsatellite‐stable status. Patient Characteristics A Total of 41 patients who were eligible for HER3 expression analysis after chemotherapy were included in this study (Figure and Table ). In addition, for five patients, pre‐chemotherapy tissue samples were available in sufficient amounts for HER3 expression analysis. Of 41 post‐chemotherapy tissue samples, HER3 expression IHC scores ≥ 1+ were observed in 40 patients (98%), while HER3 scores ≥ 2+ were observed in 26 patients (63%) (Figures and ). Clinicopathological features of 41 patients are summarized in Table , where patients were divided, on the basis of their HER3 expression status, into two groups: 2+/3+ and 0/1+. Overall, the primary site was the pancreatic head in 68% of the patients, and 93% of the patients were diagnosed as showing adenocarcinoma on histopathological assessments. Specimens were mainly obtained by surgery (81%), while a few were obtained by biopsy (19%). There was no major difference in patient background between the HER3 (2+/3+) group and the HER3 (0/1+) group, but the median tumor size of the primary lesion was 30 mm in the HER3 (2+/3+) group and 35 mm in the HER3 (0/1+) group. Approximately 40% of the patients showed a locally advanced stage at diagnosis in both groups; 46% of the tumors were resectable and 12% were metastatic in the HER3 (2+/3+) group, and 20% were resectable and 33% were metastatic in the HER3 (0/1+) group. Chemotherapy administered prior to specimen collection was often gemcitabine plus S‐1 for patients with resectable disease and gemcitabine plus nab‐paclitaxel (GnP) or FOLFIRINOX (oxaliplatin, irinotecan, leucovorin, and fluorouracil) for patients with locally advanced disease or distant metastases. With respect to differences in HER3 expression by chemotherapy regimen, GnP showed HER3 (2+/3+) in all 5 patients, followed by GEM+S‐1 and FOLFIRINOX, which showed HER3 (2+/3+) in 73.3% and 50% of patients, respectively (Table ). Association of HER3 Expression After Chemotherapy With Overall Survival In 38 cases of adenocarcinoma, excluding three cases of rare subtypes, the median OS was 21.0 months (95% CI: 15.6–32.2) in the HER3 (2+/3+) group and 17.1 months (95% CI: 8.9–27.8) in the HER3 (0/1 +) group. No significant difference in OS ( p = 0.602) was observed between the HER3 (2+/3+) and HER3 (0/1+) groups (Figure ). Subgroup analysis by disease stage also showed no significant difference in OS by HER3 expression (Figure ). Differences in HER3 Expression Pre‐ and Post‐Chemotherapy Pre‐chemotherapy HER3 status was analyzed in five cases, allowing the comparison of HER3 expression pre‐ and post‐chemotherapy (Figure ). Only one case showed an increase in the IHC score for HER3 expression after chemotherapy, while the remaining cases showed no change in the IHC score (one case) or a reduction in the score (three cases; Table ). When HER3 expression scores were categorized as 2+/3+ or 0/1+, the scores changed from 2+/3+ to 0/1+ in one case, from 0/1+ to 2+/3+ in another case, and remained at 2+/3+ in three cases. Assessments of the efficacy of chemotherapy among these five patients revealed that a partial response according to RECIST 1.1 was observed in three patients, and the carbohydrate antigen 19‐9 (CA19‐9) level was lower than the pretreatment level in four patients. Among the three cases showing partial response, two showed altered HER3 IHC expression (one showed an increase in the score and the other showed a decrease). HER3 Expression and Genomic Alterations Using the results of the CGP test in eight cases, we examined the association between HER3 expression by IHC and genomic alterations (Figure ). HER3 expression was observed in all eight cases, with an IHC score of 3+ in three cases, 2+ in one case, and 1+ in four cases. A tissue‐based CGP test was performed in seven patients, five of whom underwent testing with FoundationOne CDx and two with the OncoGuide NCC Oncopanel System, which were performed on the same samples tested for HER3 expression. The remaining patient was tested using the liquid‐based FoundationOne Liquid CDx, which was performed at a different timepoint from tissue collection. No cases of HER3 amplification or mutation were identified. No amplifications or mutations were observed in HER family members EGFR and HER2. Seven of the eight cases were adenocarcinomas, and both KRAS and TP53 mutations were observed in these seven cases. The remaining case was an acinar cell carcinoma that showed a RAF1 fusion, no KRAS or TP53 mutations, and an IHC score of 2+. All patients had a low tumor mutation burden and showed a microsatellite‐stable status. Discussion In the present study, we report HER3 expression after chemotherapy in pancreatic cancer. Only a few studies have investigated HER3 overexpression in pancreatic cancer . In these previous reports, IHC scores ≥ 1+ for HER3 expression were found in 62% to 94% of cases; specifically, IHC scores of 2+ were found in 17%–31% of cases, and IHC scores of 3+ were found in 9%–24% of cases. When IHC scores of 3+ and 2+ were defined as HER3 overexpression , the incidence of HER3 overexpression in the previous studies ranged from 26% to 41%. While these previous studies have addressed HER3 expression at initial diagnosis, to the best of our knowledge, this is the first study that investigates HER3 expression after chemotherapy in pancreatic cancers. In our study, the rates of IHC scores of 2+ and 3+ were 34% and 29%, respectively, and that of HER3 (2+/3+) was 63% in post‐chemotherapy tissue samples. As the antibodies and protocols used for IHC analysis differ among studies, comparisons of HER3 expression rates between studies need to be carefully considered. Additionally, to the best of our knowledge, this is the first study to compare the changes in HER3 expression before and after chemotherapy in pancreatic cancer. Only one case showed no change in HER3 expression, whereas the remaining four cases showed a change, including one case with a significant change in HER3 expression from an IHC score of 3+ to 0. Although HER3 is not a known resistance mechanism to EGFR kinase inhibitors, EGFR inhibition leads to increased HER3 membrane expression . HER3 expression is associated with resistance to targeted therapies, including HER2 , ALK , and BRAF inhibitors; hormonal therapies, including tamoxifen and fulvestrant ; and chemotherapeutic agents, including paclitaxel . In our study, we assumed that chemotherapy treatment regimens and treatment efficacy may be associated with changes in HER3 expression; however, no consistent relationship was observed in the pre‐ and post‐treatment comparisons. In contrast, all patients who underwent GnP treatment showed an HER3 expression IHC score of 2+/3+. Rabia et al. demonstrated that resistance to gemcitabine is mainly associated with HER2 and HER3 overexpression in preclinical models of pancreatic cancer and in some cells with HER ligand expression . In gemcitabine‐resistant patient‐derived xenograft models of pancreatic cancer, acquired gemcitabine resistance was efficiently overcome by a pan‐HER antibody mixture, which was a cocktail of anti‐EGFR, anti‐HER2, and anti‐HER3 antibodies. Camblin et al. suggested that standard‐of‐care chemotherapy regimens, such as GnP, increase the expression and activation of insulin‐like growth factor receptor 1 and HER3 in pancreatic cancer cells, rendering the cells tolerant to cytotoxic therapies . Although our study is limited because of the small number of cases evaluated, there might be an association between GnP treatment and HER3 expression in our study as well. HER3 expression may change before and after chemotherapy. Although only 19% of specimens were obtained from biopsies in our study, heterogeneity within the tumor may also affect the assessment of HER3 expression, particularly when biopsy specimens are used. A Detailed investigation of whether chemotherapy induces HER3 expression is needed in the future. In clinical practice, the collection of pre‐ and post‐chemotherapy samples may be limited; therefore, preclinical models such as patient‐derived xenograft models may be useful to confirm changes in HER3 expression pre‐ and post‐chemotherapy. Prognostic significance of HER3 overexpression in pancreatic cancer remains controversial. On the basis of their analysis of 126 resected pancreatic cancer specimens, Hirakawa et al. reported that the median survival time of patients showing HER3 overexpression was 37.2 months, while that of patients with HER3‐negative samples was 58.6 months ( p = 0.008), and they concluded that HER3 was an independent predictor of poor prognosis based on multivariate survival analysis . In addition, several studies have reported that HER3 overexpression is associated with an advanced tumor stage and shorter postoperative survival in resected pancreatic cancer . However, Kawesha et al. reported that HER3 overexpression was found in 57% (89/157) of resected pancreatic cancers and that HER3 overexpression in cancer cells showed no relationship with patient survival . Using univariate and multivariate analyses, Velde et al. also found no significant association between HER3 overexpression and survival in patients with resectable pancreatic cancer . Our study showed no significant difference in survival between the HER3 (2+/3+) and HER3 (0/1+) groups. These results should be interpreted with caution because they may show some bias related to the patient background characteristics (such as the proportion of patients with resectable and metastatic tumors) in the HER3 (2+/3+) and (0/1+) groups. Although no significant difference was observed in OS between the HER3 (2+/3+) and HER3 (0/1+) patients in the resectable, locally advanced, or metastatic cohorts, the number of cases was very small (Figure ). Thus, further studies on HER3 expression and its prognosis are needed in a larger number of cases to reach a definite conclusion. Few reports have evaluated the association between HER3 expression and genomic alterations; therefore, an additional analysis was performed in this study in order to find if HER3 expression is associated with any gene alterations, especially with HER3 amplification, despite the small number of cases with available genomic data. From our analysis, HER3 amplification was not detected by next‐generation sequencing in any case showing HER3 expression on IHC. HER3 amplification is a very rare gene alteration found in 0.2% of pancreatic cancers, according to the Center for Cancer Genomics and Advanced Therapeutics (C‐CAT) database, which consists of data from cancer gene panel tests covered by health insurance in Japan . Furthermore, regardless of HER3 expression, a high proportion of KRAS and TP53 mutations were observed, which are commonly observed in typical pancreatic adenocarcinomas. Additionally, similar results were obtained from the profiling of mutations in SMAD4 and CDKN2A , which are frequently observed in patients with regular pancreatic adenocarcinoma. No distinctive genetic mutations related to HER3 expression were found in the current study. Thus, the presence of distinctive genetic mutations associated with HER3 expression remains a subject of future investigation. Owing to the widespread expression of membrane HER3 in most patients with pancreatic cancer, HER3 is an appealing molecular target for therapeutic interventions. Patritumab deruxtecan (HER3‐DXd; U3‐1402) is an HER3‐directed antibody–drug conjugate composed of patritumab, a cleavable tetrapeptide‐based linker, and a topoisomerase I inhibitor payload (MAAA‐1181a, an exatecan derivative) . In a phase I study of EGFR inhibitor‐resistant, EGFR ‐mutated NSCLC, patritumab deruxtecan showed a response rate of 39.2% . In another phase II study of EGFR ‐mutated NSCLC previously treated with EGFR TKI therapy, patritumab deruxtecan showed an objective response rate of 29.8% . Both studies showed no clear association with clinical response to patritumab deruxtecan and HER3 expression. Notably, 3 out of 5 patients with NSCLC harboring KRAS or NRAS driver mutations responded to patritumab deruxtecan in another trial, despite the small number of cases evaluated . Patritumab deruxtecan also demonstrated preclinical efficacy in colorectal cancer xenografts, showing a dependence on HER3 expression rather than on KRAS mutations . Therapeutic development of compounds targeting HER3, including patritumab deruxtecan, is expected for pancreatic cancer, which is often characterized by KRAS mutations. This study has some limitations. First, the number of cases was limited; therefore caution is needed for the interpretability of the survival analysis. Second, this study was limited to cases with post‐chemotherapy specimens, including resectable cases and those that underwent conversion surgery or had distant metastases. Additionally, when biopsy specimens are used to evaluate HER3 expression, the results of small biopsy specimens may not reflect the overall tumor status in cases with heterogeneous HER3 expression. Multisite biopsies to improve sample availability are needed to resolve these limitations caused by small sample sizes. Finally, we evaluated HER3 expression on the basis of the evaluation of HER2 in gastric cancer, which is a relatively widely used method but is not clearly defined as a criterion for evaluating HER3 expression. Criteria for evaluating HER3 expression are expected to be established in the future. In conclusion, our study showed a high prevalence of HER3 expression in pancreatic cancer after chemotherapy treatment. The HER3 expression status of pancreatic cancer is of great interest as a therapeutic target, and further research with a larger sample size is warranted in the future. Tomoyuki Satake: writing – original draft (equal). Chigusa Morizane: writing – original draft (equal). Mao Okada: writing – original draft (equal). Mariko Nishioka: writing – original draft (equal). Nobuyoshi Hiraoka: writing – original draft (equal). Satoshi Nara: writing – original draft (equal). Tomoya Kakegawa: writing – original draft (equal). Maki Kobayashi: writing – original draft (equal). Kumiko Koyama: writing – original draft (equal). Minoru Esaki: writing – original draft (equal). Takuji Okusaka: writing – original draft (equal). This study was approved by the Institutional Review Board of the National Cancer Center, Tokyo, Japan (Approval Number: 2018–149). Written informed consent was obtained from all participants. Chigusa Morizane has received research funding from Eisai, Yakult Honsha, ONO Pharmaceutical, Taiho Pharmaceutical, J‐Pharma, AstraZeneca, Merck Biopharma, Daiichi Sankyo, Boehringer Ingelheim, Daiichi Sankyo RD Novare, Labcorp, Hitachi, and MSD K.K.; has consulted for Yakult Honsha, MSD K.K., SERVIER, Boehringer Ingelheim, Taiho, Novartis, AstraZeneca, MSD, and Merck Biopharma; and received speaker honoraria from Novartis, Yakult Honsha, SERVIER, Taiho Pharmaceutical, Eisai, MSD K.K., AstraZeneca, TORAY, Guardant, and Myriad Genetics. Takuji Okusaka has received research funding from Chugai Pharmaceutical, Eisai, Novartis Pharma, Bristol‐Myers Squibb Company, AstraZeneca, MSD, Chiome Bioscience, Syneos, Incyte jp, and SYSMEX; has consulted for AstraZeneca, Eisai, Ono Pharmaceutica, FUJIFILM, Toyama Chemical, Daiichi Sankyo, Dainippon Sumitomo Pharma, Nihon Servier, Chugai Pharmaceutical, and Novartis Pharma; received speaker honoraria from AstraZeneca, Syneos, Eisai, Ono Pharmaceutical, Johnson & Johnson, Taiho Pharmaceutical, Chugai Pharmaceutical, Nihon Servier, Novartis Pharma, Myriad Genetics, and Yakult Honsha, and Daiichi Sankyo; and is an editorial board member of Cancer Science. Kumiko Koyama, Tomoya Kakegawa, and Maki Kobayashi are employees of Daiichi Sankyo Co. Ltd. The other authors declare no conflicts of interest. Figure S1. Patients flow diagram. Overall survival according to HER3 expression and disease stage. (A) Overall survival for resectable stage. (B) Overall survival for locally advanced stage. (C) Overall survival for metastatic stage. Table S1. Clinical and therapeutic details and the outcome of patients included in the study. Table S2. |
Metaphyseal sleeve and straight stem fixation with or without screws for bone defect in complex primary total knee arthroplasty in Eastern Asian populations | 641dd5e7-b476-409f-89ab-b3dfb59a70b0 | 11837594 | Surgical Procedures, Operative[mh] | Massive bone defects present a formidable challenge for surgeons performing total knee arthroplasty (TKA), particularly in revision TKA and complex primary TKA, the latter of which often arise from diverse etiologies such as rheumatoid arthritis (RA), post-traumatic arthritis, Charcot knee arthropathy, and others . In cases of complex primary TKA where substantial bone defects exist in the epiphysis and metaphysis, coupled with significant deformities, eroded ligaments, severe osteoporosis (OP), flexion contractures, and other atypical complications, the surgical interventions closely resemble those of revision surgeries rather than routine primary procedures typically performed for common osteoarthritis. In such instances, specialized surgical techniques, metal augments, and modular prosthesis components for revision TKA must also be considered to address the challenges posed by massive bone defects. The concept of “zonal fixation” (zone 1: epiphysis, zone 2: metaphysis, zone 3: diaphysis), introduced by Haddad et al. , emphasizes the necessity of fixation that spans at least two zones to ensure the long-term survival of the revision implant. This strategy has gained widespread acceptance and is also applicable to complex primary TKA procedures. Currently, porous-coated metaphyseal sleeves (MS) are increasingly utilized for the reconstruction of significant bone defects during revision TKA, demonstrating several advantages , including rapid achievement of weight-bearing and rotational stability in metaphysis (zone 2), a stepped structure designed to minimize shear forces, favorable conditions for bone integration, reduced bone resorption, and a straightforward standardized operation mode. To our knowledge, there are currently few reports assessing the mid- to long-term follow-up outcomes of MS in complex primary TKA within East Asian populations, especially large sample studies; most existing literature predominantly stems from Caucasian cohorts, such as studies in Spain (Martín-Hernández C et al. , 2018) and the Netherlands (Van Rensch et al. , 2022), which indicate that MS can significantly enhance early and mid-term joint function, quality of life, and radiographic metrics for patients with complex knee etiologies. It is well established that knee prostheses and components are generally designed based on Caucasian knee anatomy . Several studies have identified notable differences in knee anatomy between East Asian populations and Caucasians . Specifically, MS is often employed alongside a straight stem, and the ideal positioning of the “MS-straight-stem” system should be centrally placed to ensure optimal bone contact in all directions. However, the location of the tibial axis in East Asian individuals differs significantly from that in Caucasian populations . Thus, a critical question arises: can the off-the-shelf “MS-straight-stem” system, designed based on Caucasian anatomical principles, achieve the intended “central position” placement in East Asian individuals while ensuring sufficient tibial plateau coverage and satisfactory clinical outcomes? We noted that one study involving Chinese patients reported excellent mid-term outcomes of MS with straight stems in complex primary TKA, suggesting its potential application in this context . However, the sample size of primary TKA in that study was limited (seven cases), underscoring the need for comprehensive cohort studies with larger sample sizes to evaluate the feasibility and survivorship of MS for bone defect repair in this population. Furthermore, previous reports on the application of MS for bone defect repair in both revision TKA and complex primary TKA have mainly concentrated on fixations in zones 2 and 3, where MS and cemented or press-fit stems are used in combination to achieve adequate stability . However, one or more relatively small peripheral defects may frequently persist in zone 1 despite MS placement in complex primary TKA cases, necessitating additional repair procedures . While bone grafts (autologous or allograft) have been considered for addressing the “residual defects” in revision TKA characterized by a well-vascularized cancellous base, it may not be easy to realized in complex primary TKA, where osteosclerosis typifies the base of these defects (Fig. ), requiring significant modifications to facilitate osseointegration between the graft and host bone. The preliminary treatment of bony osteosclerosis at the defect site, along with graft trimming and fixation, presents several technical challenges and is often time-consuming. What is more, studies have raised the risks of bone resorption and collapse associated with grafting .The optimal approach for addressing these defects warrants further investigation. Bone cement and screws, representing a useful, simple, and cost-effective technique for repairing minor defects in both primary and revision TKA , may offer a viable solution to these challenges; however, a comparative study addressing this issue is currently lacking. Therefore, the objective of the present study is to evaluate the clinical outcomes and survivorship of MS used for reconstructing massive bone defects in complex primary TKA among East Asians, with or without the utilization of screws. Study design This retrospective analysis examined the hospitalization and follow-up data of patients utilizing MS for the reconstruction of significant bone defects during primary TKAs at a large academic urban center. The study spanned from January 2016 to December 2020, with the patient selection process illustrated in Fig. . The analysis included multiple surgeons and received approval from the ethics committee of Guangzhou First People’s Hospital (ethics number: K-2021-036-02). Written informed consent was obtained from all patients. Inclusion criteria encompassed: (1) patients who underwent unilateral or bilateral massive bone defect reconstruction using MS during primary TKAs; (2) a minimum follow-up period of three years post-surgery. Exclusion criteria included: (1) patients with oncological diagnoses; (2) incomplete hospitalization and follow-up data; (3) knees that received bone grafts (either autologous or allograft) during the TKA procedures; and (4) cases in which MS was utilized for bilateral bone defects but screws were employed in only one knee. Preoperative assessment All patients presented with significant preoperative pain and functional impairment, severely compromising their quality of life. Conservative treatment lasting at least six months proved ineffective, leading to a strong desire for surgical intervention. X-ray examinations revealed severe bone destruction in all cases. Dual-energy X-ray absorptiometry (DEXA) scans were routinely performed to evaluate bone quality, and CT or MRI imaging was conducted when necessary for a comprehensive assessment of bone and soft tissue conditions. The evaluation of significant bone defects primarily used the Anderson Orthopedic Research Institute (AORI) classification , which categorizes bone defects into three types: type 1 refers to minor or contained defects in the epiphysis; type 2 is divided into type 2 A, involving metaphyseal bone damage and cancellous bone loss in one femoral condyle or tibial plateau, and type 2B, involving similar damage in both femoral condyles and tibial plateau; type 3 indicates extensive cancellous bone loss affecting a substantial portion of either the femoral condyle or tibial plateau, occasionally accompanied by collateral ligament damage. Indications for the use of MS included: (1) AORI type 3 bone defects of any etiology; (2) AORI type 2 bone defects in specific conditions characterized by extremely poor bone quality, particularly in zones 1 and 2, such as rheumatoid arthritis, post-traumatic arthritis, and Charcot knee arthropathy. Surgical procedures The surgical procedure was performed under general or spinal anesthesia through a midline incision extending from the superior patella to the medial tibial condyle, providing access to the joint. The joint was meticulously cleared of pathological synovium and osteophytes. Following tibial canal expansion, sleeve tibial preparation was conducted using a broach, with subsequent leveling of the tibial platform and selection of an appropriate tibial tray based on measured data. After opening the femoral canal, adjustments were made to the femoral condyles, and appropriate measurements for the femoral distal data were obtained. Osteotomies were then performed on the anterior and posterior condyles, the anterior sloping surface, and the intercondylar area. Additional expansion of the femoral canal was executed, followed by sleeve femoral preparation (if necessary) and selection of suitable femoral components, ensuring the balance of flexion-extension gaps and joint stability. All prosthetic components were assembled based on trial results, with all patients receiving implants from DePuy Synthes Inc., Warsaw, Indiana. In cases where the residual defects surrounding the metaphyseal sleeve were considerable (mainly in tibial sites in the present study), and filling with cement alone was inadequate, the cement-screw technique was employed. The definitive tibial tray, connected to a metaphyseal sleeve and a straight stem, alongside the femoral components (also connected to a metaphyseal sleeve and a straight stem if indicated), and a rotational polyethylene liner, were installed. Cement was applied to the undersurfaces of the tibial tray and femoral component, avoiding use on the surface of the MS. Drains were routinely placed, and the incision was closed with the knee joint flexed at 30 degrees. A schematic overview of the procedure is presented in Fig. . Perioperative management and follow-up Postoperative care included side-specific knee joint ice packs and elastic bandage wrapping. Routine treatments such as antimicrobial prophylaxis, pain medication, and prevention of lower limb deep vein thrombosis were provided. Patients with OP were given routine pharmacologic treatment once the diagnosis was ensured in the perioperative and follow-up period. The drainage tube was removed within 24 h after surgery. On the first day after surgery, ankle pumps, quadriceps muscle strength training, and knee joint flexion-extension exercises were initiated. Early mobilization was encouraged with partial weight-bearing using assistive devices. Gradual weaning from assistive devices for walking was initiated 2–4 weeks postoperatively. Long-term lifelong follow-up was scheduled, and routine outpatient visits were conducted to guide patients in standardized postoperative rehabilitation. Preoperative assessments and routine follow-up included measurements using the numerical rating scale (NRS) for pain (0–10 scale, where 0 indicates no pain and 10 indicates the worst pain), the range of motion (ROM) of the knee, and the Hospital for Special Surgery (HSS) knee score . Anteroposterior, lateral, and standing full-length lower limb X-ray images were obtained preoperatively and during routine follow-ups. The hip-knee-ankle (HKA) angle was measured, amd the absolute value of HKA was recorded as HKA deviation (HKAD, HKAD= | HKA | ). Monitored complications included intraoperative fractures, DVT, cardiac events, pulmonary complications, postoperative wound issues, periprosthetic joint infection (PJI), joint instability, aseptic loosening of the prosthesis, and end-of-stem pain. Standard measurement of HKA can be referred to the study of Cooke et al. . Osteointegration was defined as an increase in osteosclerosis at the bone-prosthesis interface without radiolucent lines between the host bone and prosthesis . Joint instability was defined as as any abnormal or excessive displacement of the articular elements that leads to clinical failure of the arthroplasty , while prosthetic loosening was defined as displacement or radiolucent lines exceeding 2 mm around the prosthesis . Data analysis Data entry and statistical analyses were performed using SPSS version 13.0. Descriptive statistics are presented as means ± standard deviations (SD) for continuous variables and as n for categorical variables. Group comparisons were conducted using a two-sample independent t-test or a general linear model for continuous variables, a chi-square test, continuous calibration chi-square test, or Fisher’s exact test for unordered categorical variables, and a Mann-Whitney U test for ordered categorical variables. Statistical significance was defined as P < 0.05. This retrospective analysis examined the hospitalization and follow-up data of patients utilizing MS for the reconstruction of significant bone defects during primary TKAs at a large academic urban center. The study spanned from January 2016 to December 2020, with the patient selection process illustrated in Fig. . The analysis included multiple surgeons and received approval from the ethics committee of Guangzhou First People’s Hospital (ethics number: K-2021-036-02). Written informed consent was obtained from all patients. Inclusion criteria encompassed: (1) patients who underwent unilateral or bilateral massive bone defect reconstruction using MS during primary TKAs; (2) a minimum follow-up period of three years post-surgery. Exclusion criteria included: (1) patients with oncological diagnoses; (2) incomplete hospitalization and follow-up data; (3) knees that received bone grafts (either autologous or allograft) during the TKA procedures; and (4) cases in which MS was utilized for bilateral bone defects but screws were employed in only one knee. All patients presented with significant preoperative pain and functional impairment, severely compromising their quality of life. Conservative treatment lasting at least six months proved ineffective, leading to a strong desire for surgical intervention. X-ray examinations revealed severe bone destruction in all cases. Dual-energy X-ray absorptiometry (DEXA) scans were routinely performed to evaluate bone quality, and CT or MRI imaging was conducted when necessary for a comprehensive assessment of bone and soft tissue conditions. The evaluation of significant bone defects primarily used the Anderson Orthopedic Research Institute (AORI) classification , which categorizes bone defects into three types: type 1 refers to minor or contained defects in the epiphysis; type 2 is divided into type 2 A, involving metaphyseal bone damage and cancellous bone loss in one femoral condyle or tibial plateau, and type 2B, involving similar damage in both femoral condyles and tibial plateau; type 3 indicates extensive cancellous bone loss affecting a substantial portion of either the femoral condyle or tibial plateau, occasionally accompanied by collateral ligament damage. Indications for the use of MS included: (1) AORI type 3 bone defects of any etiology; (2) AORI type 2 bone defects in specific conditions characterized by extremely poor bone quality, particularly in zones 1 and 2, such as rheumatoid arthritis, post-traumatic arthritis, and Charcot knee arthropathy. The surgical procedure was performed under general or spinal anesthesia through a midline incision extending from the superior patella to the medial tibial condyle, providing access to the joint. The joint was meticulously cleared of pathological synovium and osteophytes. Following tibial canal expansion, sleeve tibial preparation was conducted using a broach, with subsequent leveling of the tibial platform and selection of an appropriate tibial tray based on measured data. After opening the femoral canal, adjustments were made to the femoral condyles, and appropriate measurements for the femoral distal data were obtained. Osteotomies were then performed on the anterior and posterior condyles, the anterior sloping surface, and the intercondylar area. Additional expansion of the femoral canal was executed, followed by sleeve femoral preparation (if necessary) and selection of suitable femoral components, ensuring the balance of flexion-extension gaps and joint stability. All prosthetic components were assembled based on trial results, with all patients receiving implants from DePuy Synthes Inc., Warsaw, Indiana. In cases where the residual defects surrounding the metaphyseal sleeve were considerable (mainly in tibial sites in the present study), and filling with cement alone was inadequate, the cement-screw technique was employed. The definitive tibial tray, connected to a metaphyseal sleeve and a straight stem, alongside the femoral components (also connected to a metaphyseal sleeve and a straight stem if indicated), and a rotational polyethylene liner, were installed. Cement was applied to the undersurfaces of the tibial tray and femoral component, avoiding use on the surface of the MS. Drains were routinely placed, and the incision was closed with the knee joint flexed at 30 degrees. A schematic overview of the procedure is presented in Fig. . Postoperative care included side-specific knee joint ice packs and elastic bandage wrapping. Routine treatments such as antimicrobial prophylaxis, pain medication, and prevention of lower limb deep vein thrombosis were provided. Patients with OP were given routine pharmacologic treatment once the diagnosis was ensured in the perioperative and follow-up period. The drainage tube was removed within 24 h after surgery. On the first day after surgery, ankle pumps, quadriceps muscle strength training, and knee joint flexion-extension exercises were initiated. Early mobilization was encouraged with partial weight-bearing using assistive devices. Gradual weaning from assistive devices for walking was initiated 2–4 weeks postoperatively. Long-term lifelong follow-up was scheduled, and routine outpatient visits were conducted to guide patients in standardized postoperative rehabilitation. Preoperative assessments and routine follow-up included measurements using the numerical rating scale (NRS) for pain (0–10 scale, where 0 indicates no pain and 10 indicates the worst pain), the range of motion (ROM) of the knee, and the Hospital for Special Surgery (HSS) knee score . Anteroposterior, lateral, and standing full-length lower limb X-ray images were obtained preoperatively and during routine follow-ups. The hip-knee-ankle (HKA) angle was measured, amd the absolute value of HKA was recorded as HKA deviation (HKAD, HKAD= | HKA | ). Monitored complications included intraoperative fractures, DVT, cardiac events, pulmonary complications, postoperative wound issues, periprosthetic joint infection (PJI), joint instability, aseptic loosening of the prosthesis, and end-of-stem pain. Standard measurement of HKA can be referred to the study of Cooke et al. . Osteointegration was defined as an increase in osteosclerosis at the bone-prosthesis interface without radiolucent lines between the host bone and prosthesis . Joint instability was defined as as any abnormal or excessive displacement of the articular elements that leads to clinical failure of the arthroplasty , while prosthetic loosening was defined as displacement or radiolucent lines exceeding 2 mm around the prosthesis . Data entry and statistical analyses were performed using SPSS version 13.0. Descriptive statistics are presented as means ± standard deviations (SD) for continuous variables and as n for categorical variables. Group comparisons were conducted using a two-sample independent t-test or a general linear model for continuous variables, a chi-square test, continuous calibration chi-square test, or Fisher’s exact test for unordered categorical variables, and a Mann-Whitney U test for ordered categorical variables. Statistical significance was defined as P < 0.05. Patients’ demographics and preoperative conditions A total of 87 patients (106 knees) undergoing MS reconstruction for substantial bone defects in primary TKAs were included in the study. Among these, 46 patients (55 knees) received screws for the repair of tibial residual bone defects, while 41 patients (51 knees) did not. The distribution of knee etiologies included 53 patients with RA, 25 with post-traumatic arthritis, 6 with osteoarthritis, and 3 diagnosed with Charcot knee arthropathy. The demographics and preoperative conditions of the included patients are detailed in Table . Patients who received screws exhibited poorer bone quality (t = 3.095, P = 0.003), and a higher prevalence of osteoporosis was observed in the screw group (χ²=5.892, P = 0.015). Operation details A total of 138 metaphyseal sleeves and 140 straight stems were utilized in the TKA procedures. Intraoperative assessments revealed that knees in the screw group presented with more severe bone defects (Z=-3.590, P < 0.001). Additionally, the screw group demonstrated a statistically significant increase in operative time (t = 3.024, P = 0.003), a higher utilization of femoral MS (χ² = 12.641, P < 0.001), greater use of femoral stems (χ² = 12.118, P < 0.001), and an increased frequency of constrained prostheses (Z=-2.423, P = 0.015). Detailed intraoperative data are included in Table . General survivorship and outcomes Overall, all cases included in the study displayed successful integration of the prostheses with the host bone, as evidenced by postoperative X-rays that indicated effective healing of the bone defect at the metaphysis. Implant survival rates, measured by endpoints of reoperation and revision for any reason, were both recorded at 100% during the mean follow-up period of 57.86 months (ranging from 39 to 80 months, SD: 9.47). The mean NRS for pain significantly decreased ( P < 0.05) from 6.83 preoperatively to 1.67 postoperatively, while the mean HKAD reduced from 24.02 preoperatively to 2.75 postoperatively. Additionally, both ROM and HSS scores significantly improved ( P < 0.05) at the final follow-up, measuring 103.09° and 82.36 respectively, compared to preoperative values of 58.80° and 56.98. Based on HSS scores, 35 participants were categorized as excellent, 48 as good, and 4 as fair at the last follow-up, resulting in an excellent and good rate of 95.40% in terms of knee function. Representative cases are illustrated in Fig. (screw group) and Fig. (non-screw group). Comparison between the two groups in clinical and radiographic outcomes Specifically, knees that received screws exhibited poorer preoperative ROM (F = 11.670, P < 0.001), diminished knee function (F = 8.289, P = 0.005), and significantly greater preoperative deformities (F = 34.761, P < 0.001) compared to knees without screws, indicating a worse initial condition for the screw group. Nonetheless, postoperative outcomes revealed that the screw group attained comparable knee ROM, functional status, and radiographic alignment to the non-screw group ( P > 0.05). Detailed comparisons of clinical and radiographic outcomes between the two groups are presented in Table , with trends illustrated in Fig. . Complications Tibial end-of-stem pain appeared in five cases in the non-screw group, while none of the screw group developed such discomfort, the incidence of the pain occurs relatively more frequently in the non-screw group (9.8% VS 0%, χ² = 3.688, P = 0.0548, marginal significance). Patients suffering from this mild pain did not show radiolucency or loosening at the follow-up (Fig. ), and the pain can be relieved by routine NSAID and generally disappeared at about 3–6 months after the surgery. Incision liquefaction occurred in seven knees (four cases in the screw group and three cases in the non-screw group) postoperatively, but all cases resolved following dressing changes and infrared therapy, without the need for additional surgery. Two patients required a postoperative blood transfusion of 200 ml; however, this did not affect their length of hospital stay or time to suture removal. One readmission involved a patient with Charcot knee arthropathy who developed DVT, which was successfully treated with routine anticoagulation. Throughout the perioperative period and follow-up, no intraoperative fractures, cardiac events, pulmonary issues, joint instability, aseptic loosening, radiolucency, or PJI were reported. A total of 87 patients (106 knees) undergoing MS reconstruction for substantial bone defects in primary TKAs were included in the study. Among these, 46 patients (55 knees) received screws for the repair of tibial residual bone defects, while 41 patients (51 knees) did not. The distribution of knee etiologies included 53 patients with RA, 25 with post-traumatic arthritis, 6 with osteoarthritis, and 3 diagnosed with Charcot knee arthropathy. The demographics and preoperative conditions of the included patients are detailed in Table . Patients who received screws exhibited poorer bone quality (t = 3.095, P = 0.003), and a higher prevalence of osteoporosis was observed in the screw group (χ²=5.892, P = 0.015). A total of 138 metaphyseal sleeves and 140 straight stems were utilized in the TKA procedures. Intraoperative assessments revealed that knees in the screw group presented with more severe bone defects (Z=-3.590, P < 0.001). Additionally, the screw group demonstrated a statistically significant increase in operative time (t = 3.024, P = 0.003), a higher utilization of femoral MS (χ² = 12.641, P < 0.001), greater use of femoral stems (χ² = 12.118, P < 0.001), and an increased frequency of constrained prostheses (Z=-2.423, P = 0.015). Detailed intraoperative data are included in Table . Overall, all cases included in the study displayed successful integration of the prostheses with the host bone, as evidenced by postoperative X-rays that indicated effective healing of the bone defect at the metaphysis. Implant survival rates, measured by endpoints of reoperation and revision for any reason, were both recorded at 100% during the mean follow-up period of 57.86 months (ranging from 39 to 80 months, SD: 9.47). The mean NRS for pain significantly decreased ( P < 0.05) from 6.83 preoperatively to 1.67 postoperatively, while the mean HKAD reduced from 24.02 preoperatively to 2.75 postoperatively. Additionally, both ROM and HSS scores significantly improved ( P < 0.05) at the final follow-up, measuring 103.09° and 82.36 respectively, compared to preoperative values of 58.80° and 56.98. Based on HSS scores, 35 participants were categorized as excellent, 48 as good, and 4 as fair at the last follow-up, resulting in an excellent and good rate of 95.40% in terms of knee function. Representative cases are illustrated in Fig. (screw group) and Fig. (non-screw group). Specifically, knees that received screws exhibited poorer preoperative ROM (F = 11.670, P < 0.001), diminished knee function (F = 8.289, P = 0.005), and significantly greater preoperative deformities (F = 34.761, P < 0.001) compared to knees without screws, indicating a worse initial condition for the screw group. Nonetheless, postoperative outcomes revealed that the screw group attained comparable knee ROM, functional status, and radiographic alignment to the non-screw group ( P > 0.05). Detailed comparisons of clinical and radiographic outcomes between the two groups are presented in Table , with trends illustrated in Fig. . Tibial end-of-stem pain appeared in five cases in the non-screw group, while none of the screw group developed such discomfort, the incidence of the pain occurs relatively more frequently in the non-screw group (9.8% VS 0%, χ² = 3.688, P = 0.0548, marginal significance). Patients suffering from this mild pain did not show radiolucency or loosening at the follow-up (Fig. ), and the pain can be relieved by routine NSAID and generally disappeared at about 3–6 months after the surgery. Incision liquefaction occurred in seven knees (four cases in the screw group and three cases in the non-screw group) postoperatively, but all cases resolved following dressing changes and infrared therapy, without the need for additional surgery. Two patients required a postoperative blood transfusion of 200 ml; however, this did not affect their length of hospital stay or time to suture removal. One readmission involved a patient with Charcot knee arthropathy who developed DVT, which was successfully treated with routine anticoagulation. Throughout the perioperative period and follow-up, no intraoperative fractures, cardiac events, pulmonary issues, joint instability, aseptic loosening, radiolucency, or PJI were reported. The main findings of this study are as follows: (1) MS is effective for reconstructing significant bone defects in complex primary TKA among East Asian populations, demonstrating satisfactory short- to mid-term implant survivorship; and (2) the use of screws proved beneficial in addressing tibial “residual defects” around the MS placement, potentially reducing the risk of postoperative end-of-stem pain in complex primary TKA utilizing the MS-stem system. In typical cases of primary TKA, osteoarthritis is the chief underlying condition, accounting for over 90% of cases . However, in the current study, the distribution of etiologies among patients undergoing complex primary TKA with MS was as follows: rheumatoid arthritis, post-traumatic arthritis, osteoarthritis, and Charcot knee arthropathy, with more than 90% of patients presenting conditions other than osteoarthritis. This notable distribution reflects the unique and complex nature of the cases included in this study. These patients often exhibit more significant bone defects, poorer bone quality, more challenging deformities, and worse soft tissue conditions compared to the majority of primary TKA recipients. These special factors may occur either individually or in combination among the individuals enrolled in the study. Given these complexities, we speculated that conventional approaches to primary TKA are likely insufficient to address the aforementioned challenges, and employing standard repair techniques might lead to suboptimal outcomes, such as surgical failure and the need for early revision. Consequently, we opted for the use of MS, which incorporates a metaphyseal metal augmentation strategy to address these issues. As a new modular prosthetic component designed for revision TKA, the suitability of MS for complex primary TKA in East Asian populations remains uncertain and warrants rigorous retrospective evaluation. It is well established that current TKA prostheses and component designs are mainly based on anatomical data from Caucasians; however, numerous studies indicate significant anatomical differences between Caucasian and East Asian knee joints. Research has shown that Caucasians exhibit a higher tibial torsion angle, lower varus angle, and larger valgus angle compared to Japanese individuals. Furthermore, Caucasians have larger measures for distal femur and proximal tibia diameters as well as a higher femoral-tibial ratio compared to Chinese individuals . Some scholars have suggested that existing knee prosthesis designs may not adequately match the anatomy of East Asians . Particularly, metaphyseal sleeves are often used in combination with straight stems, which are commonly employed in complex primary TKAs or revisions to help resolve anatomical mismatches, reduce malalignment, and improve gap balancing. The ideal placement of a MS-straight-stem system should be central to ensure optimal bone contact in all directions . Failure to achieve these objectives may significantly increase the risk of surgical failure . While studies suggest the tibial axis in East Asian individuals is consistently anterolateral to the plateau center , Caucasian populations exhibit less anterolateral offset and may even demonstrate anteromedial positioning . Theoretically, the use of off-set stems in complex knee surgeries in East Asian populations may enhance tibial plateau coverage; however, this raises questions about potential mismatches when applying MS-straight-stem systems in this demographic. Therefore, the ability of off-the-shelf, Caucasian-based MS-straight-stem systems to achieve ideal “central” placement and deliver satisfactory mid to long-term clinical outcomes in East Asian individuals necessitates validation through clinical studies involving larger patient samples. The results of this study compellingly demonstrate that the use of MS in complex primary TKA for managing bone defects in East Asian populations yields early to mid-term clinical outcomes that are comparable to those reported in Caucasian patients . Radiographic evaluations (Figs. and ) indicated optimal “central position” placement of the implants, satisfactory coverage of the tibial plateau, and significant improvement in the correction of the gravity line.In a prospective study conducted by Matin-Hernandez et al. in Spain , which involved 25 patients with post-traumatic arthritis of the knee undergoing primary TKA using MS, the authors reported that the mean Knee Society Score improved from 29 preoperatively to 78 postoperatively. Additionally, the Western Ontario and McMaster Universities Osteoarthritis Index pain score improved significantly from 12 preoperatively to 3 postoperatively. Notably, all patients achieved osseous integration at a mean follow-up of 79 months, with a survival rate of 100%. Similarly, a cohort study conducted by Van Rensch et al. in the Netherlands , which included 28 primary TKA recipients following high tibial osteotomy and tibial plateau fractures, demonstrated favorable outcomes with the use of MS. With a final follow-up surpassing 2 years, the study reported significant improvements in overall health scores and the NRS, with scores increasing from 63 preoperatively to 70 postoperatively, and from 8 preoperatively to 3 postoperatively. The implant survival rate for all reasons was reported at 96.4% at the 2-year follow-up. In the present study, all 87 patients (106 knees) exhibited significant improvements in the NRS for pain, ROM, and HSS at the final follow-up after surgery. Notably, the HKAD declined dramatically from 24.02 preoperatively to 2.75 at the last follow-up, with no knees demonstrated varus or valgus deformities exceeding 4° at postoperative 2 year. During a mean follow-up period of 4.8 years, we reported no cases of joint instability, aseptic loosening, PJI, or the need for revision surgery, resulting in an excellent and good rate of 95.40% based on HSS. Furthermore, both the overall survival rates with respect to reoperation and revision endpoints were 100% at the final follow-up. Our study represents the first focused observation of MS utilized in complex primary TKA, with a sample size exceeding 100 knees (87 cases, 106 knees). The findings indicate that metaphyseal sleeves, with or without accompanying screws, demonstrate encouraging early to mid-term clinical effectiveness in addressing significant bone defects in complex primary TKA among East Asian patients, showcasing excellent applicability and implant survivorship.Importantly, as the MS positioning is guided by an intramedullary guide and the metaphyseal bone is gradually removed based on the sinking of the MS, the potential impact of tibial shaft axis offset variations - including those associated with anatomical differences - may be partially mitigated. This approach facilitates optimal tibial tray coverage while demonstrating particular advantages in complex primary TKA scenarios requiring straight extended stems. The intramedullary-guided bone resection combined with progressive MS settling appears to minimize anatomical variability challenges, representing a previously unreported benefit of sleeve implementation that merits further exploration. Another notable aspect of this study is that it is the first to describe the role of screws for tibial “residual bone defects” in the complex primary TKA procedure using MS. The concept of using bone cement and screws to repair bone defects during total knee arthroplasty was first introduced by Freeman in 1982. Since then, this method has been widely adopted in clinical practice and has demonstrated effectiveness and cost-efficiency for independently addressing small bone defects , specifically those with a depth of less than 10 mm. However, in the case of substantial bone defects exceeding 30 mm in depth, cement screws are typically employed as an adjunct to primary reconstruction techniques, such as bone grafting, metal augmentation, and constrained prostheses. During the surgical procedure, following the implantation of the MS-stem system in zones 2 and 3, successful reconstruction of the central region and the main body of substantial bone defects is typically achieved. Nevertheless, some patients are left with irregular small peripheral bone defects in zone 1 , and there is currently no dedicated research addressing the repair of these “residual defects” It has been suggested that in revision cases, bone grafting (either autologous or allogenic) may be employed to address these residual defects. In revision TKA cases, the bone defects left after implant removal (including the aforementioned residual defects) typically expose well-vascularized cancellous bone, making it conducive to successful bone grafting that provides reliable support. Conversely, in primary TKA cases, the base of the residual defects often exhibits sclerosis. Unless further interventions, such as freshening or osteotomy, are performed on these defects, they may hinder the graft’s viability, resulting in higher risks of graft failure, bone resorption, or collapse . These complications can adversely affect joint function and the long-term survival of the prosthesis. The use of bone cement and screws offers a reliable approach to rapidly fill small bone defects and provide long-term support, addressing these challenges effectively. In this study involving 87 patients (106 knees) of primary TKA with bone defects reconstructed using MS, 46 patients (55 knees) employed bone cement and screws to repair the residual defects. The clinical outcomes and prosthesis survival rates in these patients were comparable to those of the group that did not use screws (41 patients, 51 knees), demonstrating the feasibility and efficacy of this approach. Additionally, this study indicates that, compared to the non-screw group, the screw group presented with significantly greater preoperative deformity angles, more severe bone defect conditions, and poorer bone density differences that were anticipated. After the MS was placed in the central metaphysis, patients with worse knee conditions were more likely to have small residual defects needing further repair, necessitating more operation time, more MS and stems in the distal femur, and more implant constraint. The differences in bone density may also be attributed to the higher proportion of RA patients within the screw group, as it is well-known that individuals with RA are at a significant risk for OP due to the disease’s invasiveness and the effects of corticosteroids .Poor bone quality, especially severe osteoporosis is a strong indication for screw in these cases. An unexpected finding of this study was that none of the patients in the screw group experienced end-of-stem pain, whereas five knees in the non-screw group reported mild tibial end-of-stem pain; the incidence of end-of-stem pain in the screw group was less than that in the non-screw group, with a strong tendency towards statistical significance (0/55 vs. 5/51, P = 0.0548). Although radiography conducted during follow-ups (ranging from 3.2 to 6.4 years) for these patients experiencing pain did not reveal signs of implant subsidence, periprosthetic radiolucent line, or distal stem displacement, the patients still required intermittent analgesic use to manage their discomfort, which is noteworthy. End-of-stem pain is relatively common in revision cases involving MS, with an incidence ranging from 10 to 23% . In a previous study by Matin-Hernandez et al. , the incidence of end-of-stem pain in primary TKA cases utilizing MS was documented at 3.7% (1/27).It is generally believed that this discomfort is associated with the use of stems during TKA, which can result in stress shielding in the proximal tibia and potentially induce bone resorption; furthermore, the use of stems may concentrate stress around the distal tip, potentially leading to para-stem pain and an increased risk of fracture . In this study, the overall incidence of end-of-stem pain was found to be 4.72% (5/106), which aligns with the findings of Matin-Hernandez et al. , despite a significantly higher utilization of stems. Notably, we observed no occurrences of end-of-stem pain in the screw group, a result that deviates from expectations. Patients in the screw group exhibited considerably worse knee pathology and theoretically had a greater risk of postoperative complications; however, the incidence of end-of-stem pain in this group was lower than that observed in the non-screw group. We hypothesize that this outcome may be attributed to the screws enhancing the biomechanical performance of the stems. An experimental biomechanical study has indicated that strain concentration occurs at the tip of the stem for both cemented and press-fit stems used in primary TKA . The biomechanical advantage conferred by screws is their ability to provide support to the tibial platform prosthesis from above while anchoring within the cancellous bone of the medial tibial plateau below. This design may facilitate the transmission of joint loads to the cancellous bone, potentially mitigating the stress shielding effect associated with the stem and thereby lowering the risk of distal pain. Nevertheless, this hypothesis necessitates further validation through clinical studies involving larger sample sizes and extended follow-up periods. In our study, no intra-operative fractures, aseptic loosening, or PJI were observed during the follow-up. The mean length of stay (LOS) was 10 days (minimum 7 days), which is comparable to the typical LOS for general TKA recipients at our institution. Furthermore, the incidence of readmission for complications within 90 days was relatively low (0.94%, 1/106). The sole readmission case involved a patient with Charcot knee arthropathy who developed DVT; her readmission may be partially attributed to prolonged immobilization aimed at preventing early aseptic loosening. Perioperative period and follow-up did not reveal any cases of cardiac events and pulmonary issues despite the medically frail patient population being at high risk for perioperative complications. Another main perioperative complication identified in our study was fat liquefaction at the incision site, occurring in seven knees (6.60%, 7/106) without bacterial growth, which resulted in delayed wound healing and extended LOS. We believe this type of complication primarily affects obese individuals, those with hypoproteinemia, and patients with diabetes. In our study, all cases of delayed wound healing were successfully treated with conservative management, and no additional surgeries were required for wound-related complications. Two patients required postoperative blood transfusions, both of whom had preoperative anemia. A retrospective review by Morse KW et al. demonstrated a correlation between postoperative blood transfusion and prolonged LOS ; however, in our study, transfusions did not affect LOS or time to suture removal. Considering the complexity of the surgical procedures, a staged replacement strategy was employed for the 19 participants requiring bilateral arthroplasty, which we believe contributed to favorable short-term outcomes and a reduced incidence of surgical complications. Limitation of the present study The most significant limitation of this study is its retrospective design. As a clinical investigation, the challenges of randomizing and prospectively grouping patients restrict our ability to fully evaluate the effectiveness of metaphyseal sleeves and screws in complex primary TKA. While we did perform several complex primary TKA procedures utilizing metaphyseal sleeves and bone grafts for reconstructing bone defects, these cases were not included in the current analysis due to the small sample size (12 patients), as illustrated in Fig. . Consequently, further investigation is needed to compare the efficacy of bone grafts versus cement-screws in addressing residual defects around the sleeve-stem placement in complex primary TKA procedures. Additionally, the relatively short follow-up period have surely limited our ability to detect some complications that have been reported in the literature. As a result, there may be observation bias, and comprehensive survival analyses could not be conducted. The most significant limitation of this study is its retrospective design. As a clinical investigation, the challenges of randomizing and prospectively grouping patients restrict our ability to fully evaluate the effectiveness of metaphyseal sleeves and screws in complex primary TKA. While we did perform several complex primary TKA procedures utilizing metaphyseal sleeves and bone grafts for reconstructing bone defects, these cases were not included in the current analysis due to the small sample size (12 patients), as illustrated in Fig. . Consequently, further investigation is needed to compare the efficacy of bone grafts versus cement-screws in addressing residual defects around the sleeve-stem placement in complex primary TKA procedures. Additionally, the relatively short follow-up period have surely limited our ability to detect some complications that have been reported in the literature. As a result, there may be observation bias, and comprehensive survival analyses could not be conducted. Given the excellent feasibility, encouraging outcomes, high survival rates, and low complication rates observed in our study, we believe that metaphyseal sleeve is a safe and effective option for reconstructing substantial bone defects in complex primary TKA in Eastern Asian populations. The cement-screw technique represents a viable solution for addressing tibial “remaining defects” surrounding the placement of metaphyseal sleeves, particularly in cases with significant deformity and poor bone quality. However, the potential benefits of this technique in improving load transfer and reducing stress shielding need further exploration through larger studies with extended follow-up periods. |
Morphological Variations in Placentas among Deliveries in the Department of Obstetrics and Gynaecology in a Tertiary Care Centre: A Descriptive Cross-sectional Study | 657407c7-0153-461b-aed1-ea58e3e3d573 | 9794935 | Gynaecology[mh] | The full-term human placenta is a flattened discoidal mass with an approximately circular or oval outline. In 90% of individuals a discoid or oval-shaped placenta is seen. However, variations can be present in the morphology in the form of bidiscoidal shape, lobed, diffused, fenestrated or circumvallate. Abnormal shapes are seen in 10% of women and include notched or lobed placentas, membranous placentas, and other rare variants. However, relatively less research has been done for the morphology and the variations present in its shape. So this study can be helpful in better diagnosis and management of the predisposing clinical conditions related to maternal and foetal health. - This can also help to minimize the complications related to placenta and umbilical cord and improve maternal and fetal outcome. This study was done to find out the morphological variations among deliveries in the Department of Obstetrics and Gynaecology in a tertiary care centre. This descriptive cross-sectional study was carried out in 105 placentas obtained from deliveries during the period of September 2021 to November 2021, at the Department of Obstetrics and Gynaecology of Kathmandu Medical College and Teaching Hospital after receiving ethical approval from the Institutional Review Committee (Reference number: 2308202105). Placentas were collected from the labour room of the Department of Obstetrics and Gynaecology. After separating the baby from the umbilical cord, the specimens were placed in a bucket containing 10% formalin. Samples were then washed clean of blood and stored again in formal saline for further examination for shape of each placenta was done. The normal morphology and the variations observed were recorded. All the placentas obtained from deliveries in the study duration were included except for the damaged and torn placentas, which were excluded. The sample size was calculated by using the following formula: n = Z 2 × p × q e 2 = 1.96 2 × 0.065 × 0.935 0.05 2 = 94 Where, n= minimum required sample size Z= 1.96 for 95% Confidence Interval (CI) p= prevalence taken as 6.5% from educated guess e= margin of error, 5% Hence, the minimum required sample size was 94. However, a sample size of 105 was taken. The collected data was compiled, entered and analysed in Microsoft Excel 2013. Point estimate and 95% CI were calculated. Among 105 placentas observed, morphological variations were seen in two (1.91%) (0-4.53, 95% CI). One (50%) was placenta succenturiata and one (50%) was triangular in shape . Among these, marginal insertion umbilical cord in placenta was observed in triangular placenta whereas normal eccentric umbilical cord insertion was seen in placenta succenturiata . The full term human placenta is a flattened discoidal mass with an approximately circular or oval outline. In the present study, almost 98% of the placentas were observed with discoidal shape. This finding is in accordance with a study where it is mentioned that a discoid or oval-shaped placenta is seen in about 90% of the individuals. Similar finding of 94% circular and 6% oval shape of placenta has also been reported. Meanwhile, 48% placentas with oval and 36% with circular shape has been reported in a study. However, variations can be present in the morphology in the different forms, e.g. bidiscoidal shape, lobed, diffused, fenestrated or circumvallate. Abnormal shapes are seen in 10% of women and include notched or lobed placentas, membranous placentas, and other rare variants. In this study, one case of placenta succenturiata and a triangular placenta were observed. In this study, one case of placenta succenturiata and a triangular placenta were observed. Similar finding has been reported in a study, of one case of placenta succenturiata whereas they have reported four triangular shaped placentas. Placental abnormalities are considered as uncommon finding in obstetrics and among them the succenturiate lobe of placenta is a very rare entity. Apart from these, irregular shape of placentas has also been reported in different studies. , The attachment of the umbilical cord was observed to be in the central portion of placenta in 104 (99.05%) placentas. In one placenta (0.95%), the cord was attached marginally. In a study conducted in Nepal, no abnormality regarding the attachment of cord, was recorded. Almost in consistency with the present study, 2% marginal attachment of the cord has been reported in a study conducted in Pakistan. Other studies have reported 7%, 8.97%, 25% marginally attached umbilical cord. , , These variations in placental morphology has been looked upon as being corelated to any predisposing conditions such as placenta praevia, vaginal bleeding, and premature delivery. In this present study, there were certain limitations. The occurrence of various shapes of placenta were observed, however, correlation between the morphology (shape) of placenta and any predisposing factors is yet to be studied in detail. The prevalence of morphological variants in our study was found to be higher when compared to other studies from similar settings. A sound knowledge of the abnormal morphologies may help avoiding any complications as in bilobed placenta or extra lobes being mistaken for retained placental tissues. Further studies of the shape of placenta with co-relation to predisposing conditions may also help in better diagnosis and management of the predisposing clinical conditions related to maternal and foetal health. |
Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider | 97f3a00c-0dd3-44eb-b83b-2ab7a4d5293d | 8068197 | Pathology[mh] | In this report, we seek to review the impact that respiratory viruses have had upon mankind, discuss current efforts regarding their control, and propose new strategies to detect emerging respiratory virus threats and mitigate them before they become widespread. The goal in proposing new strategies is to identify methods that will be widely embraced, preemptive, and sustainable, rather than our current practice of being surprised by new threats. The latter forces countries to invest intensive and expensive response efforts towards respiratory virus control, while enduring significant morbidity. In recent decades, the world has experienced numerous epidemics of emerging or reemerging respiratory viruses. These viruses have been responsible for much morbidity and mortality , in addition to pandemics in 2009 and 2020. While these viruses come from five different viral families ( Adenoviridae , Coronaviridae , Orthomyxoviridae , Picornaviridae, and Pneumoviridae ), they have two things in common: many are RNA viruses, and most are zoonotic. A review of these epidemics is necessary for planning better future mitigation strategies. 1.1. Human Metapneumovirus (hMPV) hMPV was first discovered in 2001 in the Netherlands, yet it is thought to have been in circulation for at least 50 years beforehand . Retrospective specimen analyses in the USA have found evidence of hMPV as far back as 1982 . Phylogenetic analyses hypothesize divergence from an avian predecessor to have occurred around 200 years ago . Serological studies show that, worldwide, nearly all children develop antibodies to this commonly circulating virus by age five, accounting for 6–40% of acute respiratory infections in this age group . Surveillance efforts are primarily targeted towards young children, but reinfection throughout adulthood is common, with elderly and immunocompromised individuals at heightened risk of severe outcomes . The virus is thought to have spilled over between avian species and man as well as between nonhuman primates and man . This RNA virus belongs to the Pneumoviridae family (new classification as of 2016, formerly a subfamily within the Paramyxoviridae family). 1.2. Rhinovirus Group C (HRV-C) HRV-C was identified in 2004, following an outbreak of influenza-like illness in New York State . In limited surveillance studies, HRV-C has since been identified as the most prevalent circulating strain of rhinovirus in the fall and winter seasons of multiple countries . HRV-C is associated with more severe respiratory disease than rhinoviruses A and B, and has been connected to asthma, bronchitis, wheezing, and pneumonia . The virus has crossed species from man to nonhuman primates . This RNA virus belongs to the Picornaviridae family. 1.3. Enterovirus A71 (EV-A71) EV-A71 infections are recognized as the cause of hand, foot, and mouth disease and various neurological conditions, especially among children under five years. The virus was first isolated from an encephalitic patient in California in 1969, and between 1972 and 1990 was associated with additional outbreaks across six countries . EV-A71 became endemic in the Asian-Pacific region in the 1990s, with major outbreaks occurring every three to four years, which affected thousands of children. Surveillance is sparse, yet the virus is estimated to be responsible for at least 12.8 M infections and 3747 deaths in Asia alone . Large outbreaks have primarily been limited to the Asian-Pacific region; however, the virus still has a global presence, as indicated by small-scale flare-ups, such as the 119 cases reported in Australia in 2012 and 34 cases seen in Colorado in 2018 . While some enteroviruses are recognized to be cross-species , this RNA virus is not thought to be zoonotic. It belongs to the Picornaviridae family. 1.4. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) SARS-CoV emerged in China in 2003, and rapidly spread to 29 countries before it was halted, with heroic public health and hospital infection control measures. Though no cases of SARS-CoV infection have been identified since 2004, it is recognized to have infected more than 8000 people and caused 774 deaths during the short time the virus was circulating . The virus is thought have originated in bats and moved to man via one or more intermediate animal hosts . This RNA virus belongs to the Coronaviridae family. 1.5. Human Adenovirus 14 Strain (HAdV14) HAdV14 reemerged in the United States in 2006, causing at least 750 illnesses and 13 deaths . As with other such viruses, surveillance for this “killer cold virus” is sparse, however, available data suggest that it has spread to at least Ireland, Canada and China, and still circulates today. While some adenoviruses have jumped species , this adenovirus is not thought to be zoonotic. This DNA virus belongs to the Adenoviridae family. 1.6. 2009 H1N1 Influenza A Pandemic (H1N1pdm09) Virus The H1N1pdm09 virus emerged in Mexico in 2009, and quickly spread throughout the world, causing an estimated 60.8 million illnesses and at least 12,469 deaths from 2009–2010 in the United States alone . This swine-like influenza virus, which continues to circulate today in both humans and pigs, served as a catalyst for more comprehensive influenza A virus surveillance strategies. This RNA virus belongs to the Orthomyxoviridae family. 1.7. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in Saudi Arabia in 2012, causing alarming respiratory illnesses. As of September 2020, MERS-CoV has been confirmed as a cause of 2494 illnesses and 858 deaths in 27 countries. It is recognized to be enzootic in camels . This RNA virus belongs to the Coronaviridae family. 1.8. Enterovirus D68 (EV-D68) EV-D68 was first isolated in California in 1962, causing infrequent cases in the USA and only minor outbreaks in Europe, Africa, and Southeast Asia. In 2014, however, a novel clade of a 2nd EV-D68 was implicated in a series of outbreaks of respiratory disease across 21 countries, resulting in at least 2529 EV-D68 illnesses and 17 deaths in that year alone. Additional international outbreaks were identified in 2016 and 2018, as the virus was implicated as the cause of severe respiratory illness and acute flaccid myelitis, a polio-like illness. This RNA virus is not thought to be zoonotic. It belongs to the Picornaviridae family. 1.9. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) SARS-CoV-2, a novel bat-like coronavirus, emerged in China in December 2019, and quickly spread worldwide. It is the cause of our current COVID-19 pandemic. Case counts and fatalities continue to increase, having reached 120 M and 2.6 M, respectively, as of 15 March 2021. The virus is thought have originated in bats and moved to man via pangolins, or possibly another, yet-unidentified, intermediate animal host. This RNA virus belongs to the Coronaviridae family. hMPV was first discovered in 2001 in the Netherlands, yet it is thought to have been in circulation for at least 50 years beforehand . Retrospective specimen analyses in the USA have found evidence of hMPV as far back as 1982 . Phylogenetic analyses hypothesize divergence from an avian predecessor to have occurred around 200 years ago . Serological studies show that, worldwide, nearly all children develop antibodies to this commonly circulating virus by age five, accounting for 6–40% of acute respiratory infections in this age group . Surveillance efforts are primarily targeted towards young children, but reinfection throughout adulthood is common, with elderly and immunocompromised individuals at heightened risk of severe outcomes . The virus is thought to have spilled over between avian species and man as well as between nonhuman primates and man . This RNA virus belongs to the Pneumoviridae family (new classification as of 2016, formerly a subfamily within the Paramyxoviridae family). HRV-C was identified in 2004, following an outbreak of influenza-like illness in New York State . In limited surveillance studies, HRV-C has since been identified as the most prevalent circulating strain of rhinovirus in the fall and winter seasons of multiple countries . HRV-C is associated with more severe respiratory disease than rhinoviruses A and B, and has been connected to asthma, bronchitis, wheezing, and pneumonia . The virus has crossed species from man to nonhuman primates . This RNA virus belongs to the Picornaviridae family. EV-A71 infections are recognized as the cause of hand, foot, and mouth disease and various neurological conditions, especially among children under five years. The virus was first isolated from an encephalitic patient in California in 1969, and between 1972 and 1990 was associated with additional outbreaks across six countries . EV-A71 became endemic in the Asian-Pacific region in the 1990s, with major outbreaks occurring every three to four years, which affected thousands of children. Surveillance is sparse, yet the virus is estimated to be responsible for at least 12.8 M infections and 3747 deaths in Asia alone . Large outbreaks have primarily been limited to the Asian-Pacific region; however, the virus still has a global presence, as indicated by small-scale flare-ups, such as the 119 cases reported in Australia in 2012 and 34 cases seen in Colorado in 2018 . While some enteroviruses are recognized to be cross-species , this RNA virus is not thought to be zoonotic. It belongs to the Picornaviridae family. SARS-CoV emerged in China in 2003, and rapidly spread to 29 countries before it was halted, with heroic public health and hospital infection control measures. Though no cases of SARS-CoV infection have been identified since 2004, it is recognized to have infected more than 8000 people and caused 774 deaths during the short time the virus was circulating . The virus is thought have originated in bats and moved to man via one or more intermediate animal hosts . This RNA virus belongs to the Coronaviridae family. HAdV14 reemerged in the United States in 2006, causing at least 750 illnesses and 13 deaths . As with other such viruses, surveillance for this “killer cold virus” is sparse, however, available data suggest that it has spread to at least Ireland, Canada and China, and still circulates today. While some adenoviruses have jumped species , this adenovirus is not thought to be zoonotic. This DNA virus belongs to the Adenoviridae family. The H1N1pdm09 virus emerged in Mexico in 2009, and quickly spread throughout the world, causing an estimated 60.8 million illnesses and at least 12,469 deaths from 2009–2010 in the United States alone . This swine-like influenza virus, which continues to circulate today in both humans and pigs, served as a catalyst for more comprehensive influenza A virus surveillance strategies. This RNA virus belongs to the Orthomyxoviridae family. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in Saudi Arabia in 2012, causing alarming respiratory illnesses. As of September 2020, MERS-CoV has been confirmed as a cause of 2494 illnesses and 858 deaths in 27 countries. It is recognized to be enzootic in camels . This RNA virus belongs to the Coronaviridae family. EV-D68 was first isolated in California in 1962, causing infrequent cases in the USA and only minor outbreaks in Europe, Africa, and Southeast Asia. In 2014, however, a novel clade of a 2nd EV-D68 was implicated in a series of outbreaks of respiratory disease across 21 countries, resulting in at least 2529 EV-D68 illnesses and 17 deaths in that year alone. Additional international outbreaks were identified in 2016 and 2018, as the virus was implicated as the cause of severe respiratory illness and acute flaccid myelitis, a polio-like illness. This RNA virus is not thought to be zoonotic. It belongs to the Picornaviridae family. SARS-CoV-2, a novel bat-like coronavirus, emerged in China in December 2019, and quickly spread worldwide. It is the cause of our current COVID-19 pandemic. Case counts and fatalities continue to increase, having reached 120 M and 2.6 M, respectively, as of 15 March 2021. The virus is thought have originated in bats and moved to man via pangolins, or possibly another, yet-unidentified, intermediate animal host. This RNA virus belongs to the Coronaviridae family. Emerging and reemerging viruses have had major economic impacts. These costs arise from missed work due to worker morbidity and care of loved ones, lost workers due to mortality, healthcare costs, and economic costs of mitigation, which can include disruptions to travel and trade, closing of public spaces and businesses, and compliance with workplace regulations. The World Bank has estimated that the SARS-CoV epidemic cost the affected nations USD 30–50 billion, and the H1N1 pandemic USD 45–55 billion . An incursion of MERS-CoV from June 2015 to June 2016 was estimated to cost South Korea USD 2.6 billion . At present, the economic impact of COVID-19 promises to exceed the combined cost of all other recent respiratory disease epidemics. A number of infectious disease modeling teams have worked to identify risk factors for emerging influenza A viruses , emerging infectious diseases , and emerging zoonotic diseases . The identified risk factors for specific viruses are often complex and sometimes disparate. However, they typically point to densely populated geographical areas in warm regions as areas of high risk. They also point to encounters with live animals, especially in markets and large domestic animal farms , as a source of human infection risk. We posit that, especially for respiratory viruses, the highest probability of a novel virus emerging occurs in large dense populations of animals, which can sustain the replication of multiple virus subtypes that can recombine. Over time, these viruses and generate new progeny viruses. We see these populations in two contexts: in domestic meat production animals, where a continued introduction of immunologically naïve animals enables multiple viral types to circulate indefinitely in the production animal population, and among mammals that live in large colonies, such as many species of bats. Although their contact with humans is less frequent than the contact domestic animals have with humans, bats often connect with domestic animals, which can serve as intermediate animal hosts in introducing novel viruses to humans. Multiple species of bats have unique immune systems capable of harboring viruses for long periods of time without causing significant illness to their bat colonies, yet they are quite capable of transmitting these same viruses to other animal species. They often do so via roosting in large colonies in habitats such as caves, where enzootic viruses are shared within their population, and by having a long lifespan, showing high geographical movement through flight, and exposing other animal species through fecal and urinary droppings . For some zoonotic wildlife viruses, another factor leading to spillover involves land use change. One recent study, for example, found that land converted to human use had a higher abundance of mammals and birds that serve as reservoirs for zoonotic diseases . Land use change may also influence the emergence of bat-borne emerging diseases through changes to bat geographic ranges, population densities, mixing of different bat species, and their use of human-dominated landscapes for foraging and roosting . Thus, human activities can also shape patterns of zoonotic disease risk in nature—including through effects on reservoir host abundance—while also increasing the human–animal contact needed for spillover to occur. More research is needed to understand how these factors influence respiratory virus spillover from wildlife. A common misunderstanding is that viral spillover to humans is rare, but when it occurs, the virus is fully capable of onward transmission in human populations. Popular novels, movies, and news media promote this view. The reality is different: most animal viruses that humans are exposed to fail to cause significant human infections. The rare virus that does cause a human infection is most often unable to transmit efficiently to other humans. The difficulty of human-to-human transmission occurs because different species often have quite different immunological protections against viral infections, along with different viral receptors on their cells. The new virus may be well-adapted to its current host species but is unlikely to have the unique constellation of viral characteristics that enable it to infect and transmit efficiently in a new host species. The phylogenetic proximity of the reservoir host and the new host affects the probability of host shifts . This occurs because more closely related hosts will have more similar viral receptors and immunological profiles, along with shared ecological characteristics that facilitate frequent contact. Thus, humans share more parasites and pathogens with the chimpanzee and gorilla than with other primates . Similarly, humans may be more likely to acquire pathogens from captive primates than from other mammals. This effect can also work in reverse, with wild great apes especially susceptible to respiratory viruses from humans . Given the many challenges in efficiently transmitting a virus to a new host, significant evolutionary change is typically required for a new virus to adapt to humans and spread from human to human. Viral changes occur via mutations, recombination, or reassortment. Viral mutations are so regular that evolutionary biologists can compare viral sequences and accurately predict how long ago two viruses shared a common ancestor. However, most changes to the viral genome do not increase viral fitness in each host; most mutations result in decreased fitness of the virus, and thus are lost through natural selection. In addition, those mutations that would increase fitness in another host are not under selection pressure until after spillover occurs, making pre-adaptation to other hosts highly unlikely in the original “source” host. For a novel virus to adapt to humans generally means that the virus must both successfully continue to replicate in the animal host while also having an affinity for cellular attachment, invasion, and replication in the new human host. Thus, it takes time and the generation of many novel viruses before a virus emerges that is effective in transmitting from human to human. We illustrate this spillover process with a graphic showing the lengthy time periods required for this incremental process. These considerations suggest that the evolution of efficient human-to-human transmission requires repeated exposure, involving many people, animals, and settings. Probability-wise, many viruses must first spill over to humans and yet not cause human-to-human transmission before a virus is selected with the fitness to cause limited human-to-human transmission. For a novel virus to become highly transmissible between humans is an extremely rare event . Again, numerous viruses with limited human-to-human transmission are likely to fail before a novel virus has the fitness to cause efficient human-to-human transmission. The basic reproductive number (R 0 ) is an ecological concept that applies to the reproduction of any organism; it measures the success of a population as the rate of births divided by the rate of deaths. In the context of infectious diseases, this concept translates to the rate at which new infections arise (as a function of contact rates, population density and transmissibility) divided by the rate at which infections are lost (due to disease associated mortality, or virulence, and recovery from infection). R 0 must be greater than 1 for a virus to invade a fully susceptible novel host population, such as humans, but the challenges just noted mean that most viruses have an R 0 less than 1 when initially introduced. Modeling research has shown that when evolution is considered, even pathogens with an R 0 that is initially less than 1 can invade a population . This research simulated the transient infections we have been discussing: the occasional spillover of a virus that is not yet adapted to its new host (and thus has an R 0 < 1). The authors assumed that mutations can occur with some probability that would boost the R 0 to be greater than 1. The simulations revealed that stuttering chains of transmission occur even when R 0 < 1, and these chains are longer (resulting in more infected individuals) as R 0 approaches 1. Moreover, a longer chain of transmissions increased the opportunities for the mutation to occur. In other words, the probability of an adaptive mutation occurring is higher as R 0 approaches 1. The main message is that anything that facilitates these initial transmission events is also facilitating the evolution of more transmissible variants. This consideration of initial transmission chains and evolution also highlights the importance of other factors that can favor the evolution of an efficient virus in humans. The larger the population of the viruses in the new host—in terms of the diversity of viruses, their prevalence in the host population, and the viral load of each host—the more opportunities for mutations to arise that will confer more efficient transmission. Similarly, any factors that increase the length of a transmission chain—such as early super-spreading events or initial infections in chains of immunosuppressed hosts —will also increase opportunities for the necessary mutations to occur, which then increases the probability of adaptive evolution and a large outbreak (or even a pandemic). While novel animal viruses are assaulting the immune systems of humans with animal exposure, the humans’ immune systems are not static. The immune system, too, is adapting to exposures and increasing immunological resistance as viral insults increase. We see this clearly in seroepidemiological studies of animal-exposed workers with respect to zoonotic influenza A exposures . Animal workers often have elevated antibodies against multiple strains of influenza A virus. Even so, sometimes the magnitude of the viral exposure (viral load) overwhelms the animal workers’ immune systems, and we see animal workers infected despite having preexisting antibodies against enzootic animal viruses . This competition between changes in animal viruses and changes in animal workers’ immune defenses is known as antagonistic coevolution . This process refers to the reciprocal changes in host and pathogen traits as the two lineages adapt to one another under negative-frequency-dependent selection. Each defensive action by the host can be countered by new mutations in the pathogen to overcome that defense, with selection thus favoring hosts that have new (rare) defenses and favoring pathogens that can infect hosts with the most common defensive strategy. This results in cycles of coevolution, with the host and pathogen continuously evolving. Given the vastly shorter life cycle of viruses, they often have the upper hand in this contest when examined genetically. However, the rapid response of the human immune system enables the animal worker to safely withstand the new viral threats. Another evolutionary question involves virulence, which is defined by evolutionary biologists as the damage caused to the host by infection with a pathogen. Traditionally, evolutionary biologists proposed that a highly virulent pathogen is a poorly adapted one. More recent work has shown that this is not the case, and that a variety of factors favor higher virulence . Virulence is now commonly understood as tradeoff between transmissibility and the death or recovery of a host, which is easily seen in the equation for R 0 described above. Thus, mutations that influence transmission more than the disease-associated death rate will increase R 0 and be favored by natural selection. This might be achieved, for example, by mutations that increase viral load, which would be expected to increase transmissibility and host death rate (through use of host resources). In the United States, public health efforts at mitigating emerging infectious disease threats often vacillate with public interest . Money will be dedicated to investigating and controlling emerging novel virus problems, but, over time, the interest and support eventually wanes . We need to find better ways to establish and sustain worldwide surveillance for novel emerging respiratory virus threats. We need innovative and cost-efficient approaches that will be embraced by both the developed and the developing world. We need to conduct rapid risk assessments when novel viruses are detected and to mitigate their threat to humans and animals once discovered. If we conduct surveillance for animal viruses among animal workers, we are likely to be more efficient in identifying pre-pandemic viruses, in contrast to attempting to study all viruses detected in many animal species. Detecting an animal virus in the upper airway of an animal worker already demonstrates some risk to man and makes focusing on further studies of that virus worthwhile. In contrast, much of the U.S. effort towards prepandemic virus detection has had a Wildlife Pathogen Discovery Focus . Examples include USAID’s PREDICT I and II programs, as well as the proposed Global Virome Project. These programs have been criticized as poorly targeted, expensive and not likely to yield human health benefit . Their costs are also not considered to be sustainable. More than USD 200 M has already been invested in the PREDICT programs since 2009. The proposed Global Virome Project has a projected cost of USD 3.4B, which is considered prohibitive . USAID’s new multimillion dollar effort (referred to as “STOP Spillover”) and newly proposed DEEP VZN surveillance and training strategies seem to again be largely focused upon the Wildlife Pathogen Discovery Focus . An alternative to the Wildlife Pathogen Discovery Focus strategy has been the Known Human Pathogen Focus , which has been embraced by leaders at the US National Institute of Allergy and Infectious Diseases (NIAID) and Centers for Disease Control and Prevention (CDC) for multiple years. NIAID has supported, and continues to support, multiple groups of researchers focused on specific pathogens in their Centers of Excellence for Influenza Research and Surveillance (CEIRS), newly promoted Centers for Research in Emerging Infectious Diseases (CREID), and the expected Centers of Excellence for Influenza Research and Response (CEIRR). NIAID leadership has also proposed large 20-year human-cohort studies, with intensive immunology and pathology research involving 120 known human pathogen groups , although senior leadership has admitted that the large amount of funding necessary to pursue this strategy is not available . The CDC has also long embraced a Known Human Pathogen Focus in the organization of its public health and research professionals. Some of these teams are quite large and have a worldwide footprint. A good example is CDC’s Influenza Division, which has more than 300 professionals working across more than 200 surveillance sites, many in international settings. While there have been benefits to the US government’s efforts in both its Wildlife Pathogen Discovery Focused and Known Human Pathogen Focused surveillance and research efforts, one can argue that these efforts were not effective in anticipating either the 2009 influenza A H1N1 pandemic or the 2020 SARS-CoV-2 pandemic. Fortunately, these efforts were very useful in responding to these pandemics. In fact, both strategies have contributed mightily to our successful responses. However, responding well is not ideal, as each pandemic has had tremendous morbidity, mortality, and economic costs. We posit that a more strategic approach to detecting and mitigating prepandemic respiratory viruses would be to concentrate more targeted surveillance at the human–animal interface, where large populations of animals come into contact with humans, and, in doing so, employ a One Health Approach in this more targeted surveillance effort . The One Health Approach involves professionals from disparate disciplines working together to solve complex health problems, such as zoonotic disease epidemics, antimicrobial resistance, and food safety. The goal is to achieve optimum health for humans, animals, and the environment. The One Health Approach has been endorsed by numerous academic and professional organizations , governments, and health-concerned multilateral organizations, including the WHO, FAO, OIE, the World Bank and the G20. We recognize that USAID, NIAID, the CDC, and most other US governmental agencies publicly embrace the One Health Approach and invest some effort in One Health activities. However, we argue that, when compared to their Wildlife Pathogen Discovery Focused or Known Human Pathogen Focused surveillance and research efforts, their investment in the One Health Approach is quite modest. By periodically examining both animals and humans using a One Health Approach, we would be able to determine which viruses are becoming successful in adapting to humans and subsequently develop interventions to stop that viral adaptation. This could save millions of lives and prevent tens of millions of infections regarding a prepandemic virus threat. Viral evolutionary theory suggests the majority of novel animal viruses often fail to cause infection in humans. It is a rarity that a virus will succeed in colonizing or infecting humans. For that virus or its progeny to cause limited human-to-human infection will be rarer still. It will be incredibly rare for that virus or its progeny to become highly transmissible among humans. The selection of such viruses is likely to occur incrementally over long periods of time. Hence, evolutionary theory supports the notion that conducting surveillance for novel viruses at the animal–human interface would be an effective strategy to detect prepandemic viruses before they fully adapt to humans and become highly transmissible. Alternatively, if One Health surveillance for prepandemic respiratory viruses is not possible at the human–animal interface, a second high-yield strategy would be to conduct surveillance for novel animal pathogens among pneumonia patients in geographical areas known to be at high risk of novel respiratory virus emergence. We have recently applied these strategies with good success and at modest cost in detecting zoonotic coronaviruses among humans hospitalized with pneumonia (Malaysia) , and recently isolated and fully sequenced a recombinant, canine-like, feline-like, alphacoronavirus from among these patients [personal communication G. Gray]. We have also detected a bat-like adenovirus in a patient with respiratory illness (Malaysia) . Other teams have similarly found evidence of animal coronaviruses in humans (USA, Haiti) . Using the One Health Approach, we have documented human metapneumovirus infections in turkeys and evidence for avian pneumovirus infections among turkey workers (USA) . We have also found molecular evidence of influenza D virus among poultry (Malaysia) , human enteroviruses in pig slurry (USA) , and equine influenza A virus spillover from horses to camels (Mongolia) . Successful international human–animal interface network studies will necessarily often require the identification of primary partners in emerging infectious disease hotspots among national or regional veterinary institutions. Such institutions often have better access to environments where animal workers are likely to be exposed to enzootic animal respiratory viruses. For pneumonia network studies, this will involve referral hospital institutions with a relatively high volume of pneumonias. Fundamental to both human–animal interface and pneumonia networks surveillance is the identification of laboratories with the molecular equipment and ultracold freezers necessary to detect and preserve respiratory viruses. Having argued that we should conduct One Health surveillance at the human–animal interface, specifically where and how should we do so? Some have argued that we should focus surveillance on novel respiratory viruses among humans who have contact with wildlife, especially bush meat hunters and wet market workers in geographical areas rich in biodiversity. However, hominids have been eating bushmeat for several hundred thousand years, and only recently have we begun to note zoonotic respiratory virus epidemics. One could argue that while such bushmeat hunters or marketers may occasionally be infected with a wildlife virus, the virus or its progeny are not likely to have the prolonged exposure to humans required to become highly transmissible to man. A more logical approach is supported by evolutionary and ecological data. For instance, in their 2009 report, Lloyd-Smith and colleagues present their concept of “spillover force of infection”, which they describe as the probability of spillover as dependent on three major components: (1) prevalence of the pathogen in the reservoir, (2) the reservoir-to-human contact rate, and (3) the probability of human infection with the animal pathogen. If one considers areas where these components are high, one may effectively select the best sites for conducting surveillance of pre-pandemic viruses. 7.1. Among Which Animals Is Prevalence of Respiratory Viruses High? If we consider the five viral families of respiratory viruses discussed earlier in this text, data are available to answer this question for, at least, influenza A viruses ( Orthomyxoviridae ) and coronaviruses ( Coronaviridae ). The published literature is not rich with prevalence data among animals for the other viruses of concern. Influenza A viruses are highly enzootic in livestock, especially poultry and swine, as well as among aquatic birds. Influenza D viruses are also thought to be highly enzootic in cattle and swine . Coronaviruses are enzootic in numerous animal species, especially in bats, birds, and livestock . Hence, studying these animal species seems an appropriate strategy. Additionally, studying dense, dynamic populations of these animals with efficient reproduction is very strategic, as such animal populations will be able to sustain respiratory virus populations via continual infections among immunologically naïve offspring. 7.2. Where Is the Animal Reservoir-Human Contact Rate High? If we consider the bats and domestic animals mentioned above, we might select sites for surveillance where these animals have frequent and close contact with man. Bats are not frequently in direct contact with humans, save for those occasional colonies which are maintained in zoos, for research purposes, or by bat enthusiasts. In contrast, domestic livestock, such as pigs, chickens, ducks, geese, cattle, goats, and sheep, are often reared in close contact with humans. Modern industrial farming efforts are increasing the size of such domestic herds or flocks, as their meat can be produced more efficiently and at lower cost compared to farms with smaller herds or flocks. The downside of these large industrialized farms is their propensity to harbor sustained animal pathogens among livestock, which may cause mild disease in the animals but, over time, can cross-over to infect animal workers. Reports of novel pathogen generation on industrialized farms and transmission to humans is becoming increasingly common for both viral and bacterial pathogens . Consistent with the logic prescribed by Lloyd-Smith and colleagues , large industrialized farms in emerging infectious disease hotspots would be strategic sites for conducting surveillance for novel prepandemic respiratory viruses. 7.3. From Which Viruses and Viral Hosts Is the Probability of Zoonotic Virus Transmission to Humans Highest? Again considering the five viral families of concern, data to support an answer to this question are a bit sparse. However, some data are available. Freidl et al. published a large review of animal to human risk for zoonotic influenza A in 2014. After reviewing 89 published reports, they concluded that the risk was greatest from human exposure to poultry and pigs. In our 2019 review of the literature for zoonotic influenza A transmission to humans , we concluded that the species barrier is lower for viruses moving from swine to humans rather than from other animals to humans. In a 2019 report, Wang et al. concluded that the probability of interspecies transmission of coronaviruses from two different coronavirus genera, alphacoronaviruses, and deltacoronaviruses are especially high. Given that pigs have experienced recent epizoonotics from five novel coronaviruses , it seems worthwhile to conduct surveillance for prepandemic coronaviruses among pigs. Our research team has recently conducted reviews of the literature regarding adenovirus and enterovirus zoonotic infections. While data are sparse, it seems relevant that nonhuman primates might be at the greatest risk of sharing adenoviruses and enteroviruses with humans. If we consider the five viral families of respiratory viruses discussed earlier in this text, data are available to answer this question for, at least, influenza A viruses ( Orthomyxoviridae ) and coronaviruses ( Coronaviridae ). The published literature is not rich with prevalence data among animals for the other viruses of concern. Influenza A viruses are highly enzootic in livestock, especially poultry and swine, as well as among aquatic birds. Influenza D viruses are also thought to be highly enzootic in cattle and swine . Coronaviruses are enzootic in numerous animal species, especially in bats, birds, and livestock . Hence, studying these animal species seems an appropriate strategy. Additionally, studying dense, dynamic populations of these animals with efficient reproduction is very strategic, as such animal populations will be able to sustain respiratory virus populations via continual infections among immunologically naïve offspring. If we consider the bats and domestic animals mentioned above, we might select sites for surveillance where these animals have frequent and close contact with man. Bats are not frequently in direct contact with humans, save for those occasional colonies which are maintained in zoos, for research purposes, or by bat enthusiasts. In contrast, domestic livestock, such as pigs, chickens, ducks, geese, cattle, goats, and sheep, are often reared in close contact with humans. Modern industrial farming efforts are increasing the size of such domestic herds or flocks, as their meat can be produced more efficiently and at lower cost compared to farms with smaller herds or flocks. The downside of these large industrialized farms is their propensity to harbor sustained animal pathogens among livestock, which may cause mild disease in the animals but, over time, can cross-over to infect animal workers. Reports of novel pathogen generation on industrialized farms and transmission to humans is becoming increasingly common for both viral and bacterial pathogens . Consistent with the logic prescribed by Lloyd-Smith and colleagues , large industrialized farms in emerging infectious disease hotspots would be strategic sites for conducting surveillance for novel prepandemic respiratory viruses. Again considering the five viral families of concern, data to support an answer to this question are a bit sparse. However, some data are available. Freidl et al. published a large review of animal to human risk for zoonotic influenza A in 2014. After reviewing 89 published reports, they concluded that the risk was greatest from human exposure to poultry and pigs. In our 2019 review of the literature for zoonotic influenza A transmission to humans , we concluded that the species barrier is lower for viruses moving from swine to humans rather than from other animals to humans. In a 2019 report, Wang et al. concluded that the probability of interspecies transmission of coronaviruses from two different coronavirus genera, alphacoronaviruses, and deltacoronaviruses are especially high. Given that pigs have experienced recent epizoonotics from five novel coronaviruses , it seems worthwhile to conduct surveillance for prepandemic coronaviruses among pigs. Our research team has recently conducted reviews of the literature regarding adenovirus and enterovirus zoonotic infections. While data are sparse, it seems relevant that nonhuman primates might be at the greatest risk of sharing adenoviruses and enteroviruses with humans. Ideally, laboratory work should be designed such that partners in conducting human-animal nexus or pneumonia etiology surveillance networks are trained with standard operating procedures (SOPs) that are adapted to their field, hospital, and laboratory environments. This will often require the shipment of quality reagents and conducting capacity-building workshops. International partners should be trained to conduct as much of the field sampling, clinical sampling, and laboratory assays as they can. As the collaboration progresses, partners may wish to become more and more autonomous. We have had considerable success in carrying out such partnered surveillance work. In general, we anticipate our field sites to collect, process, and minimally study specimens with real-time molecular assays , after which specimens are shipped to one of our three research laboratories (China, Singapore, or the United States), where additional studies are performed as described below. We approach our international partners with options regarding what type of specimens they wish to collect. For human–animal interface studies, this often involves nasal washes or nasopharyngeal swab, and sometimes fecal specimen collections from the workers’ animals. We often also collect serial serum specimens from the animal workers. We typically train international partners in collecting environmental samples, including bioaerosol, water, and animal environmental samples. Biological and environmental swabs are typically collected with a commercially available swab kit, though we recommend reducing the viral transfer medium (VTM) by up to half for samples that are suspected to have low viral presence. Following the collection of samples in the field, specimens are brought to the lab for processing, with the protocol dependent on the type of sample collected. Nasopharyngeal, rectal, and fomite swabs are generally collected in 1.5 mL viral transfer medium (VTM) or utilizing a commercially available swab kit . Around 2 mL of saliva is collected as-is. For aerosol sampling, we most often use National Institute of Occupational Safety and Health (NIOSH) two-stage bioaerosol cyclone samplers connected to a SKC AirCheck Tough personal sampling pump (Cat # 220-50000TC-K; SKC Inc. Eighty Four, PA, USA) . Samples are collected in two stages: a liquid stage (15 mL in VTM), and a dry stage, and then suspended in 1 mL PBS containing 0.5% bovine serum albumin. These samples are aliquoted, reserving 140–200 µL for viral DNA/RNA extraction, 250 µL for live virus inoculation (stored at −80 °C), and 1 mL as an archival sample (stored at −80 °C) . Viral nucleic acid extractions are subjected to a variety of molecular-detection algorithms. These assays incorporate routine assessment and pathogen discovery pathways for specific viral families. Initially, these assays use algorithms of real-time PCR (qPCR or qRT-PCR) and conventional PCR/RT-PCR assay techniques to detect previously identified viruses and novel viruses within that same group. For instance, for our influenza A virus group we often search for influenza A, B, C, and D with qRT-PCR assays; when a virus is detected, we employ conventional RT-PCR with Sanger sequencing to further characterize the virus . For some virus groups, we begin with a pan-species diagnostic approach (such as with our conventional pan- Paramyxoviridae / Pneumoviridae RT-PCR assay and then further characterize individual viruses through Sanger sequencing of the amplicon. This permits us to identify viruses of both human and animal origin . Molecular detection algorithms for adenoviruses and enteroviruses incorporate both qPCR/qRT-PCR to identify human strains of these viruses and conventional PCR in a pan-species RT-PCR approach, with sequencing to characterize novel human and animal virus strains . Similarly, for our coronavirus algorithm, we employ a battery of specific qRT-PCR assays to identify previously known seasonal or pandemic human coronaviruses and a pan-species coronavirus conventional PCR assay to detect and characterize other human or animal coronaviruses . These assays were expanded to incorporate detection of the SARS-CoV-2 virus in biological and environmental samples during the onset of the 2020 pandemic . Detailed versions of each assay can be found in the associated with this manuscript. Overall, these assays are vital in detecting and characterizing both previously recognized respiratory viruses and novel emerging viruses that would likely be missed by commercial assays. As part of our pathogen discovery pathway, field specimens with evidence of a novel virus are transported to one of our dedicated research laboratories for further study. This may involve work to determine if a novel virus meets Bradford Hill or other related criteria to be considered as a human pathogen. Often, characterization involves assessing viral infectivity in various cell lines to support hypotheses for host range and pathogenicity. From the original specimen, 250 µL of the sample is inoculated in an appropriate cell line. This procedure requires previous knowledge relating to characteristics of the viral family to determine which cell culture line would provide the optimal conditions for growing the virus. Cytopathic effects (CPE) are monitored in the inoculated cells to assess successful viral amplification, and virus in the supernatant is harvested when 80% CPE is observed. We next develop a real-time molecular assay for that pathogen. The resultant virus is confirmed to grow in cell culture using qPCR/qRT-PCR, and the virus concentration is titered. In the case of novel viruses, the DNA/RNA may be extracted and sent for whole-genome sequencing. Isolated viruses are stored for future use by our team or apportioned to other researchers through our online biorepository. If the virus is especially threatening it may be necessary to determine the virus’ geographical and host prevalence. This may involve the development of a serum neutralization assay and seroepidemiological studies of animal workers and their animals. Further concerns may lead to pathogenesis studies in animal models. In this manuscript, we endeavored to review the most common respiratory virus threats and detail their morbidity. We discussed current efforts at early emerging pathogen detection and noted their recent failure in providing early warning for the 2009 influenza A virus and 2020 coronavirus pandemics. We also proposed an alternate One Health strategy for novel respiratory virus detection and argued that it is likely to be more efficient and more sustainable than current efforts, especially when integrated with research perspectives from ecology and evolution. Finally, we provided considerable detail regarding laboratory methods in employing this One Health strategy. We proposed establishing networks for novel respiratory virus surveillance in known geographical hotspots for emerging infectious diseases. We proposed targeting sites where humans and animals interface, such as live-animal markets, large industrial farms, or alternatively, when that is not possible, large hospital sites with high rates of pneumonia admissions where the etiology of these patients might be studied for novel respiratory virus pathogens. By employing targeted, minimally invasive surveillance studies, future respiratory virus threats may be more effectively detected mitigated prior to causing tremendous human and economic costs to the affected nations. |
Fumigant dazomet induces tobacco plant growth via changing the rhizosphere microbial community | 425b0a38-61ba-46ca-b177-af26ed229b98 | 11850595 | Microbiology[mh] | Tobacco is an important economic crop in China, and the main planting areas are distributed in Yunnan, Sichuan, Guizhou, and Chongqing. Owing to the limited farmland area and cultivation style, continuous cropping is a common planting style in major tobacco planting areas . However, long-term continuous cropping causes an imbalance in soil nutrients and disorders in microbial structure, leading to the accumulation of autotoxic substances and pathogens . Indeed, continuous cropping limits the sustainable development of tobacco production, which causes several problems, such as abnormal growth of tobacco seedlings, outbreaks of soil-borne disease and decreases in crop yield . Therefore, screening for effective strategies for solving continuous-cropping obstacles is necessary. Soil microorganisms make special contributions to the life activities of plants. Microorganisms can convert organic nutrients in the soil into inorganic forms, which are available for plant absorption and utilization . During their growth and reproduction, microorganisms secrete substances such as extracellular polysaccharides. These substances can bind soil particles together, form stable aggregates, improve soil structure, and increase the soil’s aeration and water holding capacity . Roots provide plants with multiple key functions, including fixation and obtaining water and nutrients. Therefore, well developed root systems enable plants to obtain more resources and then feed back to the above ground tissues . The rhizosphere soil provides a place for communication between plants and microorganisms . Due to the intercalation of the root system, the moisture and aeration conditions in the rhizosphere are superior to those in the non-rhizosphere, thus forming a microenvironment conducive to the growth and reproduction of rhizosphere microorganisms , . At the same time, during the growth of plants, the senescent root system, as well as its shed parts and secretions, are important sources of nutrition and energy for rhizosphere microorganisms . However, in continuous cropping soil, the communication between plant roots and microorganisms is disrupted, which is usually manifested as the affected growth of crops – .Soil fumigation is an effective strategy to alleviate the continuous - cropping obstacle . Dazomet, as a broad - spectrum soil fumigant and disinfectant, has the advantages of high efficiency, low toxicity, and no residue. In production, there are two modes of soil fumigation: whole - field fumigation and local fumigation. Whole - field fumigation refers to the mode of covering and fumigating the entire field; while local fumigation refers to the mode of fumigating only the soil involved in crop planting in a field. Compared with whole - field fumigation, local fumigation can effectively reduce the use of fumigants while achieving the same fumigation effect. When dazomet is applied to moist soil, it can rapidly decompose into gaseous - methyl isothiocyanate (MITC), which can diffuse to different depths in the soil layer and effectively kill various harmful organisms in the soil .The effects of dazomet on soil microorganisms have also been studied to some extent. After dazomet fumigation, the number of soil microorganisms will first show a downward trend, and when the inhibitory effect of fumigation is no longer obvious, the soil microbial community has the ability to recover , . Although some studies have shown that after dazomet fumigation, the number of pathogenic bacteria in the soil, such as Fusarium , etc., decreases significantly, and at the same time some beneficial microorganisms occupy a dominant position in the rhizosphere soil . However, the functions of these beneficial microorganisms are only limited to discussion, lacking further evidence. We first explored the relationship between the changes of soil microbial communities after dazomet fumigation by the local fumigation mode and the promotion of plant growth, as well as the pathways by which dazomet improves plant phenotypes. Effects of different concentrations of dazomet on tobacco plant growth The survey data from the three periods revealed that different concentrations of dazomet significantly promoted the agronomic traits of tobacco plants to some extent (Fig. A, B), and this promoting effect increased significantly with increasing dazomet concentration. We found that this difference was most significant during the vigorous growth period. Compared with those of the control, the leaf length, leaf width, plant height, stem circumference, leaf area, and number of leaves of tobacco treated with D12.5 increased by 1.68, 1.97, 2.50, 1.75, 0.32, and 1.35 times, respectively. The leaf length, leaf width, plant height, stem circumference, leaf area, and number of leaves of tobacco treated with D9.5 increased by 1.65, 1.96, 3.33, 1.7, 3.24, and 1.36 times, respectively. Overall, the promoting effects of the D9.5 and D12.5 treatments were better than those of the D6.5 and D3.5 treatments, and the agronomic traits of tobacco treated with D12.5 were best. However, there was no significant difference in the agronomic traits of tobacco between the D9.5 treatment and the D12.5 treatment. We collected tobacco root samples and evaluated the effects of different doses of dazomet on tobacco root growth at 60 days after transplanting. The results revealed that tobacco plants treated with different doses of dazomet had significantly greater root weights, root volumes, and overground weights than did those in the CK treatment (Fig. ). Specifically, the root weight, root volume and overground weight of the D3.5 and D6.5 treatments increased by 1.61, 1.61, and 1.75 times and 2.24, 2.18, and 1.97 times, respectively, compared with those of the control. Moreover, we found that those of the D9.5 and D12.5 treatments were significantly better than those of the D3.5 and D6.5 treatments. Compared with those of the control, the root weight, root volume, and overground weight of the D9.5 and D12.5 treatments increased by 0.51, 3.42, and 2.84 times and 3.22, 3.11, and 0.83 times, respectively. However, the data on root length and the root-to-shoot ratio did not significantly differ (Fig. A, B). The fitting curve results of the system indicators and agronomic traits revealed that the root weight and root volume were significantly linearly related to the agronomic traits of tobacco. Specifically, the P values of the fitting curves between root weight, root volume, and leaf length, leaf height, plant height, stem circumference, leaf area, and number of leaves were all less than 0.0001 (Fig. C, D). These results indicate that different concentrations of dazomet are conducive to promoting the growth and development of the tobacco root system, and we found that the growth advantage of the overground tissues may be attributed to the developmental advantages in terms of root weight and root volume. The impact of different treatments on the α diversity of tobacco rhizosphere microbial communities The results of the soil microbial α diversity after 90 days of treatment with different doses of Dazomet are shown in Table . Compared with the control, both the D9.5 and D12.5 treatments significantly reduced the richness and diversity of the bacterial and fungal communities, with the D95 treatment having a greater effect on the diversity of the microbial communities. In terms of bacterial community diversity, the D3.5 and D6.5 treatments resulted in differences compared with the control treatment but were not significantly different. Compared with the control treatment, the D3.5 treatment significantly reduced the richness of the fungal community but did not significantly affect the fungal community diversity. However, there was no significant difference between the D6.5 treatment and the control. These results indicate that after 90 days of treatment with different doses of Dazomet, the richness and diversity of the soil microbial communities varied greatly, which could have led to differences in the composition of the root communities. The impact of different treatments on the α diversity of tobacco rhizosphere microbial communities Beta diversity can be used for preliminary evaluation of the degree of difference between different samples. As shown Fig. A, the difference in bacterial communities among the different treatments reached a significant level (R 2 = 0.4631, P < 0.001, Adonis), whereas the difference in fungal communities did not reach a significant level (R 2 = 0.3014, P = 0.66, Adonis) (Annex 3D). Annex 3B and E show the composition of the microbial communities at the phylum level under the different treatments. The results revealed that the composition of the microbial communities in the soil after dazomet treatment was similar, but the abundance changed, with some phyla showing significant differences. In the bacterial community, the D9.5 and D12.5 treatments significantly reduced the abundance of Chloroflexi, Acidobacteriota, Myxococcota, and Crenarchaeota by 0.59, 0.84, 0.47, 0.94, 0.49, 0.69, 0.41, and 0.87 times, respectively; in the D3.5 treatment, the proportion of Crenarchaeota also significantly decreased by 0.65 times. However, Actinobacteriota increased to different degrees in the rhizosphere of tobacco, with the greatest increase occurring in the D6.5 and D12.5 treatments; the increase was approximately 21.7% and 23.63% greater than that in the control treatment but did not reach a significant level. In the fungal community,, the dazomet treatment significantly affected the abundance of Ascomycota, with the D3.5, D6.5, D9.5, and D12.5 treatments reducing the abundance of Ascomycota by 0.48, 0.52, 0.70, and 0.51 times, respectively (Annex 3 F); these treatments significantly increased the abundance of Mortierellomycota, with increases of 80.73%, 1.73%, 114.89%, and 91.67%, respectively, compared with the control. These results indicate that dazomet treatment has a significant effect on the structure and composition of microbial communities, which is manifested mainly in the differences in some microbial communities at the phylum level, and these changes in the community may be the reason for the growth advantage of tobacco roots. Impact of different treatments on the microbial community composition at the genus level Composition of bacterial community in different treatments The Venn analysis results at the genus level of the bacterial community revealed th Fig. A) the diversity of bacteria in the soil decreased to some extent after dazomet treatment, as indicated by the number of unique bacterial genera in each treatment, which were 40 57, 25, 24, and 63 for the D3.5, D6.5, D9.5, 12.5, and CK treatments, respectively. However, most bacterial genera still coexisted, as shown by the 457 common bacterial genera across treatments, and the proportion of common bacterial genera in each soil treatment exceeded 62.26% of the total number of bacterial genera. The results of the bacterial community composition at the genus lev Fig. B) revealed that the top ten bacterial genera in the treatment group were similar to those in the control group, but the “others” group accounted for approximately 63% of the bacteria in each treatment group, which might play an important role in explaining the extent of the community differences. The one-way ANOVA difference comparison results reveal Fig. C) 110 bacterial genera with significant differences at the genus level, with Proteobacteria accounting for the greatest percentage (31.82%), followed by Acidobacteriota (20.91%). Moreover, according to the heatmap, the differential bacterial genera might be concentrated mainly in the D9.5 and D12.5 treatment groups. Composition of fungal community in different treatments The changes in the fungal community at the genus level are analogous to those in the bacterial community (Annex 4 A). Concerning the number of fungal genera exclusive to each treatment, the numbers of fungal genera that are unique to D3.5, D6.5, D9.5, D12.5 and CK are 18, 37, 17, 21 and 69 respectively. Among them, the proportion of fungal genera specific to each treatment in the total number of fungal genera is the highest, exceeding 53.64%. The results of the composition of the fungal community at the genus level indicate (Annex 4B) that the composition of the top - ten fungal genera in the treatment groups is similar to that of the control. In contrast to the bacterial community, the proportion of the “others” group in each treatment is relatively low. The results of the one - way - ANOVA difference comparison analysis show (Annex 4 C) that 50 fungal genera are found to have significant differences at the genus level, and the vast majority of these differential fungal genera belong to Acidobacteriota (88%). Correlations between differential microbial genera and root traits as well as different dazomet application rates Network analysis helped us screen out some data directly related to our selected indicators through ordinal analysis in the dataset without real numbers. In the above analysis, we analysed some significantly different microbial genera; therefore, we screened out these different microbial groups through the construction of co-occurrence network models and screened out microbial groups significantly related to root traits. The results of the co-occurrence network model revealed that there were 17 microbial groups related directly to root traits, 14 of which presented significant negative correlations with the phenotypic data of root trait development. Moreover, these different microbes mutually promoted each other, indicating that there may have been some synergistic effects among them, thus limiting the growth of tobacco plan Fig. A). Overall, the different bacterial genera related to root traits were divided into three main categories: those related to plant pathogens, those related to denitrification, and those related to some microbial groups. To explore the effects of dazomet on the screened differential microbial taxa, we performed a linear regression analysis between the screened microbial taxa and the application rate of dazomet (Fig. B). The results revealed that the application rate of dazomet had a significant linear relationship with 3 differential microbial taxa, among which 4 taxa increased ( g_Brevundimonas , g_Domibacillus , g_Microbacterium and Pedobacter ) and 9 taxa decreased ( g_Aspergillus , g_Microdochium , g_Scytalidium , g_Tratella , g_Nitrosomonas , g_Hyphomicrobium , g_Candidatus_Nitrososphaera , g_Bibacillus and g_Sphingomonas ). Interestingly, we found that the number of microbial taxa negatively correlated with root traits significantly decreased with increasing dazomet application rate, whereas the number of microbial taxa positively correlated with root traits significantly increased with increasing dazomet application rate. Path analysis of dazomet changing soil microorganism genus to promote plant growth The establishment of structural equation models helps us test the reliability of our assumptions. The parameters of the structural equation model were CMIN/DF1.057, RMSEA = 0.064, ITI = 0.997, TLI = 0.994, C = 0.997, DF = 11. The results Fig. show that the path coefficient from the amount of dazomet to the rising community was 0.53, P = 0.02, the hypothesis was established, the path coefficient from the rising microbial community to the root system traits 0.91, P < 0.001, the hypothesis was established; the path coefficient from the root system traits to the plant aboveground was 0.91, P < 0.001, the hypothesis was established; however, the path coefficient from the amount of dazomet the declining microbial community was − 0.60, P = 0.005, the hypothesis was established; the path coefficient from the declining microbial community to rising microbial community was − 0.28, P = 0.210, the hypothesis was not established; the path coefficient from the declining microbial community to root system traits was − 0.11, P = 0.098, the hypothesis was not established. The above results show that the dominant establishment of system traits is closely related to the rhizosphere rising microbial community. The survey data from the three periods revealed that different concentrations of dazomet significantly promoted the agronomic traits of tobacco plants to some extent (Fig. A, B), and this promoting effect increased significantly with increasing dazomet concentration. We found that this difference was most significant during the vigorous growth period. Compared with those of the control, the leaf length, leaf width, plant height, stem circumference, leaf area, and number of leaves of tobacco treated with D12.5 increased by 1.68, 1.97, 2.50, 1.75, 0.32, and 1.35 times, respectively. The leaf length, leaf width, plant height, stem circumference, leaf area, and number of leaves of tobacco treated with D9.5 increased by 1.65, 1.96, 3.33, 1.7, 3.24, and 1.36 times, respectively. Overall, the promoting effects of the D9.5 and D12.5 treatments were better than those of the D6.5 and D3.5 treatments, and the agronomic traits of tobacco treated with D12.5 were best. However, there was no significant difference in the agronomic traits of tobacco between the D9.5 treatment and the D12.5 treatment. We collected tobacco root samples and evaluated the effects of different doses of dazomet on tobacco root growth at 60 days after transplanting. The results revealed that tobacco plants treated with different doses of dazomet had significantly greater root weights, root volumes, and overground weights than did those in the CK treatment (Fig. ). Specifically, the root weight, root volume and overground weight of the D3.5 and D6.5 treatments increased by 1.61, 1.61, and 1.75 times and 2.24, 2.18, and 1.97 times, respectively, compared with those of the control. Moreover, we found that those of the D9.5 and D12.5 treatments were significantly better than those of the D3.5 and D6.5 treatments. Compared with those of the control, the root weight, root volume, and overground weight of the D9.5 and D12.5 treatments increased by 0.51, 3.42, and 2.84 times and 3.22, 3.11, and 0.83 times, respectively. However, the data on root length and the root-to-shoot ratio did not significantly differ (Fig. A, B). The fitting curve results of the system indicators and agronomic traits revealed that the root weight and root volume were significantly linearly related to the agronomic traits of tobacco. Specifically, the P values of the fitting curves between root weight, root volume, and leaf length, leaf height, plant height, stem circumference, leaf area, and number of leaves were all less than 0.0001 (Fig. C, D). These results indicate that different concentrations of dazomet are conducive to promoting the growth and development of the tobacco root system, and we found that the growth advantage of the overground tissues may be attributed to the developmental advantages in terms of root weight and root volume. The results of the soil microbial α diversity after 90 days of treatment with different doses of Dazomet are shown in Table . Compared with the control, both the D9.5 and D12.5 treatments significantly reduced the richness and diversity of the bacterial and fungal communities, with the D95 treatment having a greater effect on the diversity of the microbial communities. In terms of bacterial community diversity, the D3.5 and D6.5 treatments resulted in differences compared with the control treatment but were not significantly different. Compared with the control treatment, the D3.5 treatment significantly reduced the richness of the fungal community but did not significantly affect the fungal community diversity. However, there was no significant difference between the D6.5 treatment and the control. These results indicate that after 90 days of treatment with different doses of Dazomet, the richness and diversity of the soil microbial communities varied greatly, which could have led to differences in the composition of the root communities. Beta diversity can be used for preliminary evaluation of the degree of difference between different samples. As shown Fig. A, the difference in bacterial communities among the different treatments reached a significant level (R 2 = 0.4631, P < 0.001, Adonis), whereas the difference in fungal communities did not reach a significant level (R 2 = 0.3014, P = 0.66, Adonis) (Annex 3D). Annex 3B and E show the composition of the microbial communities at the phylum level under the different treatments. The results revealed that the composition of the microbial communities in the soil after dazomet treatment was similar, but the abundance changed, with some phyla showing significant differences. In the bacterial community, the D9.5 and D12.5 treatments significantly reduced the abundance of Chloroflexi, Acidobacteriota, Myxococcota, and Crenarchaeota by 0.59, 0.84, 0.47, 0.94, 0.49, 0.69, 0.41, and 0.87 times, respectively; in the D3.5 treatment, the proportion of Crenarchaeota also significantly decreased by 0.65 times. However, Actinobacteriota increased to different degrees in the rhizosphere of tobacco, with the greatest increase occurring in the D6.5 and D12.5 treatments; the increase was approximately 21.7% and 23.63% greater than that in the control treatment but did not reach a significant level. In the fungal community,, the dazomet treatment significantly affected the abundance of Ascomycota, with the D3.5, D6.5, D9.5, and D12.5 treatments reducing the abundance of Ascomycota by 0.48, 0.52, 0.70, and 0.51 times, respectively (Annex 3 F); these treatments significantly increased the abundance of Mortierellomycota, with increases of 80.73%, 1.73%, 114.89%, and 91.67%, respectively, compared with the control. These results indicate that dazomet treatment has a significant effect on the structure and composition of microbial communities, which is manifested mainly in the differences in some microbial communities at the phylum level, and these changes in the community may be the reason for the growth advantage of tobacco roots. Composition of bacterial community in different treatments The Venn analysis results at the genus level of the bacterial community revealed th Fig. A) the diversity of bacteria in the soil decreased to some extent after dazomet treatment, as indicated by the number of unique bacterial genera in each treatment, which were 40 57, 25, 24, and 63 for the D3.5, D6.5, D9.5, 12.5, and CK treatments, respectively. However, most bacterial genera still coexisted, as shown by the 457 common bacterial genera across treatments, and the proportion of common bacterial genera in each soil treatment exceeded 62.26% of the total number of bacterial genera. The results of the bacterial community composition at the genus lev Fig. B) revealed that the top ten bacterial genera in the treatment group were similar to those in the control group, but the “others” group accounted for approximately 63% of the bacteria in each treatment group, which might play an important role in explaining the extent of the community differences. The one-way ANOVA difference comparison results reveal Fig. C) 110 bacterial genera with significant differences at the genus level, with Proteobacteria accounting for the greatest percentage (31.82%), followed by Acidobacteriota (20.91%). Moreover, according to the heatmap, the differential bacterial genera might be concentrated mainly in the D9.5 and D12.5 treatment groups. Composition of fungal community in different treatments The changes in the fungal community at the genus level are analogous to those in the bacterial community (Annex 4 A). Concerning the number of fungal genera exclusive to each treatment, the numbers of fungal genera that are unique to D3.5, D6.5, D9.5, D12.5 and CK are 18, 37, 17, 21 and 69 respectively. Among them, the proportion of fungal genera specific to each treatment in the total number of fungal genera is the highest, exceeding 53.64%. The results of the composition of the fungal community at the genus level indicate (Annex 4B) that the composition of the top - ten fungal genera in the treatment groups is similar to that of the control. In contrast to the bacterial community, the proportion of the “others” group in each treatment is relatively low. The results of the one - way - ANOVA difference comparison analysis show (Annex 4 C) that 50 fungal genera are found to have significant differences at the genus level, and the vast majority of these differential fungal genera belong to Acidobacteriota (88%). Correlations between differential microbial genera and root traits as well as different dazomet application rates Network analysis helped us screen out some data directly related to our selected indicators through ordinal analysis in the dataset without real numbers. In the above analysis, we analysed some significantly different microbial genera; therefore, we screened out these different microbial groups through the construction of co-occurrence network models and screened out microbial groups significantly related to root traits. The results of the co-occurrence network model revealed that there were 17 microbial groups related directly to root traits, 14 of which presented significant negative correlations with the phenotypic data of root trait development. Moreover, these different microbes mutually promoted each other, indicating that there may have been some synergistic effects among them, thus limiting the growth of tobacco plan Fig. A). Overall, the different bacterial genera related to root traits were divided into three main categories: those related to plant pathogens, those related to denitrification, and those related to some microbial groups. To explore the effects of dazomet on the screened differential microbial taxa, we performed a linear regression analysis between the screened microbial taxa and the application rate of dazomet (Fig. B). The results revealed that the application rate of dazomet had a significant linear relationship with 3 differential microbial taxa, among which 4 taxa increased ( g_Brevundimonas , g_Domibacillus , g_Microbacterium and Pedobacter ) and 9 taxa decreased ( g_Aspergillus , g_Microdochium , g_Scytalidium , g_Tratella , g_Nitrosomonas , g_Hyphomicrobium , g_Candidatus_Nitrososphaera , g_Bibacillus and g_Sphingomonas ). Interestingly, we found that the number of microbial taxa negatively correlated with root traits significantly decreased with increasing dazomet application rate, whereas the number of microbial taxa positively correlated with root traits significantly increased with increasing dazomet application rate. Path analysis of dazomet changing soil microorganism genus to promote plant growth The establishment of structural equation models helps us test the reliability of our assumptions. The parameters of the structural equation model were CMIN/DF1.057, RMSEA = 0.064, ITI = 0.997, TLI = 0.994, C = 0.997, DF = 11. The results Fig. show that the path coefficient from the amount of dazomet to the rising community was 0.53, P = 0.02, the hypothesis was established, the path coefficient from the rising microbial community to the root system traits 0.91, P < 0.001, the hypothesis was established; the path coefficient from the root system traits to the plant aboveground was 0.91, P < 0.001, the hypothesis was established; however, the path coefficient from the amount of dazomet the declining microbial community was − 0.60, P = 0.005, the hypothesis was established; the path coefficient from the declining microbial community to rising microbial community was − 0.28, P = 0.210, the hypothesis was not established; the path coefficient from the declining microbial community to root system traits was − 0.11, P = 0.098, the hypothesis was not established. The above results show that the dominant establishment of system traits is closely related to the rhizosphere rising microbial community. The Venn analysis results at the genus level of the bacterial community revealed th Fig. A) the diversity of bacteria in the soil decreased to some extent after dazomet treatment, as indicated by the number of unique bacterial genera in each treatment, which were 40 57, 25, 24, and 63 for the D3.5, D6.5, D9.5, 12.5, and CK treatments, respectively. However, most bacterial genera still coexisted, as shown by the 457 common bacterial genera across treatments, and the proportion of common bacterial genera in each soil treatment exceeded 62.26% of the total number of bacterial genera. The results of the bacterial community composition at the genus lev Fig. B) revealed that the top ten bacterial genera in the treatment group were similar to those in the control group, but the “others” group accounted for approximately 63% of the bacteria in each treatment group, which might play an important role in explaining the extent of the community differences. The one-way ANOVA difference comparison results reveal Fig. C) 110 bacterial genera with significant differences at the genus level, with Proteobacteria accounting for the greatest percentage (31.82%), followed by Acidobacteriota (20.91%). Moreover, according to the heatmap, the differential bacterial genera might be concentrated mainly in the D9.5 and D12.5 treatment groups. The changes in the fungal community at the genus level are analogous to those in the bacterial community (Annex 4 A). Concerning the number of fungal genera exclusive to each treatment, the numbers of fungal genera that are unique to D3.5, D6.5, D9.5, D12.5 and CK are 18, 37, 17, 21 and 69 respectively. Among them, the proportion of fungal genera specific to each treatment in the total number of fungal genera is the highest, exceeding 53.64%. The results of the composition of the fungal community at the genus level indicate (Annex 4B) that the composition of the top - ten fungal genera in the treatment groups is similar to that of the control. In contrast to the bacterial community, the proportion of the “others” group in each treatment is relatively low. The results of the one - way - ANOVA difference comparison analysis show (Annex 4 C) that 50 fungal genera are found to have significant differences at the genus level, and the vast majority of these differential fungal genera belong to Acidobacteriota (88%). Network analysis helped us screen out some data directly related to our selected indicators through ordinal analysis in the dataset without real numbers. In the above analysis, we analysed some significantly different microbial genera; therefore, we screened out these different microbial groups through the construction of co-occurrence network models and screened out microbial groups significantly related to root traits. The results of the co-occurrence network model revealed that there were 17 microbial groups related directly to root traits, 14 of which presented significant negative correlations with the phenotypic data of root trait development. Moreover, these different microbes mutually promoted each other, indicating that there may have been some synergistic effects among them, thus limiting the growth of tobacco plan Fig. A). Overall, the different bacterial genera related to root traits were divided into three main categories: those related to plant pathogens, those related to denitrification, and those related to some microbial groups. To explore the effects of dazomet on the screened differential microbial taxa, we performed a linear regression analysis between the screened microbial taxa and the application rate of dazomet (Fig. B). The results revealed that the application rate of dazomet had a significant linear relationship with 3 differential microbial taxa, among which 4 taxa increased ( g_Brevundimonas , g_Domibacillus , g_Microbacterium and Pedobacter ) and 9 taxa decreased ( g_Aspergillus , g_Microdochium , g_Scytalidium , g_Tratella , g_Nitrosomonas , g_Hyphomicrobium , g_Candidatus_Nitrososphaera , g_Bibacillus and g_Sphingomonas ). Interestingly, we found that the number of microbial taxa negatively correlated with root traits significantly decreased with increasing dazomet application rate, whereas the number of microbial taxa positively correlated with root traits significantly increased with increasing dazomet application rate. The establishment of structural equation models helps us test the reliability of our assumptions. The parameters of the structural equation model were CMIN/DF1.057, RMSEA = 0.064, ITI = 0.997, TLI = 0.994, C = 0.997, DF = 11. The results Fig. show that the path coefficient from the amount of dazomet to the rising community was 0.53, P = 0.02, the hypothesis was established, the path coefficient from the rising microbial community to the root system traits 0.91, P < 0.001, the hypothesis was established; the path coefficient from the root system traits to the plant aboveground was 0.91, P < 0.001, the hypothesis was established; however, the path coefficient from the amount of dazomet the declining microbial community was − 0.60, P = 0.005, the hypothesis was established; the path coefficient from the declining microbial community to rising microbial community was − 0.28, P = 0.210, the hypothesis was not established; the path coefficient from the declining microbial community to root system traits was − 0.11, P = 0.098, the hypothesis was not established. The above results show that the dominant establishment of system traits is closely related to the rhizosphere rising microbial community. Effects of different dosages of dazomet on plant growth and development Some studies have shown that dazomet has a promoting effect on crop growth and yield , which is similar to our results. We found that after fumigation with dazomet, the agronomic traits of tobacco were significantly greater than those of the control during the flourishing period, with the greatest difference in agronomic traits occurring during the rosette stage. Additionally, the difference in the root traits of tobacco was greater than that in the aboveground tissues. After treatment with D9.5 and 12.5, the root weight and root volume of the tobacco plants increased to 3.1 and 3.42 times greater than those of the control plants, respectively. We believe that this promoting effect on aboveground tissues is related to the developmental advantage of underground roots. Effects of different dosages of dazomet on the rhizosphere microbial community Continuous cropping leads to poor development of underground crop roots , which may be due to changes in the soil microbiome . Some scholars believe that changes in soil microbes could be the reason that dazomet al.leviates crop growth stress and increases crop yield . Our results revealed that dazomet significantly affected the rhizosphere microbial community of plants, which reduced the diversity. In the process of soil microbial community restoration, the application amount of dazomet is the main influencing factor, which shows that fumigation with low application rate recovers faster and fumigation with high application rate recovers slower. In terms of community composition, the restored microbial community is similar to that of the soil without fumigation treatment. Focusing more on changes at the microbial genus level, we found that the microbial groups that increased after treatment were g_Pedobacter , g_Microbacterium , g_Brevundimonas and g_Domibacillus , among which g_Pedobacter , g_Microbacterium and g_Brevundimonas have been reported to be associated with plant growth-promoting bacteria, which is consistent with our study results. We found that g_Pedobacter , g_Microbacterium , g_Brevundimonas , and g_Domibacillus were significantly positively correlated with tobacco root traits ( P < 0.05). Moreover, the microbial groups negatively correlated with the application of dazomet included three categories: the first category included groups associated with plant pathogens, such as g_ Aspergillus , g_Microdochium , g_Scytalidium and g_Truncatella . The application of dazomet reduces the degree of biological stress on tobacco roots, especially some microbial groups associated with pathogens; the second category includes microbial groups related to the soil N cycle, including g_Nitrosomonas , g_Hyphomicrobium , g_Candidatus_Nitrososphaera and g_Aquicella . Among them, g_Nitrosomonas , g_Hyphomicrobium and g_Candidatus_Nitrososphaera have been reported to participate in plant denitrification, which is consistent with previous studies showing that the application of dazomet leads to a significant reduction in nitrogen-fixing bacterial genera , Interestingly, there is research showing that continuous cropping of tobacco can weaken nitrification in the soil which may be an important reason for suppressing tobacco growth . The third category includes beneficial plant microbial groups, g_ Brevibacil lus and g_Sphingomonas . The accidental killing effect of soil fumigation on beneficial microorganisms has always been the focus of controversy in fumigation research . However, our study revealed that fumigation with dazomet can cause changes in beneficial microbes, such as plant growth-promoting bacteria, but these changes were bidirectional. We found two microbial groups were reduced by fumigation, such as g_Brevibacillus and g_Sphingomonas and others were significantly increased, such as g_Pedobacter , g_Microbacterium and g_Brevundimonas , which could be related to their resistance gene levels discrepancy , . Through which microbial communities does dazomet promote plants? In previous studies, more attention was paid to how soil fumigation eliminated harmful microorganisms, and our study also obtained similar results. Interestingly, the changes in soil microorganisms were reflected in two aspects: significant up - regulation and significant down - regulation. The results of the structural equation model showed that, compared with the decrease in harmful microorganisms, the up - regulation of beneficial microorganisms contributed more to the growth traits of plants. In recent years, many scholars have begun to focus on the recovery of beneficial microorganisms after fumigation. For example, they combine fumigation with organic fertilizer or biochar to reduce its impact on beneficial soil microorganisms , .This gives us ideas for future research. That is, adding microbial agents after soil fumigation may achieve better results. At the same time, it is more appropriate to select specific beneficial microbial inoculants, such as microbial groups that recover quickly after fumigation treatment, for restoration. Some studies have shown that dazomet has a promoting effect on crop growth and yield , which is similar to our results. We found that after fumigation with dazomet, the agronomic traits of tobacco were significantly greater than those of the control during the flourishing period, with the greatest difference in agronomic traits occurring during the rosette stage. Additionally, the difference in the root traits of tobacco was greater than that in the aboveground tissues. After treatment with D9.5 and 12.5, the root weight and root volume of the tobacco plants increased to 3.1 and 3.42 times greater than those of the control plants, respectively. We believe that this promoting effect on aboveground tissues is related to the developmental advantage of underground roots. Continuous cropping leads to poor development of underground crop roots , which may be due to changes in the soil microbiome . Some scholars believe that changes in soil microbes could be the reason that dazomet al.leviates crop growth stress and increases crop yield . Our results revealed that dazomet significantly affected the rhizosphere microbial community of plants, which reduced the diversity. In the process of soil microbial community restoration, the application amount of dazomet is the main influencing factor, which shows that fumigation with low application rate recovers faster and fumigation with high application rate recovers slower. In terms of community composition, the restored microbial community is similar to that of the soil without fumigation treatment. Focusing more on changes at the microbial genus level, we found that the microbial groups that increased after treatment were g_Pedobacter , g_Microbacterium , g_Brevundimonas and g_Domibacillus , among which g_Pedobacter , g_Microbacterium and g_Brevundimonas have been reported to be associated with plant growth-promoting bacteria, which is consistent with our study results. We found that g_Pedobacter , g_Microbacterium , g_Brevundimonas , and g_Domibacillus were significantly positively correlated with tobacco root traits ( P < 0.05). Moreover, the microbial groups negatively correlated with the application of dazomet included three categories: the first category included groups associated with plant pathogens, such as g_ Aspergillus , g_Microdochium , g_Scytalidium and g_Truncatella . The application of dazomet reduces the degree of biological stress on tobacco roots, especially some microbial groups associated with pathogens; the second category includes microbial groups related to the soil N cycle, including g_Nitrosomonas , g_Hyphomicrobium , g_Candidatus_Nitrososphaera and g_Aquicella . Among them, g_Nitrosomonas , g_Hyphomicrobium and g_Candidatus_Nitrososphaera have been reported to participate in plant denitrification, which is consistent with previous studies showing that the application of dazomet leads to a significant reduction in nitrogen-fixing bacterial genera , Interestingly, there is research showing that continuous cropping of tobacco can weaken nitrification in the soil which may be an important reason for suppressing tobacco growth . The third category includes beneficial plant microbial groups, g_ Brevibacil lus and g_Sphingomonas . The accidental killing effect of soil fumigation on beneficial microorganisms has always been the focus of controversy in fumigation research . However, our study revealed that fumigation with dazomet can cause changes in beneficial microbes, such as plant growth-promoting bacteria, but these changes were bidirectional. We found two microbial groups were reduced by fumigation, such as g_Brevibacillus and g_Sphingomonas and others were significantly increased, such as g_Pedobacter , g_Microbacterium and g_Brevundimonas , which could be related to their resistance gene levels discrepancy , . In previous studies, more attention was paid to how soil fumigation eliminated harmful microorganisms, and our study also obtained similar results. Interestingly, the changes in soil microorganisms were reflected in two aspects: significant up - regulation and significant down - regulation. The results of the structural equation model showed that, compared with the decrease in harmful microorganisms, the up - regulation of beneficial microorganisms contributed more to the growth traits of plants. In recent years, many scholars have begun to focus on the recovery of beneficial microorganisms after fumigation. For example, they combine fumigation with organic fertilizer or biochar to reduce its impact on beneficial soil microorganisms , .This gives us ideas for future research. That is, adding microbial agents after soil fumigation may achieve better results. At the same time, it is more appropriate to select specific beneficial microbial inoculants, such as microbial groups that recover quickly after fumigation treatment, for restoration. Dazomet fumigation can significantly relieve the growth inhibition of tobacco caused by continuous cropping obstacles, and this effect may be achieved through beneficial bacteria that respond to dazomet fumigation. Plant material and growth conditions The experiment was performed in Runxi County, Pengshui County, Chongqing city, China. This area has a 30 year history of tobacco cultivation, and in a considerable part of the area, there are serious continuous cropping obstacles.(The inhibition test is shown in Annex 1.) The tobacco variety is K326, which is the main local tobacco variety. Tobacco seeds were provided by the Tobacco Industry Co., Ltd. in Chongqing, China. The basic physical and chemical properties of the soil were as follows: organic matter content of 26.60 g/kg, total nitrogen content of 1.66 g/kg, total phosphorus content of 1138 mg/kg, total potassium content of 20.7 g/kg, alkaline dissolved nitrogen content of 154.00 mg/kg, effective phosphorus content of 49.40 mg/kg, quick-acting potassium content of 545.00 mg/kg, and pH value of 7.16. The test fumigant 98% dazomet was purchased from Nantong Shizhuang Chemical Co., Ltd. The tobacco seedlings used in the experiment were raised by the floating method, and the demonstration areas all carried out unified field management according to the relevant technical standards, with the central flower opening and topping and the axillary buds controlled with 12.5% flumetralin EC. Effects of different concentrations of dazomet on soil sterilization The experiment consisted of five treatments and three replications, totaling 15 plots. Each plot had an area of approximately 66.7 m². Protection rows were arranged, and the area of the experimental plots was about 0.13 hectares. The medicine was applied during ridging. After that, the soil was covered with conventional plastic film and compacted around the film with new soil placed between the ridges. Then, air tight disinfection was maintained for about 20 days. Ten days before transplanting, use the transplanting hole to open the film for ventilation, and transplant tobacco seedlings to carry out germination test (Annex 2).Treatment 1 corresponded to D3.5 (dazomet 3.5 kg/666.7 m2); Treatment 2 corresponded to D6.5 (dazomet 6.5 kg/666.7 m2); Treatment 3 corresponded to D9.5 (dazomet 9.5 kg/666.7 m2); Treatment 4 corresponded to D12.5 (dazomet 12.5 kg/666.7 m2); and Treatment 5 corresponded to a blank control (CK). Effects of dazomet on tobacco plant growth Five to ten representative tobacco plants were selected for each treatment and marked with tags. In accordance with the YC/T 142–2010 “Tobacco Agronomic Trait Survey Methods”, the agronomic traits of the tobacco plants were determined at the resettling stage and 7 days after topping. The agronomic traits mainly included plant height, stem circumference, effective leaf number, maximum leaf length and maximum leaf width. Effects of dazomet on tobacco root growth Samples were taken at the doughnut stage and 7 d after topping to determine the root growth of the tobacco plants. The variables included root fresh weight, aboveground fresh weight, maximum root length and root volume. A sunny day was chosen for the measurement, and the entire experiment was performed on the same day. The sampling time was determined by more than 50% of the plots at the test site to reach the fertility stage. Each plot was sampled each time a representative tobacco plant was selected, and the entire root system was excavated from the soil. The roots were soaked in water, rinsed and washed clean, the maximum root length was measured first, the volume of the root system was measured via the drainage method, and the root system was weighed to determine the fresh weight. Structure and function of microorganisms in tobacco rhizosphere soil Tobacco rhizosphere soil samples were collected 50 days after transplanting. Each treatment had 3 sample bags, that is, 3 replicates. The rhizosphere soil samples were collected according to the principle of “random, equal and multipoint mixing”. The rhizosphere soil samples included fibrous roots in a range of 2 mm that were shaken and collected, impurities were removed, the samples were sifted through a 2 mm sieve, and each bag of soil was bagged at 5 points after mixing the soil samples. The soil samples were bagged and labeled immediately after collection, and 15 bags of soil were collected at a time. The sample numbers were D3.5, D6.5, D9.5, D12.5 and CK. All the samples were taken back to the laboratory at a low temperature and stored at -80°C. The total DNA of the soil microorganisms was extracted, 16S rRNA and ITS sequencing techniques were used to amplify and sequence the fragments, and the diversity of the soil microbial structure was determined and analysed. Extraction of soil microbial DNA The total DNA of the Soil microbial community was extracted according to the Power Soil ® DNA kit(Axygen), purified and detected by agarose gel (1%) electrophoresis. PCR amplification of V4 ~ V5 region of bacterial 16 S rRNA gene was performed using primers 515 F (5 ‘-GTGCCAGCMGCCGCGG-3’) and 806R (5′- GGACTACHVGGGTWTCTAAT-3′). ITS1F (5′- CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′- GCTGCGTTCTTCATCGATGC-3′) were used to amplify the ITS1 region of the fungal ITS gene.Each sample was repeated 3 times, and the PCR mixture of the same sample was detected by agarose gel (2%) electrophoresis, after which the amplicon was recovered . According to the instructions of Axy-Prep DNA Gel Recovery Kit (Axygen), the PCR products were purified, detected.The 16 S rRNA and ITS gene fragments were sequenced by Shanghai Majorbio Co., Ltd., China, using the Illumina MiSeqPE250 platform ( https://cloud.majorbio.com ). According to the barcode sequence corresponding to the sample, the effective sequence of the corresponding sample was obtained. Use FLASH software (v1.2.7, http://ccb.jhu.edu/ftware/FLASH/ ) splicing sequence. QIIME2 (2018.2, Quantitative Insight Into Microbial Ecology) software was used to invoke DADA2 to check and eliminate the chimera sequence. A table of amplicon sequence variant ASV(amplicon sequence variant) was constructed for the sequence obtained above, and ASV representative sequence was obtained [DADA2 clustering with 100% similarity (de-duplication), and each de-duplication sequence generated after DADA2 quality control was called ASVs]. Will get ASVs SILVA on behalf of the sequence in the database ( http://greengenes.secondgenome.com/ , Release 138) J91 annotate and to obtain its classification information. All samples in the ASVs abundance matrix were randomly resampling at the lowest sequencing depth level of 95% to correct for diversity differences caused by sequencing depth. QIME 2 software was then used to calculate the diversity index for each sample separately. Data analysis The test data were sorted via Excel 2013. The Duncan’s new complex range method in SPSS 17.0 statistical software was used to compare and analyse the differences in related data, with P ≤ 0.05 .Alpha diversity is calculated through the website https://mothur.org/wiki/calculators/ .The “vegan” package of R software was used in ADONIS analysis to explore the influence of different grouping factors on sample differences, and the displacement test was used to conduct a significant analysis of the statistical significance of the division (R 4.4.1). The “vegan” package of R software is used to analyze principal component analysis (PCoA) based on Bray-Curtis distance algorithm at the level. The GraphPad Prism8.0.2 software were used to generate a best-fit line and calculate relevant parameters, Venn chart was analyzed and drawn by R software “Venn” package, and network attributes were calculated by Rstudio “igraph” and “WGCNA”. The Cytoscape.3.10.2 software was used to visualize the co-occurrence network at the genius level. In order to reduce the network complexity, the genera whose relative abundance proportion was ≥ 01% and were present in ≥ 50% of the samples were retained. Spearman’s correlation coefficient r was ≥ 0.6, and the significance P < 0.05.Circos chart was analyzed and drawn using the R software ‘circlize’ package, and the heat map was drawn using the ‘pheatmap’ package.The structural equation model is calculated by Amos 26 Graphics. Adobe illustrator 2021 is used for graphic combination. The experiment was performed in Runxi County, Pengshui County, Chongqing city, China. This area has a 30 year history of tobacco cultivation, and in a considerable part of the area, there are serious continuous cropping obstacles.(The inhibition test is shown in Annex 1.) The tobacco variety is K326, which is the main local tobacco variety. Tobacco seeds were provided by the Tobacco Industry Co., Ltd. in Chongqing, China. The basic physical and chemical properties of the soil were as follows: organic matter content of 26.60 g/kg, total nitrogen content of 1.66 g/kg, total phosphorus content of 1138 mg/kg, total potassium content of 20.7 g/kg, alkaline dissolved nitrogen content of 154.00 mg/kg, effective phosphorus content of 49.40 mg/kg, quick-acting potassium content of 545.00 mg/kg, and pH value of 7.16. The test fumigant 98% dazomet was purchased from Nantong Shizhuang Chemical Co., Ltd. The tobacco seedlings used in the experiment were raised by the floating method, and the demonstration areas all carried out unified field management according to the relevant technical standards, with the central flower opening and topping and the axillary buds controlled with 12.5% flumetralin EC. The experiment consisted of five treatments and three replications, totaling 15 plots. Each plot had an area of approximately 66.7 m². Protection rows were arranged, and the area of the experimental plots was about 0.13 hectares. The medicine was applied during ridging. After that, the soil was covered with conventional plastic film and compacted around the film with new soil placed between the ridges. Then, air tight disinfection was maintained for about 20 days. Ten days before transplanting, use the transplanting hole to open the film for ventilation, and transplant tobacco seedlings to carry out germination test (Annex 2).Treatment 1 corresponded to D3.5 (dazomet 3.5 kg/666.7 m2); Treatment 2 corresponded to D6.5 (dazomet 6.5 kg/666.7 m2); Treatment 3 corresponded to D9.5 (dazomet 9.5 kg/666.7 m2); Treatment 4 corresponded to D12.5 (dazomet 12.5 kg/666.7 m2); and Treatment 5 corresponded to a blank control (CK). Five to ten representative tobacco plants were selected for each treatment and marked with tags. In accordance with the YC/T 142–2010 “Tobacco Agronomic Trait Survey Methods”, the agronomic traits of the tobacco plants were determined at the resettling stage and 7 days after topping. The agronomic traits mainly included plant height, stem circumference, effective leaf number, maximum leaf length and maximum leaf width. Samples were taken at the doughnut stage and 7 d after topping to determine the root growth of the tobacco plants. The variables included root fresh weight, aboveground fresh weight, maximum root length and root volume. A sunny day was chosen for the measurement, and the entire experiment was performed on the same day. The sampling time was determined by more than 50% of the plots at the test site to reach the fertility stage. Each plot was sampled each time a representative tobacco plant was selected, and the entire root system was excavated from the soil. The roots were soaked in water, rinsed and washed clean, the maximum root length was measured first, the volume of the root system was measured via the drainage method, and the root system was weighed to determine the fresh weight. Tobacco rhizosphere soil samples were collected 50 days after transplanting. Each treatment had 3 sample bags, that is, 3 replicates. The rhizosphere soil samples were collected according to the principle of “random, equal and multipoint mixing”. The rhizosphere soil samples included fibrous roots in a range of 2 mm that were shaken and collected, impurities were removed, the samples were sifted through a 2 mm sieve, and each bag of soil was bagged at 5 points after mixing the soil samples. The soil samples were bagged and labeled immediately after collection, and 15 bags of soil were collected at a time. The sample numbers were D3.5, D6.5, D9.5, D12.5 and CK. All the samples were taken back to the laboratory at a low temperature and stored at -80°C. The total DNA of the soil microorganisms was extracted, 16S rRNA and ITS sequencing techniques were used to amplify and sequence the fragments, and the diversity of the soil microbial structure was determined and analysed. The total DNA of the Soil microbial community was extracted according to the Power Soil ® DNA kit(Axygen), purified and detected by agarose gel (1%) electrophoresis. PCR amplification of V4 ~ V5 region of bacterial 16 S rRNA gene was performed using primers 515 F (5 ‘-GTGCCAGCMGCCGCGG-3’) and 806R (5′- GGACTACHVGGGTWTCTAAT-3′). ITS1F (5′- CTTGGTCATTTAGAGGAAGTAA-3′) and ITS2R (5′- GCTGCGTTCTTCATCGATGC-3′) were used to amplify the ITS1 region of the fungal ITS gene.Each sample was repeated 3 times, and the PCR mixture of the same sample was detected by agarose gel (2%) electrophoresis, after which the amplicon was recovered . According to the instructions of Axy-Prep DNA Gel Recovery Kit (Axygen), the PCR products were purified, detected.The 16 S rRNA and ITS gene fragments were sequenced by Shanghai Majorbio Co., Ltd., China, using the Illumina MiSeqPE250 platform ( https://cloud.majorbio.com ). According to the barcode sequence corresponding to the sample, the effective sequence of the corresponding sample was obtained. Use FLASH software (v1.2.7, http://ccb.jhu.edu/ftware/FLASH/ ) splicing sequence. QIIME2 (2018.2, Quantitative Insight Into Microbial Ecology) software was used to invoke DADA2 to check and eliminate the chimera sequence. A table of amplicon sequence variant ASV(amplicon sequence variant) was constructed for the sequence obtained above, and ASV representative sequence was obtained [DADA2 clustering with 100% similarity (de-duplication), and each de-duplication sequence generated after DADA2 quality control was called ASVs]. Will get ASVs SILVA on behalf of the sequence in the database ( http://greengenes.secondgenome.com/ , Release 138) J91 annotate and to obtain its classification information. All samples in the ASVs abundance matrix were randomly resampling at the lowest sequencing depth level of 95% to correct for diversity differences caused by sequencing depth. QIME 2 software was then used to calculate the diversity index for each sample separately. The test data were sorted via Excel 2013. The Duncan’s new complex range method in SPSS 17.0 statistical software was used to compare and analyse the differences in related data, with P ≤ 0.05 .Alpha diversity is calculated through the website https://mothur.org/wiki/calculators/ .The “vegan” package of R software was used in ADONIS analysis to explore the influence of different grouping factors on sample differences, and the displacement test was used to conduct a significant analysis of the statistical significance of the division (R 4.4.1). The “vegan” package of R software is used to analyze principal component analysis (PCoA) based on Bray-Curtis distance algorithm at the level. The GraphPad Prism8.0.2 software were used to generate a best-fit line and calculate relevant parameters, Venn chart was analyzed and drawn by R software “Venn” package, and network attributes were calculated by Rstudio “igraph” and “WGCNA”. The Cytoscape.3.10.2 software was used to visualize the co-occurrence network at the genius level. In order to reduce the network complexity, the genera whose relative abundance proportion was ≥ 01% and were present in ≥ 50% of the samples were retained. Spearman’s correlation coefficient r was ≥ 0.6, and the significance P < 0.05.Circos chart was analyzed and drawn using the R software ‘circlize’ package, and the heat map was drawn using the ‘pheatmap’ package.The structural equation model is calculated by Amos 26 Graphics. Adobe illustrator 2021 is used for graphic combination. Below is the link to the electronic supplementary material. Supplementary Material 1. |
Identification of Salivary Exosome-Derived miRNAs as Potential Biomarkers of Bone Remodeling During Orthodontic Tooth Movement | ae472dd6-7138-4d76-92f6-68723317fe77 | 11818790 | Dentistry[mh] | microRNAs (miRNAs) are an integral part of gene regulatory networks, influencing a considerable number of processes both in physiological and diseased states in different tissues and organs. Alterations to their levels have been associated with many diseases and many miRNAs have been identified as biomarkers in pathological conditions . miRNAs are a set of non-coding RNAs that modulate gene expression post-transcriptionally. They are, on average, 22 nucleotides long and play an important role in biological processes . They target mRNAs by imperfect complementary binding, usually in the 3’ untranslated region (UTR), and they suppress their expression through a combination of translational inhibition and the promotion of mRNA decay. Their biogenesis involves their transcription by the RNA pol II as pri-miRNA and their cleavage to form pre-miRNA in the nucleus. Then, another cleavage occurs through the action of Dicer, leading to the formation of mature miRNA in a duplex form. The duplex miRNA is bound by Argonaute, thus forming the RISC complex, which is the effector of transcript downregulation . miRNAs have been shown to be involved in processes relevant to dentistry such as tooth development, bone remodeling, and the differentiation of dental stem cells . Many miRNAs are known to regulate osteoclast and osteoblast differentiation and their maintenance and function, which are important to bone remodeling. More specifically, regarding orthodontic tooth movement, miRNAs are involved in a process that triggers a series of biological changes . In orthodontics, tooth displacement and skeletal growth modification occur due to the bone’s capacity to remodel. Orthodontic devices exert forces on the teeth and the surrounding tissues, thereby inducing reactions to their cells and the extracellular components. Bone remodeling is regulated by a balance system of two types of cells, the osteoblasts and osteoclasts, and includes a complex network of interactions between cells and between the extracellular matrix and the cells in the presence of hormones, cytokines, growth factors, and mechanical loading . These processes heavily depend on the sterile inflammation that occurs upon the application of mechanical stress. Following the reception of mechanical cues, the signal conveying the mechanical conditions of the extracellular environment is carried towards the nucleus through MAPK kinases and, more importantly, through extracellular-signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs) . Then, to activate osteo-specific transcription factors like Runx2 and c-Jun, c-Fos stimulates the DNA-binding potential to specific genes associated with osteoblast differentiation (ALP, osteocalcin, collagen type I). All of the above ultimately lead to a change in gene expression and reprogramming towards the osteoblastic phenotype. Additionally, mature osteoblasts produce cytokines such as RANKL and OPG, the balance of which is essential for osteoclast differentiation and bone resorption . The proteins essential for these processes are targeted by miRNAs. There is already a set of miRNA molecules that have been validated to be differentially expressed during orthodontic movement. These miRNAs comprise the miRNAs 21, 27, 29, 34,146, 214, and 101 . Lately, the research focus in dentistry and orthodontics has been directed towards the development of biomarkers derived from oral fluids. This would be of interest because these biomarkers could provide information about the monitoring of physiological processes and serve as tools for the early diagnosis of diseases. Most studies regarding orthodontic tooth movement (OTM) have been performed on gingival crevicular fluid (GCF) . GCF is the fluid between a tooth and the surrounding gingival tissues, known as gingival sulcus, and it can contain cells, biomolecules, and microbiota . Apart from GCF, saliva has been identified as a potential fluid for biomarker discovery. Saliva can give a variety of information for both oral and systemic health, as its contents can alter in diseased states. Various molecules have been identified as biomarkers in saliva, in patients diagnosed with cancer or autoimmune and infectious diseases . Despite the use of GCF as a biomarker source in orthodontic tooth movement, the limited sample material that can be reliably collected in the clinic, as well as the non-invasive and cost-effective collection of saliva, could make saliva a more widespread material for biomarker discovery . In the context of using saliva as a source for biomarkers, there is increasing evidence regarding the utilization of salivary exosomes. Exosomes are small extracellular vehicles (EVs) (40–150 nm) secreted by a variety of cells, and they play a crucial role in intracellular communication, as they include various macromolecules such as RNAs, proteins, etc. . An important feature of exosomes is that they can pass through epithelial barriers, meaning that information from the underlying tissues in the mouth can eventually be found in the saliva. This phenomenon could be leveraged in order to identify biomarkers related to systemic skeletal disorders such as osteoporosis . The investigation of these salivary exosomes could clarify the molecular pathways associated with the bone-remodeling process, which occurs after the application of mechanical stress as in orthodontic treatment . As the current literature suggests, exosomal miRNAs could be a better source for biomarker studies . This study aimed to isolate salivary exosome-derived miRNAs in patients undergoing OTM and identify new biomarkers for bone remodeling. Following the isolation and analysis of salivary samples, transmission electron microscopy (TEM) confirmed the presence of EVs in the samples from patients undergoing orthodontic treatment. In the TEM analysis , the EVs appeared as spherical structures, with some exhibiting a distinct membrane-like structure. A complementary size distribution analysis via nanoparticle tracking confirmed that the majority of the EVs were between 100 and 150 nm in diameter, with a peak concentration around 100 nm . These findings confirm the successful isolation and identification of extracellular vesicles, which provide a viable source for a further miRNA analysis relevant to bone-remodeling processes during orthodontic treatment. The most variably expressed miRNAs can be viewed in the heatmap below , with no distinct clustering observed in either the samples or the miRNA expression patterns. Likewise, the MDS plot did not display distinct groupings based on time points or individual subjects, suggesting an absence of strong time-dependent or subject-specific patterns in the data. Of the comparisons made, the Day 40 vs. Day 0 contrast yielded a statistically significant result, identifying hsa-miR-4634 as differentially expressed, with its expression reduced on Day 40 compared to Day 0 (log fold change = −1.9, mean expression = 6.2 log2-transformed CPMs, adjusted p -value = 0.043) . While other miRNAs did show notable changes in expression, they did not reach statistical significance after adjusting for multiple testing to reduce the likelihood of false positives (adjusted p -value > 0.1). Because these miRNAs changed in a statistically significant way ( p -value < 0.05), it is noteworthy to mention and analyze their alterations . Using the database TarBase-v9.0, we generated a list of the genes that these miRNAs target . Afterwards, we performed a gene ontology (GO) enrichment analysis using the bioinformatics tool DAVID (database for annotation, visualization, and integrated discovery) . Of all the processes affected by the input gene set, we searched only the processes that are the most relevant to bone remodeling, such as osteoblast differentiation (GO:0001649); osteoclast differentiation (GO:0030218); the positive regulation of osteoblast differentiation (GO:0045669); and the positive regulation of bone mineralization (GO:0030501) ( , and ). The present study is the first, to our knowledge, to investigate the expression of miRNAs in salivary exosomes during OTM in patients. The foremost finding of this study concerns miRNA hsa-miR-4634, which was found to have a statistically significant altered expression. Its expression was downregulated after 40 days of treatment. The literature regarding this particular miRNA is limited. Still, the only known target for this miRNA is VAV3 . VAV3 is a Rho family guanine nucleotide exchange factor, which has been shown to be essential for stimulated osteoclast activation and bone density. Guanine nucleotide exchange factors (GEFs) mediate the activation of Rho family GTPase by exchanging GDP for GTP. This occurs in addition to GTPase activation, thus influencing downstream signaling pathways. VAV3 has been identified as an essential factor in the regulation of osteoclast function. More specifically, VAV3-deficient mice were shown to have an increased bone mass because of dysfunctional osteoclasts and exhibited protection from stimulated bone loss. Also, the authors reported that GTPase Rac1 was affected by defective VAV3, and the authors concluded that the activation of Rac1 is VAV3-dependent and the signaling after cytokine stimulation required for cytoskeletal reorganization in osteoclasts is impaired . Based on the findings of the aforementioned study, it is evident that the regulation of VAV3 could be significant in orthodontic tooth movement, in the phase where osteoclast activity is essential for bone remodeling. It is possible that, in this setting, hsa-miR-4634 is downregulated in order for an upregulation of VAV3 to occur, a hypothesis that could be addressed in a future study with the use of experimental models. Apart from miR-4634, which is statistically significant for the adjusted p -value, some noteworthy results can be extracted from the other miRNAs with a p -value < 0.05, such as hsa-miR-195-5p and hsa-miR-1246, which are known to regulate osteoblast differentiation and inhibit the osteogenic potential of progenitor cells, respectively . The GO enrichment analysis revealed that processes associated with bone remodeling were impacted, including osteoblast differentiation, the positive regulation of osteoblast differentiation, osteoclast differentiation, and the positive regulation of bone mineralization. A comparison of the three tables ( , and ) yielded significant data. The first statistical comparison was of the differential expression of miRNAs on Day 7 vs. Day 0, the second was of the expression on Day 40 vs. Day 7, and the third was of the expression on Day 40 vs. Day 0. What might be observed is that the first comparison showed genes related to processes of osteoblast differentiation, the positive regulation of osteoblast differentiation, and bone mineralization, while the other comparisons included osteoclast differentiation as well. OTM can be divided into three phases: the initial phase, characterized by rapid tooth movement immediately after force application; the lag phase, during which tooth movement temporarily halts due to the hyalinization of the periodontal ligament; and the post-lag phase, when movement resumes as necrotic tissue is cleared, allowing the tooth to continue its displacement . In our study, the selected timepoints corresponded to the initial (0 to 7 days) and the lag and post-lag phases (7 to 40 days). Current reports in the literature suggest that, in the initial phase after the application of mechanical stress, the early cellular response involves inflammation and tissue remodeling. Osteoclastogenesis, a critical process for bone resorption, tends to increase during the later stages of tooth movement, which aligns with our findings . The absence of detectable miRNAs targeting osteoclast differentiation genes early on can be attributed to the fact that osteoclast activity is still relatively low during this phase . In the lag and post-lag phases of orthodontic tooth movement, after the application of stress, alveolar bone remodeling becomes more pronounced as osteoclasts are activated on the pressure side of the tooth . At this phase, miRNAs related to osteoclast differentiation, such as those targeting the RANK, RANKL, or NFATc1 pathways, are more likely to be expressed . This molecular phase corresponds clinically with the acceleration phase of tooth movement, when bone resorption becomes more critical, facilitating tooth displacement through the alveolar bone. These reports are in accordance with our results, as in the initial phase of our research, only osteoblast differentiation and the positive regulation of osteoblast differentiation were affected, while osteoclast differentiation was affected at later time points . The detection of differentially expressed miRNAs in this final phase could be a result of the crosstalk of the periodontal ligament cells and the cells of the alveolar bones. The cellular source of EVs in the saliva is heterogenous, as various cell types can contribute . Apart from the epithelial cells and immune cells of the oral cavity, a portion of the EVs found in the saliva can originate from dental-tissue-derived cells, such as gingival mesenchymal stem cells and periodontal ligament stem cells . The periodontal ligament is mechanically stimulated during orthodontic tooth movement; it is the primary tissue that responds to mechanical signal transduction, and it conveys changes in the surrounding bone . As it is known that periodontal ligament stem cells (PDLSCs) change their EV miRNA content in response to mechanical stress in order to affect the activity of osteoblasts and osteoclasts , and preventing the release of EVs from PDLSCs results in a disrupted osteoclast function in OTM , one could hypothesize that the changes in the expression of miR-4634 and the other differentially expressed miRNAs in our study could be reflective of the response of the periodontal ligament during OTM. Summarizing the results of this study, the most significant finding was the downregulation of the miRNA hsa-miR-4634. This downregulation was found to occur on Day 0 to Day 40 of tooth movement. Even though there might be a plausible biological explanation for their altered expression that fits the setting of orthodontic stress application, none of the aforementioned miRNAs ( , and ) were found to be significant after the p -value adjustment. One of the potential limitations of this study is that the sample comprised patients in the first phase of orthodontic treatment, during which only a small number of teeth were moved. The cellular and molecular changes expected to occur might have been more pronounced if mechanical stress had been exerted on a larger number of teeth. It is important to note that the term saliva is used interchangeably with oral fluid. As the samples were collected after the patients chewed on parafilm, saliva production was stimulated . Because of this, the collected fluid was oral fluid that contained mainly saliva, but also other components of the oral cavity. Moreover, the flow rate was not recorded, but at least 5 min were required to produce 5 mL of saliva. This corresponds to a stimulated saliva flow rate of approximately 1 mL/min and aligns with the expectations in healthy adolescents . This study might serve as a starting point in investigating altered oral fluid miRNA expression during orthodontic treatment. Further studies are needed to elucidate the molecular interplay between miRNAs and their targets. The identification of potential biomarkers will be of great value to clinical orthodontics and future oral therapeutics. In molecular orthodontics, the clinician may be ultimately capable of controlling several clinical aspects, like the rate of tooth movement. The decoding of the human genome, along with new developments in molecular biology, has provided a much-anticipated boost to the biological sciences. Orthodontics, which increasingly relies on biotechnology, is expected to be significantly impacted by these advancements. 4.1. Patients This study received ethical approval from the ethical committee of the RWTH Medical University of Aachen in Germany, and informed consent was obtained from all the participants (ethical approval number: CTC-A-Nr.: 22-262). This study involved fifteen Caucasian patients aged 11–15 years (six females with a mean age of 14 ± 2.3 years and nine males with a mean age of 13 ± 1.3 years) presenting with a dental Class I or Class II malocclusion accompanied by moderate to severe crowding or spacing. When comparing the mean age of male and female patients, there were no statistically significant differences between the two groups ( p -value = 0.583), suggesting that gender did not have a significant effect on patient age. These patients were treated in a private orthodontic office in Bedburg, Germany. The age group used for this study was selected in order to minimize the likelihood of the presence of underlying conditions, such as periodontitis or systemic disorders, that could confound the results. The exclusion criteria included any history of cleft lip/palate, dentofacial deformities or syndromes, autoimmune diseases, or type 1 or type 2 diabetes; a history of drug use; and previous orthodontic treatment or intraoral/external oral surgery. All the patients underwent a thorough intraoral examination, a clinical assessment of the teeth, and a periodontal evaluation, which included the approximal plaque index (API), sulcus bleeding index (SBI), and periodontal screening index (PSI) . According to the orthodontic treatment plan, the 4 maxillary incisors and the 2 first maxillary molars were bonded with fixed pre-adjusted orthodontic appliances featuring a 0.22 slot size. From each patient, saliva samples were collected at three different time points: one week before bracket placement and archwire activation, 7 days after the treatment’s onset, and 40 days after the treatment’s onset. Thus, 45 saliva samples were finally collected. 4.2. Sample Size Calculation This study was designed as an exploratory analysis, with the primary objective of examining temporal changes in biomarkers, without testing predefined hypotheses. The sample size was determined through a power analysis based on a repeated measures design, where three measurements were collected from each participant at distinct time points: prior to bracket placement (Day 0), one week after the treatment (Day 7), and 40 days after the treatment (Day 40). Based on previous studies, a medium effect size (f = 0.30) was observed for changes in biomarkers over time . A power analysis was performed using the pwr package in R , which indicated that a minimum of 15 participants was required to achieve 40% power at an alpha level of 0.05 for detecting differences across the three time points. In exploratory analyses, the use of a lower power can be justified when the primary objective is to identify preliminary trends that may guide the design of future, more rigorous studies . Accordingly, 15 patients were recruited for this study, and saliva samples were collected from each participant at the three time points, resulting in a total of 45 samples. 4.3. Saliva Collection The saliva samples were collected in the dental office where the patients received orthodontic treatment. The collection was placed on ice in Falcon tubes previously refrigerated. The participants were instructed not to brush their teeth, chew gum, eat, or drink any liquids for at least 1.5 h before the visit. Saliva was collected upon arrival by having the patients chew a piece of parafilm for one minute while swallowing normally. The time of collection was set as late afternoon (15:00–18:00) for all the patients in order to prevent potential time-dependent changes in the saliva content. Before collection, 100 μL of PhosSTOP™ EASYpack (Roche Applied Science, Cat. No. 04 906 845 001, Mannheim, Germany) and 100 μL of cOmplete™ Mini Protease Inhibitor Cocktail Tablets (Roche Applied Science, Cat. No. 04 693 124 001, Mannheim, Germany) were added to a cold 50 mL Falcon tube. This was equivalent to ½ tablet of PhosSTOP and ½ tablet of cOmplete. The participants were instructed to begin chewing and then hold the saliva in their mouth (i.e., not swallow), and at 30-s intervals, eject the saliva into the cold 50 mL Falcon tube. The participants continued this process until a minimum of 5 mL had been obtained, or for up to 15 min of chewing and ejecting the saliva. After collection, the saliva samples were diluted with pre-cooled phosphate-buffered saline (PBS) at a ratio of 1:1. The samples were then centrifuged (Sigma 3K15, Sigma, Osterode am Harz, Germany) at 300× g for 20 min at 4 °C and the pellet was removed. Afterward, the supernatant was centrifuged further at 2000× g for 10 min at 4 °C, and the resulting pellet was discarded. This step was repeated at 5000× g for 30 min at 4 °C, and the final pellet was discarded. The remainder of the supernatant was stored at 4 °C until same-day transport and then stored at −80 °C in the lab. At the lab, the supernatant was thawed and centrifuged using an Optima LE-80K ultracentrifuge with an SW 32 Ti rotor (Beckman Coulter, Chaska, MN, USA) at 12,000× g for 20 min at 4 °C; then, the pellet was discarded. The sample was centrifuged at 120,000× g for 70 min at 4 °C, after which the supernatant was discarded. The pellet was resuspended, filtered using a 0.2 μm filter, and washed again by centrifugation at 120,000× g for 70 min at 4 °C. The final pellet was resuspended in filtered PBS and stored at −80 °C for long-term storage . 4.4. RNA Isolation RNA was isolated from 60 µL of exosome samples using the Maxwell RSC miRNA Plasma and Serum Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. This process ensured the efficient extraction of small RNAs, including miRNAs, from the exosome samples. 4.5. RNA Quantification The concentration of the isolated RNA was determined using a Promega Quantus Fluorometer (Promega, Madison, WI, USA). This quantification step was crucial to ensure sufficient RNA input for downstream library preparation. 4.6. Library Preparation Sequencing libraries were prepared using the QIAseq miRNA UDI Library Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. QIAseq miRNA Library QC spike-ins were added to each sample to monitor the library preparation process and ensure the accuracy of miRNA detection. This step facilitated the normalization and validation of the library preparation efficiency. 4.7. Library Quality Control The size distribution of the prepared libraries was assessed using the Agilent TapeStation (Agilent Technologies, Santa Clara, CA, USA). The average library size was confirmed to fall within the expected range for miRNA libraries. After size confirmation, the library concentrations were determined with the Promega Quantus Fluorometer to ensure accurate loading for sequencing. 4.8. Sequencing Sequencing was performed on an Illumina NextSeq 500/550 system using a High Output Kit v2.5 (75 cycles) (Illumina Inc., San Diego, CA, USA). The libraries were sequenced in a single-end mode for 72 cycles, with a 1% PhiX spike-in used as an internal control to monitor sequencing quality and performance. 4.9. Statistical and Bioinformatical Analysis FASTQ files were generated using bcl2fastq (Illumina). To facilitate a reproducible analysis, the samples were processed using the publicly available nf-core/smRNAseq pipeline, version 1.1.0 , implemented in Nextflow 21.10.6 using Docker 20.10.12 (Merkel 2014) with the minimal command. Out of 45 samples, 1 was eliminated from further analysis because no miRNA could be detected in the sample. Therefore, miRNA counts from the 44 samples were analyzed to identify changes in the expression levels. Two samples with library sizes (sum of miRNA counts) of less than 10,000 were excluded, resulting in 42 samples for analysis. Such a phenomenon could be a result of the various centrifugation and washing steps involved in the process of purification. Out of the 1405 miRNAs, only those with at least one read in a minimum of five samples were retained, yielding 185 miRNAs. The miRNA expression table in TMM-normalized CPMs, after count-based miRNA filtering, is included in the . To assess the overall structure and quality of the data before conducting a differential expression analysis, a multidimensional scaling (MDS) plot was generated using the plotMDS function from the “limma” R package. In this plot, each point represents a sample, with the distance between points indicating the similarity of their miRNA expression profiles. The differential expression analysis was performed using the “limma” R package once again. The “voom” function from the “limma” R package was used to transform the count data into log2-counts per million (CPM) with associated weights, accounting for mean–variance relationships. Next, a linear model was fitted to the transformed data while accounting for within-subject correlations. The “duplicateCorrelation” function was employed to estimate the correlation between measurements from the same subject, with the inter-subject correlation being incorporated into the linear model fitting process. This correlation was subsequently used in the “lmFit” function, which fitted the linear model to the data, adjusting for the block effect (i.e., subject-specific variation). To compare different time points, contrasts were defined using the “makeContrasts” function for the following comparisons: Day 7 vs. Day 0, Day 40 vs. Day 7, and Day 40 vs. Day 0. These contrasts were applied to the fitted model using the “contrasts.fit” function. Finally, moderated t -tests were computed using the “eBayes” function to determine the significance of differential expression across the specified contrasts. The R script for this analysis is provided in the . This study received ethical approval from the ethical committee of the RWTH Medical University of Aachen in Germany, and informed consent was obtained from all the participants (ethical approval number: CTC-A-Nr.: 22-262). This study involved fifteen Caucasian patients aged 11–15 years (six females with a mean age of 14 ± 2.3 years and nine males with a mean age of 13 ± 1.3 years) presenting with a dental Class I or Class II malocclusion accompanied by moderate to severe crowding or spacing. When comparing the mean age of male and female patients, there were no statistically significant differences between the two groups ( p -value = 0.583), suggesting that gender did not have a significant effect on patient age. These patients were treated in a private orthodontic office in Bedburg, Germany. The age group used for this study was selected in order to minimize the likelihood of the presence of underlying conditions, such as periodontitis or systemic disorders, that could confound the results. The exclusion criteria included any history of cleft lip/palate, dentofacial deformities or syndromes, autoimmune diseases, or type 1 or type 2 diabetes; a history of drug use; and previous orthodontic treatment or intraoral/external oral surgery. All the patients underwent a thorough intraoral examination, a clinical assessment of the teeth, and a periodontal evaluation, which included the approximal plaque index (API), sulcus bleeding index (SBI), and periodontal screening index (PSI) . According to the orthodontic treatment plan, the 4 maxillary incisors and the 2 first maxillary molars were bonded with fixed pre-adjusted orthodontic appliances featuring a 0.22 slot size. From each patient, saliva samples were collected at three different time points: one week before bracket placement and archwire activation, 7 days after the treatment’s onset, and 40 days after the treatment’s onset. Thus, 45 saliva samples were finally collected. This study was designed as an exploratory analysis, with the primary objective of examining temporal changes in biomarkers, without testing predefined hypotheses. The sample size was determined through a power analysis based on a repeated measures design, where three measurements were collected from each participant at distinct time points: prior to bracket placement (Day 0), one week after the treatment (Day 7), and 40 days after the treatment (Day 40). Based on previous studies, a medium effect size (f = 0.30) was observed for changes in biomarkers over time . A power analysis was performed using the pwr package in R , which indicated that a minimum of 15 participants was required to achieve 40% power at an alpha level of 0.05 for detecting differences across the three time points. In exploratory analyses, the use of a lower power can be justified when the primary objective is to identify preliminary trends that may guide the design of future, more rigorous studies . Accordingly, 15 patients were recruited for this study, and saliva samples were collected from each participant at the three time points, resulting in a total of 45 samples. The saliva samples were collected in the dental office where the patients received orthodontic treatment. The collection was placed on ice in Falcon tubes previously refrigerated. The participants were instructed not to brush their teeth, chew gum, eat, or drink any liquids for at least 1.5 h before the visit. Saliva was collected upon arrival by having the patients chew a piece of parafilm for one minute while swallowing normally. The time of collection was set as late afternoon (15:00–18:00) for all the patients in order to prevent potential time-dependent changes in the saliva content. Before collection, 100 μL of PhosSTOP™ EASYpack (Roche Applied Science, Cat. No. 04 906 845 001, Mannheim, Germany) and 100 μL of cOmplete™ Mini Protease Inhibitor Cocktail Tablets (Roche Applied Science, Cat. No. 04 693 124 001, Mannheim, Germany) were added to a cold 50 mL Falcon tube. This was equivalent to ½ tablet of PhosSTOP and ½ tablet of cOmplete. The participants were instructed to begin chewing and then hold the saliva in their mouth (i.e., not swallow), and at 30-s intervals, eject the saliva into the cold 50 mL Falcon tube. The participants continued this process until a minimum of 5 mL had been obtained, or for up to 15 min of chewing and ejecting the saliva. After collection, the saliva samples were diluted with pre-cooled phosphate-buffered saline (PBS) at a ratio of 1:1. The samples were then centrifuged (Sigma 3K15, Sigma, Osterode am Harz, Germany) at 300× g for 20 min at 4 °C and the pellet was removed. Afterward, the supernatant was centrifuged further at 2000× g for 10 min at 4 °C, and the resulting pellet was discarded. This step was repeated at 5000× g for 30 min at 4 °C, and the final pellet was discarded. The remainder of the supernatant was stored at 4 °C until same-day transport and then stored at −80 °C in the lab. At the lab, the supernatant was thawed and centrifuged using an Optima LE-80K ultracentrifuge with an SW 32 Ti rotor (Beckman Coulter, Chaska, MN, USA) at 12,000× g for 20 min at 4 °C; then, the pellet was discarded. The sample was centrifuged at 120,000× g for 70 min at 4 °C, after which the supernatant was discarded. The pellet was resuspended, filtered using a 0.2 μm filter, and washed again by centrifugation at 120,000× g for 70 min at 4 °C. The final pellet was resuspended in filtered PBS and stored at −80 °C for long-term storage . RNA was isolated from 60 µL of exosome samples using the Maxwell RSC miRNA Plasma and Serum Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. This process ensured the efficient extraction of small RNAs, including miRNAs, from the exosome samples. The concentration of the isolated RNA was determined using a Promega Quantus Fluorometer (Promega, Madison, WI, USA). This quantification step was crucial to ensure sufficient RNA input for downstream library preparation. Sequencing libraries were prepared using the QIAseq miRNA UDI Library Kit (QIAGEN, Hilden, Germany), following the manufacturer’s protocol. QIAseq miRNA Library QC spike-ins were added to each sample to monitor the library preparation process and ensure the accuracy of miRNA detection. This step facilitated the normalization and validation of the library preparation efficiency. The size distribution of the prepared libraries was assessed using the Agilent TapeStation (Agilent Technologies, Santa Clara, CA, USA). The average library size was confirmed to fall within the expected range for miRNA libraries. After size confirmation, the library concentrations were determined with the Promega Quantus Fluorometer to ensure accurate loading for sequencing. Sequencing was performed on an Illumina NextSeq 500/550 system using a High Output Kit v2.5 (75 cycles) (Illumina Inc., San Diego, CA, USA). The libraries were sequenced in a single-end mode for 72 cycles, with a 1% PhiX spike-in used as an internal control to monitor sequencing quality and performance. FASTQ files were generated using bcl2fastq (Illumina). To facilitate a reproducible analysis, the samples were processed using the publicly available nf-core/smRNAseq pipeline, version 1.1.0 , implemented in Nextflow 21.10.6 using Docker 20.10.12 (Merkel 2014) with the minimal command. Out of 45 samples, 1 was eliminated from further analysis because no miRNA could be detected in the sample. Therefore, miRNA counts from the 44 samples were analyzed to identify changes in the expression levels. Two samples with library sizes (sum of miRNA counts) of less than 10,000 were excluded, resulting in 42 samples for analysis. Such a phenomenon could be a result of the various centrifugation and washing steps involved in the process of purification. Out of the 1405 miRNAs, only those with at least one read in a minimum of five samples were retained, yielding 185 miRNAs. The miRNA expression table in TMM-normalized CPMs, after count-based miRNA filtering, is included in the . To assess the overall structure and quality of the data before conducting a differential expression analysis, a multidimensional scaling (MDS) plot was generated using the plotMDS function from the “limma” R package. In this plot, each point represents a sample, with the distance between points indicating the similarity of their miRNA expression profiles. The differential expression analysis was performed using the “limma” R package once again. The “voom” function from the “limma” R package was used to transform the count data into log2-counts per million (CPM) with associated weights, accounting for mean–variance relationships. Next, a linear model was fitted to the transformed data while accounting for within-subject correlations. The “duplicateCorrelation” function was employed to estimate the correlation between measurements from the same subject, with the inter-subject correlation being incorporated into the linear model fitting process. This correlation was subsequently used in the “lmFit” function, which fitted the linear model to the data, adjusting for the block effect (i.e., subject-specific variation). To compare different time points, contrasts were defined using the “makeContrasts” function for the following comparisons: Day 7 vs. Day 0, Day 40 vs. Day 7, and Day 40 vs. Day 0. These contrasts were applied to the fitted model using the “contrasts.fit” function. Finally, moderated t -tests were computed using the “eBayes” function to determine the significance of differential expression across the specified contrasts. The R script for this analysis is provided in the . The downregulation of hsa-miR-4634 may play a role in modulating osteoclast function, possibly through the regulation of the gene VAV3. The findings suggest that salivary miRNAs, particularly those derived from exosomes, hold promise as non-invasive biomarkers for monitoring bone-remodeling processes during orthodontic tooth movement. Although other miRNAs showed changes in expression, they did not reach statistical significance after adjustment, highlighting the complexity of miRNA regulation in response to mechanical stress. Future research should focus on elucidating the molecular interactions between miRNAs and their targets, which may provide valuable insights not only for advancing orthodontic treatments, but also for enhancing our understanding of broader skeletal health issues, such as osteoporosis. Understanding these molecular mechanisms could eventually lead to more personalized approaches in orthodontics, including the modulation of tooth movement rates based on individual miRNA signatures. |
Educational needs assessment for health advocate role in family medicine residency training in Turkey: A Delphi study | e3b625be-4d93-46a4-bef2-9e3995650b9d | 11363731 | Family Medicine[mh] | Health advocacy (HA) is purposeful action by informing or initiating, mobilising and organising activities on behalf of or with them to create change towards the social determinants of health that adversely affect the health of individuals or communities . Today, HA is accepted as a fundamental component of medical practice and is considered mandatory by professional and educational bodies. In this context, it is important to include HA training in pre- and post-graduate medical education. HA training is also important in terms of the social accountability of medical schools. The Accreditation Council for Graduate Medical Education integrated HA principles into the program requirements. The Physician Charter of the American Board of Internal Medicine, and the Royal College of Physicians and Surgeons of Canada also supported this commitment . Family medicine (FM) is a specialty that provides patient-centered and continuous primary health care to individuals and families regardless of age, gender or disease. This approach not only enhances the overall healthcare experience but also fosters a collaborative and empowering relationship between healthcare providers and patients. A vital aspect of health advocacy, especially in the realm of general practice (GP), is the concept of quaternary prevention. Quaternary prevention focuses on preventing the harmful effects of medical interventions and avoiding unnecessary medicalisation. In the Family Medicine Tree published by the European Regional Branch of the World Organisation of Family Doctors (WONCA Europe) , ‘community oriented’ is defined as reconciling the health needs of individual patients with the health needs of the society in which they live, in a balance in terms of the use of existing resources. The HA role of family physicians was also mentioned in CanMEDS Physician Competency Framework and The Canadian Medical Education Directives for Specialists – Family Medicine Competency Framework . In Turkey, HA is also not included in the professional competencies of the Turkish Association of Family Physicians Qualification Board . In the FP framework education program developed by the specialty association, although HA was mentioned among the Family Physician’s (FP) characteristics, it is not included among the key competencies defined for FP, but community orientation is mentioned. Firstly, the training needs for the HA role should be contextually determined. Needs assessment will guide the development of the HA training program and the design of teaching and assessment processes. In this study, we aimed to determine the need for HA training for FPs based on expert opinions at the national level. Research questions are defined: What are the expected competencies of the Family Physician for the role of health advocacy? How can HA competencies be gained and assessed in Family Medicine Residency Training? How can HA training be integrated into Family Medicine Residency Training? Delphi technique was used to evaluate the need for HA training in Family Medicine Residency Training (FMRT). The Delphi technique is a method of collecting the opinions of experts and forming a common expert opinion without coming together for a determined research question . Delphi process Selection of expert panel Participants in the expert panel in the Delphi study are expected to be competent, sufficient and heterogeneous in their field . Definition of an expert within the scope of the research: FM academics: Faculty members involved in FMRT FPs who have been working in Family Health Centres for at least 2 years FPs who have been working actively in the Turkish Association of Family Physicians and Turkish Medical Association – Family Medicine Specialty Board Experts working in the relevant department of the Ministry of Health Public health academics Medical education academics ‘Purposive sampling’ method was used to identify potential experts. In this method, the sample is not intended to be representative of the population, but a sample is selected that is believed to provide the data needed and in the selection of the panellists in the study, care was taken to take sides regarding FM and HA. In the study, suggestions were received from the Turkish Association of Family Physicians and Turkish Medical Association- Family Medicine Specialty Board in identifying potential specialists. Questionnaire development The first round questionnaire was designed based on literature and expert (2 FM and 1 medical education expert) opinions. It was pilot tested with six FPs who would not participate for clarity. In line with the findings obtained, the first round questionnaire was composed of five open-ended questions . Data collection This study was applied in three rounds in the processes described in [ , p.69–83]. The research was carried out between February and September 2022. Data analysis First round Qualitative data obtained from the first round were analysed by content analysis method. The COREQ List, which is the checklist of items that should be included in reports of qualitative research, was added to . Qualitative content analysis is a research method used to systematically analyse and interpret the content of textual, visual, or audio data. The content analysis includes reading and re-reading of responses, developing a process of coding, categorising and conceptualising the responses . Content analysis was performed by the MD (MD) and DAB (MD) by following the steps below: Merged open-ended responses from the expert panel Researchers independently reviewed data and generated code recommendations The researchers compared their code lists, discussed the similarities and differences, and finalised the code list Researchers independently coded the answers of the first five participants. They then discussed the codes and extracts together and reached a consensus on the coding Researchers independently coded the data Researchers discussed the cods and extracts together The researchers wrote the statements together based on the code extracts The similarities in the statements were evaluated and 119 statements were created under three headings so that no view was left out. In line with the results of the content analysis, the second-round questionnaire was prepared. Second round Mean, standard deviation and percentage were calculated for each statement. In addition, the total percentage of those who scored 4 and 5 on a 5-point Likert scale was determined. The consensus level was defined as 85% (those who scored 4 and 5), and statements above this level were accepted as the statements agreed in the second round . Adjustments were made in the statements in line with the expert suggestions. Third round Descriptive statistical analyses were performed for responses to the third-round questionnaire. Statements that exceeded the consensus level with the new data were accepted as the recommendations agreed in the third-round. The statements agreed in the second and third rounds were brought together and accepted as the statements agreed in the three rounds. Statements that were below the level of consensus were added to . Selection of expert panel Participants in the expert panel in the Delphi study are expected to be competent, sufficient and heterogeneous in their field . Definition of an expert within the scope of the research: FM academics: Faculty members involved in FMRT FPs who have been working in Family Health Centres for at least 2 years FPs who have been working actively in the Turkish Association of Family Physicians and Turkish Medical Association – Family Medicine Specialty Board Experts working in the relevant department of the Ministry of Health Public health academics Medical education academics ‘Purposive sampling’ method was used to identify potential experts. In this method, the sample is not intended to be representative of the population, but a sample is selected that is believed to provide the data needed and in the selection of the panellists in the study, care was taken to take sides regarding FM and HA. In the study, suggestions were received from the Turkish Association of Family Physicians and Turkish Medical Association- Family Medicine Specialty Board in identifying potential specialists. Questionnaire development The first round questionnaire was designed based on literature and expert (2 FM and 1 medical education expert) opinions. It was pilot tested with six FPs who would not participate for clarity. In line with the findings obtained, the first round questionnaire was composed of five open-ended questions . Data collection This study was applied in three rounds in the processes described in [ , p.69–83]. The research was carried out between February and September 2022. Data analysis First round Qualitative data obtained from the first round were analysed by content analysis method. The COREQ List, which is the checklist of items that should be included in reports of qualitative research, was added to . Qualitative content analysis is a research method used to systematically analyse and interpret the content of textual, visual, or audio data. The content analysis includes reading and re-reading of responses, developing a process of coding, categorising and conceptualising the responses . Content analysis was performed by the MD (MD) and DAB (MD) by following the steps below: Merged open-ended responses from the expert panel Researchers independently reviewed data and generated code recommendations The researchers compared their code lists, discussed the similarities and differences, and finalised the code list Researchers independently coded the answers of the first five participants. They then discussed the codes and extracts together and reached a consensus on the coding Researchers independently coded the data Researchers discussed the cods and extracts together The researchers wrote the statements together based on the code extracts The similarities in the statements were evaluated and 119 statements were created under three headings so that no view was left out. In line with the results of the content analysis, the second-round questionnaire was prepared. Second round Mean, standard deviation and percentage were calculated for each statement. In addition, the total percentage of those who scored 4 and 5 on a 5-point Likert scale was determined. The consensus level was defined as 85% (those who scored 4 and 5), and statements above this level were accepted as the statements agreed in the second round . Adjustments were made in the statements in line with the expert suggestions. Third round Descriptive statistical analyses were performed for responses to the third-round questionnaire. Statements that exceeded the consensus level with the new data were accepted as the recommendations agreed in the third-round. The statements agreed in the second and third rounds were brought together and accepted as the statements agreed in the three rounds. Statements that were below the level of consensus were added to . Participants in the expert panel in the Delphi study are expected to be competent, sufficient and heterogeneous in their field . Definition of an expert within the scope of the research: FM academics: Faculty members involved in FMRT FPs who have been working in Family Health Centres for at least 2 years FPs who have been working actively in the Turkish Association of Family Physicians and Turkish Medical Association – Family Medicine Specialty Board Experts working in the relevant department of the Ministry of Health Public health academics Medical education academics ‘Purposive sampling’ method was used to identify potential experts. In this method, the sample is not intended to be representative of the population, but a sample is selected that is believed to provide the data needed and in the selection of the panellists in the study, care was taken to take sides regarding FM and HA. In the study, suggestions were received from the Turkish Association of Family Physicians and Turkish Medical Association- Family Medicine Specialty Board in identifying potential specialists. The first round questionnaire was designed based on literature and expert (2 FM and 1 medical education expert) opinions. It was pilot tested with six FPs who would not participate for clarity. In line with the findings obtained, the first round questionnaire was composed of five open-ended questions . This study was applied in three rounds in the processes described in [ , p.69–83]. The research was carried out between February and September 2022. First round Qualitative data obtained from the first round were analysed by content analysis method. The COREQ List, which is the checklist of items that should be included in reports of qualitative research, was added to . Qualitative content analysis is a research method used to systematically analyse and interpret the content of textual, visual, or audio data. The content analysis includes reading and re-reading of responses, developing a process of coding, categorising and conceptualising the responses . Content analysis was performed by the MD (MD) and DAB (MD) by following the steps below: Merged open-ended responses from the expert panel Researchers independently reviewed data and generated code recommendations The researchers compared their code lists, discussed the similarities and differences, and finalised the code list Researchers independently coded the answers of the first five participants. They then discussed the codes and extracts together and reached a consensus on the coding Researchers independently coded the data Researchers discussed the cods and extracts together The researchers wrote the statements together based on the code extracts The similarities in the statements were evaluated and 119 statements were created under three headings so that no view was left out. In line with the results of the content analysis, the second-round questionnaire was prepared. Second round Mean, standard deviation and percentage were calculated for each statement. In addition, the total percentage of those who scored 4 and 5 on a 5-point Likert scale was determined. The consensus level was defined as 85% (those who scored 4 and 5), and statements above this level were accepted as the statements agreed in the second round . Adjustments were made in the statements in line with the expert suggestions. Third round Descriptive statistical analyses were performed for responses to the third-round questionnaire. Statements that exceeded the consensus level with the new data were accepted as the recommendations agreed in the third-round. The statements agreed in the second and third rounds were brought together and accepted as the statements agreed in the three rounds. Statements that were below the level of consensus were added to . Qualitative data obtained from the first round were analysed by content analysis method. The COREQ List, which is the checklist of items that should be included in reports of qualitative research, was added to . Qualitative content analysis is a research method used to systematically analyse and interpret the content of textual, visual, or audio data. The content analysis includes reading and re-reading of responses, developing a process of coding, categorising and conceptualising the responses . Content analysis was performed by the MD (MD) and DAB (MD) by following the steps below: Merged open-ended responses from the expert panel Researchers independently reviewed data and generated code recommendations The researchers compared their code lists, discussed the similarities and differences, and finalised the code list Researchers independently coded the answers of the first five participants. They then discussed the codes and extracts together and reached a consensus on the coding Researchers independently coded the data Researchers discussed the cods and extracts together The researchers wrote the statements together based on the code extracts The similarities in the statements were evaluated and 119 statements were created under three headings so that no view was left out. In line with the results of the content analysis, the second-round questionnaire was prepared. Mean, standard deviation and percentage were calculated for each statement. In addition, the total percentage of those who scored 4 and 5 on a 5-point Likert scale was determined. The consensus level was defined as 85% (those who scored 4 and 5), and statements above this level were accepted as the statements agreed in the second round . Adjustments were made in the statements in line with the expert suggestions. Descriptive statistical analyses were performed for responses to the third-round questionnaire. Statements that exceeded the consensus level with the new data were accepted as the recommendations agreed in the third-round. The statements agreed in the second and third rounds were brought together and accepted as the statements agreed in the three rounds. Statements that were below the level of consensus were added to . One hundred and five experts were invited to the study. Fifty-eight (55.2%) experts agreed to participate in the study and answered the first round questionnaire. Forty-six experts (79.3%) participated in the second round and 41 experts (70.7%) completed the third round . As a result of the content analysis of the first round data, 119 statements were defined: 44 for HA competencies, 49 for teaching and assessment methods and 26 for the integration of HA training into FMRT. As a result of the analysis of the second round answers, 74 common statements (above the consensus level) were determined in total: 38 (86.4%) for HA competencies, 18 (36.7%) for teaching and assessment methods, and 18 (69.2%) for integration of HA training. Experts were also asked to write down their suggestions for the propositions presented in the second round. Some statements were revised in response to the suggestions. In the third round 45 statements that were below the consensus level were again presented to the experts’ opinion. As a result of the analysis, 7 statements (3 for teaching methods, 2 for assessment methods and 2 for integration of HA training) total exceeded the consensus level and a consensus was reached on 38 statements for HA competencies, 23 for teaching and assessment methods, 20 for integration of HA training . Main findings In this study, it was observed that the experts approached the HA competencies from a broad perspective. Providing patient-centred and community-focused care; prioritising health protection and promotion; effective communication and acting by ethical principles are seen as competencies related to or forming the basis of HA. HA competencies agreed in the study include ‘commonalities’ with other roles of FP, especially ‘FM expert’ (competencies 23, 28, 30, 33), ‘professional’ (competencies 6, 11, 12, 21, 24, 26), and ‘leader’ roles (competencies 8, 27, 29, 34). In the study, the approach to HA predominantly emphasises the ‘agency role’, such as directing patients to receive health services and protecting them from unnecessary examinations and procedures. In the study, a rich spectrum of teaching methods has been proposed to gain HA competencies in FM residency training. In particular, role modelling, work-based (bedside, supervision, one-to-one training, etc.), community-based, small groups practices (case-based discussion, video watching and discussion, reflection session) and simulation-based teaching methods have been suggested. Teaching methods are aligned with the recommended competencies. The assessment methods recommended included formative and summative components. Summative methods are suggested as written (case-based, clinical reasoning, written exam), simulated (OSCE) and on-the-job assessment (mini clinical exam). The findings indicate that the panel experts emphasise that assessment of knowledge acquisition, as well as performance, is considered valuable. In the study, recommendations for integrating HA training mainly included program development processes and the provision of purposeful learning-teaching opportunities. The recommendation agreed upon by all panellists is to train FM trainers on HA. In the study, it is also suggested that integration should be done through inclusion in the national FM core curriculum with the leadership of the Turkish Association of Family Physicians and studies to reveal the need for training (evaluation of graduates’ HA competencies and practices, discussion with stakeholders, examination of program examples). Comparison with the literature The HA competencies agreed in the study are in line with the literature. In traditional medical care, the physician’s role is perceived as problem solving a biomedical problem on its own rather than addressing it in partnership . HA is commonly conceptualised as an essential components of ‘good care’ . In this study, competencies related to ‘patient-centred care’ and ‘being good physician’ were emphasised for HA. The role of HA includes ‘agency’ and ‘activist’ sub-roles. The agentic physician acts on behalf of the patient to secure access to resources, institutions, and supports, while the activist physician attempts to change certain practices or policies for a group or community . In the study, activism has been limited to a general expression such as defining and meeting the health needs of the community served. Perceiving the activist sub-role as a disruptive and risky political activity for compelling reasons, such as the required certain degree of power and privilege, fear of burnout, perceived institutional risks and professional boundaries may affect HA practices and result in inexperience/avoidance. In the study, ‘agency role’ was predominant in the approach to the HA role. The experiences and perceptions of the Delphi experts may have been influential in this finding. However, further research is needed to evaluate the possible effects of the existing literature findings on the activist role. The study proposes a wide range of teaching methods (role modelling, work based, community-based learning etc.) to develop HA competencies. Various pedagogical approaches have been proposed and piloted in HA training, including positive role modelling, reflective learning, transformative learning, experiential learning, interprofessional learning, research-based health activism, partnership with community organisations and service-based learning . The findings of the study are in line with the literature. The Royal College of Physicians and Surgeons of Canada has published six preferred tools for assessing HA in residency training: essays, short-answer questions, direct observation and In-Training Evaluation Report (ITER), objective structured clinical assessment (OSCE), multi-source feedback, peer assessment and portfolio . Written exercises, assessments by clinical trainers, clinical simulations, multi-source feedback , formal assessments with MCQ and OSCE and self-assessment (such as log books, portfolios and self-reflection) are used to assess HA. The lack of clarity regarding the HA role, ‘commonalities’ with other roles, and the tendency for advocacy actions to take place away from the bedside are factors that make assessment difficult . In the study, it is recommended that HA education should be integrated into the national core curriculum under the leadership of the Turkish Association of Family Physicians and trainers of FMRT should be trained on HA. This finding is in line with literature findings that many medical educators lack the social sciences and humanities background necessary to support the learning and practice of HA ; and do not have much insight into how to teach and assess the role of the health advocate; and report faculty expertise as a barrier in education . McDonald et al.'s systematic review of HA training found that (i) the concept of HA is shifting towards doing ‘with’ rather than ‘for’ patients, populations and communities; (ii) longitudinal curricula are less common, but most promising, often linked to academic or policy goals; (iii) hands-on, immersive opportunities build competence and confidence; (iv) community-identified needs and partnerships are increasingly taken into account when designing curricula; and (v) resident-led and motivated programs engage residents and achieve targeted outcomes. In the literature, ‘longitudinal’ , ‘interprofessional’ , ‘collaborative’ and ‘sliding scale’ program models were reported. Addressing the determinants of health and advocating to improve them requires FPs to work with their patients, community or society; to engage and collaborate with other stakeholders within the health system; and to understand the dynamics of relationships . However, university partnerships with communities and stakeholders and institutional advocacy activities and support were not emphasised in the study. Limitations and strengths The study is valuable in terms of revealing the views of the experts on HA training for FPs in Turkey. However, the study has some limitations. Although there is no consensus in the literature on the definition and number of ‘experts’ in the Delphi process, it has been reported that more than 30 experts seldom improve the results . To increase the validity and reliability of our study , it aimed to ensure heterogeneity by reaching competent FPs and FM academics from different institutions as much as possible. Experts were purposively selected, but the participation of FPs working in the field remained at a lower level. The low participation and their perspective on HA training can be considered as an important finding in terms of experts’ educational background and HA experiences. In order to increase the validity of the study, the first round questionnaire was developed based on literature and the opinions of the field experts and piloted. Delphi panellists participated in the study voluntarily. The panellists were informed about the purpose and process of the research in the invitation letter and an informed consent form was presented. In all stages of Delphi, the identity of the experts was only made known to the researchers to allow them to participate in subsequent rounds. It was ensured that the panellists did not know each other’s identity information and opinions. In each round, the panellists were informed about the process of the research and the next step/s. In this study, it was observed that the experts approached the HA competencies from a broad perspective. Providing patient-centred and community-focused care; prioritising health protection and promotion; effective communication and acting by ethical principles are seen as competencies related to or forming the basis of HA. HA competencies agreed in the study include ‘commonalities’ with other roles of FP, especially ‘FM expert’ (competencies 23, 28, 30, 33), ‘professional’ (competencies 6, 11, 12, 21, 24, 26), and ‘leader’ roles (competencies 8, 27, 29, 34). In the study, the approach to HA predominantly emphasises the ‘agency role’, such as directing patients to receive health services and protecting them from unnecessary examinations and procedures. In the study, a rich spectrum of teaching methods has been proposed to gain HA competencies in FM residency training. In particular, role modelling, work-based (bedside, supervision, one-to-one training, etc.), community-based, small groups practices (case-based discussion, video watching and discussion, reflection session) and simulation-based teaching methods have been suggested. Teaching methods are aligned with the recommended competencies. The assessment methods recommended included formative and summative components. Summative methods are suggested as written (case-based, clinical reasoning, written exam), simulated (OSCE) and on-the-job assessment (mini clinical exam). The findings indicate that the panel experts emphasise that assessment of knowledge acquisition, as well as performance, is considered valuable. In the study, recommendations for integrating HA training mainly included program development processes and the provision of purposeful learning-teaching opportunities. The recommendation agreed upon by all panellists is to train FM trainers on HA. In the study, it is also suggested that integration should be done through inclusion in the national FM core curriculum with the leadership of the Turkish Association of Family Physicians and studies to reveal the need for training (evaluation of graduates’ HA competencies and practices, discussion with stakeholders, examination of program examples). The HA competencies agreed in the study are in line with the literature. In traditional medical care, the physician’s role is perceived as problem solving a biomedical problem on its own rather than addressing it in partnership . HA is commonly conceptualised as an essential components of ‘good care’ . In this study, competencies related to ‘patient-centred care’ and ‘being good physician’ were emphasised for HA. The role of HA includes ‘agency’ and ‘activist’ sub-roles. The agentic physician acts on behalf of the patient to secure access to resources, institutions, and supports, while the activist physician attempts to change certain practices or policies for a group or community . In the study, activism has been limited to a general expression such as defining and meeting the health needs of the community served. Perceiving the activist sub-role as a disruptive and risky political activity for compelling reasons, such as the required certain degree of power and privilege, fear of burnout, perceived institutional risks and professional boundaries may affect HA practices and result in inexperience/avoidance. In the study, ‘agency role’ was predominant in the approach to the HA role. The experiences and perceptions of the Delphi experts may have been influential in this finding. However, further research is needed to evaluate the possible effects of the existing literature findings on the activist role. The study proposes a wide range of teaching methods (role modelling, work based, community-based learning etc.) to develop HA competencies. Various pedagogical approaches have been proposed and piloted in HA training, including positive role modelling, reflective learning, transformative learning, experiential learning, interprofessional learning, research-based health activism, partnership with community organisations and service-based learning . The findings of the study are in line with the literature. The Royal College of Physicians and Surgeons of Canada has published six preferred tools for assessing HA in residency training: essays, short-answer questions, direct observation and In-Training Evaluation Report (ITER), objective structured clinical assessment (OSCE), multi-source feedback, peer assessment and portfolio . Written exercises, assessments by clinical trainers, clinical simulations, multi-source feedback , formal assessments with MCQ and OSCE and self-assessment (such as log books, portfolios and self-reflection) are used to assess HA. The lack of clarity regarding the HA role, ‘commonalities’ with other roles, and the tendency for advocacy actions to take place away from the bedside are factors that make assessment difficult . In the study, it is recommended that HA education should be integrated into the national core curriculum under the leadership of the Turkish Association of Family Physicians and trainers of FMRT should be trained on HA. This finding is in line with literature findings that many medical educators lack the social sciences and humanities background necessary to support the learning and practice of HA ; and do not have much insight into how to teach and assess the role of the health advocate; and report faculty expertise as a barrier in education . McDonald et al.'s systematic review of HA training found that (i) the concept of HA is shifting towards doing ‘with’ rather than ‘for’ patients, populations and communities; (ii) longitudinal curricula are less common, but most promising, often linked to academic or policy goals; (iii) hands-on, immersive opportunities build competence and confidence; (iv) community-identified needs and partnerships are increasingly taken into account when designing curricula; and (v) resident-led and motivated programs engage residents and achieve targeted outcomes. In the literature, ‘longitudinal’ , ‘interprofessional’ , ‘collaborative’ and ‘sliding scale’ program models were reported. Addressing the determinants of health and advocating to improve them requires FPs to work with their patients, community or society; to engage and collaborate with other stakeholders within the health system; and to understand the dynamics of relationships . However, university partnerships with communities and stakeholders and institutional advocacy activities and support were not emphasised in the study. The study is valuable in terms of revealing the views of the experts on HA training for FPs in Turkey. However, the study has some limitations. Although there is no consensus in the literature on the definition and number of ‘experts’ in the Delphi process, it has been reported that more than 30 experts seldom improve the results . To increase the validity and reliability of our study , it aimed to ensure heterogeneity by reaching competent FPs and FM academics from different institutions as much as possible. Experts were purposively selected, but the participation of FPs working in the field remained at a lower level. The low participation and their perspective on HA training can be considered as an important finding in terms of experts’ educational background and HA experiences. In order to increase the validity of the study, the first round questionnaire was developed based on literature and the opinions of the field experts and piloted. Delphi panellists participated in the study voluntarily. The panellists were informed about the purpose and process of the research in the invitation letter and an informed consent form was presented. In all stages of Delphi, the identity of the experts was only made known to the researchers to allow them to participate in subsequent rounds. It was ensured that the panellists did not know each other’s identity information and opinions. In each round, the panellists were informed about the process of the research and the next step/s. In line with the findings of the study, it is recommended that qualitative studies be conducted to investigate how the related parties (FM residents, patients, faculty administrators, public health specialists, health politicians, etc.) perceive HA, factors underlying their perceptions, their experiences and expectations. The structure and functioning of the national health system and the roles of FPs within these systems should also be evaluated. The study findings revealed that the competencies expected for the HA role of the FP are very broad in perspective and show commonalities with the FP's predominantly ‘professional’, ‘FM expert’ and ‘leader’ roles. HA competencies are more focused on patient-centred care and individual-level advocacy. The proposed teaching and assessment approaches are consistent with the competencies and show diversity. It was seen as important to longitudinally integrate HA training into the FMRT core curriculum at the national level through participatory processes and to train FM trainers on HA. Ethical approval was obtained from Hacettepe University Faculty of Medicine Non-Interventional Clinical Research Ethics Committee for the conduct of the study with the code 2021/18-02 dated. Supplemental Material |
The relationship between parental health literacy and primary school students’ anthropometric measurements and general health status | 69ba065f-b231-4fdd-b5a2-9e5d1cb2d45a | 11844121 | Health Literacy[mh] | Health literacy (HL) is a concept that refers to the ability of individuals to protect and improve their health and prevent disease by effectively to effectively accessing, understanding, evaluate, and applying health information in daily life . This competency includes the critical evaluation of health-related information, the optimal use of health services, adoption of healthy lifestyles, and making important health-related decisions . The level of HL of individuals has a significant impact on their health status . Low levels of HL often limit their ability to make healthy decisions and effectively manage their health behaviors. This can lead to risky health behaviors, unhealthy choices, and increased hospitalizations . On the other hand, high HL offers benefits such as greater life satisfaction and more effective use of health services, as well as reduced health expenditures . Parents’ HL encompasses their knowledge and skills in supporting and promoting their children’s health while preventing potential health risks. Parents with adequate HL contribute significantly to the healthy development of their children . They are able to identify potential health problems early and ensure that their children receive timely and appropriate treatment . Research has shown several negative health outcomes have been observed in children of parents with low health literacy, including poor eating habits, increased risk of obesity, chronic kidney disease, diabetes, and oral health problems . Therefore, the adoption of informed and knowledge-based approaches by parents can have a positive impact on the overall health of their children . Anthropometric measurements are widely used to assess children’s physical development. These measurements are based on parameters such as height, weight, and body mass index (BMI) . The use of anthropometric measures plays an important role in understanding children’s health and guiding health services. By analyzing the relationships between child health and related health conditions through these measurements allows for more effective planning and delivery of health services . HL encompasses factors such as parents’ knowledge about health, ability to access health services, awareness of healthy lifestyles, and ability to implement behaviors in this direction. This study is unique in its detailed examination the relationship between parental HL and anthropometric measures and health outcomes in primary school children aged 6–10 years. The findings discussed in this article will enhance our understanding of the impact of HL on children’s health by offering a valuable perspective. In this context, this article was conducted to reveal the relationship between the level of HL of parents and anthropometric measurements and general health status of primary school students. Research questions What are parents’ health literacy levels? What are the factors affecting the health literacy levels of parents? Is there a relationship between the health literacy levels of parents have an effect on children’s anthropometric measurements? What are parents’ health literacy levels? What are the factors affecting the health literacy levels of parents? Is there a relationship between the health literacy levels of parents have an effect on children’s anthropometric measurements? Research design and sample This descriptive and correlational study was conducted between 15 February and 31 June 2023 in two primary schools in the Fethiye district, employing a simple random sampling method. The population of the study consists of a total of 1753 people consisting of parents of 1st-4th grade students in Fethiye. Using G-Power software program, it was calculated that the sample size required to reflect the results with 5% sampling error and 80% power at 95% confidence interval was 568 . Considering the possible problems that may occur during the data collection process (for example, participants leaving the study, providing incomplete information or not being able to reach the participants), the following formula was applied to compensate for the losses while planning the sample size: 𝑁 required = 𝑁 calculated × (1 + 0.20) . As a result of the initial power analysis, the required sample size was calculated as 568, but after a 20% increase, a total of 681 children and parents were planned to be included in the study sample. The study was successfully completed with 681 children and their parents as planned. Ethical considerations This study was conducted with ethical approval (230002/28) from Mugla Sitki Kocman University Health Sciences Ethics Committee. Permission to conduct the study was obtained from the competent authority. Written permission to use the scales used was obtained from the respective authors. Parents who participated in the study were informed in detail about the aims and objectives of the study and their written consent was obtained, and ethical principles were followed. Data collection tools Socio-demographic Data Form, School Child Follow-Up Form, and European Health Literacy Scale Short Form (EHSLSI-TRQ16) were used as data collection tools in the study. Socio-demographic data form The questionnaire consisted of 21 questions including demographic characteristics (age, gender, marital status, education level), general health status, employment and economic status, child growth and development, anthropometric measurements (weight and height), oral and dental health, hearing, and vision . School child follow-up form The school child follow-up form was developed based on the literature and consists of 21 questions. The form focuses on the health status of children and includes topics such as disability status (e.g., visual or hearing impairment), chronic diseases (e.g., musculoskeletal, or gastrointestinal problems), oral and dental health problems, frequent infections, immunization status, and growth and development status . European health literacy scale short form (EHSL-TRQ16) The Turkish Health Literacy Scale was developed by Emiral et al. in 2018 as a Turkish adaptation of the European Health Literacy Scale and validity and reliability studies were conducted in Turkey . This scale was designed to assess individuals’ knowledge and skills in using health services, disease prevention, and health promotion. The reliability of the 16-question, five-point Likert scale was found to be high with a Cronbach’s alpha coefficient of 0.89. The questions are scored from 1 (“very easy”) to 4 (“very difficult”) and the number 5 is used for the answer “I don’t know”. The collected scores are evaluated between 0 and 50, and those with a score of 33 or more are considered to have adequate HL . Data collection process The requisite approval documentation pertaining to the research process, participant information and consent forms, and questionnaire forms were delivered to the administrative units of the schools in which the study was to be conducted. With the support of the school administrations, these forms were delivered to the parents and subsequently collected from those who had agreed to participate in the study at the conclusion of one week. In accordance with the written consent obtained from the parents, anthropometric measurements of the children were carried out in an office provided by the school administration. The researchers measured the weight and height of the children using a weight scale and a height meter provided by the participants. During the measurement process, each student was invited in turn, and confidentiality and hygiene standards were meticulously observed. The measurement of a child’s height and weight took approximately 5–8 min, while the time for parents to fill in the data collection form was approximately 10 min. The data obtained were then evaluated in accordance with the age-appropriate anthropometric measurements of Turkish children and categorized by calculating standard deviation scores (SDS) for height, weight and body mass index (BMI) . The SDS calculations according to age were assessed as follows : SDS for weight by age : <-3 and − 2 underweight; > 3 overweight; 0, 1, 2 and <-1 normal. SDS for height by age : <-3 dwarf; <-2 short; >3 tall; 0, 1, 2 and <-1 normal. SDS for body mass index by age : >3 obese; >2 overweight; <-2 and − 3 underweight; 0, 1 and <-1 normal. Data analysis The data obtained in the research were analyzed using the Statistical Package for the Social Sciences (SPSS 22) program. In addition to frequencies and percentages, data analysis included chi-squared test and binary regression analysis to examine the relationship between participants’ HL and parents’ sociodemographic characteristics and children’s anthropometric measurements. In statistical analyses, p < 0.05 was accepted as the level of significance. This descriptive and correlational study was conducted between 15 February and 31 June 2023 in two primary schools in the Fethiye district, employing a simple random sampling method. The population of the study consists of a total of 1753 people consisting of parents of 1st-4th grade students in Fethiye. Using G-Power software program, it was calculated that the sample size required to reflect the results with 5% sampling error and 80% power at 95% confidence interval was 568 . Considering the possible problems that may occur during the data collection process (for example, participants leaving the study, providing incomplete information or not being able to reach the participants), the following formula was applied to compensate for the losses while planning the sample size: 𝑁 required = 𝑁 calculated × (1 + 0.20) . As a result of the initial power analysis, the required sample size was calculated as 568, but after a 20% increase, a total of 681 children and parents were planned to be included in the study sample. The study was successfully completed with 681 children and their parents as planned. This study was conducted with ethical approval (230002/28) from Mugla Sitki Kocman University Health Sciences Ethics Committee. Permission to conduct the study was obtained from the competent authority. Written permission to use the scales used was obtained from the respective authors. Parents who participated in the study were informed in detail about the aims and objectives of the study and their written consent was obtained, and ethical principles were followed. Socio-demographic Data Form, School Child Follow-Up Form, and European Health Literacy Scale Short Form (EHSLSI-TRQ16) were used as data collection tools in the study. The questionnaire consisted of 21 questions including demographic characteristics (age, gender, marital status, education level), general health status, employment and economic status, child growth and development, anthropometric measurements (weight and height), oral and dental health, hearing, and vision . The school child follow-up form was developed based on the literature and consists of 21 questions. The form focuses on the health status of children and includes topics such as disability status (e.g., visual or hearing impairment), chronic diseases (e.g., musculoskeletal, or gastrointestinal problems), oral and dental health problems, frequent infections, immunization status, and growth and development status . The Turkish Health Literacy Scale was developed by Emiral et al. in 2018 as a Turkish adaptation of the European Health Literacy Scale and validity and reliability studies were conducted in Turkey . This scale was designed to assess individuals’ knowledge and skills in using health services, disease prevention, and health promotion. The reliability of the 16-question, five-point Likert scale was found to be high with a Cronbach’s alpha coefficient of 0.89. The questions are scored from 1 (“very easy”) to 4 (“very difficult”) and the number 5 is used for the answer “I don’t know”. The collected scores are evaluated between 0 and 50, and those with a score of 33 or more are considered to have adequate HL . The requisite approval documentation pertaining to the research process, participant information and consent forms, and questionnaire forms were delivered to the administrative units of the schools in which the study was to be conducted. With the support of the school administrations, these forms were delivered to the parents and subsequently collected from those who had agreed to participate in the study at the conclusion of one week. In accordance with the written consent obtained from the parents, anthropometric measurements of the children were carried out in an office provided by the school administration. The researchers measured the weight and height of the children using a weight scale and a height meter provided by the participants. During the measurement process, each student was invited in turn, and confidentiality and hygiene standards were meticulously observed. The measurement of a child’s height and weight took approximately 5–8 min, while the time for parents to fill in the data collection form was approximately 10 min. The data obtained were then evaluated in accordance with the age-appropriate anthropometric measurements of Turkish children and categorized by calculating standard deviation scores (SDS) for height, weight and body mass index (BMI) . The SDS calculations according to age were assessed as follows : SDS for weight by age : <-3 and − 2 underweight; > 3 overweight; 0, 1, 2 and <-1 normal. SDS for height by age : <-3 dwarf; <-2 short; >3 tall; 0, 1, 2 and <-1 normal. SDS for body mass index by age : >3 obese; >2 overweight; <-2 and − 3 underweight; 0, 1 and <-1 normal. The data obtained in the research were analyzed using the Statistical Package for the Social Sciences (SPSS 22) program. In addition to frequencies and percentages, data analysis included chi-squared test and binary regression analysis to examine the relationship between participants’ HL and parents’ sociodemographic characteristics and children’s anthropometric measurements. In statistical analyses, p < 0.05 was accepted as the level of significance. Descriptive results 75.2% of the parents were female and the mean age was 37.40 ± 5.89 years. 89.1% of the parents were married and 34.7% were high school graduates. 56.1% of the parents were employed and 64.6% of them had income equal to expenses. About half of the parents (50.7%) had 2 children. While 49% of the parents rated their own general health as good, 47.3% of them rated their children’s health as good. 59.6% of the parents preferred the Family Health Center when they first applied for health services. In addition, 88.7% of them follow health news (Table ). Factors affecting health literacy of parental Single parents demonstrated significantly higher health literacy (HL) levels ( p = 0.012) (Table ). Additionally, education level was significantly associated with HL ( p = 0.001). University graduates had higher HL levels compared to both middle school ( p = 0.004) and primary school graduates ( p < 0.000). Similarly, high school graduates exhibited significantly higher HL levels than primary school graduates ( p = 0.001) (Table ). A statistically significant relationship was observed between economic status and health literacy (HL) levels ( p = 0.033) (Table ), but pairwise comparisons revealed no significant differences ( p > 0.05). Conversely, a strong association was found between parents’ general health assessment and HL levels ( p < 0.000) (Table ). Pairwise comparisons showed that parents who rated their general health as “quite good” had significantly higher HL levels than those rating their health as “good” ( p = 0.001) or “not bad” ( p < 0.001). Similarly, those who rated their health as “excellent” or “good” ( p < 0.000) had higher HL levels than those rating their health as “not bad” ( p < 0.000). Parents whose first visit to a health facility was to a hospital had significantly higher health literacy (HL) levels compared to those who first visited a family health center ( p = 0.011) (Table ). Parental health literacy and children’s health outcomes A significant relationship was found between parents’ perceptions of their children’s general health and their health literacy (HL) levels ( p < 0.001) (Table ). Pairwise comparisons revealed that parents who rated their children’s health as “excellent” had significantly higher HL levels than those who rated it as “good” ( p < 0.001) or “fair” ( p < 0.001). Additionally, parents who rated their children’s health as “fair” demonstrated significantly higher HL levels compared to those who rated it as “not bad” ( p < 0.000) (Table ). The HL level of children without oral health problems was higher and statistically significant ( p < 0.004) (Table ). In addition, the HL level of children without dental caries was also higher and statistically significant ( p < 0.009) (Table ). The analysis of the factors associated with the determinants of HL levels in the study is presented in Table . As a result of the analysis, it was found that men (OR: 1.76, 95% CI: 1.050–2.953), single people (OR: 2.47, 95% CI: 1.209–5.055), high school graduates (OR: 2.04, 95% CI: 1.210–3.470), university graduates (OR: 2.66, 95% CI: 1.492–4.762), those who reported that their parents’ general health status was quite good (OR: 2. 33, 95% CI: 1.211–4.514), those who reported their children’s general health status as good (OR: 1.96, 95% CI: 1.034–3.730) and excellent (OR: 5.79, 95% CI: 2.020- 16.601), and parents who had been hospitalized (OR: 1.60, 95% CI: 1.089–2.353) were found to have adequate HL(Table ). 75.2% of the parents were female and the mean age was 37.40 ± 5.89 years. 89.1% of the parents were married and 34.7% were high school graduates. 56.1% of the parents were employed and 64.6% of them had income equal to expenses. About half of the parents (50.7%) had 2 children. While 49% of the parents rated their own general health as good, 47.3% of them rated their children’s health as good. 59.6% of the parents preferred the Family Health Center when they first applied for health services. In addition, 88.7% of them follow health news (Table ). Single parents demonstrated significantly higher health literacy (HL) levels ( p = 0.012) (Table ). Additionally, education level was significantly associated with HL ( p = 0.001). University graduates had higher HL levels compared to both middle school ( p = 0.004) and primary school graduates ( p < 0.000). Similarly, high school graduates exhibited significantly higher HL levels than primary school graduates ( p = 0.001) (Table ). A statistically significant relationship was observed between economic status and health literacy (HL) levels ( p = 0.033) (Table ), but pairwise comparisons revealed no significant differences ( p > 0.05). Conversely, a strong association was found between parents’ general health assessment and HL levels ( p < 0.000) (Table ). Pairwise comparisons showed that parents who rated their general health as “quite good” had significantly higher HL levels than those rating their health as “good” ( p = 0.001) or “not bad” ( p < 0.001). Similarly, those who rated their health as “excellent” or “good” ( p < 0.000) had higher HL levels than those rating their health as “not bad” ( p < 0.000). Parents whose first visit to a health facility was to a hospital had significantly higher health literacy (HL) levels compared to those who first visited a family health center ( p = 0.011) (Table ). A significant relationship was found between parents’ perceptions of their children’s general health and their health literacy (HL) levels ( p < 0.001) (Table ). Pairwise comparisons revealed that parents who rated their children’s health as “excellent” had significantly higher HL levels than those who rated it as “good” ( p < 0.001) or “fair” ( p < 0.001). Additionally, parents who rated their children’s health as “fair” demonstrated significantly higher HL levels compared to those who rated it as “not bad” ( p < 0.000) (Table ). The HL level of children without oral health problems was higher and statistically significant ( p < 0.004) (Table ). In addition, the HL level of children without dental caries was also higher and statistically significant ( p < 0.009) (Table ). The analysis of the factors associated with the determinants of HL levels in the study is presented in Table . As a result of the analysis, it was found that men (OR: 1.76, 95% CI: 1.050–2.953), single people (OR: 2.47, 95% CI: 1.209–5.055), high school graduates (OR: 2.04, 95% CI: 1.210–3.470), university graduates (OR: 2.66, 95% CI: 1.492–4.762), those who reported that their parents’ general health status was quite good (OR: 2. 33, 95% CI: 1.211–4.514), those who reported their children’s general health status as good (OR: 1.96, 95% CI: 1.034–3.730) and excellent (OR: 5.79, 95% CI: 2.020- 16.601), and parents who had been hospitalized (OR: 1.60, 95% CI: 1.089–2.353) were found to have adequate HL(Table ). In this study, which revealed the relationship between parents’ HL and anthropometric measurements and general health status of primary school students, it was found that 3/4 of parents’ HL was at an adequate level. This rate was higher compared to previous studies on similar topics in Europe and Turkey . Among the possible reasons for the high level of HL in this study, factors such as the high rate of schooling in the area where the study was conducted and the age range of the participating parents, who were generally between 30 and 40 years old, may have played a role. This study shows that men have higher levels of HL than women. This finding contradicts studies in the literature , which generally show that women have higher HL levels than men. In this context, considering gender differences in HL and designing education and information programs in a gender-specific manner can help develop more effective approaches by addressing these differences. This study shows that single parents have higher levels of HL than married parents. Therefore, this study differs from other studies that found that there is no significant difference between marital status and health literacy. In addition, there are also studies that found that married individuals have higher levels of HL . In this context, certain social or regional factors may influence the relationship between marital status and health literacy. This study demonstrates that parents with higher economic status tend to exhibit higher levels of health literacy. These findings align with previous research , which has similarly indicated that individuals with greater economic prosperity generally possess higher levels of health literacy. Economic well-being likely exerts a positive influence on health literacy, as improved financial status enables individuals to access health-related information more effectively and evaluate it with greater ease. In this study, the level of HL increased as the level of parental education increased. Another study also found that literacy levels increased as the level of education increased . Increasing the level of education contributes to the development of basic reading, comprehension, and communication skills of individuals and strengthens their scientific thinking skills. These developments pave the way for strengthening skills that support better understanding of health-related issues, effective management of health processes, and making appropriate health decisions . In this study, it was found that as the perceived general health level of parents increased, the level of HL also increased. Similarly, it was found that adults with good general health have higher levels of HL . Other studies have found that individuals who rate their general health as poor have inadequate HL and that negative health perceptions may lead to lack of knowledge or misinformation about health issues . These findings highlight that increasing and improving HL is an effective way to improve the overall health status of the community. In this study, the HL of parents who visited hospitals was higher than that of parents who visited family health centers. In another study, contrary to the results of this study, the HL of individuals who applied to family health centers was higher . Another study found that there was no significant difference between the first health facility visited and HL . It is believed that this inconsistency between health institutions as a reason for the lack of difference may be related to the variability of the time allocated to patients in the provision of health services and whether informational activities are carried out to improve HL skills. In this study, the HL of parents with children without oral and dental health problems was higher than that of parents with children with problems. These findings are consistent with the literature . The level of knowledge and education of families has a significant impact on children’s diet and tooth brushing habits. This suggests that families’ level of HL has a positive effect on children’s oral and dental health and may contribute to the acquisition of healthy habits. Top of Form. Strengths and limitations The results show that the study represents a specific sample of students and parents. This sample does not include all primary school students and their parents, which limits the generalizability of the results obtained. In addition, this study represents one of the few studies in the national and international literature that has examined the relationship between the level of HL of parents and the anthropometric measurements and general health status of primary school students aged 6–10 years. In this context, the ability of the study to reach a large sample group stands out as one of the strengths of the study. The results show that the study represents a specific sample of students and parents. This sample does not include all primary school students and their parents, which limits the generalizability of the results obtained. In addition, this study represents one of the few studies in the national and international literature that has examined the relationship between the level of HL of parents and the anthropometric measurements and general health status of primary school students aged 6–10 years. In this context, the ability of the study to reach a large sample group stands out as one of the strengths of the study. This study examined the relationship between parental HL and anthropometric measures and general health status in primary school children. The results provide an important perspective on the impact of parents’ HL on children’s health status. Demographic and socioeconomic factors such as gender, marital status, economic status, educational level, and general health perception were observed to have an impact on HL levels. It was observed that single parents, high school or university graduates, parents who rated their general health status as high, parents who perceived their children’s health status as good or excellent, parents who had been hospitalized, and parents who did not have oral and dental health problems had higher levels of health literacy. In this context, considering the findings, the development and implementation of tailored awareness and education programs for target groups, considering demographic and socioeconomic factors, is recommended as a strategic approach to increase and improve health literacy. Practical implications This study underscores the importance of improving parental health literacy (HL) to enhance both parents’ and children’s health outcomes. While there is limited research specifically addressing parental HL, these findings highlight its significant impact on child health, particularly in areas such as oral hygiene and preventive care. Tailored educational programs targeting parents with lower educational attainment or socioeconomic challenges are essential to bridge gaps in HL. Given the scarcity of studies focusing on parental HL, this research provides valuable insights and emphasizes the need for further exploration to develop effective, evidence-based strategies that improve health literacy and, consequently, the well-being of families. This study underscores the importance of improving parental health literacy (HL) to enhance both parents’ and children’s health outcomes. While there is limited research specifically addressing parental HL, these findings highlight its significant impact on child health, particularly in areas such as oral hygiene and preventive care. Tailored educational programs targeting parents with lower educational attainment or socioeconomic challenges are essential to bridge gaps in HL. Given the scarcity of studies focusing on parental HL, this research provides valuable insights and emphasizes the need for further exploration to develop effective, evidence-based strategies that improve health literacy and, consequently, the well-being of families. |
Understanding characteristics of internal medicine residents matching into pulmonary critical care medicine fellowships | ce4d426c-61d0-43e7-9d84-3194593d6691 | 11577943 | Internal Medicine[mh] | Approximately 6,000 internal medicine (IM) residents apply for a subspecialty fellowship yearly. With 5,000 fellowship positions available, one in six will not match. Residents often develop career interests before residency training, and early exposure to specialty rotations may further impact their career decisions. Alternatively, some residents’ career choices may evolve later in training. IM residents who pursue fellowship training may have higher general medical knowledge than those who do not; however, some competitive subspecialties may require early specialization leading to decreased general medical knowledge. Scholastic accomplishments such as Alpha-Omega-Alpha (AOA) membership, scholarship, and class rank have been shown to predict future performance in training; yet, resident publications may be a poor predictor of fellowship publications. Recently, authors identified relationships between IM residents’ medical knowledge, early career intentions, rotation evaluations, and pre-residency characteristics and matriculation into cardiology fellowships.. Pulmonary and Critical Care Medicine (PCCM) is the second largest IM fellowship in the US with 629 training positions. More than 80% of intensivists are IM-trained. This fellowship has become competitive with a 24.8% unmatched rate between 2004 and 2019. As the US population ages, projections suggest a shortage of critical care providers in the US. Trainees pursing PCCM must develop confidence leading multidisciplinary teams during stressful situations, demonstrate empathy with distressed patients and family members, and learn psychomotor skills for numerous bedside procedures. Considering the growing importance of work-life balance on IM resident career selection, residents may feel disinclined to pursue careers in PCCM.. No studies have demonstrated how residents matching into PCCM fellowships differ from their peers. Furthermore, understanding unique characteristics of residents who match into PCCM should help mentor residents with interests in PCCM, assist with attracting residents into PCCM, and alert PCCM fellowship program directors regarding features of successful applicants. This study sought to compare IM residents entering PCCM fellowships with other IM residents based on standardized measures of performance, stated career interests on electronic residency application service (ERAS) personal statements, and exposures to PCCM rotations. This study’s methods reflect a modification of a prior study from our group focused on internal medicine residents entering cardiology fellowships. While methodological approaches of the current and prior studies are similar, we have revised the methods section to reflect changes relevant to the current study. This study included residents who matched to the Rochester, Minnesota Mayo Clinic categorical IM residency from 2007 to 2018; thus completing residency between 2010 and 2021. Those who completed residency in under 3 years or left the program were excluded. The primary outcome was matching into PCCM fellowship within a year of completing residency to account for those who completed a chief medical resident year and those who delayed fellowship match for 1 year. Residents who matched into PCCM were compared to all other graduating residents. Performance, career intent, and PCCM exposure were compared with pre-residency variables, characteristics of PCCM experiences, and global performance markers during residency. Only information available by the time of fellowship applications was included. After 2011, the fellowship match moved from June of PGY2 year to December of PGY3 year. As such, residents entering between 2007–2009 had data through the first half of PGY2; residents starting after 2009 had data included through the end of PGY2. Pre-residency variables included declaration of PCCM intent, United States Medical Licensing Examination (USMLE) Step 1 and Step 2 Clinical Knowledge scores, pre-residency PubMed® publications (noting first author), AOA membership, and US News & World Report (USNWR) medical school research ranking. Study team members (MC, TB, DK) reviewed personal statements for declaration of a PCCM subspecialty intent. Data on resident PCCM experiences included previously validated faculty assessments for required PCCM rotations. We differentiated the timing of a resident’s first PCCM experience into the first versus second half of the PGY1 year, and assessed the choice to complete an elective pulmonary rotation. Similar to other evaluations, PCCM evaluation scores were deemed “highly professional” if in the top 20th percentile of their class. [8, 20] The percentile score on PCCM-specific content areas of the ITE was included as a measure of PCCM knowledge. Our analysis incorporated data from any PCCM rotations completed by the time of fellowship application for each resident in the study. Residency clinical performance was assessed with rotation evaluations and mini-clinical evaluation exercise (mini-CEX) scores completed prior to fellowship application. Clinical evaluations were considered “highly professional” if in the top 20th percentile of their class. In-training examination (ITE) was included as a marker of medical knowledge. These scored were restricted to the PGY2 year in order to ensure variance inflation factor (VIF) < 3 for all covariates in the multiple logistic regression model. The total number of PubMed®-indexed publications during residency served as a reflection of academic performance. This is the only variable with data across a resident’s entire residency rather than up to the time of fellowship application. Publications were analyzed as discrete variables with odds ratios calculated for zero publications vs. one, two, or greater than or equal to three publications during residency. REDCap (Research Electronic Data Capture) data tools, hosted at Mayo Clinic, was used to collect and manage study data abstracted from residency application materials. This is a secure, web-based application designed to support research study data capture. One author (AJH), who has no evaluative role within the residency, merged data. After merging data by resident, the data was deidentified prior to analysis to protect confidentiality. Independent variables distribution was reported as mean (standard deviation) for continuous variables and n for categorical variables. Relationships between independent variables and the binary primary outcome variable were assessed using logistic regression models. We visually examined functional form for continuous-valued covariates using Loess plots and objectively by Hosmer & Lemeshow goodness-of-fit tests, with those deviating from the assumption of linearity in the logit categorized by logical breakpoints. Potential multicollinearity among covariates was assessed using the VIF, with the highest VIF-valued covariate being excluded and re-assessing until all VIF < 3. A multivariable logistic regression model for the primary outcome adjusted for all modifiable covariates simultaneously. The threshold for statistical significance was set at α = 0.01. All analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC). The Mayo Clinic Institutional Review Board (IRB) and Internal Medicine Residency Education Group (IMREG) approved this study. The IRB and IMREG groups served as the ethics committees that waived the need for informed consent. Over this study period, 550 residents matched into the categorical program. Twenty-eight of them did not complete the program or graduated after only 2 years, leaving 522 residents for inclusion. Much of this population has been previously described. We identified no significant differences in demographic or pre-residency characteristics between those included and excluded from our study (Table ). Summaries of the study variables, for the entire population and the PCCM-matched cohort, are seen in Table . Of the 522 included residents, 54 (10.3%) matched into PCCM. 187 (35.8%) residents had a publication at the time of residency application including 86 (16.5%) with a first author publication. One-hundred and eighty-nine (36.2%) graduated from a top 50 medical school as ranked by USNWR, and 142 (27.2%) were AOA members. Seventeen (3.3%) declared an intent to pursue PCCM in their residency application personal statement. Mean (SD) USMLE scores for Step 1 were 238.7 (15.6) and for Step 2 CK were 250.1 (14.8). Eighty-six (16.5%) of the residents studied completed a pulmonary elective rotation prior to fellowship application. During their residency, 204 (39.1%) authored ≥ 3 publications. The mean percentile (SD) on the PGY-2 ITE was 79.7 (18.3) overall and 62.4 (24.6) for the PCCM portion of the exam. Bivariate logistic regression found a positive association with declaring PCCM in residency personal statements ( p = 0.002); however, it was no longer significant in the multivariable model after accounting for all other modifiable independent variables (OR 4.28 (0.85–21.55); p = 0.02). Multivariable logistic regression found completion of a pulmonary elective rotation was significantly associated with matching into a PCCM fellowship (odds ratio (OR) 7.78, 99% confidence interval (CI) 3.10–19.53, p < 0.0001). PCCM matriculants were more likely to have < 3 publications than 3 + publications (OR 3.51 (1.20–10.25), p = 0.003). We endeavored to understand how residents who match in PCCM fellowship differ from their peers. Completing a pulmonary elective was significantly associated with matching into PCCM. Such PCCM electives are essential for residents advancing their skills in managing PCCM patients, meeting faculty members, and obtaining letters of recommendation. Additionally, it is likely that, in many cases, these elective rotations alter residents’ pre-existing career plans. Matriculants into PCCM in this study were more likely to have < 3 publications than ≥ 3 publications. Our institution trains many residents pursuing cardiology, gastroenterology, or hematology/oncology fellowship training and we believe that these highly competitive groups are more likely to have ≥ 3 publications. Supporting this, a recent similar study at our institution found that twice as many residents matched into cardiology than PCCM and that those pursuing cardiology fellowship had more publications during residency than their peers. In the current study, the average number of publications in the PCCM-matched group was 1.56, indicating that residents choosing PCCM are more focused on clinical skill enhancement than research. Furthermore, PCCM fellowships may place less emphasis on publications when ranking applicants for selection. Adjusted analysis did not reveal significant associations between stated career intent in personal statements with matching into PCCM. Many programs may place lesser emphasis on the personal statement vis-a-vis selection decisions; however, this same association was significant in previous research and deserves further study in the field of PCCM. . Strengths of this study include large sample size over a long timeframe and inclusion of both subjective and objective variables. Limitations include the single institution as a large academic medical center, which may reduce generalizability. That said, the study cohort included diverse residents from over 140 different medical schools. This study incorporated residents who trained before the Covid-19 pandemic, which potentially constrains conclusions about fellowship selection since the pandemic. Finally, this was a purely quantitative study; therefore, future qualitative research should examine reasons why residents choose to enter PCCM and other specialties. To our knowledge, this is the first study on characteristics of residents who match into PCCM fellowship training. This study identified the importance of PCCM elective rotations during residency training on entering PCCM fellowships. While scholarship is an important feature among all fellowship applicants, this study suggests that residents entering PCCM training may place greater emphasis on clinical skills enhancement, and that PCCM program directors may focus more on other qualities, such as clinical experience. These findings should assist in mentoring residents who select careers in PCCM. These results can offer valuable guidance to trainees as they prepare for fellowship applications and prioritize their tasks. By providing general benchmarks, trainees may also be motivated to engage more actively in research by recognizing its differentiating value. Future research should further explore the potential value of personal statements and expressions of career intent, potentially through qualitative inquiry, to better understand the motivation and characteristics of residents who match into PCCM. We found that completion of a pulmonary elective in residency was significantly associated with matching into PCCM. Additionally, PCCM-bound residents were less likely to achieve high numbers of publications suggesting a preference for clinical exposure over scholarship. This data may help provide insight into residents who match in PCCM and aid in mentoring these residents. |
Identifying barriers and opportunities to facilitate the uptake of whole genome sequencing in paediatric haematology and oncology practice | ef2f0566-92d7-465e-8537-0911f87b9bae | 11542304 | Internal Medicine[mh] | Since early 2021, NHS England (NHSE) has commissioned paired whole genome sequencing (WGS) for all patients < 25 years of age with cancer . Whilst some centres around the world offer genome sequencing to children, these efforts are typically single institution projects or confined to those with high-risk tumours . In a model similarly adopted by the Genomic Medicine Sweden Childhood Cancer project , the NHSE programme provides nationwide equitable access to WGS, which represents the single most informative assay for cancer to aid patient care . However, there is a perception that the uptake of WGS for children with cancer has not being accessed routinely, with notable regional variation. WGS is a test that is accessible for all clinicians involved in the care of children with malignancies. After parental consent is obtained, samples are tested via a decentralised network of seven Genomic Laboratory Hubs (GLHs) that serve the 15 paediatric haematology and oncology units (Principal Treatment Centres; PTCs) in the NHS in England. Successful implementation, however, depends on the requisite infrastructure and multiple health care professionals, including paediatric haematologists and oncologists, pathologists, clinical geneticists, clinical scientists, and technical laboratory staff, comprising genetic technologists and biomedical scientists. The reasons for incomplete uptake of WGS for childhood cancer are not fully understood, but likely relate to the complexities around aligning infrastructure needs, capacity building, and clinical workflows across multiple hospital sites. To obtain a more systematic understanding of barriers to offering WGS for children with cancer, and to identify potential solutions, an in-person workshop was convened that brought together key stakeholders from across the United Kingdom (UK; England, Wales, Scotland, Northern Ireland), and the Republic of Ireland (ROI), as well as some international participants. The event was hosted by Wellcome Connecting Science in collaboration with the University of Cambridge Department of Paediatrics, Cambridge University Hospital NHS Foundation Trust, East-GLH, Genomics England, and NHSE. Prior to the workshop, a pre-meeting survey was conducted amongst all delegates that confirmed notable regional variation in uptake of WGS for children with cancer within the GLHs which comprise NHSE, in the devolved nations of Scotland, Wales, and Northern Ireland, and ROI. A key component of the event itself was a structured workshop, used to elucidate current and anticipated challenges and opportunities from end users. Here, we provide a qualitative themed analysis of the main barriers and possible solutions identified by participants, to help ensure that every child with cancer in the UK and ROI may benefit from WGS. Workshop overview This purpose of the one-day workshop, held on 20 October 2022, was to bring together key stakeholders from all four countries of the UK, and ROI. The day was structured with a morning session, comprised of a series of didactic lectures, and the afternoon session where participants were allocated to small groups to work through a structured questionnaire. The groups then convened for a round-table discussion before the day closed. To allow for free communication, the afternoon workshop followed ‘Chatham House Rules’ , namely that neither the identity nor the affiliation of the speakers/participants may be revealed in the dissemination of the findings. The running of this workshop, and collection of data, was approved by the Research Governance Committee (which oversees the Research Ethics Committee) at the Wellcome Sanger Institute, UK. Attendees Using purposive sampling principles, the attendee list was assembled to ensure representation from the PTCs as well as the seven GLHs, coverage of all healthcare professionals involved in the clinical pathway, and input from across the UK and ROI, as well as centres of excellence internationally. Personal invitations were sent by PT, SB, and MJM via email. Invitees who were unable to attend due to scheduling conflicts were asked to identify a suitable alternative. An a priori decision was made to cap the number of attendees to 60. This balanced the need for sufficient representation from key organisations with the practicalities of capturing feedback from a diverse audience. Data collection In the afternoon session, attendees in breakout groups of seven or eight worked through a series of structured questions (see Supplementary Table ). These questions were informed by the COM-B model of behaviour (see Table ) , where capability (C), opportunity (O), and motivation (M) are perceived as the three key factors capable of changing behaviour (B). The COM-B model is widely used to identify factors that need to be considered for any behaviour change intervention to be effective , such as the implementation of WGS into clinical care. The questions that explored capability were focussed on identifying where education and training could facilitate uptake of WGS. That is, they were to identify education and training needs and not intended to be a subjective assessment of workforce competence. Responses to the structured questions were captured by a scribe in each group. During the round-table discussion, field notes were taken by MB to capture key discussion points and verified via an audiotape of the session. The findings presented were de-identified, removing any information around an individual’s clinical profession or place of work, to align with Chatham House Rules. Data analysis The written responses from the groups and the notes from the group discussion were typed and uploaded to NVivo 12, the qualitative data analysis computer software package. The text was analysed through deductive content analysis initially by MB and reviewed by MJM, AV, and PT. The predefined categories were informed by the COM-B model and coded as facilitators or barriers. This purpose of the one-day workshop, held on 20 October 2022, was to bring together key stakeholders from all four countries of the UK, and ROI. The day was structured with a morning session, comprised of a series of didactic lectures, and the afternoon session where participants were allocated to small groups to work through a structured questionnaire. The groups then convened for a round-table discussion before the day closed. To allow for free communication, the afternoon workshop followed ‘Chatham House Rules’ , namely that neither the identity nor the affiliation of the speakers/participants may be revealed in the dissemination of the findings. The running of this workshop, and collection of data, was approved by the Research Governance Committee (which oversees the Research Ethics Committee) at the Wellcome Sanger Institute, UK. Using purposive sampling principles, the attendee list was assembled to ensure representation from the PTCs as well as the seven GLHs, coverage of all healthcare professionals involved in the clinical pathway, and input from across the UK and ROI, as well as centres of excellence internationally. Personal invitations were sent by PT, SB, and MJM via email. Invitees who were unable to attend due to scheduling conflicts were asked to identify a suitable alternative. An a priori decision was made to cap the number of attendees to 60. This balanced the need for sufficient representation from key organisations with the practicalities of capturing feedback from a diverse audience. In the afternoon session, attendees in breakout groups of seven or eight worked through a series of structured questions (see Supplementary Table ). These questions were informed by the COM-B model of behaviour (see Table ) , where capability (C), opportunity (O), and motivation (M) are perceived as the three key factors capable of changing behaviour (B). The COM-B model is widely used to identify factors that need to be considered for any behaviour change intervention to be effective , such as the implementation of WGS into clinical care. The questions that explored capability were focussed on identifying where education and training could facilitate uptake of WGS. That is, they were to identify education and training needs and not intended to be a subjective assessment of workforce competence. Responses to the structured questions were captured by a scribe in each group. During the round-table discussion, field notes were taken by MB to capture key discussion points and verified via an audiotape of the session. The findings presented were de-identified, removing any information around an individual’s clinical profession or place of work, to align with Chatham House Rules. The written responses from the groups and the notes from the group discussion were typed and uploaded to NVivo 12, the qualitative data analysis computer software package. The text was analysed through deductive content analysis initially by MB and reviewed by MJM, AV, and PT. The predefined categories were informed by the COM-B model and coded as facilitators or barriers. Sixty people attended the workshop. Of those that attended, the largest single group ( n = 26) were paediatric oncologists, including those that had an academic appointment, making up 43% of attendees. The remaining 34 (57%) attendees included clinical scientists and bioinformaticians ( n = 11), paediatric pathologists ( n = 8), paediatric haematologists ( n = 5) and clinical geneticists ( n = 5). The other five individuals were from key organisations and/or speakers. While all countries in the UK and ROI were represented, the vast majority (83%) were from England. The morning session included international representation from Hong Kong to provide a case study of successful implementation of WGS in a non-NHS setting. Key education and training targets to support uptake of WGS While all attendees recognised the work of NHSE in providing equity of access to WGS, the group felt the same could not be said about the knowledge base of the workforce. This was felt even at the level of understanding the clinical utility of WGS. Whilst the group recognised many oncologists and haematologists are “ very invested in WGS” this was not universal, with others “not even aware of the existence of WGS”. Attendees acknowledged the disparity in the availability of local expertise. The phrase “lack of knowledge equality” was used to describe the difference in knowledge (and skills) between NHS Trusts, departments and even colleagues within the same department. All attendees believed this inequality would, inevitably, impact the equity of access to WGS. Several education and training needs were identified by the group. This included process knowledge as well as clinical skills. Full details are outlined in Table . A small group of attendees (representing approximately 20% of the audience) challenged the status quo on the current model of workforce development in genomics. There was a suggestion that some clinical tasks, such as taking and recording consent for genomic tests, should require mandatory (or, as stated “ formal ”) training and assessment of competence, which is in place in some NHS Trusts, but is not routine practice. Whilst all attendees agreed that workforce development activities were needed, and wanted by healthcare professionals, about half acknowledged that clinicians may already possess relevant knowledge and skills but lack confidence and clinical experience. This group felt that any education and training activities should also provide clinicians with protected time in which to practise their skills by, for example, through role-play. Throughout the discussion, it became clear to both workshop attendees, and facilitators, that access to education and training differed depending on where healthcare professionals worked, with some GLH/Genomic Medicine Service Alliances (GMSA) and Trusts providing more workforce development opportunities around consent and variant interpretation (amongst other topics) than others ( “we have sessions on this , don’t you?”) . Other barriers impeding implementation and/or uptake of WGS The commissioning of WGS for paediatric malignancies by NHSE was lauded by all attending the workshop. However, for those working in Scotland, Wales, Northern Ireland, and the ROI, funding this service was seen as the priority before anything else could, or should, be considered. For those working in England, the perpetuating theme from both the group work and subsequent discussion was that opportunities to implement WGS at a Trust and/or pathway level have, from the clinician’s perspective, not been built into existing systems/infrastructures. This includes a lack of systematic clinical pathways and sufficient resources - both financial and people. When the question around resources was put to the groups, the consensus view was there was just simply not enough, with the clear message from one group being “No , a resounding no” . The perception is that most of the current activity is happening due to the ‘goodwill’ of a few people. As summed up by one of the groups, the view from those involved with implementing this test is that: “The test is funded but the clinical process is not.” Specific examples included staff capacity across the pathway ( “not enough time for interpretation , not enough scientists full stop”) and properly resourcing Genomic Tumour Advisory Boards (GTABs) “we have established specific GTABs , but clinician time to attend GTABs (is) not funded”) . Not having sufficient time was a recurring theme, with a particular emphasis on the consent process. This was both in how long it took to obtain consent (or was perceived to take) and which, if any, healthcare professionals then have the time to obtain and record consent. If these issues were not addressed, all participants felt this would create an insurmountable barrier to ubiquitous uptake of WGS across the NHS. The oncologists and haematologists were perplexed as to why the consent process for WGS needs to be separate to standard diagnostic tests and, as such, take additional time to complete. For many of these clinicians, their patients are unwell and simultaneously having other tests, procedures, and/or being enrolled for clinical trials - all of which require verbal and/or formal written consent. This means that clinicians and parents are faced with multiple consent processes and forms at a very stressful time for parents/guardians. It was felt that parents/guardians would benefit from simplification of the consent process, or for consent to occur in a staged manner. For example, one group commented it would be easier for the parents to provide generic consent for collection of the germline sample and then have the consent conversation for analysis after treatment has commenced. As they stated, “you can have a much more sensible conversation four weeks into treatment. This way you prioritise consent for tests that will impact first line treatment , rather than do everything at once”. For other attendees, specifically those from a genetic/genomic professional background, the recognition that WGS is the only tumour assay where germline variants are routinely explored justified having separate consent processes, although recognition of overburdening parents at such a stressful time was appreciated. Despite some tension regarding single or multiple consent processes, attendees all agreed that obtaining consent for WGS significantly impacted clinicians’ ability to deliver other aspects of patient care at the critical diagnostic timepoint. A suggested solution was to employ dedicated staff for this role. However, there was caution in how this should be implemented. As stated by one attendee, “There needs to be investment in staffing , not just taking resources from another part of the service.” Time was also seen as a barrier for the interpretation of variants identified by WGS, which had a knock-on effect on the turnaround time (TAT) for reporting. It was recognised that this is most likely a capacity issue within the laboratories. However, some of the clinical scientists in the room reported an additional physical barrier that would also impact TAT; that of not being able to access key academic journals through their institution (“It just takes more time , time to find a colleague who has an academic appointment who has the time to resource the article for you. It just all adds to the delay in returning results”) . This delay in access to critical data/resources that may influence variant interpretation will inevitably have an impact on TAT. There was agreement from all non-clinical genetics attendees that lack of access to specialist genetic clinical services was also a major factor in the ability to universally implement WGS. This ranged from the limited ability of clinical geneticists to provide input into GTABs, to the very long waiting lists for clinical appointments after the detection of a germline variant (“the waiting list can be up to a year”). For some oncologists and haematologists at the workshop, this meant that they feared receiving a result which reported a germline variant, as they knew how long it could be until the parents/guardians were able to access specialist clinical genetics support (“ should we be offering this test if we can’t get appropriate clinical support to deal with the results?”) . The final area that was discussed were processes and systems that would not only operationalise the end-to-end pathway, but also facilitate implementation of WGS. This included: developing user-friendly digital systems that supported the ordering of tests and tracked samples as well as having a record of consent ( “need better information technology to flag missing consent , samples”); defined clinical pathways (“Have proper process maps and clinical pathways so we all know who is responsible for the task”) and national guidance for clinical trials; and inter- and intra-Trust tissue transport logistics that ensured samples arrived at the relevant laboratory in a safe and timely manner (“ in some cases consultants are transporting samples between hospitals” ). There was a strong sense from all of those at the workshop that it will be much easier to onboard clinicians to offer and consent for WGS when all the pathways, systems, and guidelines are embedded in hospital Trusts. Motivation to offer WGS As a group, the attendees at the workshop considered introducing WGS as a positive step and that the implementation of the test will achieve desired outcomes. However, there was a recognition by the oncologists and haematologists in the room that some of their colleagues do not have the same vision. Several theories were suggested as to why this may be this case. These included: A general lack of awareness that WGS is an option. Attendees suggested WGS needs to be included in relevant clinical checklists to remind people of the availability of the test and therefore ensure it is considered (“Suggest including in a checklist or making this part of tumour board discussions for each new patient. Could be included in ward rounds , pre-clinic meetings , pre-ward round meetings”) . Their colleagues’ current level of trust and ‘comfort’ with standard-of-care (SOC) testing – and questioning the benefits of WGS over SOC. This was seen as an issue that needed to be addressed as the tumour samples for this patient cohort are often small. Therefore, it was felt that there needs to be a push to prioritise WGS for testing rather than an optional extra if there was sufficient tissue to test. Finite resources within the NHS. There may be a tension between taking resources from other services to implement WGS, when SOC testing is already being undertaken. A perceived lack of support from Trust management (in some Trusts) which may influence the distribution of resources and provide a subliminal message on the importance of WGS (“Our Trust has not bought into NGS (Next Generation Sequencing) , they have different priorities) . Some of the oncologists felt that there needs to be a shift in mindset as WGS is still seen by many in the NHS “as research & discovery rather than clinical test” . As such, WGS needs to be ‘marketed’ to clinicians as a routine SOC clinical test that is available now. There was also universal recognition that people will be influenced by the views of others within the GTAB, whether they be positive or negative, towards the use of WGS. Therefore, it was felt that there is a need for a widespread campaign illustrating what WGS can offer compared with SOC testing. This could include, for example, the sharing of good practice through knowledge exchange activities like, for example, virtual ‘Grand Rounds’ or national GTABs. While all attendees recognised the work of NHSE in providing equity of access to WGS, the group felt the same could not be said about the knowledge base of the workforce. This was felt even at the level of understanding the clinical utility of WGS. Whilst the group recognised many oncologists and haematologists are “ very invested in WGS” this was not universal, with others “not even aware of the existence of WGS”. Attendees acknowledged the disparity in the availability of local expertise. The phrase “lack of knowledge equality” was used to describe the difference in knowledge (and skills) between NHS Trusts, departments and even colleagues within the same department. All attendees believed this inequality would, inevitably, impact the equity of access to WGS. Several education and training needs were identified by the group. This included process knowledge as well as clinical skills. Full details are outlined in Table . A small group of attendees (representing approximately 20% of the audience) challenged the status quo on the current model of workforce development in genomics. There was a suggestion that some clinical tasks, such as taking and recording consent for genomic tests, should require mandatory (or, as stated “ formal ”) training and assessment of competence, which is in place in some NHS Trusts, but is not routine practice. Whilst all attendees agreed that workforce development activities were needed, and wanted by healthcare professionals, about half acknowledged that clinicians may already possess relevant knowledge and skills but lack confidence and clinical experience. This group felt that any education and training activities should also provide clinicians with protected time in which to practise their skills by, for example, through role-play. Throughout the discussion, it became clear to both workshop attendees, and facilitators, that access to education and training differed depending on where healthcare professionals worked, with some GLH/Genomic Medicine Service Alliances (GMSA) and Trusts providing more workforce development opportunities around consent and variant interpretation (amongst other topics) than others ( “we have sessions on this , don’t you?”) . The commissioning of WGS for paediatric malignancies by NHSE was lauded by all attending the workshop. However, for those working in Scotland, Wales, Northern Ireland, and the ROI, funding this service was seen as the priority before anything else could, or should, be considered. For those working in England, the perpetuating theme from both the group work and subsequent discussion was that opportunities to implement WGS at a Trust and/or pathway level have, from the clinician’s perspective, not been built into existing systems/infrastructures. This includes a lack of systematic clinical pathways and sufficient resources - both financial and people. When the question around resources was put to the groups, the consensus view was there was just simply not enough, with the clear message from one group being “No , a resounding no” . The perception is that most of the current activity is happening due to the ‘goodwill’ of a few people. As summed up by one of the groups, the view from those involved with implementing this test is that: “The test is funded but the clinical process is not.” Specific examples included staff capacity across the pathway ( “not enough time for interpretation , not enough scientists full stop”) and properly resourcing Genomic Tumour Advisory Boards (GTABs) “we have established specific GTABs , but clinician time to attend GTABs (is) not funded”) . Not having sufficient time was a recurring theme, with a particular emphasis on the consent process. This was both in how long it took to obtain consent (or was perceived to take) and which, if any, healthcare professionals then have the time to obtain and record consent. If these issues were not addressed, all participants felt this would create an insurmountable barrier to ubiquitous uptake of WGS across the NHS. The oncologists and haematologists were perplexed as to why the consent process for WGS needs to be separate to standard diagnostic tests and, as such, take additional time to complete. For many of these clinicians, their patients are unwell and simultaneously having other tests, procedures, and/or being enrolled for clinical trials - all of which require verbal and/or formal written consent. This means that clinicians and parents are faced with multiple consent processes and forms at a very stressful time for parents/guardians. It was felt that parents/guardians would benefit from simplification of the consent process, or for consent to occur in a staged manner. For example, one group commented it would be easier for the parents to provide generic consent for collection of the germline sample and then have the consent conversation for analysis after treatment has commenced. As they stated, “you can have a much more sensible conversation four weeks into treatment. This way you prioritise consent for tests that will impact first line treatment , rather than do everything at once”. For other attendees, specifically those from a genetic/genomic professional background, the recognition that WGS is the only tumour assay where germline variants are routinely explored justified having separate consent processes, although recognition of overburdening parents at such a stressful time was appreciated. Despite some tension regarding single or multiple consent processes, attendees all agreed that obtaining consent for WGS significantly impacted clinicians’ ability to deliver other aspects of patient care at the critical diagnostic timepoint. A suggested solution was to employ dedicated staff for this role. However, there was caution in how this should be implemented. As stated by one attendee, “There needs to be investment in staffing , not just taking resources from another part of the service.” Time was also seen as a barrier for the interpretation of variants identified by WGS, which had a knock-on effect on the turnaround time (TAT) for reporting. It was recognised that this is most likely a capacity issue within the laboratories. However, some of the clinical scientists in the room reported an additional physical barrier that would also impact TAT; that of not being able to access key academic journals through their institution (“It just takes more time , time to find a colleague who has an academic appointment who has the time to resource the article for you. It just all adds to the delay in returning results”) . This delay in access to critical data/resources that may influence variant interpretation will inevitably have an impact on TAT. There was agreement from all non-clinical genetics attendees that lack of access to specialist genetic clinical services was also a major factor in the ability to universally implement WGS. This ranged from the limited ability of clinical geneticists to provide input into GTABs, to the very long waiting lists for clinical appointments after the detection of a germline variant (“the waiting list can be up to a year”). For some oncologists and haematologists at the workshop, this meant that they feared receiving a result which reported a germline variant, as they knew how long it could be until the parents/guardians were able to access specialist clinical genetics support (“ should we be offering this test if we can’t get appropriate clinical support to deal with the results?”) . The final area that was discussed were processes and systems that would not only operationalise the end-to-end pathway, but also facilitate implementation of WGS. This included: developing user-friendly digital systems that supported the ordering of tests and tracked samples as well as having a record of consent ( “need better information technology to flag missing consent , samples”); defined clinical pathways (“Have proper process maps and clinical pathways so we all know who is responsible for the task”) and national guidance for clinical trials; and inter- and intra-Trust tissue transport logistics that ensured samples arrived at the relevant laboratory in a safe and timely manner (“ in some cases consultants are transporting samples between hospitals” ). There was a strong sense from all of those at the workshop that it will be much easier to onboard clinicians to offer and consent for WGS when all the pathways, systems, and guidelines are embedded in hospital Trusts. As a group, the attendees at the workshop considered introducing WGS as a positive step and that the implementation of the test will achieve desired outcomes. However, there was a recognition by the oncologists and haematologists in the room that some of their colleagues do not have the same vision. Several theories were suggested as to why this may be this case. These included: A general lack of awareness that WGS is an option. Attendees suggested WGS needs to be included in relevant clinical checklists to remind people of the availability of the test and therefore ensure it is considered (“Suggest including in a checklist or making this part of tumour board discussions for each new patient. Could be included in ward rounds , pre-clinic meetings , pre-ward round meetings”) . Their colleagues’ current level of trust and ‘comfort’ with standard-of-care (SOC) testing – and questioning the benefits of WGS over SOC. This was seen as an issue that needed to be addressed as the tumour samples for this patient cohort are often small. Therefore, it was felt that there needs to be a push to prioritise WGS for testing rather than an optional extra if there was sufficient tissue to test. Finite resources within the NHS. There may be a tension between taking resources from other services to implement WGS, when SOC testing is already being undertaken. A perceived lack of support from Trust management (in some Trusts) which may influence the distribution of resources and provide a subliminal message on the importance of WGS (“Our Trust has not bought into NGS (Next Generation Sequencing) , they have different priorities) . Some of the oncologists felt that there needs to be a shift in mindset as WGS is still seen by many in the NHS “as research & discovery rather than clinical test” . As such, WGS needs to be ‘marketed’ to clinicians as a routine SOC clinical test that is available now. There was also universal recognition that people will be influenced by the views of others within the GTAB, whether they be positive or negative, towards the use of WGS. Therefore, it was felt that there is a need for a widespread campaign illustrating what WGS can offer compared with SOC testing. This could include, for example, the sharing of good practice through knowledge exchange activities like, for example, virtual ‘Grand Rounds’ or national GTABs. The aim of this workshop was to identify the current and anticipated challenges and opportunities to facilitate the uptake of WGS for children with malignancies. Through this process, we have been able to categorise the findings as those where education and training can be used to overcome identified barriers, and where further consideration may need to be made around the operationalising this clinical pathway (see Fig. ). Where education and training can make a difference Despite WGS being routinely available in this context in the NHS in England for over 18 months at the time of the workshop, our findings suggest there is still a requirement for raising awareness amongst clinicians. Surprisingly, the view of attendees was that their colleagues do not view WGS as SOC, even though it is commissioned and resident in the cancer test directory . There is a sense that many clinicians still see WGS as belonging solely in the domain of research projects and not as a test routinely in the clinical setting. If this is truly the view of even a minority of NHS clinicians, this misconception about the availability of WGS needs to be addressed so that clinicians appreciate that this test that is available within the NHS as SOC for patients < 25 years with cancer and can deliver clinically insightful variants inaccessible via alternate assays. Attendees also felt that there is a clear ‘lack of knowledge equity’ across England and the UK more widely. Knowledge deficits need to be addressed to ensure all relevant healthcare professionals’ base-line knowledge is sufficient to ensure patients receive the same access to diagnostic tests regardless of geographical location. Alongside this, it is believed that many clinicians lack confidence and clinical experience, and traditional education and training events may not meet these training needs. Education and training providers need to provide a way for clinicians to ‘practice’ in a safe environment where they can make mistakes - such as using role-play or standardised patients - either in person or in a virtual learning environment . While not explicitly stated by workshop attendees, it is recognised that a lack of data on the clinical utility of WGS versus SOC may also be a barrier to uptake. WGS analysis is evolving, and the algorithms and interfaces which provide the results are under active and ongoing development. These enhancements will expedite and improve the scientific analysis and clinical interpretation of what is perceived as a complex assay, and disseminating this information to the clinical workforce can only expedite uptake. The suggestion of a national standardised programme of mandatory training and assessment of competence would provide a structured way to address these training needs. This will also provide clinicians (and patients) with the assurance that any healthcare professional providing this service, regardless of professional background or geographical location, is equipped with the relevant knowledge and skills. To deliver this programme of training, there will need to be a corresponding equity of access to education and training across the board. Alongside work that is being coordinated at a national level in England to facilitate equity of access to education and training resources through the work of NHS England Genomics Education , GLHs/GMSAs and Trusts in the devolved nations will need to work together and share resources to ensure no clinician is disadvantaged in accessing appropriate training due to their location. Leveraging existing education and training frameworks, such as those for paediatric oncologists, will effectively disseminate crucial information about WGS. A centralised resource, such as a national standardised GTAB, may be a consideration for analysis, interpretation of results, and clinical recommendations to increase access to WGS as an important clinical and research tool. Indeed, such national tumour boards have been suggested for different paediatric cancers to provide treatment recommendations. However, these boards would carry substantial resource implications, including the need for appropriate financing to support time and infrastructure requirements. Based on clinician dedication and goodwill, in the UK there are existing Children’s Cancer and Leukaemia Group (CCLG) National Advisory Panels (NAPS) which provide treatment suggestions, but which are not formally funded. Thus, at present, without more dedicated funding, the centralised sequencing approach with devolved analysis/interpretation is the most pragmatic model, alongside the development of education workshops/events. Other areas that need to be addressed to improve uptake For the other UK nations (outside of England) and ROI it was clear that the first barrier that must be addressed is sustainable funding for the service. For those working in England, attendees identified their perception that investing in the test will not correlate with adoption into clinical practice unless investment is also made for the clinical process. Otherwise, it was felt that the service relies on ‘goodwill’ which is not sustainable and will not translate into an equitable service. Our findings indicate attendees wanted resource investment across the end-to-end pathway including capacity to take consent and through to increased access to clinical genetics services. While the conversations on consent during the workshop focused on consent for the clinical test, WGS also provides an opportunity for patients/parents/guardians in England to consent for their data and samples to be included within the National Genomic Research Library (NGRL), which is overseen by Genomics England. Consenting to the NGRL is not mandatory, and indeed around 3% of cancer patients who receive WGS via the NHS Genomic Medicine Service decline to sign the research consent forms (personal communication, Parker Moss). The rationale for obtaining ‘research consent’ is that beyond the clinical purpose of the test, consented patient genomic and clinical data are de-identified and stored in the NGRL. Through linking national health datasets, this research resource follows the patient’s clinical results for the rest of their lives, enabling the study of longitudinal outcomes by genotype and by intervention. This creates material research upside to the test beyond its clinical benefit, but this upside does require the patients to be informed and to consent to the storage and follow-up of their data, further complicating the research conversation. Next steps The workshop findings identified several actions that can be taken to improve uptake of WGS. Firstly, the topics outlined in Table provide a broad curriculum for education and training that providers developing and delivering sessions around WGS for the different health professional groups can use to ensure coverage of relevant knowledge and skills within any education sessions. Although the authors’ goal was a coordinated education program within 12 months of the workshop, successful implementation depends on collaboration with partners with differing priorities. Nonetheless, significant strides have been made; SB, MJM, PT, and AV delivered a teaching session to the CCLG Paediatric Oncology Trainee Group (POTG) in May 2023 as part of their scheduled learning programme, using case studies to cover the topics in Table . Efforts will continue with key stakeholders including NHS England Genomics Education and the Royal Colleges who are charged with delivering training curriculum for paediatric oncologists and haematologists to ensure the relevant health care professionals receive timeline and pertinent education and training in this topic. Regarding the other barriers identified, there were many providers within the room that are achieving excellence in certain parts of the clinical pathway, even if no institutions appear to have a perfect end-to-end process. For example, through the workshop, participants shared examples of best practice and potential ways of working, such as using centralised model for analysis an interpretation of results as well as and regional tumour boards to support implementation. This sharing of this type of information was seen by many as an unexpected benefit of attending the workshop, and as such participants requested annual or bi-annual meetings. Bringing this groups of attendees together on a regular basis would provide an avenue for collective problem solving, essentially establishing a UK and ROI community of practice in this area . The effectiveness of these interventions will be evaluated based on the volume of WGS tests ordered and the equitable access provided across the UK. Data from NHS England, along with counterparts in Scotland, Wales, and Northern Ireland, as well as the ROI, will track test uptake, offering a valuable objective measure to assess behavioural changes over time. Despite WGS being routinely available in this context in the NHS in England for over 18 months at the time of the workshop, our findings suggest there is still a requirement for raising awareness amongst clinicians. Surprisingly, the view of attendees was that their colleagues do not view WGS as SOC, even though it is commissioned and resident in the cancer test directory . There is a sense that many clinicians still see WGS as belonging solely in the domain of research projects and not as a test routinely in the clinical setting. If this is truly the view of even a minority of NHS clinicians, this misconception about the availability of WGS needs to be addressed so that clinicians appreciate that this test that is available within the NHS as SOC for patients < 25 years with cancer and can deliver clinically insightful variants inaccessible via alternate assays. Attendees also felt that there is a clear ‘lack of knowledge equity’ across England and the UK more widely. Knowledge deficits need to be addressed to ensure all relevant healthcare professionals’ base-line knowledge is sufficient to ensure patients receive the same access to diagnostic tests regardless of geographical location. Alongside this, it is believed that many clinicians lack confidence and clinical experience, and traditional education and training events may not meet these training needs. Education and training providers need to provide a way for clinicians to ‘practice’ in a safe environment where they can make mistakes - such as using role-play or standardised patients - either in person or in a virtual learning environment . While not explicitly stated by workshop attendees, it is recognised that a lack of data on the clinical utility of WGS versus SOC may also be a barrier to uptake. WGS analysis is evolving, and the algorithms and interfaces which provide the results are under active and ongoing development. These enhancements will expedite and improve the scientific analysis and clinical interpretation of what is perceived as a complex assay, and disseminating this information to the clinical workforce can only expedite uptake. The suggestion of a national standardised programme of mandatory training and assessment of competence would provide a structured way to address these training needs. This will also provide clinicians (and patients) with the assurance that any healthcare professional providing this service, regardless of professional background or geographical location, is equipped with the relevant knowledge and skills. To deliver this programme of training, there will need to be a corresponding equity of access to education and training across the board. Alongside work that is being coordinated at a national level in England to facilitate equity of access to education and training resources through the work of NHS England Genomics Education , GLHs/GMSAs and Trusts in the devolved nations will need to work together and share resources to ensure no clinician is disadvantaged in accessing appropriate training due to their location. Leveraging existing education and training frameworks, such as those for paediatric oncologists, will effectively disseminate crucial information about WGS. A centralised resource, such as a national standardised GTAB, may be a consideration for analysis, interpretation of results, and clinical recommendations to increase access to WGS as an important clinical and research tool. Indeed, such national tumour boards have been suggested for different paediatric cancers to provide treatment recommendations. However, these boards would carry substantial resource implications, including the need for appropriate financing to support time and infrastructure requirements. Based on clinician dedication and goodwill, in the UK there are existing Children’s Cancer and Leukaemia Group (CCLG) National Advisory Panels (NAPS) which provide treatment suggestions, but which are not formally funded. Thus, at present, without more dedicated funding, the centralised sequencing approach with devolved analysis/interpretation is the most pragmatic model, alongside the development of education workshops/events. For the other UK nations (outside of England) and ROI it was clear that the first barrier that must be addressed is sustainable funding for the service. For those working in England, attendees identified their perception that investing in the test will not correlate with adoption into clinical practice unless investment is also made for the clinical process. Otherwise, it was felt that the service relies on ‘goodwill’ which is not sustainable and will not translate into an equitable service. Our findings indicate attendees wanted resource investment across the end-to-end pathway including capacity to take consent and through to increased access to clinical genetics services. While the conversations on consent during the workshop focused on consent for the clinical test, WGS also provides an opportunity for patients/parents/guardians in England to consent for their data and samples to be included within the National Genomic Research Library (NGRL), which is overseen by Genomics England. Consenting to the NGRL is not mandatory, and indeed around 3% of cancer patients who receive WGS via the NHS Genomic Medicine Service decline to sign the research consent forms (personal communication, Parker Moss). The rationale for obtaining ‘research consent’ is that beyond the clinical purpose of the test, consented patient genomic and clinical data are de-identified and stored in the NGRL. Through linking national health datasets, this research resource follows the patient’s clinical results for the rest of their lives, enabling the study of longitudinal outcomes by genotype and by intervention. This creates material research upside to the test beyond its clinical benefit, but this upside does require the patients to be informed and to consent to the storage and follow-up of their data, further complicating the research conversation. The workshop findings identified several actions that can be taken to improve uptake of WGS. Firstly, the topics outlined in Table provide a broad curriculum for education and training that providers developing and delivering sessions around WGS for the different health professional groups can use to ensure coverage of relevant knowledge and skills within any education sessions. Although the authors’ goal was a coordinated education program within 12 months of the workshop, successful implementation depends on collaboration with partners with differing priorities. Nonetheless, significant strides have been made; SB, MJM, PT, and AV delivered a teaching session to the CCLG Paediatric Oncology Trainee Group (POTG) in May 2023 as part of their scheduled learning programme, using case studies to cover the topics in Table . Efforts will continue with key stakeholders including NHS England Genomics Education and the Royal Colleges who are charged with delivering training curriculum for paediatric oncologists and haematologists to ensure the relevant health care professionals receive timeline and pertinent education and training in this topic. Regarding the other barriers identified, there were many providers within the room that are achieving excellence in certain parts of the clinical pathway, even if no institutions appear to have a perfect end-to-end process. For example, through the workshop, participants shared examples of best practice and potential ways of working, such as using centralised model for analysis an interpretation of results as well as and regional tumour boards to support implementation. This sharing of this type of information was seen by many as an unexpected benefit of attending the workshop, and as such participants requested annual or bi-annual meetings. Bringing this groups of attendees together on a regular basis would provide an avenue for collective problem solving, essentially establishing a UK and ROI community of practice in this area . The effectiveness of these interventions will be evaluated based on the volume of WGS tests ordered and the equitable access provided across the UK. Data from NHS England, along with counterparts in Scotland, Wales, and Northern Ireland, as well as the ROI, will track test uptake, offering a valuable objective measure to assess behavioural changes over time. This workshop provided an opportunity and forum for change leaders in the UK and ROI to have a focused discussion on the implementation of WGS in paediatric haematology and oncology practice. Using structured questions within a group discussion ensured that all areas of the clinical pathway and implementation process were covered, and exposed many perceived barriers which were not commonly known across the whole group. Outcomes from this workshop have identified where education and training can improve uptake. However, for education and training to have an impact on behavioural change, consideration also needs to be made for appropriate operational changes. This will facilitate the most widespread and equitable uptake of WGS in this cohort, with the aim of allowing real-time changes in clinical management and ultimately improving patient outcomes. Below is the link to the electronic supplementary material. Supplementary Material 1 |
The discrepancy of antemortem clinical diagnosis and postmortem autopsy diagnosis of lung pathologies in under-five deaths and the reasons for discrepancies: a case series analysis | 05582d09-2b85-4366-a93c-036293aa27eb | 11131180 | Forensic Medicine[mh] | Diagnostic errors contribute to as much as 10% of adverse events occurring in healthcare practice globally . This rate is higher in countries like Ethiopia, where there is limited diagnostic capacity and shortages of trained health professionals . Diagnostic errors often result from diagnostic uncertainty, the complex nature of making accurate diagnoses in clinical practice, limitations in the availability and accuracy of some diagnostic tests, and time constraints encountered by clinicians . Moreover, greater workload burdens, rising quality expectations, cognitive biases, and insufficient health system infrastructure may also contribute to diagnostic errors . The increasing complexity in health care can be attributed to multiple factors such as a wide range of options for diagnostic testing and treatment, extensive and continually growing biomedical and clinical evidence influencing clinical practice, along with a higher occurrence of comorbidities among patients . Diagnostic errors can lead to improper treatment and deaths that could potentially be averted if the correct diagnosis had been made . Lung diseases, with their broad clinical and histological spectrum, present unique challenges as radiological and clinical signs are often non-specific . Perinatal and under-five deaths due to lung diseases are a significant global burden leading to over 800,000 deaths each year among children aged < 5 years . Detailed postmortem examinations of lung tissue yield the most accurate results when ascertaining the cause of death . However, postmortem examinations are infrequently conducted in low- and middle-income countries, where the disease burden of lung pathology is highest among young children , leaving a gap in true understanding of causes of death and accuracy of antemortem clinical diagnoses for lung diseases. Clinicopathologic discrepancy studies aim to evaluate the accuracy of clinical diagnoses by comparing them with the reference standard of postmortem autopsy diagnoses . Despite advances in medical technology, significant discrepancies between antemortem clinical diagnoses and complete diagnostic autopsies (CDA) have been observed in as much as 45% of childhood deaths . CDAs have long been considered the benchmark in establishing the cause of death (CoD). By conducting a comprehensive postmortem analysis, which involves examining internal organs and tissues, pathologists can gather insightful information regarding diverse diseases and their manifestations in the body, which can be applied to enhance the diagnosis, treatment, and prevention of these ailments for future patients. CDA improves the identification of underlying causes and mechanisms of disease leading to death . However, the resources and expertise required for CDAs are often lacking in developing countries like Ethiopia . Understanding which diagnoses are missed most often prior to a child’s death and the reasons for these diagnostic errors is crucial to identifying gaps in clinical diagnosis to refine diagnostic algorithms for improved treatment outcomes . However, studies evaluating the accuracy of antemortem clinical diagnoses in Ethiopia are lacking but are essential to further guide policy and programs to reduce antemortem diagnostic errors, which, in turn, may reduce childhood mortality in regions with high rates of childhood mortality. As an alternative to CDA, minimally invasive tissue sampling (MITS) has emerged as a viable method for ascertaining causes of death , and has demonstrated high concordance with CDA . Due to its acceptability and feasibility in resource-limited settings, MITS has been implemented by the Child Health and Mortality Prevention Surveillance (CHAMPS) network in nine countries across sub-Saharan Africa and South Asia. Given the high burden of disease of lung diseases among young children and prior studies suggesting high rates of diagnostic errors in similar settings, our objective was to determine the frequency of, and reasons for, diagnostic discrepancies between antemortem clinical diagnoses and postmortem causes of death determined by lung pathology, in order to provide insight for health professionals and policymakers in the goal of reducing childhood mortality. Study area and design The study was conducted within the Health and Demographic Surveillance Sites (HDSS) areas of Kersa, Harar, and Haramaya, under Haramaya University in Ethiopia, the largest HDSS site in Africa . A clinical case series design was employed. The timeframe for data collection was extended from October 2019 through April 2022. This study used data from the Child Health and Mortality Prevention Surveillance (CHAMPS) study. CHAMPS utilizes high-quality data collection methodologies, including MITS, extensive pathogen testing, histopathology evaluation, clinical data extraction, and verbal autopsies . Population and selection criteria This study focused on the examination of deaths among neonates, infants, and children under the age of five that occurred within the health facilities situated in the HDSS area . Stillbirths were excluded because antemortem diagnoses are not made in this population. There were three inclusion criteria for this study: Enrollment in CHAMPS, a MITS autopsy completed, and availability of dedicated clinical data for the deceased neonate, infant, or child within the healthcare facility where the death occurred. To maintain the integrity of the study and ensure the completeness and reliability of the data, cases, where antemortem clinical data was absent from the corresponding health facilities where the death occurred, were omitted from the study. Data collection procedures A comprehensive and systematic approach was adopted for data collection, which included several sources of clinical data including documented history and physical examination, progress notes, nursing notes, order sheets, laboratory tests, radiological examinations, pathological examinations, and operation notes. The extraction of data was systematically carried out by qualified specialist physicians using a structured Excel sheet. Physicians who collected data were not involved in the clinical care of enrolled cases. To ensure the integrity of the data collected, all clinical data, including antemortem diagnoses, was verified independently by two additional physicians. The antemortem clinical diagnoses were systematically extracted from all available clinical data sources, including documented history, physical examination findings, progress notes, nursing notes, order sheets, laboratory tests, radiological examinations, and, where appropriate, operative reports. The data extraction was conducted by qualified specialist physicians using a structured data collection tool. To ensure accuracy and completeness, the extracted antemortem diagnoses were independently verified by two additional physicians who were not involved in the clinical care of the cases. While conducting MITS, postmortem biopsy needles were used to gather tissue samples, which underwent analysis using multiple testing methodologies. Specimens from MITS were utilized for histopathologic assessment, molecular analysis, and different microbial testing methodologies . Samples underwent evaluation where multiplex molecular testing was conducted using TaqMan® Array Card (TAC) on fresh tissues to test for 126 pathogens. Furthermore, histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) were carried out on formalin-fixed paraffin-embedded (FFPE) tissues . The highly sensitive detection of pathogens via TAC is possible on refrigerated MITS samples within a time window of three days following the occurrence of death. Moreover, gene Xpert testing, a fast molecular assay capable of detecting Mycobacterium tuberculosis and its rifampicin resistance was also used. Result agreement between TAC, IHC, and PCR methods varies, making it important to interpret all diagnostic tests in aggregate to establish overall case diagnoses and maximize the utility of these testing methods in MITS . Additionally, clinical and epidemiologic data were considered, which has demonstrated enhanced interpretation of MITS findings . The MITS process involved both local and international pathologists who reviewed histopathology results. A thorough assessment was conducted on both the obtained samples and locally produced slides within the CHAMPS network to ensure that they met all requirements . The final cause of death was determined by a panel of local experts (the Determination of Cause of Death [DeCoDe] panel) that included obstetricians, pediatricians, pathologists, microbiologists, and epidemiologists who reviewed all clinical, verbal autopsy, and histopathological data. The DeCoDe determined cause of death served as the reference standard for this study. Determination of diagnostic discrepancies The antemortem clinical diagnosis and postmortem pathological diagnosis of the lung were compared for each case, and two physicians evaluated the result separately for concordance of diagnoses. These physicians were trained on the World Health Organization (WHO) classification approach of cases as concordant and discordant and assigning of reason for diagnostic discrepancy prior to commencement of the research. They used a structured Excel sheet to assign concordance and discordance and reason for all discrepancies. If there was a discrepancy between the physicians, a third physician was invited as an arbiter. The physicians classified the comparison of antemortem clinical diagnosis and postmortem causes of death into one of four categories according to predetermined categories: (1) diagnosed by both clinical and autopsy, (2) diagnosed by clinical and not by autopsy, (3) diagnosed by autopsy, not by clinical, and (4) not diagnosed by either. The reason for the diagnostic discrepancy was ascertained for each discrepant case by conducting an in-depth review of all available clinical data for each case in line with WHO guidelines on diagnostic discrepancy . The concordance analysis focused specifically on comparing antemortem clinical diagnoses with postmortem pathological diagnoses related to lung pathologies. Extrapulmonary findings identified during the postmortem evaluations were not considered in the concordance analysis, as they were not directly relevant to the primary objective of assessing diagnostic discrepancies in lung pathologies. Statistical analyses Descriptive statistics were calculated and the McNemar’s test was utilized to identify any statistically significant discrepancies between antemortem and postmortem diagnoses. A p-value threshold of < 0.05 was set to determine statistical significance. In cases where discrepancies were identified, these were thoroughly discussed and pertinent recommendations for each discordant case were subsequently made. All analyses were conducted using Stata version 17.0 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC). The study was conducted within the Health and Demographic Surveillance Sites (HDSS) areas of Kersa, Harar, and Haramaya, under Haramaya University in Ethiopia, the largest HDSS site in Africa . A clinical case series design was employed. The timeframe for data collection was extended from October 2019 through April 2022. This study used data from the Child Health and Mortality Prevention Surveillance (CHAMPS) study. CHAMPS utilizes high-quality data collection methodologies, including MITS, extensive pathogen testing, histopathology evaluation, clinical data extraction, and verbal autopsies . This study focused on the examination of deaths among neonates, infants, and children under the age of five that occurred within the health facilities situated in the HDSS area . Stillbirths were excluded because antemortem diagnoses are not made in this population. There were three inclusion criteria for this study: Enrollment in CHAMPS, a MITS autopsy completed, and availability of dedicated clinical data for the deceased neonate, infant, or child within the healthcare facility where the death occurred. To maintain the integrity of the study and ensure the completeness and reliability of the data, cases, where antemortem clinical data was absent from the corresponding health facilities where the death occurred, were omitted from the study. A comprehensive and systematic approach was adopted for data collection, which included several sources of clinical data including documented history and physical examination, progress notes, nursing notes, order sheets, laboratory tests, radiological examinations, pathological examinations, and operation notes. The extraction of data was systematically carried out by qualified specialist physicians using a structured Excel sheet. Physicians who collected data were not involved in the clinical care of enrolled cases. To ensure the integrity of the data collected, all clinical data, including antemortem diagnoses, was verified independently by two additional physicians. The antemortem clinical diagnoses were systematically extracted from all available clinical data sources, including documented history, physical examination findings, progress notes, nursing notes, order sheets, laboratory tests, radiological examinations, and, where appropriate, operative reports. The data extraction was conducted by qualified specialist physicians using a structured data collection tool. To ensure accuracy and completeness, the extracted antemortem diagnoses were independently verified by two additional physicians who were not involved in the clinical care of the cases. While conducting MITS, postmortem biopsy needles were used to gather tissue samples, which underwent analysis using multiple testing methodologies. Specimens from MITS were utilized for histopathologic assessment, molecular analysis, and different microbial testing methodologies . Samples underwent evaluation where multiplex molecular testing was conducted using TaqMan® Array Card (TAC) on fresh tissues to test for 126 pathogens. Furthermore, histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) were carried out on formalin-fixed paraffin-embedded (FFPE) tissues . The highly sensitive detection of pathogens via TAC is possible on refrigerated MITS samples within a time window of three days following the occurrence of death. Moreover, gene Xpert testing, a fast molecular assay capable of detecting Mycobacterium tuberculosis and its rifampicin resistance was also used. Result agreement between TAC, IHC, and PCR methods varies, making it important to interpret all diagnostic tests in aggregate to establish overall case diagnoses and maximize the utility of these testing methods in MITS . Additionally, clinical and epidemiologic data were considered, which has demonstrated enhanced interpretation of MITS findings . The MITS process involved both local and international pathologists who reviewed histopathology results. A thorough assessment was conducted on both the obtained samples and locally produced slides within the CHAMPS network to ensure that they met all requirements . The final cause of death was determined by a panel of local experts (the Determination of Cause of Death [DeCoDe] panel) that included obstetricians, pediatricians, pathologists, microbiologists, and epidemiologists who reviewed all clinical, verbal autopsy, and histopathological data. The DeCoDe determined cause of death served as the reference standard for this study. The antemortem clinical diagnosis and postmortem pathological diagnosis of the lung were compared for each case, and two physicians evaluated the result separately for concordance of diagnoses. These physicians were trained on the World Health Organization (WHO) classification approach of cases as concordant and discordant and assigning of reason for diagnostic discrepancy prior to commencement of the research. They used a structured Excel sheet to assign concordance and discordance and reason for all discrepancies. If there was a discrepancy between the physicians, a third physician was invited as an arbiter. The physicians classified the comparison of antemortem clinical diagnosis and postmortem causes of death into one of four categories according to predetermined categories: (1) diagnosed by both clinical and autopsy, (2) diagnosed by clinical and not by autopsy, (3) diagnosed by autopsy, not by clinical, and (4) not diagnosed by either. The reason for the diagnostic discrepancy was ascertained for each discrepant case by conducting an in-depth review of all available clinical data for each case in line with WHO guidelines on diagnostic discrepancy . The concordance analysis focused specifically on comparing antemortem clinical diagnoses with postmortem pathological diagnoses related to lung pathologies. Extrapulmonary findings identified during the postmortem evaluations were not considered in the concordance analysis, as they were not directly relevant to the primary objective of assessing diagnostic discrepancies in lung pathologies. Descriptive statistics were calculated and the McNemar’s test was utilized to identify any statistically significant discrepancies between antemortem and postmortem diagnoses. A p-value threshold of < 0.05 was set to determine statistical significance. In cases where discrepancies were identified, these were thoroughly discussed and pertinent recommendations for each discordant case were subsequently made. All analyses were conducted using Stata version 17.0 (StataCorp. 2021. Stata Statistical Software: Release 17. College Station, TX: StataCorp LLC). Sociodemographic characteristics of the participants There was a total of 5,485 death notifications received by CHAMPS Ethiopia during the study period. Among those, 687 had MITS completed. Among the cases enrolled for MITS, 612 (89.1%) lacked antemortem clinical data. This was primarily due to the majority being stillbirths (46%), with the remaining cases comprising deaths that occurred at home, on the way to a healthcare facility, or at the facility where medical records were lost or not documented. Seventy-five (10.9%) cases met inclusion criteria. 73% ( n = 55) of participants were males, and 26.7% ( n = 20) were females. Neonates aged 0–28 days at the time of death accounted for 86.7% ( n = 65) of the study participants, with the majority of neonatal deaths occurring within the first week of delivery (70.7%, n = 53) (Table ). A total of 206 different antemortem clinical diagnoses were recorded. The most common clinical diagnosis among the enrolled cases was early onset neonatal sepsis (30/206, 14.6%), followed by perinatal asphyxia (24/206, 11.7%) and preterm baby (23/206, 11.1%) (Table ). Other common diagnoses included low birth weight (21/206,10.2%) and hypothermia (18/206, 8.7%). There was a total of 147 postmortem pathologic diagnoses for the immediate, underlying, and comorbid conditions of the 75 cases (Table ). The most common postmortem pathologic diagnosis observed was extramedullary hematopoiesis, (32/147, 21.7%), followed by hyaline membrane disease (17/147, 11.6%) and sepsis (13/147, 8.8%). Pneumonia and meconium aspiration syndrome also each contributed to 8.8% of the total cases, with 13 instances each. Antemortem clinical and postmortem autopsy diagnosis concordance Of the 75 included cases included, 52.0% ( n = 39) were accurately diagnosed by antemortem clinical diagnosis compared to postmortem diagnoses (Table ). There was a diagnostic discordance in 37.3% ( n = 28) cases. Overall, there was a 35% (95% CI: 20%, 47%) diagnostic discrepancy between antemortem and postmortem diagnoses ( p < 0.0001) (Table ). There were 31 (41.3%) cases in which the clinical antemortem and postmortem pathology causes of death were discordant. Three (4.0%) were detected by clinical diagnosis but not by pathologic diagnosis, and 28 (37.3%) were detected by pathologic diagnosis but not by clinical diagnosis. Reasons for diagnostic discrepancies The majority of diagnostic discrepancies (71%, n = 44/62) were attributed to case analysis problems and deficiencies in consultation and teamwork. Other issues including documentation errors, unavailability of diagnostic investigations, communication gaps, limited access to health facilities, and existing work culture were also identified (Table ). The analysis of reasons for the diagnostic gap in cases undiagnosed by antemortem clinical diagnosis and diagnosed by postmortem autopsy are elaborated in Table and each reason for the diagnostic discrepancy is described with illustrative case as an example. There was a total of 5,485 death notifications received by CHAMPS Ethiopia during the study period. Among those, 687 had MITS completed. Among the cases enrolled for MITS, 612 (89.1%) lacked antemortem clinical data. This was primarily due to the majority being stillbirths (46%), with the remaining cases comprising deaths that occurred at home, on the way to a healthcare facility, or at the facility where medical records were lost or not documented. Seventy-five (10.9%) cases met inclusion criteria. 73% ( n = 55) of participants were males, and 26.7% ( n = 20) were females. Neonates aged 0–28 days at the time of death accounted for 86.7% ( n = 65) of the study participants, with the majority of neonatal deaths occurring within the first week of delivery (70.7%, n = 53) (Table ). A total of 206 different antemortem clinical diagnoses were recorded. The most common clinical diagnosis among the enrolled cases was early onset neonatal sepsis (30/206, 14.6%), followed by perinatal asphyxia (24/206, 11.7%) and preterm baby (23/206, 11.1%) (Table ). Other common diagnoses included low birth weight (21/206,10.2%) and hypothermia (18/206, 8.7%). There was a total of 147 postmortem pathologic diagnoses for the immediate, underlying, and comorbid conditions of the 75 cases (Table ). The most common postmortem pathologic diagnosis observed was extramedullary hematopoiesis, (32/147, 21.7%), followed by hyaline membrane disease (17/147, 11.6%) and sepsis (13/147, 8.8%). Pneumonia and meconium aspiration syndrome also each contributed to 8.8% of the total cases, with 13 instances each. Of the 75 included cases included, 52.0% ( n = 39) were accurately diagnosed by antemortem clinical diagnosis compared to postmortem diagnoses (Table ). There was a diagnostic discordance in 37.3% ( n = 28) cases. Overall, there was a 35% (95% CI: 20%, 47%) diagnostic discrepancy between antemortem and postmortem diagnoses ( p < 0.0001) (Table ). There were 31 (41.3%) cases in which the clinical antemortem and postmortem pathology causes of death were discordant. Three (4.0%) were detected by clinical diagnosis but not by pathologic diagnosis, and 28 (37.3%) were detected by pathologic diagnosis but not by clinical diagnosis. The majority of diagnostic discrepancies (71%, n = 44/62) were attributed to case analysis problems and deficiencies in consultation and teamwork. Other issues including documentation errors, unavailability of diagnostic investigations, communication gaps, limited access to health facilities, and existing work culture were also identified (Table ). The analysis of reasons for the diagnostic gap in cases undiagnosed by antemortem clinical diagnosis and diagnosed by postmortem autopsy are elaborated in Table and each reason for the diagnostic discrepancy is described with illustrative case as an example. Our investigation elucidated the accuracy of antemortem clinical diagnoses and their alignment with postmortem pathologic diagnoses in childhood mortality cases in regions with high childhood mortality in Ethiopia. This investigation revealed that 37.3% of cases had discrepant antemortem clinical diagnoses and postmortem pathologic diagnoses among children with positive lung pathology findings. Most diagnostic errors were attributed to gaps in clinical knowledge and a lack of collaboration and specialist consultation. The degree of diagnostic discrepancy observed in this investigation aligns with those reported in prior studies. The rate of diagnostic discrepancy manifested in the current study also corresponds with global rates. However, there appears to be a pronounced pattern of higher diagnostic discrepancies in resource-constrained nations relative to those in high-income settings . This highlights an important systemic issue in low- and middle-income countries, accentuating the diagnostic challenges posed by resource limitations. For instance, research conducted in Mozambique revealed a diagnostic discrepancy rate of 63% , one of the highest reported in the sub-Saharan African region. Contrasting this, a study in Germany demonstrated a progressive decline in diagnostic discrepancy rates over a ten-year period, from 43% in 1998 to 27% in 2008 . This downward trend suggests a steady advancement in diagnostic capabilities over time and underscores the disparities in diagnostic certainty across varied socioeconomic contexts given higher rates of diagnostic errors in sub-Saharan Africa. Furthermore, in the Netherlands, a mere 35% of cases demonstrated complete accuracy in the clinical determination of the cause of death and a Brazilian investigation indicated an agreement between clinical and pathological diagnoses in 58% of cases . The patterns unearthed in these studies imply that, even within the confines of high-income nations, significant diagnostic discrepancies endure. These observations suggest an urgent need to address prevalent diagnostic errors, regardless of the country’s economic status. This need is particularly pronounced in countries characterized by limited resources . Concerted efforts towards the enhancement of clinical diagnostic procedures are needed to reduce antemortem diagnostic errors, ensuring the provision of more accurate diagnoses and corresponding treatments to reduce mortality . The principal contributor to diagnostic discrepancies in our study was issues pertaining to case analysis, accounting for more than one in three of all instances. These problems often originate from a deficiency in knowledge concerning disease pathology, clinical presentation, and interpretation of commonplace scenarios in routine practice . This highlights the imperative for continuous professional education and dissemination of the latest clinical knowledge, enabling healthcare professionals to stay abreast of advancements in their fields. Similarly, issues related to consultation and teamwork constituted another 35.5% of the discrepancies. Deficient communication amongst healthcare professionals, limited involvement of senior clinicians, and ineffective teamwork were identified as key factors precipitating these challenges, underscoring the crucial role of proficient communication and collaboration within healthcare settings in Ethiopia . Furthermore, gaps in documentation contributed to 10% of diagnostic discrepancies. Inaccurate or incomplete patient records not only disrupt the continuity of patient care but also impede inter-professional communication, thereby leading to avoidable medical errors. This indicates an urgent need for health systems to refine their documentation practices, ensure meticulous maintenance of patient records . The unavailability of diagnostic investigations factored into 8% of the discrepancies. In low- and middle-income countries, a lack of access to diagnostic tools poses a significant problem , emphasizing the need to bolster healthcare infrastructure, particularly regarding diagnostic modalities. Previous studies have described numerous contributing factors to diagnostic discrepancies, with a significant emphasis on the influence of systemic, cognitive, and logistical components . Lapses in clinical diagnoses may potentially precipitate adverse patient outcomes. Prior studies suggest the causes of diagnostic discrepancies are often multifaceted . Hence, a comprehensive and multidimensional approach is imperative to mitigate such diagnostic discrepancies. This investigation emphasizes the importance of sustained monitoring of clinical diagnostic accuracy and calls for systemic enhancements in various facets of healthcare practice. The highlighted areas of concern necessitate targeted interventions and further research to mitigate diagnostic discrepancies and improve patient outcomes. Strengths of the study include the use of postmortem autopsies as the benchmark for cause-of-death determination, facilitating precise comparisons between antemortem clinical and postmortem pathological diagnoses of lung pathologies. The first of its kind, this study provides an initial investigation of clinicopathologic diagnosis discrepancies in Ethiopia among young children, laying the groundwork for further research to elucidate reasons for diagnostic errors and efforts to mediate these errors. The comprehensive dataset amplifies the study’s validity. The meticulous evaluation performed by two senior physicians, with a third involved in the event of disagreement, ensures rigor in the study. However, some limitations merit consideration. The sole focus on lung pathologies potentially restricts the study’s insights into other body systems. The modest sample size reduces statistical power and restricts the application of advanced statistical methods. Being a case series study, the lack of a control group inhibits direct comparisons and controls for confounding variables. However, conducting lung biopsies on living children in which such a procedure is not indicated would be unethical. There was a high proportion of cases that did not have antemortem clinical diagnoses available so were excluded from our analyses, which may reflect inadequate record keeping in clinical care. This could potentially lead to an underestimation of the prevalence of diagnostic errors. It is important to note that our study did not analyze the potential relationship between the level of healthcare facilities (e.g., primary care centers, district hospitals, referral hospitals) and the occurrence of diagnostic discrepancies. Exploring this aspect could provide valuable insights into the specific challenges faced by healthcare providers at different levels of the healthcare system. Future research should consider examining the distribution of diagnostic discrepancies across various levels of healthcare facilities to inform targeted interventions and resource allocation strategies aimed at reducing diagnostic errors. There was substantial discordance between antemortem clinical diagnoses and postmortem autopsy results in this study, which points to a critical need for comprehensive reform in several facets of clinical practice in Ethiopia. The identified contributing factors – including gaps in clinical knowledge and understanding, limitations in teamwork and consultation processes, problems related to patient documentation, and scarcity of diagnostic investigations– illustrate complex, multifaceted issues that contribute to diagnostic errors. The direct implication of these findings is an urgent call for robust, systemic interventions to enhance diagnostic accuracy and patient care outcomes. It is paramount that future efforts focus on reducing these discrepancies through improved provider pre-service and in-service education, communication, and resource allocation within the healthcare sector. |
Silencing METTL3 Increases HSP70 Expression and Alleviates Fibrosis in Keratocytes | 04825c9b-8621-488c-920a-c7c779ea4be0 | 11562871 | Anatomy[mh] | Corneal Alkali Burn Model of C57BL/6 Mice This research used C57BL/6 mice (male, 6–8 weeks old). The mice were obtained from Weitonghua Company (Tianjin, China) and were raised in the Experimental Center of Nankai Hospital in Tianjin. In all animal-related procedures, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research was followed. All animal experiments received approval from the experimental animal management organization of Nankai Center in Tianjin (approval no. NKYY-DWLL-2023-051). Briefly, general anesthesia was administered to mice using 1% sodium pentobarbital (40–50 mg/kg) intraperitoneally. Surface anesthesia was achieved by applying proparacaine drops to the ocular surface. The mouse cornea was injured using a 1-N NaOH-soaked filter paper with a diameter of 2 mm. The filter paper was placed on the ocular surface for 30 seconds and then removed. Subsequently, mouse eyes were rinsed with 25 mL of saline solution. Tobramycin eye ointment was applied after the injury. The ocular surfaces of the mice were observed using a slit-lamp microscope at 4, 7, 10, 14, 16, 19, 21, 25, 28, and 35 days post-injury, and they were photographed and documented. The mice were euthanized at 4, 14, 21, 28, and 35 days post-injury, and the corneas were collected for analyses. Completely normal and uninjured eyes were used as negative controls. Evaluation of Corneal Opacity Degree Mice were anesthetized, and the extent of corneal clouding following alkali burns was evaluated using a slit-lamp microscope according to established criteria. Specifically, corneal opacity was graded from 0 to 4, where 0 = completely clear; 1 = slightly hazy, iris and pupil easily visible; 2 = slightly opaque, iris and pupil still detectable; 3 = opaque, pupils hardly detectable; and 4 = completely opaque with no view of the pupil. Three experimenters independently scored opacity, and the final score was the average of their scores. Cell Culture and TGF-β1 Stimulation Human keratocytes were obtained from ScienCell Research Laboratories (#6520; Carlsbad, CA, USA). The medium composition for keratocytes was Gibco Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; #11320082; Thermo Fisher Scientific, Waltham, MA, USA), 2% to 5% Gibco fetal bovine serum (#10099141; Thermo Fisher Scientific), and 1% Gibco penicillin/streptomycin (#15140122; Thermo Fisher Scientific). Culture conditions were set at 37°C with 5% carbon dioxide. The keratocytes from the third to fifth passages were used for the subsequent experiments. Keratocytes were stimulated with TGF-β1 (10 ng/mL). Cell Transfection For gene silencing of METTL3, FTO, and YTHDF2, small interfering RNAs (siRNAs) targeting the three genes were purchased from Genepharma Co., Ltd. (Shanghai, China). These included METTL3 siRNAs 1 to 4, FTO siRNAs 1 to 3, and YTHDF2 siRNAs 1 to 4. Additionally, a negative control siRNA (si-NC) and a positive control siRNA (si-GAPDH) were also obtained. Transfection was performed utilizing Lipofectamine RNAiMax Transfection Reagent (#13778150; Life Technologies, Carlsbad, CA, USA). The siRNA sequences were as follows: METTL3 siRNA1, 5′-CCUGCAAGUAUGUUCACUATT-3′ METTL3 siRNA2, 5′-GCUACCUGGACGUCAGUAUTT-3′ METTL3 siRNA3, 5′-GCUCAACAUACCCGUACUATT-3′ METTL3 siRNA4, 5′-GGUUGGUGUCAAAGGAAAUTT-3′ FTO siRNA1, 5′-ACACUUGGCUCCCUUAUCUTT-3′ FTO siRNA2, 5′-GUGGCAGUGUACAGUUAUATT-3′ FTO siRNA3, 5′-GGCAAUCGAUACAGAAAGUTT-3′ YTHDF2 siRNA1, 5′-GCGGGUCCAUUACUAGUAATT-3′ YTHDF2 siRNA2, 5′-GCAGUGGGUUCGGUCAUAATT-3′ YTHDF2 siRNA3, 5′-GCACAGAGCAUGGUAACAATT-3′ YTHDF2 siRNA4, 5′-GGACGUUCCCAAUAGCCAATT-3′ Real-Time Quantitative Reverse-Transcription PCR Corneas were dissected at the limbus, and the iris tissue attached to the corneal endothelium was removed. RNA was obtained using an RNA extraction kit (#DP430, Tiangen Biotech, Tianjin, China). The concentration of total RNA was quantified by spectrophotometry (Thermo Fisher Scientific); reference standards were 1.8 < OD260/OD280 < 2.0 and OD260/OD230 > 2.0, where OD is optical density. Complementary DNA (cDNA) was reverse transcribed using Invitrogen M-MLV Reverse Transcriptase (#M1701; Promega, Madison, WI, USA). TB Green Premix Ex Taq II (#RR820A; Takara Bio Inc., Beijing, China) was used for real-time quantitative reverse-transcription PCR (RT-qPCR). The sequences of the primers are provided in the . Gene expression levels were standardized relative to the GAPDH mRNA levels. m6A Modification Content Quantification Total RNA was obtained using the method described above. The m6A content was quantified utilizing the m6A RNA Methylation Quantification Kit (Colorimetric) (#ab185912; Abcam, Cambridge, UK). Specifically, RNA (200 ng) was encapsulated into the wells and incubated with capture and detection antibodies. The absorbance at 450 nm was quantified utilizing a microplate reader (Bio-Tek, Winooski, VT, USA), and the content of m6A was quantified using a colorimetric method. The formula is as follows: m6A% = [(Sample OD – NC OD) ÷ S/(PC OD – NC OD) ÷ P] × 100%, where P refers to the quantity of input positive control (ng), S refers to the quantity of input sample (ng), NC refers to negative control, and PC refers to positive control. Cell Viability The viability of keratocytes was analyzed utilizing the Cell Counting Kit-8 (CCK-8; MedChemExpress, Shanghai, China). Specifically, keratocytes were seeded in 96-well plates (Corning, Corning, NY, USA). The cell density was 5 × 10 3 cells per well. After adding CCK-8, the plates were then incubated for 1 hour under standard keratocyte culture conditions. The OD values of each well were measured at a wavelength of 450 nm using a Bio-Tek microplate reader. Hematoxylin and Eosin Staining Mouse ocular globes were excised and subsequently immersed in paraformaldehyde (4%) solution for 12 hours, dehydrated using graded ethanol, and then encased in paraffin wax at 60°C in the sagittal position. The paraffin blocks containing the embedded eyes were sliced into 5-µm sections. These sections were subjected to a baking process at 60°C for 24 hours. Subsequently, the specimens were subjected to xylene treatment, then rehydrated, and finally stained with hematoxylin and eosin (H&E). These sections were observed with a microscope (DS-Ri2; Nikon, Tokyo, Japan), and images were obtained. Immunohistochemistry Staining Paraffin sections were dewaxed and hydrated. Antigen retrieval was performed using microwave radiation with EDTA buffer (pH 6.0) and allowed to cool naturally. Non-specific antigens were eliminated by 3% hydrogen peroxide. After blocking with normal goat serum for 2 hours, sections were treated overnight at 4°C with primary antibodies against alpha smooth muscle actin (α-SMA, 1:500 dilution; #ab124964; Abcam), fibronectin (1:500 dilution; #ab268020; Abcam), vimentin (1:500 dilution; #ab16700; Abcam), and collagen III (COL3A1, 1:500 dilution; #ab7778; Abcam). The sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG (GK600705; Gene Tech, Shanghai, China) at 37°C for 30 minutes, followed by a 5-minute incubation with 3,3′-diaminobenzidine (DAB; Gene Tech. Positive staining was observed with a Nikon DS-Ri2 microscope, and images were obtained. Western Blotting Corneal tissue and keratocytes were lysed utilizing a radioimmunoprecipitation assay (RIPA) lysis solution (Solarbio Science and Technology, Beijing, China), which contained a mixture of protease inhibitors. Protein concentrations were measured by the Lowry protein assay (Bio-Rad). Equal amounts of proteins (30 µg) were separated by SDS-PAGE on 6% to 12% polyacrylamide gels with running buffer (Tris/glycine/SDS), and transferred to polyvinylidene fluoride (PVDF) membrane (#IPVH00010; Merck, Rahway, NJ, USA) with transfer buffer (Tris/glycine/methanol). The membrane was blocked with 5% skim milk before incubation with Abcam primary antibodies: rabbit anti-α-SMA (1:10000; #ab124964), rabbit anti-COL3A1 (1:1000; #ab7778), rabbit anti-METTL3 (1:1000; #ab195352), rabbit anti-FTO (1:1000; #ab280081), rabbit anti-HSP70 (1:1000; #ab181606), rabbit anti-fibronectin (1:1000; #ab268020), rabbit anti-YTHDF2 (1:1000; #ab220163), and rabbit anti-GAPDH (1:1000; #ab128915). In addition, rabbit anti-HSP90 (1:1000; #4877; Cell Signaling Technology, Danvers, MA, USA), was also used. This incubation was carried out for 12 hours at a temperature of 4°C on a shaker. Subsequently, the PVDF membranes were incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG, 1:10000; #ab205718; Abcam) for 1 hour at 37°C. Blot bands were observed using a chemiluminescence imaging instrument (Tanon, Shanghai, China). Original blots were displayed in the . The bands were quantified using densitometry with ImageJ (National Institutes of Health, Bethesda, MD, USA). The grayscale value for each protein was normalized to that of GAPDH. Differential Gene Analysis The dataset GSE6676, which compared corneas from wild-type mice to those from mice under the influence of high doses of TGF-β, was used to perform a differential analysis of HSP-related genes. The analysis data derived from this dataset was included in . The judgment criteria used were P < 0.05 and |log FC| ≥ 1. m6A Methylation Site Prediction The m6A modification sites of the HSPA1B , HSPA1L , HSP90AA1 , and HSP90AB1 mRNA were predicted using SRAMP (Sequence-Based RNA Adenosine Methylation Sites Predictor). The input sequences were RNA sequences, and the possible m6A modification sites and confidence levels were obtained. Statistical Analysis The data used for the analyses were derived from at least three separate experiments. The differences between the two groups were evaluated using Student's t -test, and one-way ANOVA, in conjunction with the Tukey multiple comparison test, was applied for analyses involving more than two groups. The data are displayed as mean ± SEM. Statistics were deemed significant at P < 0.05. This research used C57BL/6 mice (male, 6–8 weeks old). The mice were obtained from Weitonghua Company (Tianjin, China) and were raised in the Experimental Center of Nankai Hospital in Tianjin. In all animal-related procedures, the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research was followed. All animal experiments received approval from the experimental animal management organization of Nankai Center in Tianjin (approval no. NKYY-DWLL-2023-051). Briefly, general anesthesia was administered to mice using 1% sodium pentobarbital (40–50 mg/kg) intraperitoneally. Surface anesthesia was achieved by applying proparacaine drops to the ocular surface. The mouse cornea was injured using a 1-N NaOH-soaked filter paper with a diameter of 2 mm. The filter paper was placed on the ocular surface for 30 seconds and then removed. Subsequently, mouse eyes were rinsed with 25 mL of saline solution. Tobramycin eye ointment was applied after the injury. The ocular surfaces of the mice were observed using a slit-lamp microscope at 4, 7, 10, 14, 16, 19, 21, 25, 28, and 35 days post-injury, and they were photographed and documented. The mice were euthanized at 4, 14, 21, 28, and 35 days post-injury, and the corneas were collected for analyses. Completely normal and uninjured eyes were used as negative controls. Mice were anesthetized, and the extent of corneal clouding following alkali burns was evaluated using a slit-lamp microscope according to established criteria. Specifically, corneal opacity was graded from 0 to 4, where 0 = completely clear; 1 = slightly hazy, iris and pupil easily visible; 2 = slightly opaque, iris and pupil still detectable; 3 = opaque, pupils hardly detectable; and 4 = completely opaque with no view of the pupil. Three experimenters independently scored opacity, and the final score was the average of their scores. Human keratocytes were obtained from ScienCell Research Laboratories (#6520; Carlsbad, CA, USA). The medium composition for keratocytes was Gibco Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; #11320082; Thermo Fisher Scientific, Waltham, MA, USA), 2% to 5% Gibco fetal bovine serum (#10099141; Thermo Fisher Scientific), and 1% Gibco penicillin/streptomycin (#15140122; Thermo Fisher Scientific). Culture conditions were set at 37°C with 5% carbon dioxide. The keratocytes from the third to fifth passages were used for the subsequent experiments. Keratocytes were stimulated with TGF-β1 (10 ng/mL). For gene silencing of METTL3, FTO, and YTHDF2, small interfering RNAs (siRNAs) targeting the three genes were purchased from Genepharma Co., Ltd. (Shanghai, China). These included METTL3 siRNAs 1 to 4, FTO siRNAs 1 to 3, and YTHDF2 siRNAs 1 to 4. Additionally, a negative control siRNA (si-NC) and a positive control siRNA (si-GAPDH) were also obtained. Transfection was performed utilizing Lipofectamine RNAiMax Transfection Reagent (#13778150; Life Technologies, Carlsbad, CA, USA). The siRNA sequences were as follows: METTL3 siRNA1, 5′-CCUGCAAGUAUGUUCACUATT-3′ METTL3 siRNA2, 5′-GCUACCUGGACGUCAGUAUTT-3′ METTL3 siRNA3, 5′-GCUCAACAUACCCGUACUATT-3′ METTL3 siRNA4, 5′-GGUUGGUGUCAAAGGAAAUTT-3′ FTO siRNA1, 5′-ACACUUGGCUCCCUUAUCUTT-3′ FTO siRNA2, 5′-GUGGCAGUGUACAGUUAUATT-3′ FTO siRNA3, 5′-GGCAAUCGAUACAGAAAGUTT-3′ YTHDF2 siRNA1, 5′-GCGGGUCCAUUACUAGUAATT-3′ YTHDF2 siRNA2, 5′-GCAGUGGGUUCGGUCAUAATT-3′ YTHDF2 siRNA3, 5′-GCACAGAGCAUGGUAACAATT-3′ YTHDF2 siRNA4, 5′-GGACGUUCCCAAUAGCCAATT-3′ Corneas were dissected at the limbus, and the iris tissue attached to the corneal endothelium was removed. RNA was obtained using an RNA extraction kit (#DP430, Tiangen Biotech, Tianjin, China). The concentration of total RNA was quantified by spectrophotometry (Thermo Fisher Scientific); reference standards were 1.8 < OD260/OD280 < 2.0 and OD260/OD230 > 2.0, where OD is optical density. Complementary DNA (cDNA) was reverse transcribed using Invitrogen M-MLV Reverse Transcriptase (#M1701; Promega, Madison, WI, USA). TB Green Premix Ex Taq II (#RR820A; Takara Bio Inc., Beijing, China) was used for real-time quantitative reverse-transcription PCR (RT-qPCR). The sequences of the primers are provided in the . Gene expression levels were standardized relative to the GAPDH mRNA levels. Total RNA was obtained using the method described above. The m6A content was quantified utilizing the m6A RNA Methylation Quantification Kit (Colorimetric) (#ab185912; Abcam, Cambridge, UK). Specifically, RNA (200 ng) was encapsulated into the wells and incubated with capture and detection antibodies. The absorbance at 450 nm was quantified utilizing a microplate reader (Bio-Tek, Winooski, VT, USA), and the content of m6A was quantified using a colorimetric method. The formula is as follows: m6A% = [(Sample OD – NC OD) ÷ S/(PC OD – NC OD) ÷ P] × 100%, where P refers to the quantity of input positive control (ng), S refers to the quantity of input sample (ng), NC refers to negative control, and PC refers to positive control. The viability of keratocytes was analyzed utilizing the Cell Counting Kit-8 (CCK-8; MedChemExpress, Shanghai, China). Specifically, keratocytes were seeded in 96-well plates (Corning, Corning, NY, USA). The cell density was 5 × 10 3 cells per well. After adding CCK-8, the plates were then incubated for 1 hour under standard keratocyte culture conditions. The OD values of each well were measured at a wavelength of 450 nm using a Bio-Tek microplate reader. Mouse ocular globes were excised and subsequently immersed in paraformaldehyde (4%) solution for 12 hours, dehydrated using graded ethanol, and then encased in paraffin wax at 60°C in the sagittal position. The paraffin blocks containing the embedded eyes were sliced into 5-µm sections. These sections were subjected to a baking process at 60°C for 24 hours. Subsequently, the specimens were subjected to xylene treatment, then rehydrated, and finally stained with hematoxylin and eosin (H&E). These sections were observed with a microscope (DS-Ri2; Nikon, Tokyo, Japan), and images were obtained. Paraffin sections were dewaxed and hydrated. Antigen retrieval was performed using microwave radiation with EDTA buffer (pH 6.0) and allowed to cool naturally. Non-specific antigens were eliminated by 3% hydrogen peroxide. After blocking with normal goat serum for 2 hours, sections were treated overnight at 4°C with primary antibodies against alpha smooth muscle actin (α-SMA, 1:500 dilution; #ab124964; Abcam), fibronectin (1:500 dilution; #ab268020; Abcam), vimentin (1:500 dilution; #ab16700; Abcam), and collagen III (COL3A1, 1:500 dilution; #ab7778; Abcam). The sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG (GK600705; Gene Tech, Shanghai, China) at 37°C for 30 minutes, followed by a 5-minute incubation with 3,3′-diaminobenzidine (DAB; Gene Tech. Positive staining was observed with a Nikon DS-Ri2 microscope, and images were obtained. Corneal tissue and keratocytes were lysed utilizing a radioimmunoprecipitation assay (RIPA) lysis solution (Solarbio Science and Technology, Beijing, China), which contained a mixture of protease inhibitors. Protein concentrations were measured by the Lowry protein assay (Bio-Rad). Equal amounts of proteins (30 µg) were separated by SDS-PAGE on 6% to 12% polyacrylamide gels with running buffer (Tris/glycine/SDS), and transferred to polyvinylidene fluoride (PVDF) membrane (#IPVH00010; Merck, Rahway, NJ, USA) with transfer buffer (Tris/glycine/methanol). The membrane was blocked with 5% skim milk before incubation with Abcam primary antibodies: rabbit anti-α-SMA (1:10000; #ab124964), rabbit anti-COL3A1 (1:1000; #ab7778), rabbit anti-METTL3 (1:1000; #ab195352), rabbit anti-FTO (1:1000; #ab280081), rabbit anti-HSP70 (1:1000; #ab181606), rabbit anti-fibronectin (1:1000; #ab268020), rabbit anti-YTHDF2 (1:1000; #ab220163), and rabbit anti-GAPDH (1:1000; #ab128915). In addition, rabbit anti-HSP90 (1:1000; #4877; Cell Signaling Technology, Danvers, MA, USA), was also used. This incubation was carried out for 12 hours at a temperature of 4°C on a shaker. Subsequently, the PVDF membranes were incubated with secondary antibodies (HRP-conjugated goat anti-rabbit IgG, 1:10000; #ab205718; Abcam) for 1 hour at 37°C. Blot bands were observed using a chemiluminescence imaging instrument (Tanon, Shanghai, China). Original blots were displayed in the . The bands were quantified using densitometry with ImageJ (National Institutes of Health, Bethesda, MD, USA). The grayscale value for each protein was normalized to that of GAPDH. The dataset GSE6676, which compared corneas from wild-type mice to those from mice under the influence of high doses of TGF-β, was used to perform a differential analysis of HSP-related genes. The analysis data derived from this dataset was included in . The judgment criteria used were P < 0.05 and |log FC| ≥ 1. The m6A modification sites of the HSPA1B , HSPA1L , HSP90AA1 , and HSP90AB1 mRNA were predicted using SRAMP (Sequence-Based RNA Adenosine Methylation Sites Predictor). The input sequences were RNA sequences, and the possible m6A modification sites and confidence levels were obtained. The data used for the analyses were derived from at least three separate experiments. The differences between the two groups were evaluated using Student's t -test, and one-way ANOVA, in conjunction with the Tukey multiple comparison test, was applied for analyses involving more than two groups. The data are displayed as mean ± SEM. Statistics were deemed significant at P < 0.05. Corneal Pathological Fibrosis Process Following Alkali Burn First, we evaluated the pathological process following alkali burn. Under the slit-lamp microscope, we observed corneal opacity and neovascularization in the corneas of mice following an alkali burn ( A). The corneal opacity score showed a significant increase starting on the fourth day after the alkali burn and continued to rise until reaching its maximum at 21 days. Afterward, it gradually decreased ( B). To clarify the molecular mechanisms underlying corneal fibrosis following an alkali burn, we evaluated the expression of key markers associated with myofibroblast transdifferentiation. These markers included α-SMA, fibronectin, vimentin, and COL3A1. We analyzed corneas at days 4, 14, 21, 28, and 35 after the alkali burn. After an alkali burn injury, upregulated expression of key markers associated with fibroblast activation was observed. Vimentin mRNA expression reached its maximum level on the 14th day and subsequently declined. Western blot and qRT-PCR assays yielded consistent results for both protein and mRNA levels, revealing that the levels of α-SMA, fibronectin, and COL3A1 gradually increased following injury, peaking at day 21 before subsequently declining ( C, D). Furthermore, we conducted H&E staining and immunohistochemical staining on paraffin sections of healthy corneas, as well as corneas at 14 and 21 days after an alkali burn. We found that there was almost no expression of α-SMA, vimentin, fibronectin, or COL3A1 proteins in the normal corneal stroma. However, at 14 and 21 days, the levels of α-SMA, vimentin, COL3A1, and fibronectin were upregulated ( E). These findings suggest that the fibrotic reaction following a corneal alkali burn in mice is primarily observed between 14 and 21 days post-burn, with the strongest fibrotic response occurring at 21 days. Elevated m6A Content and Increased Expression of METTL3 and FTO in Corneal Fibrosis To investigate the changes in m6A modification during the process of corneal fibrosis, we analyzed the corneal m6A content in total RNA at three specific time points: 4, 14, and 21 days after the alkali burn injury. A quantitative analysis of m6A content in the cornea revealed a significant increase following an alkali burn compared to the undamaged cornea ( A). To investigate the key regulatory factors of changes in m6A modification content at various time points following alkali burn injury, we conducted an analysis of the expression of several well-known methyltransferases and demethylases in the cornea at 4 days, 14 days, and 21 days post-injury. On the fourth day after a corneal alkali burn, the mRNA expression levels of Mettl3 and Fto were decreased. On the 14th day after a corneal alkali burn, the mRNA levels of Fto were increased. On the 21st day after a corneal alkali burn, the mRNA levels of Mettl3 and Fto were increased ( B, C). Furthermore, the expression of Ythdf1 showed a decrease on the fourth day, followed by a subsequent significant increase on the 21st day in comparison to the undamaged cornea. Ythdf2 exhibited a significant upregulation in expression levels on the 14th day, and Ythdf3 exhibited a significant reduction in expression levels on the 4th day ( D). METTL3 and FTO had significantly increased protein levels after a corneal alkali burn ( E). YTHDF2 protein expression significantly increased on the 14th day after injury ( E). Increased m6A Content and Variations in m6A-Related Gene Expression in Keratocytes Stimulated by TGF-β1 Research has demonstrated that the expression of TGF-β1 is upregulated in corneal tissue following alkali burn, contributing to corneal repair. , We detected the mRNA expression of Tgf -β1 in corneal tissue at 14 and 21 days post-injury and observed an increase in Tgf-β1 expression ( S1). Consequently, we treated keratocytes with 10-ng/mL TGF- β1 to investigate the role of m6A modification in corneal fibrosis. Microscopic analysis revealed that the untreated group showed keratocytes with a morphology characterized by spindle-shaped or polygonal fibroblast-like cells. After 24 and 48 hours of TGF-β1 stimulation, the keratocytes exhibited a progressive transition toward a flattened and multi-protrusion morphology ( A). The viability of keratocytes decreased after the stimulation of TGF-β1 ( B). We examined the expression of fibrosis-associated marker genes in keratocytes treated with TGF-β1 for 24 and 48 hours. Compared to the control group, the levels of α-SMA, fibronectin, COL3A1, and vimentin significantly increased in the 24-hour and 48-hour groups ( C). A quantitative analysis of m6A content revealed a significant increase in keratocytes stimulated with TGF-β1 for 24 and 48 hours ( D). After a 24-hour period of stimulation, the expression of KIAA1429 in keratocytes increased. After a 48-hour period of stimulation, the expression of methyltransferases and demethylases increased in keratocytes ( E, F). The expression levels of recognition enzymes ( YTHDF1-3 ) in keratocytes remained unchanged before and after stimulation with TGF-β1, as indicated by the lack of significant changes in their expression levels ( G). METTL3 and FTO in keratocytes stimulated with TGF-β1 showed significantly increased protein levels ( H). Differential Analysis of HSPs in Corneal Fibrosis Given the role of HSP70 in modulating the fibrotic response, we analyzed the differential gene expression profiles in the GSE6676 database. Compared to the control group, the cornea of mice overexpressing TGF-β showed upregulation of three heat shock proteins ( Dnajc6 , Hspa12a , Hsp90aa1 ) and downregulation of one heat shock protein ( Hspa1l ). Hspa1b and Hsp90ab1 showed no differential expression ( A, B). The analysis results suggest that proteins from the HSP70 and HSP90 families may be involved in corneal fibrosis, though further research is needed to elucidate differentially expressed genes with FDR (false discovery rate) multiple testing correction. The Expression of HSP70 Is Reduced in Corneal Fibrosis To further study the changes in HSP70 and HSP90 in corneal fibrosis, we first evaluated the mRNA levels of HSP70 and HSP90 after corneal alkali burn. The mRNA levels of Hspa1b and Hspa1l were decreased on days 14 and 21 following the injury ( A); however, the changes in Hsp90aa1 and Hsp90ab1 were not statistically significant ( B). The protein expression of HSP70 at 14 and 21 days was decreased after the alkali burn ( C). The changes in HSP90 protein expression were not significant following the injury ( C). After 24 hours of TGF-β1 stimulation, HSPA1L expression in human keratocytes decreased; after 48 hours, both HSPA1B and HSPA1L levels significantly declined ( D). The levels of HSP90AA1 and HSP90AB1 did not show any statistically significant alteration ( E). Similarly, the level of HSP70 was decreased after TGF-β1 stimulation ( F). The changes in HSP90 protein expression were not significant after TGF-β1 stimulation ( F). These experimental results suggest that the decrease in HSP70 may contribute to the progression of corneal fibrosis. Silencing of METTL3 in Keratocytes Results in Increased Expression of HSP70 To explore the regulatory impact of m6A modification on HSP70 and HSP90 expression, we used the SRAMP to predict m6A sites on the gene sequences of HSPA1B , HSPA1L , HSP90AA1 , and HSP90AB1 . The study findings revealed that the HSPA1B RNA (NM_005346.6) had seven potential m6A sites ( A, ). HSPA1L RNA (NM_005527.4) had eight potential m6A sites ( A). HSP90AA1 RNA (NM_005348.4) had 11 potential m6A sites. HSP90AB1 RNA (NM_001271972.2) had seven potential m6A sites ( B, ). To investigate the correlation between METTL3 and HSP70 and HSP90, we designed four distinct siRNAs specifically targeting METTL3. Compared to the si-NC group, METTL3 expression was significantly decreased in the si-METTL3 (1–4) group. Among these, METTL3 siRNA3, which exhibited the most effective silencing capability, was chosen for subsequent analysis ( C). After downregulating METTL3 expression, a slight decrease in the viability of keratocytes was observed ( D). The qRT-PCR analysis revealed that the downregulation of METTL3 resulted in upregulation of HSPA1B , HSPA1L , and HSP90AB1 mRNA expression while causing a decrease in HSP90AA1 mRNA expression ( E, F). Because both HSPA1B and HSPA1L are genes that encode the HSP70 family, we validated the expression of HSP70 at the protein level. This validation yielded consistent results with the mRNA level ( G). This study indicates that METTL3 may regulate the levels of HSP70 in keratocytes. Silencing of METTL3 Attenuated the Transformation of Keratocytes to Myofibroblasts Based on the above research, we hypothesized that inhibiting METTL3 may potentially suppress the corneal fibrosis by increasing the expression of HSP70. METTL3 mRNA expression increased after TGF-β1 stimulation compared to the control group, whereas silencing METTL3 inhibited this increase ( A). Knockdown of METTL3 resulted in a partial reversal of the decrease in HSPA1B and HSPA1L levels following TGF-β1 stimulation ( B). Additionally, knocking down METTL3 reduced the mRNA expression of α-SMA in keratocytes stimulated with TGF-β1 ( C). The protein level was found to be consistent with the mRNA level ( D– G). The results indicate that downregulating METTL3 increases HSP70 expression in keratocytes, and decreasing COL3A1, α-SMA, and fibronectin expression in TGF-β1 stimulated keratocytes. Effect of Silencing YTHDF2 or FTO on HSP70 Expression and Fibrosis To further investigate the impact of m6A modification on HSP70 expression and corneal fibrosis, we developed siRNAs that specifically target YTHDF2. Among these siRNAs, YTHDF2 siRNA4, which exhibited the highest efficiency in silencing, was chosen for further analysis ( A). Our study showed that, under normal conditions, silencing YTHDF2 did not produce a notable impact on the expression levels of HSP70 in keratocytes ( B, C). However, in keratocytes stimulated with TGF-β1, the knockdown of YTHDF2 resulted in an increase in HSP70 expression, along with a decrease in fibronectin and COL3A1 expression ( D). Meanwhile, we also designed siRNAs that specifically target FTO and chose FTO siRNA2, which exhibited the highest efficiency in silencing, for further study ( E). The inhibition of FTO did not produce a notable impact on the levels of HSP70 in keratocytes ( F– H). Additionally, the expression of key indicators associated with myofibroblast activation in keratocytes stimulated with TGF-β1 after silencing of FTO showed no significant change ( H). First, we evaluated the pathological process following alkali burn. Under the slit-lamp microscope, we observed corneal opacity and neovascularization in the corneas of mice following an alkali burn ( A). The corneal opacity score showed a significant increase starting on the fourth day after the alkali burn and continued to rise until reaching its maximum at 21 days. Afterward, it gradually decreased ( B). To clarify the molecular mechanisms underlying corneal fibrosis following an alkali burn, we evaluated the expression of key markers associated with myofibroblast transdifferentiation. These markers included α-SMA, fibronectin, vimentin, and COL3A1. We analyzed corneas at days 4, 14, 21, 28, and 35 after the alkali burn. After an alkali burn injury, upregulated expression of key markers associated with fibroblast activation was observed. Vimentin mRNA expression reached its maximum level on the 14th day and subsequently declined. Western blot and qRT-PCR assays yielded consistent results for both protein and mRNA levels, revealing that the levels of α-SMA, fibronectin, and COL3A1 gradually increased following injury, peaking at day 21 before subsequently declining ( C, D). Furthermore, we conducted H&E staining and immunohistochemical staining on paraffin sections of healthy corneas, as well as corneas at 14 and 21 days after an alkali burn. We found that there was almost no expression of α-SMA, vimentin, fibronectin, or COL3A1 proteins in the normal corneal stroma. However, at 14 and 21 days, the levels of α-SMA, vimentin, COL3A1, and fibronectin were upregulated ( E). These findings suggest that the fibrotic reaction following a corneal alkali burn in mice is primarily observed between 14 and 21 days post-burn, with the strongest fibrotic response occurring at 21 days. To investigate the changes in m6A modification during the process of corneal fibrosis, we analyzed the corneal m6A content in total RNA at three specific time points: 4, 14, and 21 days after the alkali burn injury. A quantitative analysis of m6A content in the cornea revealed a significant increase following an alkali burn compared to the undamaged cornea ( A). To investigate the key regulatory factors of changes in m6A modification content at various time points following alkali burn injury, we conducted an analysis of the expression of several well-known methyltransferases and demethylases in the cornea at 4 days, 14 days, and 21 days post-injury. On the fourth day after a corneal alkali burn, the mRNA expression levels of Mettl3 and Fto were decreased. On the 14th day after a corneal alkali burn, the mRNA levels of Fto were increased. On the 21st day after a corneal alkali burn, the mRNA levels of Mettl3 and Fto were increased ( B, C). Furthermore, the expression of Ythdf1 showed a decrease on the fourth day, followed by a subsequent significant increase on the 21st day in comparison to the undamaged cornea. Ythdf2 exhibited a significant upregulation in expression levels on the 14th day, and Ythdf3 exhibited a significant reduction in expression levels on the 4th day ( D). METTL3 and FTO had significantly increased protein levels after a corneal alkali burn ( E). YTHDF2 protein expression significantly increased on the 14th day after injury ( E). Research has demonstrated that the expression of TGF-β1 is upregulated in corneal tissue following alkali burn, contributing to corneal repair. , We detected the mRNA expression of Tgf -β1 in corneal tissue at 14 and 21 days post-injury and observed an increase in Tgf-β1 expression ( S1). Consequently, we treated keratocytes with 10-ng/mL TGF- β1 to investigate the role of m6A modification in corneal fibrosis. Microscopic analysis revealed that the untreated group showed keratocytes with a morphology characterized by spindle-shaped or polygonal fibroblast-like cells. After 24 and 48 hours of TGF-β1 stimulation, the keratocytes exhibited a progressive transition toward a flattened and multi-protrusion morphology ( A). The viability of keratocytes decreased after the stimulation of TGF-β1 ( B). We examined the expression of fibrosis-associated marker genes in keratocytes treated with TGF-β1 for 24 and 48 hours. Compared to the control group, the levels of α-SMA, fibronectin, COL3A1, and vimentin significantly increased in the 24-hour and 48-hour groups ( C). A quantitative analysis of m6A content revealed a significant increase in keratocytes stimulated with TGF-β1 for 24 and 48 hours ( D). After a 24-hour period of stimulation, the expression of KIAA1429 in keratocytes increased. After a 48-hour period of stimulation, the expression of methyltransferases and demethylases increased in keratocytes ( E, F). The expression levels of recognition enzymes ( YTHDF1-3 ) in keratocytes remained unchanged before and after stimulation with TGF-β1, as indicated by the lack of significant changes in their expression levels ( G). METTL3 and FTO in keratocytes stimulated with TGF-β1 showed significantly increased protein levels ( H). Given the role of HSP70 in modulating the fibrotic response, we analyzed the differential gene expression profiles in the GSE6676 database. Compared to the control group, the cornea of mice overexpressing TGF-β showed upregulation of three heat shock proteins ( Dnajc6 , Hspa12a , Hsp90aa1 ) and downregulation of one heat shock protein ( Hspa1l ). Hspa1b and Hsp90ab1 showed no differential expression ( A, B). The analysis results suggest that proteins from the HSP70 and HSP90 families may be involved in corneal fibrosis, though further research is needed to elucidate differentially expressed genes with FDR (false discovery rate) multiple testing correction. To further study the changes in HSP70 and HSP90 in corneal fibrosis, we first evaluated the mRNA levels of HSP70 and HSP90 after corneal alkali burn. The mRNA levels of Hspa1b and Hspa1l were decreased on days 14 and 21 following the injury ( A); however, the changes in Hsp90aa1 and Hsp90ab1 were not statistically significant ( B). The protein expression of HSP70 at 14 and 21 days was decreased after the alkali burn ( C). The changes in HSP90 protein expression were not significant following the injury ( C). After 24 hours of TGF-β1 stimulation, HSPA1L expression in human keratocytes decreased; after 48 hours, both HSPA1B and HSPA1L levels significantly declined ( D). The levels of HSP90AA1 and HSP90AB1 did not show any statistically significant alteration ( E). Similarly, the level of HSP70 was decreased after TGF-β1 stimulation ( F). The changes in HSP90 protein expression were not significant after TGF-β1 stimulation ( F). These experimental results suggest that the decrease in HSP70 may contribute to the progression of corneal fibrosis. To explore the regulatory impact of m6A modification on HSP70 and HSP90 expression, we used the SRAMP to predict m6A sites on the gene sequences of HSPA1B , HSPA1L , HSP90AA1 , and HSP90AB1 . The study findings revealed that the HSPA1B RNA (NM_005346.6) had seven potential m6A sites ( A, ). HSPA1L RNA (NM_005527.4) had eight potential m6A sites ( A). HSP90AA1 RNA (NM_005348.4) had 11 potential m6A sites. HSP90AB1 RNA (NM_001271972.2) had seven potential m6A sites ( B, ). To investigate the correlation between METTL3 and HSP70 and HSP90, we designed four distinct siRNAs specifically targeting METTL3. Compared to the si-NC group, METTL3 expression was significantly decreased in the si-METTL3 (1–4) group. Among these, METTL3 siRNA3, which exhibited the most effective silencing capability, was chosen for subsequent analysis ( C). After downregulating METTL3 expression, a slight decrease in the viability of keratocytes was observed ( D). The qRT-PCR analysis revealed that the downregulation of METTL3 resulted in upregulation of HSPA1B , HSPA1L , and HSP90AB1 mRNA expression while causing a decrease in HSP90AA1 mRNA expression ( E, F). Because both HSPA1B and HSPA1L are genes that encode the HSP70 family, we validated the expression of HSP70 at the protein level. This validation yielded consistent results with the mRNA level ( G). This study indicates that METTL3 may regulate the levels of HSP70 in keratocytes. Based on the above research, we hypothesized that inhibiting METTL3 may potentially suppress the corneal fibrosis by increasing the expression of HSP70. METTL3 mRNA expression increased after TGF-β1 stimulation compared to the control group, whereas silencing METTL3 inhibited this increase ( A). Knockdown of METTL3 resulted in a partial reversal of the decrease in HSPA1B and HSPA1L levels following TGF-β1 stimulation ( B). Additionally, knocking down METTL3 reduced the mRNA expression of α-SMA in keratocytes stimulated with TGF-β1 ( C). The protein level was found to be consistent with the mRNA level ( D– G). The results indicate that downregulating METTL3 increases HSP70 expression in keratocytes, and decreasing COL3A1, α-SMA, and fibronectin expression in TGF-β1 stimulated keratocytes. To further investigate the impact of m6A modification on HSP70 expression and corneal fibrosis, we developed siRNAs that specifically target YTHDF2. Among these siRNAs, YTHDF2 siRNA4, which exhibited the highest efficiency in silencing, was chosen for further analysis ( A). Our study showed that, under normal conditions, silencing YTHDF2 did not produce a notable impact on the expression levels of HSP70 in keratocytes ( B, C). However, in keratocytes stimulated with TGF-β1, the knockdown of YTHDF2 resulted in an increase in HSP70 expression, along with a decrease in fibronectin and COL3A1 expression ( D). Meanwhile, we also designed siRNAs that specifically target FTO and chose FTO siRNA2, which exhibited the highest efficiency in silencing, for further study ( E). The inhibition of FTO did not produce a notable impact on the levels of HSP70 in keratocytes ( F– H). Additionally, the expression of key indicators associated with myofibroblast activation in keratocytes stimulated with TGF-β1 after silencing of FTO showed no significant change ( H). Excessive fibrotic repair following severe injury can result in permanent corneal opacity. In this study, we investigated the potential role of m6A and its regulatory factors in the corneal fibrosis response, both in vivo and in vitro. Modification of m6A is a dynamic and reversible process involving methyltransferases and demethylases. A comprehensive analysis of the various regulatory factors of m6A is essential to understanding its role in corneal injury repair. By day 4 following alkali burn of the cornea, the expression of both methyltransferase and demethylase was downregulated, with a more pronounced decrease observed in demethylase. This may help explain the increased content of m6A modification at this stage. During the wound remodeling phase of corneal alkali burn, m6A content and METTL3 expression increased. Moreover, TGF-β1 stimulation in keratocytes increased m6A content and METTL3 expression. This suggests that METTL3 likely plays an important role, and it is also likely that a number of additional factors (e.g., METTL16, RBM15, ZC3H13) contribute to the increase in m6A during corneal wound healing. Previous research has shown that silencing METTL3 in retinal pigment epithelial cells effectively reduces subretinal fibrosis. In this investigation, we observed that METTL3 was significantly upregulated in corneas undergoing a fibrotic response and in keratocytes stimulated with TGF-β1. Additionally, silencing of METTL3 reduced the levels of COL3A1, α-SMA, and fibronectin in keratocytes stimulated by TGF-β1. Although FTO expression was also elevated in corneal fibrosis, this trend did not correspond to changes in m6A modification. Additionally, silencing FTO had no significant effect on the levels of the above-mentioned fibrosis-related indicators in keratocytes stimulated by TGF-β1. In light of the aforementioned findings, we speculate that METTL3 is an important factor responsible for the elevated content of m6A modification in corneal fibrosis and plays a role as a factor promoting corneal fibrosis. The increase in FTO may be a compensatory result of increased modification of m6A content. Research shows that overexpression of HSP70 binds to SMAD2 and SMAD3, restricting their phosphorylation and nuclear translocation. HSP70 can bind to TGF-βI and II receptors (TβRⅠ/TβRⅡ), preventing them from functioning properly. In this research, we found that the levels of HSP70 were significantly reduced in corneas undergoing fibrotic reaction and in keratocytes stimulated by TGF-β1. Additionally, we found that the expression of HSP70 was negatively correlated with the levels of corneal fibrotic markers. This suggests that the decreased expression of HSP70 in the cornea may be one of the factors that promote corneal fibrosis. HSP90 promotes fibrosis by facilitating the folding of TGF-β receptor proteins and protecting them, thereby regulating TGF-β signal transduction. , In TGF-β1–stimulated keratocytes, the mRNA levels of HSP90AA1 and HSP90AB1 decreased; the protein expression of HSP90 increased but without a significant difference. This suggests the need for further research into factors such as post-transcriptional regulation, translation efficiency, protein stability and degradation, post-translational modifications, and feedback mechanisms. Further investigating these factors would provide a comprehensive explanation for the discrepancy between mRNA and protein levels for HSP90 in this study. In addition to HSP90AA1 and HSP90AB1 , human genes encoding HSP90 include HSP90AA2 , HSP90B1 , and HSP90L . Additional studies are needed to explore the relationship between the increased HSP90 protein levels observed in this study and the expression levels of these genes. We identified potential sites for m6A modification on the mRNA sequences of HSP70. Silencing METTL3 in keratocytes increased HSP70 expression and reduced the expression of fibronectin, COL3A1, and α-SMA in keratocytes stimulated with TGF-β1. YTHDF2 specifically recognizes and disrupts the stability of mRNA that contain m6A. , In our study, silencing YTHDF2 in keratocytes stimulated with TGF-β1 increased HSP70 expression and reduced fibronectin and COL3A1 expression in keratocytes. We conclude that the role of METTL3 in corneal fibrosis may be partially attributed to its function in regulating HSP70 expression . Meanwhile, in this experiment, we observed that the downregulation of METTL3 could result in increased expression of KIAA1429 in the methylase complex ( A). It may be related to the compensatory increase in the linkage of the methyltransferase complex to maintain the level of m6A modification that is necessary for the cell. We also observed that downregulation of FTO could lead to elevated ALKBH5 expression ( B). One possible explanation is that the downregulation of FTO results in a significant increase in m6A modification. This situation may stimulate the upregulation or enhanced activity of m6A methylases and ALKBH5 as a form of feedback and compensatory regulation. However, more in-depth research is being conducted to understand the function of m6A methylation-modifying enzymes in corneal fibrosis. In addition, the increase in HSP70 levels may be one of the reasons why METTL3 inhibits fibrosis after downregulation. At present, the treatment of corneal pathological fibrosis remains a challenging issue in clinical practice. METTL3 is the important regulatory factor that causes changes in m6A content. Downregulation of METTL3 can increase the expression of HSP70 and inhibit corneal fibrosis. This discovery could lead to new insights into the mechanism of corneal pathological fibrosis. Our findings indicate that corneal fibrosis after alkali burns is predominantly concentrated between 14 and 21 days post-burn. The most significant corneal fibrosis was observed on the 21st day. METTL3 serves as an important regulatory factor for m6A modification changes in corneal fibrosis. The downregulation of HSP70 expression is a contributing factor in the advancement of corneal fibrosis. Silencing of METTL3 reduces the levels of fibrosis related indicators in keratocytes stimulated by TGF-β1. Meanwhile, YTHDF2 is involved in mediating the expression of HSP70 in corneal fibrosis, and silencing of YTHDF2 reduces the expression of COL3A1 and fibronectin in keratocytes stimulated by TGF-β1. Therefore, METTL3 modulates corneal fibrosis by regulating the levels of HSP70 in a YTHDF2-dependent manner, thereby playing a role in the corneal injury repair process. This research offers novel avenues for preventing pathological corneal fibrosis. Supplement 1 Supplement 2 Supplement 3 |
Effectiveness of Gastric Cancer Endoscopic Screening in Intermediate-Risk Countries: Protocol for a Systematic Review and Meta-Analysis | c8c9bcad-4f38-4afa-a860-f2208f2683e9 | 11833270 | Surgical Procedures, Operative[mh] | Gastric adenocarcinoma, also known as gastric cancer (GC), is a malignant neoplasm resulting from anarchic growth of gastric mucosal gland cells . It is a heterogeneous disease with multiple clinical, histological, and molecular variables that influence its presentation . According to data from The Global Cancer Observatory 2022, GC is the fifth most common and the fifth most lethal neoplasm worldwide . Its geographical distribution is not homogeneous: there are high-risk areas with incidence 20 and more (age-standardized rate measured per 100,000 people-year; eg, Japan, South Korea, or Mongolia); intermediate-risk (IR) areas with age-standardized rate 10 and more and less than 20 (eg, Portugal or China); and low-risk areas with age-standardized rate 10 or less (United States, United Kingdom, Switzerland, or Germany) . Diagnosis at an advanced stage and the aggressiveness of the disease result in a 5-year survival rate of between 20% and 40% . In contrast, early-stage GC has an excellent prognosis, with a 5-year survival rate of greater than 90%, and can often be treated with minimally invasive and organ-sparing techniques such as endoscopic resection . Therefore, early detection of cancer greatly increases the chances of successful treatment. The 2 components of cancer screening are early diagnosis and screening. The former focuses on detecting symptomatic patients as early as possible, while the latter involves testing healthy individuals to identify those with the disease before symptoms occur . The symptoms of stomach cancer are nonspecific and usually develop late, meaning that detection based on symptoms would not detect the disease in its early stages. On the other hand, screening programs should be implemented when: their effectiveness has been demonstrated; the resources needed to implement them are sufficient to cover the target population; there are facilities to confirm the diagnosis, treatment, and follow-up of those with positive results; and the prevalence of the disease is high enough to justify the effort and cost of the screening program . Therefore, the decision to implement the screening program requires that these conditions are met. Several methods have been proposed to screen for GC, namely serological markers, biomarkers, molecular or genetic tests, or more invasive techniques such as upper gastrointestinal endoscopy . The latter, which allows direct visualization of the gastric mucosa and collection of biopsies for histological examination, is considered the gold standard technique for definitive diagnosis of GC . In some high-risk countries, such as Japan and South Korea, GC screening programs have been in place for several years and have been shown to reduce mortality and increase early detection and 5-year survival rates . The same has been demonstrated in screening programs developed in China . In Western IR countries, several studies have been developed to determine the relevance of screening and how it might be developed. Of particular importance was the study developed by Areia et al , which showed that endoscopic screening for GC in IR countries can be cost-effective when combined with endoscopic screening for colorectal cancer. This has been included in the recommendations of the United European Gastroenterology and the European Society of Gastrointestinal Endoscopy . Despite this evidence and the recommendations, most Western Societies continue to recommend screening only in selected populations with high-risk factors for GC . Furthermore, and to the best of our knowledge, there are no systematic reviews on GC endoscopic screening in IR countries. With this systematic literature review, we aim to analyze the scientific evidence published until September 2024 on the cost-effectiveness of endoscopic screening for GC in IR countries. This study aims to determine the effectiveness and economic viability of endoscopic GC screening in IR countries , which will answer the research question, “What is the effectiveness of endoscopic screening for GC in IR countries?” These objectives are defined according to the Population, Intervention, Comparison, Outcome and Study Design (PICOS) framework . Population, Intervention, Comparison, Outcome and Study Design (PICOS) framework for the systematic review and meta-analysis. PICOS question: What is the effectiveness of endoscopic screening for gastric cancer (GC) in intermediate-risk countries? Population: Asymptomatic population of intermediated-risk countries (countries with incidence age-standardized rate 10-20 per 100.000 person/years: Tajikistan, Iran, Azerbaijan, Kyrgyzstan, Bhutan, Belarus, Peru, Mali, Chile, Costa Rica, Democratic People Republic of Korea, China, Kazakhstan, Russian Federation, Viet Nam, Estonia, Colombia, Portugal, Ecuador, Albania, Guadeloupe [France], Guatemala, Latvia, Armenia, Turkmenistan, Myanmar, Samoa, Turkey, Lithuania, Lao People’s Democratic Republic, Sao Tome and Principe, Afghanistan, Martinique [France], Brunei Darussalam, Zimbabwe, and Uzbekistan), between 40 and 80 years of age, without diagnostic of GC or precancerous lesions. Intervention: Endoscopic screening for GC. Comparison: No screening for GC. Outcome: The effectiveness of endoscopic screening of GC is defined as the detection rate of Helicobacter pylori; detection rate of precancer lesions; detection rate of GC; detection rate of early GC; stage at diagnosis; mortality rate of GC of screened versus nonscreened patients; 5-year survival rate of GC screened; and costs of screening program. Study designs: Randomized controlled trials, nonrandomized controlled trials, cohort studies, case-control studies, cross-sectional studies, and cost-effectiveness studies. This study will follow the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and PICOS criteria for comprehensive assessment. Eligibility Criteria The inclusion criteria include studies published as free full papers in English, Portuguese, or Spanish until September 2024, from countries with an IR for GC (countries with incidence age-standardized rate 10-20 per 100,000 person/years: Tajikistan, Iran, Azerbaijan, Kyrgyzstan, Bhutan, Belarus, Peru, Mali, Chile, Costa Rica, Democratic People Republic of Korea, China, Kazakhstan, Russian Federation, Viet Nam, Estonia, Colombia, Portugal, Ecuador, Albania, Guadeloupe [France], Guatemala, Latvia, Armenia, Turkmenistan, Myanmar, Samoa, Turkey, Lithuania, Lao People’s Democratic Republic, Sao Tome and Principe, Afghanistan, Martinique [France], Brunei Darussalam, Zimbabwe, Uzbekistan); eligible study designs are randomized controlled trials, nonrandomized controlled trials, cohort studies, case-control studies, cross-sectional studies, and cost-effectiveness studies; and no filters or restrictions related to year of publication or publication status, will be applied. The exclusion criteria include systematic reviews and other types of reviews, meta-analyses, case series, case reports, and other publication types such as editorials, commentaries, notes, letters, and opinions. Information Sources The information sources for this systematic review are electronic databases (MEDLINE, SCOPUS, Embase, and Web of Science). To capture additional studies (gray literature), manual searches will include published abstracts from the most relevant international gastroenterology and endoscopy conferences, clinical trial registries for ongoing studies, reference lists of included studies or other published reviews or meta-analyses. Authors of unpublished studies or published studies in which data are missing will be contacted to confirm eligibility. Search Strategy The search strategy follows the Peer Review of Electronic Search Strategies (PRESS) guidelines . The search will be conducted in MEDLINE, SCOPUS, Embase, and Web of Science and the search strategy will be tailored to each database using database-specific search terms . Selection Process The references, including the abstract of studies retrieved after searching each database, will be imported to Rayyan (Rayyan Systems, Inc), an open-source software that allows several reviewers to blindly access the inclusion or exclusion of studies in literature reviews registering the entire process. This software will analyze and merge the potential duplicates, under operator validation. The initial selection process will be carried out by 2 independent reviewers (MBM and NP) based on the title and abstract. Studies will be classified as “included,” “excluded,” or “maybe.” The selection process will be blinded and supervised by 2 other independent authors (FT and ABP). Conflicts and “maybe” assessments will be resolved between the 2 reviewers with the support of 2 authors (FT and ABP). After the final list of included studies is obtained, the full texts are retrieved. The full text of each study will be analyzed by 3 independent reviewers (MBM, NP, and ABP), and a decision taken on inclusion or exclusion. The list of items included is exported to a Microsoft Excel database where the data to be analyzed are entered. The agreement rate will be calculated using Cohen κ, Egger regression, and Begg regression and will be reported at all stages of the selection process (title screening, abstract screening, and full-text screening). Data Collection Process Data from included studies will be collected by 2 reviewers (MBM and NP) and inserted into a Microsoft Excel database with data coding. The data collected will be cataloged in 2 categories: efficacy or effectiveness data and economic data, and separate meta-analyses will be performed for each of these categories. For the statistical analyses and meta-analysis of the data collected, the authors will use SPSS (version 29.0.2.0; IBM Corp) and Jamovi (version 2.5; The jamovi Project). Bibliographic references will be managed in Zotero (Corporation for Digital Scholarship) software. Artificial intelligence tools may be used to extract and analyze data. Data Items The data collection form will include the items or variables defined in outcomes and other variables . Risk of Bias in Individual Studies To minimize the risk of bias, the included studies will undergo a quality analysis according to Cochrane risk of bias tools, RoB 2 of randomized trials and ROBINS-I, for nonrandomized trials; Newcastle-Ottawa Quality Assessment Scale for case-control and cohort studies; and National Heart, Lung and Blood Institute study quality assessment tools for cross-sectional studies. The Consensus on Health Economic Criteria (CHEC) list will be used for the assessment of the methodological quality of cost-effectiveness studies. Studies with low quality or high risk of bias will be reported and not be used for meta-analysis. Data Synthesis If it is possible to collect quantitative data from the selected studies, we will perform 2 separate meta-analysis: one to report the effect size of 5 outcomes in studies reporting efficacy or effectiveness of endoscopic screening for GC (detection rate of premalignant lesions, detection rate of GC, detection rate of early GC, and 5-year survival rate and mortality of GC diagnosed in screening programs); other to report the effect size of cost-effectiveness of endoscopic screening for GC in IR countries. If appropriate (at least 2 studies per outcome) , pooled rates and odds ratio along with 95% CIs will be calculated for GC detection, early GC detection, adherence to the screening program, and GC mortality using random-effects model, using SPSS, Jamovi, and Meta-Essentials for Microsoft Excel (Erasmus Research Institute of Management). The I 2 homogeneity will also be performed to check the need for subgroup analysis (moderation analysis). If it is not possible to carry out a meta-analysis due to insufficient data, an executive summary is prepared that summarizes the data from the studies included in the systematic review. Meta-Bias(es) After evaluating the biases of individual studies and ensuring that all studies that may be of interest for their conclusions are integrated into the review, a summary of the risks of bias in the meta-analysis or meta-synthesis will be presented. Confidence in Cumulative Evidence The strength of the body of evidence will be assessed with Grading of Recommendations, Assessment, Development and Evaluation (GRADE) . The inclusion criteria include studies published as free full papers in English, Portuguese, or Spanish until September 2024, from countries with an IR for GC (countries with incidence age-standardized rate 10-20 per 100,000 person/years: Tajikistan, Iran, Azerbaijan, Kyrgyzstan, Bhutan, Belarus, Peru, Mali, Chile, Costa Rica, Democratic People Republic of Korea, China, Kazakhstan, Russian Federation, Viet Nam, Estonia, Colombia, Portugal, Ecuador, Albania, Guadeloupe [France], Guatemala, Latvia, Armenia, Turkmenistan, Myanmar, Samoa, Turkey, Lithuania, Lao People’s Democratic Republic, Sao Tome and Principe, Afghanistan, Martinique [France], Brunei Darussalam, Zimbabwe, Uzbekistan); eligible study designs are randomized controlled trials, nonrandomized controlled trials, cohort studies, case-control studies, cross-sectional studies, and cost-effectiveness studies; and no filters or restrictions related to year of publication or publication status, will be applied. The exclusion criteria include systematic reviews and other types of reviews, meta-analyses, case series, case reports, and other publication types such as editorials, commentaries, notes, letters, and opinions. The information sources for this systematic review are electronic databases (MEDLINE, SCOPUS, Embase, and Web of Science). To capture additional studies (gray literature), manual searches will include published abstracts from the most relevant international gastroenterology and endoscopy conferences, clinical trial registries for ongoing studies, reference lists of included studies or other published reviews or meta-analyses. Authors of unpublished studies or published studies in which data are missing will be contacted to confirm eligibility. The search strategy follows the Peer Review of Electronic Search Strategies (PRESS) guidelines . The search will be conducted in MEDLINE, SCOPUS, Embase, and Web of Science and the search strategy will be tailored to each database using database-specific search terms . The references, including the abstract of studies retrieved after searching each database, will be imported to Rayyan (Rayyan Systems, Inc), an open-source software that allows several reviewers to blindly access the inclusion or exclusion of studies in literature reviews registering the entire process. This software will analyze and merge the potential duplicates, under operator validation. The initial selection process will be carried out by 2 independent reviewers (MBM and NP) based on the title and abstract. Studies will be classified as “included,” “excluded,” or “maybe.” The selection process will be blinded and supervised by 2 other independent authors (FT and ABP). Conflicts and “maybe” assessments will be resolved between the 2 reviewers with the support of 2 authors (FT and ABP). After the final list of included studies is obtained, the full texts are retrieved. The full text of each study will be analyzed by 3 independent reviewers (MBM, NP, and ABP), and a decision taken on inclusion or exclusion. The list of items included is exported to a Microsoft Excel database where the data to be analyzed are entered. The agreement rate will be calculated using Cohen κ, Egger regression, and Begg regression and will be reported at all stages of the selection process (title screening, abstract screening, and full-text screening). Data from included studies will be collected by 2 reviewers (MBM and NP) and inserted into a Microsoft Excel database with data coding. The data collected will be cataloged in 2 categories: efficacy or effectiveness data and economic data, and separate meta-analyses will be performed for each of these categories. For the statistical analyses and meta-analysis of the data collected, the authors will use SPSS (version 29.0.2.0; IBM Corp) and Jamovi (version 2.5; The jamovi Project). Bibliographic references will be managed in Zotero (Corporation for Digital Scholarship) software. Artificial intelligence tools may be used to extract and analyze data. The data collection form will include the items or variables defined in outcomes and other variables . To minimize the risk of bias, the included studies will undergo a quality analysis according to Cochrane risk of bias tools, RoB 2 of randomized trials and ROBINS-I, for nonrandomized trials; Newcastle-Ottawa Quality Assessment Scale for case-control and cohort studies; and National Heart, Lung and Blood Institute study quality assessment tools for cross-sectional studies. The Consensus on Health Economic Criteria (CHEC) list will be used for the assessment of the methodological quality of cost-effectiveness studies. Studies with low quality or high risk of bias will be reported and not be used for meta-analysis. If it is possible to collect quantitative data from the selected studies, we will perform 2 separate meta-analysis: one to report the effect size of 5 outcomes in studies reporting efficacy or effectiveness of endoscopic screening for GC (detection rate of premalignant lesions, detection rate of GC, detection rate of early GC, and 5-year survival rate and mortality of GC diagnosed in screening programs); other to report the effect size of cost-effectiveness of endoscopic screening for GC in IR countries. If appropriate (at least 2 studies per outcome) , pooled rates and odds ratio along with 95% CIs will be calculated for GC detection, early GC detection, adherence to the screening program, and GC mortality using random-effects model, using SPSS, Jamovi, and Meta-Essentials for Microsoft Excel (Erasmus Research Institute of Management). The I 2 homogeneity will also be performed to check the need for subgroup analysis (moderation analysis). If it is not possible to carry out a meta-analysis due to insufficient data, an executive summary is prepared that summarizes the data from the studies included in the systematic review. After evaluating the biases of individual studies and ensuring that all studies that may be of interest for their conclusions are integrated into the review, a summary of the risks of bias in the meta-analysis or meta-synthesis will be presented. The strength of the body of evidence will be assessed with Grading of Recommendations, Assessment, Development and Evaluation (GRADE) . Our initial search of the 4 electronic databases, using descriptors adapted for each database, identified 1615 studies of potential interest . After excluding duplicates, 969 studies were screened for title and abstract. Of these, 75 were selected for full-text screening. We retained 44 studies for data analysis and the remaining 31 studies were excluded for the following reasons: wrong population (n=2), same population as another study (n=2), wrong screening method (n=6), wrong outcome (n=18), wrong study design (n=2), and wrong publication type (n=1). In addition, our manual search identified 23 publications of potential interest, of which 2 were selected for the data extraction phase. Currently, the study is in the early stages of data extraction and risk of bias assessment and is expected to be published in the first quarter of 2025. This protocol outlines the methods for a systematic literature review and meta-analysis of published primary scientific studies on the effectiveness of endoscopic GC screening in IR countries. This study will provide results on the following outcomes: frequency of screening, age range covered by screening, screening adherence rate, number of biopsies, Helicobacter pylori diagnosis rate, detection rate of premalignant lesions, GC detection rate, early GC detection rate, lethality rate, 5-year survival rate in patients diagnosed with GC at screening, and incremental cost-effectiveness ratio. To the best of our knowledge, this is the first systematic review of the effectiveness of endoscopic screening for GC in IR countries and it is expected that the presentation of these results will shed light on the relevance of endoscopic GC screening in these populations. The benefit of performing screening upper endoscopy for asymptomatic individuals for GC remains controversial . Due to the high burden of GC, countries in East Asia such as Japan and Korea have implemented nationwide population-based GC screening strategies to reduce incidence and mortality . Other studies have shown that endoscopic screening for GC is cost-effective in the IR to high-risk population . However, to date, we have not found a systematic review that synthesizes the results of all studies conducted in IR countries, and for this reason, our systematic review is of particular interest to inform health policy makers in IR countries about the effectiveness of endoscopic screening for GC in this type of population. An extensive search of databases and gray literature will be conducted to identify all relevant studies. However, this review may have some important limitations. First, it is important to bear in mind that the majority of IR countries, especially Western countries, do not yet have population-based screening for this pathology, which may make it difficult to obtain primary studies on this topic from these countries. Second, although China is an IR country with endoscopic screening for GC, some studies reporting the results of these screening programs are written in Chinese and cannot be included in our review. Nevertheless, we will conduct an intensive search of the databases to ensure that we obtain studies written in English, Portuguese, or Spanish that report the results of these screening programs. Third, on the other hand, the Chinese territory is so large that risk varies greatly between different areas of the country, which may introduce a significant bias if some included studies are based only on results of high-risk areas, as this type of population may have different characteristics from those observed in other areas of China or other IR countries. These limitations will be reported in the final paper of the systematic review so that our results can be interpreted rigorously and truthfully. The final paper of the systematic review and meta-analysis will be published in the first quarter of 2025. With this systematic review and meta-analysis, we hope to contribute to the design of GC screening strategies in IR countries, with the primary goal of reducing mortality from this disease. In the future, it may be necessary to update this systematic review as a new consortium. Towards Gastric Cancer Screening Implementation in the European Union has recently been established in Europe to study and implement GC screening in this area. |
Spatial distribution of | 1c2c28f7-3802-4b18-b4df-df02fd8fd98f | 11233921 | Anatomy[mh] | The scarcity of tools for consistent detection of intact Mtb bacilli, bacillary remnants, or secreted antigens within human tissue hampers investigation into Mtb physiology in vivo and the spatial distribution of bacilli within host cells and TB lesions. For diagnostic and research purposes, identification of Mtb in sputum, needle biopsies, resected specimens, or postmortem tissue relies on microscopic observation of Ziehl-Neelsen (ZN)-stained, acid-fast Mtb bacilli (AFB). However, acid fastness in Mtb is highly variable, and ZN staining is not Mtb -specific. We used RNAscope, an RNA in situ hybridisation (RISH) technique, to detect Mtb mRNA within intact and disintegrating bacilli in ZN-positive and -negative human TB tissue. Employing RNAscope and dual ZN/immunohistochemistry (IHC) staining, we found that Mtb mRNA and secreted antigens accumulate within cells in histologically normal and diseased airways, including bronchiolar epithelial cells. This finding enhances our understanding of the historical imprints left by Mtb in vivo and sheds light on clinically significant mechanisms of immune subversion. Further, the identification of two phenotypically distinct Mtb populations based on differential Ag85B expression may provide unique insight into efficacy of anti-TB drugs, transmission dynamics, and immune mechanisms involved in bacillary clearance. Importantly, we identified Mtb mRNA in a ZN-negative antemortem biopsy from a patient that was initially diagnosed with histoplasmosis but was diagnosed with TB following autopsy. Our findings have important implications for the study of Mtb physiology in vivo and for TB diagnosis in complex cases where biopsy material is ZN-negative. Firstly, despite assumptions that Mtb mRNA instability would limit its use as a marker of infection, we observed intra- and extracellular RNAscope signal patterns in antemortem and postmortem human TB tissues. Secondly, detection of subsets of Mtb mRNAs in tuberculous tissue may facilitate assessment of Mtb viability, alternate physiological states, or treatment efficacy and is expected to contribute to a more nuanced understanding of the temporal aspects of Mtb infection. Thirdly, our observation that discrete bacilli in close proximity exhibit differential Ag85B expression suggests that host environmental signals are unlikely to influence expression of this antigen, and that Ag85B immunostaining alone would underestimate bacillary burden. Finally, the diagnostic capacity inherent in the detection of Mtb mRNA in ZN-negative human tissue implies that RNAscope holds promise as a diagnostic tool across a diverse spectrum of human pathogens. Tuberculosis (TB) continues to be a threat to global health, with significant morbidity and mortality. Accordingly, the capacity to detect Mtb bacilli accurately and consistently is critical for definitive TB diagnosis and research efforts. Culturing of clinical isolates remains the gold standard for TB diagnosis but takes weeks to complete. Alternatively, interferon gamma release assays are widely used to diagnose active or latent TB using whole blood. , , More rapid diagnostic approaches that rely on PCR amplification of specific Mtb genomic sequences are highly sensitive and are well-suited for detection of Mtb in paucibacillary specimens. , , , , However, despite the important diagnostic utility of these and other molecular approaches, few methods exist that allow consistent spatial detection of Mtb bacilli, bacillary remnants, or secreted antigens within human tissue, which limits investigation of Mtb physiology and bacillary distribution. Detection of Mtb bacilli in tissue samples has traditionally relied on microscopic observation of acid-fast bacilli (AFB) following Ziehl-Neelsen (ZN) histochemical staining. However, since Mtb can be cultured from ZN-negative tissue specimens, ZN staining can produce false negatives, delaying therapeutic intervention. , Further, ZN staining provides no insight into the physiological state of Mtb, i.e ., whether the bacillus is alive or dead. Importantly, studies in the 1940s showed that single Mtb colonies are stratified into three layers containing non-acid-fast, weakly acid-fast, and strongly acid-fast bacilli. Also, in the 1950s, Canetti reported the presence of “ghost” bacilli in the caseum characterized as translucent, disintegrating cells with variable acid-fastness, which became scarcer as the age of necrosis increased. Unfortunately, the implications of this important biological property, i.e ., acid-fastness, have been underappreciated. Since Mtb can transition from ZN-positive to ZN-negative in vivo and in response to drug therapy, there is a strong unmet need for improved detection of Mtb in human specimens. One approach used to bypass the limitations of ZN and Auramine O staining is detection of Mtb protein antigens in clinical samples by immunohistochemistry (IHC). , , , Mtb has highly regulated secretion systems that contribute to its virulence and secreted antigens play diverse roles in promoting TB disease. Mtb in culture has been shown to secrete over one thousand protein antigens. These include abundant, well-studied CFP-10, ESAT-6, , MPT64 , and the antigen 85 complex comprised of Ag85A, B and C proteins. , However, while these antigens are expressed within Mtb -infected host cells, little is known about the distribution of Mtb antigens in human TB lesions. , , , , Thus, despite the utility of antibodies for detecting Mtb surface or secreted antigens, , , , , diagnosis of TB based on IHC alone is problematic due to insufficient specificity and challenging standardisation. Strategies based on in situ hybridisation (ISH) using oligonucleotide probes have been used to detect Mtb DNA , , , rRNA , , , or mRNA in clinical specimens. However, studies comparing RNA in situ hybridisation (RISH) methods with routine histochemistry and/or immunohistochemistry (IHC) for the spatial identification of Mtb and Mtb mRNA are lacking, especially in human paucibacillary and abacillary tissues. A novel RISH platform, referred to as RNAscope, was recently developed. RNAscope uses an innovative probe design to increase specificity and the signal-to-noise ratio to allow visualisation of single mRNA molecules (as puncta) within cells in formalin-fixed paraffin-embedded (FFPE) tissue. , , , , Since the quantity of a specific Mtb mRNA molecule can exceed that of its corresponding gene by orders of magnitude, RNAscope allows assessment of the spatial and microenvironmental distribution of mRNA in vivo , thereby providing further insight into mycobacterial physiology and heterogeneity. Therefore, the goal of this study was to determine the spatial distribution of Mtb bacilli and/or bacillary remnants, including mRNA and secreted antigens, within distinct microenvironments in human TB lesions. To this end, we adapted RNAscope to detect Mtb in ZN-positive human pulmonary and extrapulmonary TB tissue and in ZN-negative ante- and postmortem tissue with proven active TB. Further, we employed IHC to detect Mtb antigens to investigate whether Mtb bacilli in human tissue exist as a homogenous population regarding antigen production. Ethics and human subjects This study was approved by the University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC; class approval study number BCA 535/16, and BE019/13) and the University of Alabama at Birmingham (UAB) Institutional Review Board (IRB; study numbers IRB-300008174 and IRB-300008174-2). Consent for research use of autopsy material is included in the UAB authorization for autopsy consent form signed by the decedent's next of kin. Mtb -infected lung tissues were obtained from three HIV-positive patients with TB undergoing lung resection for removal of irreversibly damaged lobes or lungs (bronchiectasis and/or cavitary lung disease) at King DinuZulu Hospital Complex, a tertiary center for patients with TB in Durban, South Africa. Written informed consent was obtained from all participants. All patients undergoing lung resection for TB had completed a full 6- to 9-month course of anti-TB treatment or up to two years of treatment for drug-resistant TB. Patients were assessed for the extent of pulmonary disease (cavitation and/or bronchiectasis) via high-resolution computed tomography (HRCT). The fitness of each patient to withstand a thoracotomy and lung resection was determined by using the Karnofsky score, 6-min-walk test, spirometry, and arterial blood gas measurement. Assessment of patients with massive hemoptysis included their general condition, effort tolerance before hemoptysis, arterial blood gas measurement, serum albumin concentration, and HRCT imaging of the chest. On gross assessment, all pneumonectomies or lobectomies were bronchiectatic, hemorrhagic, variably fibrotic, and atelectatic and contained visible tubercles. Mtb -infected testicular tissue with features of tuberculous epididymo-orchitis was obtained from a HIV-positive patient with TB following resection. Granulomas showed caseous necrosis and numerous AFB were identified. Lung tissue from an HIV-negative neonate was obtained postmortem from each lobe of the left and right lung. No infectious pathogens, granulomata, or neoplastic infiltrates were observed. Antemortem tissues from CT-guided needle biopsies (inguinal lymph node, retroperitoneal lymph node, and bone marrow) and postmortem tissues (lung, periaortic lymph node, liver, and bone marrow) were obtained from an HIV-negative patient undergoing treatment at the University of Alabama at Birmingham Hospital in Birmingham, Alabama. Given the small number of surgically resected and postmortem tissue specimens, sex and gender were not taken into consideration in the study design. Sex, stated as male and female, was self-reported. See for additional details regarding human subjects and tissues. Histology Human tissue specimens processed at the Africa Health Research Institute (AHRI) were aseptically removed and fixed in 10% neutral buffered formalin (Sigma–Aldrich, cat # HT501850). Specimens were processed in a Sakura Tissue-Tek VIP6 vacuum filtration tissue processor using a xylene-free protocol and embedded in paraffin using a Thermo Fisher Histostar embedding station. For H&E staining, the formalin-fixed, paraffin-embedded (FFPE) tissue blocks were cut into 4 μm sections, mounted on Superfrost Plus charged slides (Thermo Fisher cat # 22-037-246), heated at 56 °C for 15 min, deparaffinised through 2 changes of xylene and rehydrated through descending grades of alcohol to water. H&E staining was performed by placing slides in hematoxylin for 5 min, washing in tap water for 2 min, bluing in lithium carbonate for 1 min, rinsing in tap water for 2 min and counter staining with eosin for 5 min before a final rinse in tap water for 2 min. Slides were dehydrated in ascending grades of alcohol, cleared in xylene, and the coverslip was mounted using DPX (Distyrene, Plasticizer, and Xylene, Sigma–Aldrich, cat # 06522). For ZN staining, FFPE tissue blocks were cut into 2 μm sections, mounted on charged slides, and heated at 56 °C for 15 min. Mounted tissue sections were dewaxed in xylene followed by rinsing in 100% ethanol and 1 change of 95% ethanol. Slides were incubated with heated carbol fuchsin (MediaMage, cat #M00385) for 15 min and then washed in running tap water. Three percent acid alcohol was applied to the slide for 30 s to decolorize or until sections appeared clear. Slides were then washed in running tap water for 2 min, then counterstained with methylene blue. Slides were rinsed under running water, dehydrated, and the coverslip was mounted using DPX. Human tissue specimens processed at UAB were fixed in 10% neutral buffered formalin and embedded in paraffin. H&E, ZN, and Grocott-Gomori Methenamine Silver (GMS) staining were performed in the UAB Anatomic Pathology Laboratory according to standard clinical laboratory protocols. Prior to staining, formalin-fixed, paraffin-embedded (FFPE) tissue blocks were cut into 4 μm sections and mounted on glass slides. H&E staining was performed using a Leica Autostainer XL automated slide stainer that performs deparaffinisation, dehydration, staining, dehydration, and clearing steps using VENTANA reagents (Roche Diagnostics). Following deparaffinisation, ZN staining was performed essentially as detailed above, except slides were incubated with carbol fuchsin for 1 h. Following deparaffinisation, GMS staining was performed by hydrating sections in distilled water followed by exposure to 5% chromic acid for 30 min at RT. After rinsing in tap water, slides were placed in a 2% sodium bisulfite solution for 5 min and rinsed with distilled water. Slides were then transferred to methenamine-silver nitrate solution (a combination of 20 mL of 5% silver nitrate solution, 400 mL of a 3% methenamine solution, and 2 mL of 3% sodium borate) for 30 min at 56 °C, or until full color development. Slides were then placed in a 0.5% gold chloride solution for 5 min, rinsed with distilled water, and transferred into 5% sodium thiosulfate for 5 min. After rinsing with distilled water, slides were counterstained with 0.1% light green solution for 3 min, rinsed, and dehydrated. Coverslips were mounted with Richard-Allan Scientific Cytoseal XYL (Thermo Fisher cat # 22-050-262). Immunohistochemistry FFPE tissues were cut into 2 μm thick sections, mounted on charged slides, and heated at 56 °C for 15 min on a hotplate. Mounted sections were dewaxed in two changes of xylene followed by rinsing in 2 changes of 100% ethanol and 1 change of 95% ethanol. Slides were then rinsed in tap water for 2 min followed by antigen retrieval via Heat Induced Epitope Retrieval (HIER) in trisodium citrate (pH 6.0) for 30 min. Slides were cooled for 15 min and rinsed in tap water for 2 min. Endogenous peroxide activity was blocked using 3% hydrogen peroxide (Leica Biosystems Novolink Polymer Detection Systems, cat # RE7157) for 10 min at room temperature (RT). Slides were then rinsed in phosphate-buffered saline containing 0.1% Tween 20 (PBST) and blocked with protein block (Leica Novolink Systems, cat # RE7158) for 5 min at RT. Sections were incubated with unconjugated primary antibody directed against Mtb antigen 85B (Ag85B; Abcam cat # ab43019, RRID: AB_776575 , 1:500), Mtb Early Secreted Antigenic Target 6 (ESAT-6; Abcam cat # ab26246, RRID: AB_449032 , 1:500) or Mtb Uncharacterised Surface Protein (USP; Lifespan Bioscience cat # LS-C683286, 1:500) followed by rinsing in PBST and incubated with enzyme-linked polymer (Leica Novolink Systems, cat # RE7161) for 30 min at RT. Slides were then rinsed and stained with 3,3′-Diaminobenzidine (DAB) chromogen (Leica Novolink Systems, cat # RE7162) for 5 min, rinsed under running water and counterstained with hematoxylin for 2 min. Slides were rinsed in tap water, blued in 3% ammoniated water for 30 s, rinsed in tap water, dehydrated in ascending grades of alcohol, cleared in xylene, and the coverslip was mounted using DPX. RNAscope The RNAscope 2.5 High Definition (HD)-Red assay kit (Advanced Cell Diagnostics [ACD] cat # 322350) employed here uses a red chromogen to visualise target mRNA. All RNAscope probe sets were purchased from ACD. To ensure probe set specificity, a design algorithm was used to examine each probe for potential cross-reactivity against the host cell transcriptome (for detecting single mRNAs within eukaryotic or prokaryotic cells) or against transcriptomes of related organisms (for identifying specific organisms). We used a positive control RNAscope probe set comprised of 16 probe pairs directed against bp 139–989 of human peptidylprolyl isomerase B (PPIB) mRNA (ACD cat # 313901). The negative control probe set consists of 10 probe pairs directed against bp 414–862 of Bacillus subtilis dihydrodipicolinate reductase ( dapB ) mRNA (ACD cat # 310043). The Mtb -specific probe set (ACD cat # 552911) consists of 120 probe pairs directed against mRNAs from six genes (20 probe pairs per gene): secreted l -alanine dehydrogenase ( ald ), catalase-peroxidase-peroxynitritase T ( katG ), trehalose-6-phosphate phosphatase ( otsB1 ), resuscitation-promoting factor A ( rpfA ), resuscitation-promoting factor B ( rpfB ), and ATP-binding protein (ABC transporter) ( irtB ). RNAscope 2.5 High Definition (HD)-Red assays were performed essentially as recommended by the manufacturer, with modifications as stated below. Briefly, FFPE human lung, testicle, and lymph node tissue blocks were cut into 4 μm sections and mounted on Superfrost Plus charged slides. Control RNAscope slides (ACD cat # 310045) premounted with thin sections of a FFPE HeLa cell pellet were also used. Slides were heated at 60 °C for 1 h on a hotplate and deparaffinised at RT in two changes of xylene followed by two changes of 100% ethanol to remove the xylene. Tissue sections were pretreated with hydrogen peroxide (ACD cat # 322381) for 10 min and rinsed with distilled water. Target retrieval was performed by incubating the tissue sections in 1x RNAscope target retrieval reagent (ACD cat # 322000) at 100 °C for 30 min (lung, testicular and lymph node tissue) or 15 min (control HeLa cell pellet). Sections were immediately rinsed in distilled water, then in 100% ethanol, and air died. A hydrophobic barrier was created around the tissue section using a ImmEdge hydrophobic barrier pen (ACD cat # 310018). All tissues underwent protease treatment by applying RNAscope Protease Plus solution (ACD cat # 322381) onto each tissue section at 40 °C for 30 min (lung, testicular and lymph node tissue) or 20 min (control HeLa cell pellet) in a HybEZ oven (ACD cat# 321711) to permeabilise cells and increase probe accessibility. Prior to addition of RNAscope probe sets, the solubilized probes were heated at 40 °C for 10 min and cooled to RT. RNAscope probes were dropped onto the tissue sections which were placed in a HybEZ oven (ACD cat # 321711) for 2 h at 40 °C. Slides were then washed in 2 changes of fresh 1x RNAscope wash buffer (ACD cat # 310091) for 2 min at RT. A series of six signal amplification steps followed, each comprised of dropping the amplifier reagent on the section followed by incubation for 15 or 30 min at either RT or 40 °C, with each step followed by 2 rinses in 1x wash buffer according to the manufacturer's protocol. Following amplification, 120 μl of red chromogen solution (a 1:60 ratio of RED-B and RED-A) was dropped onto the sections and left at RT for 10 min. Excess chromogen was then tilted off the slides followed by rinsing in 1 change of distilled water and 1 change of tap water. Tissue sections were then counterstained with a 50% hematoxylin and water solution at RT for 2 min followed by 3 dips in 0.02% ammonia water to visualise cell nuclei. Slides were then washed in 2 changes of tap water followed by drying at 60 °C for 15 min. Coverslips were mounted using RNAscope VectaMount medium (ACD cat # 321584) which is compatible with the red chromogen. Imaging was performed on a slide scanner as stated below. Microscopy and imaging Resected lung specimens (tuberculous and neonatal), testicle, and antemortem inguinal lymph node needle biopsy specimens examined by H&E, ZN, IHC ( Mtb Ag85B, ESAT-6, USP) or RNAScope were imaged at AHRI using a Hamamatsu NDP slide scanner (NanoZoomer RS2, Model C10730-12) using its NDP.View2 viewing software. Antemortem inguinal and retroperitoneal lymph node specimens and postmortem lung, liver, bone marrow and periaortic lymph node specimens examined by H&E, ZN, and GMS staining were imaged at UAB using an Olympus BX50 microscope with a DP23 digital microscope camera with Olympus cellSens Entry 3.2 image acquisition software (Build 23706). Automatic exposure settings were used after manual white balance. Image resolution was set at “Full 3088 x 2076”. HALO® analysis Whole-slide image analysis was performed using Halogen-Assisted Light Optimisation (HALO) v3.6.4134 (Indica Labs, Corrales, NM). HALO uses image analysis algorithms to automate the analysis and quantitation of RNAscope Mtb mRNA puncta in TB tissue specimens. HALO was used to calculate the number of mRNA puncta, measure puncta intensity, and assess spatial distribution. Regions of interest (ROIs) were drawn using the annotation tools provided in the HALO platform. The ISH module v4.2.3 (Indica Labs, Corrales, NM) was used to detect RNAscope signal puncta. For visualisation, the Area Quantification module v2.4 was used. For both modules, the magnification was set to “1” with the parameters described in . Statistics Results are shown as Mean ± SD. All graphs were plotted using GraphPad Prism v6.04 (GraphPad Software Inc., USA) and the statistical significance was calculated by applying Student's t-test or One-way ANOVA. Differences were considered statistically significant at P values less than 0.05. Exact P values are included in the data plots for statistical evaluation. Role of funders The funders of this work played no part in the experimental design, data collection, data analysis and/or interpretation, or creation of this manuscript. This study was approved by the University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC; class approval study number BCA 535/16, and BE019/13) and the University of Alabama at Birmingham (UAB) Institutional Review Board (IRB; study numbers IRB-300008174 and IRB-300008174-2). Consent for research use of autopsy material is included in the UAB authorization for autopsy consent form signed by the decedent's next of kin. Mtb -infected lung tissues were obtained from three HIV-positive patients with TB undergoing lung resection for removal of irreversibly damaged lobes or lungs (bronchiectasis and/or cavitary lung disease) at King DinuZulu Hospital Complex, a tertiary center for patients with TB in Durban, South Africa. Written informed consent was obtained from all participants. All patients undergoing lung resection for TB had completed a full 6- to 9-month course of anti-TB treatment or up to two years of treatment for drug-resistant TB. Patients were assessed for the extent of pulmonary disease (cavitation and/or bronchiectasis) via high-resolution computed tomography (HRCT). The fitness of each patient to withstand a thoracotomy and lung resection was determined by using the Karnofsky score, 6-min-walk test, spirometry, and arterial blood gas measurement. Assessment of patients with massive hemoptysis included their general condition, effort tolerance before hemoptysis, arterial blood gas measurement, serum albumin concentration, and HRCT imaging of the chest. On gross assessment, all pneumonectomies or lobectomies were bronchiectatic, hemorrhagic, variably fibrotic, and atelectatic and contained visible tubercles. Mtb -infected testicular tissue with features of tuberculous epididymo-orchitis was obtained from a HIV-positive patient with TB following resection. Granulomas showed caseous necrosis and numerous AFB were identified. Lung tissue from an HIV-negative neonate was obtained postmortem from each lobe of the left and right lung. No infectious pathogens, granulomata, or neoplastic infiltrates were observed. Antemortem tissues from CT-guided needle biopsies (inguinal lymph node, retroperitoneal lymph node, and bone marrow) and postmortem tissues (lung, periaortic lymph node, liver, and bone marrow) were obtained from an HIV-negative patient undergoing treatment at the University of Alabama at Birmingham Hospital in Birmingham, Alabama. Given the small number of surgically resected and postmortem tissue specimens, sex and gender were not taken into consideration in the study design. Sex, stated as male and female, was self-reported. See for additional details regarding human subjects and tissues. Human tissue specimens processed at the Africa Health Research Institute (AHRI) were aseptically removed and fixed in 10% neutral buffered formalin (Sigma–Aldrich, cat # HT501850). Specimens were processed in a Sakura Tissue-Tek VIP6 vacuum filtration tissue processor using a xylene-free protocol and embedded in paraffin using a Thermo Fisher Histostar embedding station. For H&E staining, the formalin-fixed, paraffin-embedded (FFPE) tissue blocks were cut into 4 μm sections, mounted on Superfrost Plus charged slides (Thermo Fisher cat # 22-037-246), heated at 56 °C for 15 min, deparaffinised through 2 changes of xylene and rehydrated through descending grades of alcohol to water. H&E staining was performed by placing slides in hematoxylin for 5 min, washing in tap water for 2 min, bluing in lithium carbonate for 1 min, rinsing in tap water for 2 min and counter staining with eosin for 5 min before a final rinse in tap water for 2 min. Slides were dehydrated in ascending grades of alcohol, cleared in xylene, and the coverslip was mounted using DPX (Distyrene, Plasticizer, and Xylene, Sigma–Aldrich, cat # 06522). For ZN staining, FFPE tissue blocks were cut into 2 μm sections, mounted on charged slides, and heated at 56 °C for 15 min. Mounted tissue sections were dewaxed in xylene followed by rinsing in 100% ethanol and 1 change of 95% ethanol. Slides were incubated with heated carbol fuchsin (MediaMage, cat #M00385) for 15 min and then washed in running tap water. Three percent acid alcohol was applied to the slide for 30 s to decolorize or until sections appeared clear. Slides were then washed in running tap water for 2 min, then counterstained with methylene blue. Slides were rinsed under running water, dehydrated, and the coverslip was mounted using DPX. Human tissue specimens processed at UAB were fixed in 10% neutral buffered formalin and embedded in paraffin. H&E, ZN, and Grocott-Gomori Methenamine Silver (GMS) staining were performed in the UAB Anatomic Pathology Laboratory according to standard clinical laboratory protocols. Prior to staining, formalin-fixed, paraffin-embedded (FFPE) tissue blocks were cut into 4 μm sections and mounted on glass slides. H&E staining was performed using a Leica Autostainer XL automated slide stainer that performs deparaffinisation, dehydration, staining, dehydration, and clearing steps using VENTANA reagents (Roche Diagnostics). Following deparaffinisation, ZN staining was performed essentially as detailed above, except slides were incubated with carbol fuchsin for 1 h. Following deparaffinisation, GMS staining was performed by hydrating sections in distilled water followed by exposure to 5% chromic acid for 30 min at RT. After rinsing in tap water, slides were placed in a 2% sodium bisulfite solution for 5 min and rinsed with distilled water. Slides were then transferred to methenamine-silver nitrate solution (a combination of 20 mL of 5% silver nitrate solution, 400 mL of a 3% methenamine solution, and 2 mL of 3% sodium borate) for 30 min at 56 °C, or until full color development. Slides were then placed in a 0.5% gold chloride solution for 5 min, rinsed with distilled water, and transferred into 5% sodium thiosulfate for 5 min. After rinsing with distilled water, slides were counterstained with 0.1% light green solution for 3 min, rinsed, and dehydrated. Coverslips were mounted with Richard-Allan Scientific Cytoseal XYL (Thermo Fisher cat # 22-050-262). FFPE tissues were cut into 2 μm thick sections, mounted on charged slides, and heated at 56 °C for 15 min on a hotplate. Mounted sections were dewaxed in two changes of xylene followed by rinsing in 2 changes of 100% ethanol and 1 change of 95% ethanol. Slides were then rinsed in tap water for 2 min followed by antigen retrieval via Heat Induced Epitope Retrieval (HIER) in trisodium citrate (pH 6.0) for 30 min. Slides were cooled for 15 min and rinsed in tap water for 2 min. Endogenous peroxide activity was blocked using 3% hydrogen peroxide (Leica Biosystems Novolink Polymer Detection Systems, cat # RE7157) for 10 min at room temperature (RT). Slides were then rinsed in phosphate-buffered saline containing 0.1% Tween 20 (PBST) and blocked with protein block (Leica Novolink Systems, cat # RE7158) for 5 min at RT. Sections were incubated with unconjugated primary antibody directed against Mtb antigen 85B (Ag85B; Abcam cat # ab43019, RRID: AB_776575 , 1:500), Mtb Early Secreted Antigenic Target 6 (ESAT-6; Abcam cat # ab26246, RRID: AB_449032 , 1:500) or Mtb Uncharacterised Surface Protein (USP; Lifespan Bioscience cat # LS-C683286, 1:500) followed by rinsing in PBST and incubated with enzyme-linked polymer (Leica Novolink Systems, cat # RE7161) for 30 min at RT. Slides were then rinsed and stained with 3,3′-Diaminobenzidine (DAB) chromogen (Leica Novolink Systems, cat # RE7162) for 5 min, rinsed under running water and counterstained with hematoxylin for 2 min. Slides were rinsed in tap water, blued in 3% ammoniated water for 30 s, rinsed in tap water, dehydrated in ascending grades of alcohol, cleared in xylene, and the coverslip was mounted using DPX. The RNAscope 2.5 High Definition (HD)-Red assay kit (Advanced Cell Diagnostics [ACD] cat # 322350) employed here uses a red chromogen to visualise target mRNA. All RNAscope probe sets were purchased from ACD. To ensure probe set specificity, a design algorithm was used to examine each probe for potential cross-reactivity against the host cell transcriptome (for detecting single mRNAs within eukaryotic or prokaryotic cells) or against transcriptomes of related organisms (for identifying specific organisms). We used a positive control RNAscope probe set comprised of 16 probe pairs directed against bp 139–989 of human peptidylprolyl isomerase B (PPIB) mRNA (ACD cat # 313901). The negative control probe set consists of 10 probe pairs directed against bp 414–862 of Bacillus subtilis dihydrodipicolinate reductase ( dapB ) mRNA (ACD cat # 310043). The Mtb -specific probe set (ACD cat # 552911) consists of 120 probe pairs directed against mRNAs from six genes (20 probe pairs per gene): secreted l -alanine dehydrogenase ( ald ), catalase-peroxidase-peroxynitritase T ( katG ), trehalose-6-phosphate phosphatase ( otsB1 ), resuscitation-promoting factor A ( rpfA ), resuscitation-promoting factor B ( rpfB ), and ATP-binding protein (ABC transporter) ( irtB ). RNAscope 2.5 High Definition (HD)-Red assays were performed essentially as recommended by the manufacturer, with modifications as stated below. Briefly, FFPE human lung, testicle, and lymph node tissue blocks were cut into 4 μm sections and mounted on Superfrost Plus charged slides. Control RNAscope slides (ACD cat # 310045) premounted with thin sections of a FFPE HeLa cell pellet were also used. Slides were heated at 60 °C for 1 h on a hotplate and deparaffinised at RT in two changes of xylene followed by two changes of 100% ethanol to remove the xylene. Tissue sections were pretreated with hydrogen peroxide (ACD cat # 322381) for 10 min and rinsed with distilled water. Target retrieval was performed by incubating the tissue sections in 1x RNAscope target retrieval reagent (ACD cat # 322000) at 100 °C for 30 min (lung, testicular and lymph node tissue) or 15 min (control HeLa cell pellet). Sections were immediately rinsed in distilled water, then in 100% ethanol, and air died. A hydrophobic barrier was created around the tissue section using a ImmEdge hydrophobic barrier pen (ACD cat # 310018). All tissues underwent protease treatment by applying RNAscope Protease Plus solution (ACD cat # 322381) onto each tissue section at 40 °C for 30 min (lung, testicular and lymph node tissue) or 20 min (control HeLa cell pellet) in a HybEZ oven (ACD cat# 321711) to permeabilise cells and increase probe accessibility. Prior to addition of RNAscope probe sets, the solubilized probes were heated at 40 °C for 10 min and cooled to RT. RNAscope probes were dropped onto the tissue sections which were placed in a HybEZ oven (ACD cat # 321711) for 2 h at 40 °C. Slides were then washed in 2 changes of fresh 1x RNAscope wash buffer (ACD cat # 310091) for 2 min at RT. A series of six signal amplification steps followed, each comprised of dropping the amplifier reagent on the section followed by incubation for 15 or 30 min at either RT or 40 °C, with each step followed by 2 rinses in 1x wash buffer according to the manufacturer's protocol. Following amplification, 120 μl of red chromogen solution (a 1:60 ratio of RED-B and RED-A) was dropped onto the sections and left at RT for 10 min. Excess chromogen was then tilted off the slides followed by rinsing in 1 change of distilled water and 1 change of tap water. Tissue sections were then counterstained with a 50% hematoxylin and water solution at RT for 2 min followed by 3 dips in 0.02% ammonia water to visualise cell nuclei. Slides were then washed in 2 changes of tap water followed by drying at 60 °C for 15 min. Coverslips were mounted using RNAscope VectaMount medium (ACD cat # 321584) which is compatible with the red chromogen. Imaging was performed on a slide scanner as stated below. Resected lung specimens (tuberculous and neonatal), testicle, and antemortem inguinal lymph node needle biopsy specimens examined by H&E, ZN, IHC ( Mtb Ag85B, ESAT-6, USP) or RNAScope were imaged at AHRI using a Hamamatsu NDP slide scanner (NanoZoomer RS2, Model C10730-12) using its NDP.View2 viewing software. Antemortem inguinal and retroperitoneal lymph node specimens and postmortem lung, liver, bone marrow and periaortic lymph node specimens examined by H&E, ZN, and GMS staining were imaged at UAB using an Olympus BX50 microscope with a DP23 digital microscope camera with Olympus cellSens Entry 3.2 image acquisition software (Build 23706). Automatic exposure settings were used after manual white balance. Image resolution was set at “Full 3088 x 2076”. Whole-slide image analysis was performed using Halogen-Assisted Light Optimisation (HALO) v3.6.4134 (Indica Labs, Corrales, NM). HALO uses image analysis algorithms to automate the analysis and quantitation of RNAscope Mtb mRNA puncta in TB tissue specimens. HALO was used to calculate the number of mRNA puncta, measure puncta intensity, and assess spatial distribution. Regions of interest (ROIs) were drawn using the annotation tools provided in the HALO platform. The ISH module v4.2.3 (Indica Labs, Corrales, NM) was used to detect RNAscope signal puncta. For visualisation, the Area Quantification module v2.4 was used. For both modules, the magnification was set to “1” with the parameters described in . Results are shown as Mean ± SD. All graphs were plotted using GraphPad Prism v6.04 (GraphPad Software Inc., USA) and the statistical significance was calculated by applying Student's t-test or One-way ANOVA. Differences were considered statistically significant at P values less than 0.05. Exact P values are included in the data plots for statistical evaluation. The funders of this work played no part in the experimental design, data collection, data analysis and/or interpretation, or creation of this manuscript. RNAscope cellular and tissue controls RNAscope is a highly specific and sensitive RISH technique that combines multiple paired “Z” oligonucleotide probes with signal amplification steps to allow visual detection of single mRNA molecules in tissue samples as colored dots, or puncta, while preserving tissue architecture ( a). Since a single mRNA molecule can be bound by one probe pair resulting in a faint punctum, or by several probe pairs resulting in an intensely-colored punctum, it is the number of puncta, not the color intensity of individual puncta, that reflects the abundance of target mRNAs. We posited that RNAscope could detect mRNA in bacilli, infected host cells, and antemortem and/or postmortem human specimens ( b). We first validated RNAscope assays by exposing thin sections of a HeLa cell pellet to a positive control probe set directed against human peptidylprolyl isomerase B ( PPIB ) mRNA or a negative control probe set specific for Bacillus subtilis dihydrodipicolinate reductase ( dapB ) mRNA resulting in robust signals and no signal, respectively. The Mtb -specific RNAscope probe set (120 probe pairs) targets six mRNAs to provide specificity and sensitivity. We tested the Mtb probe set against human neonatal lung tissue, which showed no signal as expected . Lastly, to verify the integrity of human specimens for RNAscope analysis, we applied the PPIB positive control probe set to testicular tissue from a patient with TB which showed robust signals . Overall, these control assays demonstrate that RNAscope is compatible with the tissue processing protocols for our human specimens. RNAscope detects intact and disintegrated Mtb bacilli, and mRNA in human tissue To compare the ability of RNAscope and ZN staining to detect Mtb bacilli in situ , we examined seminiferous tubules containing AFB obtained from a patient with active TB. Since the structurally complex Mtb cell wall is considered a barrier , to oligonucleotide probes, conditions were optimised to allow Mtb RNAscope probes to enter intact bacilli. RNAscope identified Mtb bacillary rods inside and outside the seminiferous tubules ( b, d, f) that are visually similar to ZN-stained bacillary rods ( a, c, e). RNAscope yielded a spectrum of signal shapes: bacilli with halos, bacillus-sized puncta (consistent with the diameter of a vertical bacillus within the cut plane), diffused halos, and smaller puncta ( f, g, h–m), which were also observed within host cells ( g). In summary, improved permeabilisation permits RNAscope probes to detect Mtb bacillary rods. Secondly, a halo surrounding bacilli suggests mRNA leakage, indicating cellular disintegration that supports Canetti's findings of bacillary disintegration and loss of acid-fastness in vivo . Thirdly, our findings show that Mtb mRNA is stable enough for detection with RNAscope in numerous microenvironments. These findings provide new biological insight into bacillary morphology in vivo and cell death, which cannot be obtained through ZN staining. RNAscope detects Mtb mRNA in ZN-negative human TB lung tissue We next examined the utility of RNAscope for identifying Mtb or remnants of infection, i.e ., mRNA, in ZN-negative human TB tissue. ZN staining of formalin-fixed, paraffin-embedded (FFPE) lung tissue from a patient with active TB showed numerous extracellular and intracellular bacilli ( a). However, a different FFPE lung tissue specimen from the same patient was ZN-negative. This is unsurprising as most human pulmonary TB granulomas contain few, if any, ZN-positive Mtb , which is consistent with historical studies. In contrast, RNAscope analysis of this ZN-negative tissue revealed numerous RNAscope signal puncta within necrotic granulomas ( b and c). RNAscope puncta were also abundant in bronchiolar epithelial cells ( d), which were ZN-negative ( e) and antigen 85B (Ag85B)-positive ( f). These data demonstrate that Mtb mRNA is abundantly present in ZN-negative lung tissue sections of a confirmed case of pulmonary TB and that Mtb mRNA is sufficiently stable for detection by RNAscope. RNAscope and Ag85B immunostaining suggest the bronchiolar epithelial layer as an overlooked entry portal for Mtb infection. Mtb mRNA and secreted antigens accumulate in the cytoplasm of host cells In a study of individuals who died from causes other than TB, Mtb DNA was detected in alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts via in situ PCR analysis. Even though AFB were not shown, these findings are in accord with early reports , , , that Mtb can persist in lung tissue without histological evidence of TB lesions. Consistent with those studies, we identified accumulated Mtb mRNA in the cytoplasm of several alveolar epithelial cells ( a–d) in a signal pattern distinct from the intracellular puncta that represent single Mtb mRNA molecules. We refer to such cells as host-cell accumulated Mtb RNA (HAMR) cells. Mtb mRNA is present in HAMR cells within adjacent regions ( b and c), consolidated alveolar areas ( d), and within lymphocytic aggregates at the periphery of necrotic granulomas ( e). The functional significance of Mtb mRNA within HAMR cells is unknown; however, these mRNAs may act as pathogen-associated molecular patterns (PAMPs) as has been shown for mRNAs in conventional innate immune cells , , , , to modulate immunity. Secreted Mtb antigens have been identified via IHC staining in Mtb -infected human lung tissue. , , , , Similarly, we used IHC to detect Mtb secreted antigen 85B (Ag85B) in the same pulmonary and testicular specimens used for RNAscope analysis . We observed Ag85B accumulation in numerous cell types ( f) including alveolar macrophages and epithelioid histiocytes ( g). In summary, our data demonstrate that cells from histologically normal and diseased airways accumulate Mtb mRNA and secreted antigens. Spatial distribution of Mtb RNA in host microenvironments Our findings show that RNAscope can identify Mtb mRNA to reveal prior history of infection, which is not possible with ZN or Auramine O staining. Since each RNAscope punctum represents one Mtb mRNA molecule, we used the HALO® image analysis platform to count Mtb mRNA puncta, expressed as the number of Mtb mRNA puncta per square micron, in necrotic and non-necrotic granulomas, terminal and pulmonary bronchi, lymphocytic aggregates, and adjacent tissue from resected TB lung tissue ( a). To avoid potentially confounding variables such age, sex, tissue pathology bias, and experimental variability, we selected a single ZN-negative TB tissue specimen containing the aforementioned microenvironments. We reasoned that quantifying Mtb mRNA puncta within each of the four distinct regions of the necrotic granuloma (necrotic, granulomatous inflammatory, fibrotic, and lymphocytic; b) could help inform the pathophysiology of granuloma formation. We found that the necrotic zone contains the fewest Mtb mRNA puncta per square micron, followed by increases in the number of puncta within the granulomatous inflammatory, fibrotic, and lymphocytic zones ( c), indicating that Mtb mRNA is distributed in accordance with discrete pathophysiological zones that may contribute to granuloma formation. We also classified non-necrotic granulomas ( d) into lymphocytic and non-necrotic zones and found that the lymphocytic zone exhibited significantly more Mtb mRNA puncta than the non-necrotic zone ( e). We next quantified Mtb mRNA puncta in terminal and respiratory bronchioles, adjacent tissue, and lymphocytic aggregates distant from necrotic or non-necrotic granulomas and compared these values to those from regions within necrotic and non-necrotic lesions. We found that the number of Mtb mRNA puncta in the terminal bronchioles, lymphocytic aggregates, and adjacent tissue were the highest of all areas examined ( f). Further, the terminal bronchioles exhibited the highest average puncta optical density (OD) ( g), suggesting the presence of longer, more intact Mtb mRNAs containing more RNAscope probe binding sequences. In short, the zonal distribution of Mtb mRNA within the granuloma offers a new perspective into the evolution of granuloma formation and clinical course of disease that ZN or other bacillary staining methods cannot. Lastly, the spatial distribution of Mtb mRNA provides new insight into the previous impact of Mtb within human tissue. Identification of phenotypically distinct populations of Mtb in vivo Continuously changing microenvironments within the tuberculous lung, the specific bacillary load, and a variable spectrum of TB lesions , , , , , strongly suggest that at least two distinct Mtb populations, live and dead, must exist in vivo . There is currently no method that accurately distinguishes metabolically living from dead Mtb bacilli within tissue specimens; however, since MPT64 is secreted during active Mtb growth, the presence of MPT64 in sputum has been suggested as a marker of Mtb viability. Here, we tested the hypothesis that ZN staining combined with IHC for secreted Mtb antigens can identify phenotypically diverse Mtb populations in vivo . We examined testicular tissue whose seminiferous tubules contain numerous bacilli and are bordered by a basement membrane that separates the tubule from the sparsely-infected surrounding tissue, which aids in evaluating the specificity of Mtb antigen immunostaining. H&E histology revealed the presence of substantial cellular debris which overlaps with Mtb RNAscope signals . Mtb bacilli colocalise with strong Ag85B, ESAT-6, and Uncharacterised Surface Protein (USP) positivity in sequential tissue sections . While these antigens are typically associated with the Mtb cell wall, clear delineation of the bacillary rod shape is not always possible. In some cases ( e.g. , USP, ESAT-6), positivity was seen as scattered, brown patches, whereas some rod-like shapes were observed, consistent with previous studies. , , , These findings suggest that Ag85B, ESAT-6, and USP staining patterns may not always match the rod-like morphology of Mtb . To determine whether distinct microenvironments can contribute to differential antigen production, we examined whether all AFB produce Ag85B. Intriguingly, our dual ZN/Ag85B IHC stain demonstrated that not all AFB produce Ag85B. This was evident by numerous ZN-positive/Ag85B-negative bacilli close to Ag85B-positive bacilli ( a–d, ). Ag85-positive and ZN-positive/Ag85B-negative bacilli were observed extracellularly and within host cells that did, or did not, accumulate Ag85B ( e–i). Similar to Canetti, we also identified “ghost” bacilli that are weakly acid fast ( f). Taken together, these data demonstrate that two phenotypically distinct Mtb populations exist in human TB tissue: Ag85B-positive and Ag85-negative bacilli. This finding strongly suggests that Ag85B IHC alone may underestimate the number of bacilli. This has important implications for TB diagnosis and pathogenesis since mixed populations of Mtb may differently influence diagnosis, pathogenesis, and immunity. Using RNAscope and secreted Mtb antigens as tools for guiding TB therapy To evaluate the potential of RNAscope to guide clinical intervention, we examined tissue specimens from a 61-year-old female initially diagnosed with disseminated histoplasmosis. Biopsies of inguinal and retroperitoneal lymph nodes were performed 414 and 13 days before hospitalisation, respectively ( a). Both biopsy specimens were ZN-negative and revealed granulomatous inflammation . On hospital day 18, bronchoscopy with bronchoalveolar lavage (BAL) showed a negative gram stain and AFB smear. The patient died on hospital day 19 with the cause of death attributed to complications of sepsis. Autopsy revealed disseminated granulomatous inflammation of the lung, liver, bone marrow, and periaortic lymph node . ZN staining showed diffuse involvement by AFB , and the cause of death was amended to be complications from disseminated TB. Culture of antemortem BAL fluid revealed the presence of pansensitive Mtb complex 47 days after hospital admission ( a). With longitudinal specimens available, we subjected the ZN-negative inguinal lymph node biopsy obtained 414 days prior to hospital admission ( b) to RNAscope analysis and found numerous Mtb mRNA transcripts ( c). Quantitation of Mtb mRNA puncta using HALO® analysis showed a higher number of puncta around and inside giant cells and lymphocytic aggregates compared to adjacent lymphoid tissue and the whole biopsy ( d) with no difference in average puncta optical density (OD) ( e). This suggests that the number of Mtb mRNA puncta can help predict pathophysiological abnormalities in human TB tissue. The detection of Mtb via RNAscope is further supported by strong Ag85B positivity in giant cells ( f and g) and surrounding lymphocytes ( h) in the antemortem inguinal lymph node specimen. ZN-negative antemortem bone marrow biopsy ( i and j) and ZN-positive postmortem periaortic lymph node ( k) specimens also demonstrated Ag85B positivity. In this clinical scenario, application of RNAscope detection of Mtb mRNA and Ag85 IHC to biopsy tissue could have positively identified Mtb , providing much earlier diagnosis of disseminated TB, possibly enabling effective TB treatment. These findings suggest that detection of Mtb mRNA and antigens has diagnostic value in complex TB cases where sputum or biopsy material is ZN-negative. RNAscope is a highly specific and sensitive RISH technique that combines multiple paired “Z” oligonucleotide probes with signal amplification steps to allow visual detection of single mRNA molecules in tissue samples as colored dots, or puncta, while preserving tissue architecture ( a). Since a single mRNA molecule can be bound by one probe pair resulting in a faint punctum, or by several probe pairs resulting in an intensely-colored punctum, it is the number of puncta, not the color intensity of individual puncta, that reflects the abundance of target mRNAs. We posited that RNAscope could detect mRNA in bacilli, infected host cells, and antemortem and/or postmortem human specimens ( b). We first validated RNAscope assays by exposing thin sections of a HeLa cell pellet to a positive control probe set directed against human peptidylprolyl isomerase B ( PPIB ) mRNA or a negative control probe set specific for Bacillus subtilis dihydrodipicolinate reductase ( dapB ) mRNA resulting in robust signals and no signal, respectively. The Mtb -specific RNAscope probe set (120 probe pairs) targets six mRNAs to provide specificity and sensitivity. We tested the Mtb probe set against human neonatal lung tissue, which showed no signal as expected . Lastly, to verify the integrity of human specimens for RNAscope analysis, we applied the PPIB positive control probe set to testicular tissue from a patient with TB which showed robust signals . Overall, these control assays demonstrate that RNAscope is compatible with the tissue processing protocols for our human specimens. Mtb bacilli, and mRNA in human tissue To compare the ability of RNAscope and ZN staining to detect Mtb bacilli in situ , we examined seminiferous tubules containing AFB obtained from a patient with active TB. Since the structurally complex Mtb cell wall is considered a barrier , to oligonucleotide probes, conditions were optimised to allow Mtb RNAscope probes to enter intact bacilli. RNAscope identified Mtb bacillary rods inside and outside the seminiferous tubules ( b, d, f) that are visually similar to ZN-stained bacillary rods ( a, c, e). RNAscope yielded a spectrum of signal shapes: bacilli with halos, bacillus-sized puncta (consistent with the diameter of a vertical bacillus within the cut plane), diffused halos, and smaller puncta ( f, g, h–m), which were also observed within host cells ( g). In summary, improved permeabilisation permits RNAscope probes to detect Mtb bacillary rods. Secondly, a halo surrounding bacilli suggests mRNA leakage, indicating cellular disintegration that supports Canetti's findings of bacillary disintegration and loss of acid-fastness in vivo . Thirdly, our findings show that Mtb mRNA is stable enough for detection with RNAscope in numerous microenvironments. These findings provide new biological insight into bacillary morphology in vivo and cell death, which cannot be obtained through ZN staining. Mtb mRNA in ZN-negative human TB lung tissue We next examined the utility of RNAscope for identifying Mtb or remnants of infection, i.e ., mRNA, in ZN-negative human TB tissue. ZN staining of formalin-fixed, paraffin-embedded (FFPE) lung tissue from a patient with active TB showed numerous extracellular and intracellular bacilli ( a). However, a different FFPE lung tissue specimen from the same patient was ZN-negative. This is unsurprising as most human pulmonary TB granulomas contain few, if any, ZN-positive Mtb , which is consistent with historical studies. In contrast, RNAscope analysis of this ZN-negative tissue revealed numerous RNAscope signal puncta within necrotic granulomas ( b and c). RNAscope puncta were also abundant in bronchiolar epithelial cells ( d), which were ZN-negative ( e) and antigen 85B (Ag85B)-positive ( f). These data demonstrate that Mtb mRNA is abundantly present in ZN-negative lung tissue sections of a confirmed case of pulmonary TB and that Mtb mRNA is sufficiently stable for detection by RNAscope. RNAscope and Ag85B immunostaining suggest the bronchiolar epithelial layer as an overlooked entry portal for Mtb infection. mRNA and secreted antigens accumulate in the cytoplasm of host cells In a study of individuals who died from causes other than TB, Mtb DNA was detected in alveolar and interstitial macrophages, type II pneumocytes, endothelial cells, and fibroblasts via in situ PCR analysis. Even though AFB were not shown, these findings are in accord with early reports , , , that Mtb can persist in lung tissue without histological evidence of TB lesions. Consistent with those studies, we identified accumulated Mtb mRNA in the cytoplasm of several alveolar epithelial cells ( a–d) in a signal pattern distinct from the intracellular puncta that represent single Mtb mRNA molecules. We refer to such cells as host-cell accumulated Mtb RNA (HAMR) cells. Mtb mRNA is present in HAMR cells within adjacent regions ( b and c), consolidated alveolar areas ( d), and within lymphocytic aggregates at the periphery of necrotic granulomas ( e). The functional significance of Mtb mRNA within HAMR cells is unknown; however, these mRNAs may act as pathogen-associated molecular patterns (PAMPs) as has been shown for mRNAs in conventional innate immune cells , , , , to modulate immunity. Secreted Mtb antigens have been identified via IHC staining in Mtb -infected human lung tissue. , , , , Similarly, we used IHC to detect Mtb secreted antigen 85B (Ag85B) in the same pulmonary and testicular specimens used for RNAscope analysis . We observed Ag85B accumulation in numerous cell types ( f) including alveolar macrophages and epithelioid histiocytes ( g). In summary, our data demonstrate that cells from histologically normal and diseased airways accumulate Mtb mRNA and secreted antigens. Mtb RNA in host microenvironments Our findings show that RNAscope can identify Mtb mRNA to reveal prior history of infection, which is not possible with ZN or Auramine O staining. Since each RNAscope punctum represents one Mtb mRNA molecule, we used the HALO® image analysis platform to count Mtb mRNA puncta, expressed as the number of Mtb mRNA puncta per square micron, in necrotic and non-necrotic granulomas, terminal and pulmonary bronchi, lymphocytic aggregates, and adjacent tissue from resected TB lung tissue ( a). To avoid potentially confounding variables such age, sex, tissue pathology bias, and experimental variability, we selected a single ZN-negative TB tissue specimen containing the aforementioned microenvironments. We reasoned that quantifying Mtb mRNA puncta within each of the four distinct regions of the necrotic granuloma (necrotic, granulomatous inflammatory, fibrotic, and lymphocytic; b) could help inform the pathophysiology of granuloma formation. We found that the necrotic zone contains the fewest Mtb mRNA puncta per square micron, followed by increases in the number of puncta within the granulomatous inflammatory, fibrotic, and lymphocytic zones ( c), indicating that Mtb mRNA is distributed in accordance with discrete pathophysiological zones that may contribute to granuloma formation. We also classified non-necrotic granulomas ( d) into lymphocytic and non-necrotic zones and found that the lymphocytic zone exhibited significantly more Mtb mRNA puncta than the non-necrotic zone ( e). We next quantified Mtb mRNA puncta in terminal and respiratory bronchioles, adjacent tissue, and lymphocytic aggregates distant from necrotic or non-necrotic granulomas and compared these values to those from regions within necrotic and non-necrotic lesions. We found that the number of Mtb mRNA puncta in the terminal bronchioles, lymphocytic aggregates, and adjacent tissue were the highest of all areas examined ( f). Further, the terminal bronchioles exhibited the highest average puncta optical density (OD) ( g), suggesting the presence of longer, more intact Mtb mRNAs containing more RNAscope probe binding sequences. In short, the zonal distribution of Mtb mRNA within the granuloma offers a new perspective into the evolution of granuloma formation and clinical course of disease that ZN or other bacillary staining methods cannot. Lastly, the spatial distribution of Mtb mRNA provides new insight into the previous impact of Mtb within human tissue. Mtb in vivo Continuously changing microenvironments within the tuberculous lung, the specific bacillary load, and a variable spectrum of TB lesions , , , , , strongly suggest that at least two distinct Mtb populations, live and dead, must exist in vivo . There is currently no method that accurately distinguishes metabolically living from dead Mtb bacilli within tissue specimens; however, since MPT64 is secreted during active Mtb growth, the presence of MPT64 in sputum has been suggested as a marker of Mtb viability. Here, we tested the hypothesis that ZN staining combined with IHC for secreted Mtb antigens can identify phenotypically diverse Mtb populations in vivo . We examined testicular tissue whose seminiferous tubules contain numerous bacilli and are bordered by a basement membrane that separates the tubule from the sparsely-infected surrounding tissue, which aids in evaluating the specificity of Mtb antigen immunostaining. H&E histology revealed the presence of substantial cellular debris which overlaps with Mtb RNAscope signals . Mtb bacilli colocalise with strong Ag85B, ESAT-6, and Uncharacterised Surface Protein (USP) positivity in sequential tissue sections . While these antigens are typically associated with the Mtb cell wall, clear delineation of the bacillary rod shape is not always possible. In some cases ( e.g. , USP, ESAT-6), positivity was seen as scattered, brown patches, whereas some rod-like shapes were observed, consistent with previous studies. , , , These findings suggest that Ag85B, ESAT-6, and USP staining patterns may not always match the rod-like morphology of Mtb . To determine whether distinct microenvironments can contribute to differential antigen production, we examined whether all AFB produce Ag85B. Intriguingly, our dual ZN/Ag85B IHC stain demonstrated that not all AFB produce Ag85B. This was evident by numerous ZN-positive/Ag85B-negative bacilli close to Ag85B-positive bacilli ( a–d, ). Ag85-positive and ZN-positive/Ag85B-negative bacilli were observed extracellularly and within host cells that did, or did not, accumulate Ag85B ( e–i). Similar to Canetti, we also identified “ghost” bacilli that are weakly acid fast ( f). Taken together, these data demonstrate that two phenotypically distinct Mtb populations exist in human TB tissue: Ag85B-positive and Ag85-negative bacilli. This finding strongly suggests that Ag85B IHC alone may underestimate the number of bacilli. This has important implications for TB diagnosis and pathogenesis since mixed populations of Mtb may differently influence diagnosis, pathogenesis, and immunity. Mtb antigens as tools for guiding TB therapy To evaluate the potential of RNAscope to guide clinical intervention, we examined tissue specimens from a 61-year-old female initially diagnosed with disseminated histoplasmosis. Biopsies of inguinal and retroperitoneal lymph nodes were performed 414 and 13 days before hospitalisation, respectively ( a). Both biopsy specimens were ZN-negative and revealed granulomatous inflammation . On hospital day 18, bronchoscopy with bronchoalveolar lavage (BAL) showed a negative gram stain and AFB smear. The patient died on hospital day 19 with the cause of death attributed to complications of sepsis. Autopsy revealed disseminated granulomatous inflammation of the lung, liver, bone marrow, and periaortic lymph node . ZN staining showed diffuse involvement by AFB , and the cause of death was amended to be complications from disseminated TB. Culture of antemortem BAL fluid revealed the presence of pansensitive Mtb complex 47 days after hospital admission ( a). With longitudinal specimens available, we subjected the ZN-negative inguinal lymph node biopsy obtained 414 days prior to hospital admission ( b) to RNAscope analysis and found numerous Mtb mRNA transcripts ( c). Quantitation of Mtb mRNA puncta using HALO® analysis showed a higher number of puncta around and inside giant cells and lymphocytic aggregates compared to adjacent lymphoid tissue and the whole biopsy ( d) with no difference in average puncta optical density (OD) ( e). This suggests that the number of Mtb mRNA puncta can help predict pathophysiological abnormalities in human TB tissue. The detection of Mtb via RNAscope is further supported by strong Ag85B positivity in giant cells ( f and g) and surrounding lymphocytes ( h) in the antemortem inguinal lymph node specimen. ZN-negative antemortem bone marrow biopsy ( i and j) and ZN-positive postmortem periaortic lymph node ( k) specimens also demonstrated Ag85B positivity. In this clinical scenario, application of RNAscope detection of Mtb mRNA and Ag85 IHC to biopsy tissue could have positively identified Mtb , providing much earlier diagnosis of disseminated TB, possibly enabling effective TB treatment. These findings suggest that detection of Mtb mRNA and antigens has diagnostic value in complex TB cases where sputum or biopsy material is ZN-negative. Major unmet needs in the TB field are the ability to consistently identify Mtb bacilli and the means to gain a clear understanding of how Mtb contributes to human tissue pathology. We have taken initial steps toward addressing these needs by adapting the RNAscope platform to identify intact and disintegrating Mtb bacilli and single Mtb transcripts in human TB tissues. We show that Mtb mRNA puncta are highly abundant in ZN-negative lung tissue specimens from a confirmed pulmonary TB case, that Mtb mRNA is found intracellularly and extracellularly, and that Mtb mRNA and antigens accumulate in cells from histologically normal and abnormal tissue. We also provide evidence of two phenotypically distinct Mtb cell populations in vivo . Lastly, analysis of an antemortem biopsy provides evidence that RNAscope can help guide therapeutic intervention in TB cases where small biopsy material is ZN-negative. Overall, the ability of RNAscope to detect Mtb bacilli in diverse morphological states and identify molecular remnants in vivo advances our understanding of TB pathophysiology and diagnosis. RNAscope has diagnostic promise due to its highly sensitive and specific probe design. In particular, the detection of clearly-defined bacillary rod shapes that are visually similar to positive ZN staining as well as puncta indicating single transcripts makes it an attractive diagnostic platform. Current dogma asserts that bacterial mRNA is an unlikely detection marker due to its susceptibility to degradation. Like other bacteria, Mtb employs a multienzyme RNA degradome comprised of endo- and exoribonucleases, an RNA helicase, and a polynucleotide phosphorylase that controls mRNA decay rates during growth and in response to environmental cues. , While this degradome effectively degrades intrabacillary mRNA, very little is known about extrabacillary stability of Mtb mRNA within host cells, which is likely influenced by the specific cellular compartment ( e.g ., phagosome, phagolysosome, or cytoplasm) in which it is contained. As RNAscope is effective in detecting intra- and extracellular Mtb mRNA, our findings suggest that mRNA released from the bacterial cell is sufficiently stable to be detected by this platform. Pathologists are frequently confronted with diagnostic challenges in TB cases since most human necrotic granulomas contain few, if any bacilli. This is consistent with comprehensive human pathology studies conducted by Canetti that suggest the formation of necrotic granulomas stems not from bacillary replication, but from bacillary destruction accompanied by exaggerated inflammatory responses induced by Mtb antigens. Subsequent studies in animal models affirmed that TB is primarily an outcome of persistent host responses to bacillary products rather than bacillary proliferation. Since RNAscope detects Mtb mRNA in AFB-negative lung and lymph node specimens from patients with confirmed TB, this technique could assist pathologists in confirming TB diagnosis to guide treating physicians. Furthermore, the interventional value of RNAscope was demonstrated by confirming the presence of Mtb mRNA in a ZN-negative antemortem biopsy from a patient initially diagnosed with histoplasmosis. Hence, RNAscope analysis could have guided potentially life-saving therapy. Mtb is highly adaptive to changing host immune signaling, the onset of hypoxia, variable nutrient availability, and distinct lesion microenvironments, , and therefore bacilli exist in diverse phenotypic states in vivo . However, there are few methods to identify specific phenotypes in TB tissue. , Mtb naturally exists in fully, partially, and non-acid fast states and loses its acid fastness during disintegration. , Hence, acid fastness is not an accurate indicator of viability. Several studies have shown that some Mtb surface and/or secreted antigens can be detected in human sputum and in human , , , , and guinea pig tissue specimens. We identified two distinct populations of Mtb in human specimens: those that produce Ag85B and those that do not. Since many of these bacilli are in close proximity, differential ag85B expression in response to different environmental signals is unlikely. Since Ag85B secretion is not universal, Ag85B IHC alone is likely to underestimate bacillary burden, consistent with prior studies. An unexpected discovery was the accumulation of Mtb mRNA in the cytoplasm of HAMR cells. This important finding is reminiscent of studies that demonstrated the presence of Mtb DNA in host cells. , We were unable to identify discernible bacilli inside these cells, suggesting that the source of mRNA is disintegrated intracellular bacilli. An alternative explanation is that HAMR cells take up exogenous Mtb mRNA and/or secreted antigens. Recent studies have shown that Mtb can produce extracellular vesicles (EVs) that contain hundreds of proteins, including Ag85A, secrete RNA in vitro and release mRNA into the cytosol in macrophages. Further, EVs from Mtb -infected macrophages contain Mtb mRNA. , Hence, uptake of Mtb -derived EVs by HAMR cells could account for the accumulation of mRNA and Ag85B. RNA from Streptococcus agalactiae (group B streptococcus) has been implicated as an immunomodulatory PAMP that induces IFN-β production in dendritic cells, suggesting that Mtb nucleic acids and/or secreted antigens may exert similar effects. Indeed, Mtb RNA is sensed in a manner dependent on melanoma differentiation factor 5 (MDA-5), an RNA sensor in the RIG-I-like 3 receptor family, resulting in increased IL-1β production, inflammasome activation and attenuation of autophagy. Notably, these effects culminate in increased Mtb survival in macrophages. Overall, these studies suggest mechanisms by which HAMR cells in patients with TB may accumulate Mtb mRNA and/or secreted antigens and provide context for investigating the fate and immunomodulatory function of these cells. Our finding of Mtb mRNA in bronchiolar epithelial cells provides new insight into Mtb tropism, as this microenvironment is not routinely examined by pathologists. The accumulation of Mtb mRNA inside bronchiolar epithelium indicates significant bacillary destruction in this environment, which was confirmed by Ag85B positivity, and is consistent with our finding that Mtb can cross the bronchial epithelium. We have demonstrated the potential of RNAscope to detect Mtb mRNA within intact bacilli, disintegrating bacilli and single Mtb transcripts in a range of human tissues including pulmonary, lymphatic, and testicular specimens. Our case study involving ante- and postmortem biopsy specimens provides compelling evidence that RNAscope has the potential to reduce clinical time-to-diagnosis in TB cases in which histopathologic findings can neither confirm nor rule out the presence of Mtb . Positive Ag85B immunostaining confirmed the RNAscope results, increasing diagnostic confidence. In cases where clinical suspicion remains high despite negative AFB staining, RNAscope has the potential to confirm or exclude the diagnosis of TB. Our study has limitations that could affect its application. Firstly, distinguishing viable from nonviable bacilli remains to be demonstrated since the presence of intrabacillary mRNA and presence of Ag85B is not necessarily an indication of viability. Secondly, the design and synthesis of RNAscope probes is relatively expensive, which may limit its initial clinical application. In conclusion, our findings have important implications for TB pathophysiology and diagnosis. RNAscope detected Mtb mRNA in vivo , which was previously thought to be unstable, as well as in intact and disintegrating bacilli. We also detected Mtb mRNA within ZN-negative human tissues obtained from pulmonary and extrapulmonary sites and found that Mtb mRNA accumulates within some host cells but is also present outside host cells. Additionally, we show that antigen secretion is not universal, demonstrating two diverse populations of Mtb in vivo . These findings imply applications that could have considerable impact, such as the early identification of individuals who are at risk of dying from TB-related causes and tracking the clinical course of disease via the historical imprints of Mtb . Conceptualisation and Design: KN, TN, PVB, MM, AJCS. Lung tissue preparation: MM, TN, KN, KL, PVB. Pathology: TN, RLH, PVB. Histopathology: KN, TN, MM, RLH, PVB. Data integration: GW, KL, SN, AH, AJCS. Verification of underlying data: all authors. Writing initial draft: JNG, TN, AJCS. Editing: AH, JNG, SN, GW, AJCS. Final draft: All authors. Figure preparation: SN, JNG, KN, AJCS. All authors have read and approved the final version of the manuscript. High-resolution RNAscope and/or IHC images generated in the study are available from the corresponding author ( [email protected] ) on request, or can be viewed at: https://www.ahri.org/scientist/adrie-steyn/ . The authors have no competing interests or disclosures. |
Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war – a 30-year review | 7aa9a0a8-aaa9-4210-bc60-296dd6e35df2 | 11157258 | Forensic Medicine[mh] | The dissolution of Yugoslavia at the end of the 20th century triggered a range of war activities, which were centralized mainly in Croatia and B&H between 1991 and 1995, as well as in Kosovo in 1999. These wars resulted in over 200 000 deaths and an estimated 40 000 missing individuals . During the Homeland War in Croatia, more than 20 000 persons lost their lives . Due to the efforts of the relevant authorities of the Republic of Croatia, the fate of most of the registered missing persons has been resolved. However, 1418 persons are still missing, and the burial place of 394 deceased persons remains unknown, which adds to 1812 unsolved cases from the Croatian Homeland War . During the war in B&H, close to 100 000 individuals lost their lives . Around 80% of all missing individuals have been successfully identified, but almost 7600 people are still missing . Identification of war victims presents a great challenge for many reasons, especially since the remains of tens or, even hundreds, of individuals may be found within a single mass grave. Therefore, multiple methods were used in the identification efforts . These methodologies encompass various techniques, such as dental analyses, fingerprinting, and direct facial recognition by a living person. They also involve examining skeletal remains by forensic anthropologists (including estimation of age, biological sex, time of death, stature, and population affinity), autopsy, reconstruction of facial features from the skull, identifying personal traits like tattoos and scars, comparing hair samples, recognition of personal effects such as clothing and attire through witness testimony, and DNA analysis. The selection of an appropriate method and its effectiveness depend on the circumstances and condition of the remains being examined. These methods and challenges were discussed in the first publications on the identification efforts in Croatia and B&H . Strinović et al reported on the first identification in pre-DNA time in 1994. It was performed at the Institute of Forensic Medicine and Criminology in Zagreb, Croatia, resulting in 73/110 successful identifications. Some bodies were delivered in groups, while others were delivered individually. The largest group consisted of 20 members of the Croatian army killed in the village of Kusonje; they were delivered five months following their deaths . The researchers compared fingerprints, dental records, special body features and items (tattoos, jewelry), available documents, and visual recognition . However, none of these methods was completely suitable, and most had significant drawbacks when applied to war victim identification. Another group of scientists reported on the identification of the very first casualties of the war in Bosnia and Herzegovina in 1995 . They examined 59 victims from the mass graves located in the Kupres area, West Bosnia . The identification methods employed were similar to those described above, including recognizing personal items (clothes, footwear, jewelry, documents), stature and hair analysis, body marks, dental records, x-ray analysis, and video superimposition . Although as a result of such a multidimensional approach, 35 persons were positively identified, 24 remained unidentified . The limitations of the methods used at the time were pointed out clearly, especially in the case of decomposed or skeletonized human remains. From both articles, one could sense that such human remains might only be identified by applying, at that point still insufficiently developed, methods of DNA identification, which was later proven to be correct. Unidentified skeletal remains from the Kupres area were selected for one of the very first DNA identifications of skeletal remains from mass graves performed globally . These identifications were entirely performed in the local DNA facilities by the local scientists, with substantial help from their international collaborators. The importance of this effort was evidenced by several articles on this topic appearing in prominent scientific publications, including Science and JAMA . After these early successes achieved in collaboration with eminent American forensic scientists, new developments unfolded rapidly. Very early on, PCR-based techniques entered forensic genetics and found numerous applications, including war victim identification, disputed paternity testing, and crime scene evidence characterization. A turning point in human identification was the introduction of short tandem repeat (STR) markers, which shifted the power of exclusion in the testing of disputed paternity to a minimum of 99.999% and achieved high levels of discrimination in biological evidence discovered at crime scenes. It became evident that this novel multiplex system was substantially more informative in human identification than the early non-STR systems. In the analysis of more than 90 samples, the AmpliType ® PM + DQA1 systems successfully identified only 20%-25% of cases. The introduction of multiplex STR analysis and improvement of the original DNA isolation procedure increased the identification rate to 85% . However, new challenges regarding statistical calculations arose. The main question was whether the set of only nine STR loci would be sufficient to attain a high degree of statistical confidence for identification purposes given the magnitude of human remains . Therefore, Croatian scientists began successfully employing more enhanced multiplex STR systems. Also, they soon realized that long bones produced extracts of higher quality compared with samples isolated from skulls or ribs . Another important objective established around this time was to obtain relevant population data in Croatia and B&H to determine the frequency of allelic variants within the populations. The data are necessary to enable more accurate statistical calculations associated with genetic associations. Consequently, several studies addressed the genetic diversity of these populations, including the first one performed in collaboration with the FBI . At the time, mitochondrial DNA (mtDNA) and Y-STRs were employed with different success rates. These markers were used in certain cases, depending on the context and the quantity and quality of the DNA. An innovative approach to simplifying this analysis was immobilized sequence-specific oligonucleotide (SSO) probe analysis of mtDNA hypervariable regions 1 and 2, again performed by a team of Croatian and US scientists . SSO probe analysis was used to determine the population variation of human mtDNA HV I and II in 105 Croatian individuals . The study reported successful SSO hybridization in 78% of the cases and a positive identification of one sample that was identical to a unique mtDNA sequence in a population of 105 randomly selected Croatians. This work confirmed that mtDNA analysis using immobilized SSO probes had its place in forensic DNA analysis of mass disaster remains of single and mass graves, particularly when the quality and quantity of remains did not yield full or nearly complete nuclear DNA profiles . A limitation of this type of analysis is the lower power of discrimination due to the few common HVI/HVII sequences in European populations, a lower effective population size compared with autosomal markers, and a lack of recombination in the mtDNA genome. To address these problems, an improved linear array assay, mt HV+ HaploArray, was created. It targets additional polymorphic regions in both, non-coding and coding regions . The assay was validated for mtDNA analysis on bone samples, as they are often challenged forensic samples and the most common type of material in human remains. Additional population studies have contributed to confirming the improvement of the assay. The results showed a better distribution of mtDNA types (mitotypes) in the Croatian population . These studies showed that mtDNA typing can be a powerful identification tool in cases where nDNA analysis is not possible due to degradation or low copy. In 2005, the Government of the Republic of Croatia officially published that, by the end of 1992, over 11 000 were reported missing in Croatia as a direct consequence of the war. This number corresponds with records indicating that 11 834 persons lost their lives. As a continuation to this report in 2005, experts who were included in the process from the very beginning published an article reviewing the identification efforts in Croatia after the war . Out of 3502 exhumed bodies, 2944 were identified and 558 remained unidentified. Overall, 1160 persons were still considered missing in 2005. (These numbers are updated with the more recent information presented at the beginning of this article.) The paper also listed the non-DNA and DNA-based methods of human identification that were used . Furthermore, the authors emphasized that in war circumstances, with many victims lacking sufficient ante-mortem data and mostly buried in shared mass graves, identification becomes exceedingly complex and demanding. Traditional, non-DNA identification methods succeeded in identifying around 60% of the victims, while the identification of the remaining 40% required DNA analysis. Since non-DNA methods are getting less and less powerful as time passes, it was predicted that over the ensuing years, DNA analysis would become more important for victim identification . Modern B&H is a multinational and multireligious country that has witnessed many conflicts throughout history, but at the same time, it is an interesting area regarding population genetics, history, and structure. According to the Research and Documentation Center project from 2007, a total of 97 207 individuals lost their lives during the war in B&H from 1992 to 1995. These casualties comprised 64 036 Bosniaks, 24 905 Serbs, 7788 Croats, and 478 Others. Sarajevo during the four-year siege bore the brunt in terms of the number of victims, with 13 756 people killed, including 1601 children. It was followed by Srebrenica (8372 genocide victims), Prijedor, Zvornik, Bratunac, Foča, Vlasenica, Mostar, Doboj, Višegrad, Banja Luka, Brčko, and Rogatica. Immediately following the end of the war, approximately 30 000 persons were estimated to be missing. According to the International Commission on Missing Persons (ICMP), the leading authority for this problem, 75% of these missing persons were accounted for, which is a ratio that has not been equaled in any other post-war country . Bosnian scientists, with the help of international colleagues and financial support from the international community, established one of the most efficient systems for DNA identification in the world. All these efforts were coordinated by the ICMP. Before ICMP established its own laboratories for DNA identification, it cooperated with the B&H institutions with DNA identification capacities, for example, the Institute for Genetic Engineering and Biotechnology (INGEB) from Sarajevo, the Clinical Center of the University of Tuzla, and individuals from Banja Luka. The ICMP’s efforts in the former Yugoslavia countries after the war resulted in an unparalleled accomplishment – the identification of over 27 000 (70%) of all missing persons (most cases being in B&H, where more than 30 000 persons were missing) . However, starting the process was challenging. Due to the destroyed infrastructure and many scientists from the field having left the country, it was necessary to create a completely new scientific team that would run the mission of identifying war victims. The first DNA laboratories were created in pre-existing buildings of scientific institutions. These spaces needed to be renovated to accommodate extremely restrictive laboratory conditions required for DNA analysis of skeletal remains . Additionally, in the late 1990s, DNA purification and analysis from skeletal remains was a challenge on its own. However, such difficulties motivated these scientists to innovate and optimize the existing protocols. The first of these breakthrough papers was published in 2007, describing their original and highly effective DNA isolation protocol from the skeletal remains after years of optimization . This paper also overviewed the lab system for processing skeletal remains developed primarily at the INGEB, Sarajevo, and later, applied in a newly established ICMP laboratory. While this article presents the results of 20 femur samples, it is a summary of experience gained by processing thousands of skeletal remains in the years before the publication. Two other papers from the ICMP team presented unusual DNA typing results and analyzed the overall success rates of DNA typing . The same group of Slovenian, Croatian, and B&H scientists drew upon their experience with war victims in their effort to identify the remains from the Second World War (WWII) . The paper DNA Identification of Skeletal Remains from the World War II Mass Graves Uncovered in Slovenia reported one of the initial outcomes of human identification of old skeletal remains in this area . Considering the gap of 60 years between the body deposition and the time of excavation and analysis, this effort was a challenge due to low DNA quantity and quality and the presence of PCR inhibitors. However, the authors demonstrated that DNA identification was the only viable method of identification for these remains. Following a protocol optimization across all DNA typing steps, this study was one of the first identification studies of civilian WWII victims from a mass grave. As an area with a turbulent history, the countries of the former Yugoslavia were inevitably impacted by WWII. Tens of thousands of people were reported missing during the war, as well as after it due to numerous mass executions carried out by the Yugoslav communists. With the establishment of democratic governments in these countries and following the requests from the missing persons’ relatives, efforts were made to identify the recovered remains from WWII. These efforts included the identification of mass grave victims in Škofja Loka, Slovenia . Two small mass graves were confirmed to contain the remains of 27 persons: 20 in the larger and seven in the smaller grave . According to the eyewitnesses’ testimony, the larger grave contained the bodies of Slovenian home guardsmen (German collaborators), while the smaller one contained the remains of seven German soldiers executed as war prisoners. The German soldiers, reportedly, buried the executed Slovenians and then excavated another grave for themselves. In total, 15 full and 12 partial DNA profiles were generated. A comparison of victims’ profiles against collected family reference samples resulted in four strong associations and subsequent positive identification of the remains. Moreover, five other profiles were possibly associated to the reference samples, though with a lower probability . This study illustrated that the knowledge and proficiency acquired from the identification projects following the war in B&H and Croatia could effectively be applied to identify skeletal remains deposited in humid soil for a significantly longer time period. Fortunately, technologies available to forensic genetics are consistently being developed and enhanced so the power of DNA analysis as a human identification tool continues to improve. Soon after, new DNA analysis tools, namely lineage markers Y-STRs and miniSTRs, became available for the analysis of WWII skeletal remains . Y-STR typing was used to identify two samples from Škofja Loka that were linked to family reference samples with insufficient probability of meeting an identification declaration . Y-STR analysis was used to assess if the two samples belonged to a paternal relative of two reference persons, since previously provided autosomal analysis presumed possible grandparent-grandson relationship. One case met the criterion for a positive match, but in the other, the paternal relation was not supported . The identification of Škofja Loka mass grave victims also included a successful use of MiniSTR analysis. This type of analysis is especially useful in assaying markers in shorter amplicons that may recover information from degraded DNA samples. It was used to positively identify a woman who went missing in the autumn of 1942 based on the reference samples from her two sons . A summary of performed DNA analyses for the World War II skeletal remains from Slovenia is presented in . Taken together, these results demonstrated that both Y-STR and miniSTR analyses were valuable additional tools for human identification, especially in cases where autosomal STR analysis was insufficient. A few years later, almost the same team of scientists finalized the identification process of WWII skeletal remains from mass graves in Ljubuški, Herzegovina . The success rate of DNA profiling of 10 persons was ~ 90%. Six positive identifications were made, all of them for skeletal remains of male individuals. These findings underscore the significance of a timely, targeted, and efficient sample collection from living relatives, as well as the deep emotional commitment of community members in identification of the remains. The techniques outlined above, or their minor modifications, were also used in the examination of archeological skeletal remains. Archeological material analysis is vital to understanding the human population’s history. Previous experience of local DNA experts and additional protocol optimizations were applied in the DNA analysis of bone fragments and teeth from ancient graves located in several archeological sites across B&H. The most important advantage of employing new multiplex STR systems was the capability to analyze minute quantities of degraded DNA and traces characterized by the existence of a high amount of PCR inhibitors. Archaeological cases, such as those of medieval remains from Zgošća and Bobovac in B&H and the case of Sister Marija Krucifiksa Kozulić in Croatia , showed the power of new STR analysis systems in the analysis of human remains several centuries old. This review article summarizes the expertise gained over the past three decades through identifying missing individuals in the former Yugoslav countries. These findings and experiences could be routinely applied without substantial alterations to analyzing skeletal remains from different eras and for current missing persons cases around the world. DNA analysis successfully identified remains from the 1990s, but also those retrieved almost seven decades earlier. Over the last three decades, genetic typing by STR analysis has emerged as the predominant method for human remains identification, thus evolving from an adjunct method to the choice forensic identification method, especially when other identification methods fail to provide viable lead data . Nowadays, the adoption of enhanced or entirely novel methodologies increases the likelihood of successful nuclear DNA profiling of degraded skeletal remains, thereby augmenting the success rate of DNA profiling in such cases. Other methods, such as Y chromosome and mtDNA analysis and autosomal miniSTR systems, and particularly SNP analysis, allow the analysis even of the most challenging samples, such as those retrieved from archeological sites. Next-generation sequencing, high-volume SNP panels, and genealogical tools are likely the next tools that will increase the success of human identifications. Once again, forensic science helped to bring closure to families who had spent decades searching for their missing loved ones and enabled a dignified burial for the victims. As many of the closest relatives of the missing individuals are aging, it becomes urgent to collect samples from them and invest in technologies that improve the chances of typing success. Without their DNA profiles as the reference samples, the entire identification process would be significantly more complex, if not entirely unfeasible. |
Immediate risk of cervical intraepithelial neoplasia and diagnostic value of colposcopy among cytology-negative women with oncogenic HPV: a retrospective study | c4c87bc9-f052-4552-99e4-c2f5e685eafa | 11267838 | Pathology[mh] | Cervical cancer, which is the third most common cancer and the third leading cause of cancer death in females, poses a serious threat to women’s health . In China, the incidence and mortality of cervical cancer are 7.5/100,000 and 3.4/100,000, respectively . Thus, it remains an important public health problem. Persistent infection with high-risk human papillomavirus (HR-HPV) plays a crucial role in causing cervical intraepithelial neoplasia (CIN) and cervical cancer . It often takes 10–15 years for CIN to develop into cervical cancer after persistent infection with HR-HPV . Thus, it provides many opportunities to detect and treat precancerous lesions. Cervical cytology, which is a routine and widely used cervical cancer screening method, has a sensitivity of only approximately 50% . In contrast, HPV-based screening provides 60-70% greater protection against cervical cancer . Due to the relatively low sensitivity of cervical cytology, discordant cotesting, which is defined as being cytology negative but HR-HPV-positive, is common when using combined screening . However, managing women with discordant cotesting remains a challenge. An estimated 12% of cytology-negative women have HR-HPV infection . In addition, approximately 89% of cervical cancer patients are HR-HPV-positive . According to the current guidelines , approximately 10% of cervical high-grade lesions caused by HR-HPV other than HPV16 and 18 might be missed, particularly in countries with limited professional cytologists . Additionally, managing women with such cases is controversial. Owing to the low risk of immediate cancer for patients who are cytology negative but HR-HPV-positive, excessive intervention or overreferral to colposcopy are not recommended . Based on these observations, the use of colposcopy might help identify precancerous lesions and cervical cancer in these patients. In addition, unnecessary invasive cervical biopsies might be reduced if colposcopy is appropriately used. Therefore, this study aimed to explore the discrepancies between colposcopy diagnosis results and cervical biopsy results in diagnosing precancerous lesions and to investigate the immediate risk of CIN in patients with cytology-negative but HR-HPV-positive results. Study population This retrospective study included women who underwent HPV tests and liquid-based cytology (LBC) tests for cervical screening and who were referred for colposcopy examination between January 2022 and August 2023 at Fujian Medical University Union Hospital, Fuzhou, China. The following patients were eligible for inclusion in this study: (1) women who were older than 16 years and had a history of sexual activity, (2) patients who tested positive for HR-HPV and negative for the LBC test, (3) patients who underwent colposcopy-guided biopsy once they tested positive for HR-HPV, and (4) patients who had pathological resuslts of biopsy tissue. The exclusion criteria were as follows: (1) pregnant; (2) had a history of surgical treatment for CIN or cervical cancer, radiotherapy or chemotherapy; (3) had undergone colposcopy but without cervical biopsy; and (4) had a history of persistent or transient HR-HPV infection. To mitigate the impact of posttreatment pathological result reversals, we opted to rely on the initial colposcopy findings. The study was approved by the Medical Ethics Committee of Fujian Medical University Union Hospital (Date15/1/2020/No2020QH023). Informed consent was obtained from all participants before the study. Examination method The LBC test was used to perform cervical cytology analysis. The results were interpreted by experienced pathologists and classified according to the 2014 Bethesda System . Cervical cytology negative indicated no intraepithelial lesions or malignancy (NILM). HPV genotypes were examined by an HPV GenoArray test kit (HybriBio Ltd), which is capable of identifying seventeen HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) and six low-risk HPV types (6, 11, 42, 43, 81, and 83). Patients infected with HR-HPV other than HPV16/18 were classified into the “non-16/18 HR-HPV” group. Colposcopic examinations were conducted in accordance with the 2011 International Federation of Cervical Pathology and Colposcopy (IFCPC) by different colposcopists. Senior colposcopists were defined as colposcopists who had more than ten years of working experience, while others were defined as junior colposcopists. Multipoint cervical biopsies were taken at abnormal imaging sites. When no suspicious lesions were observed, routine biopsy was performed at points 3, 6, 9 and 12. Endocervical curettage (ECC) was performed when the lesions had spread into the cervical canal or were not fully visible. The pathological biopsy results included chronic cervicitis, CIN1, CIN2, CIN3 and invasive carcinoma. The colposcopy results included a normal impression, a low-grade impression, and a high-grade impression (including carcinoma). If colposcopy suggested a normal impression and biopsy suggested chronic cervicitis, the two test results were considered consistent. If colposcopy suggested a low-grade lesion and biopsy suggested CIN1, as well as when colposcopy suggested a high-grade lesion and biopsy suggested CIN2, CIN3 or invasive carcinoma, the two test results were considered consistent. Otherwise, the two tests were considered inconsistent. This retrospective study included women who underwent HPV tests and liquid-based cytology (LBC) tests for cervical screening and who were referred for colposcopy examination between January 2022 and August 2023 at Fujian Medical University Union Hospital, Fuzhou, China. The following patients were eligible for inclusion in this study: (1) women who were older than 16 years and had a history of sexual activity, (2) patients who tested positive for HR-HPV and negative for the LBC test, (3) patients who underwent colposcopy-guided biopsy once they tested positive for HR-HPV, and (4) patients who had pathological resuslts of biopsy tissue. The exclusion criteria were as follows: (1) pregnant; (2) had a history of surgical treatment for CIN or cervical cancer, radiotherapy or chemotherapy; (3) had undergone colposcopy but without cervical biopsy; and (4) had a history of persistent or transient HR-HPV infection. To mitigate the impact of posttreatment pathological result reversals, we opted to rely on the initial colposcopy findings. The study was approved by the Medical Ethics Committee of Fujian Medical University Union Hospital (Date15/1/2020/No2020QH023). Informed consent was obtained from all participants before the study. The LBC test was used to perform cervical cytology analysis. The results were interpreted by experienced pathologists and classified according to the 2014 Bethesda System . Cervical cytology negative indicated no intraepithelial lesions or malignancy (NILM). HPV genotypes were examined by an HPV GenoArray test kit (HybriBio Ltd), which is capable of identifying seventeen HR-HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82) and six low-risk HPV types (6, 11, 42, 43, 81, and 83). Patients infected with HR-HPV other than HPV16/18 were classified into the “non-16/18 HR-HPV” group. Colposcopic examinations were conducted in accordance with the 2011 International Federation of Cervical Pathology and Colposcopy (IFCPC) by different colposcopists. Senior colposcopists were defined as colposcopists who had more than ten years of working experience, while others were defined as junior colposcopists. Multipoint cervical biopsies were taken at abnormal imaging sites. When no suspicious lesions were observed, routine biopsy was performed at points 3, 6, 9 and 12. Endocervical curettage (ECC) was performed when the lesions had spread into the cervical canal or were not fully visible. The pathological biopsy results included chronic cervicitis, CIN1, CIN2, CIN3 and invasive carcinoma. The colposcopy results included a normal impression, a low-grade impression, and a high-grade impression (including carcinoma). If colposcopy suggested a normal impression and biopsy suggested chronic cervicitis, the two test results were considered consistent. If colposcopy suggested a low-grade lesion and biopsy suggested CIN1, as well as when colposcopy suggested a high-grade lesion and biopsy suggested CIN2, CIN3 or invasive carcinoma, the two test results were considered consistent. Otherwise, the two tests were considered inconsistent. Data analysis was performed using SPSS software version 25.0. Normally distributed data are presented as the means ± standard deviations (mean ± SD). Categorical data are expressed as n and were analysed using the chi-square test. Univariate and multivariate logistic regression analyses were used to evaluate independent factors associated with the outcomes. A P value < 0.05 was considered to indicate statistical significance. The characteristics of the patients included in this study are presented in Table . In total, 372 patients who were cytology negative but HR-HPV-positive and who underwent colposcopy and cervical biopsy were included in the study. The included patients ranged in age from 16 to 79 years. Of the patients, 168 (45.16%) had HPV16 infection, 98 (26.34%) had HPV18 infection, and 106 (28.5%) had non-HPV16/18 infection. Pathology revealed 195 (52.42%) chronic cervicitis patients, 131 (35.21%) CIN1 patients, 37 (9.95%) CIN2/3 patients and nine (2.42%) invasive carcinoma patients. Normal colposcopy impressions were the most common colposcopy diagnosis (47.31%), followed by low-grade impressions (43.82%) and high-grade impressions (8.87%). The consistency between the pathological biopsy results and colposcopy diagnosis results is depicted in Table . With pathological biopsy results as the gold standard, 227 (61.02%) patients had consistent colposcopy results. Among patients with CIN2 + lesions (including CIN2, CIN3 and invasive carcinoma), the accuracy was high (91.1%), and the sensitivity was 50.0%, while the specificity was 96.9%. With respect to normal colposcopy images, the accuracy was 67.5%, the sensitivity was 64.1%, and the specificity was 71.2%. The sensitivity for detecting CIN1 was 60.3%, the specificity was 65.1%, and the accuracy was 63.4%. The associations between the clinical factors and the accuracy of colposcopy diagnosis according to univariate and multivariate logistic regression analyses are presented in Table . Since 48 years of age is the general age at which individuals enter perimenopause in China, the effect was analysed before and after 48 years of age . Univariate analysis revealed that the type of cervical transformation zone affected the accuracy of the colposcopy diagnosis ( P < 0.001). Multivariate analysis demonstrated that age and the type of cervical transformation zone affected the accuracy of the colposcopy diagnosis ( P = 0.028 and < 0.001, respectively). HPV genotype and colposcopist skill had no significant effect on the accuracy of the colposcopy diagnosis. According to the pathological biopsy results, the percentages of immediate CIN2 + lesions in patients with HPV16 infection, HPV18 infection and non-HPV16/18 infection were 15.48%, 8.16% and 11.32%, respectively ( P = 0.202), while the percentages of immediate CIN3 + lesions were 5.95%, 8.16% and 2.83%, respectively ( P = 0.263)(Table ). The immediate rates of invasive carcinoma in patients with HPV16 and HPV18 infections were 2.38% and 5.10%, respectively ( P = 0.405). No cases of carcinoma were found in the non-16/18 HR-HPV group. No significant differences were found when comparing the immediate incidence of CIN2 + lesions between the HPV16/18 group and the non-HPV16/18 HR-HPV group ( P = 0.699). Comparisons of the percentages of patients with CIN2 + lesions in the < 30 years, 30–39 years, 40–49 years, and ≥ 50 years age groups are shown in Table . In the < 30 years age group, statistical evaluation could not be performed because there were too few patients in the non-16/18 HR-HPV-positive subgroup, and no patients with CIN2 + lesions were found in this subgroup (0/4). In the 40–49 years age group, the percentage of CIN2 + lesions was significantly greater in the non-16/18 HR-HPV group than in the HPV16/18 group ( p = 0.022). No significant differences were found in the 30–39 years age group or the ≥ 50 years age group. When the patients diagnosed with CIN2 + lesions were evaluated according to age group, as demonstrated in Fig. , there were 8 (17.39%) patients in the < 30 years age group. In the < 30 years age subgroup, 5 patients were aged < 25 years, and 3 patients were aged 25–29 years. In regard to invasive carcinoma, there were 3 cases, 1 case and 5 cases in the 30–39 years age group, 40–49 years age group and ≥ 50 years age group, respectively. However, no cases of carcinoma were found in the < 30 years age group. Cervical cytology, HPV testing, and colposcopy are screening methods capable of detecting cervical dysplasia and early-stage cervical carcinoma. These diagnostic tools help guide patients towards appropriate management modalities and subsequent follow-up, ensuring timely intervention and care . Recently, studies have shown that cytology-negative but HR-HPV-positive results are common when using combined screening tests, and the rates of high-grade CIN and cervical cancer are significantly greater in women who are cytology-negative but HR-HPV-positive than in those who are cytology-negative only . However, the best management for women who are cytology negative but HR-HPV-positive, particularly those who are non-16/18 HR-HPV-positive, is still controversial. Colposcopy is reported to be the main procedure and cost-effective examination for accurate diagnosis of high-grade CIN . Nevertheless, in clinical practice, the consistency and accuracy of colposcopy for diagnosing high-grade squamous intraepithelial lesion (HSIL) still need to be improved. Based on these observations, the immediate risk of CIN in patients who were cytology negative but HR-HPV-positive and the diagnostic accuracy of colposcopy were investigated in this study. In this study, of the 372 patients who were cytology negative but HR-HPV-positive, 131 had CIN1, 37 had CIN2/3, and nine had carcinoma. The percentages of CIN2 + lesions and CIN3 + lesions in patients who were not HR-HPV 16/18-positive were comparable to those in patients who were HPV16- or HPV18-positive. In addition, among patients diagnosed with CIN2 + lesions, 8 (17.39%) patients were women aged < 30 years. When pathological results were used as a reference, the consistency rate of colposcopy was 61.0% (227/372). These findings suggested that, in countries with limited resources, it is feasible to recommend immediate colposcopy referral in patients who are cytology negative but HR-HPV-positive (including non-16/18-positive patients). In addition, cervical cancer screening by cotesting in women aged < 30 years should be suggested in countries with poor resources. Previous studies have shown that approximately 1.9–9.8% of women over 30 years old are cytology negative but HR-HPV-positive . Studies have demonstrated that the 1-year risk of CIN3 + lesions is less than 4%, while the 5-year risk of CIN3 + lesions is 6.4% in these patients . Follow-up after 1 year is suggested for patients with no history of HPV positivity according to the most recent American Society of Colposcopy and Cervical Pathology (ASCCP) guidelines (2019) . The immediate risks of CIN2 + lesions and CIN3 + lesions were 4.99% and 2.13%, respectively, in these patients . In addition, in women who were HPV16-positive but cytology-negative, the immediate risks of CIN2 + lesions and CIN3 + lesions were 7.82% and 5.30%, respectively. For patients who were HPV18-positive but cytology-negative, the immediate risks of CIN2 + lesions and CIN3 + lesions were 5.56% and 3%, respectively . The immediate risk of CIN2 + lesions for patients who were non-16/18 HR-HPV-positive and cytology-negative ranged from 1.1% to 6.5% . In this study, the percentages of patients with immediate CIN2 + lesions were 15.48%, 8.16% and 11.32% among cytology-negative patients who were HPV16-positive, HPV18-positive and non-16/18 HR-HPV-positive, respectively ( P = 0.202), while the percentages of patients with immediate CIN3 + lesions were 5.95%, 8.16% and 2.83%, respectively ( P = 0.263). Hence, this study demonstrated that the incidence of CIN2 + lesions and CIN3 + lesions in non-16/18 HR-HPV-positive patients was comparable to that in HPV16- or HPV18-positive patients. This result is consistent with the studies of Kabaca et al. and Athena HPV study group. . When referred to invasive carcinoma, the percentages were 2.38%, 5.10% and 0% among cytology-negative patients who were HPV-16 positive, HPV-18 positive and non-16/18 HR-HPV-positive in this study, respectively ( P = 0.060). The present study did not find cervical carcinoma in patients with cytology-negative but non-16/18 HR-HPV-positive, which was in consistent with previous studies . This result might be attributed to the limited sample size and relatively low incidence of carcinoma in this particular patient cohort. As reported by Kabaca et al., only one (0.1%) (HPV-39 positive) of seven hundred and fifty-two patients with cytology-negative but non-16/18 HR-HPV-positive had invasive carcinoma . Zappacosta et al. reported a case of HPV-53-related cervical cancer in a 79-year-old woman with cytology-negative, which might be due to the underutilization of screening methods and the low sensitivity of cervical cytology test . Besides, before the publication of Zappacosta et al.’s findings, HPV53 infection had never been reported in patients with cervical cancer. These researches have suggested that despite the low incidence of cervical carcinoma, carcinoma has indeed been detected in patients with cytology-negative but non-16/18 HR-HPV-positive. Recently, HPV31/33/39 genotyping and multiple HPV31/32/52 infections were proposed to be added to the previous recommended HPV16/18 genotyping triage for colposcopy . Based on the aforementioned observations that the incidence of CIN2 + lesions and CIN3 + lesions in non-16/18 HR-HPV-positive patients was comparable to that in HPV16- or HPV18-positive patients, along with the detection of cervical carcinoma in non-16/18 HR-HPV-positvie patients, it could be feasible to perform colposcopy in cytology-negative patients who are non-16/18 HR-HPV-positive, as in those who are HPV16/18-positive. The implementation of repeated screening by cotesting in women who are cytology negative but HR-HPV-positive remains a considerable challenge in limited resource areas such as China. Thus, regardless of the low immediate risk of CIN3 + lesions in HR-HPV-positive and cytology-negative patients, the recommendation of immediate colposcopy referral should be suggested for these patients. Although cervical cancer is rare in women younger than 30 years, an increasing trend in the incidence of cervical cancer has been demonstrated in young women . Gumpeny N et al. reported that 13.2% of pathologically diagnosed CIN2 + lesions (including carcinoma) were observed in women aged 21–30 years . Tidy JA et al. reported that 14.2% of CIN2 + lesions were detected in women aged 25–34 years who were cytology-negative but persistently HR-HPV-positive . In this study, 17.39% (8/46) of CIN2 + lesions were detected in cytology-negative women aged < 30 years with HPV16/18 positivity, and 10.87% (5/46) of CIN2 + lesions were detected in women aged < 25 years. Although some cases of CIN2 might regress spontaneously in young women, a large cohort study of pathological results of CIN showed that 34.6% of patients in the 25- to 30-year-old age group experienced persistent disease, and 13.1% of patients in the 25- to 30-year-old age group experienced lesion progression . Approximately 36% of women < 25 years with CIN2 would experience persistent disease without treatment within 2 years, while the percentage would increase to 69% among those who were HPV16-positive . A retrospective survey of a sample of 2966 patients who underwent conization for high-grade cervical lesions revealed that patients with 12-month HPV persistence had a 2-fold greater 5-year recurrence rate than did those with 6-month HPV persistence . The crude recurrence rates were approximately 7.46%, 13.1%, and 10.3% for patients with 6-, 12-, and 24-month HPV persistence, respectively . These studies shed light on the fact that high-grade cervical lesions do occur in women < 30 years of age, including those who are cytology negative but HR-HPV-positive, as well as the potential impact of HPV persistence on long-term outcomes. Patients < 30 years of age with CIN2 + lesions are likely to be missed or progress to malignancy if they are screened by cervical cytology alone according to the American College of Obstetricians and Gynaecologists and the United States Preventive Services Task Force recommendation or are managed under the most recent ASCCP guidelines (2019) . Thus, cervical cancer screening by cotesting in women aged < 30 years should be suggested, and immediate colposcopy referral should be recommended. As a subjective examination method, colposcopy can detect more CIN lesions. Studies have shown that the accuracy of colposcopy can be influenced by several factors, such as the expertise of the colposcopist, HPV genotype, HPV viral load, cytology results, transformation zone type and age . The overall accuracy of colposcopy in detecting CIN2 + lesions ranges from 69.7% to 89%, the sensitivity ranges from 30% to 90%, and the specificity ranges from 44% to 97% . This study demonstrated a comparable accuracy of colposcopy diagnosis (91.1%), with relatively lower sensitivity (50.0%) and increased specificity (96.9%) when CIN2 + lesions was the threshold. Moreover, this study suggested that age and the type of cervical transformation zone were the factors affecting the accuracy of colposcopy diagnosis, which is consistent with the findings of Liu et al. . This may be due to low oestrogen levels and incomplete exposure of the cervical transformation zone. Notably, nearly half of the CIN2 + lesions patients were missed at initial colposcopy based on the results of this study and Bangladesh’s results . Therefore, in the event of a suspicious colposcopy result, age and the transformation zone should be taken into consideration along with clinical symptoms to reduce the risk of missed cervical lesions. In conclusion, this study revealed that the percentages of immediate CIN2 + lesions and CIN3 + lesions in cytology-negative patients who were non-16/18 HR-HPV-positive were comparable to those who were HPV16/18-positive. Follow-up after 1 year might be unsafe for these patients, as a majority of the screening patients would have insufficient follow-up in countries such as China. High-grade cervical lesions occur in women < 30 years of age, including those who are cytology negative but HR-HPV-positive. Thus, it is feasible to recommend immediate colposcopy referral in patients who are cytology negative but HR-HPV-positive (including non-16/18 positive) in countries with limited resources. In addition, cervical cancer screening by cotesting in women aged < 30 years should be suggested in countries with poor resources. Colposcopy has moderate diagnostic value and can be affected by age and the type of cervical transformation zone. The strength of the study is the use of total genotyping, which allowed us to also study the frequency of non-16/18 HPV genotypes in CIN2 + lesions. This is important because it has been demonstrated that CIN2 + lesions are frequently associated with non-16/18 HPV genotypes in older women. This study had several limitations. First, this was a retrospective study with a small sample size. We anticipate that larger sample sizes and prospective, randomized, and multicentre research findings will yield a better clinical assessment of patients and a better choice in terms of the type of therapy. Second, it was difficult to determine whether the influencing factors were independently associated with outcomes in this retrospective study. Third, long-term follow-up could not be estimated in this study because the study only evaluated the immediate risk of CIN2 + lesions, and whether the duration of HPV persistence might impact the risk of recurrence could not be evaluated. The long-term risk of CIN2 + lesions during follow-up and the risk of recurrence in patients with persistent HPV might be evaluated in future studies. Finally, as this study was limited by the small sample size of the Chinese population, further research is necessary to ascertain the applicability and generalizability of these findings to other populations. |
The phospho-ferrozine assay: a tool to study bacterial redox-active metabolites produced at the plant root | 70a740d9-1fb0-4429-be91-cf5d8f475583 | 11784245 | Microbiology[mh] | Microbial communities living at the plant root can make important contributions to plant health, and these communities are frequently controlled by exchanges of small molecules or secondary metabolites. These molecules can shape community composition, regulate commensal and pathogenic plant–microbe relationships, and aid in nutrient acquisition . Although secondary metabolites are increasingly appreciated for their role in microbe–microbe and plant–microbe interactions in soil, the sheer diversity of structures and reactivities is staggering, and we lack a fundamental understanding of which metabolites and metabolite producers are most influential in the rhizosphere. The question of how to identify secondary metabolites that govern microbial communities is a long-standing one across natural and biomedical contexts . A key part of the problem is that while secondary metabolite biosynthetic gene clusters are ubiquitous in microbial genomes from diverse environments , most are not produced under standard laboratory conditions . The difficulty in activating small molecule biosynthesis is as fascinating as it is frustrating and suggests that a better understanding of the regulation of secondary metabolite production might hold the key to understanding the function of these molecules in the wild. Indeed, low doses of antibiotics have emerged as some of the most effective activators of small molecule biosynthesis , implying that the production of many secondary metabolites is tuned by growth with other microbes. Nutrient stress can also lead to production of small molecules and in some cases has offered clues as to environmental function. The most notable example of this is the production of siderophores, iron-binding small molecules that are produced under iron limitation and aid bacteria in accessing this nutrient . We recently reported a link between redox-active secondary metabolites and limitation for the macronutrient phosphorus (P) that is analogous to that between siderophores and iron and may help us better understand the function of these molecules in the rhizosphere. Redox-active metabolites (RAMs) are a class of small, secreted molecules defined by their ability to perform electron-transfer reactions. One of the best-studied groups of RAMs are molecules called phenazines, which are commonly produced by pseudomonads and can take on a variety of roles including antibiotic activity, energy conservation, and nutrient solubilization . The P-controlled PhoBR two-component system regulates the biosynthesis of phenazines in many species of Pseudomonas and is also predicted to regulate the production of secondary metabolites in other bacteria . While difficult to understand in infection contexts, where phosphate concentrations are often quite high , this regulatory response may be especially useful in soils where phosphate is often immobilized on the surface of minerals like iron(III) oxides . The interaction between phosphate and soil minerals contributes to low P bioavailability across both natural and agricultural systems, leading to dissolved phosphorus concentrations in the micromolar range, which can make it difficult for plants to access P even in heavily fertilized soils . Owing to their capacity to reduce iron(III) to iron(II), we previously found that RAMs can increase the bioavailability of mineral-associated phosphorus . Here, we explore this link between RAMs and P stress as a tool for secondary metabolite discovery and a means to better understand how microbes use these molecules in soils and how this may contribute to plant health. We reasoned that regulation by P might serve as an expedient way to stimulate RAM production, while redox activity could act as a convenient chemical hook that can be easily detected through the interactions of RAMs with iron. If RAMs are poised at an appropriate redox potential, their presence should be detectable by monitoring the reduction of iron(III) to iron(II) via the ferrozine assay, a well-established colorimetric technique for quantifying iron(II) . We combined these two ideas to yield the phospho-ferrozine assay, which we vetted using synthetic RAMs and cultured pseudomonads. To determine the frequency of P-regulated RAM production in the rhizosphere, we generated a library of 557 root-associated bacterial isolates from sites across the United States. By applying the phospho-ferrozine assay, we found that 28% of isolates across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes likely produce P-regulated RAMs. We focused further genomic and metabolomic investigations on small molecule production in a soil isolate identified as Pseudomonas kielensis . Contrary to expectations, this organism does not appear to synthesize a known phenazine but instead makes a different RAM under P stress. Overall, this work highlights the prevalence of P-regulated RAM production in soil bacteria, provides a rapid and efficient approach to screen for this trait, and suggests that RAMs may play an important role in shaping plant and microbial access to P. Development of the phospho-ferrozine assay as a screen for bacteria that produce RAMs As a proof of concept, we first determined whether the phospho-ferrozine assay could be used to detect the production of RAMs in bacteria known to make these small molecules . We chose to focus on Pseudomonas synxantha as this bacterium is frequently found in soil and is well studied for the production of phenazines , which are synthesized under P limitation . We verified that a purified standard of phenazine-1-carboxylic acid (PCA), a RAM made by P. synxantha , as well as numerous soil-dwelling pseudomonads , can facilitate iron reduction and lead to a positive ferrozine signal. Consistent with previous findings, PCA produces a ferrozine signal when mixed with iron(III) . Next, we sought to translate our abiotic findings to whole-cell assays. To do so, we aimed to define a P concentration that would stimulate RAM production but maintain robust growth. To ensure that RAM production is specific to P limitation, we also optimized growth under nitrogen (N) limitation, a condition that is not expected to stimulate RAM production . We found that 100 µM P and 2 mM N were optimal concentrations to limit bacterial growth given that P. synxantha obtained similar optical densities in both conditions ( , purple lines). We next tested if the ferrozine assay could detect P-regulated RAM production from pseudomonads. Our assay depends on electron transfer from RAMs to iron(III) and therefore requires that RAMs be in their reduced, electron-carrying state . In the case of phenazines and pseudomonads, the mechanism of metabolite reduction is an open area of research, but is thought to be facilitated by the bacterial cell . In addition, phenazines are easily oxidized by oxygen in air, rendering them unable to perform iron(III) reduction . To both facilitate phenazine reduction and minimize phenazine oxidation, we incubated cultures in an anaerobic chamber for 1 h prior to performing the ferrozine assay. With this approach, the ferrozine signal in two phenazine-producing pseudomonads, P. synxantha and Pseudomonas aeruginosa , was significantly higher under P limitation as compared with N limitation . Importantly, the ferrozine signal produced in cultures of a P. aeruginosa strain with deletions in phenazine biosynthetic genes (Δ phz , was low and did not change in response to P-limitation . Using liquid-chromatography mass spectrometry (LC-MS), we verified that our growth conditions led to changes in phenazine production . P limitation stimulated PCA production in both P. synxantha and P. aeruginosa, but not the P. aeruginosa Δ phz strain. In P. aeruginosa, we also observed enhanced production of pyocyanin, another phenazine commonly made by this organism, under P limitation . No phenazines were detected when any strain was limited for N . The production of phenazines in the presence of 100 µM phosphate is consistent with recent work on the sensitivity of P. synxantha PhoBR to phosphate , and previous literature findings on the regulation of secondary metabolites by phosphate where medium phosphate concentrations above a few hundred micromolar often lead to repression (see, for example, references , ). This value is also within the range of reported dissolved phosphorus concentrations in soils . Altogether, these experiments indicate that the ferrozine assay in combination with P limitation can reliably be used to identify bacteria that produce RAMs and that 100 µM is a phosphorus concentration relevant for screening soil-dwelling bacteria. Application of the phospho-ferrozine assay to a diverse panel of root-associated bacteria To extend our findings beyond strains in current culture collections, we generated a library of root-associated bacteria. Isolates were obtained from three sites and 20 plants collected across the United States: Brachyelytrum aristosum grasses in Harvard Forest, Panicum virgatum grasses in Konza Prairie Biological Station, Digitaria californica grasses in Santa Rita Experimental Range, and commercial tomato plants . A total of 557 strains of at least 48 genera were collected across all sampling sites. A comparison of the bacterial composition of root-adhered soil, plated soil ( via scraping the entire plate, see Methods), and our final isolate collection showed that diversity decreased between whole soil and plates, reflecting the bias known to be introduced through media choice and plating. However, diversity was maintained between plated soil and our final isolate collection, with both being enriched for Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes ( ; , and ). We used the phospho-ferrozine assay to screen all 557 soil isolates for the ability to produce RAMs in response to P limitation. One hundred one soil isolates showed poor growth (OD 562 < 0.10) and were excluded from further analysis. P limitation increased the ferrozine signal by at least twofold in 128 of the remaining 456 (28%) soil isolates (31 Harvard Forest isolates, 34 Konza Prairie isolates, 33 Santa Rita isolates, and 30 tomato plant isolates, ; ). At least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes showed an increased ferrozine signal in response to P limitation . The majority of the positive hits identified were Proteobacteria (61%), followed by Actinobacteria (11%), Bacteroidetes (6%), and lastly, Firmicutes (5%) . Despite multiple attempts to PCR amplify the 16S region, 21 positive hits remained unidentified . The highest phospho-ferrozine signal for each location was from isolates identified at the genus level as Citrobacter (tomato plants), Stenotrophomonas (Harvard Forest), Pseudomonas (Konza Prairie), and Bacillus (Santa Rita). Isolates from Konza Prairie and Santa Rita showed the highest overall phospho-ferrozine signal, increasing by ~20-fold when limited for phosphorus . These results suggest that a diverse array of soil bacteria across multiple phyla can produce RAMs in response to P limitation. Verification of diffusible redox-active metabolite production using cyclic voltammetry When conducted with whole cells, the phospho-ferrozine screen cannot distinguish between iron(III) reduction by cellular machinery or cell-associated metabolites vs iron(III) reduction by diffusible RAMs. As our primary interest was in the latter, we investigated whether the ferrozine signal we observed from whole cells was in fact due to diffusible metabolites by conducting the phospho-ferrozine assay on filter-sterilized supernatants from a subset of P-limited soil isolates. We prioritized isolates that showed a strong response in the whole-cell assay and aimed to capture broad taxonomic diversity. Of the 48 soil isolate supernatants tested, the ferrozine signal was significantly increased under low P in 22 soil isolate supernatants . Supernatants from a Konza Prairie Pseudomonas isolate showed the highest ferrozine signal, which was 8.1-fold greater than P. aeruginosa Δ phz . These results suggest that the whole-cell ferrozine assay detects iron(III) reduction by diffusible RAMs in our soil isolates. To further confirm the presence of redox-active molecules in supernatants, we employed cyclic voltammetry, a standard electrochemical method often used to characterize redox activity . Using cyclic voltammetry, we were clearly able to detect the presence of phenazines in supernatants from P-limited wild-type P. synxantha and P. aeruginosa cultures, but we did not observe any signal in P. aeruginosa Δ phz strains . We also detected redox-active compounds in the supernatants of multiple soil isolates including Chromobacterium , Paenarthrobacter, Arthrobacter , and Pseudomonas . However, not all supernatants that showed an elevated phospho-ferrozine signal showed clear evidence for the presence of redox-active compounds , potentially due to concentrations being too low or molecule damaged during drying. Of note, a Pseudomonas isolate from Konza Prairie showed robust P-regulated RAM production from whole-cell and supernatant ferrozine assays as well as cyclic voltammetry analysis ; we chose to focus further efforts on understanding RAM production in this organism. Pseudomonas kielensis produces a non-canonical redox-active metabolite under low phosphorus To investigate the biosynthetic capacity of our Pseudomonas isolate, we conducted Illumina whole genome sequencing. Using a full length 16S sequence, this isolate was identified to the species level as Pseudomonas kielensis with 99.4% 16S identity to its closest relative by best BLAST. As pseudomonads frequently produce phenazines, we suspected this organism might be synthesizing some variant of this type of RAM. However, a search of the genome using antiSMASH failed to identify any phenazine biosynthetic gene clusters. A BLAST search using the phenazine biosynthetic genes phzA-F from Pseudomonas fluorescens (tblastn, e-value cutoff of 0.001) revealed only one hit to a gene annotated as phzF , which was not co-located with other phenazine genes suggesting that this organism does not encode phenazine biosynthetic gene clusters. To check the functionality of the phzF homolog, we created a clean deletion of the gene. The ∆ phzF mutant showed a small decrease in the phospho-ferrozine signal, but the majority of the signal was maintained , suggesting that this gene was not a major contributor to RAM production. In addition, investigations of the redox potential of this new metabolite suggested that it was not a canonical phenazine. Cyclic voltammetry experiments conducted across pH values (5.5–7.5) showed that the oxidation and reduction peaks from the P. kielensis supernatant shifted with pH, consistent with a proton-coupled redox reaction . However, the calculated midpoint potential of the P. kielensis supernatants ranged from −153 mV to −248 mV (vs NHE), which is far more negative than the potentials reported for PCA across this pH range (−1mV to −116mV vs NHE) as well as most well-studied phenazines . We next used metabolomics to search for phenazines and other metabolites in N- and P-limited P. kielensis supernatants. To ensure optimal conditions for metabolomic studies of metabolite production, we verified that when grown on 100 µM phosphorus P. kielensis was indeed limited for this nutrient . In addition, we measured changes in the ferrozine signal in response to a gradient of phosphorus concentrations ranging from 12.5 µM to 400 µM. Both analyses suggested that 100 µM was an optimal condition for phosphorus limitation and small molecule production in this organism . Under these phosphorus-limited conditions, we were unable to detect any phenazines using searches for the masses of PCA, pyocyanin, 1-hydroxy-phenazine, or phenazine-1-carboxamide (m/z = 225.2, 211.2, 197.2, 224.2, ([M + H] + ). A comparison of P. kielensis supernatants to purified standards of the phenazines pyocyanin and PCA also showed no clear overlap. Instead, our untargeted analysis revealed several peaks in the total ion chromatogram that were present under low P but not low N . These corresponded to m/z = 273.1, 285.1, 287.1, and 289.1 ([M + H] + ). All four peaks were also present in supernatants from the ∆ phzF mutant , further supporting the hypothesis that this gene does not contribute to RAM production. Altogether, our data suggest that P. kielensis produces a RAM (or RAMs) with reversible and proton-coupled redox activity, but that these molecules are distinct from well-known phenazines, possibly representing a different class of compounds. Investigations into the structures and biosynthetic pathways of these molecules are currently underway. Application and broader implications of the phospho-ferrozine assay Here, we show that the phospho-ferrozine assay detects the presence of RAMs in pseudomonads known to make these molecules and that this screen can be applied to a variety of soil isolates. As in any screen, the fidelity between the observed signal and RAM production must be independently verified. One key concern is the possibility of false positives resulting from cellular iron reduction instead of RAM production. Our experiments with P. aeruginosa phenazine-null mutants show that in this organism, non-specific cellular iron(III) reduction does not lead to a positive ferrozine signal . Nonetheless, it is notable that in some cases, cell-free supernatants showed a drop in ferrozine activity or failed to show a clear signal when analyzed with cyclic voltammetry. As we only performed cyclic voltammetry on samples that showed positive supernatant ferrozine assays, the lack of small molecule detection via this method is most likely due to low concentrations of RAMs or molecule damage during sample dry-down and resuspension. The discrepancy between whole-cell and supernatant ferrozine assay is more complex. One potential culprit could be dissimilatory iron reduction, a specialized anaerobic metabolism in which iron(III) replaces oxygen as the terminal electron acceptor . However, this process is not known to be regulated by P and is typically repressed by oxygen . Our experiments were conducted under aerobic conditions with only a 1-h anaerobic incubation in which isolates might activate dissimilatory iron reduction machinery, making it unlikely that this process is a major contributor to false positives. Another consideration is that in order to reduce iron(III), RAMs must be maintained in their reduced state, and this process may require cellular reduction . As such, instead of false positives in our whole-cell assays, some of our supernatant results may be false negatives driven by incomplete metabolite reduction. It is also possible that some of our isolates produce RAMs that are tightly cell-associated and are removed during filtration. Our primary interest has been in diffusible metabolites, but the screen could easily be adapted to detect these types of cell-associated RAMs. While care must be taken to validate results and some false positives and negatives are to be expected, the phospho-ferrozine screen represents a rapid, high-throughput, and relatively inexpensive way to screen for RAM production. The screen is comparable to the Chrome-Azurol-S (CAS) assay , which selects for a specific chemical feature (iron binding) and uses nutrient (iron) stress to stimulate production. Independent verification is also needed for the CAS assay, but it has been of extraordinary utility in detecting siderophores and in activity-guided studies to isolate these molecules. We expect the phospho-ferrozine screen to facilitate similar advances in isolation and characterization of RAMs as well as studies to determine the prevalence and patterning of P-regulated RAM production across microbial communities. A link between secondary metabolites and P stress has been well-documented in the literature . However, previous studies have mostly focused on strains in culture collections, leaving open the question of whether this is truly widespread in soils. Our finding that 28% of isolates produced a positive phospho-ferrozine signal suggests that this feature is common across genera and environments. Small modifications to the screen, such as storing isolate libraries in an array format in 96-well plates could allow the assay to be applied at a very large scale and open the door for more biogeographic studies probing the distribution and frequency of RAM production across environments. Indeed, previous studies focused on phenazines have shown that the capacity to synthesize these molecules can be present in 1%–2% of bacteria . Future studies that expand the search to all RAMs and incorporate regulatory logic and soil phosphorus availability will be an exciting next step for the study of RAMs as well as secondary metabolites in general. Finally, our findings underscore the importance of considering nutrient stress when studying secondary metabolites and highlight an opportunity to fuse these types of studies with investigations of small molecule elicitors of secondary metabolite biosynthesis. While challenging, this endeavor holds great promise for understanding the environment experienced by microbes in the wild and will contribute to applied efforts to manipulate microbes across biomedical and soil contexts. We report here that many soil-dwelling organisms activate RAM biosynthesis in response to phosphorus stress imposed by 100 µM P. However, the speciation of phosphorus in terrestrial systems is complex, and there is still much to learn about bioavailability of soil phosphorus. This element may occur in organic forms, such as phytate or phosphonate, as hydroxyapatite and as phosphate or organic phosphate adsorbed to mineral surfaces. Despite high extractable phosphorus values, readily available dissolved phosphate is often present in micromolar concentrations . Most studies suggest that small molecule biosynthesis responds more strongly to phosphate (as opposed to organic P), and these responses become active when external phosphate is below a few hundred micromolar . Our own investigations of our P. kielensis isolate were remarkably consistent with previous findings, suggesting phosphorus repression of RAM production above ~100 µM . Future quantitative studies of the external phosphorus concentrations that activate small molecule biosynthesis may help us to understand secondary metabolite functions and contribute to our understanding of the range of phosphorus concentrations and species a microbe might encounter in the environment. Many soil-dwelling microbes, including pseudomonads, can also live as opportunistic pathogens. In human hosts, phosphate is expected to be maintained at millimolar concentrations , raising the question of what role the link between phosphorus and RAMs may play in infection contexts. One partial explanation comes through consideration of the multilayered regulation of RAM and other small molecule biosynthesis. As is common for secondary metabolites, phenazines are also regulated by small molecule elicitors , including quorum sensing molecules. This type of combined nutrient and small-molecule-based regulation occurs for siderophores and we expect it will also occur in our newly discovered RAM producers. In some pseudomonads, it appears that nutrient stress can activate quorum sensing at relatively low cells densities, suggesting that density-dependent behaviors are tuned by environmental nutrient availability . In the future, it will be important to blend studies of small molecule and nutrient stress regulation. Such combinatorial studies may aid the discovery of novel metabolites that might act as therapeutics and help us understand when and how they might be produced. Finally, there is also growing momentum for using secondary metabolites to manage microbial functions in ways that promote plant health – either through the suppression of pathogens or enhancement of nutrient access . Phosphorus bioavailability is of paramount importance in natural and agricultural systems , and there is a precedent for the promotion of plant access to phosphorus by microbes . The extent to which RAMs may participate in nutrient access or pathogen suppression at plant root remains unknown. The phospho-ferrozine assay offers a powerful tool to begin addressing these questions and our findings that numerous bacteria produce RAMs in response to P stress bodes well for future work aimed at understanding P cycling in natural systems and at leveraging these small molecules in managed ones. Conclusion We developed a tool to rapidly identify RAM-producing bacteria from the soil by utilizing P limitation to induce RAM biosynthesis and measuring redox activity through the ferrozine assay. We show that multiple genera across four different phyla secrete RAMs in response to P limitation, suggesting that this regulatory mechanism is widespread. Further, we show that despite the frequency of phenazine biosynthesis in pseudomonads, P. kielensis most likely does not synthesize these metabolites and instead makes a different RAM. Our work opens the door for studies investigating how the production of these small molecules is distributed across different environments, the multilayered molecular mechanisms microbes use to regulate their biosynthesis, and the overall role RAMs play within microbial communities living at plant roots. As a proof of concept, we first determined whether the phospho-ferrozine assay could be used to detect the production of RAMs in bacteria known to make these small molecules . We chose to focus on Pseudomonas synxantha as this bacterium is frequently found in soil and is well studied for the production of phenazines , which are synthesized under P limitation . We verified that a purified standard of phenazine-1-carboxylic acid (PCA), a RAM made by P. synxantha , as well as numerous soil-dwelling pseudomonads , can facilitate iron reduction and lead to a positive ferrozine signal. Consistent with previous findings, PCA produces a ferrozine signal when mixed with iron(III) . Next, we sought to translate our abiotic findings to whole-cell assays. To do so, we aimed to define a P concentration that would stimulate RAM production but maintain robust growth. To ensure that RAM production is specific to P limitation, we also optimized growth under nitrogen (N) limitation, a condition that is not expected to stimulate RAM production . We found that 100 µM P and 2 mM N were optimal concentrations to limit bacterial growth given that P. synxantha obtained similar optical densities in both conditions ( , purple lines). We next tested if the ferrozine assay could detect P-regulated RAM production from pseudomonads. Our assay depends on electron transfer from RAMs to iron(III) and therefore requires that RAMs be in their reduced, electron-carrying state . In the case of phenazines and pseudomonads, the mechanism of metabolite reduction is an open area of research, but is thought to be facilitated by the bacterial cell . In addition, phenazines are easily oxidized by oxygen in air, rendering them unable to perform iron(III) reduction . To both facilitate phenazine reduction and minimize phenazine oxidation, we incubated cultures in an anaerobic chamber for 1 h prior to performing the ferrozine assay. With this approach, the ferrozine signal in two phenazine-producing pseudomonads, P. synxantha and Pseudomonas aeruginosa , was significantly higher under P limitation as compared with N limitation . Importantly, the ferrozine signal produced in cultures of a P. aeruginosa strain with deletions in phenazine biosynthetic genes (Δ phz , was low and did not change in response to P-limitation . Using liquid-chromatography mass spectrometry (LC-MS), we verified that our growth conditions led to changes in phenazine production . P limitation stimulated PCA production in both P. synxantha and P. aeruginosa, but not the P. aeruginosa Δ phz strain. In P. aeruginosa, we also observed enhanced production of pyocyanin, another phenazine commonly made by this organism, under P limitation . No phenazines were detected when any strain was limited for N . The production of phenazines in the presence of 100 µM phosphate is consistent with recent work on the sensitivity of P. synxantha PhoBR to phosphate , and previous literature findings on the regulation of secondary metabolites by phosphate where medium phosphate concentrations above a few hundred micromolar often lead to repression (see, for example, references , ). This value is also within the range of reported dissolved phosphorus concentrations in soils . Altogether, these experiments indicate that the ferrozine assay in combination with P limitation can reliably be used to identify bacteria that produce RAMs and that 100 µM is a phosphorus concentration relevant for screening soil-dwelling bacteria. To extend our findings beyond strains in current culture collections, we generated a library of root-associated bacteria. Isolates were obtained from three sites and 20 plants collected across the United States: Brachyelytrum aristosum grasses in Harvard Forest, Panicum virgatum grasses in Konza Prairie Biological Station, Digitaria californica grasses in Santa Rita Experimental Range, and commercial tomato plants . A total of 557 strains of at least 48 genera were collected across all sampling sites. A comparison of the bacterial composition of root-adhered soil, plated soil ( via scraping the entire plate, see Methods), and our final isolate collection showed that diversity decreased between whole soil and plates, reflecting the bias known to be introduced through media choice and plating. However, diversity was maintained between plated soil and our final isolate collection, with both being enriched for Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes ( ; , and ). We used the phospho-ferrozine assay to screen all 557 soil isolates for the ability to produce RAMs in response to P limitation. One hundred one soil isolates showed poor growth (OD 562 < 0.10) and were excluded from further analysis. P limitation increased the ferrozine signal by at least twofold in 128 of the remaining 456 (28%) soil isolates (31 Harvard Forest isolates, 34 Konza Prairie isolates, 33 Santa Rita isolates, and 30 tomato plant isolates, ; ). At least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes showed an increased ferrozine signal in response to P limitation . The majority of the positive hits identified were Proteobacteria (61%), followed by Actinobacteria (11%), Bacteroidetes (6%), and lastly, Firmicutes (5%) . Despite multiple attempts to PCR amplify the 16S region, 21 positive hits remained unidentified . The highest phospho-ferrozine signal for each location was from isolates identified at the genus level as Citrobacter (tomato plants), Stenotrophomonas (Harvard Forest), Pseudomonas (Konza Prairie), and Bacillus (Santa Rita). Isolates from Konza Prairie and Santa Rita showed the highest overall phospho-ferrozine signal, increasing by ~20-fold when limited for phosphorus . These results suggest that a diverse array of soil bacteria across multiple phyla can produce RAMs in response to P limitation. When conducted with whole cells, the phospho-ferrozine screen cannot distinguish between iron(III) reduction by cellular machinery or cell-associated metabolites vs iron(III) reduction by diffusible RAMs. As our primary interest was in the latter, we investigated whether the ferrozine signal we observed from whole cells was in fact due to diffusible metabolites by conducting the phospho-ferrozine assay on filter-sterilized supernatants from a subset of P-limited soil isolates. We prioritized isolates that showed a strong response in the whole-cell assay and aimed to capture broad taxonomic diversity. Of the 48 soil isolate supernatants tested, the ferrozine signal was significantly increased under low P in 22 soil isolate supernatants . Supernatants from a Konza Prairie Pseudomonas isolate showed the highest ferrozine signal, which was 8.1-fold greater than P. aeruginosa Δ phz . These results suggest that the whole-cell ferrozine assay detects iron(III) reduction by diffusible RAMs in our soil isolates. To further confirm the presence of redox-active molecules in supernatants, we employed cyclic voltammetry, a standard electrochemical method often used to characterize redox activity . Using cyclic voltammetry, we were clearly able to detect the presence of phenazines in supernatants from P-limited wild-type P. synxantha and P. aeruginosa cultures, but we did not observe any signal in P. aeruginosa Δ phz strains . We also detected redox-active compounds in the supernatants of multiple soil isolates including Chromobacterium , Paenarthrobacter, Arthrobacter , and Pseudomonas . However, not all supernatants that showed an elevated phospho-ferrozine signal showed clear evidence for the presence of redox-active compounds , potentially due to concentrations being too low or molecule damaged during drying. Of note, a Pseudomonas isolate from Konza Prairie showed robust P-regulated RAM production from whole-cell and supernatant ferrozine assays as well as cyclic voltammetry analysis ; we chose to focus further efforts on understanding RAM production in this organism. produces a non-canonical redox-active metabolite under low phosphorus To investigate the biosynthetic capacity of our Pseudomonas isolate, we conducted Illumina whole genome sequencing. Using a full length 16S sequence, this isolate was identified to the species level as Pseudomonas kielensis with 99.4% 16S identity to its closest relative by best BLAST. As pseudomonads frequently produce phenazines, we suspected this organism might be synthesizing some variant of this type of RAM. However, a search of the genome using antiSMASH failed to identify any phenazine biosynthetic gene clusters. A BLAST search using the phenazine biosynthetic genes phzA-F from Pseudomonas fluorescens (tblastn, e-value cutoff of 0.001) revealed only one hit to a gene annotated as phzF , which was not co-located with other phenazine genes suggesting that this organism does not encode phenazine biosynthetic gene clusters. To check the functionality of the phzF homolog, we created a clean deletion of the gene. The ∆ phzF mutant showed a small decrease in the phospho-ferrozine signal, but the majority of the signal was maintained , suggesting that this gene was not a major contributor to RAM production. In addition, investigations of the redox potential of this new metabolite suggested that it was not a canonical phenazine. Cyclic voltammetry experiments conducted across pH values (5.5–7.5) showed that the oxidation and reduction peaks from the P. kielensis supernatant shifted with pH, consistent with a proton-coupled redox reaction . However, the calculated midpoint potential of the P. kielensis supernatants ranged from −153 mV to −248 mV (vs NHE), which is far more negative than the potentials reported for PCA across this pH range (−1mV to −116mV vs NHE) as well as most well-studied phenazines . We next used metabolomics to search for phenazines and other metabolites in N- and P-limited P. kielensis supernatants. To ensure optimal conditions for metabolomic studies of metabolite production, we verified that when grown on 100 µM phosphorus P. kielensis was indeed limited for this nutrient . In addition, we measured changes in the ferrozine signal in response to a gradient of phosphorus concentrations ranging from 12.5 µM to 400 µM. Both analyses suggested that 100 µM was an optimal condition for phosphorus limitation and small molecule production in this organism . Under these phosphorus-limited conditions, we were unable to detect any phenazines using searches for the masses of PCA, pyocyanin, 1-hydroxy-phenazine, or phenazine-1-carboxamide (m/z = 225.2, 211.2, 197.2, 224.2, ([M + H] + ). A comparison of P. kielensis supernatants to purified standards of the phenazines pyocyanin and PCA also showed no clear overlap. Instead, our untargeted analysis revealed several peaks in the total ion chromatogram that were present under low P but not low N . These corresponded to m/z = 273.1, 285.1, 287.1, and 289.1 ([M + H] + ). All four peaks were also present in supernatants from the ∆ phzF mutant , further supporting the hypothesis that this gene does not contribute to RAM production. Altogether, our data suggest that P. kielensis produces a RAM (or RAMs) with reversible and proton-coupled redox activity, but that these molecules are distinct from well-known phenazines, possibly representing a different class of compounds. Investigations into the structures and biosynthetic pathways of these molecules are currently underway. Here, we show that the phospho-ferrozine assay detects the presence of RAMs in pseudomonads known to make these molecules and that this screen can be applied to a variety of soil isolates. As in any screen, the fidelity between the observed signal and RAM production must be independently verified. One key concern is the possibility of false positives resulting from cellular iron reduction instead of RAM production. Our experiments with P. aeruginosa phenazine-null mutants show that in this organism, non-specific cellular iron(III) reduction does not lead to a positive ferrozine signal . Nonetheless, it is notable that in some cases, cell-free supernatants showed a drop in ferrozine activity or failed to show a clear signal when analyzed with cyclic voltammetry. As we only performed cyclic voltammetry on samples that showed positive supernatant ferrozine assays, the lack of small molecule detection via this method is most likely due to low concentrations of RAMs or molecule damage during sample dry-down and resuspension. The discrepancy between whole-cell and supernatant ferrozine assay is more complex. One potential culprit could be dissimilatory iron reduction, a specialized anaerobic metabolism in which iron(III) replaces oxygen as the terminal electron acceptor . However, this process is not known to be regulated by P and is typically repressed by oxygen . Our experiments were conducted under aerobic conditions with only a 1-h anaerobic incubation in which isolates might activate dissimilatory iron reduction machinery, making it unlikely that this process is a major contributor to false positives. Another consideration is that in order to reduce iron(III), RAMs must be maintained in their reduced state, and this process may require cellular reduction . As such, instead of false positives in our whole-cell assays, some of our supernatant results may be false negatives driven by incomplete metabolite reduction. It is also possible that some of our isolates produce RAMs that are tightly cell-associated and are removed during filtration. Our primary interest has been in diffusible metabolites, but the screen could easily be adapted to detect these types of cell-associated RAMs. While care must be taken to validate results and some false positives and negatives are to be expected, the phospho-ferrozine screen represents a rapid, high-throughput, and relatively inexpensive way to screen for RAM production. The screen is comparable to the Chrome-Azurol-S (CAS) assay , which selects for a specific chemical feature (iron binding) and uses nutrient (iron) stress to stimulate production. Independent verification is also needed for the CAS assay, but it has been of extraordinary utility in detecting siderophores and in activity-guided studies to isolate these molecules. We expect the phospho-ferrozine screen to facilitate similar advances in isolation and characterization of RAMs as well as studies to determine the prevalence and patterning of P-regulated RAM production across microbial communities. A link between secondary metabolites and P stress has been well-documented in the literature . However, previous studies have mostly focused on strains in culture collections, leaving open the question of whether this is truly widespread in soils. Our finding that 28% of isolates produced a positive phospho-ferrozine signal suggests that this feature is common across genera and environments. Small modifications to the screen, such as storing isolate libraries in an array format in 96-well plates could allow the assay to be applied at a very large scale and open the door for more biogeographic studies probing the distribution and frequency of RAM production across environments. Indeed, previous studies focused on phenazines have shown that the capacity to synthesize these molecules can be present in 1%–2% of bacteria . Future studies that expand the search to all RAMs and incorporate regulatory logic and soil phosphorus availability will be an exciting next step for the study of RAMs as well as secondary metabolites in general. Finally, our findings underscore the importance of considering nutrient stress when studying secondary metabolites and highlight an opportunity to fuse these types of studies with investigations of small molecule elicitors of secondary metabolite biosynthesis. While challenging, this endeavor holds great promise for understanding the environment experienced by microbes in the wild and will contribute to applied efforts to manipulate microbes across biomedical and soil contexts. We report here that many soil-dwelling organisms activate RAM biosynthesis in response to phosphorus stress imposed by 100 µM P. However, the speciation of phosphorus in terrestrial systems is complex, and there is still much to learn about bioavailability of soil phosphorus. This element may occur in organic forms, such as phytate or phosphonate, as hydroxyapatite and as phosphate or organic phosphate adsorbed to mineral surfaces. Despite high extractable phosphorus values, readily available dissolved phosphate is often present in micromolar concentrations . Most studies suggest that small molecule biosynthesis responds more strongly to phosphate (as opposed to organic P), and these responses become active when external phosphate is below a few hundred micromolar . Our own investigations of our P. kielensis isolate were remarkably consistent with previous findings, suggesting phosphorus repression of RAM production above ~100 µM . Future quantitative studies of the external phosphorus concentrations that activate small molecule biosynthesis may help us to understand secondary metabolite functions and contribute to our understanding of the range of phosphorus concentrations and species a microbe might encounter in the environment. Many soil-dwelling microbes, including pseudomonads, can also live as opportunistic pathogens. In human hosts, phosphate is expected to be maintained at millimolar concentrations , raising the question of what role the link between phosphorus and RAMs may play in infection contexts. One partial explanation comes through consideration of the multilayered regulation of RAM and other small molecule biosynthesis. As is common for secondary metabolites, phenazines are also regulated by small molecule elicitors , including quorum sensing molecules. This type of combined nutrient and small-molecule-based regulation occurs for siderophores and we expect it will also occur in our newly discovered RAM producers. In some pseudomonads, it appears that nutrient stress can activate quorum sensing at relatively low cells densities, suggesting that density-dependent behaviors are tuned by environmental nutrient availability . In the future, it will be important to blend studies of small molecule and nutrient stress regulation. Such combinatorial studies may aid the discovery of novel metabolites that might act as therapeutics and help us understand when and how they might be produced. Finally, there is also growing momentum for using secondary metabolites to manage microbial functions in ways that promote plant health – either through the suppression of pathogens or enhancement of nutrient access . Phosphorus bioavailability is of paramount importance in natural and agricultural systems , and there is a precedent for the promotion of plant access to phosphorus by microbes . The extent to which RAMs may participate in nutrient access or pathogen suppression at plant root remains unknown. The phospho-ferrozine assay offers a powerful tool to begin addressing these questions and our findings that numerous bacteria produce RAMs in response to P stress bodes well for future work aimed at understanding P cycling in natural systems and at leveraging these small molecules in managed ones. We developed a tool to rapidly identify RAM-producing bacteria from the soil by utilizing P limitation to induce RAM biosynthesis and measuring redox activity through the ferrozine assay. We show that multiple genera across four different phyla secrete RAMs in response to P limitation, suggesting that this regulatory mechanism is widespread. Further, we show that despite the frequency of phenazine biosynthesis in pseudomonads, P. kielensis most likely does not synthesize these metabolites and instead makes a different RAM. Our work opens the door for studies investigating how the production of these small molecules is distributed across different environments, the multilayered molecular mechanisms microbes use to regulate their biosynthesis, and the overall role RAMs play within microbial communities living at plant roots. Bacterial strains and growth conditions Strains, plasmids, and primers used in this study are listed in . A list of soil isolates and their closest 16S identity is provided in . Unless stated otherwise, all bacterial strains were grown at 30°C on Luria Broth (LB) agar plates prior to experiments. Liquid cultures were grown at 30°C with continuous shaking at 200 rpm except for Escherichia coli strains which were grown at 37°C. Bacterial strains were grown in defined media, (8.22 mM glucose, 12.33 mM succinate, 16.44 mM pyruvate, 16 mM NH 4 Cl, 4.70 mM K 2 HPO 4 , 2.30 mM KH 2 PO 4 , 0.68 mM CaCl 2 , 0.41 mM MgSO 4 , and Aquil trace metals supplemented with 10 µM iron, buffered at pH 5.5 with 25 mM MES). For P limitation, P was adjusted to 100 µM (K 2 HPO 4 = 33 µM and KH 2 PO 4 = 67 µM), while N was maintained at 16 mM. For N limitation, NH 4 Cl was adjusted to 2 mM, and P was maintained at 7 mM. For growth gradient experiments, cultures were grown overnight in defined media, washed and resuspended at OD 1.0 (500 nm) in either P-free or N-free defined media, and inoculated at an OD 500 of 0.01 in 96-well plates. Growth was tracked via absorbance at 500 nm every 30 min for 24 h using a BioTek Epoch 2 microplate reader with constant shaking. Abiotic ferrozine assays Abiotic ferrozine assays were performed in potassium chloride (KCl) buffer (1 M KCl buffered with 10 mM ammonium acetate-MOPS at pH 6) using a final concentration of 200 µM FeCl 3 and 2 mM ferrozine and the ferrozine–Fe(II) complex was quantified using absorbance at 562 nm. PCA was reduced electrochemically under an N 2 headspace as previously described , and experiments were performed anaerobically (95% N 2 , 5% H 2 ) to minimize PCA oxidation by oxygen. Liquid-chromatography mass spectrometry For metabolomics studies, filtered supernatants were analyzed directly on an Agilent 6125B single quadrupole mass spectrometer fitted with a diode array detector. Briefly, 2 µL of sample was injected into a C18 column (Agilent Poroshell 120: 50 mm length, 2.1 mm internal diameter, 2.7 µm particle size). Separation was achieved using a gradient from acidified (0.1% formic acid) 90% water/10% acetonitrile to 100% acetonitrile over 6 min. PCA and pyocyanin peaks were identified by comparison to purchased standards (Sigma) and quantified using a standard curve and the extracted absorbance at 315 nm. Isolation of root-associated bacteria from plants Five individual plants of the grass species Brachyelytrum aristosum , Panicum virgatum , and Digitaria californica were collected from National Ecological Observatory Network (NEON) sites at Harvard Forest, Konza Prairie, and Santa Rita, respectively (see for GPS coordinates and sampling dates). “Big Beef” variety tomato plants were purchased. NEON samples were transported on ice and processed within 3 days of harvest. To isolate root-associated bacteria, soil was shaken off the plant until only soil adhered to plant roots remained. Roots were cut near the base of the stem, placed in conical tubes with sterile wash buffer (0.40 mM MgSO 4 and 1.7 mM NaCl, pH 6.0), vortexed for 10 s, sonicated for 1 min, and vortexed again for an additional 10 s. The resulting slurry was diluted 10–100×, plated onto agar plates supplemented with 50 µg/mL nystatin, and incubated for 48 h at 25°C. Preliminary experiments used plates that contained glucose, succinate, pyruvate, or yeast extract and tryptone as carbon sources (10 mM defined carbon or 1 g/L yeast extract and 1 g/L tryptone) in addition to 0.5 mM NH 4 Cl, 0.5 mM K 2 HPO 4 , 1 mM MgSO 4 , 2 mM NaCl, MEM essential amino acids (1.6 ml/L, Sigma M5550), and Aquil trace metals supplemented with 10 µM Fe. However, comparisons between these different carbon sources showed that yeast extract and tryptone maintained the highest levels of diversity. As such, yeast extract-tryptone plates were used for all subsequent analyses. Colonies that appeared morphologically distinct were picked and re-streaked three times to purity before being freezer stocked. To ensure the sterility of the process, sterile wash buffer was also plated and showed no microbial growth. 16S amplicon sequencing of soil and plate samples Bacterial diversity on plates was determined by scraping the entire plate. Plates were incubated for 24 h, 2 mL phosphate-buffered saline solution (8 g/L NaCl, 0.2 g/L, 1.44 g/L Na 2 HPO 4 , 0.24 g/L KH 2 PO 4 ) was applied to the plate, cells were homogenized using a sterile scraper, collected in an Eppendorf tube, centrifuged at 10,000× g for 5 min, and pellets were stored at −80°C. For soils, a slurry (described above) was centrifuged at 10,000× g for 20 min, supernatants were decanted, and soil pellets were stored at −80°C. Genomic DNA from plates and soil samples was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and the DNeasy PowerLyzer Power Soil Kit (Qiagen), respectively. 16S paired-end amplicon sequencing was performed by SeqCenter (Pittsburgh, PA, USA) using the 341F (CCTACGGGDGGCWGCAG, CCTAYGGGGYGCWGCAG) and 806R(GACTACNVGGGTMTCTAATCC) primers. Amplicons were sequenced on a P1 600cyc NextSeq2000 Flowcell. Bioinformatic analysis was conducted using a standard QIIME2 pipeline . Adapters were removed using CutAdapt. Denoising and trimming was performed with DADA2: forward and reverse reads were trimmed to 250 bp, >100,000 reads were analyzed for each sample, except for tomato plant plates and soils samples where only ~60–77K reads were retained. Taxonomy was assigned using the Silva classifier. Identification of soil isolates The taxonomic identity of purified isolates was determined via colony PCR. Colonies resuspended in nuclease-free water and lysed by sonicating for 5 min at 40 kHz, followed by boiling for 10 min at 100°C. Cell lysates were pelleted, and clarified lysates were used for PCR reactions. The 16S rRNA gene was amplified using 1492R (TACGGYTACCTTGTTACGACTT) and 27F (AGAGTTTGATCMTGGCTCAG) primers . PCR products were run on an agarose gel to confirm the presence of a single amplicon at the expected size (~1,500 bp) and Sanger sequenced with the 27F primer at Epoch Life Science (Missouri City, TX, USA). Strain identity was obtained through a BLAST search against the NCBI 16S rRNA sequence database . Subsequently, 220 of 370 16S amplicon sequences were deposited in GenBank under the accession numbers PQ223443-PQ223663. The remaining 16S amplicon sequences failed to meet the requirements for submission to GenBank. Whole-cell phospho-ferrozine screen Pseudomonas cultures or soil isolates were grown for 24 h in 200 µL P- and N-replete media in a 96-well plate, transferred (1:100) to new 96-well plates containing either 200 µL P-limited or N-limited media, and grown for an additional 24 h. Plates were then incubated statically for one hour in an anaerobic chamber (95% N 2 and 5% H 2 ) to remove residual oxygen . Subsequently, 500 µM of FeCl 3 (maintained as a concentrated stock in 0.1 N HCl) followed by 2 mM ferrozine (in 2 M MOPS, pH 7) was added, and the ferrozine-Fe(II) complex was detected immediately via absorbance at 562 nm using a BioTek Synergy HTX plate reader. Ferrozine stock solutions were degassed in the anaerobic chamber for at least 3 days prior to experiments. To account for differences in growth that might contribute to the OD 562 signal, absorbances at 562 nm for each isolate were recorded prior to the addition of FeCl 3 .This background number was subtracted from the final ferrozine signal (denoted as ΔOD 562 ). The ferrozine signal for each soil isolate is provided in . Supernatant phospho-ferrozine assays Cultures were grown overnight in defined media, diluted to an OD 500 of 0.01 in 10 mL P-limited media and grown for an additional 24 h. P-limited cultures were then incubated statically for 1 h in an anaerobic chamber to deplete oxygen, and supernatants were filter sterilized using a 0.22 µm Spin-X column (Corning). Then, 200 µL of filter-sterilized supernatant was transferred to a 96-well plate and mixed with 500 µM FeCl 3 and 2 mM ferrozine. After 40 min, the ferrozine–Fe(II) complex was detected at 562 nm (BioTek Synergy HTX plate reader). The remaining volume of supernatant was used for cyclic voltammetry experiments below. For ferrozine assay gradients with P. kielensis , the protocol above was followed except P. kielensis cultures that were grown overnight in defined media were washed in P-free media, then diluted to an OD 500 of 0.01 in 10 mL P-limited media that ranged from 12.5 μM to 400 μM P. P-limited cultures were grown for 24 h, and growth (OD 500 ) was measured before transferring the cultures to the anaerobic chamber to measure ferrozine signal from filter-sterilized supernatants. Cyclic voltammetry Supernatants from P-limited cultures were filter-sterilized (0.22 µm), concentrated under a stream of N 2 gas, and resuspended in 1 mL KCl buffer. Cyclic voltammetry measurements were conducted using a BioLogic SP-300 potentiostat at a scan rate of 50 mV/s using a platinum wire counter electrode, an Ag/AgCl reference electrode and a 3 mm gold working electrode. All experiments with coumarin and PCA were conducted with 1 mM solutions in KCl buffer. pH gradient experiments were conducted under a constant stream of N 2 gas. Midpoint redox potentials were calculated as the average of the potentials at minimal and maximal current (vs Ag/AgCl) +207 mV for comparison to the NHE. Genomic sequencing and annotation P. kielensis genomic DNA from an LB liquid overnight culture was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Sequencing, assembly, and gene annotation were performed by SeqCenter (Pittsburgh, PA, USA) using an Illumina NovaSeq X Plus sequencer. Construction of P. kielensis deletion strains In-frame deletions were constructed by allelic exchange using the pMQ30 shuttle vector containing ~1 kb flanking regions, which were built by Gibson Assembly . Colony PCR and Sanger sequencing were used to confirm plasmid construction. Plasmids were introduced to P. kielensis by triparental conjugation and merodiploids were selected on Vogel–Bonner (VBMM) plates containing 50 µg/mL gentamycin . Transconjugants were counter selected on 5% sucrose LB medium with no added salt. Deletions were confirmed by colony PCR. Strains, plasmids, and primers used in this study are listed in . A list of soil isolates and their closest 16S identity is provided in . Unless stated otherwise, all bacterial strains were grown at 30°C on Luria Broth (LB) agar plates prior to experiments. Liquid cultures were grown at 30°C with continuous shaking at 200 rpm except for Escherichia coli strains which were grown at 37°C. Bacterial strains were grown in defined media, (8.22 mM glucose, 12.33 mM succinate, 16.44 mM pyruvate, 16 mM NH 4 Cl, 4.70 mM K 2 HPO 4 , 2.30 mM KH 2 PO 4 , 0.68 mM CaCl 2 , 0.41 mM MgSO 4 , and Aquil trace metals supplemented with 10 µM iron, buffered at pH 5.5 with 25 mM MES). For P limitation, P was adjusted to 100 µM (K 2 HPO 4 = 33 µM and KH 2 PO 4 = 67 µM), while N was maintained at 16 mM. For N limitation, NH 4 Cl was adjusted to 2 mM, and P was maintained at 7 mM. For growth gradient experiments, cultures were grown overnight in defined media, washed and resuspended at OD 1.0 (500 nm) in either P-free or N-free defined media, and inoculated at an OD 500 of 0.01 in 96-well plates. Growth was tracked via absorbance at 500 nm every 30 min for 24 h using a BioTek Epoch 2 microplate reader with constant shaking. Abiotic ferrozine assays were performed in potassium chloride (KCl) buffer (1 M KCl buffered with 10 mM ammonium acetate-MOPS at pH 6) using a final concentration of 200 µM FeCl 3 and 2 mM ferrozine and the ferrozine–Fe(II) complex was quantified using absorbance at 562 nm. PCA was reduced electrochemically under an N 2 headspace as previously described , and experiments were performed anaerobically (95% N 2 , 5% H 2 ) to minimize PCA oxidation by oxygen. For metabolomics studies, filtered supernatants were analyzed directly on an Agilent 6125B single quadrupole mass spectrometer fitted with a diode array detector. Briefly, 2 µL of sample was injected into a C18 column (Agilent Poroshell 120: 50 mm length, 2.1 mm internal diameter, 2.7 µm particle size). Separation was achieved using a gradient from acidified (0.1% formic acid) 90% water/10% acetonitrile to 100% acetonitrile over 6 min. PCA and pyocyanin peaks were identified by comparison to purchased standards (Sigma) and quantified using a standard curve and the extracted absorbance at 315 nm. Five individual plants of the grass species Brachyelytrum aristosum , Panicum virgatum , and Digitaria californica were collected from National Ecological Observatory Network (NEON) sites at Harvard Forest, Konza Prairie, and Santa Rita, respectively (see for GPS coordinates and sampling dates). “Big Beef” variety tomato plants were purchased. NEON samples were transported on ice and processed within 3 days of harvest. To isolate root-associated bacteria, soil was shaken off the plant until only soil adhered to plant roots remained. Roots were cut near the base of the stem, placed in conical tubes with sterile wash buffer (0.40 mM MgSO 4 and 1.7 mM NaCl, pH 6.0), vortexed for 10 s, sonicated for 1 min, and vortexed again for an additional 10 s. The resulting slurry was diluted 10–100×, plated onto agar plates supplemented with 50 µg/mL nystatin, and incubated for 48 h at 25°C. Preliminary experiments used plates that contained glucose, succinate, pyruvate, or yeast extract and tryptone as carbon sources (10 mM defined carbon or 1 g/L yeast extract and 1 g/L tryptone) in addition to 0.5 mM NH 4 Cl, 0.5 mM K 2 HPO 4 , 1 mM MgSO 4 , 2 mM NaCl, MEM essential amino acids (1.6 ml/L, Sigma M5550), and Aquil trace metals supplemented with 10 µM Fe. However, comparisons between these different carbon sources showed that yeast extract and tryptone maintained the highest levels of diversity. As such, yeast extract-tryptone plates were used for all subsequent analyses. Colonies that appeared morphologically distinct were picked and re-streaked three times to purity before being freezer stocked. To ensure the sterility of the process, sterile wash buffer was also plated and showed no microbial growth. Bacterial diversity on plates was determined by scraping the entire plate. Plates were incubated for 24 h, 2 mL phosphate-buffered saline solution (8 g/L NaCl, 0.2 g/L, 1.44 g/L Na 2 HPO 4 , 0.24 g/L KH 2 PO 4 ) was applied to the plate, cells were homogenized using a sterile scraper, collected in an Eppendorf tube, centrifuged at 10,000× g for 5 min, and pellets were stored at −80°C. For soils, a slurry (described above) was centrifuged at 10,000× g for 20 min, supernatants were decanted, and soil pellets were stored at −80°C. Genomic DNA from plates and soil samples was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and the DNeasy PowerLyzer Power Soil Kit (Qiagen), respectively. 16S paired-end amplicon sequencing was performed by SeqCenter (Pittsburgh, PA, USA) using the 341F (CCTACGGGDGGCWGCAG, CCTAYGGGGYGCWGCAG) and 806R(GACTACNVGGGTMTCTAATCC) primers. Amplicons were sequenced on a P1 600cyc NextSeq2000 Flowcell. Bioinformatic analysis was conducted using a standard QIIME2 pipeline . Adapters were removed using CutAdapt. Denoising and trimming was performed with DADA2: forward and reverse reads were trimmed to 250 bp, >100,000 reads were analyzed for each sample, except for tomato plant plates and soils samples where only ~60–77K reads were retained. Taxonomy was assigned using the Silva classifier. The taxonomic identity of purified isolates was determined via colony PCR. Colonies resuspended in nuclease-free water and lysed by sonicating for 5 min at 40 kHz, followed by boiling for 10 min at 100°C. Cell lysates were pelleted, and clarified lysates were used for PCR reactions. The 16S rRNA gene was amplified using 1492R (TACGGYTACCTTGTTACGACTT) and 27F (AGAGTTTGATCMTGGCTCAG) primers . PCR products were run on an agarose gel to confirm the presence of a single amplicon at the expected size (~1,500 bp) and Sanger sequenced with the 27F primer at Epoch Life Science (Missouri City, TX, USA). Strain identity was obtained through a BLAST search against the NCBI 16S rRNA sequence database . Subsequently, 220 of 370 16S amplicon sequences were deposited in GenBank under the accession numbers PQ223443-PQ223663. The remaining 16S amplicon sequences failed to meet the requirements for submission to GenBank. Pseudomonas cultures or soil isolates were grown for 24 h in 200 µL P- and N-replete media in a 96-well plate, transferred (1:100) to new 96-well plates containing either 200 µL P-limited or N-limited media, and grown for an additional 24 h. Plates were then incubated statically for one hour in an anaerobic chamber (95% N 2 and 5% H 2 ) to remove residual oxygen . Subsequently, 500 µM of FeCl 3 (maintained as a concentrated stock in 0.1 N HCl) followed by 2 mM ferrozine (in 2 M MOPS, pH 7) was added, and the ferrozine-Fe(II) complex was detected immediately via absorbance at 562 nm using a BioTek Synergy HTX plate reader. Ferrozine stock solutions were degassed in the anaerobic chamber for at least 3 days prior to experiments. To account for differences in growth that might contribute to the OD 562 signal, absorbances at 562 nm for each isolate were recorded prior to the addition of FeCl 3 .This background number was subtracted from the final ferrozine signal (denoted as ΔOD 562 ). The ferrozine signal for each soil isolate is provided in . Cultures were grown overnight in defined media, diluted to an OD 500 of 0.01 in 10 mL P-limited media and grown for an additional 24 h. P-limited cultures were then incubated statically for 1 h in an anaerobic chamber to deplete oxygen, and supernatants were filter sterilized using a 0.22 µm Spin-X column (Corning). Then, 200 µL of filter-sterilized supernatant was transferred to a 96-well plate and mixed with 500 µM FeCl 3 and 2 mM ferrozine. After 40 min, the ferrozine–Fe(II) complex was detected at 562 nm (BioTek Synergy HTX plate reader). The remaining volume of supernatant was used for cyclic voltammetry experiments below. For ferrozine assay gradients with P. kielensis , the protocol above was followed except P. kielensis cultures that were grown overnight in defined media were washed in P-free media, then diluted to an OD 500 of 0.01 in 10 mL P-limited media that ranged from 12.5 μM to 400 μM P. P-limited cultures were grown for 24 h, and growth (OD 500 ) was measured before transferring the cultures to the anaerobic chamber to measure ferrozine signal from filter-sterilized supernatants. Supernatants from P-limited cultures were filter-sterilized (0.22 µm), concentrated under a stream of N 2 gas, and resuspended in 1 mL KCl buffer. Cyclic voltammetry measurements were conducted using a BioLogic SP-300 potentiostat at a scan rate of 50 mV/s using a platinum wire counter electrode, an Ag/AgCl reference electrode and a 3 mm gold working electrode. All experiments with coumarin and PCA were conducted with 1 mM solutions in KCl buffer. pH gradient experiments were conducted under a constant stream of N 2 gas. Midpoint redox potentials were calculated as the average of the potentials at minimal and maximal current (vs Ag/AgCl) +207 mV for comparison to the NHE. P. kielensis genomic DNA from an LB liquid overnight culture was extracted using the DNeasy Blood & Tissue Kit (Qiagen). Sequencing, assembly, and gene annotation were performed by SeqCenter (Pittsburgh, PA, USA) using an Illumina NovaSeq X Plus sequencer. P. kielensis deletion strains In-frame deletions were constructed by allelic exchange using the pMQ30 shuttle vector containing ~1 kb flanking regions, which were built by Gibson Assembly . Colony PCR and Sanger sequencing were used to confirm plasmid construction. Plasmids were introduced to P. kielensis by triparental conjugation and merodiploids were selected on Vogel–Bonner (VBMM) plates containing 50 µg/mL gentamycin . Transconjugants were counter selected on 5% sucrose LB medium with no added salt. Deletions were confirmed by colony PCR. |
Incidence in pharmacoepidemiology: A conceptual framework for incidence of a single substance or group of substances with statins as an example | 0487a1b9-97ae-40a3-bbe6-2abab15396f0 | 10107903 | Pharmacology[mh] | INTRODUCTION In pharmacoepidemiology, the concept of incidence—a new case of drug use—is important from several different perspectives. A new case of drug use defines the start of a specific period of drug exposure. It also represents a decision by the prescriber to either treat a patient for the first time with a specific substance or group of substances (the first‐ever case of drug treatment with this substance of this patient) or to initiate a new period of drug treatment. In pharmacoepidemiology, dispensations of drugs are commonly used as a proxy for actual drug use over the period covered by the amount dispensed. The first dispensation of a drug is probably more sensitive to changes in prescribing habits than subsequent prescriptions or successive dispensations of the same prescription. Repeated treatment episodes with the substance, or a group of substances, over time with periods without treatment in between have to be analysed when studying incidence in pharmacoepidemiology. For instance, a new case of drug treatment should be differentiated from continuing treatment. In addition, first‐ever use has to be distinguished from a recurrent treatment episode. In epidemiology, measures of disease frequency such as incidence and prevalence are well defined, based initially on a simple illness–death model (also known as the disability model). Drug use is often intermittent for chronic diseases, either due to changes in the severity of the disease or non‐compliance. Drugs are mainly used to treat a disease or as secondary prevention in order to prevent possible future complications of a disease. However, they are also used for primary prevention of future disease in otherwise healthy individuals with an increased risk of becoming ill. The original simple model of incidence based on infectious diseases with immunity thus needs to be extended to be applicable for drug treatment where we consider treatment status instead of disease status (see Figure ). The definition of incidence is made more complicated because multiple drugs can be combined or used consecutively to treat a disease. It is essential to consider whether a new case of drug use representing a new case of treatment with the specific substance is preceded or not by other possible substitutes within or outside a specific pharmacological group defined, for instance, by the ATC system. A switch from one substance to another may have many different reasons. For the lipid‐lowering groups of statins (HMG‐CoA reductase inhibitors), the reasons might, for instance, be adverse drug reactions, an unsatisfactory lowering of blood lipid levels, or an increased risk for the patient of cardiovascular events. Other factors might be changes in the costs for the society or the patient, new generic competition, and changes in the pharmaceutical benefit scheme. With a strict definition of different types of new cases of drug use and a well‐defined methodology, it is possible to report incidence not only in studies of drug utilization but also as a standard measure in routine statistics of drug use. Incidence is already part of national standard annual drug utilization statistics from the National Board of Health and Welfare of Sweden, albeit only for some groups of substances. A more stringent methodology and a standardized mode of reporting the different incidences are essential when incidence becomes more widely adopted as a standard measure in publicly available drug utilization statistics. AIM This article aims to explore incidence as new cases of treatment with a specific drug or group of drugs and to develop a corresponding methodology and terminology for consistent reporting in drug utilization studies and national drug statistics. An additional aim is to illustrate this by analysing the initiation of treatment with statins in Sweden 2019. MATERIAL AND METHODS The Swedish Prescribed Drug Registry data were extracted as patient‐level data, fully anonymized and classified as statistics by the National Board of Health and Welfare. Substances were classified according to the Anatomical Therapeutic Chemical (ATC) classification system in 2020. , All first individual occurrences of the dispensation of C10AA HMG‐CoA reductase inhibitors and fixed combinations of HMG‐CoA reductase inhibitors in C10BA in Sweden for both sexes and all ages during 2019 were extracted, together with the ATC code and the number of days since the last dispensation of the same ATC code (total population = 10 230 185 with n of individuals with a dispensation of at least one statin = 1 017 058 corresponding to a 1‐year prevalence of 9.9%). In addition, the number of days since the last dispensation of any other studied substances was obtained with information on ATC code, gender, age (5‐year intervals up to ≥85) and Swedish citizen status on 1 January in 2009 and 2019. Stata was used for all data analyses. Simvastatin, pravastatin, atorvastatin and rosuvastatin in monotherapy constituted >99.9% of the 1‐year prevalence for all statins in C10AA during 2010–2019 in Sweden. The available fixed‐combination products C10BA02 simvastatin + ezetimibe and C10BA05 atorvastatin + ezetimibe represented 0.31% and 0.07% of the sale of respective statins in monotherapy (0.29% and 0.14% in 1‐year prevalence). The incidence proportion was calculated with the number of new cases (first‐ever or recurrent treatment) defined by different run‐in periods as the numerator and the population at the beginning of the year as the denominator. The positive predictive value was calculated as the ratio between the incidence proportions for different lengths of the run‐in compared with a run‐in of 10 years. It can be interpreted as the fraction of the new cases given a specific run‐in that represents first‐ever use, that is, no dispensation 10 years before the index dispensation. Using a 10‐year run‐in as a reference represented a pragmatic approximation defining users as actual first‐ever users of statins. The reason for this approach is the limitation of data available over time in many countries with national prescription databases covering individual‐level patient data of dispensations. Extending the run‐in from 10 to 13 years (the longest possible for dispensations in 2019 in Sweden) had a minimal impact on the incidence proportion (see Section ). 3.1 Methodological considerations when defining a new case of drug use Before we consider the main problem of patients being new to a specific substance or a group of substances, we briefly review the concepts of a run‐in period and incidence rates versus incidence proportion, as these are fundamental for analysing treatment initiation. 3.1.1 The effects of run‐in on different misclassifications There are several possible misclassifications when studying incidence. We have previously explored the concepts of a new case, first‐ever use and recurrent treatment and different types of misclassifications of incidence associated with varying the length of the run‐in period. A run‐in period (sometimes also called a washout period) is commonly used to differentiate between a dispensation indicating a new case of drug use and one representing a continuation of treatment. A short run‐in period will not differentiate well between first‐ever use and recurrence of treatment. With a long run‐in, a more significant fraction of new cases of drug use will represent first‐ever use. The run‐in consists of the total period without treatment and the assumed duration of the last dispensation. This pragmatic practice in register‐based studies will not be influenced by previous hoarding, change in dosage or a decision to end the treatment early (either by the prescriber or the patient). When comparing the incidence of drug use between countries and clinical settings, the assumed duration without treatment, and not the actual run‐in, must be considered since the average treatment duration of a dispensation varies between countries due to clinical practice and regulations. Suppose the average duration is 3–4 months as in Sweden due to the rules of the pharmaceutical benefit scheme. In that case, a 12‐month run‐in will usually represent a period between 8 and 12 months without treatment, while a 16‐month run‐in will represent at least a full year without treatment. If the average duration of a dispensation is 1 month, then the same run‐in period of 12 months in most cases will correspond to 11–12 months without treatment. 3.1.2 Incidence, incidence rate and proportion The incidence, the number of new cases in a defined population, is often presented as a rate or a proportion. In incidence rate, the denominator is the aggregated study time contributed by each studied individual (actual person‐time). The denominator in incidence proportion (also called the cumulative incidence) is the population at risk at the beginning of a time interval, for instance, a calendar year. For incidence proportions, individuals that emigrate or die during the studied interval will still contribute to the denominator for the entire interval. Thus, all other things being equal, the incidence proportion will be lower than the incidence rate in a population with high mortality, such as the elderly population at risk, if defined as the population at the beginning of a time interval. With a high level of immigration, the incidence proportion, all other things being equal, will be higher than the incidence rate if immigrants are not censored. If censoring for immigration, each individual should be censored in the numerator and the denominator for the length of the run‐in after the date of immigration since a prevalent user otherwise would be potentially misclassified as a new case of drug treatment. The traditional definition of incidence rate and incidence proportion in epidemiology focuses on persons at risk as the denominator. In pharmacoepidemiology (whether or not a cohort in rate or a population in a proportion), that would represent only those not classified as prevalent users. However, in drug utilization studies reporting incidence proportion, the whole population is often the denominator (see also Section ). 3.1.3 Substance or condition The reason for prescribing the substance might be considered when studying new cases of drug use if the information is available. However, this information is not registered in large claims or population databases in most instances. Where reasons for prescribing are available, they are not always reliable due to external factors such as reimbursement rules or a heavy workload influencing reporting. Linking prescriptions to specific diagnoses for the same or earlier healthcare episodes is possible in limited situations but creates considerable methodological challenges. Each prescription might be made for several different reasons, which might change over time. A disease such as depression often fluctuates in severity over several years. A new prescription leading to a dispensation, that is, a case of recurrent treatment, can then represent either a repeat treatment for the same reason or treatment with the same substance or group of substances for other reasons. 3.1.4 New on a drug or new on a group of drugs New cases of drug use can relate to a single substance or a group of substances. However, the number of new cases of a group of substances does not equal the sum of the number of new users of each substance since a patient that starts treatment with one substance might have been treated with another substance belonging to the same group earlier. When placing both individual substances and groups of these substances into a simple two‐level model, four different situations can be described: New on a group regardless of the substance— NoG New on a specified substance, whether treated earlier with another substance in the group or not— NoS New on a specified substance and new on the group— NoS_and_NoG New on specified substance and not new on group— NoS_not_NoG This classification can be exemplified as an analysis with two levels for a group with four different substances (see Table and Figure ). During the studied period of 2009–2019, with 10 years of run‐in for dispensations during 2019, only four different statins were dispensed in Sweden (Table ). Methodological considerations when defining a new case of drug use Before we consider the main problem of patients being new to a specific substance or a group of substances, we briefly review the concepts of a run‐in period and incidence rates versus incidence proportion, as these are fundamental for analysing treatment initiation. 3.1.1 The effects of run‐in on different misclassifications There are several possible misclassifications when studying incidence. We have previously explored the concepts of a new case, first‐ever use and recurrent treatment and different types of misclassifications of incidence associated with varying the length of the run‐in period. A run‐in period (sometimes also called a washout period) is commonly used to differentiate between a dispensation indicating a new case of drug use and one representing a continuation of treatment. A short run‐in period will not differentiate well between first‐ever use and recurrence of treatment. With a long run‐in, a more significant fraction of new cases of drug use will represent first‐ever use. The run‐in consists of the total period without treatment and the assumed duration of the last dispensation. This pragmatic practice in register‐based studies will not be influenced by previous hoarding, change in dosage or a decision to end the treatment early (either by the prescriber or the patient). When comparing the incidence of drug use between countries and clinical settings, the assumed duration without treatment, and not the actual run‐in, must be considered since the average treatment duration of a dispensation varies between countries due to clinical practice and regulations. Suppose the average duration is 3–4 months as in Sweden due to the rules of the pharmaceutical benefit scheme. In that case, a 12‐month run‐in will usually represent a period between 8 and 12 months without treatment, while a 16‐month run‐in will represent at least a full year without treatment. If the average duration of a dispensation is 1 month, then the same run‐in period of 12 months in most cases will correspond to 11–12 months without treatment. 3.1.2 Incidence, incidence rate and proportion The incidence, the number of new cases in a defined population, is often presented as a rate or a proportion. In incidence rate, the denominator is the aggregated study time contributed by each studied individual (actual person‐time). The denominator in incidence proportion (also called the cumulative incidence) is the population at risk at the beginning of a time interval, for instance, a calendar year. For incidence proportions, individuals that emigrate or die during the studied interval will still contribute to the denominator for the entire interval. Thus, all other things being equal, the incidence proportion will be lower than the incidence rate in a population with high mortality, such as the elderly population at risk, if defined as the population at the beginning of a time interval. With a high level of immigration, the incidence proportion, all other things being equal, will be higher than the incidence rate if immigrants are not censored. If censoring for immigration, each individual should be censored in the numerator and the denominator for the length of the run‐in after the date of immigration since a prevalent user otherwise would be potentially misclassified as a new case of drug treatment. The traditional definition of incidence rate and incidence proportion in epidemiology focuses on persons at risk as the denominator. In pharmacoepidemiology (whether or not a cohort in rate or a population in a proportion), that would represent only those not classified as prevalent users. However, in drug utilization studies reporting incidence proportion, the whole population is often the denominator (see also Section ). 3.1.3 Substance or condition The reason for prescribing the substance might be considered when studying new cases of drug use if the information is available. However, this information is not registered in large claims or population databases in most instances. Where reasons for prescribing are available, they are not always reliable due to external factors such as reimbursement rules or a heavy workload influencing reporting. Linking prescriptions to specific diagnoses for the same or earlier healthcare episodes is possible in limited situations but creates considerable methodological challenges. Each prescription might be made for several different reasons, which might change over time. A disease such as depression often fluctuates in severity over several years. A new prescription leading to a dispensation, that is, a case of recurrent treatment, can then represent either a repeat treatment for the same reason or treatment with the same substance or group of substances for other reasons. 3.1.4 New on a drug or new on a group of drugs New cases of drug use can relate to a single substance or a group of substances. However, the number of new cases of a group of substances does not equal the sum of the number of new users of each substance since a patient that starts treatment with one substance might have been treated with another substance belonging to the same group earlier. When placing both individual substances and groups of these substances into a simple two‐level model, four different situations can be described: New on a group regardless of the substance— NoG New on a specified substance, whether treated earlier with another substance in the group or not— NoS New on a specified substance and new on the group— NoS_and_NoG New on specified substance and not new on group— NoS_not_NoG This classification can be exemplified as an analysis with two levels for a group with four different substances (see Table and Figure ). During the studied period of 2009–2019, with 10 years of run‐in for dispensations during 2019, only four different statins were dispensed in Sweden (Table ). The effects of run‐in on different misclassifications There are several possible misclassifications when studying incidence. We have previously explored the concepts of a new case, first‐ever use and recurrent treatment and different types of misclassifications of incidence associated with varying the length of the run‐in period. A run‐in period (sometimes also called a washout period) is commonly used to differentiate between a dispensation indicating a new case of drug use and one representing a continuation of treatment. A short run‐in period will not differentiate well between first‐ever use and recurrence of treatment. With a long run‐in, a more significant fraction of new cases of drug use will represent first‐ever use. The run‐in consists of the total period without treatment and the assumed duration of the last dispensation. This pragmatic practice in register‐based studies will not be influenced by previous hoarding, change in dosage or a decision to end the treatment early (either by the prescriber or the patient). When comparing the incidence of drug use between countries and clinical settings, the assumed duration without treatment, and not the actual run‐in, must be considered since the average treatment duration of a dispensation varies between countries due to clinical practice and regulations. Suppose the average duration is 3–4 months as in Sweden due to the rules of the pharmaceutical benefit scheme. In that case, a 12‐month run‐in will usually represent a period between 8 and 12 months without treatment, while a 16‐month run‐in will represent at least a full year without treatment. If the average duration of a dispensation is 1 month, then the same run‐in period of 12 months in most cases will correspond to 11–12 months without treatment. Incidence, incidence rate and proportion The incidence, the number of new cases in a defined population, is often presented as a rate or a proportion. In incidence rate, the denominator is the aggregated study time contributed by each studied individual (actual person‐time). The denominator in incidence proportion (also called the cumulative incidence) is the population at risk at the beginning of a time interval, for instance, a calendar year. For incidence proportions, individuals that emigrate or die during the studied interval will still contribute to the denominator for the entire interval. Thus, all other things being equal, the incidence proportion will be lower than the incidence rate in a population with high mortality, such as the elderly population at risk, if defined as the population at the beginning of a time interval. With a high level of immigration, the incidence proportion, all other things being equal, will be higher than the incidence rate if immigrants are not censored. If censoring for immigration, each individual should be censored in the numerator and the denominator for the length of the run‐in after the date of immigration since a prevalent user otherwise would be potentially misclassified as a new case of drug treatment. The traditional definition of incidence rate and incidence proportion in epidemiology focuses on persons at risk as the denominator. In pharmacoepidemiology (whether or not a cohort in rate or a population in a proportion), that would represent only those not classified as prevalent users. However, in drug utilization studies reporting incidence proportion, the whole population is often the denominator (see also Section ). Substance or condition The reason for prescribing the substance might be considered when studying new cases of drug use if the information is available. However, this information is not registered in large claims or population databases in most instances. Where reasons for prescribing are available, they are not always reliable due to external factors such as reimbursement rules or a heavy workload influencing reporting. Linking prescriptions to specific diagnoses for the same or earlier healthcare episodes is possible in limited situations but creates considerable methodological challenges. Each prescription might be made for several different reasons, which might change over time. A disease such as depression often fluctuates in severity over several years. A new prescription leading to a dispensation, that is, a case of recurrent treatment, can then represent either a repeat treatment for the same reason or treatment with the same substance or group of substances for other reasons. New on a drug or new on a group of drugs New cases of drug use can relate to a single substance or a group of substances. However, the number of new cases of a group of substances does not equal the sum of the number of new users of each substance since a patient that starts treatment with one substance might have been treated with another substance belonging to the same group earlier. When placing both individual substances and groups of these substances into a simple two‐level model, four different situations can be described: New on a group regardless of the substance— NoG New on a specified substance, whether treated earlier with another substance in the group or not— NoS New on a specified substance and new on the group— NoS_and_NoG New on specified substance and not new on group— NoS_not_NoG This classification can be exemplified as an analysis with two levels for a group with four different substances (see Table and Figure ). During the studied period of 2009–2019, with 10 years of run‐in for dispensations during 2019, only four different statins were dispensed in Sweden (Table ). RESULTS Table shows the incidence proportion with the total population as the denominator in 2019 and a different run‐in for new on statins as a group (NoG); new on each statin whether treated earlier with another statin or not (NoS); new on each statin and new on statins (NoS_and_NoG); and new on each statin and not new on group (NoS_not_NoG). For a run‐in of 12 months, the incidence of new on statins (NoG) was 13.39 new cases per 1000 inhabitants, with a positive predictive value for first‐ever use of 63%. New on a specified statin and new on statins (NoS_and_NoG) varied between 9.99 new cases per 1000 inhabitants for atorvastatin and 0.06 for pravastatin. New on a specified statin, but not new on statins (NoS_not_NoG), varied between 0.70 for atorvastatin and 0.03 for pravastatin. In addition, 1.27 per 1000 inhabitants started treatment with any statin but had been treated with another statin during the run‐in (the difference between the sum of NoS_not_NoG for the individual substances and NoG). This corresponded to 9.5% of the individuals being new on statins (NoG). Extending the run‐in from 10 to 13 years (the longest possible run‐in for dispensations in 2019 in Sweden) had a minimal impact on the incidence proportion. For new on statins as a group, the decrease was less than 1% (from 8.40 to 8.34 new cases per 1000 inhabitants) in 2019. With increasing length of the run‐in period, the incidences for new on statins (NoG) and new both on a specified statin and on statins (NoS_and_NoG) decreased as expected, while their respective positive predictive value compared with a run‐in of 10 years increased. Concurrently, the incidence of new on a specified statin but not new on statins (NoS_not_NoG) increased since the observed time during which another statin might have been dispensed lengthened. DISCUSSION The focus of this study is the distinction between new cases of drug use in analyses for groups of substances (NoG) and the individual substances of the group defined in three different ways (NoS, NoS_and_NoG, NoS_not_NoG). The incidence and prevalence of statin use have been studied in different countries, periods and age groups. There is a significant variation in methodology between studies of statin incidence. Both incidence rates , , and incidence proportions , , , , are used when studying incidence. Individuals not at risk , , or the total population regardless of treatment status , were used as denominators for incidence proportions in the different studies. Studying only individuals at risk as a rate (per person‐time) or a proportion (during a defined period) describes the introduction of the drug among those not treated and thus available to become treated. Relating the new cases to all individuals is more straightforward in a study based on population registers. The latter approach is often the preferred choice for the incidence proportion based on register data since there is often no need to adjust for the prevalence in a simple time‐trend analysis. When comparing incidence proportion based on the total population between early and later phases of the introduction of a drug or between high‐ and low‐prevalence populations, it is advisable to assess the incidence in relation to the prevalence. With a commonly used group of substances such as statins, the difference in incidence between using persons at risk and the total population as the denominator will be significant if the prevalence is high. This is relevant for statins in Sweden, where the 1‐year prevalence in the whole population is 9.9%. This article calculates the reported incidence proportions of statins with the total population as the denominator. Correcting for a 1‐year prevalence of 9.9% would result in an approximately 11% higher incidence proportion for the non‐prevalent population. This could be further studied for different subpopulations. There is a wide variation in handling the length of the run‐in in reports of incidence treatment with statins. For statins, a fixed run‐in of 12 months is common, , , but it can vary between 6 months and several years. The run‐in length should be defined based on the clinical question and whether the focus is on all new cases, only first‐ever use, or recurrence of treatment. In several studies, the length of the run‐in is not fixed based on the index date. Instead, the first dispensation during a calendar year is considered a new case of statin prescription if the individual had no dispensation during the preceding calendar year. In these cases, the chosen run‐in varies between 12 and 24 months depending on the date of the first dispensation. , , , , Well‐defined incidence measures are needed not only for studies of drug utilization but also as a part of general drug statistics. Changes in incidence could be used as an indicator of possible future changes in prevalence but also for more sensitive studies of the effects of interventions through, for instance, interrupted time‐series analyses of incidence instead of the number of dispensations or defined daily doses. For statins, an increased incidence has been reported related in time to the results of the 4S trial in 1994 and to both 4S and the West of Scotland Coronary Prevention Study (WOSCOPS). Kildemoes et al studied the relationship between the incidence of statins according to indication in Denmark in the period of 1996–2009 and several external factors such as evolving clinical evidence, international guidelines on CVD prevention, national CVD guidelines and healthcare policies and statin costs. There is a need for further development of methodology and terminology for incidence rates or proportions when presented in studies of drug utilization or introduced as a measure in regular aggregated statistics of drug use. In addition, the estimated misclassification depending on the length of run‐in and which types of new cases are studied (all new cases, first‐ever use or recurrent treatment) should be presented. Table summarizes suggestions for presenting incidence for a drug utilization review. CONCLUSIONS When studying new cases of drug treatment, it is essential to differentiate between those new to both the substance and possible substitutes (NoS_and_NoG) and those new to the substance but who have been treated earlier with substitutes during the chosen run‐in (NoS_not_NoG). In order to allow for consistent comparisons over time and between populations, new incidence measures with validated methodology and descriptions of the degree of misclassification are needed both for scientific studies of drug utilization and when introducing incidence as a measure in aggregated drug statistics. The authors report no conflicts of interest according to the ICMJE Disclosure Form. |
Clinical Pharmacogenetic Testing and Application: 2024 Updated Guidelines by the Korean Society for Laboratory Medicine | 893c053e-8d8a-433c-9a37-06e1c87b7afe | 11788703 | Pharmacology[mh] | This revised guideline is an update of the Clinical Pharmacogenetic Testing and Application guidelines issued by the Korean Society for Laboratory Medicine (KSLM) in 2016. The current guideline was developed by professionals within the society to reflect changes in the academic and medical fields until 2024. We incorporated recent advances in pharmacogenetic testing technology, changes in clinical fields and laboratory environments, and new evidence on pharmacogenetic testing to facilitate the practical application of the latest pharmacogenetic knowledge in clinical settings. Recently, various international consortia and expert groups have made practical joint recommendations based on the integration of databases and consensus guidelines . However, testing laboratory environments and clinical practices vary by country, and the frequency of specific genetic variants differs by ethnicity. Therefore, the spectrum of recommended pharmacogenetic tests also varies, implying a need for updated domestic clinical guidelines that reflect recent evidence and criteria for the effective utilization of pharmacogenetic testing. This guideline aims to provide principles and recommendations based on the latest insights to ensure that clinical laboratories currently performing or planning to implement pharmacogenetic testing can perform these tests effectively and apply them appropriately in clinical practice. The guideline includes updates on the nomenclature for pharmacogenetic testing, evidence supporting clinically useful test items, and strategies for applying clinical pharmacogenetic testing. This guideline includes changes from the previous version, presenting updates on nomenclature, the latest evidence for target gene–drug combinations, and an explanation of current genetic testing methods, including next-generation sequencing (NGS). In addition, the guideline describes strategies for preemptive testing and integration into clinical decision-making systems, which were not covered in the previous version. The current guideline also includes detailed updates on domestic and international external quality assessment programs and reference materials. Finally, the guideline has been revised to reflect recent changes in domestic accreditation programs and the latest information on healthcare reimbursement costs. We publish an English version of the guideline for more readers to understand, with the translated Korean language version of this article simultaneously published in Laboratory Medicine Online. The pharmacogenetic tests discussed in these guidelines are limited to the clinical tests performed at medical institutions. The basic concepts and terminologies for pharmacogenetic tests can be found in the previous guidelines . This section focuses on the interpretative process. The general process of interpreting clinical pharmacogenetic tests is shown in . The first step is to detect genetic variants using molecular diagnostic methods. The next step is to determine a diplotype consisting of the two haplotypes. Phenotypes are derived from diplotypes. When describing phenotypes, the use of standardized terms based on the functional category of the genes is recommended . For drug-metabolizing enzymes, such as CYP2C9 and CYP2C19 , the use of terms such as “ultrarapid metabolizer,” “rapid metabolizer,” “normal metabolizer,” “intermediate metabolizer,” and “poor metabolizer” in decreasing order of enzyme activity, is recommended. For drug transporters, such as SLCO1B1 , terms such as “increased function,” “normal function,” “decreased function,” and “poor function” are recommended. “Positive” and “negative” can be used to describe high-risk genotypes, such as HLA-B * 15:02 . In the final step, clinical recommendations are established based on the phenotypes. Diplotype–phenotype relationships and clinical recommendations can be obtained from authorized guidelines, such as the Clinical Pharmacogenetics Implementation Consortium (CPIC, https://cpicpgx.org/guidelines/ ) and the Dutch Pharmacogenetics Working Group (DPWG, https://www.pharmgkb.org/page/dpwgMapping ) guidelines . Various genetic biomarkers are used to determine the eligibility of chemotherapeutic agents for cancer treatment. Typical examples include somatic variants in oncogenes such as EGFR , BRAF , and ERBB2 , which are used to predict responses to targeted agents. Although this category of biomarkers has been considered in the previous guidelines, in the present version, the scope is limited to genes for which germline variants have been investigated for inborn variability in drug responses. Since the first publication of the clinical pharmacogenetic testing guidelines by the KSLM , various target gene–drug pairs have been comprehensively evaluated for clinical utility. PharmGKB ( https://www.pharmgkb.org/ ) and CPIC ( https://cpicpgx.org/ ) are widely used pharmacogenetic databases for systematical evaluation of accumulated evidence. They offer objective gene–drug combinations based on reviews of international guidelines and the literature, curated by experts, and presented in a web-based format. summarizes the gene–drug combinations with sufficient clinical evidence based on the two databases. These combinations correspond to CPIC A (final/provisional) and B (final) ratings and PharmGKB Levels 1A and 1B, respectively, indicating sufficient clinical evidence to assist in dosage adjustments, efficacy, metabolic/pharmacokinetic changes, and toxicity prediction through pharmacogenetic testing. Clinical laboratories can use to introduce pharmacogenetic tests that meet the requirements of their healthcare institutions. Gene–drug combinations with currently insufficient evidence are listed in . These combinations may be introduced into clinical testing as evidence accumulates in forthcoming clinical and experimental studies. In addition to the CPIC and PharmGKB databases, professional sources, such as drug labels from the U.S. Food and Drug Administration (FDA) with PharmGKB annotation for drug pharmacogenetics (PGx) levels and recommendations from national or disease-specific expert groups, such as DPWG, provide drug–gene information. However, the terms and evidence presented in the different guidelines vary, and new information and recommendations are continuously emerging. Therefore, when considering the introduction of specific pharmacogenetic tests in clinical practice, clinical pathologists should comprehensively review various sources and the latest information. Currently available pharmacogenetic testing methods and examples of platforms are listed in . The methods can be classified into two main categories: targeted genotyping and sequencing. In targeted genotyping, the genotypes to be evaluated are pre-defined and typically limited to the more common variants. Common methods include real-time PCR, microarray, and single-base extension. In contrast, sequencing allows for the detection of all variants within the genomic region analyzed. Sanger sequencing is the most widely used sequencing method; however, the use of NGS is increasing because of its ability to analyze multiple genes simultaneously. Long-read sequencing is also gaining attention in pharmacogenetics, particularly for genes with complex structures, such as CYP2D6 . Structural complexities in CYP2D6 , caused by pseudogenes, copy number variants (CNVs), and hybrid genes, have posed challenges to its clinical implementation. Long-read sequencing is expected to overcome these limitations. CNVs can also be analyzed using methods such as real-time PCR, microarrays, and NGS. More accurate approaches for CNV detection include multiplex ligation-dependent probe amplification and long-range PCR, which can be used to confirm CNVs identified using other methods. For a deeper understanding of the structure of CYP2D6 and the strategies used to analyze it, we highly recommend the tutorial on structural variants recently published by the Pharmacogene Variation Consortium (PharmVar; https://www.pharmvar.org/ ) . Clinical laboratory test results are critical for patient care and should, therefore, be appropriate, accurate, and reliable. However, various factors, ranging from systematic or random errors to clerical mistakes in genotyping or interpretation, can compromise the quality of the results, potentially placing patients at risk. Therefore, risk management is essential at each stage. Genotypes are inherent and remain unchanged throughout life; therefore, an error in testing can lead to a lifelong misinterpretation of results. Moreover, incorrect interpretation and dosage adjustments may increase the risks of toxicity and treatment failure. Therefore, clinical laboratories must implement quality improvement plans to mitigate potential risks and ensure the safe and effective application of pharmacogenetic testing in clinical practice. Pharmacogenetic testing can be performed either retrospectively to identify genetic predispositions in patients who have exhibited adverse reactions to conventional drug treatments or prospectively to predict therapeutic responses and side effects before initiating treatment, enabling safe and effective dosing. The indications, conditions, and points of consideration for clinical pharmacogenetic testing are summarized in . The major purpose of pharmacogenetic testing is to predict treatment failure or the risk of drug toxicity, helping clinicians select appropriate medications and determine optimal dosing regimens. Clinical pharmacogenetic testing is particularly valuable when supported by solid scientific evidence and demonstrated clinical relevance and when the results can guide the selection of alternative medications or dose adjustments. The presence of standardized guidelines further enhances its usability as a clinical tool. When interpreting genotyping results, considering factors such as age, weight, disease status, detailed medication history of the drug and co-administered drugs, various other test findings, drug or metabolite concentrations, clinical therapeutic responses, and side effects is essential. This comprehensive approach helps to determine the most appropriate drug and dosage for each patient. The test report should be carefully designed to effectively communicate the pharmacogenetic test results with clinicians. The report should clearly explain the results and minimize the use of specialized or technical jargon to facilitate smooth communication between laboratory specialists and clinicians. The final report should include a summary of test result interpretation, along with a context-based analysis that considers the clinical information provided by the requesting physician. This approach ensures that the report is both clinically relevant and easily understandable, helping guide appropriate treatment decisions. The contents that should be included in a pharmacogenetic test report are summarized in . Clinical laboratories can develop effective clinical application strategies tailored to specific situations within healthcare institutions by accurately understanding and assessing the advantages and disadvantages of preemptive testing, clinical decision support systems (CDSSs), and harmonization with therapeutic drug monitoring (TDM). To accomplish successful clinical application strategies, clinical pathologists with professional knowledge of both laboratory testing and clinical pharmacology/genomics encompassing laboratory medicine should take full responsibility and control of pharmacogenetic testing. Preemptive testing Preemptive pharmacogenetic testing is a proactive test performed before the initiation of drug therapy to predict therapeutic responses and potential side effects, allowing for safe and effective dosing. While this generally refers to checking results before drug administration, it can also include testing in the general population, regardless of the presence of disease or the possibility of drug administration. Considering the risks of adverse drug reactions, treatment failure, and associated costs, preemptive pharmacogenetic testing using NGS-based multidrug gene tests has been identified as a cost-effective strategy . Clinically significant and actionable variants are frequently observed in the general population . With the aging and extended life expectancy of the general population, individuals are more likely to be prescribed drugs that are targets of pharmacogenetic testing, and the prescription frequency for related drugs tends to increase in Korea . Therefore, a preemptive pharmacogenetic testing strategy is expected to be useful. Multitarget gene analysis in preemptive testing is anticipated to be cost-effective and to aid in preventing severe side effects of drugs. Considerations for preemptive testing include the selection of target genes and analytical methods, result interpretation and reporting, legal and ethical issues, training of relevant healthcare providers, and issues related to fees and healthcare reimbursements . Recent studies evaluating the clinical utility of preemptive pharmacogenetic testing through randomized clinical trials in several European countries reported that genotype-based treatment strategies using 12-drug–gene panels reduced the incidence of drug side effects by 6.7%–7.1% . However, some preemptive strategies for anticoagulant-related pharmacogenetic testing did not decrease major cardiovascular side effects , indicating the need for a careful review of focused applications and testing strategies. Integration into CDSSs To effectively adjust drug dosages and select alternative medications based on pharmacogenetic test results, practical solutions must be developed to overcome the complexity of CDSSs, from genetic testing results to actual drug adjustment processes. In addition to the Pharmacogenomics Clinical Annotation Tool , developed by PharmGKB and PGRN, bioinformatics tools for integrating and applying pharmacogenetic test results into CDSSs include FARMAPRICE, GeneSight, RIGHT, PREDICT, and PG4KDS . Although the content presented by the CDSS platforms varies, they commonly provide alert messages, recommendations for alternative drugs or dosage adjustments, and evidence levels. Implementing a CDSS is an effective strategy for enhancing the activation and clinical utility of pharmacogenetic testing. Establishing core information delivery systems, setting appropriate information limits, and continuously checking and updating the CDSS using the latest databases are necessary prerequisites. Comprehensive application of pharmacogenetic testing in combination with TDM Pharmacogenetic testing is crucial for predicting drug metabolism or responsiveness; however, it cannot replace the clinical evaluation of drug responses or TDM. Therefore, pharmacogenetic testing and TDM are complementary. Pharmacogenetic testing typically provides information that can help assess the appropriateness and risks of a drug before starting treatment, whereas TDM, which includes drug concentration measurements, is essential during the treatment process after the drug or dosage has been selected. The half-life of drug elimination can vary depending on the genotype, and the duration until the steady-state concentration is reached may be prolonged or shortened. Genotype affecting drug receptor sensitivity may alter drug responsiveness, and the therapeutic range might have to be adjusted for the given genotype. Therefore, considering the genotype is necessary for the optimization of TDM in clinical practice. TDM provides valuable information from a pharmacokinetic perspective, as it evaluates changes in drug concentration over time, and from a pharmacodynamic perspective, as it assesses the relationship between the drug concentration and its effect. The relationship between the genotype and phenotype is not always perfect or predictable. TDM helps identify influencing factors beyond the genotype, such as co-administered drugs or liver function. TDM can also aid in understanding patient compliance and the causes of drug resistance. Furthermore, because pharmacogenetic tests do not screen for all variants or may detect novel variants, the measurement of drug concentrations and metabolites can complement pharmacogenetic testing. Preemptive pharmacogenetic testing is a proactive test performed before the initiation of drug therapy to predict therapeutic responses and potential side effects, allowing for safe and effective dosing. While this generally refers to checking results before drug administration, it can also include testing in the general population, regardless of the presence of disease or the possibility of drug administration. Considering the risks of adverse drug reactions, treatment failure, and associated costs, preemptive pharmacogenetic testing using NGS-based multidrug gene tests has been identified as a cost-effective strategy . Clinically significant and actionable variants are frequently observed in the general population . With the aging and extended life expectancy of the general population, individuals are more likely to be prescribed drugs that are targets of pharmacogenetic testing, and the prescription frequency for related drugs tends to increase in Korea . Therefore, a preemptive pharmacogenetic testing strategy is expected to be useful. Multitarget gene analysis in preemptive testing is anticipated to be cost-effective and to aid in preventing severe side effects of drugs. Considerations for preemptive testing include the selection of target genes and analytical methods, result interpretation and reporting, legal and ethical issues, training of relevant healthcare providers, and issues related to fees and healthcare reimbursements . Recent studies evaluating the clinical utility of preemptive pharmacogenetic testing through randomized clinical trials in several European countries reported that genotype-based treatment strategies using 12-drug–gene panels reduced the incidence of drug side effects by 6.7%–7.1% . However, some preemptive strategies for anticoagulant-related pharmacogenetic testing did not decrease major cardiovascular side effects , indicating the need for a careful review of focused applications and testing strategies. To effectively adjust drug dosages and select alternative medications based on pharmacogenetic test results, practical solutions must be developed to overcome the complexity of CDSSs, from genetic testing results to actual drug adjustment processes. In addition to the Pharmacogenomics Clinical Annotation Tool , developed by PharmGKB and PGRN, bioinformatics tools for integrating and applying pharmacogenetic test results into CDSSs include FARMAPRICE, GeneSight, RIGHT, PREDICT, and PG4KDS . Although the content presented by the CDSS platforms varies, they commonly provide alert messages, recommendations for alternative drugs or dosage adjustments, and evidence levels. Implementing a CDSS is an effective strategy for enhancing the activation and clinical utility of pharmacogenetic testing. Establishing core information delivery systems, setting appropriate information limits, and continuously checking and updating the CDSS using the latest databases are necessary prerequisites. Pharmacogenetic testing is crucial for predicting drug metabolism or responsiveness; however, it cannot replace the clinical evaluation of drug responses or TDM. Therefore, pharmacogenetic testing and TDM are complementary. Pharmacogenetic testing typically provides information that can help assess the appropriateness and risks of a drug before starting treatment, whereas TDM, which includes drug concentration measurements, is essential during the treatment process after the drug or dosage has been selected. The half-life of drug elimination can vary depending on the genotype, and the duration until the steady-state concentration is reached may be prolonged or shortened. Genotype affecting drug receptor sensitivity may alter drug responsiveness, and the therapeutic range might have to be adjusted for the given genotype. Therefore, considering the genotype is necessary for the optimization of TDM in clinical practice. TDM provides valuable information from a pharmacokinetic perspective, as it evaluates changes in drug concentration over time, and from a pharmacodynamic perspective, as it assesses the relationship between the drug concentration and its effect. The relationship between the genotype and phenotype is not always perfect or predictable. TDM helps identify influencing factors beyond the genotype, such as co-administered drugs or liver function. TDM can also aid in understanding patient compliance and the causes of drug resistance. Furthermore, because pharmacogenetic tests do not screen for all variants or may detect novel variants, the measurement of drug concentrations and metabolites can complement pharmacogenetic testing. QM Laboratories performing pharmacogenetic testing should have in-house QM programs. The QM program should cover general issues, such as sample collection, identification, preparation, assay validation, and sample storage. Considering the importance of databases for pharmacogenetic testing, a database management plan should be implemented. Data on allele definition, allele functionality, diplotype–phenotype relationship, and allele frequency of the major pharmacogenes can be downloaded from the CPIC or PharmGKB website. In addition, clinical guidelines published by the CPIC and DPWG should be used in test report generation . As the above databases and guidelines are frequently updated, laboratory databases should be updated at least once every six months. Result accumulation and a review of the distribution of the reported genotypes in each laboratory are recommended. The distribution should be compared with known genotype frequencies. When the distribution deviates from that of the population group, the potential causes of bias should be investigated. Reference materials for validation and QC can be purchased from the Get-RM project of the Coriell Institute ( https://www.coriell.org/1/NIGMS/Additional-Resources/Pharmacogenetics ). The consensus genotypes of the materials can be found in the articles provided on the website; however, they may not reflect an updated database. In such cases, the latest publications can be searched for true genotypes. External quality assessment Laboratories should participate in proficiency testing for each item. An alternative program must be prepared in the absence of proficiency testing for a specific item. In Korea, a pharmacogenetic testing program is provided by the Korean Association of External Quality Assessment Service ( https://keqas.org/ ). The genotyping of six genes, including CYP2C19 , CYP2C9 , VKORC1 , CYP2D6 , TPMT , and NUDT15 , can be evaluated using this program. Internationally, the College of American Pathologists (CAP) provides three pharmacogenetic programs in which laboratories can participate: PGX ( CYP2C19 , CYP2C9 , CYP2B6 , CYP2D6 , CYP3A4 , CYP3A5 , CYP4F2 , SLCO1B1 , and VKORC1 ), PGX1 ( IL28B , COMT , G6PD , and OPRM1 ), and PGX3 ( DPYD , NUDT15 , TPMT , and UGT1A1 ) ( https://www.cap.org/laboratory-improvement/proficiency-testing ). Additionally, the European Molecular Genetics Quality Network provides a pharmacogenetic program that evaluates DPYD and UGT1A1 genotyping ( https://www.emqn.org/ ). Laboratory accreditation Participation in laboratory accreditation programs can drive improvements in laboratory QM systems. CAP is a global organization of board-certified pathologists that aims to foster excellence in laboratory medicine and pathology ( https://www.cap.org/ ). CAP accreditation can be achieved by inspection every 2 years by a CAP-assigned inspection team that determines compliance with specific laboratory standards regarding QM systems and safety measures. In Korea, two accreditation programs are available: the Outstanding Laboratory Accreditation Program (OLAP) of the Laboratory Medicine Foundation ( https://www.lmf.or.kr/ ) and the Korea Institute of Genetic Testing Evaluation (KIGTE) ( http://www.kigte.org/ ). The OLAP, performed as a peer-review by a team of laboratory medicine specialists, investigates the statuses of internal/external QC, facilities, equipment, personnel, and environment. Two categories of laboratory pharmacogenetic tests must be evaluated: laboratory management and molecular diagnostics. KIGTE accreditation is mandatory for laboratories performing any type of genetic testing, including pharmacogenetic testing. However, clinical laboratories in medical institutions that have achieved OLAP accreditation can be exempted from KIGTE accreditation. Laboratories performing pharmacogenetic testing should have in-house QM programs. The QM program should cover general issues, such as sample collection, identification, preparation, assay validation, and sample storage. Considering the importance of databases for pharmacogenetic testing, a database management plan should be implemented. Data on allele definition, allele functionality, diplotype–phenotype relationship, and allele frequency of the major pharmacogenes can be downloaded from the CPIC or PharmGKB website. In addition, clinical guidelines published by the CPIC and DPWG should be used in test report generation . As the above databases and guidelines are frequently updated, laboratory databases should be updated at least once every six months. Result accumulation and a review of the distribution of the reported genotypes in each laboratory are recommended. The distribution should be compared with known genotype frequencies. When the distribution deviates from that of the population group, the potential causes of bias should be investigated. Reference materials for validation and QC can be purchased from the Get-RM project of the Coriell Institute ( https://www.coriell.org/1/NIGMS/Additional-Resources/Pharmacogenetics ). The consensus genotypes of the materials can be found in the articles provided on the website; however, they may not reflect an updated database. In such cases, the latest publications can be searched for true genotypes. Laboratories should participate in proficiency testing for each item. An alternative program must be prepared in the absence of proficiency testing for a specific item. In Korea, a pharmacogenetic testing program is provided by the Korean Association of External Quality Assessment Service ( https://keqas.org/ ). The genotyping of six genes, including CYP2C19 , CYP2C9 , VKORC1 , CYP2D6 , TPMT , and NUDT15 , can be evaluated using this program. Internationally, the College of American Pathologists (CAP) provides three pharmacogenetic programs in which laboratories can participate: PGX ( CYP2C19 , CYP2C9 , CYP2B6 , CYP2D6 , CYP3A4 , CYP3A5 , CYP4F2 , SLCO1B1 , and VKORC1 ), PGX1 ( IL28B , COMT , G6PD , and OPRM1 ), and PGX3 ( DPYD , NUDT15 , TPMT , and UGT1A1 ) ( https://www.cap.org/laboratory-improvement/proficiency-testing ). Additionally, the European Molecular Genetics Quality Network provides a pharmacogenetic program that evaluates DPYD and UGT1A1 genotyping ( https://www.emqn.org/ ). Participation in laboratory accreditation programs can drive improvements in laboratory QM systems. CAP is a global organization of board-certified pathologists that aims to foster excellence in laboratory medicine and pathology ( https://www.cap.org/ ). CAP accreditation can be achieved by inspection every 2 years by a CAP-assigned inspection team that determines compliance with specific laboratory standards regarding QM systems and safety measures. In Korea, two accreditation programs are available: the Outstanding Laboratory Accreditation Program (OLAP) of the Laboratory Medicine Foundation ( https://www.lmf.or.kr/ ) and the Korea Institute of Genetic Testing Evaluation (KIGTE) ( http://www.kigte.org/ ). The OLAP, performed as a peer-review by a team of laboratory medicine specialists, investigates the statuses of internal/external QC, facilities, equipment, personnel, and environment. Two categories of laboratory pharmacogenetic tests must be evaluated: laboratory management and molecular diagnostics. KIGTE accreditation is mandatory for laboratories performing any type of genetic testing, including pharmacogenetic testing. However, clinical laboratories in medical institutions that have achieved OLAP accreditation can be exempted from KIGTE accreditation. Pharmacogenetic tests covered by the National Health Insurance Service (NHIS) in Korea are listed in ( https://repository.hira.or.kr/handle/2019.oak/3201 ). For items covered by the NHIS, following the general rule for genetic tests, pharmacogenetic tests that guide therapeutic decisions or predict critical side effects are reimbursable. Additionally, gene-specific rules exist for HLA-B * 58:01 and TPMT . Nucleic acid amplification tests and sequencing of HLA-B * 58:01 are reimbursable when used before allopurinol treatment for hyperuricemia (uric acid level ≥9 mg/dL) in a patient with chronic kidney disease. Otherwise, only the first nucleic acid amplification test performed before allopurinol treatment is reimbursed. Although TPMT tests are fully reimbursable when used for patients with symptoms of adverse drug reactions to thiopurines, 80% deductibility is applied when used to predict the drug response before treatment. MTHFR , G6PD , and CFTR are established pharmacogenes; however, they also are causative genes of the inherited diseases homocystinuria, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and cystic fibrosis, respectively. Tests for MTHFR are reimbursable only when used to diagnose inherited diseases, not when used as a pharmacogenetic test. In contrast, tests for G6PD and CFTR are reimbursable for both indications. The reimbursement criteria were determined based on both clinical indications and test methods. The diagnostic methods covered by the NHIS of Korea include nucleic acid amplification-based methods, such as real-time PCR and sequencing. For sequencing codes, the insurance fee is determined based on the number of sequencifng reactions. Notably, either of the two codes, for ≤10 and ≤20 reactions, can be used for CYP2C9 , CYP2C19 , TPMT , and UGT1A1 , according to the number of targeted exons. This guideline provides practical information on clinical pharmacogenetic testing and its application, covering updates on nomenclature, target gene–drug combinations, laboratory methods for pharmacogenetic testing, strategies for efficient clinical application, QM and accreditation programs, and current reimbursement issues. Clinical pathologists have a pivotal role in ensuring the reliability and professionalism of clinical pharmacogenetic testing. Appropriate application of clinical pharmacogenetic testing is an essential prerequisite for precision medicine. We suggest clinical practice guidelines for the clinical application, interpretation, and reporting of clinically useful pharmacogenetic tests with updated evidence and the current status. |
Evaluation of Two Anorganic Bovine Xenogenous Grafts in Bone Healing of Critical Defect in Rats Calvaria | ecc747f6-c6a0-478e-8875-676a6caa6a5a | 11506241 | Anatomy[mh] | Bone substitutes play a significant role in implant dentistry, providing crucial support for bone healing, reconstruction, and augmentation . In cases of bone loss or damage, the use of bone graft substitutes is essential to ensure proper support as a scaffold and to stimulate the new bone formation . Bone graft substitutes encompass a range of substances used to fill bone defects and induce new bone formation . Biomaterials can be obtained from various sources, including the patient's own body called autografts, from humans or animals termed allografts and xenografts, or from synthetic compounds named alloplastic . Each of these alternatives has specific characteristics, making them suitable for different clinical scenarios . Various biomaterials have been proposed as alternatives to autogenous bone grafts since they require additional surgery and possible donor-site morbidity . Thus, widely used alternatives include anorganic xenogenous bovine bones . Bio-Oss® (BO) is considered one of the most effective and widely used anorganic bovine xenogenous grafts . This biomaterial is commonly used as a reference in studies comparing other bone substitutes when autografts are not used . The efficiency and compatibility of BO have been extensively studied in surgical procedures, such as sinus floor elevation and implant placement , . BO is a medullary bovine bone substitute designed to mimic the properties of natural human bone, enabling integration and bone regeneration . BO particles are meticulously crafted through a precise heating process at 300°C, and then undergo a thorough treatment with sodium hydroxide . BO particles stimulate the deposition of calcium and phosphate, promoting the formation of hydroxyapatite, a key inorganic component of the bone matrix . BO has a favorable environment for bone regeneration with interconnected porosity, allowing the ingrowth of blood vessels and cellular infiltration . Despite BO being frequently recommended, there is a vast array of other bone substitutes available, and their possible prominent worth needs to be explored. Bonefill® (BF) is an anorganic bovine xenogenous graft derived from the cortically bovine femurs . BF is manufactured through a sequential bath process designed to effectively solubilize the organic component from its particles . It has been widely discussed in the literature with promising results , . Different studies including histomorphometry and physicochemical analysis showed its composition and potential to serve as an alternative bone substitute. The use of BF has been investigated in comparison to other bone substitutes such as BO , , . Previous studies have analyzed BF in various preclinical applications, including bone healing in rabbits calvaria , calvaria critical-size defects in rats and alveolar bone healing in rats . The results demonstrated that BF exhibits favorable osteoconductive properties, mainly providing a scaffold for osteogenesis . Beside animal studies, clinical reports also demonstrated convincing findings of the BF in the form of blocks for maxilla reconstruction or granule particles for sinus augmentation . Moreover, studies assessing the long-term stability of BF have reported that this bone substitute maintains its structural integrity over a long time and under dissolution conditions . According to the available literature, there is a need for further investigations evaluating these bone biomaterial substitutes in different scenarios , . Although there is wide use of BO, some findings indicate superior outcomes when using BO in comparison with other bone substitutes , while others have not reported additional benefits , . Understanding the intricate nuances of bone biomaterial substitutes is crucial for clinicians and researchers. It supports them in making informed decisions regarding the most suitable preferences for patients with different bone defects and treatment requirements. Therefore, this article aimed to evaluate and compare through histomorphometry and immunohistochemical analysis the characteristics of two anorganic bovine xenogenous grafts (BO and BF) by examining their regenerative properties using a standardized model of bone healing in critical-sized defects in rats calvaria. Animals The study was approved by the Ethics Committee on Animal Experimentation of the Faculty of Dentistry (process 1200-03/2011), in accordance with the current norms adopted by the Brazilian College of Animal Experimentation. Additionally, all steps of the experiment carefully followed the Arrive Guidelines 2.0 Sample size A total of thirty male rats (Rattus norvegicus Albinus, Wistar), 3 to 4 months of age, weighing approximately 250 to 300g, kept in an environment with stable temperature (22 ± 2°C), with water and food ad libitum, were used for this study. The sample size was estimated according to the previous literature, practice, and performance of a pilot study. It was assumed, as established by Grossi-Oliveira et al. , that four animals were adequate to ensure statistical significance for histological analysis. Based on this, and to compensate for possible dropouts, while minimizing the number of animals, the sample size for this parameter was n=5. Previous experience of our research group using the same rodent model determined that n = 10 per group was sufficient to reject the null hypothesis in the other analyses. The estimation for each outcome measure was performed to achieve a 0.8 power and 0.05 alpha error. Study design Simple randomization of the animals (1:1 allocation ratio) into groups was performed through a computer-generated random number table by a blinded external member of the study. Numerals from 1 to 30 were labelled on the tails of the rats with a drawing pen. The number order was uploaded to the software (Minitab® 17 Minitab Inc., State College, PA, USA). The animals were randomly assigned into three experimental groups of ten animals per group. Next, each animal received one of the following treatments: Control group (n=10), defects were filled with blood clot; BO group (n=10), the defects were filled with inorganic hydroxyapatite extracted from the medullary portion of the tibia bovine (particles of 0.25-1 mm in diameter; Bio-Oss®, Geistlich Pharma AG, Switzerland); BF group (n=10), the defects were filled with anorganic bone matrix extracted from cortical bovine femur bone (particles of 0.6-1.5 mm in diameter, Bonefill®, Bionnovation Produtos Biomédicos S.A., Bauru, SP, Brazil). Both bone biomaterial substitutes were inserted carefully into the bone defects until complete filling, without excessive condensation. All defects were covered with a collagen membrane of demineralized bovine cortical bone (GenDerm®, Baumer, SP, Brazil). Both bone biomaterial substitutes were hydrated with saline solution (0.9%) before implantation in the bone defect. Soft tissue sutures were performed with resorbable sutures (Vicryl 4-0, Ethicon, Johnson & Johnson, Somerville, NJ, USA). All evaluations were performed following calibration and blinding examination. Surgery All animals received general anesthesia with 80 mg/kg of ketamine hydrochloride (Cetamin, Syntec do Brasil Ltd., Cotia, SP, Brazil) and 60 mg/kg of Xylazine Hydrochloride (Xilazin, Syntec do Brasil Ltd., Cotia, SP, Brazil) administered intramuscularly to the right leg of the animal. When supplementation was needed for anesthesia, a dose equivalent to 50% of the initial dose was applied. After asepsis and trichotomy of the calvaria, a semilunar incision was performed, and a full-thickness flap was raised. Next, a single bone defect of critical size was created (Ø=5mm) in the central portion of the calvaria, performed with a trephine drill (Dentoflex, System of Implantes, São Paulo, SP, Brazil) mounted on an implant engine with reduced speed and under abundant irrigation with sterilize saline solution (0.9%). It was carried out twice ¨L¨-shaped markings from the edge of the defect created using a drill spherical no. 2 mm posterior and 2 mm anterior to the margin of the defect, and its major axis followed the median sagittal plane of the animal and served to guide the histological processing and evaluation. The technique for creating the bone defects followed a previously described and validated protocol as demonstrated in the . Euthanasia and histology process Five animals from each group were euthanized at 30 and 45 days after surgery. The calvaria of these animals, containing the created bone defects, were removed and preserved for 48 hours in a 4% formaldehyde solution. They were then washed in running water for 24 hours, and the demineralization process started, carried out in a solution of Acid 16% ethylenediaminetetraacetic (EDTA) in the proportion of 250 mg per 1750 ml of distilled water. After the parts were washed and embedded in paraffin blocks. Six serial sections were made 5 µm thick from the center of the bone defect. Half of these sections were stained by the Hematoxylin and Eosin (HE) technique and served for the histomorphometry analysis, while the others underwent processing for immunohistochemical analysis. Histomorphometry analysis The histomorphometry analysis was performed using a computer image evaluation system, ImageLab 2000 software (Diracon Bio Informática Ltda., Vargem Grande do Sul, SP, Brazil), by a single examiner, calibrated and blinded to the periods and treatments. The technique for histomorphometry analysis followed previous established methods . Histological sections were selected from the central area of each specimen's surgical defect in a sagittal direction. Each section was captured using a digital camera coupled to an optical microscope and saved on a computer. In each image, a delimitation of the analyzed area was performed, corresponding to the region of the calvaria where the defect was created, defined as the total area. This area was first determined by the identification of the external and internal surfaces of the original calvaria on the right and left margins of the surgical defect. These surfaces are related to drawn lines following their respective curvatures. Considering the total length of the histological specimen, 2 mm were measured from the right and left extremities of the specimen, towards the center of the defect, to identify the margins of the surgical defect. The area of new bone formation occupied over the remnants of the implanted bone biomaterial substitutes, BO and BF, was delineated within the limits of the total area. The new bone formation of the respective specimens was evaluated three times by the same examiner and on different days. The three measurements obtained were statistically analyzed and a significant level was set at 5% using the Kappa test. Mean values were ascertained and statistically compared. Digital images were created with a combination of three images, because of the impossibility of capturing the entire defect in only one image due to the magnification used. The image was created using Adobe Photoshop® software (Adobe, San Jose, CA) concerning anatomical structures such as blood vessels and bone trabeculae in each of the histological sections. Immunohistochemical analysis For immunohistochemical reactions, the slides were treated by indirect immunoperoxidase technique employing the primary polyclonal antibodies to bone morphogenetic protein 2/4 (1:100, anti-BMP2/4, sc137087, Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (1:100, anti-OCN, sc365797, Santa Cruz Biotechnology, Santa Cruz, CA) and tartrate-resistant acid phosphatase (1:100, anti-TRAP, sc376875, Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies were diluted in bovine serum albumin (BSA) with diluent (DAKO - Carpinteria, CA, USA) and normal serum (3%, Sigma, CA, USA). Initially, the histological sections were deparaffinized at 56 °C for 30 min, and a second cycle of deparaffinization started with xylol baths, followed by rehydration in decreasing solutions of alcohols, and finally washed in successive baths in sodium phosphate buffer (SPB). After that, the slides were placed in a solution containing 198 ml of distilled water with 2 ml of 100X citrate buffer at 95 °C for 5 min for antigen retrieval. The histological sections were treated for blockade of the endogenous peroxidase employing 3% hydrogen peroxide in SPB for 1 h and then washed again with SPB. Endogenous biotin blockade was performed with a solution containing SBP and skimmed milk powder 3% for 1 h. Blocking of non-specific sites was also performed with a solution of bovine serum albumin (BSA) overnight. Thereafter, the sections were incubated with the above-mentioned primary antibodies at room temperature for 18-24 hours and washed with SPB. A second incubation was performed using a universal biotinylated secondary antibody (Anti-Goat made in Horse, DAKO-Carpinteria, CA, USA) for 2 h at room temperature, followed by a wash with SPB. A third incubation was performed with a solution containing streptavidin conjugated to peroxidase (DAKO - Carpinteria, CA, USA) at room temperature for 2 h. Immunoperoxidase reaction was performed with buffer (DAB-Substrate, DAKO - Carpinteria, CA, USA) and diaminobenzidine (DAB-Chromogen, DAKO - Carpinteria, CA, USA) for 5 min for BMP 2/4 and OCN, 60 s for TRAP at room temperature. Finally, histological sections were washed several times in SPB and counterstained for 15 seconds with hematoxylin. All immunoperoxidase reactions were accompanied by a negative control when primary antibodies were omitted. Immunohistochemical analysis followed a previously establish method . Thus, immunolabelling located at both margins and the center of the defect was analyzed at 400X magnification by light microscopy. The expression of BMP 2/4 and OCN were measured semi-quantitatively using scores from 1 to 4 assigned as 1= absent, 2=mild, 3=moderate and 4=intense. TRAP-positive cells were counted, and the results were expressed in units. In order to be considered TRAP-positive cells, mature osteoclasts should contain three or more nuclei. Statistical analysis Statistical analysis of the histometric and immunohistochemical data was performed by GraphPad Prism 9 software (GraphPad Software; La Jolla; CA; USA). The hypothesis that there was no statistically significant difference among the different groups and periods was tested. The normality of the data was evaluated by the Shapiro-Wilk test. A parametric normal distribution of the data for new bone formation and TRAP immunolabelling was observed. Therefore, their statistical test was performed by parametric analysis of variance ANOVA with Tukey complementation at p < 0.05. Otherwise, BMP2/4 and OCN obtained a nonparametric distribution, thus it was assumed Kruskal Wallis Analysis of Variance; Student Newman-Keuls post-test at p<0.05. The study was approved by the Ethics Committee on Animal Experimentation of the Faculty of Dentistry (process 1200-03/2011), in accordance with the current norms adopted by the Brazilian College of Animal Experimentation. Additionally, all steps of the experiment carefully followed the Arrive Guidelines 2.0 A total of thirty male rats (Rattus norvegicus Albinus, Wistar), 3 to 4 months of age, weighing approximately 250 to 300g, kept in an environment with stable temperature (22 ± 2°C), with water and food ad libitum, were used for this study. The sample size was estimated according to the previous literature, practice, and performance of a pilot study. It was assumed, as established by Grossi-Oliveira et al. , that four animals were adequate to ensure statistical significance for histological analysis. Based on this, and to compensate for possible dropouts, while minimizing the number of animals, the sample size for this parameter was n=5. Previous experience of our research group using the same rodent model determined that n = 10 per group was sufficient to reject the null hypothesis in the other analyses. The estimation for each outcome measure was performed to achieve a 0.8 power and 0.05 alpha error. Simple randomization of the animals (1:1 allocation ratio) into groups was performed through a computer-generated random number table by a blinded external member of the study. Numerals from 1 to 30 were labelled on the tails of the rats with a drawing pen. The number order was uploaded to the software (Minitab® 17 Minitab Inc., State College, PA, USA). The animals were randomly assigned into three experimental groups of ten animals per group. Next, each animal received one of the following treatments: Control group (n=10), defects were filled with blood clot; BO group (n=10), the defects were filled with inorganic hydroxyapatite extracted from the medullary portion of the tibia bovine (particles of 0.25-1 mm in diameter; Bio-Oss®, Geistlich Pharma AG, Switzerland); BF group (n=10), the defects were filled with anorganic bone matrix extracted from cortical bovine femur bone (particles of 0.6-1.5 mm in diameter, Bonefill®, Bionnovation Produtos Biomédicos S.A., Bauru, SP, Brazil). Both bone biomaterial substitutes were inserted carefully into the bone defects until complete filling, without excessive condensation. All defects were covered with a collagen membrane of demineralized bovine cortical bone (GenDerm®, Baumer, SP, Brazil). Both bone biomaterial substitutes were hydrated with saline solution (0.9%) before implantation in the bone defect. Soft tissue sutures were performed with resorbable sutures (Vicryl 4-0, Ethicon, Johnson & Johnson, Somerville, NJ, USA). All evaluations were performed following calibration and blinding examination. All animals received general anesthesia with 80 mg/kg of ketamine hydrochloride (Cetamin, Syntec do Brasil Ltd., Cotia, SP, Brazil) and 60 mg/kg of Xylazine Hydrochloride (Xilazin, Syntec do Brasil Ltd., Cotia, SP, Brazil) administered intramuscularly to the right leg of the animal. When supplementation was needed for anesthesia, a dose equivalent to 50% of the initial dose was applied. After asepsis and trichotomy of the calvaria, a semilunar incision was performed, and a full-thickness flap was raised. Next, a single bone defect of critical size was created (Ø=5mm) in the central portion of the calvaria, performed with a trephine drill (Dentoflex, System of Implantes, São Paulo, SP, Brazil) mounted on an implant engine with reduced speed and under abundant irrigation with sterilize saline solution (0.9%). It was carried out twice ¨L¨-shaped markings from the edge of the defect created using a drill spherical no. 2 mm posterior and 2 mm anterior to the margin of the defect, and its major axis followed the median sagittal plane of the animal and served to guide the histological processing and evaluation. The technique for creating the bone defects followed a previously described and validated protocol as demonstrated in the . Five animals from each group were euthanized at 30 and 45 days after surgery. The calvaria of these animals, containing the created bone defects, were removed and preserved for 48 hours in a 4% formaldehyde solution. They were then washed in running water for 24 hours, and the demineralization process started, carried out in a solution of Acid 16% ethylenediaminetetraacetic (EDTA) in the proportion of 250 mg per 1750 ml of distilled water. After the parts were washed and embedded in paraffin blocks. Six serial sections were made 5 µm thick from the center of the bone defect. Half of these sections were stained by the Hematoxylin and Eosin (HE) technique and served for the histomorphometry analysis, while the others underwent processing for immunohistochemical analysis. The histomorphometry analysis was performed using a computer image evaluation system, ImageLab 2000 software (Diracon Bio Informática Ltda., Vargem Grande do Sul, SP, Brazil), by a single examiner, calibrated and blinded to the periods and treatments. The technique for histomorphometry analysis followed previous established methods . Histological sections were selected from the central area of each specimen's surgical defect in a sagittal direction. Each section was captured using a digital camera coupled to an optical microscope and saved on a computer. In each image, a delimitation of the analyzed area was performed, corresponding to the region of the calvaria where the defect was created, defined as the total area. This area was first determined by the identification of the external and internal surfaces of the original calvaria on the right and left margins of the surgical defect. These surfaces are related to drawn lines following their respective curvatures. Considering the total length of the histological specimen, 2 mm were measured from the right and left extremities of the specimen, towards the center of the defect, to identify the margins of the surgical defect. The area of new bone formation occupied over the remnants of the implanted bone biomaterial substitutes, BO and BF, was delineated within the limits of the total area. The new bone formation of the respective specimens was evaluated three times by the same examiner and on different days. The three measurements obtained were statistically analyzed and a significant level was set at 5% using the Kappa test. Mean values were ascertained and statistically compared. Digital images were created with a combination of three images, because of the impossibility of capturing the entire defect in only one image due to the magnification used. The image was created using Adobe Photoshop® software (Adobe, San Jose, CA) concerning anatomical structures such as blood vessels and bone trabeculae in each of the histological sections. For immunohistochemical reactions, the slides were treated by indirect immunoperoxidase technique employing the primary polyclonal antibodies to bone morphogenetic protein 2/4 (1:100, anti-BMP2/4, sc137087, Santa Cruz Biotechnology, Santa Cruz, CA), osteocalcin (1:100, anti-OCN, sc365797, Santa Cruz Biotechnology, Santa Cruz, CA) and tartrate-resistant acid phosphatase (1:100, anti-TRAP, sc376875, Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies were diluted in bovine serum albumin (BSA) with diluent (DAKO - Carpinteria, CA, USA) and normal serum (3%, Sigma, CA, USA). Initially, the histological sections were deparaffinized at 56 °C for 30 min, and a second cycle of deparaffinization started with xylol baths, followed by rehydration in decreasing solutions of alcohols, and finally washed in successive baths in sodium phosphate buffer (SPB). After that, the slides were placed in a solution containing 198 ml of distilled water with 2 ml of 100X citrate buffer at 95 °C for 5 min for antigen retrieval. The histological sections were treated for blockade of the endogenous peroxidase employing 3% hydrogen peroxide in SPB for 1 h and then washed again with SPB. Endogenous biotin blockade was performed with a solution containing SBP and skimmed milk powder 3% for 1 h. Blocking of non-specific sites was also performed with a solution of bovine serum albumin (BSA) overnight. Thereafter, the sections were incubated with the above-mentioned primary antibodies at room temperature for 18-24 hours and washed with SPB. A second incubation was performed using a universal biotinylated secondary antibody (Anti-Goat made in Horse, DAKO-Carpinteria, CA, USA) for 2 h at room temperature, followed by a wash with SPB. A third incubation was performed with a solution containing streptavidin conjugated to peroxidase (DAKO - Carpinteria, CA, USA) at room temperature for 2 h. Immunoperoxidase reaction was performed with buffer (DAB-Substrate, DAKO - Carpinteria, CA, USA) and diaminobenzidine (DAB-Chromogen, DAKO - Carpinteria, CA, USA) for 5 min for BMP 2/4 and OCN, 60 s for TRAP at room temperature. Finally, histological sections were washed several times in SPB and counterstained for 15 seconds with hematoxylin. All immunoperoxidase reactions were accompanied by a negative control when primary antibodies were omitted. Immunohistochemical analysis followed a previously establish method . Thus, immunolabelling located at both margins and the center of the defect was analyzed at 400X magnification by light microscopy. The expression of BMP 2/4 and OCN were measured semi-quantitatively using scores from 1 to 4 assigned as 1= absent, 2=mild, 3=moderate and 4=intense. TRAP-positive cells were counted, and the results were expressed in units. In order to be considered TRAP-positive cells, mature osteoclasts should contain three or more nuclei. Statistical analysis of the histometric and immunohistochemical data was performed by GraphPad Prism 9 software (GraphPad Software; La Jolla; CA; USA). The hypothesis that there was no statistically significant difference among the different groups and periods was tested. The normality of the data was evaluated by the Shapiro-Wilk test. A parametric normal distribution of the data for new bone formation and TRAP immunolabelling was observed. Therefore, their statistical test was performed by parametric analysis of variance ANOVA with Tukey complementation at p < 0.05. Otherwise, BMP2/4 and OCN obtained a nonparametric distribution, thus it was assumed Kruskal Wallis Analysis of Variance; Student Newman-Keuls post-test at p<0.05. The histomorphometry showed that the new bone formation at 30 days was statistically higher in the BO group than in the BF group (24.8% ± 2.1 vs 13.6± 1.8; p=0.0071). The Control group presented a percentage of 19.9%±2.8 ( A). The new bone formation at 45 days was statistically greater in the Control group compared to the BF group (37.7± 1.7 vs 18.9±1.6; P=0.0486). The BO group presented a percentage of 27.5±1.5 of new bone formation at 45 days ( B). As seen in , a narrow band of newly formed bone tissue along the edges of the surgical wound was observed. The patterns of new bone in the BO and BF groups were similar. However, the Control group exhibited a greater amount of bone compared to the BF group at 45 days. All bone defects in each group were filled with thin layer dense connective tissue. This connective tissue layer had a thin amount of collagen fibers aligned parallel to the wound surface, along with a small number of inflammatory cells, fibroblasts, and blood vessels. The immunohistochemical technique used to detect BMP2/4, OCN and TRAP showed high specificity, which was confirmed by the absence of total labelling in the negative control of the reaction. The immunoreactive cells showed a dark brown coloration confined exclusively to the cytoplasm, in the case of TRAP, and confined to the cytoplasm and, to a lesser extent, to the extracellular matrix, in the case of BMP2/4 and OCN. Analysis of the immunostaining of the BMP2/4 and the OCN revealed that the medians of specimens from the Control and BO groups demonstrated the similarity of results of BMP 2/4 and OCN in both evaluation periods ( A and 4B). During the 30 and 45-day period, the BO group showed slightly more BMP2/4 immunostaining compared to the BF group. At 30 days, the BF group had lower levels of OCN expression compared to BO group, but there was no significant difference observed at 45 days. In the analysis of the TRAP immunostaining, the specimens from the BF group showed the highest means of immunostaining positive cells in both periods (33.2 ± 4.0; 37.2 ± 6.4; C); when compared to the BO and Control groups during the same periods. Various bone biomaterials have been proposed as alternatives to autogenous bone grafts in oral surgeries . Some of these alternatives include xenogenous biomaterials such as the products tested in this study, since these biomaterials aim to provide a suitable scaffold for bone regeneration by their osteoconductive properties . The choice of a bone substitute depends on several factors, including the specific clinical application, patient characteristics, and the surgeon preference . It is important to note that biocompatibility and effectiveness can vary depending on consolidation with progressive apposition of new bone followed by resorption and replacement by bone . Additionally, it is necessary to understand and evaluate the osteoconductive and osteoinductive properties as a guidance to select the most suitable bone substitute . The main results from the histomorphometry and immunological analysis demonstrated that BO and BF presented equivalent quantitative amount of new bone formation at the final evaluated period. However, BO has revealed greater expression of bone proteins of OCN at 30 days and BMP2/4 at 45 days, and lower number of TRAP-positive cells in both periods. On the other hand, the BF group exhibited a delay in bone formation compared to the BO group within a 30-day period. The results obtained in the present study showed that immunostaining for BMP2/4 and OCN in the BO group was equivalent with those of the control group in both evaluation periods. This finding highlights the importance of neo-angiogenesis to promote platelet adhesion and stimulates mesenchymal cells for bone formation through the presence of blood. Bone cell precursors from angiogenesis are crucial factors for bone healing and its continuous remodeling . In the comparative analysis of the biomaterials, it was observed that both products demonstrated capacity for bone neoformation, however, BO demonstrated immunostaining of BMP2/4 and OCN slightly higher than that of BF group, standing out in the period of 45 days. Then, it was observed that BO led to a positive induction of proteins that signaled the process of osteoblastic differentiation and maturation. The difference can be directly attributed to the temperature process of the bovine bone denaturation. BO undergoes high-temperature preparation at 300ºC, while BF is crafted at a lower temperature . This can result in a higher purification of BO and consequently lead to improved immune responses of bone proteins . The findings also showed that BF exhibited prolonged osteoclast activity, characterized by the higher number of TRAP-positive cells in both evaluation periods. These outcomes suggest that BF is capable to promote reabsorption and bone formation due to its osteoconductive properties. An earlier study comparing BF and BO in rabbits through histometric and immunohistochemical analysis found that BF and BO both showed similar bone formation and integration . There were no significant differences observed in bone density and inflammation between the two products . However, it was concluded that further research and clinical trials were still necessary to fully evaluate the long-term compatibility and performance of these materials . Another clinical study revealed effective outcomes of implant osseointegration after implant placement and simultaneous sinus augmentation with BF . Furthermore, a prior animal study in rats found no significant differences in bone volume and density of BF associated or not with active oxygen-based oral gel . Regarding biocompatibility, both have previously demonstrated safe biocompatibility, meaning they are well-tolerated and capable of promoting bone regeneration , . In the applications aspect, BF has been effective in challenging bone healing situations, including rats submitted to experimental alcoholism , regenerative treatment in human mandibular class II furcation defects and sinus augmentation . The comparable features of the two tested materials might also be attributed to the porosity present in both materials as mentioned before . Both biomaterials exhibit an external surface of microporous , , which enhances the superficial area for angiogenesis and bone regeneration. The two grafts have different particle sizes, which are important factors for cell adherence and cytokine release , . Interestingly, the BO graft has nanopores of 15-nm hydroxyapatite crystallites, and BO particles are smaller than BF granules. This might allow a larger surface area for bone cell adherence of endogenous proteins related to bone formation . Additionally, the manufacturing process significantly influences the osteoconductive features of these biomaterials. BO is manufactured by heating at 300 °C followed by a treatment with sodium hydroxide, while BF receives only a sequential bath to solubilize the organic part . The heating treatment results in a large surface area and a polyhydric format, consequently it can enhance bone formation by BO . BO has shown a higher dissolution ratio in acid conditions, which contributes to its resorption and bone formation . Moreover, BO was found to be free of organic parts , while BF possibly contain fragments of organic particles , . However, this did not compromise the biological effects of bone healing in our present study. Furthermore, the various sources of the bovine graft, whether from cortical or medullary, may have affected the results of this study. BF is manufactured from the cortical bone might explain the more intensified TRAP-positive cells involved in reabsorption of this biomaterial and its slower bone formation. Further research is warranted to evaluate both biomaterials including molecular analysis, to shed light on the strengths and limitations of BO and BF. Regarding the limitations of this study, it should be stated that this is a rodent animal model which only partially simulate the clinical condition. Clinical studies using a split mouth design with a large sample should be conducted to reflect the clinical scenario in humans. Ultimately, these future studies can promote insights in the way for more effective bone grafting interventions, improving patient outcomes and quality of life. In conclusion, even with the higher expression of proteins related to bone formation, there was no difference in new bone formation at 45 days when both types of anorganic bovine xenogenous grafts were evaluated. |
Using a modified nominal group technique to develop general practice | bdbc611b-ea6a-425f-92b1-e7513ce89e4e | 6052560 | Preventive Medicine[mh] | General practitioners (GPs) regularly make difficult choices about treatment options. Guidelines are one way of assisting GPs in decision-making and, in an ideal world, guidelines would be based on evidence derived from rigorously conducted empirical studies. In practice, there are few areas of health care where sufficient research-based evidence exists or can even be produced , and this is especially so within primary healthcare . In such situations, the development of assisted support will inevitably be based, largely or in part, on the opinions and experience of clinicians and others with knowledge of the relevant field . Since 2013, a group of Danish GPs has worked on producing systematic and knowledge-based electronic health records for the seven preventive child health examinations (PCHEs) held in general practice. The Danish National Board of Health provides guidelines for PCHEs, but the recommendations are extensive and cover all aspects of a child’s health . There is no structured and systematic process in place to determine which of the comprehensive recommendations is the most important to focus on during the limited time available to carry out the PCHEs, nor is there a nationally aligned process for keeping journal notes. The vision is to make new electronic journal records available to all Danish GPs and the idea is that GPs’ use of an electronic health record, with its potential for decision support and easy access to previous findings, will support their work and make it easier to keep an overview of the patient’s case history. The development of electronic records therefore holds the potential for a quality development in child healthcare in Denmark. Given the likely diversity of opinion that any group of people may display when considering a topic, formalised methods, such as consensus techniques, are essential for organising subjective judgments in group work. Consensus techniques have been successfully used by several research groups in their work to develop quality markers in complex clinical areas, such as angina , emergency care , cancer , and also within the field of child healthcare . The three most common consensus methods used for medical and health services research are the Delphi method, the consensus development conference, and the nominal group technique (NGT) . The Delphi method is a forecasting method based on several rounds of questionnaires sent to a panel of experts. The anonymous, written responses are aggregated and shared with the group after each round . The consensus development conference brings together practitioners, researchers, and consumers over a period of several days to seek general agreement, or consensus, on the efficacy, safety, and appropriate conditions for the use of various medical and surgical procedures, drugs, and devices . The third method, and the one we selected in the present study, is the NGT. The NGT is a structured, well-established, multistep, facilitated, group meeting technique used to generate and prioritise responses to a specific question by a group of people who have expert insight into a particular area of interest . It is an organised process that gives participants an equal opportunity to contribute their personal views before inviting them to build on the reflections of others to develop their own thoughts, and finally to reach consensus about the issues raised in the original question . The NGT has been applied on several occasions for projects in general practice . It has an advantage in that its format resembles the way Danish GPs are accustomed to collaborating in network groups, where experiences and challenges from everyday working life in practice are shared and discussed . In this study the NGT was used with a twofold purpose. First, it was a way to systematise and develop the content of PCHEs; and second, it was a method to develop the format of electronic health records to be used in PCHEs. These two parallel purposes were strongly interlinked. To bridge the gap between research and practice, evidence as well as its applicability should be considered when formulating recommendations. It is important that recommendations are compatible with existing norms and values and it is therefore essential that practitioners, in this case the future users of electronic health records, participate in the development of practice . In this paper we explore how the NGT can be used to establish consensus in a complex clinical field by analysing how it supported the development of structured electronic health records for PCHEs in Danish general practice. This study applied group discussions based on the NGT method in an adapted serial meeting design. The adapted design complies with the checklist created by Humphrey-Murto et al. to ensure methodological rigour when using consensus group methods, with only one deviation concerning anonymous re-ranking of feedback . In-depth descriptions of the original steps in the NGT method have been reported extensively elsewhere . The project was conceived and designed by KL and RE, who are both experienced GPs specialising in research on children’s health. During the meetings, KL was in the facilitator’s role and RE participated as a member of the NGT panel. RE participated on the same terms as the other panel members, meaning that e.g. she waited for her turn to speak in the rounds, and her opinion carried no more weight than any of the other participants. More importantly, RE was aware of her double role in the project and its potential downfalls. This demanded a continuous reflection on her position, which we shall return to in the discussion section. As well as RE, we purposively identified four GPs known for their broad knowledge and expertise in general practice and their specific interest in the PCHEs. We invited them to constitute the expert panel. Verbal informed consent was given before the four recruited GPs freely and informed chose to participate in the project. All five participating GPs worked either in Region Zealand or in the capital area of Copenhagen in Denmark. Prior to the first meeting the GPs were asked to read the report: Evaluation of the Preventive Child Consultations in General Practice and before each meeting they were also asked to read the chapters in the Danish National Board of Health’s guidelines on PCHEs relevant for that particular meeting’s focus. In this way we pursued a systematic method combining evidence and expert opinion. In addition to background reading, we asked the participating GPs to be extra observant when carrying out PCHEs in the period leading up to the first meeting. We encouraged them to ask parents about their needs and expectations during the PCHEs. Throughout the working period of four months, the expert panel met three times. The meetings lasted five hours, five hours, and two-and-a-half hours respectively, and the four invited participants were offered compensation for their time. All meetings took place at facilities convenient to the practice of two of the participants. The main aim of NGT is to generate themes and issues, which are discussed and ranked by the group. At the first meeting, KL described the NGT as a method to the panel members who had the opportunity to ask questions. This introduction was a factual description of the method’s different steps and did not have any content or comment that would influence participants and the task in hand. After the introduction, KL asked the panel the nominal question: What do you consider important to prioritise in the preventive child health examination at five weeks, and what do parents think is important, according to your experience and knowledge ? The question was developed based on KL’s extensive work with the PCHEs and RE’s previous experience with developing electronic health records for antenatal care visits in general practice in Denmark. At this stage, the panel was given no guidance on how broad or narrow their focus should be. The original plan was to work on the first three PCHEs, which take place when a baby is five weeks, five months, and twelve months old; one PCHE per scheduled meeting. However, while working through the steps of the NGT during the first meeting, the group found it necessary to make adjustments and deviations to the original model, outlined by Gallagher et al. . The five hours allocated to develop a health record for the first PCHE were not enough to meet the project’s combined objective: exploratory research involving a qualitative understanding of the priorities; and the development of a concrete product in the form of a systematic electronic health record. As a result, it was agreed in plenum that KL and RE should work with the draft produced by the panel between the first and the second meeting. This work solely concerned linguistic and structural aspects and a conscious effort was made to keep the content unchanged. The re-edited draft was then presented to participants at the second meeting, where it was critically evaluated, adjusted, and approved in plenum. In this way, a mutual understanding was secured in a forum in which the participants were both informants and collaborators. This pattern was repeated between the second and third meetings and became the model for working with drafts of the succeeding electronic health records (Fig. ). Consensus was defined as having been achieved when there were no further comments or suggestions for corrections from any of the participants. Achieved consensus determined the process. The continual and circular re-evaluation of the drafts enhanced the process and secured communicative validity . The group’s experience from working with the content and structure of the first electronic health record was used strategically at the next meeting when the focus shifted to the succeeding PCHE. During the meetings, the atmosphere was jovial and enthusiastic. The participation of KL and RE as the project’s initiators did not seem to affect or influence the four invited GPs. All participants took part in the discussions equally and appeared confident in their roles as well as eager to contribute with their individual perspectives on the work. In addition to working papers from the meeting rounds and the different draft versions of the electronic health records for the first three PCHEs, material for the present article also consisted of field notes produced during one of the meetings by ES, who participated as an observer. Having an observer in the research project enhanced opportunities for noticing aspects of interpersonal communication and group dynamic that are taken for granted or missed by participants due to their immediate obviousness . The observations and field notes permitted an extra level of abstraction in the discussion of the group’s use of the NGT, particularly with regard to the steps of the model where the group deviated from the original structure. Based on the output of the meetings, including descriptive field notes, we conducted a thematic text analysis to identify important areas of new knowledge and to better understand what the modified version of the NGT meant for the validity of the method. The use of the NGT made it possible to combine idea generation and problem solving as two complementary parts of the same process. This makes the method well suited for development work in general practice with its complex characteristics and demands for applicability. Three main categories of experience were identified and these are described and appraised below. For clarity, the third category is divided into four sub-categories. Keeping focus and supporting equal opportunities to speak The structured and stepwise process of the NGT ensured that the energetic expert panel kept focus on the defined purpose, while the repeated table rounds supported opportunities for participants to be equally heard. The method’s face-to-face approach integrated non-verbal communication, such as laughter; while the structured design minimised potential power structures that can appear when participants already know each other, or when one of the panelists is also the initiator of the project, which was our experience on this project. Generating new perspectives on clinical practice During steps 4 and 5 (Table ) the participants became aware of the potential to re-use knowledge previously obtained about the patient (Table , column 3). Prior to the seven PCHEs, the Danish preventive healthcare programme has three antenatal care visits and, in principle, all ten visits are conducted by the same GP. Data from antenatal care visits are recorded in the mother’s journal, which is not automatically consulted in the PCHEs that follow. The process of the group discussions generated an awareness of the prospective re-use of knowledge gathered during the antenatal care visits, such as the pregnancy’s development or the family’s socioeconomic situation. Flexibility of the NGT model The NGT proved to be a highly flexible model well suited to the complex research question we asked, and conducive to detailed discussion and elucidation of themes and issues. Discussions and thematic classification in pairs The panel recognised early in the working process that it would not be favourable to strictly follow the model’s original outline. For example, the discussions and clarifications carried out in step 4 (Table ) revealed that it did not make sense to produce a prioritised list, as the model prescribes. Since all the suggested themes were important, the expert panel found it more relevant to organise them into broader thematic categories and line them up in that way. The work with these categories was carried out first as a group and then the panel divided into pairs to further discuss the categories. This resulted in an outline of the first draft of the electronic health record (Table , column 2). Serial meetings Serial meetings provided time for continual evaluation and the search for more information. The original time allocated to work with electronic health records turned out to be too short to produce adequate content and a format for each record. As a consequence, drafts were linguistically and structurally reorganised by KL and RE between meetings (Fig. ). At the same time, participants had opportunities to test in their practices aspects that had been discussed during the meeting, and to return to the next session with new experience-based knowledge. Participants became aware that some instructions in the guidelines from the National Board of Health were not fully up-to-date; the recommendations for congenital cataract, for example. The serial nature of the meetings meant that such questions of doubt could be checked in the interim and discussed at the next meeting. Reflections and ethical considerations The serial character of the meetings and the continual re-evaluation of the drafts (Fig. ) made room for participants to further reflect between meetings on the topics discussed. During the first meeting, the idea that information collected at the mother’s antenatal care visits could automatically be transferred to the child’s health record was presented and calmly received in the group. However, at the third meeting an intense discussion arose concerning ethical issues raised by the idea of transferring certain kinds of information to the child’s record, e.g. alcohol abuse in the family. The serial application of the method provided time for important critical reflection on themes and, in this case, the ethical challenges around a potential transfer of data. Adjustable template Although the group did not succeed at the first meeting in producing a final model for the PCHE at five weeks, the NGT secured the production of a fruitful draft which was applied as a model for the first and all succeeding PCHEs (See Table ). The flexibility of the NGT therefore led to the production of a template that could be used and tailored during the development of systematic electronic health records for all seven PCHEs. The template was based on the experience of frontline professionals, the guidelines from the National Board of Health, and best evidence. The findings were practice-near, experience-based, and therefore directly applicable to PCHE work in general practice. The structured and stepwise process of the NGT ensured that the energetic expert panel kept focus on the defined purpose, while the repeated table rounds supported opportunities for participants to be equally heard. The method’s face-to-face approach integrated non-verbal communication, such as laughter; while the structured design minimised potential power structures that can appear when participants already know each other, or when one of the panelists is also the initiator of the project, which was our experience on this project. During steps 4 and 5 (Table ) the participants became aware of the potential to re-use knowledge previously obtained about the patient (Table , column 3). Prior to the seven PCHEs, the Danish preventive healthcare programme has three antenatal care visits and, in principle, all ten visits are conducted by the same GP. Data from antenatal care visits are recorded in the mother’s journal, which is not automatically consulted in the PCHEs that follow. The process of the group discussions generated an awareness of the prospective re-use of knowledge gathered during the antenatal care visits, such as the pregnancy’s development or the family’s socioeconomic situation. The NGT proved to be a highly flexible model well suited to the complex research question we asked, and conducive to detailed discussion and elucidation of themes and issues. Discussions and thematic classification in pairs The panel recognised early in the working process that it would not be favourable to strictly follow the model’s original outline. For example, the discussions and clarifications carried out in step 4 (Table ) revealed that it did not make sense to produce a prioritised list, as the model prescribes. Since all the suggested themes were important, the expert panel found it more relevant to organise them into broader thematic categories and line them up in that way. The work with these categories was carried out first as a group and then the panel divided into pairs to further discuss the categories. This resulted in an outline of the first draft of the electronic health record (Table , column 2). Serial meetings Serial meetings provided time for continual evaluation and the search for more information. The original time allocated to work with electronic health records turned out to be too short to produce adequate content and a format for each record. As a consequence, drafts were linguistically and structurally reorganised by KL and RE between meetings (Fig. ). At the same time, participants had opportunities to test in their practices aspects that had been discussed during the meeting, and to return to the next session with new experience-based knowledge. Participants became aware that some instructions in the guidelines from the National Board of Health were not fully up-to-date; the recommendations for congenital cataract, for example. The serial nature of the meetings meant that such questions of doubt could be checked in the interim and discussed at the next meeting. Reflections and ethical considerations The serial character of the meetings and the continual re-evaluation of the drafts (Fig. ) made room for participants to further reflect between meetings on the topics discussed. During the first meeting, the idea that information collected at the mother’s antenatal care visits could automatically be transferred to the child’s health record was presented and calmly received in the group. However, at the third meeting an intense discussion arose concerning ethical issues raised by the idea of transferring certain kinds of information to the child’s record, e.g. alcohol abuse in the family. The serial application of the method provided time for important critical reflection on themes and, in this case, the ethical challenges around a potential transfer of data. Adjustable template Although the group did not succeed at the first meeting in producing a final model for the PCHE at five weeks, the NGT secured the production of a fruitful draft which was applied as a model for the first and all succeeding PCHEs (See Table ). The flexibility of the NGT therefore led to the production of a template that could be used and tailored during the development of systematic electronic health records for all seven PCHEs. The template was based on the experience of frontline professionals, the guidelines from the National Board of Health, and best evidence. The findings were practice-near, experience-based, and therefore directly applicable to PCHE work in general practice. The panel recognised early in the working process that it would not be favourable to strictly follow the model’s original outline. For example, the discussions and clarifications carried out in step 4 (Table ) revealed that it did not make sense to produce a prioritised list, as the model prescribes. Since all the suggested themes were important, the expert panel found it more relevant to organise them into broader thematic categories and line them up in that way. The work with these categories was carried out first as a group and then the panel divided into pairs to further discuss the categories. This resulted in an outline of the first draft of the electronic health record (Table , column 2). Serial meetings provided time for continual evaluation and the search for more information. The original time allocated to work with electronic health records turned out to be too short to produce adequate content and a format for each record. As a consequence, drafts were linguistically and structurally reorganised by KL and RE between meetings (Fig. ). At the same time, participants had opportunities to test in their practices aspects that had been discussed during the meeting, and to return to the next session with new experience-based knowledge. Participants became aware that some instructions in the guidelines from the National Board of Health were not fully up-to-date; the recommendations for congenital cataract, for example. The serial nature of the meetings meant that such questions of doubt could be checked in the interim and discussed at the next meeting. The serial character of the meetings and the continual re-evaluation of the drafts (Fig. ) made room for participants to further reflect between meetings on the topics discussed. During the first meeting, the idea that information collected at the mother’s antenatal care visits could automatically be transferred to the child’s health record was presented and calmly received in the group. However, at the third meeting an intense discussion arose concerning ethical issues raised by the idea of transferring certain kinds of information to the child’s record, e.g. alcohol abuse in the family. The serial application of the method provided time for important critical reflection on themes and, in this case, the ethical challenges around a potential transfer of data. Although the group did not succeed at the first meeting in producing a final model for the PCHE at five weeks, the NGT secured the production of a fruitful draft which was applied as a model for the first and all succeeding PCHEs (See Table ). The flexibility of the NGT therefore led to the production of a template that could be used and tailored during the development of systematic electronic health records for all seven PCHEs. The template was based on the experience of frontline professionals, the guidelines from the National Board of Health, and best evidence. The findings were practice-near, experience-based, and therefore directly applicable to PCHE work in general practice. Our main findings from working with the NGT relate to its flexibility and modifiability. The flexibility of the method confirmed its suitability for complex research questions, such as ours; while the production of an adjustable template with consensus results made the meetings’ outcomes both manageable and tangible. The functionality of the modified serial meeting design provided fruitful time for continued reflection on the results and previous discussions, as well as providing opportunities for relevant checks between meetings where a lack of knowledge or doubts had become apparent. The latter provided openings for systematic development of knowledge. Some of our results concur with findings from previous projects working with the NGT and the model’s flexibility has been recognised by other studies that also successfully modified the original NGT design and experienced an improvement . One study had difficulty with the ranking in step 5 (Table ) , which we also report in our findings. They ended up voting when consensus could not be reached through ranking, while the present study chose to divide the panel into pairs to work with the themes, before returning once more to discussion in plenum. In line with our findings, other studies have also found that the original NGT structure, with one meeting allocated to reach final recommendations, was not sufficient for an in-depth elaboration of themes . The serial character of the meetings in this study is comparable to the Delphi method where consensus is obtained through evaluation of written documents that are sent back and forth among participants a number of times until consensus is reached. Therefore, in the present study we incorporated strength from the Delphi method into the modified version of the NGT. Experiences emerged during the working processes that, to our knowledge, have not previously been reported by other studies. A central feature of using the NGT is that a question is investigated extensively from a broad spectrum of viewpoints and thereby creates awareness of overlaps, knowledge-sharing, gaps in knowledge, or unproductive working patterns. In this project, our participants became aware of the possibility of using existing knowledge about their patients, but also about the potential ethical downside of such a practice. By documenting all proposals and ideas, the NGT model ensures that no insights are lost through the potential uncertainty of some participants, while at the same time tangible products in the form of written documents are produced. Implication of findings for future research Related to the specific research project It is anticipated that the possibility of using electronic health records as a support when carrying out PCHEs in the future will systematise and develop both the structure and the content of the PCHEs in general practice. However, experience and intuition are fundamental and effective elements of everyday working life in general practice, not least when it comes to diagnosing children . It is therefore crucial that the electronic health records do not compromise this practice, which is why the use of the records will be a supportive option and not a mandatory practice. Lippert et al. have pointed to a need for further discussion about the relationship between situatedness and standardisation in primary care and for further empirical investigations of the possible consequences of standardisation processes . DanChild, of which the present study is a part, has a combined vision to investigate GPs’ responses to electronic health records as well as to develop child health through cohort research. While the NGT as a method encourages consensus and practice-near solutions, it is important to emphasise that the success of the electronic health records is dependent on a continued and reciprocal collaboration with general practice . Related to the applicability of the modified method The modifications we made to the NGT were feasible and did not lose the method’s advantageous structure. We believe this was because the participants and the facilitator shared a common professional background as GPs, limiting the perspectives to one professional grouping. Participants had been asked to read relevant chapters in the Danish National Board of Health’s guidelines on PCHEs as well as a thematically relevant report, further enhancing a mutual starting point. However, we did not check whether or not they had read the documents. We felt that this would unnecessarily highlight the fact that one of the participants, RE, was also one of the project’s initiators. Therefore we cannot guarantee that all participants had a common starting point for discussion. Finally, the project had a well-defined goal, namely the production of content and a format for electronic health records supporting the PCHEs, and that concrete purpose enabled a softening of the original NGT model’s mechanical steps, without the group losing its focus. Based on our experience with the modified NGT all three aspects are crucial for future researchers implementing similar changes to the original NGT model. Strengths and limitations of the study By asking highly professionally engaged GPs with a specific interest in the PCHEs to reach consensus and suggest a way forward for all GPs to follow, our results may be ambiguous for the average practitioner. The project group is aware of this risk and will incorporate it into their continued work with electronic health records by pilot testing the product and by giving individual practitioners flexibility to use the record in their own way. In this project two of the article’s authors participated either as facilitator (KL) or as a member of the panel (RE), and they worked with the drafts between the meetings. Double roles like these are not uncommon in similar projects , but still worth critical reflection and consideration. Any data collection, analysis, and conclusion are inextricably entwined with the researcher’s presuppositions as well as the positions adopted while collecting the data . According to Skjervheim, researchers can only gain access to social phenomena of interest by recognising themselves as a contributing participant . In this case, RE is an experienced GP with a known research interest in child health, and took part in the panel as an equal to the other participants. RE was, however, aware of her double role in the process and continuously reflected on the effect it might have on the way her suggestions and comments were received by the group, and, ultimately, on how it might have affected consensus. One could discuss if KL’s and RE’s work between the meetings minimised the democratic process, by giving them more influence than the rest of the group. This risk was reduced as much as possible, by the process of repeatedly evaluating their work in plenum at subsequent meetings. During these evaluations the other participants actively suggested critical corrections and this leads us to believe that the final product meets a satisfactory degree of representativeness. Furthermore, we attempted to minimise bias by enrolling co-authors who were not actively implicated in the data producing process. One of them even participated as an observer at one of the meetings. Finally, the authors recognise that objective knowledge in the form of true consensus is a naïve understanding of reality. Following Haraway, it might be more fruitful to think of knowledge as situated within a context . While the point of view within a context has a more limited range than disembodied objectivity, situated points of view are richer in content as they take into account the numerous bits of information constituting the context and the environment of that point of view. For the present project, it means acknowledging the influence KL and RE had on consensus, while at the same time recognising this as a given condition that simultaneously supported the success of the group’s progressive work. Related to the specific research project It is anticipated that the possibility of using electronic health records as a support when carrying out PCHEs in the future will systematise and develop both the structure and the content of the PCHEs in general practice. However, experience and intuition are fundamental and effective elements of everyday working life in general practice, not least when it comes to diagnosing children . It is therefore crucial that the electronic health records do not compromise this practice, which is why the use of the records will be a supportive option and not a mandatory practice. Lippert et al. have pointed to a need for further discussion about the relationship between situatedness and standardisation in primary care and for further empirical investigations of the possible consequences of standardisation processes . DanChild, of which the present study is a part, has a combined vision to investigate GPs’ responses to electronic health records as well as to develop child health through cohort research. While the NGT as a method encourages consensus and practice-near solutions, it is important to emphasise that the success of the electronic health records is dependent on a continued and reciprocal collaboration with general practice . Related to the applicability of the modified method The modifications we made to the NGT were feasible and did not lose the method’s advantageous structure. We believe this was because the participants and the facilitator shared a common professional background as GPs, limiting the perspectives to one professional grouping. Participants had been asked to read relevant chapters in the Danish National Board of Health’s guidelines on PCHEs as well as a thematically relevant report, further enhancing a mutual starting point. However, we did not check whether or not they had read the documents. We felt that this would unnecessarily highlight the fact that one of the participants, RE, was also one of the project’s initiators. Therefore we cannot guarantee that all participants had a common starting point for discussion. Finally, the project had a well-defined goal, namely the production of content and a format for electronic health records supporting the PCHEs, and that concrete purpose enabled a softening of the original NGT model’s mechanical steps, without the group losing its focus. Based on our experience with the modified NGT all three aspects are crucial for future researchers implementing similar changes to the original NGT model. It is anticipated that the possibility of using electronic health records as a support when carrying out PCHEs in the future will systematise and develop both the structure and the content of the PCHEs in general practice. However, experience and intuition are fundamental and effective elements of everyday working life in general practice, not least when it comes to diagnosing children . It is therefore crucial that the electronic health records do not compromise this practice, which is why the use of the records will be a supportive option and not a mandatory practice. Lippert et al. have pointed to a need for further discussion about the relationship between situatedness and standardisation in primary care and for further empirical investigations of the possible consequences of standardisation processes . DanChild, of which the present study is a part, has a combined vision to investigate GPs’ responses to electronic health records as well as to develop child health through cohort research. While the NGT as a method encourages consensus and practice-near solutions, it is important to emphasise that the success of the electronic health records is dependent on a continued and reciprocal collaboration with general practice . The modifications we made to the NGT were feasible and did not lose the method’s advantageous structure. We believe this was because the participants and the facilitator shared a common professional background as GPs, limiting the perspectives to one professional grouping. Participants had been asked to read relevant chapters in the Danish National Board of Health’s guidelines on PCHEs as well as a thematically relevant report, further enhancing a mutual starting point. However, we did not check whether or not they had read the documents. We felt that this would unnecessarily highlight the fact that one of the participants, RE, was also one of the project’s initiators. Therefore we cannot guarantee that all participants had a common starting point for discussion. Finally, the project had a well-defined goal, namely the production of content and a format for electronic health records supporting the PCHEs, and that concrete purpose enabled a softening of the original NGT model’s mechanical steps, without the group losing its focus. Based on our experience with the modified NGT all three aspects are crucial for future researchers implementing similar changes to the original NGT model. By asking highly professionally engaged GPs with a specific interest in the PCHEs to reach consensus and suggest a way forward for all GPs to follow, our results may be ambiguous for the average practitioner. The project group is aware of this risk and will incorporate it into their continued work with electronic health records by pilot testing the product and by giving individual practitioners flexibility to use the record in their own way. In this project two of the article’s authors participated either as facilitator (KL) or as a member of the panel (RE), and they worked with the drafts between the meetings. Double roles like these are not uncommon in similar projects , but still worth critical reflection and consideration. Any data collection, analysis, and conclusion are inextricably entwined with the researcher’s presuppositions as well as the positions adopted while collecting the data . According to Skjervheim, researchers can only gain access to social phenomena of interest by recognising themselves as a contributing participant . In this case, RE is an experienced GP with a known research interest in child health, and took part in the panel as an equal to the other participants. RE was, however, aware of her double role in the process and continuously reflected on the effect it might have on the way her suggestions and comments were received by the group, and, ultimately, on how it might have affected consensus. One could discuss if KL’s and RE’s work between the meetings minimised the democratic process, by giving them more influence than the rest of the group. This risk was reduced as much as possible, by the process of repeatedly evaluating their work in plenum at subsequent meetings. During these evaluations the other participants actively suggested critical corrections and this leads us to believe that the final product meets a satisfactory degree of representativeness. Furthermore, we attempted to minimise bias by enrolling co-authors who were not actively implicated in the data producing process. One of them even participated as an observer at one of the meetings. Finally, the authors recognise that objective knowledge in the form of true consensus is a naïve understanding of reality. Following Haraway, it might be more fruitful to think of knowledge as situated within a context . While the point of view within a context has a more limited range than disembodied objectivity, situated points of view are richer in content as they take into account the numerous bits of information constituting the context and the environment of that point of view. For the present project, it means acknowledging the influence KL and RE had on consensus, while at the same time recognising this as a given condition that simultaneously supported the success of the group’s progressive work. To our knowledge, this study is the first to report on Danish GPs using the NGT to identify key areas of focus and to structure quality marker development in general practice. The structured interactive process used in this study supported equal opportunities for experienced professionals to significantly contribute to the development of electronic health records to support PCHEs in Danish general practice. By using the modified NGT, participating GPs actively expressed their views through structured discussions as a group, through working in pairs, and through the process of reaching final consensus. In accordance with previous studies we therefore argue that the original NGT model developed in the late 1960s can be modified advantageously and used to explore developmental work and changes in general practice. Due to the integration of experienced professionals from the very beginning of the process the results are practice-based and applicable. We are confident that the NGT model can be useful for capturing group perspectives in complex working areas such as general practice, and we recommend the NGT as a working tool in general practice development in the future. |
Evaluation of microbial diversity in the formation water of the producer and marginal wells in bokaro coal field | b53d9210-77d7-4824-894f-90391c1bb93b | 11605091 | Microbiology[mh] | Regardless of efforts made globally to lessen reliance on fossil fuels, coal remains a major fuel for energy production. Since coal is the primary source of CO 2 emissions and electricity generation, switching to low-carbon energy systems presents a unique challenge. Coal Bed Methane (CBM) is an unconventional form of natural gas formed inside coal seams . CBM is a clean form of energy; therefore, its development and utilization carry great social and economic benefits and provide a way forward for the global energy transition . Considering the surge in energy demand and environmental perspective, CBM is a better alternative to fulfill domestic and industrial energy demands . Production of methane from coal beds is biogenic and thermogenic . Biogenic methane production involves complex metabolism performed by a consortium of indigenous microbes . The coal seam ecosystem is an example of synchronizing hydrolyzing and fermenting microbes with methanogens. The methane generation process involves the degeneration of complex hydrocarbons to lower molecular weight organic compounds, which act as a substrate for the methanogens and are finally converted into methane gas . Methanogens can be divided into three categories according to the metabolic pathway followed in methane production. Hydrogenotrophic methanogens utilize hydrogen as an electron donor to reduce carbon dioxide to methane, e.g., Methanobacterium. The acetotrophic methanogens convert acetate to methane and CO 2, e.g., Methanosarcina and Methanosaeta. Lastly, the methylotrophic methanogens convert methanol and methylamine into methane . Recently, the development of biogenic CBM has picked up and is gathering more attention. Researchers are focusing on microbial diversity to understand the microbial ecosystem and their functioning in the production of CBM . A recent study explored seasonal variation in coal bed water in China and reported the prevalence of phyla Proteobacteria, Bacteroidetes, and Firmicutes . The comparative analysis of coal bed diversity of Gunnedah, Sydney, and Surat coals shows the abundance of Firmicutes, Proteobacteria, Euryarchaeota, Bacteroidetes, and Actinobacteria . Another study exploring the Jharia coal mine's formation water diversity revealed Proteobacteria, Bacteroidetes, Actinobacteria, Verrucomicrobia, and Firmicutes as the dominant phyla in the formation water . These studies established the significant role of these members in the production of biogenic methane by degradation of complex polymers and providing substrate for methane production. Worldwide efforts are being made to enhance the production of biogenic CBM. Coal provides over 58% of India's energy demands, followed by hydrocarbons at 38% and nuclear and hydroelectric power at 4% each. The high reliance creates immense pressure to limit the coal dependency . A recent report suggested that harnessing 10% of coal bed methane reserves can cut India's energy import bill by two billion US dollars ( https://energy.economictimes.indiatimes.com/news/coal/harnesing-10-of-coal-bed-methane-reserves-can-cut-indias-energy-import-bill-by-2-billion-experts/100237602 ). Therefore, a deeper understanding of the microbial diversity and environmental factors that drives the CBM production process is essential. Despite the various research in this area, there is a void in understanding variation in microbial diversity of CBM wells differing in their gas production . The present study utilizes metagenomics to decipher microbial communities in the Producing and the Marginal groups of CBM well in Bokaro, India. The present investigation is the first to demonstrate microbial distinctiveness in the CBM wells according to the gas production performance. The study will facilitate a better understanding of the roles of the microbes in the production of biogenic methane, which may further help in implementing field jobs for its augmentation in the wells. Sample area description and collection The formation water and coal samples were collected from CBM wells, namely Well01, Well02, Well15, Well16, Well27, Well29, and Well47. The well belongs to the Bokaro coal bed methane block of Oil and Natural Gas Corp. in eastern India. The location and grouping details of the samples are shown in Table and Fig. . The Bokaro Block, extending over 95 km 2 , consists of three disconnected sectors. Central Patch (Patch-A covering 66 km 2 area), Western Patch (Patch B covering 18 km 2 area), and Eastern Patch (Patch-C covering 11 km 2 area). The Central Patch comprises three distinct geographic areas viz. (i) Saram-Gumia in the adjoining eastern part of the Lugu hill, (ii) Jarangdih Deep beyond the Saram-Gumia area in the further east separated by the Bokaro River, and (iii) Daniya to the west of Lugu hill. The western patch lies in the Hesagara-Kasikhap area, whereas the Eastern Patch lies in the Chalkari-Phusro area. The Bokaro Coalfield forms a part of the Damodar Valley coalfields and is situated in the Bokaro and Hazaribagh districts of Jharkhand. This coalfield is one of the country's major producers of medium-cooking blendable coals. Lugu Hill, a major topographic high of the area, divides the coalfield into two parts. East Bokaro Coalfield and West Bokaro Coalfield. The Damodar River flows along the southern margin of East Bokaro Coalfield, and the Bokaro River forms the main drainage of West Bokaro Coalfield. The samples are grouped according to the wells' annual gas production. Wells with gas production > 500 m 3 /d belong to the Producer groups, and the wells with production < 500 m 3 /d belong to the Marginal group. The sampling of all the wells was done in April 2022. The sample collection device consisted of a 10 cm diameter, 1 m long polyvinyl chloride pipe sealed at the bottom and capped with an open steel mesh at the top. The pipe and mesh enclosure were sterilized with a bleach solution before being lowered to the well's bottom on a wireline . Samples were collected in triplicate in 1000 ml of anaerobic sterilized serum bottles and analyzed for pH and conductivity on site. All the samples were transported to the laboratory within 48 h at 4 °C and processed immediately for activity measurement and microbial analysis . The indigenous coal samples of a CBM well were obtained from the core house and transferred in sterile, airtight containers. Physio-chemical analysis of formation water and coal Physio-chemical analysis of the formation water samples from the Bokaro CBM well was performed for pH, Total dissolved solids (TDS), Total suspended solids (TSS), and Electrical conductivity using the pH conductivity meter instrument (Seven Compact pH meter S220, Metler Toledo, USA). Heavy metals such as arsenic, cadmium, chromium, copper, zinc, nickel, silver, and total iron were estimated per the standard methods mentioned in Table . The analysis of the water samples was done using atomic absorption spectroscopy (Thermo Scientific Model-AA301, USA). The presence of cation and anions calcium, sodium, flouride, and sulphate was examined following the standard method mentioned in Table . The detailed evaluation of coal in terms of proximate and ultimate analysis, such as ash, moisture, volatile matter, and fixed carbon, along with the specific carbon, hydrogen, nitrogen, sulphur, and oxygen content, was estimated as per the standard guidelines Table . The calorific value of the coal sample was analyzed using a bomb calorimeter (Ferrotek equipment FE-255A, Ghaziabad, India). DNA extraction from the formation water and amplicon sequencing In order to assess the native microbial diversity of the formation water of CBM wells, genomic DNA extraction was performed. The formation water from each well was collected in triplicate for the extraction of microbial DNA. For this, the triplicate water samples from each well were pooled together, and then 500 ml of pooled water sample was passed through a membrane filter (90 mm, 0.22 μm). The formation water contained suspended coal particles. To avoid losing those microorganisms attached to coal particles, all that was retained on the filter was then processed for DNA extraction using DNeasy PowerWater Kit (Qiagen, Germany). DNA was extracted as per the manufacturer's protocol. Following extraction, DNA samples were quantified and evaluated. The extracted DNA sample with good quality (A260/A280: 1.8–2.0) and concentrations (more than 50 ng/μl) was taken up further for sequencing . Polymerase chain reaction (PCR) amplification was done using primers for 16S rRNA (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT, targeting the V3-V4 region) . Amplicon libraries were prepared with high-quality DNA and Nextera Index Kit 16S metagenomic sequencing library preparation protocol. Libraries were sequenced using the Illumina MiSeq platform with 2*300 base-pair chemistry at Medgenome Pvt. Ltd., Bangalore, India. Sequence pre-processing, OTU picking, and downstream processing FASTQC tool v 0.11.8 (Babraham bioinformatics) verified the quality of sequences after demultiplexing and adaptor/primer/ barcodes sequence removal from raw reads. Merging of paired-end reads of each sample was carried out using FLASH v 1.2.11 software . Quantitative Insights Into Microbial Ecology, QIIME 2 standard protocol was followed . For sequence quality control, the denoise_paired action in the dada2 plugin was performed. This did quality filtering, chimera checking, and paired-end read joining. Operational Taxonomic Units (OTU) picking was done against SILVA db version 138 database , . Cumulative sum scaling , low variance, and low count filtering were carried out before downstream analysis. These pre-processing steps enabled the avoidance of sequencing depth bias for better comparative analysis. Metagenomic analysis-culture-independent approach Comparative analysis of microbiomes was done at alpha and beta diversity levels. At the alpha level, the analysis was done using the Chao diversity index. The pie charts were generated using Microbiome Analyst software . Core Microbiome and Random Forest analysis in the Microbiome Analysts platform were used to identify signature microbes in the river systems . Canonical Correspondence analysis (CCA) was conducted to determine the relationship between physicochemical factors and normalized abundances of major taxonomic groups (genus) using PAST v4.03 software . The formation water and coal samples were collected from CBM wells, namely Well01, Well02, Well15, Well16, Well27, Well29, and Well47. The well belongs to the Bokaro coal bed methane block of Oil and Natural Gas Corp. in eastern India. The location and grouping details of the samples are shown in Table and Fig. . The Bokaro Block, extending over 95 km 2 , consists of three disconnected sectors. Central Patch (Patch-A covering 66 km 2 area), Western Patch (Patch B covering 18 km 2 area), and Eastern Patch (Patch-C covering 11 km 2 area). The Central Patch comprises three distinct geographic areas viz. (i) Saram-Gumia in the adjoining eastern part of the Lugu hill, (ii) Jarangdih Deep beyond the Saram-Gumia area in the further east separated by the Bokaro River, and (iii) Daniya to the west of Lugu hill. The western patch lies in the Hesagara-Kasikhap area, whereas the Eastern Patch lies in the Chalkari-Phusro area. The Bokaro Coalfield forms a part of the Damodar Valley coalfields and is situated in the Bokaro and Hazaribagh districts of Jharkhand. This coalfield is one of the country's major producers of medium-cooking blendable coals. Lugu Hill, a major topographic high of the area, divides the coalfield into two parts. East Bokaro Coalfield and West Bokaro Coalfield. The Damodar River flows along the southern margin of East Bokaro Coalfield, and the Bokaro River forms the main drainage of West Bokaro Coalfield. The samples are grouped according to the wells' annual gas production. Wells with gas production > 500 m 3 /d belong to the Producer groups, and the wells with production < 500 m 3 /d belong to the Marginal group. The sampling of all the wells was done in April 2022. The sample collection device consisted of a 10 cm diameter, 1 m long polyvinyl chloride pipe sealed at the bottom and capped with an open steel mesh at the top. The pipe and mesh enclosure were sterilized with a bleach solution before being lowered to the well's bottom on a wireline . Samples were collected in triplicate in 1000 ml of anaerobic sterilized serum bottles and analyzed for pH and conductivity on site. All the samples were transported to the laboratory within 48 h at 4 °C and processed immediately for activity measurement and microbial analysis . The indigenous coal samples of a CBM well were obtained from the core house and transferred in sterile, airtight containers. Physio-chemical analysis of the formation water samples from the Bokaro CBM well was performed for pH, Total dissolved solids (TDS), Total suspended solids (TSS), and Electrical conductivity using the pH conductivity meter instrument (Seven Compact pH meter S220, Metler Toledo, USA). Heavy metals such as arsenic, cadmium, chromium, copper, zinc, nickel, silver, and total iron were estimated per the standard methods mentioned in Table . The analysis of the water samples was done using atomic absorption spectroscopy (Thermo Scientific Model-AA301, USA). The presence of cation and anions calcium, sodium, flouride, and sulphate was examined following the standard method mentioned in Table . The detailed evaluation of coal in terms of proximate and ultimate analysis, such as ash, moisture, volatile matter, and fixed carbon, along with the specific carbon, hydrogen, nitrogen, sulphur, and oxygen content, was estimated as per the standard guidelines Table . The calorific value of the coal sample was analyzed using a bomb calorimeter (Ferrotek equipment FE-255A, Ghaziabad, India). In order to assess the native microbial diversity of the formation water of CBM wells, genomic DNA extraction was performed. The formation water from each well was collected in triplicate for the extraction of microbial DNA. For this, the triplicate water samples from each well were pooled together, and then 500 ml of pooled water sample was passed through a membrane filter (90 mm, 0.22 μm). The formation water contained suspended coal particles. To avoid losing those microorganisms attached to coal particles, all that was retained on the filter was then processed for DNA extraction using DNeasy PowerWater Kit (Qiagen, Germany). DNA was extracted as per the manufacturer's protocol. Following extraction, DNA samples were quantified and evaluated. The extracted DNA sample with good quality (A260/A280: 1.8–2.0) and concentrations (more than 50 ng/μl) was taken up further for sequencing . Polymerase chain reaction (PCR) amplification was done using primers for 16S rRNA (338F: ACTCCTACGGGAGGCAGCA, 806R: GGACTACHVGGGTWTCTAAT, targeting the V3-V4 region) . Amplicon libraries were prepared with high-quality DNA and Nextera Index Kit 16S metagenomic sequencing library preparation protocol. Libraries were sequenced using the Illumina MiSeq platform with 2*300 base-pair chemistry at Medgenome Pvt. Ltd., Bangalore, India. FASTQC tool v 0.11.8 (Babraham bioinformatics) verified the quality of sequences after demultiplexing and adaptor/primer/ barcodes sequence removal from raw reads. Merging of paired-end reads of each sample was carried out using FLASH v 1.2.11 software . Quantitative Insights Into Microbial Ecology, QIIME 2 standard protocol was followed . For sequence quality control, the denoise_paired action in the dada2 plugin was performed. This did quality filtering, chimera checking, and paired-end read joining. Operational Taxonomic Units (OTU) picking was done against SILVA db version 138 database , . Cumulative sum scaling , low variance, and low count filtering were carried out before downstream analysis. These pre-processing steps enabled the avoidance of sequencing depth bias for better comparative analysis. Comparative analysis of microbiomes was done at alpha and beta diversity levels. At the alpha level, the analysis was done using the Chao diversity index. The pie charts were generated using Microbiome Analyst software . Core Microbiome and Random Forest analysis in the Microbiome Analysts platform were used to identify signature microbes in the river systems . Canonical Correspondence analysis (CCA) was conducted to determine the relationship between physicochemical factors and normalized abundances of major taxonomic groups (genus) using PAST v4.03 software . Physico-chemical analysis of formation water and coal A physio-chemical analysis of the formation water collected for this study is shown in Table . The pH ranges from 7.14 to 7.87. The TDS of the formation water ranges from 320 to 4438 mg/l. Also, sulphate, chloride, fluoride, and iron were found (Table ). The physicochemical analysis shows differences in various parameters in the formation water of two groups. The analysis shows high sodium, TDS, and conductivity in the Producer wells. Previous studies have shown that high TDS, salinity, and conductivity are indicative of high coal bed methane in the wells – . The gas phase desorption of methane can be accelerated by salinity, or the TDS in the formation water, as it can compete with methane for coal surface adsorption sites . The concentration of sodium was found to be significantly higher in the Producer wells. This finding relates to another study that reported high sodium concentrations in the formation water of CBM wells . Research reveals that high sodium and low calcium, magnesium, and sulfate concentrations in groundwater typically point to a high CBM enrichment potential, which is favorable for high production outcomes . High sodium and TDS in the Producing wells may contribute to high CBM production in the wells. Further, most of the heavy metals in the samples were found below the detection limit or in very low concentrations, suggesting ambient conditions for the growth of the microbes. The proximate and ultimate analysis of coal samples of the seams from the Bokaro CBM wells was carried out (Table ). The coal contains low moisture (0.07–0.24%), medium ash (22.35–36.3%) with 21.08–23.04% volatile matter, and 40.59–56.27% fixed carbon. Ultimate analysis results indicated low sulfur content, carbon ranging from 53.19 to 67.12%, and hydrogen at 3.08–4.09% (Table ). The calorific value of the coal sample was found to be 5006–6387 kcal/kg. The analysis of all the coal samples indicates that the ASTM rank of the coal is high volatile 'A' bituminous (HVAB). The analysis showed the same category of coal in all the selected wells. General statistics for 16S rRNA sequencing and microbial diversity analysis in the producer wells and marginal wells After pre-processing and quality filtering, the resulting library size included a total of 2,435,998 sequences. A total library size of 274,498 sequences was further utilized for analysis after OTU picking. The average library size of samples was 39,214, which was further rarefied to the minimum library size. The rarefaction curve based on the observed OTU shows that the produced sequence adequately represents the present microbial communities in the wells. The rarefaction curve also depicts higher species richness in Well 47 in Producing wells, while in the Marginal wells, the highest species richness was found in Well 27 (Figure S1). The amplicon sequencing reveals the abundance of the diverse microbiome in the formation water of Producers and Marginal wells. At the domain level, both bacterial and archaeal groups were present (Fig. a). The Producer wells show an abundance of Bacteria —at 99% and Archaea at 1%, whereas the Marginal wells show an abundance of Bacteria —at 97.4% and Archaea at 2.6%. The archaeal groups comprise members of the class Methanobacteria belonging to the Euryarchaeota phylum, suggesting hydrogenotrophic as the primary pathway for generating biogenic methane in the wells . According to the sequence read classification, the relative abundance analysis shows the variance in the abundance of dominant phyla in the two groups (Fig. b). The dominant phyla in the Producer group were Proteobacteria ( recently named Pseudomonadota), Epsilonbacteraeota, Firmicutes ( recently named Bacillota), Dictyoglomi ( recently named Dictyoglomerota), and Spirochaetes ( recently named Spirochaetota) . In contrast, the dominant members in the Marginal groups were Proteobacteria, Actinobacteria ( recently named Actinomycetota), Armanimondetes, Bacteroidetes ( recently named Bacteroidota ), and Spirochaetes . Phyla Proteobacteria, Firmicutes , and Bacteroidetes are the commonly identified bacterial phyla in the anaerobic digestive systems . At the class level, Proteobacteria consists of Gammaproteobacteria, Alphaproteobacteria , and Deltaproteobacteria (Fig. a). Studies have shown Gammaproteobacteria's important role in mediating the utilization of methyl-, sulfur- and petroleum organic compounds in deep ocean hydrothermal plumes . Members of Deltaproteobacteria have been studied for their hydrocarbon-degrading capabilities in anaerobic conditions . Figure b shows the abundance of phylum Firmicutes in Producer wells. This phylum comprises members of the class Clostridia, Erysipelotrichia, and BRH_c20a . Members of the class Clostridia are known to be involved in hydrogen-producing mechanisms , . Hydrogen acts as the limiting factor in the hydrogenotrophic methanogenesis pathway; therefore, the presence of these members may immensely impact the gas production in the wells . Members of class Dictyoglomus are abundantly present in producing wells. Dictyoglomus are extremely thermophilic, chemoorganotrophic, and obligate anaerobes . Members of class Spirochaetia are involved in syntrophic acetate oxidation in anaerobic methanogenesis . These variations in microbial diversity and other parameters may contribute to the difference in biogenic methane production. Previous studies have shown higher coal methanogenesis in samples with a high abundance of Firmicutes. Thus suggesting that coal methanogenesis is unlikely to be limited by methanogen biomass but rather by the activation and degradation of coal constituents . The phylum Actinobacteria has been recently reported for their significance as purportedly resistant organic matter decomposers, particularly lignocellulose, and consequently, their capacity to aid in the creation of bio-based goods (energy and materials) while lowering carbon emissions has been taken into account . The archaeal phyla Euryarchaeota mainly consists of class Methanobacteria , thus showing hydrogenotrophic production of methane as the prominent way of methanogenesis. This group of methanogens utilizes hydrogen as an electron donor to reduce carbon dioxide to methane . The differential abundance of microbes is further supported by the heatmap analysis at the genus level (Fig. b). The red line marks the higher abundance of genus members in the Producing wells. Genus Youngiibacter belongs to Firmicutes phylum, abundant in Producer wells, and is a newly described genus of the family Clostridiaceae . It's a strictly anaerobic bacteria that ferments a range of carbohydrates to ethanol, formate, acetate, and CO 2 . Previous studies have reported Rhizobium in coal and formation water samples , . Studies have also shown that Rhizobium members have phenol and trichloroethene degrading capabilities . The members of the genus Sulfurospirillum, Proteiniclasticum , produce hydrogen and acetate as bi-products of their metabolism , , thus responsible for methane production enhancement. Genus Soehngenia, Proteiniclasticum , and Gelria belong to the class Clostridia . Soenhgenia is a fermenting bacterium often isolated from petroleum reservoirs . Gelria is an anaerobic thermophilic bacteria obligatory syntrophic bacteria; few members are often isolated from was isolated from a propionate-oxidizing methanogenic enrichment culture . Genus JGI_0000079_D21, abundant in Producer wells, is associated with the degradation of phenols and N-heterocyclic compounds in anaerobic digestion . Members of Coprothermobacter grow in a protein-rich environment and are associated with hydrogen and methane production. They also have syntrophic relationships with hydrogenotrophic methanogenic archaea . Methanothermobacter in the Producing wells shows the presence of thermophilic methanogens, thus carrying methanogenesis at higher temperatures . The analysis also reveals the abundance of Methyloversatilis, Methylococcus, Methylocystis, and Methylobacterium in the Marginal samples. These members use methane as a substrate for their metabolism . Their abundance in the Marginal group might significantly contribute to the lesser methane production in the wells. Genus Hydrogenphaga are abundant in Marginal wells and are hydrogen-oxidizing bacteria . Therefore, their abundance may limit the availability of hydrogen to methanogens. A previous study has also reported their presence in CBM wells. However, their function in the wells needs more exploration . The heatmap analysis also reveals variation in the relative abundance of microbial groups among the wells of the Producer group. Geographical variations, location, nutrient availability, and other factors impact the relative abundance of microbes in the formation water of coal beds , . The analysis also revealed variations in the abundance of methanogens ( Methanobacterium and Methanothermobacter ) among the wells of the Producer group. The variation in methanogen abundance demonstrates how, in addition to methanogen abundance, various other factors also control CBM production. Therefore, CBM production in the wells is unlikely to be limited by methanogenic mass . Alpha and Beta Diversity Analysis The alpha level diversity shows the highest abundance and richness in Well#47 in the Producer wells, and in the case of Marginal wells, the highest abundance and richness was found in Well#27 (Figure S2). Beta diversity projection on the PCoA plot revealed significantly different communities between the Producers and Marginal wells at the genus level (Fig. , ANOSIM, p-value < 0.05). The significant variance supports the existence of diverse microbes in both wells. This signifies microbial communities' role in producing methane from the well. The distinctiveness in the microbial diversity in the two groups is also supported by the dendrogram analysis, which shows the samples of the two groups belong to two different clusters (Figure S3). Previous studies have demonstrated distinct microbial communities residing in dissimilar hydrological areas of southern Qinshui Basin coal reservoirs . Another study revealed seasonal variation in the microbial communities in the Erlian basin, China . Differentially abundant taxa in the producer and the marginal wells The differences between the microbial communities can be understood by analysis of the differentiating members of the core microbiome. A core microbiome represents those genomes or genetic markers common to all the samples studied and is critical to the genetic functions and composition of the microbial communities . Therefore, it is crucial to identify the community structures and ecological processes of the core microbiome in the CBM wells (Fig. a, b). At the class level, Gammaproteobacteria, Campylobacteria, Alphaproteobacteria, Clostridia, Spirochaetia, and Methanobacteria were present in the Producer well groups. In contrast, the Marginal group shows class Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Fimbriimonadia, and Spirochaetia . Microbes belonging to these classes are widely reported in Indian coal bed wells , . The analysis of the core microbiome suggests the presence of hydrogen-producing ( Clostridia ), syntrophic acetate oxidative microbes ( Spirochaetia ) , and hydrogenotrophic methanogens in all the samples of the Producer groups. Research has shown that syntrophic acetate oxidative microbes play a key role in hydrogenotrophic methanogenesis . The analysis shows the importance of synergy among these microbes and their critical role in the process of methanogenesis in the CBM wells. Random forest analysis shows a significant association of microbes with the Producing and the Marginal wells (Fig. c) . Gelria, Methanothermobacter, Thaurea, Proteiniclasticum, Youngiibacter, JGI_0000079_D21, Coprothermobacter, and Rhizobium were significantly associated with Producing wells. The Marginal wells were found to be significantly associated with the genera Roseomonas, Rhodobacter, Mycobacterium, Methylobacterium, Bosea, Bradyrhizobium, and Limnobacter. The role of Rhodobacter and Roseomonas member s is studied in modulating the anaerobic digestion of different substrates , . Members of Methylobacterium use methane as a substrate for their metabolism . Random forest analysis further strengthens the relationship between microbial members belonging to Clostridia and Methanothermobacter (methanogens) members with the Producer group. These findings corroborate the previous studies, which show an abundance of these microbial groups in methane-producing systems . Relationship between microbial community and physicochemical parameters of wells The relatedness among the microbial communities and the physicochemical parameters was done using CCA analysis. The CCA analysis at the genus level supports the microbial and physicochemical distinctiveness between the Producer and Marginal wells (Fig. ). Axis 1 and Axis 2 account for 63.61% of the total variance. Physicochemical parameters like sodium, conductivity, TDS, fluoride, sulfate, and chloride were found to be related to Fusibacter, Proteiniclasticum, Sulfospirillum, Youngiibacter, Coprothermobacter , Magnetospirillum . Most of these bacteria are known for their hydrocarbon-degrading capabilities. Salts can modulate the hydrocarbon-degrading ability of microbes; recent findings stated higher expression of hydrocarbon-degradation-related genes with salt addition in slurry bioreactors . A recent study has shown the relation between high bulk conductivities and TDS with enhanced mineral weathering, interlinked with the activities of hydrocarbon-degrading microbes in aquifers contaminated with hydrocarbons . The strong relationship between chromium and bacteria suggests its importance in microbial metabolism in the ecosystem. Research has shown the role of trace metals, including chromium, in predicting methanogenic community structure and moderate concentrations of trace metals, which are essential for microbial functioning . The CCA plot also reveals relatedness among members of Methanothermobacter, Dictyoglomus, Syntrophothermus, Gelria, Rhizobium, Acetothermia, Azospira, and Desulfovibrio . This indicates the presence of microbial syntrophy, which is essential for the process of methanogenesis . The CCA analysis shows an association of zinc, manganese, magnesium, copper, iron, and pH with microbial genera abundant in the Marginal wells. The analysis also revealed the relatedness among the genera Methylobacterium, Rhodobacter, Reyranella, Methylococcus, Methylocystis, Hydrogenophaga, and others. Copper and iron are widely studied to play a role in the methane oxidation mechanism of methanotrophic bacteria . The analysis shows the crucial role of physicochemical parameters of the formation water in defining the microbial composition of the wells. The findings of the study showed distinct microbial communities residing in the Producer and Marginal wells of the Bokaro region. Further, it emphasizes the role of physicochemical parameters of the formation water in framing distinct microbiomes. The results of the present study also reflect a higher diversity of degrading syntrophic, hydrogen-producing, and fermentative microbes in the Producer wells. Studies have supported the crucial role of these microbes in the process of methanogenesis , . Further, the results indicate a high abundance of various methylotrophs, which may be one of the significant parameters for decreased methane production in the wells. Previously, methylotrophs have been studied to significantly decrease methane production in different ecosystems , . Further exploration on methanotrophs is required to fully understand their role in the CBM production. A physio-chemical analysis of the formation water collected for this study is shown in Table . The pH ranges from 7.14 to 7.87. The TDS of the formation water ranges from 320 to 4438 mg/l. Also, sulphate, chloride, fluoride, and iron were found (Table ). The physicochemical analysis shows differences in various parameters in the formation water of two groups. The analysis shows high sodium, TDS, and conductivity in the Producer wells. Previous studies have shown that high TDS, salinity, and conductivity are indicative of high coal bed methane in the wells – . The gas phase desorption of methane can be accelerated by salinity, or the TDS in the formation water, as it can compete with methane for coal surface adsorption sites . The concentration of sodium was found to be significantly higher in the Producer wells. This finding relates to another study that reported high sodium concentrations in the formation water of CBM wells . Research reveals that high sodium and low calcium, magnesium, and sulfate concentrations in groundwater typically point to a high CBM enrichment potential, which is favorable for high production outcomes . High sodium and TDS in the Producing wells may contribute to high CBM production in the wells. Further, most of the heavy metals in the samples were found below the detection limit or in very low concentrations, suggesting ambient conditions for the growth of the microbes. The proximate and ultimate analysis of coal samples of the seams from the Bokaro CBM wells was carried out (Table ). The coal contains low moisture (0.07–0.24%), medium ash (22.35–36.3%) with 21.08–23.04% volatile matter, and 40.59–56.27% fixed carbon. Ultimate analysis results indicated low sulfur content, carbon ranging from 53.19 to 67.12%, and hydrogen at 3.08–4.09% (Table ). The calorific value of the coal sample was found to be 5006–6387 kcal/kg. The analysis of all the coal samples indicates that the ASTM rank of the coal is high volatile 'A' bituminous (HVAB). The analysis showed the same category of coal in all the selected wells. After pre-processing and quality filtering, the resulting library size included a total of 2,435,998 sequences. A total library size of 274,498 sequences was further utilized for analysis after OTU picking. The average library size of samples was 39,214, which was further rarefied to the minimum library size. The rarefaction curve based on the observed OTU shows that the produced sequence adequately represents the present microbial communities in the wells. The rarefaction curve also depicts higher species richness in Well 47 in Producing wells, while in the Marginal wells, the highest species richness was found in Well 27 (Figure S1). The amplicon sequencing reveals the abundance of the diverse microbiome in the formation water of Producers and Marginal wells. At the domain level, both bacterial and archaeal groups were present (Fig. a). The Producer wells show an abundance of Bacteria —at 99% and Archaea at 1%, whereas the Marginal wells show an abundance of Bacteria —at 97.4% and Archaea at 2.6%. The archaeal groups comprise members of the class Methanobacteria belonging to the Euryarchaeota phylum, suggesting hydrogenotrophic as the primary pathway for generating biogenic methane in the wells . According to the sequence read classification, the relative abundance analysis shows the variance in the abundance of dominant phyla in the two groups (Fig. b). The dominant phyla in the Producer group were Proteobacteria ( recently named Pseudomonadota), Epsilonbacteraeota, Firmicutes ( recently named Bacillota), Dictyoglomi ( recently named Dictyoglomerota), and Spirochaetes ( recently named Spirochaetota) . In contrast, the dominant members in the Marginal groups were Proteobacteria, Actinobacteria ( recently named Actinomycetota), Armanimondetes, Bacteroidetes ( recently named Bacteroidota ), and Spirochaetes . Phyla Proteobacteria, Firmicutes , and Bacteroidetes are the commonly identified bacterial phyla in the anaerobic digestive systems . At the class level, Proteobacteria consists of Gammaproteobacteria, Alphaproteobacteria , and Deltaproteobacteria (Fig. a). Studies have shown Gammaproteobacteria's important role in mediating the utilization of methyl-, sulfur- and petroleum organic compounds in deep ocean hydrothermal plumes . Members of Deltaproteobacteria have been studied for their hydrocarbon-degrading capabilities in anaerobic conditions . Figure b shows the abundance of phylum Firmicutes in Producer wells. This phylum comprises members of the class Clostridia, Erysipelotrichia, and BRH_c20a . Members of the class Clostridia are known to be involved in hydrogen-producing mechanisms , . Hydrogen acts as the limiting factor in the hydrogenotrophic methanogenesis pathway; therefore, the presence of these members may immensely impact the gas production in the wells . Members of class Dictyoglomus are abundantly present in producing wells. Dictyoglomus are extremely thermophilic, chemoorganotrophic, and obligate anaerobes . Members of class Spirochaetia are involved in syntrophic acetate oxidation in anaerobic methanogenesis . These variations in microbial diversity and other parameters may contribute to the difference in biogenic methane production. Previous studies have shown higher coal methanogenesis in samples with a high abundance of Firmicutes. Thus suggesting that coal methanogenesis is unlikely to be limited by methanogen biomass but rather by the activation and degradation of coal constituents . The phylum Actinobacteria has been recently reported for their significance as purportedly resistant organic matter decomposers, particularly lignocellulose, and consequently, their capacity to aid in the creation of bio-based goods (energy and materials) while lowering carbon emissions has been taken into account . The archaeal phyla Euryarchaeota mainly consists of class Methanobacteria , thus showing hydrogenotrophic production of methane as the prominent way of methanogenesis. This group of methanogens utilizes hydrogen as an electron donor to reduce carbon dioxide to methane . The differential abundance of microbes is further supported by the heatmap analysis at the genus level (Fig. b). The red line marks the higher abundance of genus members in the Producing wells. Genus Youngiibacter belongs to Firmicutes phylum, abundant in Producer wells, and is a newly described genus of the family Clostridiaceae . It's a strictly anaerobic bacteria that ferments a range of carbohydrates to ethanol, formate, acetate, and CO 2 . Previous studies have reported Rhizobium in coal and formation water samples , . Studies have also shown that Rhizobium members have phenol and trichloroethene degrading capabilities . The members of the genus Sulfurospirillum, Proteiniclasticum , produce hydrogen and acetate as bi-products of their metabolism , , thus responsible for methane production enhancement. Genus Soehngenia, Proteiniclasticum , and Gelria belong to the class Clostridia . Soenhgenia is a fermenting bacterium often isolated from petroleum reservoirs . Gelria is an anaerobic thermophilic bacteria obligatory syntrophic bacteria; few members are often isolated from was isolated from a propionate-oxidizing methanogenic enrichment culture . Genus JGI_0000079_D21, abundant in Producer wells, is associated with the degradation of phenols and N-heterocyclic compounds in anaerobic digestion . Members of Coprothermobacter grow in a protein-rich environment and are associated with hydrogen and methane production. They also have syntrophic relationships with hydrogenotrophic methanogenic archaea . Methanothermobacter in the Producing wells shows the presence of thermophilic methanogens, thus carrying methanogenesis at higher temperatures . The analysis also reveals the abundance of Methyloversatilis, Methylococcus, Methylocystis, and Methylobacterium in the Marginal samples. These members use methane as a substrate for their metabolism . Their abundance in the Marginal group might significantly contribute to the lesser methane production in the wells. Genus Hydrogenphaga are abundant in Marginal wells and are hydrogen-oxidizing bacteria . Therefore, their abundance may limit the availability of hydrogen to methanogens. A previous study has also reported their presence in CBM wells. However, their function in the wells needs more exploration . The heatmap analysis also reveals variation in the relative abundance of microbial groups among the wells of the Producer group. Geographical variations, location, nutrient availability, and other factors impact the relative abundance of microbes in the formation water of coal beds , . The analysis also revealed variations in the abundance of methanogens ( Methanobacterium and Methanothermobacter ) among the wells of the Producer group. The variation in methanogen abundance demonstrates how, in addition to methanogen abundance, various other factors also control CBM production. Therefore, CBM production in the wells is unlikely to be limited by methanogenic mass . The alpha level diversity shows the highest abundance and richness in Well#47 in the Producer wells, and in the case of Marginal wells, the highest abundance and richness was found in Well#27 (Figure S2). Beta diversity projection on the PCoA plot revealed significantly different communities between the Producers and Marginal wells at the genus level (Fig. , ANOSIM, p-value < 0.05). The significant variance supports the existence of diverse microbes in both wells. This signifies microbial communities' role in producing methane from the well. The distinctiveness in the microbial diversity in the two groups is also supported by the dendrogram analysis, which shows the samples of the two groups belong to two different clusters (Figure S3). Previous studies have demonstrated distinct microbial communities residing in dissimilar hydrological areas of southern Qinshui Basin coal reservoirs . Another study revealed seasonal variation in the microbial communities in the Erlian basin, China . The differences between the microbial communities can be understood by analysis of the differentiating members of the core microbiome. A core microbiome represents those genomes or genetic markers common to all the samples studied and is critical to the genetic functions and composition of the microbial communities . Therefore, it is crucial to identify the community structures and ecological processes of the core microbiome in the CBM wells (Fig. a, b). At the class level, Gammaproteobacteria, Campylobacteria, Alphaproteobacteria, Clostridia, Spirochaetia, and Methanobacteria were present in the Producer well groups. In contrast, the Marginal group shows class Gammaproteobacteria, Alphaproteobacteria, Actinobacteria, Fimbriimonadia, and Spirochaetia . Microbes belonging to these classes are widely reported in Indian coal bed wells , . The analysis of the core microbiome suggests the presence of hydrogen-producing ( Clostridia ), syntrophic acetate oxidative microbes ( Spirochaetia ) , and hydrogenotrophic methanogens in all the samples of the Producer groups. Research has shown that syntrophic acetate oxidative microbes play a key role in hydrogenotrophic methanogenesis . The analysis shows the importance of synergy among these microbes and their critical role in the process of methanogenesis in the CBM wells. Random forest analysis shows a significant association of microbes with the Producing and the Marginal wells (Fig. c) . Gelria, Methanothermobacter, Thaurea, Proteiniclasticum, Youngiibacter, JGI_0000079_D21, Coprothermobacter, and Rhizobium were significantly associated with Producing wells. The Marginal wells were found to be significantly associated with the genera Roseomonas, Rhodobacter, Mycobacterium, Methylobacterium, Bosea, Bradyrhizobium, and Limnobacter. The role of Rhodobacter and Roseomonas member s is studied in modulating the anaerobic digestion of different substrates , . Members of Methylobacterium use methane as a substrate for their metabolism . Random forest analysis further strengthens the relationship between microbial members belonging to Clostridia and Methanothermobacter (methanogens) members with the Producer group. These findings corroborate the previous studies, which show an abundance of these microbial groups in methane-producing systems . The relatedness among the microbial communities and the physicochemical parameters was done using CCA analysis. The CCA analysis at the genus level supports the microbial and physicochemical distinctiveness between the Producer and Marginal wells (Fig. ). Axis 1 and Axis 2 account for 63.61% of the total variance. Physicochemical parameters like sodium, conductivity, TDS, fluoride, sulfate, and chloride were found to be related to Fusibacter, Proteiniclasticum, Sulfospirillum, Youngiibacter, Coprothermobacter , Magnetospirillum . Most of these bacteria are known for their hydrocarbon-degrading capabilities. Salts can modulate the hydrocarbon-degrading ability of microbes; recent findings stated higher expression of hydrocarbon-degradation-related genes with salt addition in slurry bioreactors . A recent study has shown the relation between high bulk conductivities and TDS with enhanced mineral weathering, interlinked with the activities of hydrocarbon-degrading microbes in aquifers contaminated with hydrocarbons . The strong relationship between chromium and bacteria suggests its importance in microbial metabolism in the ecosystem. Research has shown the role of trace metals, including chromium, in predicting methanogenic community structure and moderate concentrations of trace metals, which are essential for microbial functioning . The CCA plot also reveals relatedness among members of Methanothermobacter, Dictyoglomus, Syntrophothermus, Gelria, Rhizobium, Acetothermia, Azospira, and Desulfovibrio . This indicates the presence of microbial syntrophy, which is essential for the process of methanogenesis . The CCA analysis shows an association of zinc, manganese, magnesium, copper, iron, and pH with microbial genera abundant in the Marginal wells. The analysis also revealed the relatedness among the genera Methylobacterium, Rhodobacter, Reyranella, Methylococcus, Methylocystis, Hydrogenophaga, and others. Copper and iron are widely studied to play a role in the methane oxidation mechanism of methanotrophic bacteria . The analysis shows the crucial role of physicochemical parameters of the formation water in defining the microbial composition of the wells. The findings of the study showed distinct microbial communities residing in the Producer and Marginal wells of the Bokaro region. Further, it emphasizes the role of physicochemical parameters of the formation water in framing distinct microbiomes. The results of the present study also reflect a higher diversity of degrading syntrophic, hydrogen-producing, and fermentative microbes in the Producer wells. Studies have supported the crucial role of these microbes in the process of methanogenesis , . Further, the results indicate a high abundance of various methylotrophs, which may be one of the significant parameters for decreased methane production in the wells. Previously, methylotrophs have been studied to significantly decrease methane production in different ecosystems , . Further exploration on methanotrophs is required to fully understand their role in the CBM production. The enhancement of microbial-produced coal bed methane from marginal wells carries great environmental and economic benefits. Various factors like geography, coal rank, hydrology, and microbial composition influence the coal bed methane production. The present study revealed how microbial composition and environmental factors can significantly impact methane gas production in different CBM wells within a reservoir. The findings indicate a high abundance of methane-oxidizing bacteria in the Marginal wells, while Producer wells show a high abundance of hydrogen-producing and hydrocarbon-degrading microbes. A deeper knowledge of the microbial composition in CBM wells will help better strategize improvement in gas production in the marginal wells. The findings will help design optimized biostimulation and bioaugmentation jobs in the Marginal wells that can improve their CBM production efficiency. However, further investigation utilizing transcriptomics and proteomics analysis is required for a profound understanding of microbial metabolisms in CBM wells. Supplementary Information. |
Challenges to | 41c1200e-514c-4fe9-b3d1-d8f39e9a2623 | 10779078 | Pathology[mh] | Microbial plant pathogens, which include fungi, bacteria, phytoplasmas, viruses, and viroids, affect a broad range of plant species worldwide, causing significant losses in the growth and quality of both food and nonfood plants. Effective management of these pathogens requires a timely diagnosis to confirm their involvement in any disease symptoms, which frequently can be problematic where plants fail to show classic visible symptoms, yet show a decline in growth and productivity over time due to underlying infections. Accurate diagnosis in most cases has relied on the utility of molecular diagnostic approaches, most of which are based on the polymerase chain reaction (PCR). In 2018, the approval of the Farm Bill in the USA that allowed the production of hemp (containing <0.3% THC), and the legalization of cannabis (marijuana) (representing high-THC genotypes) in Canada for medicinal and recreational purposes, sparked interest in expanding the commercial production of these plants, both designated as Cannabis sativa L. and members of the Cannabaceae family. To date, more than 100 pathogens/diseases have been identified to cause problems on these two crops , the majority of which are fungi and oomycetes, followed by viruses and viroids. In this study, we illustrate how a range of PCR-based diagnostic approaches can be used to identify the pathogens of importance affecting cannabis and hemp in North America. We additionally have applied methods in whole-genome sequencing and bioinformatics to better understand the diversity and impact of specific pathogen groups affecting these plants. The diseases and pathogens which are currently of most significant economic concern for cannabis and hemp producers include fusarium root rot ( Fusarium oxysporum ), pythium root rot ( Pythium myriotylum ), powdery mildew ( Golovinomyces ambrosiae ), botrytis bud rot ( Botrytis cinerea ), hop latent viroid, and beet curly top virus . These six pathogens were included in this study as examples of how molecular diagnostic approaches can provide rapid identification and can be used to understand pathogen dynamics and spread from an epidemiological perspective. In addition, a more detailed investigation on the distribution and levels of hop latent viroid (HLVd) within cannabis plant tissues was conducted using several different molecular methods, and the results are presented. The results presented demonstrate how whole-genome sequencing (NGS) approaches applied to cannabis and hemp plants have revealed insights into the virome of these two crops and illustrate the complex group of viruses and a viroid that are present. Additional studies are likely to reveal additional pathogens that belong to this group. The complex of fungal/oomycete pathogens recovered from symptomatic plant tissues has been identified using PCR with a universal set of primers, and the methods are described. Additional diagnostic approaches based on RT-PCR, RT-qPCR, and ddPCR, as well as LAMP assays, have shown how these methods can be used to characterize the incidence and severity of Hop latent viroid (HLVd) affecting cannabis plants. Lastly, this study illustrates the application of molecular diagnostics and bioinformatics to characterize populations of HLVd and Beet curly top virus affecting cannabis and hemp crops. 2.1. Detection of Fungal and Oomycete Pathogens on Cannabis 2.1.1. Symptoms and Pathogen Isolation The symptoms observed on the cannabis plants that were included for analysis are shown in . They included stunting and yellowing on vegetative and flowering plants and internal stem discoloration ( a–c). In some instances, visible growth of the fungal mycelium could be seen on inflorescence tissues ( d–f). These symptoms were observed on a range of different cannabis genotypes that were cultivated during experiments conducted in 2020–2022. Following surface sterilization and plating onto potato dextrose agar containing 140 mg/L streptomycin sulfate, emerging colonies were purified through subculturing and examined for spore morphology and other features to identify them to genus and species. The appearance of the colonies of the fungi recovered from symptomatic tissues are shown in g–l. They confirm the presence of Fusarium , Pythium , and Botrytis species, which was confirmed by molecular analysis. 2.1.2. Molecular Detection by PCR The primers UN-UP18S42 and UN-LO28S576B amplified a region of the internal transcribed region (ITS1-5.8S-ITS2) of ribosomal DNA and produced bands of different molecular weight sizes when the DNA extracted from diseased and healthy leaf and inflorescence tissues was used . Sequencing of these bands confirmed the presence of Golovinomyces ambrosiae ( a) and Botrytis cinerea ( b) in the affected tissues, as well as the cannabis DNA sequence. When these primers were used with DNA extracted from pure cultures recovered from symptomatic tissues, a range of band sizes was observed, depending on the culture . Sequencing of these bands and comparison to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST confirmed the identification of various species using cut-off values >99%. The pathogens identified were Fusarium oxysporum ( g), F. proliferatum ( h), Pythium myriotylum ( i), G. ambrosiae ( j), B. cinerea ( k), and F. sporotrichiodes ( l). Two additional fungi— Trichoderma asperellum and Penicillium olsonii —recovered from diseased root and stem tissues, respectively, were also identified. 2.2. Detection of Viral Pathogens on Cannabis 2.2.1. Symptoms Leaves displaying symptoms of mosaic, mottling, and line patterns that superficially resembled those caused by viral infections were collected from greenhouse-grown cannabis plants during 2021–2022. Representative symptoms on leaf samples on several genotypes are shown in . These symptoms were consistently observed on vegetative and flowering plants of four genotypes—‘OG Kush’ (OG) ( a), ‘Headband’ (HB) ( b), ‘Golden Papaya’ (GP) ( c), and ‘Motor Breath’ (MB) ( d). The leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if viral pathogens were present. By comparison, leaves with chlorotic sectors and loss of chlorophyll characteristic of somatic mutations are shown in e. 2.2.2. PCR with Broad-Spectrum and Specific Primer Sets Several broad-spectrum degenerate primer sets were tested following the conditions described in . As well, specific primers designed to detect turnip ringspot virus, alfalfa mosaic virus (AMV), yobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) were utilized. Leaves of cannabis genotypes OG and HB, with symptoms as shown in a,b, were included in the analysis. None of the primer sets produced a band in the samples originating from cannabis leaf tissues, while the positive controls of the infected tobacco leaves showed the expected band size for these viruses. The RT-PCR results for TMV and AMV are shown in a–c. These results indicate these specific viruses were absent in these leaf cannabis tissues. 2.2.3. Transmission Electron Microscopy (TEM) Under the TEM, rod-shaped particles of TMV were observed in leaf tissues from infected positive-control tobacco plants ( d), while cannabis leaf tissues showed an absence of any particles resembling viruses in the samples examined. 2.2.4. Host Range Studies Following inoculation of nine host plant species using ground leaf extracts originating from cannabis genotypes OG and HB with mosaic and line-pattern symptoms, and monitoring plants for symptoms over a period of 3 weeks, no symptoms were observed on any of the host plants tested . 2.3. High-Throughput Sequencing (HTS) The HTS analysis was initially conducted on leaf samples originating from the genotypes OG and HB with symptoms of mosaic and line patterns ( a,b), and was then extended to an additional six cannabis genotypes (PD, Mac-1, PK, BC, CBD, G54-2). Some of these latter genotypes (PD, Mac-1) showed distinct symptoms of stunted and reduced inflorescence growth ( a,b). These symptoms were also apparent after the inflorescences were trimmed and dried ( c,d). A comparison of the fresh weights of the inflorescences from healthy (asymptomatic) and symptomatic plants of five genotypes included in the HTS analysis is shown in e. The genotypes that were the most significantly affected were PD, Mac-1, and BC, while PK and CBD did not show a significant reduction in the fresh weight of inflorescence tissues or any obvious symptoms. Following Illumina sequencing and assembly, only two confirmed pathogen sequences were detected in the genotypes HB and OG—hop latent viroid (HLVd) and Cannabis sativa mitovirus 1 (CasaMV1). The results are summarized in . The sequences of HLVd were 100% identical at the nucleotide level to each other and to those in GenBank. The sequences of CasaMV1 were 99.6% identical to the sequences found in GenBank. The sequences from this study have been deposited in GenBank (accession OQ420426 for HLVd from HB and accession OQ420425 for CasaMV1 from HB). When the HTS analysis was conducted on the leaf tissues of six additional genotypes of cannabis (PD, Mac-1, PK, BC, CBD, G54-2), the results were similar, revealing only HLVd and CasaMV1 to be present in all genotypes except for PK. 2.4. Molecular Assays for Viroid and Other Viral Pathogens 2.4.1. RNA Extractions, cDNA Preparation, and RT-PCR Hop Latent Viroid The results from the RNA extractions from the leaf tissues of seven cannabis genotypes, and a check for the RNA quality, showed that all samples yielded satisfactory RNA for conducting RT-PCR . Extracted RNA from leaf tissues was subjected to cDNA preparation and RT-PCR using primers that were developed in this study (see . Primers for the HLVd amplified bands of sizes 256 bp and 512 bp from the leaf tissues of plants of genotype PD showed symptoms of stunted growth and reduced inflorescence development . Sequencing results confirmed that both bands represented HLVd. Tissues from genotype PK did not show a band and there were no symptoms on this genotype . When the RT-PCR analysis was conducted on an additional six genotypes of cannabis, HLVd was shown to be present in PD, Mac, BC, and HB . Three of these four genotypes previously displayed symptoms of stunting and had reduced inflorescence growth, as shown in . These results suggest that HLVd infection had impacted their growth. The corresponding band size (256 bp) in genotypes G54-2 and CBD was very faint . Genotype CBD did not display any symptoms attributed to HLVd, and its growth was not affected, as shown in . The growth of genotype G54-2 was not measured. Mitovirus To assess the extent to which CasaMV1 is present in cannabis tissues, leaves from 34 genotypes originating from 20 indoor-grown plants and 14 from outdoor-grown plants were sampled and used for RNA extraction and cDNA synthesis, as described previously. The forward primer: 5′ CGGTAGGATTGCTCAGTCGG 3′ and reverse primer: 5′ CGAACATGCGGTTCATAGGC 3′ were used to amplify a region of the RNA-dependent RNA polymerase enzyme to produce a 998 bp size product. The PCR conditions were the same as those used for HLVd detection (see ). The results showed that CasaMV1 was present in 18 out of 20 cannabis genotypes grown indoors ( a) and in 10 out of 14 genotypes grown outdoors ( b). Sequence analysis indicated the bands were 100% identical to each other. These sequences showed 99.7% sequence homology to Colorado isolate MT878084 and 99.4% to BK010437.1 from Purple Kush. For an additional four cannabis genotypes, samples of roots, petioles, leaves, and flower tissues were assayed for the presence of CasaMV1, and it was found to be present in all the tissue types analyzed ( c,d). 2.4.2. Sequence Diversity and Molecular Phylogeny of Hop Latent Viroid A phylogenetic analysis of HLVd was conducted using seven sequences from hops, two from hemp, as well as four from cannabis (selected from GenBank to represent full-length sequences), and included six from the current study. The analysis showed that no significant sequence diversity was present ( a). However, one SNP was observed between sequences originating from cannabis genotypes ‘Mac’ and G54-2, where a ‘G’ was ‘A’ ( b). 2.4.3. Droplet Digital PCR Quantification of HLVd levels (titer) in the leaves of eight cannabis genotypes was conducted using ddPCR. The genotypes PD, Mac, BC, PK, CBD, G54-2, HB, and OG were the same as those used previously for the RT-PCR . The results are shown in . The highest titers (>10,000 genomes per reaction) were seen in PD, Mac, and BC, followed by PDH (healthy) and HB, and a low level (10 genomes) was observed in G54-2. Genotypes PK, CBD, and OG had no detectable levels of viroid. The genotypes PD, Mac, and BC also displayed symptoms of HLVd infection, that included stunting and reduced inflorescence growth, and a reduction in inflorescence fresh weight, as shown previously . By comparison, the genotypes PK and CBD showed no symptoms. In comparing these results to those for the same genotypes obtained by RT-PCR, no band was observed in PK and CBD, and a very faint band was seen in G54-2 . The remaining genotypes (PD, Mac, BC) produced a strong band following RT-PCR. Genotype PDH was confirmed to contain HLVd by both RT-PCR and ddPCR despite being asymptomatic. These results show a positive correlation between the results obtained by RT-PCR and ddPCR for all genotypes tested. 2.4.4. Multiplex Taqman RT-qPCR Inflorescence tissues from symptomatic plants of genotypes PD and Mac, subjected to the RT-PCR and ddPCR methods above, were also tested using a Taqman RT-qPCR method. In both samples, the C T cycle thresholds were around 18, indicating a high level of viroid titre was present in the inflorescences . 2.4.5. LAMP Assays The tissues used for this analysis were comprised of 30 leaf, 15 petiole, and 30 root samples taken from 6-week to 10-week-old stock plants representing 11 genotypes of cannabis. These samples were tested by LAMP, and showed that 9 leaf, 5 petiole, and 24 root samples tested positive for HLVd . There was no case in which a plant that tested negative in the root tissues was found to be positive in the leaves or petioles. Conversely, many samples that were positive for HLVd in the roots were found to be negative in the leaves or petioles . These results show that root samples are the most consistent tissue source for detection of HLVd by LAMP in 6-to-10-week-old plants. All genotypes tested showed the highest frequency of samples to be positive for HLVd from root tissues. 2.5. Monitoring Distribution of HLVd in Various Tissues of Cannabis Plants 2.5.1. Distribution within Stock Plants Leaf samples were obtained from various positions within the canopy of individual stock plants, as well as from roots ( a,b), and subjected to RT-PCR ( c,d) as well as ddPCR ( e). The RT-PCR results showed the presence of multiple bands in most samples tested, varying in size from 256 bp, 512 bp, to 768 bp . Sequencing of these bands confirmed they were all HLVd. In genotype G54-2, the viroid was present in the roots, bottom-canopy leaves, and at the top of the plant, but was barely detectable in the middle-canopy leaves ( c). In genotype PD, the viroid was present in the roots, bottom-canopy leaves, middle-canopy leaves, and in the top-canopy leaves ( d). Using ddPCR for the same two genotypes, HLVd was detected in the roots and bottom-canopy leaves of genotype G54-2, but not in the middle- or top-canopy leaves. In genotype PD, HLVd was present in the roots, lower-canopy leaves, and middle- and top-canopy leaves at high concentrations (>10,000 genomes) ( e). 2.5.2. Distribution of HLVd within Inflorescence Tissues To quantify the distribution of HLVd within cannabis inflorescences, symptomatic ‘Mac-1’ plants were compared with asymptomatic (healthy) plants ( a). These plants were selected based on the presence/absence of HLVd as confirmed by RT-PCR (see ). The terminal inflorescence on symptomatic and asymptomatic plants were each removed and brought back to the laboratory ( a). Using a scalpel, the fan leaves (FL) and inflorescence leaves (IL) were dissected, leaving the central stripped flower (SF) exposed ( b). A sample of foliage leaves (PL) taken from the bottom canopy of each plant was included for analysis as well. HLVd presence was confirmed using ddPCR in the small inflorescence leaves (IL), the stripped flower (SF), and in the foliage leaves (PL) of symptomatic ‘Mac’, but not in the fan leaves (FL) ( d). The highest accumulation of HLVd was seen in the stripped flowers, which are made up of clusters of pistils surrounded by the inflorescence leaves ( b). In the healthy ‘Mac’ plant, none of the inflorescence samples showed HLVd to be present, but a low level was detected in the foliage leaves by ddPCR ( d). 2.5.3. Distribution of HLVd within Cuttings from Infected Stock Plants Vegetative cuttings taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd were rooted and tested for HLVd using RT-PCR and RT-qPCR after 14 days ( a). The results consistently showed that all cuttings were infected with the viroid ( b), as shown by a band of 256 bp size observed in seven infected cuttings ( b). This was confirmed by RT-qPCR that showed that four out of seven cuttings tested originating from the infected stock plant contained the viroid at a high titre, with C T values of 15–18 ( c). 2.5.4. Detection of Pathogens in Fresh and Dried Cannabis Inflorescences Primers for the ITS1-5.8S-ITS2 region of ribosomal DNA were used to detect fungal pathogens, and primers for HLVd were used on tissue samples obtained from freshly harvested inflorescences and from dried cannabis flowers destined for commercial sale ( a). Out of 20 samples, 7 were positive for HLVd, 4 samples contained B. cinerea , 3 had F. oxysporum , and 2 had a powdery mildew ( b). RT-PCR confirmed the presence of HLVd in 7/10 dried flower samples ( c). 2.5.5. Detection of HLVd and CasaMV1 in Cannabis Seed Seeds derived from a cross made between an infected ‘Mac1′ female plant and pollen from a male plant of genotype ‘GPie’ were ground individually, and the extracted RNA was subjected to RT-PCR. The results showed that 14/16 seeds (87.5%) were positive for the presence of HLVd ( a) and 5/5 seeds tested also contained CasaMV1 ( b). A sample of 10 seeds from the same batch used in was also subjected to Taqman RT-qPCR using the methods described in . The results are presented in . They show that viroid levels, as estimated by C T values, differed significantly from one seed and the next, and ranged from 17.1 to 33.66, with a mean of 24.07. The infection rate was 70%, as three out of ten seeds did not contain detectable HLVd . 2.6. Detection of Beet Curly Top Virus in Cannabis Plants The symptoms observed on cannabis plants characteristic of infection by BCTV are shown in . The uppermost leaves on the plants were curled and twisted and deformed ( a,b). Similar symptoms were observed on plants in the field ( c). On some plants, leaf curl symptoms were very severe, causing the plants to appear malformed ( d–f). RT-PCR with BCTV-specific primers showed the presence of a 1 kb band, confirmed to be the Worland strain in the greenhouse-grown plants . 2.7. Diagnostic Approaches to Detect Viral and Viroid Pathogens Affecting Low-THC-Containing Cannabis sativa L. (hemp) 2.7.1. Next-Generation Sequencing (NGS) Symptomatic field-grown plants, from which leaves were collected for shotgun metagenomic analysis, are shown in . These samples were collected from various locations in Colorado during 2019, 2021, and 2022. Among these samples, four viruses and one viroid were identified in 2019 . In general, the number of pathogens recovered varied by sampling location, and ranged from two to five. Cannabis sativa mitovirus (CasaMV1) and beet curly top virus (BCTV) were the most commonly found in each year of sampling. HLVd was only present in certain fields sampled in 2019. Citrus yellow-vein-associated virus (CYVaV) and Tobacco streak virus (TSV) were also only detected in 2019 . In samples collected in 2021, the viruses that were found Cannabis cryptic virus (CanCV), Alfalfa mosaic virus (AMV), BCTV, and CasaMV1 . In 2022, the viruses detected were CasaMV1, BCTV, CanCV, and Tomato bushy stunt virus . 2.7.2. RT-PCR with Specific Primer Sets The majority of the viruses identified through NGS had a high percentage nucleotide identity (>90%; ). Only three of the viruses, CasaMV1, TSV, and CYVaV, were below this threshold. The presence of these low-percentage nucleotide-identity (<90%) viruses was confirmed in the respective samples with RT-PCR analysis . These results confirmed the presence of each of the three viruses in the hemp samples. 2.7.3. Sequence Diversity and Molecular Phylogeny of Beet Curly Top Virus Phylogenetic analysis based on a fragment of the BCTV coat protein (CP) sequences from the 2019 hemp samples had nucleotide identities to one another between 98.99 and 98.24%. Sequences from this study had a 97–99% sequence identity with cannabis BCTV sequences from Colorado (isolate BCTV-Can; MK803280) and Arizona (isolate BCTV-Can-AZ; MW182244). Phylogenetic analysis revealed that sequences of specific strains of BCTV from hemp formed a distinct cluster that separated BCTV-Wor and BCTV-CO sequences . Strains from other locations in Colorado and elsewhere, available in GenBank, are also shown. 2.7.4. Detection of HLVd in Hemp Seeds and on Thrips A sample of hemp seeds from a commercial supplier ( a) was shown to contain HLVd on/in the seed tissues following grinding. The RT-PCR analysis showed the expected band size of 256 bp was present in four out of eight seeds tested ( b). Another batch of seeds from the same sample was germinated, and the developing seedlings were also determined to be infected with HLVd when both the cotyledons and true leaves were sampled ( c). In addition, a sample containing eight adult thrips, identified as onion thrips ( Thrips tabaci ) ( d), observed feeding on the infected plants and causing significant damage ( e) were also shown to contain the viroid. The viroid was likely located on the mouthparts and on the external surface of the insects ( b, lane ‘T’). When also assayed by the RT-qPCR method described in this study, the thrips sample was confirmed to be HLVd-positive (C T value of 30.3). Thrips are commonly found within greenhouses in large numbers, and can be detected and quantified using yellow sticky cards placed adjacent to plants ( f). 2.1.1. Symptoms and Pathogen Isolation The symptoms observed on the cannabis plants that were included for analysis are shown in . They included stunting and yellowing on vegetative and flowering plants and internal stem discoloration ( a–c). In some instances, visible growth of the fungal mycelium could be seen on inflorescence tissues ( d–f). These symptoms were observed on a range of different cannabis genotypes that were cultivated during experiments conducted in 2020–2022. Following surface sterilization and plating onto potato dextrose agar containing 140 mg/L streptomycin sulfate, emerging colonies were purified through subculturing and examined for spore morphology and other features to identify them to genus and species. The appearance of the colonies of the fungi recovered from symptomatic tissues are shown in g–l. They confirm the presence of Fusarium , Pythium , and Botrytis species, which was confirmed by molecular analysis. 2.1.2. Molecular Detection by PCR The primers UN-UP18S42 and UN-LO28S576B amplified a region of the internal transcribed region (ITS1-5.8S-ITS2) of ribosomal DNA and produced bands of different molecular weight sizes when the DNA extracted from diseased and healthy leaf and inflorescence tissues was used . Sequencing of these bands confirmed the presence of Golovinomyces ambrosiae ( a) and Botrytis cinerea ( b) in the affected tissues, as well as the cannabis DNA sequence. When these primers were used with DNA extracted from pure cultures recovered from symptomatic tissues, a range of band sizes was observed, depending on the culture . Sequencing of these bands and comparison to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST confirmed the identification of various species using cut-off values >99%. The pathogens identified were Fusarium oxysporum ( g), F. proliferatum ( h), Pythium myriotylum ( i), G. ambrosiae ( j), B. cinerea ( k), and F. sporotrichiodes ( l). Two additional fungi— Trichoderma asperellum and Penicillium olsonii —recovered from diseased root and stem tissues, respectively, were also identified. The symptoms observed on the cannabis plants that were included for analysis are shown in . They included stunting and yellowing on vegetative and flowering plants and internal stem discoloration ( a–c). In some instances, visible growth of the fungal mycelium could be seen on inflorescence tissues ( d–f). These symptoms were observed on a range of different cannabis genotypes that were cultivated during experiments conducted in 2020–2022. Following surface sterilization and plating onto potato dextrose agar containing 140 mg/L streptomycin sulfate, emerging colonies were purified through subculturing and examined for spore morphology and other features to identify them to genus and species. The appearance of the colonies of the fungi recovered from symptomatic tissues are shown in g–l. They confirm the presence of Fusarium , Pythium , and Botrytis species, which was confirmed by molecular analysis. The primers UN-UP18S42 and UN-LO28S576B amplified a region of the internal transcribed region (ITS1-5.8S-ITS2) of ribosomal DNA and produced bands of different molecular weight sizes when the DNA extracted from diseased and healthy leaf and inflorescence tissues was used . Sequencing of these bands confirmed the presence of Golovinomyces ambrosiae ( a) and Botrytis cinerea ( b) in the affected tissues, as well as the cannabis DNA sequence. When these primers were used with DNA extracted from pure cultures recovered from symptomatic tissues, a range of band sizes was observed, depending on the culture . Sequencing of these bands and comparison to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST confirmed the identification of various species using cut-off values >99%. The pathogens identified were Fusarium oxysporum ( g), F. proliferatum ( h), Pythium myriotylum ( i), G. ambrosiae ( j), B. cinerea ( k), and F. sporotrichiodes ( l). Two additional fungi— Trichoderma asperellum and Penicillium olsonii —recovered from diseased root and stem tissues, respectively, were also identified. 2.2.1. Symptoms Leaves displaying symptoms of mosaic, mottling, and line patterns that superficially resembled those caused by viral infections were collected from greenhouse-grown cannabis plants during 2021–2022. Representative symptoms on leaf samples on several genotypes are shown in . These symptoms were consistently observed on vegetative and flowering plants of four genotypes—‘OG Kush’ (OG) ( a), ‘Headband’ (HB) ( b), ‘Golden Papaya’ (GP) ( c), and ‘Motor Breath’ (MB) ( d). The leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if viral pathogens were present. By comparison, leaves with chlorotic sectors and loss of chlorophyll characteristic of somatic mutations are shown in e. 2.2.2. PCR with Broad-Spectrum and Specific Primer Sets Several broad-spectrum degenerate primer sets were tested following the conditions described in . As well, specific primers designed to detect turnip ringspot virus, alfalfa mosaic virus (AMV), yobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) were utilized. Leaves of cannabis genotypes OG and HB, with symptoms as shown in a,b, were included in the analysis. None of the primer sets produced a band in the samples originating from cannabis leaf tissues, while the positive controls of the infected tobacco leaves showed the expected band size for these viruses. The RT-PCR results for TMV and AMV are shown in a–c. These results indicate these specific viruses were absent in these leaf cannabis tissues. 2.2.3. Transmission Electron Microscopy (TEM) Under the TEM, rod-shaped particles of TMV were observed in leaf tissues from infected positive-control tobacco plants ( d), while cannabis leaf tissues showed an absence of any particles resembling viruses in the samples examined. 2.2.4. Host Range Studies Following inoculation of nine host plant species using ground leaf extracts originating from cannabis genotypes OG and HB with mosaic and line-pattern symptoms, and monitoring plants for symptoms over a period of 3 weeks, no symptoms were observed on any of the host plants tested . Leaves displaying symptoms of mosaic, mottling, and line patterns that superficially resembled those caused by viral infections were collected from greenhouse-grown cannabis plants during 2021–2022. Representative symptoms on leaf samples on several genotypes are shown in . These symptoms were consistently observed on vegetative and flowering plants of four genotypes—‘OG Kush’ (OG) ( a), ‘Headband’ (HB) ( b), ‘Golden Papaya’ (GP) ( c), and ‘Motor Breath’ (MB) ( d). The leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if viral pathogens were present. By comparison, leaves with chlorotic sectors and loss of chlorophyll characteristic of somatic mutations are shown in e. Several broad-spectrum degenerate primer sets were tested following the conditions described in . As well, specific primers designed to detect turnip ringspot virus, alfalfa mosaic virus (AMV), yobacco mosaic virus (TMV), and cucumber mosaic virus (CMV) were utilized. Leaves of cannabis genotypes OG and HB, with symptoms as shown in a,b, were included in the analysis. None of the primer sets produced a band in the samples originating from cannabis leaf tissues, while the positive controls of the infected tobacco leaves showed the expected band size for these viruses. The RT-PCR results for TMV and AMV are shown in a–c. These results indicate these specific viruses were absent in these leaf cannabis tissues. Under the TEM, rod-shaped particles of TMV were observed in leaf tissues from infected positive-control tobacco plants ( d), while cannabis leaf tissues showed an absence of any particles resembling viruses in the samples examined. Following inoculation of nine host plant species using ground leaf extracts originating from cannabis genotypes OG and HB with mosaic and line-pattern symptoms, and monitoring plants for symptoms over a period of 3 weeks, no symptoms were observed on any of the host plants tested . The HTS analysis was initially conducted on leaf samples originating from the genotypes OG and HB with symptoms of mosaic and line patterns ( a,b), and was then extended to an additional six cannabis genotypes (PD, Mac-1, PK, BC, CBD, G54-2). Some of these latter genotypes (PD, Mac-1) showed distinct symptoms of stunted and reduced inflorescence growth ( a,b). These symptoms were also apparent after the inflorescences were trimmed and dried ( c,d). A comparison of the fresh weights of the inflorescences from healthy (asymptomatic) and symptomatic plants of five genotypes included in the HTS analysis is shown in e. The genotypes that were the most significantly affected were PD, Mac-1, and BC, while PK and CBD did not show a significant reduction in the fresh weight of inflorescence tissues or any obvious symptoms. Following Illumina sequencing and assembly, only two confirmed pathogen sequences were detected in the genotypes HB and OG—hop latent viroid (HLVd) and Cannabis sativa mitovirus 1 (CasaMV1). The results are summarized in . The sequences of HLVd were 100% identical at the nucleotide level to each other and to those in GenBank. The sequences of CasaMV1 were 99.6% identical to the sequences found in GenBank. The sequences from this study have been deposited in GenBank (accession OQ420426 for HLVd from HB and accession OQ420425 for CasaMV1 from HB). When the HTS analysis was conducted on the leaf tissues of six additional genotypes of cannabis (PD, Mac-1, PK, BC, CBD, G54-2), the results were similar, revealing only HLVd and CasaMV1 to be present in all genotypes except for PK. 2.4.1. RNA Extractions, cDNA Preparation, and RT-PCR Hop Latent Viroid The results from the RNA extractions from the leaf tissues of seven cannabis genotypes, and a check for the RNA quality, showed that all samples yielded satisfactory RNA for conducting RT-PCR . Extracted RNA from leaf tissues was subjected to cDNA preparation and RT-PCR using primers that were developed in this study (see . Primers for the HLVd amplified bands of sizes 256 bp and 512 bp from the leaf tissues of plants of genotype PD showed symptoms of stunted growth and reduced inflorescence development . Sequencing results confirmed that both bands represented HLVd. Tissues from genotype PK did not show a band and there were no symptoms on this genotype . When the RT-PCR analysis was conducted on an additional six genotypes of cannabis, HLVd was shown to be present in PD, Mac, BC, and HB . Three of these four genotypes previously displayed symptoms of stunting and had reduced inflorescence growth, as shown in . These results suggest that HLVd infection had impacted their growth. The corresponding band size (256 bp) in genotypes G54-2 and CBD was very faint . Genotype CBD did not display any symptoms attributed to HLVd, and its growth was not affected, as shown in . The growth of genotype G54-2 was not measured. Mitovirus To assess the extent to which CasaMV1 is present in cannabis tissues, leaves from 34 genotypes originating from 20 indoor-grown plants and 14 from outdoor-grown plants were sampled and used for RNA extraction and cDNA synthesis, as described previously. The forward primer: 5′ CGGTAGGATTGCTCAGTCGG 3′ and reverse primer: 5′ CGAACATGCGGTTCATAGGC 3′ were used to amplify a region of the RNA-dependent RNA polymerase enzyme to produce a 998 bp size product. The PCR conditions were the same as those used for HLVd detection (see ). The results showed that CasaMV1 was present in 18 out of 20 cannabis genotypes grown indoors ( a) and in 10 out of 14 genotypes grown outdoors ( b). Sequence analysis indicated the bands were 100% identical to each other. These sequences showed 99.7% sequence homology to Colorado isolate MT878084 and 99.4% to BK010437.1 from Purple Kush. For an additional four cannabis genotypes, samples of roots, petioles, leaves, and flower tissues were assayed for the presence of CasaMV1, and it was found to be present in all the tissue types analyzed ( c,d). 2.4.2. Sequence Diversity and Molecular Phylogeny of Hop Latent Viroid A phylogenetic analysis of HLVd was conducted using seven sequences from hops, two from hemp, as well as four from cannabis (selected from GenBank to represent full-length sequences), and included six from the current study. The analysis showed that no significant sequence diversity was present ( a). However, one SNP was observed between sequences originating from cannabis genotypes ‘Mac’ and G54-2, where a ‘G’ was ‘A’ ( b). 2.4.3. Droplet Digital PCR Quantification of HLVd levels (titer) in the leaves of eight cannabis genotypes was conducted using ddPCR. The genotypes PD, Mac, BC, PK, CBD, G54-2, HB, and OG were the same as those used previously for the RT-PCR . The results are shown in . The highest titers (>10,000 genomes per reaction) were seen in PD, Mac, and BC, followed by PDH (healthy) and HB, and a low level (10 genomes) was observed in G54-2. Genotypes PK, CBD, and OG had no detectable levels of viroid. The genotypes PD, Mac, and BC also displayed symptoms of HLVd infection, that included stunting and reduced inflorescence growth, and a reduction in inflorescence fresh weight, as shown previously . By comparison, the genotypes PK and CBD showed no symptoms. In comparing these results to those for the same genotypes obtained by RT-PCR, no band was observed in PK and CBD, and a very faint band was seen in G54-2 . The remaining genotypes (PD, Mac, BC) produced a strong band following RT-PCR. Genotype PDH was confirmed to contain HLVd by both RT-PCR and ddPCR despite being asymptomatic. These results show a positive correlation between the results obtained by RT-PCR and ddPCR for all genotypes tested. 2.4.4. Multiplex Taqman RT-qPCR Inflorescence tissues from symptomatic plants of genotypes PD and Mac, subjected to the RT-PCR and ddPCR methods above, were also tested using a Taqman RT-qPCR method. In both samples, the C T cycle thresholds were around 18, indicating a high level of viroid titre was present in the inflorescences . 2.4.5. LAMP Assays The tissues used for this analysis were comprised of 30 leaf, 15 petiole, and 30 root samples taken from 6-week to 10-week-old stock plants representing 11 genotypes of cannabis. These samples were tested by LAMP, and showed that 9 leaf, 5 petiole, and 24 root samples tested positive for HLVd . There was no case in which a plant that tested negative in the root tissues was found to be positive in the leaves or petioles. Conversely, many samples that were positive for HLVd in the roots were found to be negative in the leaves or petioles . These results show that root samples are the most consistent tissue source for detection of HLVd by LAMP in 6-to-10-week-old plants. All genotypes tested showed the highest frequency of samples to be positive for HLVd from root tissues. Hop Latent Viroid The results from the RNA extractions from the leaf tissues of seven cannabis genotypes, and a check for the RNA quality, showed that all samples yielded satisfactory RNA for conducting RT-PCR . Extracted RNA from leaf tissues was subjected to cDNA preparation and RT-PCR using primers that were developed in this study (see . Primers for the HLVd amplified bands of sizes 256 bp and 512 bp from the leaf tissues of plants of genotype PD showed symptoms of stunted growth and reduced inflorescence development . Sequencing results confirmed that both bands represented HLVd. Tissues from genotype PK did not show a band and there were no symptoms on this genotype . When the RT-PCR analysis was conducted on an additional six genotypes of cannabis, HLVd was shown to be present in PD, Mac, BC, and HB . Three of these four genotypes previously displayed symptoms of stunting and had reduced inflorescence growth, as shown in . These results suggest that HLVd infection had impacted their growth. The corresponding band size (256 bp) in genotypes G54-2 and CBD was very faint . Genotype CBD did not display any symptoms attributed to HLVd, and its growth was not affected, as shown in . The growth of genotype G54-2 was not measured. Mitovirus To assess the extent to which CasaMV1 is present in cannabis tissues, leaves from 34 genotypes originating from 20 indoor-grown plants and 14 from outdoor-grown plants were sampled and used for RNA extraction and cDNA synthesis, as described previously. The forward primer: 5′ CGGTAGGATTGCTCAGTCGG 3′ and reverse primer: 5′ CGAACATGCGGTTCATAGGC 3′ were used to amplify a region of the RNA-dependent RNA polymerase enzyme to produce a 998 bp size product. The PCR conditions were the same as those used for HLVd detection (see ). The results showed that CasaMV1 was present in 18 out of 20 cannabis genotypes grown indoors ( a) and in 10 out of 14 genotypes grown outdoors ( b). Sequence analysis indicated the bands were 100% identical to each other. These sequences showed 99.7% sequence homology to Colorado isolate MT878084 and 99.4% to BK010437.1 from Purple Kush. For an additional four cannabis genotypes, samples of roots, petioles, leaves, and flower tissues were assayed for the presence of CasaMV1, and it was found to be present in all the tissue types analyzed ( c,d). The results from the RNA extractions from the leaf tissues of seven cannabis genotypes, and a check for the RNA quality, showed that all samples yielded satisfactory RNA for conducting RT-PCR . Extracted RNA from leaf tissues was subjected to cDNA preparation and RT-PCR using primers that were developed in this study (see . Primers for the HLVd amplified bands of sizes 256 bp and 512 bp from the leaf tissues of plants of genotype PD showed symptoms of stunted growth and reduced inflorescence development . Sequencing results confirmed that both bands represented HLVd. Tissues from genotype PK did not show a band and there were no symptoms on this genotype . When the RT-PCR analysis was conducted on an additional six genotypes of cannabis, HLVd was shown to be present in PD, Mac, BC, and HB . Three of these four genotypes previously displayed symptoms of stunting and had reduced inflorescence growth, as shown in . These results suggest that HLVd infection had impacted their growth. The corresponding band size (256 bp) in genotypes G54-2 and CBD was very faint . Genotype CBD did not display any symptoms attributed to HLVd, and its growth was not affected, as shown in . The growth of genotype G54-2 was not measured. To assess the extent to which CasaMV1 is present in cannabis tissues, leaves from 34 genotypes originating from 20 indoor-grown plants and 14 from outdoor-grown plants were sampled and used for RNA extraction and cDNA synthesis, as described previously. The forward primer: 5′ CGGTAGGATTGCTCAGTCGG 3′ and reverse primer: 5′ CGAACATGCGGTTCATAGGC 3′ were used to amplify a region of the RNA-dependent RNA polymerase enzyme to produce a 998 bp size product. The PCR conditions were the same as those used for HLVd detection (see ). The results showed that CasaMV1 was present in 18 out of 20 cannabis genotypes grown indoors ( a) and in 10 out of 14 genotypes grown outdoors ( b). Sequence analysis indicated the bands were 100% identical to each other. These sequences showed 99.7% sequence homology to Colorado isolate MT878084 and 99.4% to BK010437.1 from Purple Kush. For an additional four cannabis genotypes, samples of roots, petioles, leaves, and flower tissues were assayed for the presence of CasaMV1, and it was found to be present in all the tissue types analyzed ( c,d). A phylogenetic analysis of HLVd was conducted using seven sequences from hops, two from hemp, as well as four from cannabis (selected from GenBank to represent full-length sequences), and included six from the current study. The analysis showed that no significant sequence diversity was present ( a). However, one SNP was observed between sequences originating from cannabis genotypes ‘Mac’ and G54-2, where a ‘G’ was ‘A’ ( b). Quantification of HLVd levels (titer) in the leaves of eight cannabis genotypes was conducted using ddPCR. The genotypes PD, Mac, BC, PK, CBD, G54-2, HB, and OG were the same as those used previously for the RT-PCR . The results are shown in . The highest titers (>10,000 genomes per reaction) were seen in PD, Mac, and BC, followed by PDH (healthy) and HB, and a low level (10 genomes) was observed in G54-2. Genotypes PK, CBD, and OG had no detectable levels of viroid. The genotypes PD, Mac, and BC also displayed symptoms of HLVd infection, that included stunting and reduced inflorescence growth, and a reduction in inflorescence fresh weight, as shown previously . By comparison, the genotypes PK and CBD showed no symptoms. In comparing these results to those for the same genotypes obtained by RT-PCR, no band was observed in PK and CBD, and a very faint band was seen in G54-2 . The remaining genotypes (PD, Mac, BC) produced a strong band following RT-PCR. Genotype PDH was confirmed to contain HLVd by both RT-PCR and ddPCR despite being asymptomatic. These results show a positive correlation between the results obtained by RT-PCR and ddPCR for all genotypes tested. Inflorescence tissues from symptomatic plants of genotypes PD and Mac, subjected to the RT-PCR and ddPCR methods above, were also tested using a Taqman RT-qPCR method. In both samples, the C T cycle thresholds were around 18, indicating a high level of viroid titre was present in the inflorescences . The tissues used for this analysis were comprised of 30 leaf, 15 petiole, and 30 root samples taken from 6-week to 10-week-old stock plants representing 11 genotypes of cannabis. These samples were tested by LAMP, and showed that 9 leaf, 5 petiole, and 24 root samples tested positive for HLVd . There was no case in which a plant that tested negative in the root tissues was found to be positive in the leaves or petioles. Conversely, many samples that were positive for HLVd in the roots were found to be negative in the leaves or petioles . These results show that root samples are the most consistent tissue source for detection of HLVd by LAMP in 6-to-10-week-old plants. All genotypes tested showed the highest frequency of samples to be positive for HLVd from root tissues. 2.5.1. Distribution within Stock Plants Leaf samples were obtained from various positions within the canopy of individual stock plants, as well as from roots ( a,b), and subjected to RT-PCR ( c,d) as well as ddPCR ( e). The RT-PCR results showed the presence of multiple bands in most samples tested, varying in size from 256 bp, 512 bp, to 768 bp . Sequencing of these bands confirmed they were all HLVd. In genotype G54-2, the viroid was present in the roots, bottom-canopy leaves, and at the top of the plant, but was barely detectable in the middle-canopy leaves ( c). In genotype PD, the viroid was present in the roots, bottom-canopy leaves, middle-canopy leaves, and in the top-canopy leaves ( d). Using ddPCR for the same two genotypes, HLVd was detected in the roots and bottom-canopy leaves of genotype G54-2, but not in the middle- or top-canopy leaves. In genotype PD, HLVd was present in the roots, lower-canopy leaves, and middle- and top-canopy leaves at high concentrations (>10,000 genomes) ( e). 2.5.2. Distribution of HLVd within Inflorescence Tissues To quantify the distribution of HLVd within cannabis inflorescences, symptomatic ‘Mac-1’ plants were compared with asymptomatic (healthy) plants ( a). These plants were selected based on the presence/absence of HLVd as confirmed by RT-PCR (see ). The terminal inflorescence on symptomatic and asymptomatic plants were each removed and brought back to the laboratory ( a). Using a scalpel, the fan leaves (FL) and inflorescence leaves (IL) were dissected, leaving the central stripped flower (SF) exposed ( b). A sample of foliage leaves (PL) taken from the bottom canopy of each plant was included for analysis as well. HLVd presence was confirmed using ddPCR in the small inflorescence leaves (IL), the stripped flower (SF), and in the foliage leaves (PL) of symptomatic ‘Mac’, but not in the fan leaves (FL) ( d). The highest accumulation of HLVd was seen in the stripped flowers, which are made up of clusters of pistils surrounded by the inflorescence leaves ( b). In the healthy ‘Mac’ plant, none of the inflorescence samples showed HLVd to be present, but a low level was detected in the foliage leaves by ddPCR ( d). 2.5.3. Distribution of HLVd within Cuttings from Infected Stock Plants Vegetative cuttings taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd were rooted and tested for HLVd using RT-PCR and RT-qPCR after 14 days ( a). The results consistently showed that all cuttings were infected with the viroid ( b), as shown by a band of 256 bp size observed in seven infected cuttings ( b). This was confirmed by RT-qPCR that showed that four out of seven cuttings tested originating from the infected stock plant contained the viroid at a high titre, with C T values of 15–18 ( c). 2.5.4. Detection of Pathogens in Fresh and Dried Cannabis Inflorescences Primers for the ITS1-5.8S-ITS2 region of ribosomal DNA were used to detect fungal pathogens, and primers for HLVd were used on tissue samples obtained from freshly harvested inflorescences and from dried cannabis flowers destined for commercial sale ( a). Out of 20 samples, 7 were positive for HLVd, 4 samples contained B. cinerea , 3 had F. oxysporum , and 2 had a powdery mildew ( b). RT-PCR confirmed the presence of HLVd in 7/10 dried flower samples ( c). 2.5.5. Detection of HLVd and CasaMV1 in Cannabis Seed Seeds derived from a cross made between an infected ‘Mac1′ female plant and pollen from a male plant of genotype ‘GPie’ were ground individually, and the extracted RNA was subjected to RT-PCR. The results showed that 14/16 seeds (87.5%) were positive for the presence of HLVd ( a) and 5/5 seeds tested also contained CasaMV1 ( b). A sample of 10 seeds from the same batch used in was also subjected to Taqman RT-qPCR using the methods described in . The results are presented in . They show that viroid levels, as estimated by C T values, differed significantly from one seed and the next, and ranged from 17.1 to 33.66, with a mean of 24.07. The infection rate was 70%, as three out of ten seeds did not contain detectable HLVd . Leaf samples were obtained from various positions within the canopy of individual stock plants, as well as from roots ( a,b), and subjected to RT-PCR ( c,d) as well as ddPCR ( e). The RT-PCR results showed the presence of multiple bands in most samples tested, varying in size from 256 bp, 512 bp, to 768 bp . Sequencing of these bands confirmed they were all HLVd. In genotype G54-2, the viroid was present in the roots, bottom-canopy leaves, and at the top of the plant, but was barely detectable in the middle-canopy leaves ( c). In genotype PD, the viroid was present in the roots, bottom-canopy leaves, middle-canopy leaves, and in the top-canopy leaves ( d). Using ddPCR for the same two genotypes, HLVd was detected in the roots and bottom-canopy leaves of genotype G54-2, but not in the middle- or top-canopy leaves. In genotype PD, HLVd was present in the roots, lower-canopy leaves, and middle- and top-canopy leaves at high concentrations (>10,000 genomes) ( e). To quantify the distribution of HLVd within cannabis inflorescences, symptomatic ‘Mac-1’ plants were compared with asymptomatic (healthy) plants ( a). These plants were selected based on the presence/absence of HLVd as confirmed by RT-PCR (see ). The terminal inflorescence on symptomatic and asymptomatic plants were each removed and brought back to the laboratory ( a). Using a scalpel, the fan leaves (FL) and inflorescence leaves (IL) were dissected, leaving the central stripped flower (SF) exposed ( b). A sample of foliage leaves (PL) taken from the bottom canopy of each plant was included for analysis as well. HLVd presence was confirmed using ddPCR in the small inflorescence leaves (IL), the stripped flower (SF), and in the foliage leaves (PL) of symptomatic ‘Mac’, but not in the fan leaves (FL) ( d). The highest accumulation of HLVd was seen in the stripped flowers, which are made up of clusters of pistils surrounded by the inflorescence leaves ( b). In the healthy ‘Mac’ plant, none of the inflorescence samples showed HLVd to be present, but a low level was detected in the foliage leaves by ddPCR ( d). Vegetative cuttings taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd were rooted and tested for HLVd using RT-PCR and RT-qPCR after 14 days ( a). The results consistently showed that all cuttings were infected with the viroid ( b), as shown by a band of 256 bp size observed in seven infected cuttings ( b). This was confirmed by RT-qPCR that showed that four out of seven cuttings tested originating from the infected stock plant contained the viroid at a high titre, with C T values of 15–18 ( c). Primers for the ITS1-5.8S-ITS2 region of ribosomal DNA were used to detect fungal pathogens, and primers for HLVd were used on tissue samples obtained from freshly harvested inflorescences and from dried cannabis flowers destined for commercial sale ( a). Out of 20 samples, 7 were positive for HLVd, 4 samples contained B. cinerea , 3 had F. oxysporum , and 2 had a powdery mildew ( b). RT-PCR confirmed the presence of HLVd in 7/10 dried flower samples ( c). Seeds derived from a cross made between an infected ‘Mac1′ female plant and pollen from a male plant of genotype ‘GPie’ were ground individually, and the extracted RNA was subjected to RT-PCR. The results showed that 14/16 seeds (87.5%) were positive for the presence of HLVd ( a) and 5/5 seeds tested also contained CasaMV1 ( b). A sample of 10 seeds from the same batch used in was also subjected to Taqman RT-qPCR using the methods described in . The results are presented in . They show that viroid levels, as estimated by C T values, differed significantly from one seed and the next, and ranged from 17.1 to 33.66, with a mean of 24.07. The infection rate was 70%, as three out of ten seeds did not contain detectable HLVd . The symptoms observed on cannabis plants characteristic of infection by BCTV are shown in . The uppermost leaves on the plants were curled and twisted and deformed ( a,b). Similar symptoms were observed on plants in the field ( c). On some plants, leaf curl symptoms were very severe, causing the plants to appear malformed ( d–f). RT-PCR with BCTV-specific primers showed the presence of a 1 kb band, confirmed to be the Worland strain in the greenhouse-grown plants . 2.7.1. Next-Generation Sequencing (NGS) Symptomatic field-grown plants, from which leaves were collected for shotgun metagenomic analysis, are shown in . These samples were collected from various locations in Colorado during 2019, 2021, and 2022. Among these samples, four viruses and one viroid were identified in 2019 . In general, the number of pathogens recovered varied by sampling location, and ranged from two to five. Cannabis sativa mitovirus (CasaMV1) and beet curly top virus (BCTV) were the most commonly found in each year of sampling. HLVd was only present in certain fields sampled in 2019. Citrus yellow-vein-associated virus (CYVaV) and Tobacco streak virus (TSV) were also only detected in 2019 . In samples collected in 2021, the viruses that were found Cannabis cryptic virus (CanCV), Alfalfa mosaic virus (AMV), BCTV, and CasaMV1 . In 2022, the viruses detected were CasaMV1, BCTV, CanCV, and Tomato bushy stunt virus . 2.7.2. RT-PCR with Specific Primer Sets The majority of the viruses identified through NGS had a high percentage nucleotide identity (>90%; ). Only three of the viruses, CasaMV1, TSV, and CYVaV, were below this threshold. The presence of these low-percentage nucleotide-identity (<90%) viruses was confirmed in the respective samples with RT-PCR analysis . These results confirmed the presence of each of the three viruses in the hemp samples. 2.7.3. Sequence Diversity and Molecular Phylogeny of Beet Curly Top Virus Phylogenetic analysis based on a fragment of the BCTV coat protein (CP) sequences from the 2019 hemp samples had nucleotide identities to one another between 98.99 and 98.24%. Sequences from this study had a 97–99% sequence identity with cannabis BCTV sequences from Colorado (isolate BCTV-Can; MK803280) and Arizona (isolate BCTV-Can-AZ; MW182244). Phylogenetic analysis revealed that sequences of specific strains of BCTV from hemp formed a distinct cluster that separated BCTV-Wor and BCTV-CO sequences . Strains from other locations in Colorado and elsewhere, available in GenBank, are also shown. 2.7.4. Detection of HLVd in Hemp Seeds and on Thrips A sample of hemp seeds from a commercial supplier ( a) was shown to contain HLVd on/in the seed tissues following grinding. The RT-PCR analysis showed the expected band size of 256 bp was present in four out of eight seeds tested ( b). Another batch of seeds from the same sample was germinated, and the developing seedlings were also determined to be infected with HLVd when both the cotyledons and true leaves were sampled ( c). In addition, a sample containing eight adult thrips, identified as onion thrips ( Thrips tabaci ) ( d), observed feeding on the infected plants and causing significant damage ( e) were also shown to contain the viroid. The viroid was likely located on the mouthparts and on the external surface of the insects ( b, lane ‘T’). When also assayed by the RT-qPCR method described in this study, the thrips sample was confirmed to be HLVd-positive (C T value of 30.3). Thrips are commonly found within greenhouses in large numbers, and can be detected and quantified using yellow sticky cards placed adjacent to plants ( f). Symptomatic field-grown plants, from which leaves were collected for shotgun metagenomic analysis, are shown in . These samples were collected from various locations in Colorado during 2019, 2021, and 2022. Among these samples, four viruses and one viroid were identified in 2019 . In general, the number of pathogens recovered varied by sampling location, and ranged from two to five. Cannabis sativa mitovirus (CasaMV1) and beet curly top virus (BCTV) were the most commonly found in each year of sampling. HLVd was only present in certain fields sampled in 2019. Citrus yellow-vein-associated virus (CYVaV) and Tobacco streak virus (TSV) were also only detected in 2019 . In samples collected in 2021, the viruses that were found Cannabis cryptic virus (CanCV), Alfalfa mosaic virus (AMV), BCTV, and CasaMV1 . In 2022, the viruses detected were CasaMV1, BCTV, CanCV, and Tomato bushy stunt virus . The majority of the viruses identified through NGS had a high percentage nucleotide identity (>90%; ). Only three of the viruses, CasaMV1, TSV, and CYVaV, were below this threshold. The presence of these low-percentage nucleotide-identity (<90%) viruses was confirmed in the respective samples with RT-PCR analysis . These results confirmed the presence of each of the three viruses in the hemp samples. Phylogenetic analysis based on a fragment of the BCTV coat protein (CP) sequences from the 2019 hemp samples had nucleotide identities to one another between 98.99 and 98.24%. Sequences from this study had a 97–99% sequence identity with cannabis BCTV sequences from Colorado (isolate BCTV-Can; MK803280) and Arizona (isolate BCTV-Can-AZ; MW182244). Phylogenetic analysis revealed that sequences of specific strains of BCTV from hemp formed a distinct cluster that separated BCTV-Wor and BCTV-CO sequences . Strains from other locations in Colorado and elsewhere, available in GenBank, are also shown. A sample of hemp seeds from a commercial supplier ( a) was shown to contain HLVd on/in the seed tissues following grinding. The RT-PCR analysis showed the expected band size of 256 bp was present in four out of eight seeds tested ( b). Another batch of seeds from the same sample was germinated, and the developing seedlings were also determined to be infected with HLVd when both the cotyledons and true leaves were sampled ( c). In addition, a sample containing eight adult thrips, identified as onion thrips ( Thrips tabaci ) ( d), observed feeding on the infected plants and causing significant damage ( e) were also shown to contain the viroid. The viroid was likely located on the mouthparts and on the external surface of the insects ( b, lane ‘T’). When also assayed by the RT-qPCR method described in this study, the thrips sample was confirmed to be HLVd-positive (C T value of 30.3). Thrips are commonly found within greenhouses in large numbers, and can be detected and quantified using yellow sticky cards placed adjacent to plants ( f). Rapid and accurate identification of pathogens affecting cannabis and hemp crops is essential to implement the most appropriate disease-management practices. Conventional diagnostic methods, that include symptomology, microscopic observations of pathogen presence/morphology, culturing techniques for pathogen recovery, and pathogen identification and proof of pathogenicity, have all been successfully and reliably used for the diagnosis of emerging pathogens of cannabis and hemp . These methods are now being augmented with molecular advances in nucleic acid-based diagnosis for the characterization of specific pathogens based on their DNA or RNA sequences. These methods primarily involve the polymerase chain reaction (PCR), DNA barcoding, and next-generation and high-throughput sequencing. These molecular approaches are particularly useful in instances where multiple pathogens may be involved in a disease complex, as is commonly encountered in cannabis and hemp crops . They are also useful where disease symptoms may be confused with environmental stresses or damage caused by insect pests. Lastly, molecular diagnostics are important for confirming pathogen presence in instances where plants remain asymptomatic after infection, or if the pathogen cannot be recovered on culture media. This study describes a range of recently developed molecular diagnostic approaches that can be used to confirm the presence of fungal and oomycete pathogens affecting cannabis, as well as to identify a diverse group of viruses and a viroid that affect cannabis and hemp crops during commercial production, all of which are summarized in . By sampling and performing the requisite analyses over several years (2020–2023) on samples representing many different genotypes of each crop from different geographical regions, the methods described were validated in several laboratories. The most prevalent fungal and oomycete pathogens detected on the roots and stems of cannabis plants were Fusarium and Pythium spp., confirming previous reports of the widespread occurrence of these pathogens in different cannabis growing environments . On the leaves and inflorescence tissues, powdery mildew ( Golovinomyces ambrosiae ) and Botrytis cinerea were shown to be prevalent pathogens on cannabis, as previously reported . The universal eukaryotic primers also confirmed the presence of Trichoderma asperellum originating from root tissues and Penicillium olsonii originating from stem tissues. The former is a biological control agent that is frequently applied during greenhouse cannabis production, while the latter is a naturally occurring endophyte found in cannabis stems . Cannabis DNA was also amplified with this primer set, which was differentiated by its different molecular weight fragment size and unique sequence that differentiated it from the respective pathogens. The advantage of a universal primer set is that prior knowledge of which pathogens may be present is not required, overcoming a limitation to the use of species-specific primers for undetermined pathogens in a diseased sample. The primer set also confirmed that three of the four most prevalent pathogens could be detected in commercially dried cannabis flower samples following PCR amplification and sequencing. If routine testing of harvested product needs to be implemented, the universal primer set could confirm the presence of these three pathogens, and potentially others that may be present. An important component of cannabis quality assurance is the determination of total yeast and mold (TYM) levels in the final dried cannabis product that reaches the consumer . While molecular approaches were not investigated or utilized in the present study to determine how TYM could be assessed, previous whole-genome sequencing and the use of PCR approaches that targeted specific fungal species considered to be of importance have been described . These types of studies should be extended to provide a view of the microbiome within cannabis inflorescences using next-generation sequencing to identify the fungal species potentially posing the most concern for human health . Cannabis leaf samples of many genotypes, which occasionally displayed mosaic, line patterns, and mottling symptoms, particularly on younger leaves, were tested with primers to broad virus groups and to three specific viruses—tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and alfalfa mosaic virus (AMV)—as described in . None of these RT-PCR analyses yielded a PCR product that confirmed the presence of these viruses in over 30 leaf samples displaying these symptoms. Furthermore, a host range study, in which leaf extracts from three symptomatic cannabis genotypes were inoculated onto nine plant species, did not result in local lesion development or other symptoms that would indicate the transmission and presence of putative viruses. Transmission electron microscopy, furthermore, showed no virus particles were present in these tissues. Subsequently, whole-genome sequencing approaches were conducted on eight cannabis genotypes displaying these symptoms and which were sampled at different times. The results showed that the majority of the samples (95–100%) contained only hop latent viroid (HLVd) and a previously reported cannabis mitovirus (CasaMV1) . Neither of these pathogens has been demonstrated to cause foliar symptoms resembling those shown in . The characteristic symptoms of HLVd infection are stunting and reduced inflorescence growth . Righetti et al. tested leaf samples from hemp plants displaying mosaic and streak patterns using PCR with specific primers to a large group of viruses, and also performed next-generation sequencing, host range transmission studies, and electron microscopy of symptomatic tissues, similar to what was conducted in the present study. They only reported the presence of Cannabis cryptic virus (CanCV) , which was present in symptomatic and asymptomatic tissues at varying levels. Their results and ours indicate that these symptoms were not caused by any previously described virus group. In the present study, CasaMV1 was shown to be present in the leaf, petiole, and root tissues of a majority of cannabis plants (>90%) grown indoors, as well as in most cannabis plants grown outdoors. These samples represented a broad range of genotypes. It was also detected in inflorescence and seed samples derived from an infected mother plant. This confirms the previous finding of CasaMV1 reported to be present in tissues derived from the leaves, inflorescences, seeds, seedlings, and roots of C. sativa . There has been no prior research to evaluate the impact of CasaMV1 on cannabis plants, which was also detected in hemp plants in commercial fields and, in the present study, on hemp samples. Mitoviruses are commonly reported to occur in fungi and may cause changes in host physiology, in many cases by altering the mitochondrial structure and function . They have also been shown to be present in a number of plant species, including hemp and hops, without causing any apparent symptoms, and are presumed to be cryptic . In fungi, disruptions in mitochondrial function due to mitoviruses can impact growth and pathogenicity . Alterations in the levels of protein expression have been found in some plants infected by a mitovirus . Further research is needed to determine the significance of the widespread occurrence of CasaMV1 in various tissues of cannabis plants and whether it is indeed cryptic or could be causing mild symptoms. In cannabis plants grown indoors which are propagated vegetatively for successive generations, the occurrence of coinfections with viruses/viroid presents a challenge in experimentally demonstrating their individual effects. For example, both HLVd and CasaMV1 were found simultaneously in a large number of cannabis genotypes in this study, and were present in both symptomatic as well as in asymptomatic plants. Establishing the roles that each may play in symptom development requires the elimination of one/both of these entities, followed by reinoculation with individual and combined virus/viroid or infectious cDNA clones to observe symptoms and discern if there are any potential interactions between these two coinfecting agents. To date, these types of inoculation experiments have not been performed. Therefore, while the diagnostic assays described in this study provide confirmation of pathogen presence, their causation or involvement in symptom expression remains to be confirmed. In previous experimental inoculations conducted on hemp plants by Keglar and Sparr (summarized by Miotti et al. ), it was reported that a number of mechanically transmitted viruses could cause mosaic and mottling symptoms on leaves, including AMV, CMV, potato viruses X and Y, and arabis mosaic virus. None of these viruses were identified in this study on cannabis, while AMV was detected in hemp plants in 2020. In addition, despite widespread conclusions in many nonverifiable sources, such as Internet sites, stating that TMV is an important pathogen causing mosaic symptoms, it has not been demonstrated to infect cannabis or hemp plants to date, and there are no previous reports of its natural occurrence on these hosts. A possible explanation for the mosaic and mottling symptoms seen on cannabis plants is that they are the result of somatic mutations, which are commonly seen on vegetatively propagated plants . A few obvious chimeras were observed on leaves of cannabis plants during this study , some of which bore a striking resemblance to the foliar symptoms putatively attributed to virus infection. Further research is needed to establish whether these symptoms are the result of somatic mutations, which are known to occur in cannabis. Adamek et al. demonstrated the extent of intraplant variation that arises from vegetative propagation from a cannabis stock plant to give rise to genetic mosaicism. Using the deep sequencing of whole genomes, they reported a higher occurrence of variants among shoots obtained from actively growing regions of the plant compared to older shoots at the bottom of the plant. Their results showed that a large number of mutations arise as the cannabis plant grows, and is maintained for a long period of time, and, as a consequence, can potentially impact the functions of important genes . Epigenetic changes in plants and other organisms, caused by DNA methylation and histone modifications, among other mechanisms, have been proposed to be heritable, resulting in these epimutations potentially contributing to phenotypic variation in subsequent generations . In cannabis plants that are extensively propagated through vegetative means, the appearance of these variant phenotypes may be frequent and transgenerational, and may be confused with symptoms of putative infection by viruses such as TMV or CMV. Distinct symptoms of stunted plant growth, leaf distortion, chlorosis of leaves, and leaf curl have recently been associated with the presence of several viruses confirmed to be present in cannabis and hemp plants. These include Lettuce chlorosis virus (LCV) , Beet curly top virus (BCTV) , and Citrus yellow-vein-associated virus . Subsequently, diagnostic methods using RT-PCR with specific primers have been developed to detect these viruses . Recent studies have also reported spiroplasma/phytoplasma pathogens affecting hemp, including Spiroplasma citri and Candidatus Phytoplasma trifolii, which were detected using specific PCR primers . Cannabis cryptic virus (CCV) has also been reported in cannabis plants, without causing any apparent symptoms . This latter virus was not identified in cannabis samples in this study, but was detected in hemp plants sampled during 2020 and 2021. The potential origins of some of these pathogens can be attributed to infected seed/planting material and/or to influxes of insect vectors. The occurrence of BCTV, HLVd, CasaMV1, Tobacco streak virus (TSV), AMV, Citrus yellow-vein-associated virus (CYVaV), and cannabis cryptic virus (CCV) was confirmed on hemp plants through next-generation sequencing. In addition, Tomato bushy stunt virus was detected for the first time in 2023 and has not been reported previously to infect cannabis or hemp. The diversity of viruses present in commercial hemp crops was much greater compared to indoor-grown cannabis, possibly due to the activity of insect vectors, especially leafhoppers, that are prevalent in outdoor production sites and are known to be vectors of some viruses . BCTV exists as phylogenetically different strains and is reported to affect hemp grown in Colorado, Arizona, Nevada, Oregon, Washington, and California . The virus has an extremely wide host range and is considered to be a pathogen that poses a significant threat to this crop . Additional research is likely to identify more viruses and phytoplasmas occurring on both cannabis and hemp, particularly where these crops are grown in close proximity to nonhemp crops that can provide a source of inoculum for spread to cannabis and hemp plants, in addition to the presence of insect vectors that can transmit these pathogens. A multiplex RT-PCR method that is capable of detecting multiple pathogens simultaneously, including BCTV, HLVd, CYVaV, and CasaMV1, would be useful for rapid diagnostic confirmation and is currently being developed in several laboratories. The various PCR-based diagnostic assays described for these pathogens, augmented by whole-genome and high-throughput sequencing approaches, have been instrumental in confirming the presence and distribution of these pathogens. Within indoor growing environments used for the majority of commercial production of cannabis, fewer viral pathogens have been reported to date compared to hemp. Mechanical transmission and vegetative propagation are likely the key means through which viral/viroid pathogens would spread indoors if present. Many mechanically transmitted viruses can be transmitted by insect vectors in a nonpersistent manner . Therefore, there is the potential for insect pests reported to occur on indoor-grown cannabis plants, such as rice root aphids ( Rhopalosiphum rufiabdominalis ) and onion thrips ( Thrips tabaci ), to acquire HLVd during feeding. Diagnostic assays utilizing RT-qPCR and RT-PCR have demonstrated that HLVd can be acquired by root aphids ( https://www.einnews.com/pr_news/581016978/3-rivers-biotech-identifies-root-aphids-as-potential-vector-for-hop-latent-viroid-hlvd , accessed on 8 October 2023) and potentially by onion thrips, as was demonstrated in this study. A recent study also demonstrated the acquisition of the viroid by leafhoppers feeding on HLVd-infected plants , suggesting potential pathogen transmission by these insect species could occur, although the importance of this mode for pathogen transmission remains unknown. In addition, whether HLVd-infected plants serve as better hosts for colonization and development of thrips, as has been demonstrated for Tomato spotted wilt virus-infected tomato plants and western flower thrips , remains to be seen. The presence of whiteflies ( Bemesia tabaci ) in indoor cannabis growing environments was reported to result in transmission of crinviruses, such as Lettuce chlorosis virus (LCV) and Cucurbit chlorotic yellows virus (CCYV) , both first detected from cannabis farms in Israel. Transmission of viruses by insect pests affecting hemp crops under field conditions has resulted in multiple occurrences of viral pathogens in one field , and sometimes coinfections of up to three different pathogens can occur on a single plant . In Nevada, BCTV was found in association with Spiroplasma citri and Candidatus Phytoplasma trifolii on the same and on different plants . In Washington, BCTV, HLVd, and CYVaV were reported to occur in the same field and on the same plant . In Colorado and California, multiple viruses and strains have been shown to be present in hemp fields . It remains to be seen if multiple viral complexes are detected on indoor-grown cannabis plants. On outdoor-grown cannabis plants, the potential for multiple virus complex development, similar to what has been observed in hemp fields, should be monitored. The development of PCR-based molecular diagnostic assays has aided in the characterization of these pathogen complexes, since symptomology alone is inadequate to distinguish which viruses may be occurring simultaneously. A large part of this study was focused on developing molecular methods to detect and quantify HLVd in cannabis plants, due to its potential to cause significant damage and for which little is presently understood about its epidemiology and spread . Initially, primers used in RT-PCR assays demonstrated the presence of the viroid in stock plants, in rooted cuttings derived from these stock plants, and in various types of plant tissues. The distribution of the viroid was not always uniform in 3–4-month-old infected stock plants, but root tissues and youngest leaves generally tested positive for the viroid. In addition, the pathogen was detected in the inflorescence tissues of symptomatic flowering plants of several cannabis genotypes by RT-PCR. A comparison of 11 cannabis genotypes, utilizing plants ranging in age from 6 weeks to 10 weeks following the initiation of rooting from cuttings, and assessing presence/absence of HLVd using a LAMP assay, demonstrated that root tissues consistently showed the highest frequency of detection in these plants compared to the leaves and petioles. These results were confirmed by RT-PCR, and also by RT-qPCR and ddPCR, all of which confirmed the presence of HLVd in different tissues of infected cannabis plants, including inflorescences, and demonstrated consistent viroid presence in root tissues. The LAMP (loop-mediated isothermal amplification) technique was developed for the detection of plant pathogens due to its speed, high specificity, sensitivity, efficiency, and isothermal conditions suitable for field conditions . LAMP is a one-step amplification assay that amplifies the target DNA or RNA sequence and requires two or three pairs of primers to detect six distinct regions in the target sequence . Additionally, the target gene fragment is usually short, producing a series of DNA fragments that are of different sizes . In this study, LAMP was used to demonstrate the presence of HLVd in various tissues of cannabis plants (leaves, petioles, and roots). Some limitations of the LAMP technique include the high risk of cross-contamination, as well as carry-over contamination and off-target amplification, which subsequently can result in false-positives due to the high efficiency of DNA amplification in this method . In a recent study, a combined method utilizing RT-LAMP with RT-qPCR was described for HLVd detection in cannabis plants (LAMP/qPCR) . The results provided comparable results to standard RT-qPCR methods and confirmed HLVd was present in various tissues (roots, petioles, leaves) of plants varying in age from 5 weeks to 10 weeks at high levels. A sampling strategy based on leaf tissues taken from 5–10-week-old plants was recommended to be the most suitable approach for the early detection of HLVd. Our findings using RT-PCR, RT-qPCR, and ddPCR demonstrated that consistent and high levels of HLVd were present in root tissues of 6–10-week-old cannabis plants, as well as in 3–4-month-old stock plants. Leaf tissues sampled from these plants sometimes resulted in negative or low titers of HLVd, depending on the location of the samples. The results obtained from leaf samples also varied according to the genotype of the stock plant tested. The results from ddPCR showed that high levels of viroid genomes (>10,000 copies per reaction) were present in the roots of one genotype; in a second genotype, there were 10,000–100,000 copies in the roots and youngest leaves, indicating it was highly susceptible to infection. There were also significant differences in the levels to which the viroid accumulated in the leaves of flowering plants among eight cannabis genotypes grown adjacent to one another in the same environment, ranging from undetectable to >10,000 genomes, reflecting differences in susceptibility to infection or spread. The earliest detection of HLVd by RT-qPCR was observed in samples of root tissues from 2-week-old rooted cuttings in this study. Selection of the appropriate tissues, time of sampling, and cannabis genotype can influence the accuracy of the detection of HLVd. Currently, several commercial laboratories offering diagnostic services for HLVd testing have identified root tissues as the preferred sampling material based on the earlier, higher, and more consistent accumulation of viroid levels in plants ranging from 2 weeks to 3 months of age ( https://tumigenomics.com/hop-latent-viroid-information , accessed on 10 October 2023, https://medicinalgenomics.com/hop-latent-viroid-in-cannabis/ , accessed on 10 October 2023, https://3riversbiotech.com/3-rivers-biotech-identifies-root-tissue-from-mature-plants-as-the-most-reliable-to-detect-hop-latent-viroid-hlvd/ , accessed on 10 October 2023). In inflorescence tissues of cannabis genotype ‘Mac-1’, a highly susceptible genotype, HLVd was detected at the highest titer (>150,000 copies) within the central inflorescence tissues that had been stripped of the surrounding inflorescence leaves and the fan leaves. The viroid was also present at high levels (10,000 copies) in the inflorescence leaves, but not in the fan leaves. In adjacent asymptomatic plants, the viroid was absent in inflorescence tissues, but was present at low levels (five copies) in vegetative leaf tissue. When HLVd-infected ‘Mac1′ was used as a female parent and fertilized with pollen from another cannabis genotype, the resulting seeds had a high incidence of HLVd infection (70–87.5%) as determined by RT-PCR and RT-qPCR. The ddPCR method has been previously used for the quantitative assessment of a range of different plant pathogens . The main principle of ddPCR, as in other PCR-based methods, including quantitative PCR (qPCR), is the specific amplification of a nucleic acid target. The distinctive feature of dPCR is the separation of the reaction mixture into thousands to millions of partitions, which is followed by a real-time or end-point detection in each partitioned reaction. The distribution of target sequences into partitions (droplets) is described by the Poisson distribution, thus allowing the accurate and absolute quantification of the target from the ratio of positive against all partitions at the end of the reaction. This omits the need to use reference materials with known target concentrations and increases the accuracy of quantification at low target concentrations compared to qPCR. The ddPCR has also shown higher resilience to inhibitors in a number of different types of samples. This is the first application of ddPCR to detect a pathogen in cannabis plants and the first demonstration of its use for the quantification of HLVd. A comparison of the whole-genome sequences of HLVd from hop, hemp, and cannabis plants worldwide using phylogenetic analysis revealed a lack of diversity among the sequences included. Two single-nucleotide polymorphisms (SNPs) detected did not influence the overall alignment of the sequences, and all isolates were placed into one large group. In contrast to HLVd, a related viroid—potato spindle tuber viroid—shows considerably more sequence heterogeneity among isolates from different hosts and regions . Continuous monitoring for any potential changes in the genome of HLVd will be needed to detect possible variants that may arise in the near future as the pathogen continues to spread and evolve. In BCTV, the evolution of new strains (biotypes) has been shown to occur in different regions where hemp and other crops are cultivated, and currently up to 11 strains have been identified . In TSV, a variant found to be present in hemp plants had only 80–83% sequence similarity to previous strains infecting other crops . Variation in the CasaMV1 sequences from hemp plants was also observed, with 88–99% nt identity to sequences from C. sativa . The CYVaV sequences had 90% nt identity with CYVaV identified from citrus . Therefore, there is some evidence for the potential evolution of new and genetically diverse virus/viroid strains that can infect and become established in hemp and cannabis crops. Whether the virulence patterns have been altered in these strains has not been established. Similarly, whether commonly encountered and widespread viruses on other crops, such as AMV, CMV, and TMV, will become problematic on cannabis and hemp crops remains to be seen. Continuing bioinformatics studies on populations of viruses and viroids that may be present in cannabis and hemp plants are needed. As well, the impact on host plant growth and development following pathogen infection and reproduction need to be established, preferably in studies involving artificial inoculations. Current reports of virus/viroid presence need to include further investigations into their potential role in causing symptoms. An extensive review of the potential impact of viruses/viroid on cannabis and hemp (historical and current) has been recently published by Miotti et al. . Some potential aphid-transmissible viruses that can infect cannabis and hemp plants under artificial inoculation conditions, but have not yet become widespread under commercial conditions, include AMV, CMV, and PVY . Viruses vectored by thrips which can also pose a threat include Tobacco streak virus and Tomato spotted wilt virus . Many of these emerging viruses are potentially also seed-borne . These observations suggest that cannabis and hemp crops are susceptible to a range of viruses, and that the insect-vectored pathogens appear to have the greatest potential to cause damage under widespread cultivation conditions, with mechanically transmitted pathogens less so. The exception to this is HLVd, which is mechanically transmitted and is presently widespread on cannabis . Therefore, the development of diagnostic assays that can be applied in seed-testing programs will be important for the cannabis and hemp industries moving forward. Such testing programs are currently unavailable in many production areas. In the present study, HLVd presence on cannabis and hemp seeds was confirmed by RT-PCR. Given the high titer of viroid present in cannabis inflorescence tissues (which consist of clusters of pistils) of a susceptible genotype, fertilization of the ovules contained in these infected tissues by pollen originating from a male plant would likely yield a high frequency of seed-borne transmission, which was demonstrated in this study for HLVd. Infection during seed development may cause seed abortion and a reduction in the seed weight of surviving seeds. While it was not determined whether HLVd was present on the seed coat or internally within the seed, it is likely to be both. The viroid was also detected on cannabis seeds that had been stored for more than 2 years (dating back to 2021), suggesting that seed stocks of cannabis should be tested prior to widespread distribution. The viroid levels differed significantly from one seed to the next, even within the same batch of seeds. This variability should be considered in seed-testing protocols or when establishing levels of transmission of the viroid to seedlings, since the resulting levels of the viroid may be variable. Seeds of hemp infected with HLVd gave rise to infected seedlings at a high frequency in the present study, but the symptomology on these plants has not been established. The diagnostic approaches described in this study should aid in the routine screening of plants and seeds for the range of pathogens currently reported to affect cannabis and hemp crops. A comparison of the prevalence of fungal and oomycete pathogens affecting cannabis and hemp crops to the viruses/viroid disease complex indicates that, while there are new reports of the occurrence of the former group of pathogens on these hosts in different geographic regions, there is no evidence for selection of new pathogen strains that are adapted to cannabis and hemp, i.e., the pathogens are shown to have originated and spread from previous crops or adjacent crops . A similar situation may be taking place with the virus/viroid pathogens, but preliminary evidence may suggest an evolving suite of these pathogens may be found infecting these crops in the future. The applications of the molecular diagnostic and bioinformatics methods described in this study should provide useful information to address the evolving challenges facing cannabis and hemp crops as a result of these evolving multiple and assorted pathogens, which could become a limiting factor to production in certain regions . 4.1. Detection of Fungal and Oomycete Pathogens on Cannabis 4.1.1. Symptoms and Pathogen Isolation Cannabis plants displaying symptoms that included stunting and yellowing of vegetative and flowering plants and showed internal stem discoloration ( a–c), as well as visible evidence of fungal growth on the inflorescences ( d–f), were obtained from indoor production sites and greenhouses in which the cultivation of a range of different genotypes (strains) occurred. To recover fungi and oomycetes from the roots, stems, and inflorescences, tissue pieces measuring 0.5 mm 2 were taken from symptomatic plants and surface-sterilized by immersing them in 10% bleach (0.525% NaOCl) for 30 s, followed by 70% EtOH for 30 s, and three rinses in sterile distilled water. The pieces were transferred to potato dextrose agar containing 140 mg/L streptomycin sulfate and incubated at room temperature (22–25 °C) for 5–7 days. Emerging colonies were subcultured onto fresh agar medium. Morphological criteria that included colony features and spore characteristics were used to tentatively assign a genus and species identification to the various colonies that were recovered. These cultures were then used to conduct the molecular analysis, as described below. Symptomatic plant tissues were also used directly for DNA extraction, as described below, and used for PCR. DNA from noninfected cannabis tissues was included as a control. 4.1.2. PCR Analysis DNA was extracted from 50–100 mg of active growing mycelium or from 50 mg of plant tissues using the QIAGEN DNeasy Plant Mini Kit (Hilden, Germany) (cat. no. 69104). A pair of universal eukaryotic primers that amplified the ribosomal DNA in the internal transcribed region (ITS1-5.8S-ITS2) was used to amplify each sample. The primers (UN-UP18S42 (5′-CGTAACAAGGTTTCCGTAGGTGAAC-3′) and UN-LO28S576B (5′-GTTTCTTTTCCTCCGCTTATTAATATG-3′) were used with the following PCR conditions: initial denaturation at 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 7 min, followed by a 4 °C hold. PCR bands were cut from the gel, collected using the MinElute Gel Extraction Kit (Valencia, CA, USA) (cat. no. 28604), and 8 uL was sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. The resulting sequences were compared to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST to confirm species identity, using cut-off values >99%. Representative sequences were deposited in GenBank. 4.2. Detection of Viral Pathogens on Cannabis 4.2.1. Symptoms Leaves of cannabis plants displaying foliar symptoms of mosaic, mottling, chlorosis, and line patterns that resembled those caused by viral infection were collected from several genotypes. These symptoms were observed on vegetative and flowering plants of genotypes ‘OG Kush’ (OG), ‘Headband’ (HB), ‘Motor Breath’ (MB), and ‘Golden Papaya’ (GP). Leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if the observed symptoms observed were caused by viruses. The samples were collected on various times during 2021–2022. 4.2.2. PCR with Broad-Spectrum and Specific Primer Sets Leaf tissues from the plants of genotypes OG and HB were used for the total RNA extraction using an RNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD, USA). A small portion of the extracted RNA was converted to cDNA using the two-step RT-PCR system, iScriptTM Select cDNA Synthesis Kit (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). PCR was conducted on the cDNA samples generated using individual degenerate broad-spectrum primer sets under the conditions described in . These primer sets were designed to detect viruses belonging to one of the following genera: Tobamovirus, Nepovirus, Potyvirus, and Ilarvirus, or to the species Turnip ringspot virus (Comovirus), Alfalfa mosaic virus (Alfamovirus), Tobacco mosaic virus (Tobamovirus), and Cucumber mosaic virus (Cucumovirus). Control tobacco tissues ( Nicotiana tabacum cv. ‘Samsun’) infected with the tobacco mosaic virus U1 strain and alfalfa mosaic virus were also included (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada). Any amplified PCR products of the expected size for the specific primer set used were then purified with the MinElute PCR Purification Kit (Qiagen Sciences, Germantown, MD, USA) and sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. 4.2.3. Transmission Electron Microscopy Sap samples from symptomatic leaf tissues of the cannabis genotype MB, as well as from N. tabacum with symptoms of Tobacco mosaic virus previously inoculated with a confirmed TMV strain (U1 strain) (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada), were obtained by grinding leaves with a mortar and pestle. The preparation method for the crude leaf extracts was performed as per Hitchborn and Hills . The sample was observed with a Hitachi H-7100 Transmission Electron Microscope (TEM) (100 kv) and imaged in Gatan Digital Micrograph software (v. 2.31.734.0; Gatan Inc., Pleasanton, CA, USA). Virions were searched for systematically from top to bottom, from left to right. Preliminary searches were done at 5000–6000× magnification, and increased to 30,000–40,000× magnification for the imaging and measurement of individual virions. Fifty virions (if present) were imaged from each host sample, and the length and diameter of each was measured in the Gatan Digital Micrograph software. 4.2.4. Host Range Studies A mechanical transmission study utilizing select plant species was conducted by sap inoculations using symptomatic cannabis leaf tissues from genotypes OG and HB as source inoculum. The following plant species were included: Nicotiana clevelandii (Cleveland’s tobacco), N. glutinosa (Peruvian tobacco), N. tabacum ‘Samsun’ (cultivated tobacco), Chenopodium quinoa (quinoa), C. amaranticolor (goosefoot), Gomphrena globosa (globe amaranth) (all seeds were provided by Agriculture and Agri-Food Canada), Solanum lycopersicoides (tomato), Cucumis sativus (cucumber) (seeds were purchased from West Coast Seeds), and Urtica diocea (stinging nettle plants originating from rhizomes collected from an outdoor forested location). Seeds of all plants (except Urtica diocea ) were planted in a cocofibre:perlite potting medium (3:1) and placed under a 24 h photoperiod for 3 weeks, after which they were transplanted into individual pots and fertilized with a 20:20:20 (N:P:K) fertilizer. One week later, the plants were transferred to complete darkness for 24 hr prior to inoculation. Leaves of all plants were dusted with 320 grit carborundum (Thermo Fisher Scientific, Waltham, MA, USA) and gently rubbed with a leaf slurry prepared by grinding 0.5–1 g of tissue from each genotype in 1–2 mL 50 mM sodium phosphate buffer (PO 4 ) at a pH of 7.5. Inoculated and control plants ( n = 3–5 for each set of inoculated or control plants per species) were maintained under a 12:12 hr photoperiod and observed daily for symptoms for up to 3 weeks . 4.2.5. High-throughput Sequencing (HTS) Leaves from cannabis genotypes OG and HB were taken from plants displaying symptoms of mottling and mosaic , as well from plants of 6 additional genotypes, many of which exhibited stunting and reduced inflorescence growth , for HTS. These genotypes were ‘Powdered Donuts’ (PD), ‘Mac-1’ (Mac), ‘Pink Kush’ (PK), ‘Black Cherry’ (BC), CBD, and G54-2. Approximately 1 g of leaf tissue was used for virus and viroid double-stranded RNA (dsRNA) extraction. Extraction of dsRNA, removal of genomic DNA and single-stranded RNA (ssRNA), and construction of cDNA libraries were performed as described by Su et al. . The cDNA libraries were paired-end sequenced on a HiSeq 4000 platform (Illumina) by Applied Biological Materials Inc. (Richmond, BC, Canada). Sequence reads were trimmed to remove low-quality reads and the adaptor sequences, and assembled using the de novo assembly algorithm of the CLC Genomics Workbench v20 (Qiagen Sciences, Germantown, MD, USA). Assembled sequences were compared to a database of known viruses derived from the National Center for Biotechnology Information (NCBI) GenBank database. 4.3. Molecular Assays for Viroid and Other Viral Pathogens 4.3.1. RNA Extractions and cDNA Preparation In addition to the virus-like symptoms shown in , cannabis plants representing eight genotypes with symptoms of stunting, reduced stem growth, and poor development of the inflorescences were subjected to molecular analysis. These genotypes were PD, Mac, PK, BC, CBD, G54-2, HB, and OG. Leaf and flower tissues were subjected to RT-PCR using primers that were developed as described below (see ). For total RNA extractions, fresh leaf samples were placed in paper bags and freeze-dried using a Genesis 25 Freeze Drier (SP Industries Inc., Gardiner, NY, USA), after which the leaves were gently pulverized to create a semihomogenous crumble mix inside the bags. Approximately 500 mg of tissue was transferred to universal extraction bags (Bioreba AG, Reinach, Switzerland) and suspended in 5 mL of UltraPure water (Thermo Fisher Scientific, Waltham, MA, USA). The samples were ground using a HOMEX 6 homogenizer (Bioreba AG) and homogenate saps were transferred to 1.5 mL tubes using disposable transfer pipettes. From these tubes, 100 µL of homogenate sap was transferred to 2.0 mL tubes containing 300 µL of RB buffer (Omega Bio-tek, Norcross, GA, USA) with fresh 0.21% ( v / v ) β-mercaptoethanol (Thermo Fisher Scientific). Samples were loaded onto a QIAcube Connect (Qiagen, Germantown, MD, USA) for RNA extraction using an E.Z.N.A. ® Plant RNA Kit (Omega Bio-tek) and the QIAcube program for the RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). The elution volume was set to 50 µL and the eluted RNA was stored at −20 °C. RNA concentrations and purity were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) . The cDNA was generated using a SuperScript™ VILO™ Master Mix kit (Thermo Fisher Scientific) and 500 ng of template RNA per reaction. The following thermal cycling protocol was used on a T100™ Thermal Cycler (Bio-Rad, Hecules, CA, USA): lid temperature set at 105 °C; primer binding at 25 °C for 10 min; reverse transcription at 50 °C for 10 min; reaction termination at 85 °C for 5 min, then held at 10 °C until storage at −20 °C. 4.3.2. Primer Design for RT-PCR Using the HTS data from cannabis leaf samples of genotypes HB and OG, primers were either designed from this study (specifically for hop latent viroid (HLVd) and CasaMV1 mitovirus) or from previously published reports . Primers were designed using Primer-BLAST and specificity was tested against all plant, virus, bacteria, and fungal sequences in the nonredundant nucleotide collection (nr) from the National Center for Biotechnology Information (NCBI) GenBank database. Sequencing primer specificity was checked to ensure nonspecifics were not generated. To achieve coverage of the primed regions, two sequencing primer sets were evaluated: HLVd seq F1/HLVd seq R1 and HLVd quant F1/HLVd seq R2. Specificity was checked by PCR of 1 µL of generated cDNA in 20 µL reactions of the Q5 ® High-Fidelity 2X Master Mix (New England Biobabs, Ipswich, MA, USA) with sequencing primers at final concentrations of 500 nM. The following thermal cycling protocol was used: lid temp set at 105 °C, initial denaturation at 98 °C for 30 s; 30 cycles of 98 °C; denaturation for 10 s; 58 °C annealing for 30 s; 72 °C elongation for 80 s; a final elongation at 72 °C for 7 min, and held at 10 °C until stored at −20 °C. Following PCR, these were electrophoresed in 2% agarose (Thermo Fisher Scientific) at 100 V for 60 min in 0.5x TBE buffer (Thermo Fisher Scientific). The agarose gel was poststained in 1x SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific) in 0.5x TBE (Thermo Fisher Scientific) for 30 min. The stained gel was imaged using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). In the case of both primer sets, nonspecific bands were not observed, although multiple bands (multimers) were seen due to the circular nature and small size of the HLVd genome. 4.4. Sequence Diversity and Molecular Phylogeny of Hop Latent Viroid The evolutionary history was inferred by using the maximum likelihood method and Kimura 2-parameter model . The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 70% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches . Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. This analysis involved 21 nucleotide sequences from GenBank. Only full-length sequences were used, and selections were chosen to represent wide geographic regions. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 185 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 . 4.5. Droplet Digital PCR Quantitative primer specificity was also tested using the primer sets HLVd quant F1/HLVd quant F2 and Cannabis EF1α F/Cannabis EF1α R . Specificity was evaluated using only the cDNA generated (see above) from the RNA of one cannabis genotype (PD). PCR was conducted using 20 µL reactions of QX200™ ddPCR™ EvaGreen Supermix (Bio-rad) with quantitative primers at final concentrations of 150 nM, 3 µL of 10 −2 diluted PD cDNA template per reaction, and without generating droplets. The PCRs were conducted across a 5-point thermal gradient ranging from 55.8 °C to 62 °C to determine the ideal annealing temperature. Following PCR, these were electrophoresed, stained, and imaged, as described above. Neither primer set produced nonspecific bands, and both sets performed equally well across the thermal gradient. A temperature of 58 °C was selected for the subsequent ddPCR, as higher temperatures can produce inadequate separation between positive and negative droplets, termed ‘rain’. A total of five cannabis genotypes were tested by ddPCR, and the levels of HLVd present were quantified. 4.6. Multiplex Taqman RT-qPCR Tissues were obtained from the flowering plants of genotypes PD and Mac showing symptoms, as seen in . Approximately 100 mg of fresh inflorescence tissues were placed directly into 750 uL of RNAlater ® solution (Thermo Fisher Scientific) in a 2 mL screw-cap tube and placed on ice. The samples were either processed the same day or stored overnight at 4 °C and processed the following day. Total nucleic acids were extracted as described by Mark et al. , with slight modifications. The RNAlater ® was removed from the 2 mL tube followed by adding 2.3 mm diameter zirconia–silica beads and 1.0 mm diameter glass beads (BioSpec, Bartlesville, OK, USA). The tissue was then macerated using a TissueLyser (Qiagen) at 25 Hz for 1 min. Once homogenized, 1 mL of extraction buffer (2% CTAB, 2% PVP40,000, 25 mM EDTA at a pH of 8, 100 mM Tris-HCl at a pH of 8, and 2.5 mM NaCl) prewarmed at 65 °C was added to each sample and incubated at 65 °C for 10 min. The homogenate was centrifuged at max speed (>16,000× g ) for 5 min at 4 °C and the supernatant transferred to a new sterile 1.5 mL Eppendorf tube. An equal volume (750 µL) of chloroform isoamyl alcohol (24:1) was added and centrifuged at max speed (>16,000× g ) for 5 min. The supernatant (550 uL) was transferred to a new 1.5 mL Eppendorf tube, and 0.6 volumes of cold isopropanol were added and centrifuged again at max speed (>16,000× g ) for 5 min. Isopropanol was decanted and about 650 µL of 70% ethanol was added onto the pellets, centrifuged at max speed (>16,000× g ) for 3 min, and air-dried before resuspending into 40 µL of nuclease-free water. RT-qPCR was performed on a Bio-Rad CFX96 instrument using TaqPath™ 1-Step Multiplex Master Mix (No ROX) (Thermo Fisher Scientific). The reagents were brought to room temperature prior to prevent infrequent nonspecific signal increases found when master mixes are prepared on ice. Each 20 μL reaction mixture contained 5 μL of 4 × TaqPath 1-Step Multiplex Master Mix (No ROX; Thermo Fisher Scientific), 1 μL of primer/probes mix, and 4 μL of extracted total nucleic acid. The primers used are shown in . The final concentrations of primers and probes were 0.3 μM (target primers), 0.05 μM (target probes), 0.15 μM (internal control primer), and 0.05 μM (internal control probe). The cycling program on a Bio-Rad CFX96 instrument was as follows: uracil–DNA glycosylase incubation at 25 °C for 2 min; reverse transcription at 53 °C for 10 min; polymerase activation at 95 °C for 2 min; 40 cycles of PCR at 95 °C for 3 s and 60 °C for 30 s (signal acquisition). The filter combinations were 465–510 (FAM; UBQ P), 540–580 (HEX; HLVd P1), and 610–670 (Cy5; HLVd P2). 4.7. LAMP Assays Tissues were sampled from plants showing symptoms, as illustrated in , as well as from asymptomatic plants (showing no visible alteration of growth). Leaves, petioles, and roots were collected from 11 cannabis genotypes for comparison. Plants ranged in age from 6 to 10 weeks after the initiation of cuttings. Approximately 100 mg of fresh tissues were placed directly into 750 uL of RNAlater ® solution (Thermo Fisher Scientific) in a 2 mL screw-cap tube and placed on ice. The samples were either processed the same day or stored overnight at 4 °C and processed the following day. Total nucleic acids were extracted as described by Mark et al. , with slight modifications. The RNAlater ® was removed from the 2 mL tube followed by adding 2.3 mm diameter zirconia–silica beads and 1.0 mm diameter glass beads (BioSpec, Bartlesville, OK, USA). The tissue was then macerated using a TissueLyser (Qiagen, Germantown, MD, USA) at 25 Hz for 1 min. Once homogenized, 1 mL of extraction buffer (2% CTAB, 2% PVP40,000, 25 mM EDTA at a pH of 8, 100 mM Tris-HCl at a pH of 8, and 2.5 mM NaCl) prewarmed at 65 °C was added to each sample and incubated at 65 °C for 10 min. The homogenate was centrifuged at max speed (>16,000× g ) for 5 min at 4 °C and the supernatant transferred to a new sterile 1.5 mL Eppendorf tube. An equal volume (750 µL) of chloroform isoamyl alcohol (24:1) was added and centrifuged at max speed (>16,000× g ) for 5 min. The supernatant (550 uL) was transferred to a new 1.5 mL Eppendorf tube, and 0.6 volumes of cold isopropanol were added and centrifuged again at max speed (>16,000× g ) for 5 min. Isopropanol was decanted, and about 650 µL of 70% ethanol was added onto the pellets, centrifuged at max speed (>16,000× g ) for 3 min, and air-dried before resuspending into 40 µL of nuclease-free water. RT-LAMP primer sequences were designed using New England Biolabs ® (NEB) LAMP primer design tool and synthesized by Integrated DNA Technologies (Coraville, IA, USA) . RT-LAMP reactions were performed using WarmStart ® Fluorescent LAMP/RT-LAMP Kit (with UDG) (New England Biolabs, Whitby, ON, Canada; E1708) with standard primer concentrations (0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.4 μM Loop F, 0.4 μM Loop B) in 25 μL on 96-well plates at 65 °C for 30 min in a Bio-Rad CFX96 instrument. 4.8. Monitoring Distribution of HLVd in Various Plant Tissues 4.8.1. Distribution within Stock Plants To assess how specific diagnostic assays can be used to monitor the presence of HLVd in different tissues of cannabis plants, RT-PCR with primers HLVd seq F1/HLVd seq R1 were used according to the conditions specified above . Stock (mother) plants of several genotypes that were 3–4 months old were sampled at different positions on the plant—leaves were collected from the top (youngest growth), middle, and bottom (oldest growth), as well as from roots. The plants did not display any obvious symptoms of disease, such as stunting or leaf curl . Each leaf sample (approximately 5 gm fresh weight) was frozen at −80 C until used. The extractions were conducted using the Qiagen RNeasy Plant Mini Kit (cat. #74904) according to the manufacturer’s instructions. The final RNA product was eluted with 52 µL nuclease-free H 2 O. The QIAGEN OneStep RT-PCR Kit (cat. #210212) was used for reverse transcription and PCR amplification. The reaction mixture contained 14 µL of water, 5 µL of 5x reaction buffer, 1 µL of dNTPs (10 mM), 1.5 µL each of primers HLVd seq F1/HLVd seq R1, 1 µL of RNA template, and 1 µL of enzyme mix, resulting in a total volume of 25 µL. All PCR amplifications were performed in a MyCycler thermocycler (BIORAD) with the following program: 30 min at 50 °C, 15 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, 60 s at 72 °C, and final extension at 72 °C for 10 min. The resulting PCR products were run on a 1% TAE agarose gel, and images were captured with E-gel imager (Life Technologies, Carlsbad, CA, USA). Bands of the expected size (ca. 256 and 512 bp) were purified with QIAquick Gel Extraction Kit and sent to Eurofins Genomics (Eurofins MWG Operon LLC 2016, Louisville, KY, USA) for sequencing. The resulting sequences were compared to the corresponding HpLVd sequences from the National Centre for Biotechnology Information (NCBI) GenBank database to confirm identity. In addition, primers HLVd quant F1/HLVd quant F2 were also used to quantify viroid levels in the same tissues, as described in above. 4.8.2. Distribution within Inflorescence Tissues Mature inflorescences were obtained at harvest from a plant (approximately 12 weeks of age that had been grown under a photoperiod of 12:12 hr to induce flowering for 8 weeks) of genotype ‘Mac-1’ which was previously confirmed to be infected by HLVd. The leaves surrounding the inflorescence that included large fan leaves and smaller inflorescence leaves were manually dissected from the inflorescence using a scalpel and used for RNA extraction, followed by RT-PCR and ddPCR to compare the relative viroid concentrations in these tissues relative to that present in the whole inflorescence . 4.8.3. Distribution of HLVd within Cuttings from Infected Stock Plants Vegetative cuttings were taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd and were rooted and tested for HLVd presence using RT-PCR and RT-qPCR after 14 days. The conditions for rooting are described elsewhere . A total of 7 cuttings were subjected to RT-PCR, and 4 of the cuttings were also analyzed by RT-qPCR, and the results were compared. 4.8.4. Presence of Pathogens in Fresh and Dried Cannabis Inflorescences Following harvest of inflorescences from a number of genotypes of cannabis plants, both fresh and dried flowers were tested for the presence of specific pathogens by PCR with the universal primers for fungal/oomycete pathogens, and by RT-PCR for HLVd. The fresh flowers were tested immediately after they were obtained by removing inflorescence leaves and subjecting them to PCR, as described previously . For dried samples, flowers were subjected to commercial drying conditions (5 days at 20–22 °C and 50–55% relative humidity), after which the tissue samples were obtained and subjected to PCR with HLVd primers, as described in . Up to 20 fresh and dried samples were included in the analysis. Resulting bands observed in these analyses following PCR were collected and sent for sequencing to confirm which pathogens were present. 4.8.5. Detection of HLVd and Mitovirus in Cannabis Seed A cross was made between an infected ‘Mac1′ female plant and pollen from a healthy male plant of genotype ‘GPie’ by collecting pollen and manually transferring to the stigmatic surfaces. The plants were grown in isolation after crossing for 10 weeks, after which seeds that had developed were collected and frozen at −80 C. Individual seeds were ground and the RNA was extracted, as previously described, and subjected to RT-PCR using HLVd primers and CasaMV1 primers. 4.9. Detection of Beet Curly Top Virus Cannabis plants with symptoms of stunting, extensive curling of young leaves, and twisted and deformed stem growth were used. DNA from approximately 100 mg of stem tissues from these plants was extracted with a Qiagen DNeasy extraction kit and eluted into 100 uL of elution buffer. The extracted DNA was then diluted 1:10 in TE buffer before PCR. The PCR was carried out in 20 uL reactions using Thermo Fisher DreamTaq according to the manufacturer’s instructions. Primer sequences used are shown in . The PCR conditions were 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 58 °C for 60 s, 72 °C for 90 s, and a final extension of 72 °C for 10 min. The PCR products were run on a 2% TAE agarose gel at 100 V for 30 min before imaging. 4.10. Diagnostic Approaches to Detect Viral and Viroid Pathogens Affecting Low THC-Containing Cannabis sativa L. (hemp) 4.10.1. Next-Generation Sequencing (NGS) Leaf samples were obtained from several outdoor hemp fields in Colorado during 2019, 2021, and 2022, and subjected to shotgun metagenomic analysis, as described by Chiginsky et al. . In most cases, samples were taken from symptomatic plants, as shown in . In each year, total RNA was extracted from a composite of 3–5 leaves from outdoor hemp-production fields in Colorado. Approximately 100 mg of leaf tissue samples was placed in a 2 mL microcentrifuge tube and stored at −20 °C until nucleic acid extraction was performed. Briefly, total RNA was extracted using the Qiagen Plant RNeasy kit and checked for the concentration using a Nanodrop One spectrophotometer (Thermo Fisher Scientific), and for quality using a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Approximately 2 μg of RNA was submitted to the Colorado State University Next Generation Sequencing Facility, where library preparation, quality measurements, and sequencing were performed. Briefly, RNA quality was confirmed using an Agilent Tapestation instrument. Shotgun RNA libraries were constructed using the Kapa Biosystems RNA HyperPrep kit (Roche, IN, USA) according to the manufacturer’s instructions. Pooled libraries were sequenced on an Illumina NextSeq 500 instrument (Baltimore, MD, USA) to produce single-end 150 nucleotide (nt) reads. Bioinformatic analysis for the 2019 hemp samples are described in Chiginsky et al. . Bioinformatic analysis for the 2021 and 2022 hemp samples was performed using CLC Genomics Work Bench (Qiagen). Reads were mapped to the hemp reference genome (assembly accession GCF_900626175.1). Remaining nonhost reads were assembled through the de novo assembly algorithm from the unmapped reads. Contigs and nonassembling reads were taxonomically categorized first by nucleotide-level alignment to the NCBI nucleotide (nt) database using Blastn, and then by protein-level alignment to the NCBI protein (nr) database. This produced a comprehensive classification of all nonhost reads. Candidate virus sequences were manually validated by aligning reads to draft genome sequences using bowtie2. Lastly, the raw sequence data were deposited in the NCBI Sequence Read Archive (SRA). 4.10.2. RT-PCR with Specific Primer Sets The universal BCTV primers, BCTV2- F, and BCTV2-R were used to amplify a 496 bp fragment of the coat protein (CP) gene, a region that is conserved among BCTV strains . The amplification cycle consisted of a 94 °C initial denaturation for 5 min, 25 cycles of denaturation at 94 °C for 1 min, 58 °C annealing for 2 min, and 72 °C extension for 2 min, followed by a 10 min final extension. The GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) was used. All PCR products were visualized on 1% agarose gel. The PCR products were excised from the agarose gels and purified using the DNA Clean and Concentrator™-5 (Zymo Research, Irvine, CA, USA). One to two PCR products were randomly selected and submitted for Sanger sequencing at Genewiz Inc. to confirm the virus identity. The sequences for each BCTV strain were checked for identity against the nonredundant (nr) database using Blastn in the NCBI database. To confirm the presence of low-percentage nucleotide (nt)-identity viruses (<90%), 1 μg of total RNA was used to synthesize the cDNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific) according to manufacturer’s instructions. For additional viruses, including Cannabis sativa mitovirus (CasaMV1), citrus yellow-vein-associated virus (CYVaV), and tobacco streak virus (TSV), the primers used are shown in . The RT-PCR was performed using GoTaq R Flexi DNA polymerase (Promega, Madison, WI, USA). The amplification cycle consisted of 2 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 35 s at 72 °C, followed by 5 min at 72 °C for all viruses except for citrus yellow-vein-associated virus (CYVaV), which had Tm of 51 °C. 4.10.3. Sequence Diversity and Molecular Phylogeny of Beet Curly Top Virus All currently available nucleotide sequences of BCTV capsid proteins (BCTV-CP) were retrieved from the GenBank database, after which their strains and sources of origins (hosts where viruses were identified) were determined according to the featured information in GenBank. Sequences representing each of the 11 BCTV strains were selected based on Strausbaugh et al. , and included 10 California/Logan, 8 Colorado, 2 Kimberly1, 10 Leafhopper71, 5 Mild, 10 Severe, 8 Worland, 6 Spanish curly top, 1 Pepper yellow dwarf, 2 Pepper curly top, and 1 Severe pepper. A total of 35 BCTV-CP sequences were found to be identified from Cannabis sativa , and all were included in the phylogenetic analysis. Multiple sequence alignments (total 98 sequences) were performed using MAFFT v7.505 , and the poorly aligned regions were trimmed using TrimAI v1.2.59 . The phylogenetic tree was constructed using the maximum likelihood method implemented in the RAxML v8.2.12 with the GTR+G+I model for nucleotide substitution through the CIPRES Science Gateway Environment . The robustness of each internal branch was estimated with 1000 bootstrap replications. The phylogenetic tree was visualized using FigTree v1.4.4 . 4.10.4. Detection of HLVd on Hemp Seeds and on Thrips A sample of hemp seeds from a commercial supplier was frozen at −80 °C, and then individual seeds were used to extract total RNA, as described in above. In addition, a sample containing 8 adult thrips found feeding on the leaves of plants derived from these seeds at the true leaf stage were collected using a pair of forceps, and also frozen at −80 °C prior to analysis. 4.1.1. Symptoms and Pathogen Isolation Cannabis plants displaying symptoms that included stunting and yellowing of vegetative and flowering plants and showed internal stem discoloration ( a–c), as well as visible evidence of fungal growth on the inflorescences ( d–f), were obtained from indoor production sites and greenhouses in which the cultivation of a range of different genotypes (strains) occurred. To recover fungi and oomycetes from the roots, stems, and inflorescences, tissue pieces measuring 0.5 mm 2 were taken from symptomatic plants and surface-sterilized by immersing them in 10% bleach (0.525% NaOCl) for 30 s, followed by 70% EtOH for 30 s, and three rinses in sterile distilled water. The pieces were transferred to potato dextrose agar containing 140 mg/L streptomycin sulfate and incubated at room temperature (22–25 °C) for 5–7 days. Emerging colonies were subcultured onto fresh agar medium. Morphological criteria that included colony features and spore characteristics were used to tentatively assign a genus and species identification to the various colonies that were recovered. These cultures were then used to conduct the molecular analysis, as described below. Symptomatic plant tissues were also used directly for DNA extraction, as described below, and used for PCR. DNA from noninfected cannabis tissues was included as a control. 4.1.2. PCR Analysis DNA was extracted from 50–100 mg of active growing mycelium or from 50 mg of plant tissues using the QIAGEN DNeasy Plant Mini Kit (Hilden, Germany) (cat. no. 69104). A pair of universal eukaryotic primers that amplified the ribosomal DNA in the internal transcribed region (ITS1-5.8S-ITS2) was used to amplify each sample. The primers (UN-UP18S42 (5′-CGTAACAAGGTTTCCGTAGGTGAAC-3′) and UN-LO28S576B (5′-GTTTCTTTTCCTCCGCTTATTAATATG-3′) were used with the following PCR conditions: initial denaturation at 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 7 min, followed by a 4 °C hold. PCR bands were cut from the gel, collected using the MinElute Gel Extraction Kit (Valencia, CA, USA) (cat. no. 28604), and 8 uL was sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. The resulting sequences were compared to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST to confirm species identity, using cut-off values >99%. Representative sequences were deposited in GenBank. Cannabis plants displaying symptoms that included stunting and yellowing of vegetative and flowering plants and showed internal stem discoloration ( a–c), as well as visible evidence of fungal growth on the inflorescences ( d–f), were obtained from indoor production sites and greenhouses in which the cultivation of a range of different genotypes (strains) occurred. To recover fungi and oomycetes from the roots, stems, and inflorescences, tissue pieces measuring 0.5 mm 2 were taken from symptomatic plants and surface-sterilized by immersing them in 10% bleach (0.525% NaOCl) for 30 s, followed by 70% EtOH for 30 s, and three rinses in sterile distilled water. The pieces were transferred to potato dextrose agar containing 140 mg/L streptomycin sulfate and incubated at room temperature (22–25 °C) for 5–7 days. Emerging colonies were subcultured onto fresh agar medium. Morphological criteria that included colony features and spore characteristics were used to tentatively assign a genus and species identification to the various colonies that were recovered. These cultures were then used to conduct the molecular analysis, as described below. Symptomatic plant tissues were also used directly for DNA extraction, as described below, and used for PCR. DNA from noninfected cannabis tissues was included as a control. DNA was extracted from 50–100 mg of active growing mycelium or from 50 mg of plant tissues using the QIAGEN DNeasy Plant Mini Kit (Hilden, Germany) (cat. no. 69104). A pair of universal eukaryotic primers that amplified the ribosomal DNA in the internal transcribed region (ITS1-5.8S-ITS2) was used to amplify each sample. The primers (UN-UP18S42 (5′-CGTAACAAGGTTTCCGTAGGTGAAC-3′) and UN-LO28S576B (5′-GTTTCTTTTCCTCCGCTTATTAATATG-3′) were used with the following PCR conditions: initial denaturation at 94 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 45 s, extension at 72 °C for 2 min, and a final extension at 72 °C for 7 min, followed by a 4 °C hold. PCR bands were cut from the gel, collected using the MinElute Gel Extraction Kit (Valencia, CA, USA) (cat. no. 28604), and 8 uL was sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. The resulting sequences were compared to ITS1-5.8S-ITS2 sequences from the National Center for Biotechnology Information (NCBI) GenBank database using BLAST to confirm species identity, using cut-off values >99%. Representative sequences were deposited in GenBank. 4.2.1. Symptoms Leaves of cannabis plants displaying foliar symptoms of mosaic, mottling, chlorosis, and line patterns that resembled those caused by viral infection were collected from several genotypes. These symptoms were observed on vegetative and flowering plants of genotypes ‘OG Kush’ (OG), ‘Headband’ (HB), ‘Motor Breath’ (MB), and ‘Golden Papaya’ (GP). Leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if the observed symptoms observed were caused by viruses. The samples were collected on various times during 2021–2022. 4.2.2. PCR with Broad-Spectrum and Specific Primer Sets Leaf tissues from the plants of genotypes OG and HB were used for the total RNA extraction using an RNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD, USA). A small portion of the extracted RNA was converted to cDNA using the two-step RT-PCR system, iScriptTM Select cDNA Synthesis Kit (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). PCR was conducted on the cDNA samples generated using individual degenerate broad-spectrum primer sets under the conditions described in . These primer sets were designed to detect viruses belonging to one of the following genera: Tobamovirus, Nepovirus, Potyvirus, and Ilarvirus, or to the species Turnip ringspot virus (Comovirus), Alfalfa mosaic virus (Alfamovirus), Tobacco mosaic virus (Tobamovirus), and Cucumber mosaic virus (Cucumovirus). Control tobacco tissues ( Nicotiana tabacum cv. ‘Samsun’) infected with the tobacco mosaic virus U1 strain and alfalfa mosaic virus were also included (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada). Any amplified PCR products of the expected size for the specific primer set used were then purified with the MinElute PCR Purification Kit (Qiagen Sciences, Germantown, MD, USA) and sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. 4.2.3. Transmission Electron Microscopy Sap samples from symptomatic leaf tissues of the cannabis genotype MB, as well as from N. tabacum with symptoms of Tobacco mosaic virus previously inoculated with a confirmed TMV strain (U1 strain) (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada), were obtained by grinding leaves with a mortar and pestle. The preparation method for the crude leaf extracts was performed as per Hitchborn and Hills . The sample was observed with a Hitachi H-7100 Transmission Electron Microscope (TEM) (100 kv) and imaged in Gatan Digital Micrograph software (v. 2.31.734.0; Gatan Inc., Pleasanton, CA, USA). Virions were searched for systematically from top to bottom, from left to right. Preliminary searches were done at 5000–6000× magnification, and increased to 30,000–40,000× magnification for the imaging and measurement of individual virions. Fifty virions (if present) were imaged from each host sample, and the length and diameter of each was measured in the Gatan Digital Micrograph software. 4.2.4. Host Range Studies A mechanical transmission study utilizing select plant species was conducted by sap inoculations using symptomatic cannabis leaf tissues from genotypes OG and HB as source inoculum. The following plant species were included: Nicotiana clevelandii (Cleveland’s tobacco), N. glutinosa (Peruvian tobacco), N. tabacum ‘Samsun’ (cultivated tobacco), Chenopodium quinoa (quinoa), C. amaranticolor (goosefoot), Gomphrena globosa (globe amaranth) (all seeds were provided by Agriculture and Agri-Food Canada), Solanum lycopersicoides (tomato), Cucumis sativus (cucumber) (seeds were purchased from West Coast Seeds), and Urtica diocea (stinging nettle plants originating from rhizomes collected from an outdoor forested location). Seeds of all plants (except Urtica diocea ) were planted in a cocofibre:perlite potting medium (3:1) and placed under a 24 h photoperiod for 3 weeks, after which they were transplanted into individual pots and fertilized with a 20:20:20 (N:P:K) fertilizer. One week later, the plants were transferred to complete darkness for 24 hr prior to inoculation. Leaves of all plants were dusted with 320 grit carborundum (Thermo Fisher Scientific, Waltham, MA, USA) and gently rubbed with a leaf slurry prepared by grinding 0.5–1 g of tissue from each genotype in 1–2 mL 50 mM sodium phosphate buffer (PO 4 ) at a pH of 7.5. Inoculated and control plants ( n = 3–5 for each set of inoculated or control plants per species) were maintained under a 12:12 hr photoperiod and observed daily for symptoms for up to 3 weeks . 4.2.5. High-throughput Sequencing (HTS) Leaves from cannabis genotypes OG and HB were taken from plants displaying symptoms of mottling and mosaic , as well from plants of 6 additional genotypes, many of which exhibited stunting and reduced inflorescence growth , for HTS. These genotypes were ‘Powdered Donuts’ (PD), ‘Mac-1’ (Mac), ‘Pink Kush’ (PK), ‘Black Cherry’ (BC), CBD, and G54-2. Approximately 1 g of leaf tissue was used for virus and viroid double-stranded RNA (dsRNA) extraction. Extraction of dsRNA, removal of genomic DNA and single-stranded RNA (ssRNA), and construction of cDNA libraries were performed as described by Su et al. . The cDNA libraries were paired-end sequenced on a HiSeq 4000 platform (Illumina) by Applied Biological Materials Inc. (Richmond, BC, Canada). Sequence reads were trimmed to remove low-quality reads and the adaptor sequences, and assembled using the de novo assembly algorithm of the CLC Genomics Workbench v20 (Qiagen Sciences, Germantown, MD, USA). Assembled sequences were compared to a database of known viruses derived from the National Center for Biotechnology Information (NCBI) GenBank database. Leaves of cannabis plants displaying foliar symptoms of mosaic, mottling, chlorosis, and line patterns that resembled those caused by viral infection were collected from several genotypes. These symptoms were observed on vegetative and flowering plants of genotypes ‘OG Kush’ (OG), ‘Headband’ (HB), ‘Motor Breath’ (MB), and ‘Golden Papaya’ (GP). Leaf tissues from these symptomatic plants were subjected to several diagnostic approaches, as described below, to determine if the observed symptoms observed were caused by viruses. The samples were collected on various times during 2021–2022. Leaf tissues from the plants of genotypes OG and HB were used for the total RNA extraction using an RNeasy Plant Mini Kit (Qiagen Sciences, Germantown, MD, USA). A small portion of the extracted RNA was converted to cDNA using the two-step RT-PCR system, iScriptTM Select cDNA Synthesis Kit (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada). PCR was conducted on the cDNA samples generated using individual degenerate broad-spectrum primer sets under the conditions described in . These primer sets were designed to detect viruses belonging to one of the following genera: Tobamovirus, Nepovirus, Potyvirus, and Ilarvirus, or to the species Turnip ringspot virus (Comovirus), Alfalfa mosaic virus (Alfamovirus), Tobacco mosaic virus (Tobamovirus), and Cucumber mosaic virus (Cucumovirus). Control tobacco tissues ( Nicotiana tabacum cv. ‘Samsun’) infected with the tobacco mosaic virus U1 strain and alfalfa mosaic virus were also included (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada). Any amplified PCR products of the expected size for the specific primer set used were then purified with the MinElute PCR Purification Kit (Qiagen Sciences, Germantown, MD, USA) and sent to Eurofins Genomics ( https://www.eurofinsgenomics.com/en/home/ , accessed on 1 October 2023) for sequencing. Sap samples from symptomatic leaf tissues of the cannabis genotype MB, as well as from N. tabacum with symptoms of Tobacco mosaic virus previously inoculated with a confirmed TMV strain (U1 strain) (provided by the Canadian Plant Virus Collection at Agriculture and Agri-Food Canada), were obtained by grinding leaves with a mortar and pestle. The preparation method for the crude leaf extracts was performed as per Hitchborn and Hills . The sample was observed with a Hitachi H-7100 Transmission Electron Microscope (TEM) (100 kv) and imaged in Gatan Digital Micrograph software (v. 2.31.734.0; Gatan Inc., Pleasanton, CA, USA). Virions were searched for systematically from top to bottom, from left to right. Preliminary searches were done at 5000–6000× magnification, and increased to 30,000–40,000× magnification for the imaging and measurement of individual virions. Fifty virions (if present) were imaged from each host sample, and the length and diameter of each was measured in the Gatan Digital Micrograph software. A mechanical transmission study utilizing select plant species was conducted by sap inoculations using symptomatic cannabis leaf tissues from genotypes OG and HB as source inoculum. The following plant species were included: Nicotiana clevelandii (Cleveland’s tobacco), N. glutinosa (Peruvian tobacco), N. tabacum ‘Samsun’ (cultivated tobacco), Chenopodium quinoa (quinoa), C. amaranticolor (goosefoot), Gomphrena globosa (globe amaranth) (all seeds were provided by Agriculture and Agri-Food Canada), Solanum lycopersicoides (tomato), Cucumis sativus (cucumber) (seeds were purchased from West Coast Seeds), and Urtica diocea (stinging nettle plants originating from rhizomes collected from an outdoor forested location). Seeds of all plants (except Urtica diocea ) were planted in a cocofibre:perlite potting medium (3:1) and placed under a 24 h photoperiod for 3 weeks, after which they were transplanted into individual pots and fertilized with a 20:20:20 (N:P:K) fertilizer. One week later, the plants were transferred to complete darkness for 24 hr prior to inoculation. Leaves of all plants were dusted with 320 grit carborundum (Thermo Fisher Scientific, Waltham, MA, USA) and gently rubbed with a leaf slurry prepared by grinding 0.5–1 g of tissue from each genotype in 1–2 mL 50 mM sodium phosphate buffer (PO 4 ) at a pH of 7.5. Inoculated and control plants ( n = 3–5 for each set of inoculated or control plants per species) were maintained under a 12:12 hr photoperiod and observed daily for symptoms for up to 3 weeks . Leaves from cannabis genotypes OG and HB were taken from plants displaying symptoms of mottling and mosaic , as well from plants of 6 additional genotypes, many of which exhibited stunting and reduced inflorescence growth , for HTS. These genotypes were ‘Powdered Donuts’ (PD), ‘Mac-1’ (Mac), ‘Pink Kush’ (PK), ‘Black Cherry’ (BC), CBD, and G54-2. Approximately 1 g of leaf tissue was used for virus and viroid double-stranded RNA (dsRNA) extraction. Extraction of dsRNA, removal of genomic DNA and single-stranded RNA (ssRNA), and construction of cDNA libraries were performed as described by Su et al. . The cDNA libraries were paired-end sequenced on a HiSeq 4000 platform (Illumina) by Applied Biological Materials Inc. (Richmond, BC, Canada). Sequence reads were trimmed to remove low-quality reads and the adaptor sequences, and assembled using the de novo assembly algorithm of the CLC Genomics Workbench v20 (Qiagen Sciences, Germantown, MD, USA). Assembled sequences were compared to a database of known viruses derived from the National Center for Biotechnology Information (NCBI) GenBank database. 4.3.1. RNA Extractions and cDNA Preparation In addition to the virus-like symptoms shown in , cannabis plants representing eight genotypes with symptoms of stunting, reduced stem growth, and poor development of the inflorescences were subjected to molecular analysis. These genotypes were PD, Mac, PK, BC, CBD, G54-2, HB, and OG. Leaf and flower tissues were subjected to RT-PCR using primers that were developed as described below (see ). For total RNA extractions, fresh leaf samples were placed in paper bags and freeze-dried using a Genesis 25 Freeze Drier (SP Industries Inc., Gardiner, NY, USA), after which the leaves were gently pulverized to create a semihomogenous crumble mix inside the bags. Approximately 500 mg of tissue was transferred to universal extraction bags (Bioreba AG, Reinach, Switzerland) and suspended in 5 mL of UltraPure water (Thermo Fisher Scientific, Waltham, MA, USA). The samples were ground using a HOMEX 6 homogenizer (Bioreba AG) and homogenate saps were transferred to 1.5 mL tubes using disposable transfer pipettes. From these tubes, 100 µL of homogenate sap was transferred to 2.0 mL tubes containing 300 µL of RB buffer (Omega Bio-tek, Norcross, GA, USA) with fresh 0.21% ( v / v ) β-mercaptoethanol (Thermo Fisher Scientific). Samples were loaded onto a QIAcube Connect (Qiagen, Germantown, MD, USA) for RNA extraction using an E.Z.N.A. ® Plant RNA Kit (Omega Bio-tek) and the QIAcube program for the RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). The elution volume was set to 50 µL and the eluted RNA was stored at −20 °C. RNA concentrations and purity were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) . The cDNA was generated using a SuperScript™ VILO™ Master Mix kit (Thermo Fisher Scientific) and 500 ng of template RNA per reaction. The following thermal cycling protocol was used on a T100™ Thermal Cycler (Bio-Rad, Hecules, CA, USA): lid temperature set at 105 °C; primer binding at 25 °C for 10 min; reverse transcription at 50 °C for 10 min; reaction termination at 85 °C for 5 min, then held at 10 °C until storage at −20 °C. 4.3.2. Primer Design for RT-PCR Using the HTS data from cannabis leaf samples of genotypes HB and OG, primers were either designed from this study (specifically for hop latent viroid (HLVd) and CasaMV1 mitovirus) or from previously published reports . Primers were designed using Primer-BLAST and specificity was tested against all plant, virus, bacteria, and fungal sequences in the nonredundant nucleotide collection (nr) from the National Center for Biotechnology Information (NCBI) GenBank database. Sequencing primer specificity was checked to ensure nonspecifics were not generated. To achieve coverage of the primed regions, two sequencing primer sets were evaluated: HLVd seq F1/HLVd seq R1 and HLVd quant F1/HLVd seq R2. Specificity was checked by PCR of 1 µL of generated cDNA in 20 µL reactions of the Q5 ® High-Fidelity 2X Master Mix (New England Biobabs, Ipswich, MA, USA) with sequencing primers at final concentrations of 500 nM. The following thermal cycling protocol was used: lid temp set at 105 °C, initial denaturation at 98 °C for 30 s; 30 cycles of 98 °C; denaturation for 10 s; 58 °C annealing for 30 s; 72 °C elongation for 80 s; a final elongation at 72 °C for 7 min, and held at 10 °C until stored at −20 °C. Following PCR, these were electrophoresed in 2% agarose (Thermo Fisher Scientific) at 100 V for 60 min in 0.5x TBE buffer (Thermo Fisher Scientific). The agarose gel was poststained in 1x SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific) in 0.5x TBE (Thermo Fisher Scientific) for 30 min. The stained gel was imaged using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). In the case of both primer sets, nonspecific bands were not observed, although multiple bands (multimers) were seen due to the circular nature and small size of the HLVd genome. In addition to the virus-like symptoms shown in , cannabis plants representing eight genotypes with symptoms of stunting, reduced stem growth, and poor development of the inflorescences were subjected to molecular analysis. These genotypes were PD, Mac, PK, BC, CBD, G54-2, HB, and OG. Leaf and flower tissues were subjected to RT-PCR using primers that were developed as described below (see ). For total RNA extractions, fresh leaf samples were placed in paper bags and freeze-dried using a Genesis 25 Freeze Drier (SP Industries Inc., Gardiner, NY, USA), after which the leaves were gently pulverized to create a semihomogenous crumble mix inside the bags. Approximately 500 mg of tissue was transferred to universal extraction bags (Bioreba AG, Reinach, Switzerland) and suspended in 5 mL of UltraPure water (Thermo Fisher Scientific, Waltham, MA, USA). The samples were ground using a HOMEX 6 homogenizer (Bioreba AG) and homogenate saps were transferred to 1.5 mL tubes using disposable transfer pipettes. From these tubes, 100 µL of homogenate sap was transferred to 2.0 mL tubes containing 300 µL of RB buffer (Omega Bio-tek, Norcross, GA, USA) with fresh 0.21% ( v / v ) β-mercaptoethanol (Thermo Fisher Scientific). Samples were loaded onto a QIAcube Connect (Qiagen, Germantown, MD, USA) for RNA extraction using an E.Z.N.A. ® Plant RNA Kit (Omega Bio-tek) and the QIAcube program for the RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA). The elution volume was set to 50 µL and the eluted RNA was stored at −20 °C. RNA concentrations and purity were determined using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific) . The cDNA was generated using a SuperScript™ VILO™ Master Mix kit (Thermo Fisher Scientific) and 500 ng of template RNA per reaction. The following thermal cycling protocol was used on a T100™ Thermal Cycler (Bio-Rad, Hecules, CA, USA): lid temperature set at 105 °C; primer binding at 25 °C for 10 min; reverse transcription at 50 °C for 10 min; reaction termination at 85 °C for 5 min, then held at 10 °C until storage at −20 °C. Using the HTS data from cannabis leaf samples of genotypes HB and OG, primers were either designed from this study (specifically for hop latent viroid (HLVd) and CasaMV1 mitovirus) or from previously published reports . Primers were designed using Primer-BLAST and specificity was tested against all plant, virus, bacteria, and fungal sequences in the nonredundant nucleotide collection (nr) from the National Center for Biotechnology Information (NCBI) GenBank database. Sequencing primer specificity was checked to ensure nonspecifics were not generated. To achieve coverage of the primed regions, two sequencing primer sets were evaluated: HLVd seq F1/HLVd seq R1 and HLVd quant F1/HLVd seq R2. Specificity was checked by PCR of 1 µL of generated cDNA in 20 µL reactions of the Q5 ® High-Fidelity 2X Master Mix (New England Biobabs, Ipswich, MA, USA) with sequencing primers at final concentrations of 500 nM. The following thermal cycling protocol was used: lid temp set at 105 °C, initial denaturation at 98 °C for 30 s; 30 cycles of 98 °C; denaturation for 10 s; 58 °C annealing for 30 s; 72 °C elongation for 80 s; a final elongation at 72 °C for 7 min, and held at 10 °C until stored at −20 °C. Following PCR, these were electrophoresed in 2% agarose (Thermo Fisher Scientific) at 100 V for 60 min in 0.5x TBE buffer (Thermo Fisher Scientific). The agarose gel was poststained in 1x SYBR™ Safe DNA Gel Stain (Thermo Fisher Scientific) in 0.5x TBE (Thermo Fisher Scientific) for 30 min. The stained gel was imaged using a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). In the case of both primer sets, nonspecific bands were not observed, although multiple bands (multimers) were seen due to the circular nature and small size of the HLVd genome. The evolutionary history was inferred by using the maximum likelihood method and Kimura 2-parameter model . The bootstrap consensus tree inferred from 1000 replicates was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 70% bootstrap replicates were collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches . Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. This analysis involved 21 nucleotide sequences from GenBank. Only full-length sequences were used, and selections were chosen to represent wide geographic regions. All positions with less than 95% site coverage were eliminated, i.e., fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position (partial deletion option). There was a total of 185 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 . Quantitative primer specificity was also tested using the primer sets HLVd quant F1/HLVd quant F2 and Cannabis EF1α F/Cannabis EF1α R . Specificity was evaluated using only the cDNA generated (see above) from the RNA of one cannabis genotype (PD). PCR was conducted using 20 µL reactions of QX200™ ddPCR™ EvaGreen Supermix (Bio-rad) with quantitative primers at final concentrations of 150 nM, 3 µL of 10 −2 diluted PD cDNA template per reaction, and without generating droplets. The PCRs were conducted across a 5-point thermal gradient ranging from 55.8 °C to 62 °C to determine the ideal annealing temperature. Following PCR, these were electrophoresed, stained, and imaged, as described above. Neither primer set produced nonspecific bands, and both sets performed equally well across the thermal gradient. A temperature of 58 °C was selected for the subsequent ddPCR, as higher temperatures can produce inadequate separation between positive and negative droplets, termed ‘rain’. A total of five cannabis genotypes were tested by ddPCR, and the levels of HLVd present were quantified. Tissues were obtained from the flowering plants of genotypes PD and Mac showing symptoms, as seen in . Approximately 100 mg of fresh inflorescence tissues were placed directly into 750 uL of RNAlater ® solution (Thermo Fisher Scientific) in a 2 mL screw-cap tube and placed on ice. The samples were either processed the same day or stored overnight at 4 °C and processed the following day. Total nucleic acids were extracted as described by Mark et al. , with slight modifications. The RNAlater ® was removed from the 2 mL tube followed by adding 2.3 mm diameter zirconia–silica beads and 1.0 mm diameter glass beads (BioSpec, Bartlesville, OK, USA). The tissue was then macerated using a TissueLyser (Qiagen) at 25 Hz for 1 min. Once homogenized, 1 mL of extraction buffer (2% CTAB, 2% PVP40,000, 25 mM EDTA at a pH of 8, 100 mM Tris-HCl at a pH of 8, and 2.5 mM NaCl) prewarmed at 65 °C was added to each sample and incubated at 65 °C for 10 min. The homogenate was centrifuged at max speed (>16,000× g ) for 5 min at 4 °C and the supernatant transferred to a new sterile 1.5 mL Eppendorf tube. An equal volume (750 µL) of chloroform isoamyl alcohol (24:1) was added and centrifuged at max speed (>16,000× g ) for 5 min. The supernatant (550 uL) was transferred to a new 1.5 mL Eppendorf tube, and 0.6 volumes of cold isopropanol were added and centrifuged again at max speed (>16,000× g ) for 5 min. Isopropanol was decanted and about 650 µL of 70% ethanol was added onto the pellets, centrifuged at max speed (>16,000× g ) for 3 min, and air-dried before resuspending into 40 µL of nuclease-free water. RT-qPCR was performed on a Bio-Rad CFX96 instrument using TaqPath™ 1-Step Multiplex Master Mix (No ROX) (Thermo Fisher Scientific). The reagents were brought to room temperature prior to prevent infrequent nonspecific signal increases found when master mixes are prepared on ice. Each 20 μL reaction mixture contained 5 μL of 4 × TaqPath 1-Step Multiplex Master Mix (No ROX; Thermo Fisher Scientific), 1 μL of primer/probes mix, and 4 μL of extracted total nucleic acid. The primers used are shown in . The final concentrations of primers and probes were 0.3 μM (target primers), 0.05 μM (target probes), 0.15 μM (internal control primer), and 0.05 μM (internal control probe). The cycling program on a Bio-Rad CFX96 instrument was as follows: uracil–DNA glycosylase incubation at 25 °C for 2 min; reverse transcription at 53 °C for 10 min; polymerase activation at 95 °C for 2 min; 40 cycles of PCR at 95 °C for 3 s and 60 °C for 30 s (signal acquisition). The filter combinations were 465–510 (FAM; UBQ P), 540–580 (HEX; HLVd P1), and 610–670 (Cy5; HLVd P2). Tissues were sampled from plants showing symptoms, as illustrated in , as well as from asymptomatic plants (showing no visible alteration of growth). Leaves, petioles, and roots were collected from 11 cannabis genotypes for comparison. Plants ranged in age from 6 to 10 weeks after the initiation of cuttings. Approximately 100 mg of fresh tissues were placed directly into 750 uL of RNAlater ® solution (Thermo Fisher Scientific) in a 2 mL screw-cap tube and placed on ice. The samples were either processed the same day or stored overnight at 4 °C and processed the following day. Total nucleic acids were extracted as described by Mark et al. , with slight modifications. The RNAlater ® was removed from the 2 mL tube followed by adding 2.3 mm diameter zirconia–silica beads and 1.0 mm diameter glass beads (BioSpec, Bartlesville, OK, USA). The tissue was then macerated using a TissueLyser (Qiagen, Germantown, MD, USA) at 25 Hz for 1 min. Once homogenized, 1 mL of extraction buffer (2% CTAB, 2% PVP40,000, 25 mM EDTA at a pH of 8, 100 mM Tris-HCl at a pH of 8, and 2.5 mM NaCl) prewarmed at 65 °C was added to each sample and incubated at 65 °C for 10 min. The homogenate was centrifuged at max speed (>16,000× g ) for 5 min at 4 °C and the supernatant transferred to a new sterile 1.5 mL Eppendorf tube. An equal volume (750 µL) of chloroform isoamyl alcohol (24:1) was added and centrifuged at max speed (>16,000× g ) for 5 min. The supernatant (550 uL) was transferred to a new 1.5 mL Eppendorf tube, and 0.6 volumes of cold isopropanol were added and centrifuged again at max speed (>16,000× g ) for 5 min. Isopropanol was decanted, and about 650 µL of 70% ethanol was added onto the pellets, centrifuged at max speed (>16,000× g ) for 3 min, and air-dried before resuspending into 40 µL of nuclease-free water. RT-LAMP primer sequences were designed using New England Biolabs ® (NEB) LAMP primer design tool and synthesized by Integrated DNA Technologies (Coraville, IA, USA) . RT-LAMP reactions were performed using WarmStart ® Fluorescent LAMP/RT-LAMP Kit (with UDG) (New England Biolabs, Whitby, ON, Canada; E1708) with standard primer concentrations (0.2 μM F3, 0.2 μM B3, 1.6 μM FIP, 1.6 μM BIP, 0.4 μM Loop F, 0.4 μM Loop B) in 25 μL on 96-well plates at 65 °C for 30 min in a Bio-Rad CFX96 instrument. 4.8.1. Distribution within Stock Plants To assess how specific diagnostic assays can be used to monitor the presence of HLVd in different tissues of cannabis plants, RT-PCR with primers HLVd seq F1/HLVd seq R1 were used according to the conditions specified above . Stock (mother) plants of several genotypes that were 3–4 months old were sampled at different positions on the plant—leaves were collected from the top (youngest growth), middle, and bottom (oldest growth), as well as from roots. The plants did not display any obvious symptoms of disease, such as stunting or leaf curl . Each leaf sample (approximately 5 gm fresh weight) was frozen at −80 C until used. The extractions were conducted using the Qiagen RNeasy Plant Mini Kit (cat. #74904) according to the manufacturer’s instructions. The final RNA product was eluted with 52 µL nuclease-free H 2 O. The QIAGEN OneStep RT-PCR Kit (cat. #210212) was used for reverse transcription and PCR amplification. The reaction mixture contained 14 µL of water, 5 µL of 5x reaction buffer, 1 µL of dNTPs (10 mM), 1.5 µL each of primers HLVd seq F1/HLVd seq R1, 1 µL of RNA template, and 1 µL of enzyme mix, resulting in a total volume of 25 µL. All PCR amplifications were performed in a MyCycler thermocycler (BIORAD) with the following program: 30 min at 50 °C, 15 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, 60 s at 72 °C, and final extension at 72 °C for 10 min. The resulting PCR products were run on a 1% TAE agarose gel, and images were captured with E-gel imager (Life Technologies, Carlsbad, CA, USA). Bands of the expected size (ca. 256 and 512 bp) were purified with QIAquick Gel Extraction Kit and sent to Eurofins Genomics (Eurofins MWG Operon LLC 2016, Louisville, KY, USA) for sequencing. The resulting sequences were compared to the corresponding HpLVd sequences from the National Centre for Biotechnology Information (NCBI) GenBank database to confirm identity. In addition, primers HLVd quant F1/HLVd quant F2 were also used to quantify viroid levels in the same tissues, as described in above. 4.8.2. Distribution within Inflorescence Tissues Mature inflorescences were obtained at harvest from a plant (approximately 12 weeks of age that had been grown under a photoperiod of 12:12 hr to induce flowering for 8 weeks) of genotype ‘Mac-1’ which was previously confirmed to be infected by HLVd. The leaves surrounding the inflorescence that included large fan leaves and smaller inflorescence leaves were manually dissected from the inflorescence using a scalpel and used for RNA extraction, followed by RT-PCR and ddPCR to compare the relative viroid concentrations in these tissues relative to that present in the whole inflorescence . 4.8.3. Distribution of HLVd within Cuttings from Infected Stock Plants Vegetative cuttings were taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd and were rooted and tested for HLVd presence using RT-PCR and RT-qPCR after 14 days. The conditions for rooting are described elsewhere . A total of 7 cuttings were subjected to RT-PCR, and 4 of the cuttings were also analyzed by RT-qPCR, and the results were compared. 4.8.4. Presence of Pathogens in Fresh and Dried Cannabis Inflorescences Following harvest of inflorescences from a number of genotypes of cannabis plants, both fresh and dried flowers were tested for the presence of specific pathogens by PCR with the universal primers for fungal/oomycete pathogens, and by RT-PCR for HLVd. The fresh flowers were tested immediately after they were obtained by removing inflorescence leaves and subjecting them to PCR, as described previously . For dried samples, flowers were subjected to commercial drying conditions (5 days at 20–22 °C and 50–55% relative humidity), after which the tissue samples were obtained and subjected to PCR with HLVd primers, as described in . Up to 20 fresh and dried samples were included in the analysis. Resulting bands observed in these analyses following PCR were collected and sent for sequencing to confirm which pathogens were present. 4.8.5. Detection of HLVd and Mitovirus in Cannabis Seed A cross was made between an infected ‘Mac1′ female plant and pollen from a healthy male plant of genotype ‘GPie’ by collecting pollen and manually transferring to the stigmatic surfaces. The plants were grown in isolation after crossing for 10 weeks, after which seeds that had developed were collected and frozen at −80 C. Individual seeds were ground and the RNA was extracted, as previously described, and subjected to RT-PCR using HLVd primers and CasaMV1 primers. To assess how specific diagnostic assays can be used to monitor the presence of HLVd in different tissues of cannabis plants, RT-PCR with primers HLVd seq F1/HLVd seq R1 were used according to the conditions specified above . Stock (mother) plants of several genotypes that were 3–4 months old were sampled at different positions on the plant—leaves were collected from the top (youngest growth), middle, and bottom (oldest growth), as well as from roots. The plants did not display any obvious symptoms of disease, such as stunting or leaf curl . Each leaf sample (approximately 5 gm fresh weight) was frozen at −80 C until used. The extractions were conducted using the Qiagen RNeasy Plant Mini Kit (cat. #74904) according to the manufacturer’s instructions. The final RNA product was eluted with 52 µL nuclease-free H 2 O. The QIAGEN OneStep RT-PCR Kit (cat. #210212) was used for reverse transcription and PCR amplification. The reaction mixture contained 14 µL of water, 5 µL of 5x reaction buffer, 1 µL of dNTPs (10 mM), 1.5 µL each of primers HLVd seq F1/HLVd seq R1, 1 µL of RNA template, and 1 µL of enzyme mix, resulting in a total volume of 25 µL. All PCR amplifications were performed in a MyCycler thermocycler (BIORAD) with the following program: 30 min at 50 °C, 15 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 30 s at 58 °C, 60 s at 72 °C, and final extension at 72 °C for 10 min. The resulting PCR products were run on a 1% TAE agarose gel, and images were captured with E-gel imager (Life Technologies, Carlsbad, CA, USA). Bands of the expected size (ca. 256 and 512 bp) were purified with QIAquick Gel Extraction Kit and sent to Eurofins Genomics (Eurofins MWG Operon LLC 2016, Louisville, KY, USA) for sequencing. The resulting sequences were compared to the corresponding HpLVd sequences from the National Centre for Biotechnology Information (NCBI) GenBank database to confirm identity. In addition, primers HLVd quant F1/HLVd quant F2 were also used to quantify viroid levels in the same tissues, as described in above. Mature inflorescences were obtained at harvest from a plant (approximately 12 weeks of age that had been grown under a photoperiod of 12:12 hr to induce flowering for 8 weeks) of genotype ‘Mac-1’ which was previously confirmed to be infected by HLVd. The leaves surrounding the inflorescence that included large fan leaves and smaller inflorescence leaves were manually dissected from the inflorescence using a scalpel and used for RNA extraction, followed by RT-PCR and ddPCR to compare the relative viroid concentrations in these tissues relative to that present in the whole inflorescence . Vegetative cuttings were taken directly from a stock plant of genotype ‘Blue Dream’ confirmed to be infected with HLVd and were rooted and tested for HLVd presence using RT-PCR and RT-qPCR after 14 days. The conditions for rooting are described elsewhere . A total of 7 cuttings were subjected to RT-PCR, and 4 of the cuttings were also analyzed by RT-qPCR, and the results were compared. Following harvest of inflorescences from a number of genotypes of cannabis plants, both fresh and dried flowers were tested for the presence of specific pathogens by PCR with the universal primers for fungal/oomycete pathogens, and by RT-PCR for HLVd. The fresh flowers were tested immediately after they were obtained by removing inflorescence leaves and subjecting them to PCR, as described previously . For dried samples, flowers were subjected to commercial drying conditions (5 days at 20–22 °C and 50–55% relative humidity), after which the tissue samples were obtained and subjected to PCR with HLVd primers, as described in . Up to 20 fresh and dried samples were included in the analysis. Resulting bands observed in these analyses following PCR were collected and sent for sequencing to confirm which pathogens were present. A cross was made between an infected ‘Mac1′ female plant and pollen from a healthy male plant of genotype ‘GPie’ by collecting pollen and manually transferring to the stigmatic surfaces. The plants were grown in isolation after crossing for 10 weeks, after which seeds that had developed were collected and frozen at −80 C. Individual seeds were ground and the RNA was extracted, as previously described, and subjected to RT-PCR using HLVd primers and CasaMV1 primers. Cannabis plants with symptoms of stunting, extensive curling of young leaves, and twisted and deformed stem growth were used. DNA from approximately 100 mg of stem tissues from these plants was extracted with a Qiagen DNeasy extraction kit and eluted into 100 uL of elution buffer. The extracted DNA was then diluted 1:10 in TE buffer before PCR. The PCR was carried out in 20 uL reactions using Thermo Fisher DreamTaq according to the manufacturer’s instructions. Primer sequences used are shown in . The PCR conditions were 94 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 58 °C for 60 s, 72 °C for 90 s, and a final extension of 72 °C for 10 min. The PCR products were run on a 2% TAE agarose gel at 100 V for 30 min before imaging. 4.10.1. Next-Generation Sequencing (NGS) Leaf samples were obtained from several outdoor hemp fields in Colorado during 2019, 2021, and 2022, and subjected to shotgun metagenomic analysis, as described by Chiginsky et al. . In most cases, samples were taken from symptomatic plants, as shown in . In each year, total RNA was extracted from a composite of 3–5 leaves from outdoor hemp-production fields in Colorado. Approximately 100 mg of leaf tissue samples was placed in a 2 mL microcentrifuge tube and stored at −20 °C until nucleic acid extraction was performed. Briefly, total RNA was extracted using the Qiagen Plant RNeasy kit and checked for the concentration using a Nanodrop One spectrophotometer (Thermo Fisher Scientific), and for quality using a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Approximately 2 μg of RNA was submitted to the Colorado State University Next Generation Sequencing Facility, where library preparation, quality measurements, and sequencing were performed. Briefly, RNA quality was confirmed using an Agilent Tapestation instrument. Shotgun RNA libraries were constructed using the Kapa Biosystems RNA HyperPrep kit (Roche, IN, USA) according to the manufacturer’s instructions. Pooled libraries were sequenced on an Illumina NextSeq 500 instrument (Baltimore, MD, USA) to produce single-end 150 nucleotide (nt) reads. Bioinformatic analysis for the 2019 hemp samples are described in Chiginsky et al. . Bioinformatic analysis for the 2021 and 2022 hemp samples was performed using CLC Genomics Work Bench (Qiagen). Reads were mapped to the hemp reference genome (assembly accession GCF_900626175.1). Remaining nonhost reads were assembled through the de novo assembly algorithm from the unmapped reads. Contigs and nonassembling reads were taxonomically categorized first by nucleotide-level alignment to the NCBI nucleotide (nt) database using Blastn, and then by protein-level alignment to the NCBI protein (nr) database. This produced a comprehensive classification of all nonhost reads. Candidate virus sequences were manually validated by aligning reads to draft genome sequences using bowtie2. Lastly, the raw sequence data were deposited in the NCBI Sequence Read Archive (SRA). 4.10.2. RT-PCR with Specific Primer Sets The universal BCTV primers, BCTV2- F, and BCTV2-R were used to amplify a 496 bp fragment of the coat protein (CP) gene, a region that is conserved among BCTV strains . The amplification cycle consisted of a 94 °C initial denaturation for 5 min, 25 cycles of denaturation at 94 °C for 1 min, 58 °C annealing for 2 min, and 72 °C extension for 2 min, followed by a 10 min final extension. The GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) was used. All PCR products were visualized on 1% agarose gel. The PCR products were excised from the agarose gels and purified using the DNA Clean and Concentrator™-5 (Zymo Research, Irvine, CA, USA). One to two PCR products were randomly selected and submitted for Sanger sequencing at Genewiz Inc. to confirm the virus identity. The sequences for each BCTV strain were checked for identity against the nonredundant (nr) database using Blastn in the NCBI database. To confirm the presence of low-percentage nucleotide (nt)-identity viruses (<90%), 1 μg of total RNA was used to synthesize the cDNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific) according to manufacturer’s instructions. For additional viruses, including Cannabis sativa mitovirus (CasaMV1), citrus yellow-vein-associated virus (CYVaV), and tobacco streak virus (TSV), the primers used are shown in . The RT-PCR was performed using GoTaq R Flexi DNA polymerase (Promega, Madison, WI, USA). The amplification cycle consisted of 2 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 35 s at 72 °C, followed by 5 min at 72 °C for all viruses except for citrus yellow-vein-associated virus (CYVaV), which had Tm of 51 °C. 4.10.3. Sequence Diversity and Molecular Phylogeny of Beet Curly Top Virus All currently available nucleotide sequences of BCTV capsid proteins (BCTV-CP) were retrieved from the GenBank database, after which their strains and sources of origins (hosts where viruses were identified) were determined according to the featured information in GenBank. Sequences representing each of the 11 BCTV strains were selected based on Strausbaugh et al. , and included 10 California/Logan, 8 Colorado, 2 Kimberly1, 10 Leafhopper71, 5 Mild, 10 Severe, 8 Worland, 6 Spanish curly top, 1 Pepper yellow dwarf, 2 Pepper curly top, and 1 Severe pepper. A total of 35 BCTV-CP sequences were found to be identified from Cannabis sativa , and all were included in the phylogenetic analysis. Multiple sequence alignments (total 98 sequences) were performed using MAFFT v7.505 , and the poorly aligned regions were trimmed using TrimAI v1.2.59 . The phylogenetic tree was constructed using the maximum likelihood method implemented in the RAxML v8.2.12 with the GTR+G+I model for nucleotide substitution through the CIPRES Science Gateway Environment . The robustness of each internal branch was estimated with 1000 bootstrap replications. The phylogenetic tree was visualized using FigTree v1.4.4 . 4.10.4. Detection of HLVd on Hemp Seeds and on Thrips A sample of hemp seeds from a commercial supplier was frozen at −80 °C, and then individual seeds were used to extract total RNA, as described in above. In addition, a sample containing 8 adult thrips found feeding on the leaves of plants derived from these seeds at the true leaf stage were collected using a pair of forceps, and also frozen at −80 °C prior to analysis. Leaf samples were obtained from several outdoor hemp fields in Colorado during 2019, 2021, and 2022, and subjected to shotgun metagenomic analysis, as described by Chiginsky et al. . In most cases, samples were taken from symptomatic plants, as shown in . In each year, total RNA was extracted from a composite of 3–5 leaves from outdoor hemp-production fields in Colorado. Approximately 100 mg of leaf tissue samples was placed in a 2 mL microcentrifuge tube and stored at −20 °C until nucleic acid extraction was performed. Briefly, total RNA was extracted using the Qiagen Plant RNeasy kit and checked for the concentration using a Nanodrop One spectrophotometer (Thermo Fisher Scientific), and for quality using a Qubit 3.0 fluorometer (Thermo Fisher Scientific). Approximately 2 μg of RNA was submitted to the Colorado State University Next Generation Sequencing Facility, where library preparation, quality measurements, and sequencing were performed. Briefly, RNA quality was confirmed using an Agilent Tapestation instrument. Shotgun RNA libraries were constructed using the Kapa Biosystems RNA HyperPrep kit (Roche, IN, USA) according to the manufacturer’s instructions. Pooled libraries were sequenced on an Illumina NextSeq 500 instrument (Baltimore, MD, USA) to produce single-end 150 nucleotide (nt) reads. Bioinformatic analysis for the 2019 hemp samples are described in Chiginsky et al. . Bioinformatic analysis for the 2021 and 2022 hemp samples was performed using CLC Genomics Work Bench (Qiagen). Reads were mapped to the hemp reference genome (assembly accession GCF_900626175.1). Remaining nonhost reads were assembled through the de novo assembly algorithm from the unmapped reads. Contigs and nonassembling reads were taxonomically categorized first by nucleotide-level alignment to the NCBI nucleotide (nt) database using Blastn, and then by protein-level alignment to the NCBI protein (nr) database. This produced a comprehensive classification of all nonhost reads. Candidate virus sequences were manually validated by aligning reads to draft genome sequences using bowtie2. Lastly, the raw sequence data were deposited in the NCBI Sequence Read Archive (SRA). The universal BCTV primers, BCTV2- F, and BCTV2-R were used to amplify a 496 bp fragment of the coat protein (CP) gene, a region that is conserved among BCTV strains . The amplification cycle consisted of a 94 °C initial denaturation for 5 min, 25 cycles of denaturation at 94 °C for 1 min, 58 °C annealing for 2 min, and 72 °C extension for 2 min, followed by a 10 min final extension. The GoTaq Flexi DNA polymerase (Promega, Madison, WI, USA) was used. All PCR products were visualized on 1% agarose gel. The PCR products were excised from the agarose gels and purified using the DNA Clean and Concentrator™-5 (Zymo Research, Irvine, CA, USA). One to two PCR products were randomly selected and submitted for Sanger sequencing at Genewiz Inc. to confirm the virus identity. The sequences for each BCTV strain were checked for identity against the nonredundant (nr) database using Blastn in the NCBI database. To confirm the presence of low-percentage nucleotide (nt)-identity viruses (<90%), 1 μg of total RNA was used to synthesize the cDNA using the Verso cDNA synthesis kit (Thermo Fisher Scientific) according to manufacturer’s instructions. For additional viruses, including Cannabis sativa mitovirus (CasaMV1), citrus yellow-vein-associated virus (CYVaV), and tobacco streak virus (TSV), the primers used are shown in . The RT-PCR was performed using GoTaq R Flexi DNA polymerase (Promega, Madison, WI, USA). The amplification cycle consisted of 2 min at 95 °C, 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 35 s at 72 °C, followed by 5 min at 72 °C for all viruses except for citrus yellow-vein-associated virus (CYVaV), which had Tm of 51 °C. All currently available nucleotide sequences of BCTV capsid proteins (BCTV-CP) were retrieved from the GenBank database, after which their strains and sources of origins (hosts where viruses were identified) were determined according to the featured information in GenBank. Sequences representing each of the 11 BCTV strains were selected based on Strausbaugh et al. , and included 10 California/Logan, 8 Colorado, 2 Kimberly1, 10 Leafhopper71, 5 Mild, 10 Severe, 8 Worland, 6 Spanish curly top, 1 Pepper yellow dwarf, 2 Pepper curly top, and 1 Severe pepper. A total of 35 BCTV-CP sequences were found to be identified from Cannabis sativa , and all were included in the phylogenetic analysis. Multiple sequence alignments (total 98 sequences) were performed using MAFFT v7.505 , and the poorly aligned regions were trimmed using TrimAI v1.2.59 . The phylogenetic tree was constructed using the maximum likelihood method implemented in the RAxML v8.2.12 with the GTR+G+I model for nucleotide substitution through the CIPRES Science Gateway Environment . The robustness of each internal branch was estimated with 1000 bootstrap replications. The phylogenetic tree was visualized using FigTree v1.4.4 . A sample of hemp seeds from a commercial supplier was frozen at −80 °C, and then individual seeds were used to extract total RNA, as described in above. In addition, a sample containing 8 adult thrips found feeding on the leaves of plants derived from these seeds at the true leaf stage were collected using a pair of forceps, and also frozen at −80 °C prior to analysis. |
Common pitfalls, and how to avoid them, in child and adolescent psychopharmacology: Part I | a5b24d98-ef83-4583-9d58-69819e4afbbb | 11010544 | Pharmacology[mh] | As Faculty of the British Association for Psychopharmacology (BAP) course on child and adolescent psychopharmacology, we previously published a paper reporting the most common questions we have been asked in recent editions of the course, alongside evidence-based and/or expert-informed answers. Here, based on our experience during the course, we have selected what we deem are the most common pitfalls, and how to avoid them, in child and adolescent psychopharmacology, focusing on attention-deficit/hyperactivity disorder (ADHD), anxiety, bipolar disorder, depression, obsessive-compulsive disorder and related disorders, and tic disorder. We have grouped the pitfalls by disorder to which they refer, in alphabetical order. Pitfalls in relation to the treatment of other disorders (autism and intellectual disability, eating disorders, neuropsychiatric correlates of epilepsy and psychosis) are addressed in a separate paper (part II). Switching to equivalent doses of long-acting formulations of methylphenidate When switching patients from immediate- or extended-release methylphenidate (which acts by inhibiting the reuptake of dopamine and norepinephrine) to a long-acting methylphenidate formulation, prescribers should avoid a simple equivalence based on the total dose. Rather, they should use the immediate-release component of each formulation as the reference. For instance, when switching from 20 mg of Medikinet XL ® (methylphenidate extended release, ER) (50% immediate release: 10 mg) to OROS Concerta XL ® , 45 mg of OROS Concerta XL ® (22% immediate release: 9.9 mg) would give the equivalent immediate-release dose . Along the same line of reasoning, 40 mg of Equasym XL ® (methylphenidate extended release, ER) (30% immediate release: 12 mg) would be equivalent to OROS Concerta XL ® 54 mg and Equasym XL ® 50 mg to, roughly, OROS Concerta XL ® 72 mg. Optimising the treatment While the maximum licensed dose of methylphenidate for children (except for osmotic release and prolonged release formulations, see below) is 60 mg/day in many countries, some guidelines (e.g. those from the Canadian ADHD Resource Alliance (CADDRA), caddra.ca) and other documents recommend higher doses . For instance, the British National Formulary (BNF; ) recommends a dose of up to 90 mg/day, under the direction of a specialist. For osmotic-release and prolonged-release formulations of methylphenidate, the maximum license dose is 54 mg/day, but the BNF mentions a maximum of 108 mg/day for Concerta XL ® in children (as well as in adults). Some prescribers may fail to optimise the dose of treatment to reach the maximum benefit with the highest tolerated dose, being satisfied with a moderate improvement in the severity of the symptoms. Meta-analytic evidence based on flexible-dose trials for both methylphenidate and amphetamines shows increased efficacy and reduced likelihood of discontinuations for any reason with increasing stimulant doses, up to the maximum FDA-licensed dose . Also, meta-analytic evidence in adults (not available in children) shows that, at the group level, doses beyond the licensed ones are generally not associated with a favourable benefit–risk profile, even though it is important to appreciate that individual patients may benefit from, and tolerate well, doses beyond the licensed ones . While it should not be a standard practice, using doses beyond the maximum recommended ones could be considered when the patient has presented with a partial response, there is only some degree of improvement at the maximum recommended dose, and tolerability is good. It should be noted that, in some countries, special authorisation may be necessary for doses exceeding the maximum licensed amount, subject to local regulations. Drug holidays An important issue is around adherence to medication . Due to concerns about the side effects of stimulants, some prescribers may consider advising stopping the treatment during the weekend. However, to our knowledge, to date, only one randomised trial showed a trend ( p = 0.08) for an association between drug holidays on the weekend and less interference with appetite . The European ADHD Guidelines Group advised that the risk–benefit balance of drug holidays during weekends must be taken into account and better investigated. Evidence on the beneficial effects on appetite of stopping medication during longer drug holidays (e.g. summer holidays) to allow for catch-up growth is also mixed . Therefore, discontinuing stimulants during the weekend should be considered in cases where there is a clear benefit from the stimulant but serious concerns around appetite reduction, even though simulant discontinuation could not be enough to allow catching up the weight. If, despite the implementation of the previous management strategies, weight and/or height values are below critical thresholds, a referral to the paediatric endocrinologist or growth specialist is recommended . Note: General aspects of the psychopharmacology of ADHD are covered elsewhere (e.g. ; ; ). When switching patients from immediate- or extended-release methylphenidate (which acts by inhibiting the reuptake of dopamine and norepinephrine) to a long-acting methylphenidate formulation, prescribers should avoid a simple equivalence based on the total dose. Rather, they should use the immediate-release component of each formulation as the reference. For instance, when switching from 20 mg of Medikinet XL ® (methylphenidate extended release, ER) (50% immediate release: 10 mg) to OROS Concerta XL ® , 45 mg of OROS Concerta XL ® (22% immediate release: 9.9 mg) would give the equivalent immediate-release dose . Along the same line of reasoning, 40 mg of Equasym XL ® (methylphenidate extended release, ER) (30% immediate release: 12 mg) would be equivalent to OROS Concerta XL ® 54 mg and Equasym XL ® 50 mg to, roughly, OROS Concerta XL ® 72 mg. While the maximum licensed dose of methylphenidate for children (except for osmotic release and prolonged release formulations, see below) is 60 mg/day in many countries, some guidelines (e.g. those from the Canadian ADHD Resource Alliance (CADDRA), caddra.ca) and other documents recommend higher doses . For instance, the British National Formulary (BNF; ) recommends a dose of up to 90 mg/day, under the direction of a specialist. For osmotic-release and prolonged-release formulations of methylphenidate, the maximum license dose is 54 mg/day, but the BNF mentions a maximum of 108 mg/day for Concerta XL ® in children (as well as in adults). Some prescribers may fail to optimise the dose of treatment to reach the maximum benefit with the highest tolerated dose, being satisfied with a moderate improvement in the severity of the symptoms. Meta-analytic evidence based on flexible-dose trials for both methylphenidate and amphetamines shows increased efficacy and reduced likelihood of discontinuations for any reason with increasing stimulant doses, up to the maximum FDA-licensed dose . Also, meta-analytic evidence in adults (not available in children) shows that, at the group level, doses beyond the licensed ones are generally not associated with a favourable benefit–risk profile, even though it is important to appreciate that individual patients may benefit from, and tolerate well, doses beyond the licensed ones . While it should not be a standard practice, using doses beyond the maximum recommended ones could be considered when the patient has presented with a partial response, there is only some degree of improvement at the maximum recommended dose, and tolerability is good. It should be noted that, in some countries, special authorisation may be necessary for doses exceeding the maximum licensed amount, subject to local regulations. An important issue is around adherence to medication . Due to concerns about the side effects of stimulants, some prescribers may consider advising stopping the treatment during the weekend. However, to our knowledge, to date, only one randomised trial showed a trend ( p = 0.08) for an association between drug holidays on the weekend and less interference with appetite . The European ADHD Guidelines Group advised that the risk–benefit balance of drug holidays during weekends must be taken into account and better investigated. Evidence on the beneficial effects on appetite of stopping medication during longer drug holidays (e.g. summer holidays) to allow for catch-up growth is also mixed . Therefore, discontinuing stimulants during the weekend should be considered in cases where there is a clear benefit from the stimulant but serious concerns around appetite reduction, even though simulant discontinuation could not be enough to allow catching up the weight. If, despite the implementation of the previous management strategies, weight and/or height values are below critical thresholds, a referral to the paediatric endocrinologist or growth specialist is recommended . Note: General aspects of the psychopharmacology of ADHD are covered elsewhere (e.g. ; ; ). Is the diagnosis correct? A correct and complete diagnostic assessment is essential. We have evidence for what works for depressive and anxiety disorders (cognitive behavioural therapy (CBT) for both, selective serotonin reuptake inhibitors (SSRIs) for both (but probably just robust evidence for fluoxetine in depression) and interpersonal psychotherapy (IPT) for depression only , but no evidence that those same treatments work for other disorders/problems, for example, situational sadness. So, prescribers should ensure that diagnostic criteria are met. Particular caution needs to be taken around personality disorders (in particular emotionally unstable personality disorder (EUPD), also referred to as borderline personality disorder). We note that the diagnosis of personality disorders in adolescence is controversial, with some practitioners/researchers endorsing this diagnosis, and others using the term emerging personality disorder . Young people with EUPD or emerging personality disorder may appear to have depression during the first assessment, as they are most likely to present when their mood is low. A careful assessment is essential. If young people diagnosed with a depressive disorder do not respond to first-line treatment, the prescriber should go back and review the diagnosis and consider personality disorder/ emerging personality disorder (which tends to become more obvious the longer a young person is treated). Are we addressing all the problems? A complete diagnostic formulation is essential. Basic treatments aimed solely at depression or anxiety are unlikely to be effective if there are significant co-morbidities. For instance, if ADHD is causing educational failure, leading to low self-esteem, then this needs to be addressed alongside treatment for depression. Another example is when CBT for depression ignores the primary anxiety disorder, which is unlikely to lead to the resolution of the depression. We must also address social factors that contribute to depression – for example, if a young person is being bullied and becomes depressed, an SSRI will not stop the bullying which makes them feel worthless. We must enquire about social factors and do our best to address them and revisit what social stresses there may be if a young person does not respond to treatment. One should bear in mind that young people may not disclose challenging events in their lives (particularly abuse) during the first assessment, but they may feel more comfortable with the practitioner in subsequent sessions. Are they really ‘treatment resistant’? Pharmacologically treatment-resistant depression is defined, in adults, by at least two prior treatment failures with adequate dose and duration . Similarly, pharmacologically treatment-resistant anxiety has been recently defined in adults as at least two separate failed full trials of pharmacological monotherapy with first-line agents approved for those disorders by the US Food and Drug Administration (FDA) or the European Medicines Agency or other equivalent regulatory agencies, and recommended by guidelines . Treatment-resistant depression and anxiety in children and young people are difficult to treat, with little research evidence to guide the practitioner. However, we need to review the history of treatment before concluding there is treatment resistance. Notably, significant numbers of people stop antidepressants when they have minor side effects which may have worn off if they had persevered. The amount of time needed to wait until side effects go away is very variable, but we would advise patients to persevere for a month if side effects are tolerable, before stopping. It may be worth going back to re-try the antidepressant with psychoeducation and encouragement to persevere. A slower dose titration may also reduce the impact of side effects. It is also appropriate to increase the dose to BNF dose limits if tolerated. It is important to discuss with the patient and their families the risks and benefits of this strategy, and how this is outside the license for adolescents, but is an acceptable treatment for adults, and often used in adolescents. A particularly concerning side effect of SSRIs is increased suicidal thoughts. These have only been demonstrated to be significantly higher than placebo in randomised controlled trials of depression, not of anxiety , and indeed the UK 2003 MHRA and USA 2004 FDA Black Box warnings only applied to adolescent depression, not anxiety disorders ( https://journalofethics.ama-assn.org/article/antidepressants-and-fdas-black-box-warning-determining-rational-public-policy-absence-sufficient/2012-06#:~:text=The%20MHRA%27s%202003%20recommendation%2C%20based,randomized%20clinical%20trials%20%5B1%5D ). However, negative results in anxiety may reflect a lower baseline risk and fewer total participants in trials. Hence, prescribers need to watch carefully for suicidality. Due to the severe potential consequences of suicidal thoughts, many prescribers and families would choose not to continue SSRIs if they emerged. We note that it can be even more difficult to judge treatment resistance for psychological therapy. To determine true treatment resistance, we need to ensure the patient has good quality therapy at an adequate dose. This requires a fully accredited therapist, appropriate expert supervision of the therapist and an adequate number of sessions for the patient to try out techniques, as opposed to having one session and deciding it was ‘rubbish’. In addition, patients may find it hard to get on well with therapist A but get on well with (and be prepared to try therapy with) therapist B. General guidance on the pharmacological treatment of anxiety (provided, for instance, in ) and depressive disorders (e.g. ) in children and adolescents, mentioning key studies in the field such as the Treatment for Adolescents with Depression Study or the Treatment of SSRIs-resistant depression in adolescence , is beyond the scope of the current paper. A correct and complete diagnostic assessment is essential. We have evidence for what works for depressive and anxiety disorders (cognitive behavioural therapy (CBT) for both, selective serotonin reuptake inhibitors (SSRIs) for both (but probably just robust evidence for fluoxetine in depression) and interpersonal psychotherapy (IPT) for depression only , but no evidence that those same treatments work for other disorders/problems, for example, situational sadness. So, prescribers should ensure that diagnostic criteria are met. Particular caution needs to be taken around personality disorders (in particular emotionally unstable personality disorder (EUPD), also referred to as borderline personality disorder). We note that the diagnosis of personality disorders in adolescence is controversial, with some practitioners/researchers endorsing this diagnosis, and others using the term emerging personality disorder . Young people with EUPD or emerging personality disorder may appear to have depression during the first assessment, as they are most likely to present when their mood is low. A careful assessment is essential. If young people diagnosed with a depressive disorder do not respond to first-line treatment, the prescriber should go back and review the diagnosis and consider personality disorder/ emerging personality disorder (which tends to become more obvious the longer a young person is treated). A complete diagnostic formulation is essential. Basic treatments aimed solely at depression or anxiety are unlikely to be effective if there are significant co-morbidities. For instance, if ADHD is causing educational failure, leading to low self-esteem, then this needs to be addressed alongside treatment for depression. Another example is when CBT for depression ignores the primary anxiety disorder, which is unlikely to lead to the resolution of the depression. We must also address social factors that contribute to depression – for example, if a young person is being bullied and becomes depressed, an SSRI will not stop the bullying which makes them feel worthless. We must enquire about social factors and do our best to address them and revisit what social stresses there may be if a young person does not respond to treatment. One should bear in mind that young people may not disclose challenging events in their lives (particularly abuse) during the first assessment, but they may feel more comfortable with the practitioner in subsequent sessions. Pharmacologically treatment-resistant depression is defined, in adults, by at least two prior treatment failures with adequate dose and duration . Similarly, pharmacologically treatment-resistant anxiety has been recently defined in adults as at least two separate failed full trials of pharmacological monotherapy with first-line agents approved for those disorders by the US Food and Drug Administration (FDA) or the European Medicines Agency or other equivalent regulatory agencies, and recommended by guidelines . Treatment-resistant depression and anxiety in children and young people are difficult to treat, with little research evidence to guide the practitioner. However, we need to review the history of treatment before concluding there is treatment resistance. Notably, significant numbers of people stop antidepressants when they have minor side effects which may have worn off if they had persevered. The amount of time needed to wait until side effects go away is very variable, but we would advise patients to persevere for a month if side effects are tolerable, before stopping. It may be worth going back to re-try the antidepressant with psychoeducation and encouragement to persevere. A slower dose titration may also reduce the impact of side effects. It is also appropriate to increase the dose to BNF dose limits if tolerated. It is important to discuss with the patient and their families the risks and benefits of this strategy, and how this is outside the license for adolescents, but is an acceptable treatment for adults, and often used in adolescents. A particularly concerning side effect of SSRIs is increased suicidal thoughts. These have only been demonstrated to be significantly higher than placebo in randomised controlled trials of depression, not of anxiety , and indeed the UK 2003 MHRA and USA 2004 FDA Black Box warnings only applied to adolescent depression, not anxiety disorders ( https://journalofethics.ama-assn.org/article/antidepressants-and-fdas-black-box-warning-determining-rational-public-policy-absence-sufficient/2012-06#:~:text=The%20MHRA%27s%202003%20recommendation%2C%20based,randomized%20clinical%20trials%20%5B1%5D ). However, negative results in anxiety may reflect a lower baseline risk and fewer total participants in trials. Hence, prescribers need to watch carefully for suicidality. Due to the severe potential consequences of suicidal thoughts, many prescribers and families would choose not to continue SSRIs if they emerged. We note that it can be even more difficult to judge treatment resistance for psychological therapy. To determine true treatment resistance, we need to ensure the patient has good quality therapy at an adequate dose. This requires a fully accredited therapist, appropriate expert supervision of the therapist and an adequate number of sessions for the patient to try out techniques, as opposed to having one session and deciding it was ‘rubbish’. In addition, patients may find it hard to get on well with therapist A but get on well with (and be prepared to try therapy with) therapist B. General guidance on the pharmacological treatment of anxiety (provided, for instance, in ) and depressive disorders (e.g. ) in children and adolescents, mentioning key studies in the field such as the Treatment for Adolescents with Depression Study or the Treatment of SSRIs-resistant depression in adolescence , is beyond the scope of the current paper. Prescribing subtherapeutic doses of mood-stabilising agents Prescribers often report ‘breakthrough’ episodes, with symptoms emerging during treatment, despite good adherence to the regime advised. In such cases, frequently, the dose of psychotropic medication being prescribed is subtherapeutic, such as, for instance, when quetiapine (receptor antagonist (D2 and 5-HT2)) 25 mg in the morning and 50 mg at night is prescribed. It is more likely in such cases that the index episode for which the medication was being prescribed self-remitted and the improvement was not associated with medication. Therefore, prescribers should familiarise themselves with therapeutic doses of psychotropic medications and use them appropriately whilst monitoring for both efficacy and side effects. Continuing antidepressants in the case of treatment-emergent affective switch The use of antidepressants to treat unipolar depression, anxiety disorders and OCD in children and adolescents may be associated with treatment-emergent affective switch (TEAS) as described in our previous paper . Prescribers often consider a gradual tapering of antidepressants to avoid a discontinuation syndrome. However, it would actually be best practice to stop antidepressants when TEAS is evident, as continued use of antidepressants even in small doses may perpetuate the TEAS. Management of bipolar depression Bipolar depressive episodes are frequent in children and adolescents but are also frequently missed in clinical practice. When these require management with psychotropics, it is prudent to check adherence to medication and/or optimising the dose of medication. Antidepressants should be used cautiously to treat bipolar depressive episodes in children and specifically always with an appropriate mood stabilising agent(s) at a therapeutic dose . The use of antidepressants as monotherapy may be associated with a switch in polarity, emergence of mixed features and/or rapid cycling. Note: General aspects of the treatment of bipolar disorder in children and young people are reported elsewhere (e.g. ). Prescribers often report ‘breakthrough’ episodes, with symptoms emerging during treatment, despite good adherence to the regime advised. In such cases, frequently, the dose of psychotropic medication being prescribed is subtherapeutic, such as, for instance, when quetiapine (receptor antagonist (D2 and 5-HT2)) 25 mg in the morning and 50 mg at night is prescribed. It is more likely in such cases that the index episode for which the medication was being prescribed self-remitted and the improvement was not associated with medication. Therefore, prescribers should familiarise themselves with therapeutic doses of psychotropic medications and use them appropriately whilst monitoring for both efficacy and side effects. The use of antidepressants to treat unipolar depression, anxiety disorders and OCD in children and adolescents may be associated with treatment-emergent affective switch (TEAS) as described in our previous paper . Prescribers often consider a gradual tapering of antidepressants to avoid a discontinuation syndrome. However, it would actually be best practice to stop antidepressants when TEAS is evident, as continued use of antidepressants even in small doses may perpetuate the TEAS. Bipolar depressive episodes are frequent in children and adolescents but are also frequently missed in clinical practice. When these require management with psychotropics, it is prudent to check adherence to medication and/or optimising the dose of medication. Antidepressants should be used cautiously to treat bipolar depressive episodes in children and specifically always with an appropriate mood stabilising agent(s) at a therapeutic dose . The use of antidepressants as monotherapy may be associated with a switch in polarity, emergence of mixed features and/or rapid cycling. Note: General aspects of the treatment of bipolar disorder in children and young people are reported elsewhere (e.g. ). Insufficient evidence base It is a common pitfall to think that the evidence base accurately covers the full range of obsessive and compulsive conditions. This is not the case. For instance, to date, there is no published evidence supporting the role of low-dose antipsychotic augmentation in body dysmorphic disorder (BDD) nor is any clear evidence that SSRI medications are helpful in the treatment of trichotillomania . It is important therefore to be clear about the dominant aspect of the mental state in the child and make decisions on the evidence base, where it exists and, in turn, being clear where there is a lack of evidence or indeed evidence that medication is not likely to be of benefit. Co-morbidity in obsessive-compulsive disorders The issue of co-morbidity in the presentation requires careful thought. A common pitfall in treatment planning is not comprehensively assessing the full clinical presentation and then missing opportunities to design the best treatment plan. Co-morbid mood (more than 60%) and anxiety (more than 75%) diagnoses are extremely common in young people with obsessive-compulsive disorders (OCD) . These co-morbidities may require careful additional consideration, as they will undoubtedly influence the choice and sequencing of pharmacological interventions for OCD . As an example, whilst trichotillomania is unlikely to respond to treatment with SSRI medication, the presence of a co-morbid affective disorder might increase the likelihood of a better medication response, in a young person with trichotillomania. The broader neurodevelopmental profile can help guide the best choice of medications, as well as their sequencing. For instance, a common pitfall can be to attempt to treat OCD with psychological therapy in a patient with co-morbid ADHD, without adequately addressing the impact of ADHD. This can render treatment much less effective in our experience. We therefore find that ensuring ADHD symptoms are adequately treated pharmacologically can be a helpful precursor to better treatment outcomes of OCD. Some young people, however, can become more focussed on their compulsions when treated for ADHD. Dose and duration NICE guidelines encourage the adoption of the maximum tolerated dosing schedule for SSRI medication in the treatment of OCD , as high-dose treatment (i.e. 200 mg/day for sertraline) is likely to be needed to effect good clinical outcomes in the majority of patients within OCD. In our experience, one of the most common pitfalls is to use too low a dose of SSRI, for too short a duration, and to not carefully support young people and their families to increase to a high dose of SSRI medication. We believe that this pitfall can be mitigated with careful explanation, which includes sharing good quality psychoeducational materials about SSRI and other medications. This is extremely important in building a therapeutic alliance, to then attempt the necessary increase towards high-dose SSRI treatment regimens. Another common pitfall is for patients and their families to seek early discontinuation of medication when experiencing early side effects. Instead, the approach should be towards a maximum tolerated dosing approach. We think this pitfall of early discontinuations can be avoided by, again, carefully counselling young people and their families. As a result, if any treatment-emergent side effects do occur, then the practitioner will have proactively counselled that these can commonly settle in a short period of time. Most importantly, the approach incorporates each treatment trial of SSRI medication for a minimum of 10–12 weeks at the highest tolerated dose . Our experience of being clear about this is that it then avoids abrupt treatment discontinuations, with incumbent withdrawal side effects. With careful planning and explanation, patients and their families realise one can drop back to the last ‘maximum tolerated dose’ and continue the treatment trial, rather than feeling this medication does not suit and needs to be discontinued. It is important to have clinical oversight as to whether in these cases a lower dose of an SSRI is at such a low level, that it is unlikely to be efficacious. Clinical experience is necessary to decide, in this circumstance, if it will be better to cross-titrate to another SSRI medication, in the hope of achieving higher dosing and therefore a greater chance of a positive treatment response. Augmentation with antipsychotics A further pitfall in relation to medication regime for OCD relates to the use of antipsychotic augmentation of SSRI treatment. The evidence suggests that low-dose antipsychotic augmentation benefits around one-third of adult patients with OCD . This has not been replicated in studies in children and young people but is considered and supported under NICE guidelines . A common pitfall is then increasing the dose of antipsychotic medication to a higher dose, when not seeing any improvements in OCD patients. This lack of additional treatment response in augmented OCD patients can be expected in around two-thirds of patients. This pitfall can therefore be avoided by a clear explanation that the treatment trial of SSRI augmentation will be around 2 months in duration and with a clear remit to only try the low doses of antipsychotics, such as risperidone (D2, 5-HT2, and NE alpha 2 receptor antagonist) 0.5 mg, or up to aripiprazole (D2 and 5-HT1A receptor partial agonist) 5 mg daily. As mentioned, we do not recommend augmenting SSRI treatment with antipsychotics in young people with BDD. This is despite the high incidence of delusional intensity appearance-related beliefs, in patients with BDD. Indeed, evidence is clear that delusional and non-delusional patients with BDD are just as likely to respond to SSRI treatments . Paediatric acute-onset neuropsychiatric syndrome We note that, as standards of care are still being developed for Paediatric Acute-onset Neuropsychiatric Syndrome (PANS) and its treatment, and authoritative guidelines such as the NICE guidelines are not available, we have refrained from including here any recommendation on PANS. We look forward to additional high-level evidence to inform clinical decision-making. At the time of the writing, in the UK, the work of a PANS/PANDAS Working group is ongoing and there are no guidance and treatment recommendations to diagnose/treat PANS/PANDAD in the National Health System (NHS) ( https://commonslibrary.parliament.uk/research-briefings/cdp-2023-0174/#:~:text=There%20is%20currently%20no%20guidance,and%20patients%20and%20their%20families ). It is a common pitfall to think that the evidence base accurately covers the full range of obsessive and compulsive conditions. This is not the case. For instance, to date, there is no published evidence supporting the role of low-dose antipsychotic augmentation in body dysmorphic disorder (BDD) nor is any clear evidence that SSRI medications are helpful in the treatment of trichotillomania . It is important therefore to be clear about the dominant aspect of the mental state in the child and make decisions on the evidence base, where it exists and, in turn, being clear where there is a lack of evidence or indeed evidence that medication is not likely to be of benefit. The issue of co-morbidity in the presentation requires careful thought. A common pitfall in treatment planning is not comprehensively assessing the full clinical presentation and then missing opportunities to design the best treatment plan. Co-morbid mood (more than 60%) and anxiety (more than 75%) diagnoses are extremely common in young people with obsessive-compulsive disorders (OCD) . These co-morbidities may require careful additional consideration, as they will undoubtedly influence the choice and sequencing of pharmacological interventions for OCD . As an example, whilst trichotillomania is unlikely to respond to treatment with SSRI medication, the presence of a co-morbid affective disorder might increase the likelihood of a better medication response, in a young person with trichotillomania. The broader neurodevelopmental profile can help guide the best choice of medications, as well as their sequencing. For instance, a common pitfall can be to attempt to treat OCD with psychological therapy in a patient with co-morbid ADHD, without adequately addressing the impact of ADHD. This can render treatment much less effective in our experience. We therefore find that ensuring ADHD symptoms are adequately treated pharmacologically can be a helpful precursor to better treatment outcomes of OCD. Some young people, however, can become more focussed on their compulsions when treated for ADHD. NICE guidelines encourage the adoption of the maximum tolerated dosing schedule for SSRI medication in the treatment of OCD , as high-dose treatment (i.e. 200 mg/day for sertraline) is likely to be needed to effect good clinical outcomes in the majority of patients within OCD. In our experience, one of the most common pitfalls is to use too low a dose of SSRI, for too short a duration, and to not carefully support young people and their families to increase to a high dose of SSRI medication. We believe that this pitfall can be mitigated with careful explanation, which includes sharing good quality psychoeducational materials about SSRI and other medications. This is extremely important in building a therapeutic alliance, to then attempt the necessary increase towards high-dose SSRI treatment regimens. Another common pitfall is for patients and their families to seek early discontinuation of medication when experiencing early side effects. Instead, the approach should be towards a maximum tolerated dosing approach. We think this pitfall of early discontinuations can be avoided by, again, carefully counselling young people and their families. As a result, if any treatment-emergent side effects do occur, then the practitioner will have proactively counselled that these can commonly settle in a short period of time. Most importantly, the approach incorporates each treatment trial of SSRI medication for a minimum of 10–12 weeks at the highest tolerated dose . Our experience of being clear about this is that it then avoids abrupt treatment discontinuations, with incumbent withdrawal side effects. With careful planning and explanation, patients and their families realise one can drop back to the last ‘maximum tolerated dose’ and continue the treatment trial, rather than feeling this medication does not suit and needs to be discontinued. It is important to have clinical oversight as to whether in these cases a lower dose of an SSRI is at such a low level, that it is unlikely to be efficacious. Clinical experience is necessary to decide, in this circumstance, if it will be better to cross-titrate to another SSRI medication, in the hope of achieving higher dosing and therefore a greater chance of a positive treatment response. A further pitfall in relation to medication regime for OCD relates to the use of antipsychotic augmentation of SSRI treatment. The evidence suggests that low-dose antipsychotic augmentation benefits around one-third of adult patients with OCD . This has not been replicated in studies in children and young people but is considered and supported under NICE guidelines . A common pitfall is then increasing the dose of antipsychotic medication to a higher dose, when not seeing any improvements in OCD patients. This lack of additional treatment response in augmented OCD patients can be expected in around two-thirds of patients. This pitfall can therefore be avoided by a clear explanation that the treatment trial of SSRI augmentation will be around 2 months in duration and with a clear remit to only try the low doses of antipsychotics, such as risperidone (D2, 5-HT2, and NE alpha 2 receptor antagonist) 0.5 mg, or up to aripiprazole (D2 and 5-HT1A receptor partial agonist) 5 mg daily. As mentioned, we do not recommend augmenting SSRI treatment with antipsychotics in young people with BDD. This is despite the high incidence of delusional intensity appearance-related beliefs, in patients with BDD. Indeed, evidence is clear that delusional and non-delusional patients with BDD are just as likely to respond to SSRI treatments . We note that, as standards of care are still being developed for Paediatric Acute-onset Neuropsychiatric Syndrome (PANS) and its treatment, and authoritative guidelines such as the NICE guidelines are not available, we have refrained from including here any recommendation on PANS. We look forward to additional high-level evidence to inform clinical decision-making. At the time of the writing, in the UK, the work of a PANS/PANDAS Working group is ongoing and there are no guidance and treatment recommendations to diagnose/treat PANS/PANDAD in the National Health System (NHS) ( https://commonslibrary.parliament.uk/research-briefings/cdp-2023-0174/#:~:text=There%20is%20currently%20no%20guidance,and%20patients%20and%20their%20families ). Assessing the effectiveness of drug treatments Allow sufficient time : The natural course of tics is a waxing (increasing) and waning (decreasing) pattern over weeks or months. Over hours and days, tics can also worsen due to, for example, anxiety, anger, excitement or tiredness. Tics commonly also wax during stressful periods such as starting the new school year or exams. A common pitfall is to prematurely assume treatment is effective when simply observing a natural waning phase, or conversely, that a drug treatment is ineffective when observing a natural waxing phase. To avoid these pitfalls, it is important to evaluate treatment effects over a sufficiently long period, usually 2–3 months, to compare peaks (waxing phases) and throughs (waning phases) before and after treatment initiation. Is the tic diagnosis correct? Since the COVID-19 pandemic, there has been an unprecedented rise in functional tics, particularly in teenage girls . Functional tics can be difficult to differentiate from a primary tic disorder and can also co-occur with primary tics. However, as functional tics are not responsive to standard tic medications – a common pitfall when failing to observe a tic treatment response at standard doses may be to continue dose escalation, thus causing increasing adverse effects . Another common diagnostic pitfall is related to motor mannerisms and stereotypies which occur in autism and can mimic complex motor tics but are not responsive to tic treatments. Dosing Failing to ‘start low and go slow’ : When initiating medication, typically adverse effects occur before therapeutic benefits are seen – and can lead to premature discontinuation. This can be avoided by gradual titration; ‘starting low and going slow’. For example, initial syncope (due to postural hypotension) can be related to the initiation of clonidine (which stimulates postsynaptic alpha 2-adrenergic receptors) and guanfacine (which stimulates postsynaptic alpha 2A-adrenergic receptors) and sedation is potentially related to all medications for tics. These adverse effects are often encountered if the initial dose is too high. However, with careful low-dose initiation and slow titration, these adverse effects are much less likely to occur. Doses of 25 mcg of clonidine, 1 mg guanfacine and 0.5–1 mg aripiprazole once daily are suggested as appropriate initial doses, with titrations using a weekly, fortnightly or even monthly approach according to the response. However, the initial dose and the titration speed could differ according to body weight, age and other characteristics of the patient. Failing to achieve an adequate therapeutic dose : When a medication has been initiated, it is important to titrate to recommended therapeutic doses. In the case of clonidine, this relates to an average daily dose of 3–5 mcg/kg , for aripiprazole around 5–10 mg daily and for guanfacine between 1 and 4 mg in young children and up to 7 mg in older adolescents according to weight . Often young people are sub-optimally medicated to reduce potential side effects. However, it is a common pitfall to conclude that a medication is ineffective, when in fact it has been used at a sub-therapeutic dose. Drug choice – is the drug correct? Awareness of co-morbidities is important in determining the best drug choice. Commonly, young people with co-morbid tics and ADHD find noradrenergic agents (clonidine, guanfacine) beneficial for tic symptom control, whereas young people with anxiety disorders or OCD co-morbidities may find an approach using aripiprazole or treatment primarily targeting anxiety and/or OCD with an SSRI most beneficial in managing tic symptoms. Misidentifying new-onset tics as an adverse effect of stimulant medication for ADHD Tic disorders occur in around 20% of young people with ADHD. However, the onset of tics (typically around 7–8 years of age) is later than the emergence of ADHD symptoms. Therefore, a common pitfall when observing new onset tics in children receiving stimulants for ADHD is to erroneously assume that the tics are a stimulant adverse effect , leading to medication being stopped/swapped/reduced, when fact it is an emerging co-morbid tic disorder. If tics emerge very soon after starting stimulant medication – a dose reduction is warranted, to observe if tics reduce. However, if tics emerge after a stable period of ADHD treatment, and tics also require treatment, then augmentation with clonidine or guanfacine should be considered. Augmenting medication with behavioural therapy It is a common pitfall to assume that medication should only be tried after using psychological approaches such as behavioural therapy. Whilst acknowledging in reality these resources are scarce, when available, it is not always beneficial to consider medication only after first attempting psychological therapies. Often barriers to successfully implementing these therapists can relate to the age or intellectual ability of the young person, co-morbidities such as ADHD and autism, family and environmental factors, and severity/impairment of the tics. For more severe tics, medication may reduce tic intensity/frequency and make behavioural techniques easier to implement. Notably, there is no current evidence to suggest that medication reduces the efficacy/effectiveness of behavioural interventions. Allow sufficient time : The natural course of tics is a waxing (increasing) and waning (decreasing) pattern over weeks or months. Over hours and days, tics can also worsen due to, for example, anxiety, anger, excitement or tiredness. Tics commonly also wax during stressful periods such as starting the new school year or exams. A common pitfall is to prematurely assume treatment is effective when simply observing a natural waning phase, or conversely, that a drug treatment is ineffective when observing a natural waxing phase. To avoid these pitfalls, it is important to evaluate treatment effects over a sufficiently long period, usually 2–3 months, to compare peaks (waxing phases) and throughs (waning phases) before and after treatment initiation. Is the tic diagnosis correct? Since the COVID-19 pandemic, there has been an unprecedented rise in functional tics, particularly in teenage girls . Functional tics can be difficult to differentiate from a primary tic disorder and can also co-occur with primary tics. However, as functional tics are not responsive to standard tic medications – a common pitfall when failing to observe a tic treatment response at standard doses may be to continue dose escalation, thus causing increasing adverse effects . Another common diagnostic pitfall is related to motor mannerisms and stereotypies which occur in autism and can mimic complex motor tics but are not responsive to tic treatments. Failing to ‘start low and go slow’ : When initiating medication, typically adverse effects occur before therapeutic benefits are seen – and can lead to premature discontinuation. This can be avoided by gradual titration; ‘starting low and going slow’. For example, initial syncope (due to postural hypotension) can be related to the initiation of clonidine (which stimulates postsynaptic alpha 2-adrenergic receptors) and guanfacine (which stimulates postsynaptic alpha 2A-adrenergic receptors) and sedation is potentially related to all medications for tics. These adverse effects are often encountered if the initial dose is too high. However, with careful low-dose initiation and slow titration, these adverse effects are much less likely to occur. Doses of 25 mcg of clonidine, 1 mg guanfacine and 0.5–1 mg aripiprazole once daily are suggested as appropriate initial doses, with titrations using a weekly, fortnightly or even monthly approach according to the response. However, the initial dose and the titration speed could differ according to body weight, age and other characteristics of the patient. Failing to achieve an adequate therapeutic dose : When a medication has been initiated, it is important to titrate to recommended therapeutic doses. In the case of clonidine, this relates to an average daily dose of 3–5 mcg/kg , for aripiprazole around 5–10 mg daily and for guanfacine between 1 and 4 mg in young children and up to 7 mg in older adolescents according to weight . Often young people are sub-optimally medicated to reduce potential side effects. However, it is a common pitfall to conclude that a medication is ineffective, when in fact it has been used at a sub-therapeutic dose. Awareness of co-morbidities is important in determining the best drug choice. Commonly, young people with co-morbid tics and ADHD find noradrenergic agents (clonidine, guanfacine) beneficial for tic symptom control, whereas young people with anxiety disorders or OCD co-morbidities may find an approach using aripiprazole or treatment primarily targeting anxiety and/or OCD with an SSRI most beneficial in managing tic symptoms. Tic disorders occur in around 20% of young people with ADHD. However, the onset of tics (typically around 7–8 years of age) is later than the emergence of ADHD symptoms. Therefore, a common pitfall when observing new onset tics in children receiving stimulants for ADHD is to erroneously assume that the tics are a stimulant adverse effect , leading to medication being stopped/swapped/reduced, when fact it is an emerging co-morbid tic disorder. If tics emerge very soon after starting stimulant medication – a dose reduction is warranted, to observe if tics reduce. However, if tics emerge after a stable period of ADHD treatment, and tics also require treatment, then augmentation with clonidine or guanfacine should be considered. It is a common pitfall to assume that medication should only be tried after using psychological approaches such as behavioural therapy. Whilst acknowledging in reality these resources are scarce, when available, it is not always beneficial to consider medication only after first attempting psychological therapies. Often barriers to successfully implementing these therapists can relate to the age or intellectual ability of the young person, co-morbidities such as ADHD and autism, family and environmental factors, and severity/impairment of the tics. For more severe tics, medication may reduce tic intensity/frequency and make behavioural techniques easier to implement. Notably, there is no current evidence to suggest that medication reduces the efficacy/effectiveness of behavioural interventions. |
Revisión de botiquines de pacientes por las farmacias comunitarias de Álava | f4f60bd1-1db9-4164-8b67-06a2cd6ce318 | 11739903 | Patient Education as Topic[mh] | El Consejo General de Colegios Oficiales de Farmacéuticos definió la estrategia de la profesión farmacéutica basada en tres ejes: “somos asistenciales, somos sociales y somos digitales” . Esta estrategia contribuye a acelerar la transformación y retos de la “Agenda 2030”, que incluye los 17 “Objetivos de Desarrollo Sostenible” para lograr un futuro más justo, saludable y respetuoso con el medio ambiente. Desde el Colegio Oficial de Farmacéuticos (COF) de Álava diseñamos el proyecto de Revisión de Botiquines, con el que pretendimos, además de impulsar la realización de un SPFA en las farmacias comunitarias de Álava, resaltar la labor del farmacéutico como agente activo capaz de fomentar hábitos sanitarios y medioambientales en pro de la consecución de estos objetivos. En relación con nuestra labor asistencial, el objetivo del proyecto fue mejorar la utilización de los medicamentos, comprobando el conocimiento que los pacientes tenían de los mismos, garantizando la seguridad y optimizando su uso para aumentar su bienestar y salud y así evitar resultados negativos asociados a la medicación (RNM). Y con respecto a la labor social, el objetivo fue fomentar la adecuada gestión de los residuos de los medicamentos, educando a los pacientes en esta materia para que los eliminen en los puntos SIGRE de las farmacias comunitarias. Los objetivos específicos de este proyecto fueron: Comprobar si los medicamentos se almacenaban correctamente, detectando y eliminando los que estuviesen caducados, los innecesarios o en mal estado en el punto SIGRE. Formar sobre la adecuada conservación de los medicamentos y la eliminación de sus envases y restos en los puntos SIGRE de la farmacia comunitaria. Comprobar el conocimiento que tenían los pacientes de todos y cada uno de los medicamentos que conforman el botiquín, asesorándoles sobre su uso y facilitando información personalizada del medicamento (IPM). Detectar los medicamentos, productos sanitarios y/o de autocuidado que pudieran suponer un riesgo para la salud del paciente (seguridad). Detectar las incidencias: problemas relacionados con el medicamento (PRM) y RNM y realizar las correspondientes intervenciones farmacéuticas. Dar pautas para ordenar el botiquín. La revisión de botiquines tuvo lugar durante los meses de junio y julio de 2022. Se convocó a todas las farmacias de Álava a formar parte de la campaña “Pon al día tu botiquín”. La Comisión de SPFA y SIGRE impartieron un curso de formación en el que se explicaron los objetivos de la campaña, el procedimiento, registro de datos y el proceso de reciclado de medicamentos en el punto SIGRE. Ofrecimiento del servicio El servicio se ofreció a los pacientes que lo solicitaron o que el farmacéutico detectó que podrían beneficiarse del mismo siguiendo los criterios de inclusión y exclusión que se indican a continuación. Los criterios de inclusión fueron: Pacientes polimedicados. Pacientes con problemas en el manejo de la medicación. Pacientes que hubieran sufrido cambios en la medicación en los últimos meses. Pacientes que estuvieran tomando medicamentos considerados de alto riesgo. Pacientes en los que se comprobó en receta electrónica que no habían retirado prescripciones (sospecha de falta de adherencia). Quedaron excluidos: Menores de edad. Pacientes cuyas capacidades cognitivas o idiomáticas no les permitían entender la finalidad del estudio. Pacientes que hubieran sufrido cambios en la medicación en los últimos meses. Pacientes residentes en centros sociosanitarios. Citación del paciente Después que el paciente aceptó participar en el servicio, se concretó día y hora para realizar la revisión y se le comentó que acudiese con todos los medicamentos de tratamiento activo (MTA) junto con su hoja de tratamiento o informe, así como con los medicamentos no incluidos en el tratamiento activo, productos sanitarios y/o de autocuidado (MPSA) que contenía su botiquín. En caso de disponer de medicamentos termolábiles, se pidió que los trajesen anotados. Revisión del botiquín La entrevista se realizó en la zona de atención personalizada de la farmacia. Se separaron los MTA del resto. Revisión de MTA Se pidió al paciente que identificase los medicamentos uno a uno y se comprobó si había algún medicamento incluido en la hoja de tratamiento o en el informe que no hubiese traído. Se realizaron las preguntas correspondientes a los mismos y en el caso de detectar alguna incidencia, se realizó la intervención farmacéutica correspondiente y se registró (tabla 1). Revisión de MPSA Se pidió al paciente que identificase los MPSA uno a uno y se realizaron las preguntas del formulario correspondientes a los mismos y, en caso de detectar alguna incidencia, se realizó la intervención farmacéutica correspondiente y se registró (tabla 1). Repaso final Antes de finalizar la entrevista se realizó un repaso final, para asegurar que el paciente había entendido todo correctamente y que la recogida de datos había sido la adecuada. Por último, se le dieron consejos para el uso y mantenimiento del botiquín acompañado de la infografía para pacientes (figura 1) y de la infografía de eliminación de residuos en el punto SIGRE (figura 2). Formulario electrónico Una vez realizada la entrevista y ya sin el paciente en la farmacia, con toda la información recogida, se cumplimentó el formulario de forma electrónica para el procesamiento de datos. En la campaña participaron 10 farmacias comunitarias que realizaron 49 revisiones de botiquines. Los pacientes a los que se prestó el Servicio de Revisión de Botiquín fueron 49, de los cuales un 67,3 % fueron mujeres. Por rangos de edad, el 73,5 % de los pacientes eran mayores de 70 años y el 26,5 % tenían entre 30 y 70 años. No hubo pacientes menores de 30 años. El total de medicamentos y productos de botiquín analizado fue de 710, de los que 383 (53,9 %) fueron MTA y 326 (45,9 %) MPSA. El 67,3 % de los pacientes almacenaba sus medicamentos en un lugar inadecuado (cocina y/o baño), frente a un 32,7 % que lo hacía correctamente. Resultados de la revisión de MTA De los 383 MTA revisados, 101 (26,4 %) presentaron incidencias (gráfico 1), entre las que destacaron: conservación inadecuada: medicamento sin prospecto y/o sin embalaje, temperatura inadecuada, exceso de tiempo desde que se desprecintó, etc. (27,9 %), falta de conocimiento del uso del medicamento (26,5 %), falta de adherencia (20,4 %) y dosis, pauta y/o duración no adecuada (12,2 %). Como se observa en la tabla 2, de acuerdo con la prueba chi cuadrado, de las 49 personas a las que se realizó la revisión de botiquines, ni el género ni la edad son variables que influyan en el número de personas con MTA con o sin incidencias (p=0,21 para género y p= 0,99 para edad). Del mismo modo, no se puede establecer un grupo poblacional diana con incidencia en MTA en función de la edad y el sexo (p=0,72), siempre teniendo en consideración que no han participado en el estudio menores de 30 años. Sin embargo, se observa que, de las 101 incidencias detectadas en los 383 MTA, existe una tendencia a que las mujeres de más de 70 años sean las personas que mayor número de incidencias acumulan en relación con este tipo de medicamentos (p=0,08). Las intervenciones farmacéuticas realizadas (gráfico 2) para resolver estas incidencias fueron principalmente: facilitar IPM (42,0 %), informar sobre pautas de ordenación del botiquín (20,0 %) e intentar solucionar la falta de adherencia (18,0 %). Resultados de la revisión de MPSA De los 326 MPSA revisados, 157 (48,1 %) presentaron incidencias (gráfico 3), entre las que destacaron: medicamento caducado (48,0 %), medicamento no necesario (20,7 %) y falta de conocimiento del uso del medicamento (12,1 %). Tal como se muestra en la tabla 3, aplicando la prueba chi cuadrado, existen diferencias significativas, donde se destaca que las mujeres acumulan más MPSA con incidencias (p=0,03), mientras que no se observan diferencias en cuanto a la edad (p=0,17). Tampoco existen diferencias en función de la edad y el sexo (p=0,07) por lo que no se puede establecer un grupo poblacional diana con incidencias en los MPSA, siempre teniendo en consideración que no han participado en el estudio menores de 30 años. De las 157 incidencias detectadas en los MPSA, existen diferencias significativas, siendo las mujeres mayores de 70 años las que acumulan más incidencias en relación con los MPSA (p=0,001). Las intervenciones farmacéuticas realizadas (gráfico 4) para resolver estas incidencias fueron mayoritariamente: desechar al punto SIGRE (75,0 %) y facilitar IPM (13,7 %). Eliminación en el punto SIGRE Los farmacéuticos eliminaron en el punto SIGRE de sus farmacias un total de 137 medicamentos y productos sanitarios y/o autocuidado, lo que supone el 19,3 % del total de los revisados, siendo mayoritariamente los MPSA los eliminados: 125 envases (91,2 %), frente a los 12 envases de MTA (8,8 %). De los MPSA eliminados, los principales grupos fueron: analgésicos y antiinflamatorios no esteroideos (AINE) (20,6 %), antibióticos (19,8 %), antisépticos y material de cura (14,3 %) y tratamiento de patología invernal (8,7 %). La causa principal de eliminación en el punto SIGRE, tanto de los MTA como de los MPSA fue medicamento caducado, un 33,3 % y un 84,2 % respectivamente. Acumular medicamentos en el domicilio es una práctica muy extendida en los hogares y no siempre exenta de riesgos. La conservación en condiciones inadecuadas y la falta de conocimiento, entre otros motivos, pueden hacer del domicilio lugar de origen de PRM con consecuencias negativas para la salud de los pacientes y aumento del gasto sanitario. Los farmacéuticos debemos desarrollar medidas informativas y educativas que ayuden a nuestros pacientes a detectar y prevenir estos problemas. Los SPFA, especialmente los relacionados con la Atención Farmacéutica, es decir, con los medicamentos y productos sanitarios, pueden suponer una estrategia útil en la reducción de problemas del uso de la medicación. Una muestra de ello es el servicio de revisión de botiquines enmarcado por FORO AF FC entre los orientados al proceso de uso . Los datos obtenidos en este estudio muestran cómo el 67,3 % de las personas entrevistadas guardaba sus medicamentos en lugares inadecuados como la cocina o el baño, datos que concuerdan con los hallados por Hernández y cols. y Matos y cols. . Estos son lugares sometidos a grandes variaciones de humedad y temperatura, lo cual puede afectar a la estabilidad de los medicamentos. A la vista de estos datos, observamos que los pacientes necesitan tener más información sobre las condiciones adecuadas de conservación de sus botiquines. Las personas a las que se realizó la revisión de botiquines fueron mayoritariamente mujeres (67,3 %), ya que son las que generalmente se responsabilizan en mayor medida de los tratamientos farmacológicos/cuidados médicos en el hogar. En cuanto a la edad, la mayor parte de los entrevistados fueron mayores de 70 años (73,5 %). Esto puede deberse a que las personas de esta edad suelen ser pacientes pluripatológicos y polimedicados y, por tanto, acuden con mayor frecuencia a la farmacia comunitaria, datos que concuerdan con los hallados por Nuñez y cols y Flores y cols . Llama la atención el hecho de que no se contemplen pacientes menores de 30 años. La causa puede ser que en este grupo de edad es muy poco frecuente encontrar personas que cumplan con los criterios de inclusión. Dado que son las mujeres mayores de 70 años las que se relacionan con el mayor número de incidencias en los MTA y MPSA, podría ser interesante realizar futuros estudios en este grupo poblacional con el objetivo de intentar reducir su número. En un 26,4 % de los MTA se detectaron incidencias, siendo las principales: conservación inadecuada (medicamentos sin prospecto, y/o sin embalaje original, etc.), falta de conocimiento del uso del medicamento (para qué sirve, pauta de dosificación, etc.). Son datos que concuerdan con los estudios realizados por el Muy Ilustre Colegio Oficial de Farmacéuticos de Valencia . En el 48,3 % de los MPSA se detectaron incidencias, siendo la principal los medicamentos caducados, al igual que se recoge en el estudio de Berenguer y cols. . La eliminación en el punto SIGRE fue la intervención más importante, eliminándose un total de 137 medicamentos, lo que suponía entre 2 y 3 medicamentos por persona, al igual que se recoge en el estudio de Oñatibia y cols. . La eliminación de MPSA (91,2 %) frente a los MTA (8,80 %) fue muy superior. Las posibles causas serían la automedicación, tratamientos no acabados y cambios de tratamiento en los que se ha almacenado el medicamento deprescrito. De los MPSA eliminados en el punto SIGRE destacan analgésicos y AINE, antibióticos y antisépticos y material de cura, resultados similares a los del estudio de Echave y cols. . El farmacéutico intervino informando y educando en la necesidad de evitar el sobreconsumo de medicamentos y la acumulación en los hogares, así como en la necesidad de eliminar adecuadamente los medicamentos caducados y no consumidos en el punto SIGRE de las farmacias . Por todo ello, los resultados de esta campaña muestran que el farmacéutico comunitario es un agente sanitario clave en la promoción de actividades de información y educación sanitaria que orientan al paciente hacia la sensibilización del uso racional, seguro y efectivo de los medicamentos, los aspectos negativos de la automedicación no responsable, la mejora de aspectos del botiquín como son su ordenación, ubicación y revisiones periódicas, así como en la adecuada eliminación de los residuos medicamentosos en los puntos SIGRE por lo que concluimos que el Servicio de Revisión de Botiquines debería estar integrado en la cartera de SPFA. A SIGRE por su disponibilidad y su colaboración en este proyecto, a Jonatan Miranda por el tratamiento estadístico de los datos y a todas las farmacias de Álava participantes en este proyecto. Por orden alfabético: Cristina Dávila, García-Agundez, González Casi, Ibarra, Imanol Monteagudo, López de Ocáriz, Mosteiro, Mozas, Pérez Zubiaur, Villacorta. |
Impact of TDP‐43 co‐pathology on limbic cortical microstructure in Alzheimer's disease: evidence from an autopsy‐confirmed cohort with in vivo diffusion MRI | e33041f0-751d-47d4-b75a-6353f7027fe8 | 11712743 | Forensic Medicine[mh] | |
Perspectives from clinicians from different levels of care in Maputo, Mozambique: qualitative study of the barriers to and facilitators of paediatric injury care in resource-poor hospital settings | 986a7953-11f7-4f6d-aea8-1ecc876ff228 | 11590845 | Pediatrics[mh] | Globally, injuries are the leading cause of death among children aged 5 to 14 years, and 95% of those deaths occur in low- and middle-income countries (LMICs). Injuries are also the largest source of premature morbidity and mortality globally, regardless of age, sex, income or geographic region. Timely quality healthcare can significantly improve poor injury outcomes by ensuring the provision of essential emergency care services by well-trained staff. This need is particularly clear in resource-poor settings, especially Sub-Saharan Africa (SSA), where prehospital emergency response is generally underdeveloped and hospital emergency services suffer from shortages in staff, technical resources and medications for injury care. An additional set of constraints in the case of injured children is that they are typically treated without adequate regard for their specific needs by clinical staff ill-prepared or unfamiliar with paediatric injury care. This, in turn, increases the likelihood of diagnostic error and inadequate treatment with the potential of worsened outcomes and low survival rates. Another common finding in SSA is the quality gap between public and private hospitals, documented among others in South Africa, Uganda, Kenya and Rwanda, to the detriment of public hospitals. This is particularly the case at first-level (district) hospitals where critical care services are either non-existent or very resource-constrained. SSA studies investigating hospital care services typically focus on hospitals from a specific level of care across the country or, in some instances, on hospitals from several levels of care. A focus group with medical providers in a referral hospital in Uganda, for example, identified major barriers to caring for critically ill children including limited resources, staffing and training gaps. A study looking at all 30 primary and secondary hospitals in western Kenya found that the provision of core emergency care services for injuries and non-communicable diseases suffered from a range of issues including gaps in communication, infrastructure, supplies and trained human resources. In the Kingdom of Eswatini, a study evaluating emergency care capacity in three referral hospitals highlighted concerns related to infrastructure barriers for critical care (eg, inadequate physical space, electricity or water) and a lack of equipment, personnel knowledge and skills. In Mozambique, a recent study on paediatric injury care in the country’s four largest referral hospitals found that these hospitals had significant shortages of medications (eg, for infections, poisoning and cardiovascular disorders) and certain equipment (eg, for diagnosis and monitoring, patient safety and airway management). The extent of these shortages in hospitals at other levels of care in the country has not been documented, which leaves unanswered questions regarding care provision at these sites. The perception of the staff in those settings is also not known but is something that could give better insight into how problems are experienced and dealt with locally, what solutions are envisaged and whether there is consensus in those aspects. This study therefore explores how barriers to and facilitators of paediatric injury care at different levels of care are described by healthcare staff. It focuses on the context of Maputo, Mozambique, where the main referral hospital of the country is located and where several hospitals at different care levels are also located. Study design We conducted a qualitative interview-based study using one-to-one structured interviews with healthcare providers who take care of injured children to explore the barriers to and facilitators of better paediatric injury care. Inductive content analysis was used to better elicit the narrative of the health professionals. Setting and selection of hospitals In this study, a mix of all levels of care including district and referral hospitals was chosen, focused on Maputo, the capital and the country’s biggest city. From Maputo city, we chose Maputo Central Hospital, the only central one in the country—and one of the two secondary hospitals (Jose Macamo General Hospital), located in the suburbs of Maputo city. From outside the city but still in the province, we chose one tertiary hospital (Matola Provincial Hospital) and one secondary hospital (Manhiça Distrital Hospital), respectively, 18 and 82 km from Maputo Central Hospital (see ). Mozambique is a low-income SSA country with an estimated 60% of the 33.7 million inhabitants (2023 data) living in rural areas with no or limited access to healthcare. The country is divided into 10 provinces and one capital city, Maputo, with provincial status. Overall, Mozambique has around 1600 health facilities, 96% of which only deliver primary care services. Most of the population has access to free public healthcare services, organised into four levels: primary care (health centres), secondary care (rural hospitals; district or general hospitals), tertiary care (provincial hospitals) and quaternary care (central hospitals). Mozambique does not have any trauma centre, paediatric hospital or trauma system organisation. Participant selection and sample size Healthcare workers were purposively selected from Maputo Central Hospital, Jose Macamo General Hospital, Matola Provincial Hospital and Manhiça Distrital Hospital. The healthcare workers were chosen to represent those with a range of knowledge and experience in paediatric injury care. They would contribute to identifying and obtaining information related to barriers to and facilitators of better paediatric injury care. During the visit, clinicians taking care of injured children and working full time were asked to participate in an interview. We sought clinicians of different cadres, including surgery technicians, nurses and medical doctors from different categories such as general practitioners; surgery residents; paediatricians; and general, orthopaedic, neuro, plastic and paediatric surgeons. We aimed to interview around 10 clinicians per hospital, covering the most representative professional categories (surgeons, paediatricians, nurses and technicians). We proceeded until we felt thematic saturation was reached, and additional interviews would not generate new themes. None of the clinicians contacted refused participation or dropped out of the interview. Data collection A semistructured interview guide was developed in Portuguese that incorporated the areas of concern highlighted in our earlier study conducted in the central hospital. The interview guide used in this study is provided as . The clinicians were asked about the main difficulties they encountered while taking care of injured children, why they thought these difficulties existed and in what way they impeded quality care. They also answered questions on how they cope with the difficulties, what the main facilitators of injury care could be and how to move forward to make improvements. The data collection involved three people, the principal investigator (PI) and two experienced research assistants (RAs), educated in social anthropology and psychology, who received specific training for the study. The RAs performed the interviews under the coordination of the PI who also recruited the interviewees. Data collection took place over four consecutive weeks, during working hours. Before the interviews, the RA obtained written consent. All those contacted gave their consent. All interviews were face-to-face and performed in a quiet meeting room at the hospital, at a time most convenient for the interviewee. The interviews were audio-recorded for transcription, and field notes were taken. No repeat interviews were carried out. For each hospital, the PI and the two RAs discussed data saturation. The duration of the interviews varied from 35 to 60 min. No transcriptions were returned to participants for comments or correction. All material was kept anonymous. The audio recordings were transferred to a password-protected computer and stored, while the original recording was deleted. All consent forms and de-identified transcripts were stored in a protected locker belonging to the PI and first author. Ethical considerations In June 2022, the management of each targeted hospital received a request letter for participation in the study, which they all accepted. With their acceptance letter, the study protocol was submitted for ethical approval by the Mozambique Institutional Bioethics Committee of the Faculty of Medicine and Maputo Central Hospital (CIBS FM & HCM/107/2019). Once ethical approval was obtained, the director of each hospital was contacted to suggest a time suitable for data collection. The directors informed their staff about the visit, and voluntary participation was emphasised. Observations To get familiarised with the work environment of each hospital and contextualise capture the barriers to and facilitators of paediatric injury care described by the people interviewed, the PI and RAs observed for 8 hours split in two weekdays. They observed the infrastructure, equipment in place, specialised injury care delivery and how the health workers performed their tasks. Note was taken of anything related to the premises, equipment and tasks, or task division that was felt as susceptible to complicate the execution of the tasks or, rather, facilitate trauma care activities was noted. Research team and reflexivity The research team and authors include three women and two men with different levels of seniority. Two of the women are specialised surgeons from Maputo, and the third one is a senior scientist specialised in injury epidemiology on prevention studies, including in the clinical field. One of the men is an anthropologist with experience in qualitative data collection in Mozambique, and the other is a medical doctor specialised in emergency medicine with considerable international experience. This paper was an attempt to shed light on the barriers to and facilitators of paediatric trauma care experienced by clinical staff at different levels of care. The data collection was led by a paediatric surgeon—and doctoral student—very familiar with the Mozambican context, assisted by two experienced data collectors who conducted all the interviews. The recruitment of interviewees went well, and the interviewers were well-prepared in order to establish a relationship of trust before engaging in the interviews. The data analysis (that of the interview transcripts) took place in different steps, involving first to those who were directly involved in the data collection, all native Portuguese speakers who immersed themselves in the coding process. Other co-authors were involved in discussions about the subcategories and categories. Patient and public involvement Neither injured patients nor the public were involved in the study design, data collection, recruitment, conduct or analysis of this study. The research questions and outcomes were based on a gap in the literature related to how the shortage of equipment and medications found in the country’s four largest referral hospitals affect other levels of care. They also took into account the perception of the healthcare providers on the barriers to and facilitators of paediatric injury care. We hope the results will inform future researchers and help implementation researchers improve paediatric injury care in LMICs. We also plan to disseminate the information at national and international conferences. Data analysis All interviews were recorded and transcribed verbatim in Portuguese, while the study team members also took written notes in case there were problems with recordings. Transcripts were coded in NVivo, V.14. The first author and one RA coded the data, starting with familiarising themselves with and reviewing the transcriptions while listening to the audio recordings several times. Meaning units were inductively identified and translated to codes. These were then revised, organised and compared for consistency, and discrepancies were resolved among the researchers. The codes were thereafter classified into subcategories and categories based on their similarities and differences. Results were written after re-reading the categories and subcategories. We conducted a qualitative interview-based study using one-to-one structured interviews with healthcare providers who take care of injured children to explore the barriers to and facilitators of better paediatric injury care. Inductive content analysis was used to better elicit the narrative of the health professionals. In this study, a mix of all levels of care including district and referral hospitals was chosen, focused on Maputo, the capital and the country’s biggest city. From Maputo city, we chose Maputo Central Hospital, the only central one in the country—and one of the two secondary hospitals (Jose Macamo General Hospital), located in the suburbs of Maputo city. From outside the city but still in the province, we chose one tertiary hospital (Matola Provincial Hospital) and one secondary hospital (Manhiça Distrital Hospital), respectively, 18 and 82 km from Maputo Central Hospital (see ). Mozambique is a low-income SSA country with an estimated 60% of the 33.7 million inhabitants (2023 data) living in rural areas with no or limited access to healthcare. The country is divided into 10 provinces and one capital city, Maputo, with provincial status. Overall, Mozambique has around 1600 health facilities, 96% of which only deliver primary care services. Most of the population has access to free public healthcare services, organised into four levels: primary care (health centres), secondary care (rural hospitals; district or general hospitals), tertiary care (provincial hospitals) and quaternary care (central hospitals). Mozambique does not have any trauma centre, paediatric hospital or trauma system organisation. Healthcare workers were purposively selected from Maputo Central Hospital, Jose Macamo General Hospital, Matola Provincial Hospital and Manhiça Distrital Hospital. The healthcare workers were chosen to represent those with a range of knowledge and experience in paediatric injury care. They would contribute to identifying and obtaining information related to barriers to and facilitators of better paediatric injury care. During the visit, clinicians taking care of injured children and working full time were asked to participate in an interview. We sought clinicians of different cadres, including surgery technicians, nurses and medical doctors from different categories such as general practitioners; surgery residents; paediatricians; and general, orthopaedic, neuro, plastic and paediatric surgeons. We aimed to interview around 10 clinicians per hospital, covering the most representative professional categories (surgeons, paediatricians, nurses and technicians). We proceeded until we felt thematic saturation was reached, and additional interviews would not generate new themes. None of the clinicians contacted refused participation or dropped out of the interview. A semistructured interview guide was developed in Portuguese that incorporated the areas of concern highlighted in our earlier study conducted in the central hospital. The interview guide used in this study is provided as . The clinicians were asked about the main difficulties they encountered while taking care of injured children, why they thought these difficulties existed and in what way they impeded quality care. They also answered questions on how they cope with the difficulties, what the main facilitators of injury care could be and how to move forward to make improvements. The data collection involved three people, the principal investigator (PI) and two experienced research assistants (RAs), educated in social anthropology and psychology, who received specific training for the study. The RAs performed the interviews under the coordination of the PI who also recruited the interviewees. Data collection took place over four consecutive weeks, during working hours. Before the interviews, the RA obtained written consent. All those contacted gave their consent. All interviews were face-to-face and performed in a quiet meeting room at the hospital, at a time most convenient for the interviewee. The interviews were audio-recorded for transcription, and field notes were taken. No repeat interviews were carried out. For each hospital, the PI and the two RAs discussed data saturation. The duration of the interviews varied from 35 to 60 min. No transcriptions were returned to participants for comments or correction. All material was kept anonymous. The audio recordings were transferred to a password-protected computer and stored, while the original recording was deleted. All consent forms and de-identified transcripts were stored in a protected locker belonging to the PI and first author. In June 2022, the management of each targeted hospital received a request letter for participation in the study, which they all accepted. With their acceptance letter, the study protocol was submitted for ethical approval by the Mozambique Institutional Bioethics Committee of the Faculty of Medicine and Maputo Central Hospital (CIBS FM & HCM/107/2019). Once ethical approval was obtained, the director of each hospital was contacted to suggest a time suitable for data collection. The directors informed their staff about the visit, and voluntary participation was emphasised. To get familiarised with the work environment of each hospital and contextualise capture the barriers to and facilitators of paediatric injury care described by the people interviewed, the PI and RAs observed for 8 hours split in two weekdays. They observed the infrastructure, equipment in place, specialised injury care delivery and how the health workers performed their tasks. Note was taken of anything related to the premises, equipment and tasks, or task division that was felt as susceptible to complicate the execution of the tasks or, rather, facilitate trauma care activities was noted. The research team and authors include three women and two men with different levels of seniority. Two of the women are specialised surgeons from Maputo, and the third one is a senior scientist specialised in injury epidemiology on prevention studies, including in the clinical field. One of the men is an anthropologist with experience in qualitative data collection in Mozambique, and the other is a medical doctor specialised in emergency medicine with considerable international experience. This paper was an attempt to shed light on the barriers to and facilitators of paediatric trauma care experienced by clinical staff at different levels of care. The data collection was led by a paediatric surgeon—and doctoral student—very familiar with the Mozambican context, assisted by two experienced data collectors who conducted all the interviews. The recruitment of interviewees went well, and the interviewers were well-prepared in order to establish a relationship of trust before engaging in the interviews. The data analysis (that of the interview transcripts) took place in different steps, involving first to those who were directly involved in the data collection, all native Portuguese speakers who immersed themselves in the coding process. Other co-authors were involved in discussions about the subcategories and categories. Neither injured patients nor the public were involved in the study design, data collection, recruitment, conduct or analysis of this study. The research questions and outcomes were based on a gap in the literature related to how the shortage of equipment and medications found in the country’s four largest referral hospitals affect other levels of care. They also took into account the perception of the healthcare providers on the barriers to and facilitators of paediatric injury care. We hope the results will inform future researchers and help implementation researchers improve paediatric injury care in LMICs. We also plan to disseminate the information at national and international conferences. All interviews were recorded and transcribed verbatim in Portuguese, while the study team members also took written notes in case there were problems with recordings. Transcripts were coded in NVivo, V.14. The first author and one RA coded the data, starting with familiarising themselves with and reviewing the transcriptions while listening to the audio recordings several times. Meaning units were inductively identified and translated to codes. These were then revised, organised and compared for consistency, and discrepancies were resolved among the researchers. The codes were thereafter classified into subcategories and categories based on their similarities and differences. Results were written after re-reading the categories and subcategories. Characteristics of the participants A total of 40 participants were interviewed. Their characteristics are shown in . The average age varied between hospitals, from 31 years in Manhiça Distrital Hospital to 42 years in Matola Provincial Hospital. Most participants were male (n=26). Nurses were the most represented profession (62.5%), and the average length of years employed ranged from 5 to 8 years. Barriers Before moving on to the barriers, we highlighted the resources that the clinicians most reported as missing in each hospital, as these either impeded the provision of quality care on-site or necessitated sending patients to a higher level of care. Those missing resources are summarised in , ordered from the hospital at the highest level of care to the lowest, and classified into competence, equipment, paediatric intensive care (PICU) and specialised care. From the narrative of the participants, four main categories of barriers emerged (see ), labelled prehospital care constraints, shortage and limitation in child adaptability of resources, inappropriate infrastructure for emergency care and limited staff. Prehospital care constraints Clinicians from all hospitals reported as a main barrier the difficulties of transporting children from the injury site to the hospital most suited for their care, largely due to traffic jams, quality of the roads and frequent road traffic crashes. Access to the nearest hospital . Most of the clinicians talked about the frequency of road traffic injuries and the fact that several road traffic crashes involve overcrowded public transport vehicles. Those crashes lead to serious traffic jams and obstruct the transportation of victims to hospital. Injured children are often referred to the nearest hospital using private cars or evacuated in police cars without any care, arriving at hospital in a worse condition and with secondary injuries. We do not have prehospital care in which there are doctors who take care of injured patients at the scene of the accident…It takes a very long time to arrive at the Maputo Central Hospital, some arrive in a very critical condition that is perhaps impossible to recover from. (Doctor 1, Maputo Central Hospital) Caregiver’s consent . After the first assessment by the team, critical patients must be referred to a specialised hospital to continue the treatment, but generally, caregivers—mainly mothers—hesitate to give their consent either for financial concerns, because one parent would like to have the assent of the other or because they have other children to take care of. There are situations in which…we take a while to refer because the decision is not made by the person who is the hospital…the mother just says that the father is the one who can decide, the father is travelling…and he will arrive in a few hours. So, it makes us waste precious time . (Nurse 1, Manhiça Distrital Hospital) Referral . Clinicians also raised issues about the low number of ambulances available, which delays the initiation of care. Besides being few, those ambulances are poorly equipped for injury care. Injured patients compete with other emergencies and priorities. In the referral of patients, the vehicles often do not have oxygen, monitoring devices or ambulance stretchers, so a nurse must escort the patient. This reduces the number of staff available at the hospital, and, when the transport takes time, the patient may die during transport. Sometimes, we want to refer the patient in time, but the ambulance is at the Maputo Central Hospital because it was transporting another patient. When the injured child arrived, we stabilised, but we must wait for the ambulance to return. (Doctor 2, Matola Provincial Hospital) Shortage in child adaptability of resources Medication and medical supplies . Lack of medication and medical supplies was mentioned as affecting the quality of care and imposing limitations on interventions, making the work of the providers more complex. The lack of specific supplies for paediatric care such as splints, cervical collars, suitable sutures and catheters for children’s veins results in improvisation, compromising the effectiveness of procedures. Furthermore, it was mentioned that the lack of resources could affect patient safety and the quality of interventions. They are observed in the same place where adults are observed, and the same instruments are used. This is where the problem begins, the device for measuring blood pressure in adults is large and it will not be possible to measure blood pressure in a child when they enter intensive care services for adults too, if we want to insert an orotracheal tube then, the material used to insert the tube is for adults, it is not paediatric, so this leaves the child at a disadvantage . (Doctor 3, Maputo Central Hospital) Equipment . A lack of diagnosis and monitoring devices as well as anaesthesia machines adapted for paediatric patients was often reported by professionals from all level health facilities as a very common challenge for paediatric injury care and a quality deterrent. A shortage of CT scans, X-rays and anaesthesia machines limits the clinicians in performing diagnostic tests, operating in well-adapted rooms and monitoring injured victims in a critical condition. …Computerised axial tomography (CAT)…is a prior need to carry out some tests to identify the damage in detail, so we always choose to refer when it comes to moderate and severe traumatic brain injuries…because the Maputo Central Hospital is the only health unit that, until now, had CAT scans available . (Doctor 4, Matola Provincial Hospital) Inappropriate infrastructure for emergency care Respondents from all hospitals reported that the emergency department is inadequate for paediatric injury care as there is no specific place for injured patients: all emergency patients are admitted to the adult emergency department, which is not necessarily equipped or suitable for paediatric emergency care. Spaces . Clinicians mentioned that the small dimensions of the rooms where they treat injured patients obstruct the circulation of the staff and threaten the privacy of the patient (and family). The lack of space separators also limits the number of available beds (to about four to eight) when far more would be needed. So, injured children are cared for by adult clinicians, not in child-friendly spaces, mixed with adults of different sexes and with patients with other emergency conditions like malaria or tuberculosis. It is not an emergency service specific to trauma care. It is an emergency service that also treats other pathologies. So, you’re there with an injured child and with a patient with tuberculosis, COVID, e.g.…maybe the injured child runs the risk of being contaminated with other diseases, this is the risk we have because we don’t have an infrastructure for handling trauma in general . (Nurse 2, Matola Provincial Hospital) Resources . In all hospitals, respondents reported a lack of adequate facilities for paediatric injury care, with some differences based on the level of care of the hospital (see also ). In secondary and tertiary health facilities, clinicians reported a lack of PICU as one of the main reasons for referring critically injured patients to quaternary health facilities. They also mentioned that the emergency facility is unable to stabilise critically injured patients while waiting for the ambulance due to lack of oxygen, monitoring devices and fluids in this sector. …because we do not have an intensive care unit, or specialists to care for severe head trauma, we transfer these patients . (Nurse 3, Manhiça Distrital Hospital) PICU is available in the quaternary hospital, although clinicians mentioned that they do not have attached paediatric operating theatres, access to diagnostic tests like CT or even permanent surgeons to care for the injured child. An injured child is first admitted to the adult emergency by general surgeons, which results in errors in care and, when combined with a lack of access to diagnostic tests, affects the outcome. Patients are often sent from the PICU back to adult emergency for diagnostic testing, but an ambulance is required for this transfer which leads to significant delays in care. For example, if there is a traumatic brain injury, it should be evaluated by a neurosurgeon in the adult’s emergency to facilitate the diagnosis, treatment, and monitoring, before the injured child is referred to PICU. Sometimes the injured child comes without imaging exams and no care for her critical condition. Due to her critical situation, the child is not sent back to the imaging examination. So, one of our problems is that we don’t have, for example, the CAT, close to our intensive care unit and we must stabilise critical patients blinded without the auxiliary diagnosis . (Doctor 5, Matola Provincial Hospital) Limited staff available Qualifications . The paucity of staff well-trained to deal with paediatric injury patients was highlighted by clinicians from all four hospitals as was the lack of specialised clinicians (eg, paediatric surgeons, anaesthetists orthopaedics). Some health professionals reported that their previous education and experience often focused on adult care, making the transition to paediatric care difficult. Unfortunately, we had never had any paediatric training, capacity building or refresher training, we are limited, doing what we have learned at school. (Nurse 4, Manhiça Distrital Hospital) Staff . Most respondents underlined staff shortages and lack of staff training as serious challenges to quality paediatric injury care. They also report that because of the few radiologists available, they cannot do 24-hour imaging exams and in secondary care, and no X-rays are available after 3:30 pm and during the weekends. The first major limitation we have is the technical personnel, the personnel prepared to work with paediatric injury. Now we only have two surgical technicians who are working in the trauma section, and they…don’t work at the same time, one period is one…From morning until 1400 there is one, from 1400 to 2000 there is another. So, there are only two of them monitoring, so it is a huge overload for them and this huge overload ends up leading to a loss of efficiency, people with a lot of work like this are not able to be completely attentive all the time, the concern is to care in a short time the largest number of patients and it will not be based on quality, we are mainly based on the number of patients we serve. (Doctor 6, Jose Macamo General Hospital) Facilitators From the health professionals’ narrative, one main category of facilitator emerged: support and mentorship between professionals and institutions (see ). Mentorship of a qualified team . Participants reported that the emergency team contributes to facilitating the care of paediatric injuries in different ways. Both surgeons and surgical technicians gave great support to the team in serious and complex trauma situations, filling gaps and overcoming shortcomings. This cooperative environment avoids overloading health professionals and helps coordinate activities resulting in an improvement in the quality of care offered to injured children and in the solution of problems and challenges faced. The presence of a diverse and highly qualified team and the appropriate number of teams per shift facilitates injury paediatric care and allows for the exchange of experience. Collaboration of quaternary care . Participants from secondary and tertiary care have the opinion that Maputo Central Hospital facilitates their work. Communication via WhatsApp and video calls helps coordinate the referral process and allows the support of specialised clinicians with clinical advice to stabilise and manage critical patients, facilitate injury paediatric care, and exchange experience and resources. One of the facilitators is the good collaboration and relationship that we have, with the Maputo central hospital…Through phone consultation with paediatric surgeons for difficult cases and support…I think this good relationship is also one of the facilitators… (Doctor 7, Hospital Provincial da Matola) Most specialists from quaternary care consider paediatric emergency care and PICU as one of the most important facilitators for paediatric injury care as they stabilise and optimise the critical injury for further care or after surgery. When a severe traumatic brain injury child is admitted in the paediatric emergency department…it makes our work tremendously easier because as soon as the child arrives, s/he receives all the care that is set out in our protocol and paediatrics protocols in place…so when I receive the call to attend in the paediatric emergency, I go knowing that the child has received first aid is stabilised, they call me I will be more cheerful…whereas if I know that they are calling me for a child in the adult emergency room, I will already be very stressed, why? I’m scared of what I’m going to find. So, I would say that the paediatric emergency service is a great facilitator in our care for traumatic brain injury, when the child arrives, when we take the child to the operating room and return them to paediatric intensive care, ha! The care provided post-operatively is…excellent…I’m very satisfied with them (laughs). (Doctor 8, Maputo Central Hospital) Support of local and international institutions . Clinicians reported that partnerships with international organisations and local institutions facilitate the treatment of paediatric injuries by bringing new and advanced knowledge and surgical supplies and help with specialised care, ambulances and laboratory tests. Facilitators we are ourselves, with the few resources we have…we have some friends from the United States, they have helped us a lot, they come once a year in August, they are friends of the surgeon who treats the paediatric traumas, they help us a lot with material and new dynamics, also work, they learn from us, and we also learn from them… I think this is the only facilitator we have. (Nurse 5, Matola Provincial Hospital) A total of 40 participants were interviewed. Their characteristics are shown in . The average age varied between hospitals, from 31 years in Manhiça Distrital Hospital to 42 years in Matola Provincial Hospital. Most participants were male (n=26). Nurses were the most represented profession (62.5%), and the average length of years employed ranged from 5 to 8 years. Before moving on to the barriers, we highlighted the resources that the clinicians most reported as missing in each hospital, as these either impeded the provision of quality care on-site or necessitated sending patients to a higher level of care. Those missing resources are summarised in , ordered from the hospital at the highest level of care to the lowest, and classified into competence, equipment, paediatric intensive care (PICU) and specialised care. From the narrative of the participants, four main categories of barriers emerged (see ), labelled prehospital care constraints, shortage and limitation in child adaptability of resources, inappropriate infrastructure for emergency care and limited staff. Clinicians from all hospitals reported as a main barrier the difficulties of transporting children from the injury site to the hospital most suited for their care, largely due to traffic jams, quality of the roads and frequent road traffic crashes. Access to the nearest hospital . Most of the clinicians talked about the frequency of road traffic injuries and the fact that several road traffic crashes involve overcrowded public transport vehicles. Those crashes lead to serious traffic jams and obstruct the transportation of victims to hospital. Injured children are often referred to the nearest hospital using private cars or evacuated in police cars without any care, arriving at hospital in a worse condition and with secondary injuries. We do not have prehospital care in which there are doctors who take care of injured patients at the scene of the accident…It takes a very long time to arrive at the Maputo Central Hospital, some arrive in a very critical condition that is perhaps impossible to recover from. (Doctor 1, Maputo Central Hospital) Caregiver’s consent . After the first assessment by the team, critical patients must be referred to a specialised hospital to continue the treatment, but generally, caregivers—mainly mothers—hesitate to give their consent either for financial concerns, because one parent would like to have the assent of the other or because they have other children to take care of. There are situations in which…we take a while to refer because the decision is not made by the person who is the hospital…the mother just says that the father is the one who can decide, the father is travelling…and he will arrive in a few hours. So, it makes us waste precious time . (Nurse 1, Manhiça Distrital Hospital) Referral . Clinicians also raised issues about the low number of ambulances available, which delays the initiation of care. Besides being few, those ambulances are poorly equipped for injury care. Injured patients compete with other emergencies and priorities. In the referral of patients, the vehicles often do not have oxygen, monitoring devices or ambulance stretchers, so a nurse must escort the patient. This reduces the number of staff available at the hospital, and, when the transport takes time, the patient may die during transport. Sometimes, we want to refer the patient in time, but the ambulance is at the Maputo Central Hospital because it was transporting another patient. When the injured child arrived, we stabilised, but we must wait for the ambulance to return. (Doctor 2, Matola Provincial Hospital) Medication and medical supplies . Lack of medication and medical supplies was mentioned as affecting the quality of care and imposing limitations on interventions, making the work of the providers more complex. The lack of specific supplies for paediatric care such as splints, cervical collars, suitable sutures and catheters for children’s veins results in improvisation, compromising the effectiveness of procedures. Furthermore, it was mentioned that the lack of resources could affect patient safety and the quality of interventions. They are observed in the same place where adults are observed, and the same instruments are used. This is where the problem begins, the device for measuring blood pressure in adults is large and it will not be possible to measure blood pressure in a child when they enter intensive care services for adults too, if we want to insert an orotracheal tube then, the material used to insert the tube is for adults, it is not paediatric, so this leaves the child at a disadvantage . (Doctor 3, Maputo Central Hospital) Equipment . A lack of diagnosis and monitoring devices as well as anaesthesia machines adapted for paediatric patients was often reported by professionals from all level health facilities as a very common challenge for paediatric injury care and a quality deterrent. A shortage of CT scans, X-rays and anaesthesia machines limits the clinicians in performing diagnostic tests, operating in well-adapted rooms and monitoring injured victims in a critical condition. …Computerised axial tomography (CAT)…is a prior need to carry out some tests to identify the damage in detail, so we always choose to refer when it comes to moderate and severe traumatic brain injuries…because the Maputo Central Hospital is the only health unit that, until now, had CAT scans available . (Doctor 4, Matola Provincial Hospital) Respondents from all hospitals reported that the emergency department is inadequate for paediatric injury care as there is no specific place for injured patients: all emergency patients are admitted to the adult emergency department, which is not necessarily equipped or suitable for paediatric emergency care. Spaces . Clinicians mentioned that the small dimensions of the rooms where they treat injured patients obstruct the circulation of the staff and threaten the privacy of the patient (and family). The lack of space separators also limits the number of available beds (to about four to eight) when far more would be needed. So, injured children are cared for by adult clinicians, not in child-friendly spaces, mixed with adults of different sexes and with patients with other emergency conditions like malaria or tuberculosis. It is not an emergency service specific to trauma care. It is an emergency service that also treats other pathologies. So, you’re there with an injured child and with a patient with tuberculosis, COVID, e.g.…maybe the injured child runs the risk of being contaminated with other diseases, this is the risk we have because we don’t have an infrastructure for handling trauma in general . (Nurse 2, Matola Provincial Hospital) Resources . In all hospitals, respondents reported a lack of adequate facilities for paediatric injury care, with some differences based on the level of care of the hospital (see also ). In secondary and tertiary health facilities, clinicians reported a lack of PICU as one of the main reasons for referring critically injured patients to quaternary health facilities. They also mentioned that the emergency facility is unable to stabilise critically injured patients while waiting for the ambulance due to lack of oxygen, monitoring devices and fluids in this sector. …because we do not have an intensive care unit, or specialists to care for severe head trauma, we transfer these patients . (Nurse 3, Manhiça Distrital Hospital) PICU is available in the quaternary hospital, although clinicians mentioned that they do not have attached paediatric operating theatres, access to diagnostic tests like CT or even permanent surgeons to care for the injured child. An injured child is first admitted to the adult emergency by general surgeons, which results in errors in care and, when combined with a lack of access to diagnostic tests, affects the outcome. Patients are often sent from the PICU back to adult emergency for diagnostic testing, but an ambulance is required for this transfer which leads to significant delays in care. For example, if there is a traumatic brain injury, it should be evaluated by a neurosurgeon in the adult’s emergency to facilitate the diagnosis, treatment, and monitoring, before the injured child is referred to PICU. Sometimes the injured child comes without imaging exams and no care for her critical condition. Due to her critical situation, the child is not sent back to the imaging examination. So, one of our problems is that we don’t have, for example, the CAT, close to our intensive care unit and we must stabilise critical patients blinded without the auxiliary diagnosis . (Doctor 5, Matola Provincial Hospital) Qualifications . The paucity of staff well-trained to deal with paediatric injury patients was highlighted by clinicians from all four hospitals as was the lack of specialised clinicians (eg, paediatric surgeons, anaesthetists orthopaedics). Some health professionals reported that their previous education and experience often focused on adult care, making the transition to paediatric care difficult. Unfortunately, we had never had any paediatric training, capacity building or refresher training, we are limited, doing what we have learned at school. (Nurse 4, Manhiça Distrital Hospital) Staff . Most respondents underlined staff shortages and lack of staff training as serious challenges to quality paediatric injury care. They also report that because of the few radiologists available, they cannot do 24-hour imaging exams and in secondary care, and no X-rays are available after 3:30 pm and during the weekends. The first major limitation we have is the technical personnel, the personnel prepared to work with paediatric injury. Now we only have two surgical technicians who are working in the trauma section, and they…don’t work at the same time, one period is one…From morning until 1400 there is one, from 1400 to 2000 there is another. So, there are only two of them monitoring, so it is a huge overload for them and this huge overload ends up leading to a loss of efficiency, people with a lot of work like this are not able to be completely attentive all the time, the concern is to care in a short time the largest number of patients and it will not be based on quality, we are mainly based on the number of patients we serve. (Doctor 6, Jose Macamo General Hospital) From the health professionals’ narrative, one main category of facilitator emerged: support and mentorship between professionals and institutions (see ). Mentorship of a qualified team . Participants reported that the emergency team contributes to facilitating the care of paediatric injuries in different ways. Both surgeons and surgical technicians gave great support to the team in serious and complex trauma situations, filling gaps and overcoming shortcomings. This cooperative environment avoids overloading health professionals and helps coordinate activities resulting in an improvement in the quality of care offered to injured children and in the solution of problems and challenges faced. The presence of a diverse and highly qualified team and the appropriate number of teams per shift facilitates injury paediatric care and allows for the exchange of experience. Collaboration of quaternary care . Participants from secondary and tertiary care have the opinion that Maputo Central Hospital facilitates their work. Communication via WhatsApp and video calls helps coordinate the referral process and allows the support of specialised clinicians with clinical advice to stabilise and manage critical patients, facilitate injury paediatric care, and exchange experience and resources. One of the facilitators is the good collaboration and relationship that we have, with the Maputo central hospital…Through phone consultation with paediatric surgeons for difficult cases and support…I think this good relationship is also one of the facilitators… (Doctor 7, Hospital Provincial da Matola) Most specialists from quaternary care consider paediatric emergency care and PICU as one of the most important facilitators for paediatric injury care as they stabilise and optimise the critical injury for further care or after surgery. When a severe traumatic brain injury child is admitted in the paediatric emergency department…it makes our work tremendously easier because as soon as the child arrives, s/he receives all the care that is set out in our protocol and paediatrics protocols in place…so when I receive the call to attend in the paediatric emergency, I go knowing that the child has received first aid is stabilised, they call me I will be more cheerful…whereas if I know that they are calling me for a child in the adult emergency room, I will already be very stressed, why? I’m scared of what I’m going to find. So, I would say that the paediatric emergency service is a great facilitator in our care for traumatic brain injury, when the child arrives, when we take the child to the operating room and return them to paediatric intensive care, ha! The care provided post-operatively is…excellent…I’m very satisfied with them (laughs). (Doctor 8, Maputo Central Hospital) Support of local and international institutions . Clinicians reported that partnerships with international organisations and local institutions facilitate the treatment of paediatric injuries by bringing new and advanced knowledge and surgical supplies and help with specialised care, ambulances and laboratory tests. Facilitators we are ourselves, with the few resources we have…we have some friends from the United States, they have helped us a lot, they come once a year in August, they are friends of the surgeon who treats the paediatric traumas, they help us a lot with material and new dynamics, also work, they learn from us, and we also learn from them… I think this is the only facilitator we have. (Nurse 5, Matola Provincial Hospital) This study sheds light on the barriers and facilitators experienced by clinicians taking care of injured children in Maputo, Mozambique. It also provides insights that build from all levels of care, with a focus on a SSA resource-constrained setting. Not surprisingly, many challenges were identified, several internal to the unit and hospitals considered, but others related to the system including prehospital care. Prehospital challenges were presented by many as a source of barriers to the provision of timely and quality care to injured children. This, in turn, reflects the rudimentary organisation of trauma services where the prehospital phase remains underdeveloped. Consequently, as seen in other resource-poor settings, delays in receiving timely care (due to, eg, the transport of patients to the hospital) are common and put children at extra risk, not least when they get transported from the injury site to the nearest care facility in vehicles inappropriately equipped and lacking equipment or trained people. Not surprisingly, many preventable deaths occur, with estimates showing that, in LMICs, up to 80% of injury deaths occur outside the hospital, ambulance access being, in the main, restricted to transport between hospitals. Besides the shortcomings derived from prehospital conditions, the study also reveals barriers in resources within hospitals, including equipment and medication that are lacking, malfunctioning or poorly distributed, for instance, essential medications, surgical supplies and equipment for diagnosis, monitoring or surgical interventions. These findings align with several (checklist based) studies from SSA. Added to this is the fact that the resources in place are often ill-adapted to children’s specific needs/attributes. Consequently, they take more time to care for injured children, and this contributes to poor health outcomes due to the pressure of work time and overload. This is a finding common to studies from LMICs where resource allocation prioritises infectious diseases and pays less attention to epidemiological changes. Further, in all four hospitals, the adult emergency was the only place of admission for injured patients, although the three higher-level hospitals have paediatric emergency services, with paediatric emergency professionals. Inadequately organised infrastructure (eg, the layout itself, space and mixed populations) is an additional source of concern in paediatric trauma care. The lack of skills and knowledge in paediatric injury care and the absence of child-friendly resources may have several consequences that are not specific to clinicians from LMICs. One is that after stabilising the patient, most clinicians will refer the child to a specialised hospital. By doing so, additional delays in managing the patient occur, the risk of poorer outcomes rises, and specialised hospitals get overloaded. Alongside the lack of training, the shortage of specialists is also a critical issue in SSA, limiting the number of staff able to perform essential emergency care. Some countries, like Tanzania, may have staff trained in paediatric critical care but still lack adequate services and facilities. This finds an echo in the narrative of clinicians in remote areas who experience that their competence cannot be fully used because they work in resource-poor environments that do not allow them to put their skills into practice. To make things worse, transport to a more specialised hospital may be dependent on availability and priority setting and takes place in ambulances lacking resuscitation resources. Similar results were found in different studies in LMICs; a study from South Africa found that referring injured patients from district hospitals to higher levels of care delayed appropriate injury care and increased the risk of sepsis, urgent need for an intensive care unit, prolonged hospital stay and higher mortality. To counteract some of the barriers identified and facilitate the delivery of quality care, clinicians of all levels of care highlighted the mentorship of local qualified teams and support of different institutions, local and international, as the most important facilitators of paediatric injury care. Interestingly, this echoes the findings of reviews on the mentorship of health personnel in LMICs. Also, a study in Mozambique reported that ‘north-south’ academic mentorship between two institutions facilitated staff training and improved the quality of paediatric surgical care. Finally, the paediatric emergency department in the quaternary hospital is considered the most important facilitator. Clinicians at that level provide support and mentorship to those caring for children in critical condition, assisting both in diagnostic and care choices. Without this support, frontline clinicians experience stress and anxiety. In the same vein, the presence of a PICU supports clinicians, a finding that has been stressed in previous studies. Strengths and limitations One first strength of the study is that it presents the perspective of healthcare providers from several levels of care, dealing with paediatric injury in a resource-poor country, and highlights what emerges from their narrative. By doing so, it offers different perspectives on the local situation at each hospital and allows us to see how close the perceptions are within and across levels of care. As there was an important consensus that emerged regarding the barriers experienced within and across levels, this gives an important insight into what, in Maputo city and province, prevents healthcare professionals from caring for injured children in the best possible way. Whereas the perspective provided for tertiary and quaternary care is likely to give an adequate picture of the province, it might not be directly transferable to other parts of the country, where hospital services may not be equally developed, and the burden of child injury may be somewhat different. When it comes to lower-level hospitals, the information provided, although locally accurate and information, cannot be assumed to perfectly reflect all hospitals from similar levels in the province. As studies like this are rather uncommon, this is a meaningful contribution, strengthened by the fact that, in each of the four hospitals selected, all professionals accepted the invitation to participate, it was felt that they spoke openly, and the content of the interviews was rich. An additional, potential limitation is a certain desirability bias that can have been introduced, since the first author and PI of the project is a known health professional in the hospitals of the province. It is of note however that she did not take part in the interview process. This can also have influenced the high level of participation obtained. The study is silent regarding how important each barrier is within each hospital, as well as whether this picture would be the same if other—or different—hospitals had been involved. Even for hospitals from other levels of care, some discrepancy can be expected with other parts of the country, not least in remote areas. The study did not include the perspective of the leadership of the hospitals’ decision-makers; thus, it is difficult to determine an agenda for the improvement of child injury care. To that end, it would probably be important to even conduct onsite resource assessments, using standardised tools, as was done a few years ago in the country’s largest hospitals. It is also of note that the study did not consider the perspective of the injured victims or their caregivers, which is undeniably an aspect that deserves greater attention. Finally, as most of the studies evaluating emergency capacity in LMICs assess the availability of infrastructure and capacity to provide care by level but are not focused on paediatric injury care, this is the first study to help our understanding of paediatric injury care in Mozambique. Conclusion From the clinicians’ perspective, barriers to paediatric injury care are often similar across hospitals and professional groups. Barriers include inadequate prehospital care. While lack of resources and poor infrastructure were—as expected—clearly emphasised, respondents identified a clear wish for knowledge- and competence-sharing. One first strength of the study is that it presents the perspective of healthcare providers from several levels of care, dealing with paediatric injury in a resource-poor country, and highlights what emerges from their narrative. By doing so, it offers different perspectives on the local situation at each hospital and allows us to see how close the perceptions are within and across levels of care. As there was an important consensus that emerged regarding the barriers experienced within and across levels, this gives an important insight into what, in Maputo city and province, prevents healthcare professionals from caring for injured children in the best possible way. Whereas the perspective provided for tertiary and quaternary care is likely to give an adequate picture of the province, it might not be directly transferable to other parts of the country, where hospital services may not be equally developed, and the burden of child injury may be somewhat different. When it comes to lower-level hospitals, the information provided, although locally accurate and information, cannot be assumed to perfectly reflect all hospitals from similar levels in the province. As studies like this are rather uncommon, this is a meaningful contribution, strengthened by the fact that, in each of the four hospitals selected, all professionals accepted the invitation to participate, it was felt that they spoke openly, and the content of the interviews was rich. An additional, potential limitation is a certain desirability bias that can have been introduced, since the first author and PI of the project is a known health professional in the hospitals of the province. It is of note however that she did not take part in the interview process. This can also have influenced the high level of participation obtained. The study is silent regarding how important each barrier is within each hospital, as well as whether this picture would be the same if other—or different—hospitals had been involved. Even for hospitals from other levels of care, some discrepancy can be expected with other parts of the country, not least in remote areas. The study did not include the perspective of the leadership of the hospitals’ decision-makers; thus, it is difficult to determine an agenda for the improvement of child injury care. To that end, it would probably be important to even conduct onsite resource assessments, using standardised tools, as was done a few years ago in the country’s largest hospitals. It is also of note that the study did not consider the perspective of the injured victims or their caregivers, which is undeniably an aspect that deserves greater attention. Finally, as most of the studies evaluating emergency capacity in LMICs assess the availability of infrastructure and capacity to provide care by level but are not focused on paediatric injury care, this is the first study to help our understanding of paediatric injury care in Mozambique. From the clinicians’ perspective, barriers to paediatric injury care are often similar across hospitals and professional groups. Barriers include inadequate prehospital care. While lack of resources and poor infrastructure were—as expected—clearly emphasised, respondents identified a clear wish for knowledge- and competence-sharing. 10.1136/bmjopen-2024-085270 online supplemental file 1 10.1136/bmjopen-2024-085270 online supplemental file 2 |
Molecular Profiling of Sinonasal Adenoid Cystic Carcinoma | 9b41187f-756f-4da7-a025-6b85c0037f17 | 11834963 | Anatomy[mh] | Case Selection A retrospective search in the authors´ registry was conducted, and all sinonasal tumors with features of adenoid cystic carcinoma (AdCC) were evaluated histologically and immunohistochemically (A.S. and M.B.). In total, 100 cases were retrieved from the consultation files of the Tumor Registry at the Department of Pathology, Faculty of Medicine in Pilsen and Bioptic Laboratory Ltd in Pilsen, Czech Republic, and tumor registries of the coauthors. Detection of HPV DNA was performed using a set of several PCRs with different primers (Table ) to cover a wide range of high-risk and low-risk HPV types. Eleven HPV positive cases were excluded from the study and diagnosed as HPV-associated multiphenotypic carcinoma. One tumor was excluded based on additional clinical information indicating a primary AdCC in the submandibular gland with secondary involvement of the sinonasal area. Thus, a cohort of 88 cases of sinonasal AdCC was included in this study for further characterization. The tumors were examined histologically, immunohistochemically, and by NGS and/or FISH to detect MYB/MYBL1 and/or NFIB gene fusions, as well as any novel gene fusions/mutations. When available, clinical follow-up was obtained from the patients, their physicians, or referring pathologists. This study was approved by the Ethics Committee of the Faculty Hospital in Pilsen and Charles University, Faculty of Medicine in Pilsen, Czech Republic, on August 2, 2018. Informed consent was not required. Histologic and Immunohistochemical Studies For conventional microscopy, excised tissues were fixed in formalin, processed routinely, embedded in paraffin (FFPE), cut, and stained with hematoxylin and eosin. For immunohistochemistry, 4-μm-thick sections were cut from paraffin blocks and mounted on positively charged slides (TOMO; Matsunami Glass IND, Osaka, Japan). Sections were processed on a BenchMark ULTRA (Ventana Medical Systems, Tucson, AZ), deparaffinized and subjected to heat-induced epitope retrieval by immersion in a CC1 solution (pH: 8.6) at 95°C. All primary antibodies used in this study are summarized in Table . Visualization was performed using the ultraView Universal DAB Detection Kit (Roche, Tucson, AZ) and ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche). The slides were counterstained with Mayer’s hematoxylin. Appropriate positive and negative controls were used. Grading Three grading systems based on the presence of solid components have been recognized: the Perzin/Szanto system, , the Spiro system, and the van Weert system. These systems describe the presence of solid components as negative prognosticators at thresholds of 30%, 50%, and any solid component, respectively. All schemes were applied on our cohort, as depicted in Table . Molecular Studies TruSight Oncology 500 Kit (TS500) Mutation analysis and fusion-transcript detection were performed using the TruSight Oncology 500 Kit (Illumina, San Diego, CA). RNA was extracted using the Maxwell RSC DNA FFPE Kit and the Maxwell RSC Instrument (Promega, Madison, WI) according to the manufacturer’s instructions and quantified using the Qubit HS RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). DNA was extracted using the QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany) and quantified using the Qubit BR DNA Assay Kit (Thermo Fisher Scientific). The quality of DNA was assessed using the FFPE QC kit (Illumina), and the quality of RNA was assessed using Agilent RNA ScreenTape Assay (Agilent, Santa Clara, CA). DNA samples with Cq <5 and RNA samples with DV200 ≥20 were used for further analysis. After enzymatic fragmentation of DNA with KAPAFrag Kit (KAPA Biosystems, Wilmington, MA), DNA and RNA libraries were prepared using the TruSight Oncology 500 Kit (Illumina) according to the manufacturer’s protocol. Sequencing was performed on the NovaSeq. 6000 sequencer (Illumina) following manufacturer´s recommendations. Data analysis was conducted using the TruSight Oncology 500 v2.2 Local App (Illumina). Variant annotation and filtering were performed using the Omnomics NGS analysis software (Euformatics, Espoo, Finland). A custom variant filter was set up, including only nonsynonymous variants with coding sequences and a read depth >50, while benign variants according to the ClinVar database were excluded. The remaining subset of variants was checked visually, and suspected artefactual variants were excluded. Fluorescence In Situ Hybridization (FISH) Analysis Before FISH, hematoxylin and eosin-stained slides were examined to determine the areas for cell evaluation. Then, a 4-μm-thick FFPE section was placed onto a positively charged slide. The unstained slide was routinely deparaffinized and incubated in 1× Target Retrieval Solution Citrate pH 6 (Dako, Glostrup, Denmark) for 40 minutes at 95°C, subsequently cooled for 20 minutes at room temperature in the same solution and washed in deionized water for 5 minutes. The slide was digested in protease solution with pepsin (0.5 mg/mL) (Sigma Aldrich, St Louis, MO) in 0.01 mol/L HCl at 37°C for 45 to 60 minutes, depending on sample conditions. The slide was then rinsed in deionized water for 5 minutes, dehydrated in a series of ethanol solutions (70%, 85%, and 96% for 2 min each), and air-dried. The details of EWSR1 , MYB , NFIB break-apart, and MYB : NFIB fusion analysis have been described previously. , For the detection of EWSR1 : MYB fusion, custom designed EWSR1:MYB dual fusion probes comprising the catalogue 22q12.2. EWSR1 DF 498 kb probe and custom MYB probe with chromosomal location: chr6:135,271,382-135,771,382 (Agilent Technologies) were used following similar protocols. Detection of HPV For HPV studies, genomic DNA was isolated from formalin-fixed, paraffin-embedded tissue using QIAsymphony SP and special precautions were taken to prevent HPV DNA microcontamination. Briefly, five 5-μm-thick sections were cut from the blocks. A new microtome blade was used each time a new case was sectioned. DNA was extracted by the QIAsymphony DNA Mini Kit (Qiagen) according to manufacturer’s protocol. The quality of isolated DNA was checked by PCR which amplifies a set of control genes. Detection of HPV DNA was performed using a set of several PCRs with different primers (Table ) to cover a wide range of high-risk and low-risk HPV types. For all samples, the primers’ systems targeting both L1 and E1 region were used: CPSGB, GP5+/GP6+, as previously described. To avoid false negative findings (because of loss of L1 or E1 regions due to HPV integration into host genome) PCR targeting HPV oncogenes E6, and E7 of the 6 most prevalent HR-HPV types including types 16, 18, 31, 33, 35, and 45 was performed. All PCRs were run on the cycler GeneAmp PCR System 9700 (PE/Applied Biosystem, Forster City, CA). Amplicons were analyzed in 2% agarose gel with ethidium bromide. Positive PCR samples were genotyped by hybridizing them to type-specific probes, or sequenced and compared with BLAST databases. Positive and negative controls were included in every run. A retrospective search in the authors´ registry was conducted, and all sinonasal tumors with features of adenoid cystic carcinoma (AdCC) were evaluated histologically and immunohistochemically (A.S. and M.B.). In total, 100 cases were retrieved from the consultation files of the Tumor Registry at the Department of Pathology, Faculty of Medicine in Pilsen and Bioptic Laboratory Ltd in Pilsen, Czech Republic, and tumor registries of the coauthors. Detection of HPV DNA was performed using a set of several PCRs with different primers (Table ) to cover a wide range of high-risk and low-risk HPV types. Eleven HPV positive cases were excluded from the study and diagnosed as HPV-associated multiphenotypic carcinoma. One tumor was excluded based on additional clinical information indicating a primary AdCC in the submandibular gland with secondary involvement of the sinonasal area. Thus, a cohort of 88 cases of sinonasal AdCC was included in this study for further characterization. The tumors were examined histologically, immunohistochemically, and by NGS and/or FISH to detect MYB/MYBL1 and/or NFIB gene fusions, as well as any novel gene fusions/mutations. When available, clinical follow-up was obtained from the patients, their physicians, or referring pathologists. This study was approved by the Ethics Committee of the Faculty Hospital in Pilsen and Charles University, Faculty of Medicine in Pilsen, Czech Republic, on August 2, 2018. Informed consent was not required. For conventional microscopy, excised tissues were fixed in formalin, processed routinely, embedded in paraffin (FFPE), cut, and stained with hematoxylin and eosin. For immunohistochemistry, 4-μm-thick sections were cut from paraffin blocks and mounted on positively charged slides (TOMO; Matsunami Glass IND, Osaka, Japan). Sections were processed on a BenchMark ULTRA (Ventana Medical Systems, Tucson, AZ), deparaffinized and subjected to heat-induced epitope retrieval by immersion in a CC1 solution (pH: 8.6) at 95°C. All primary antibodies used in this study are summarized in Table . Visualization was performed using the ultraView Universal DAB Detection Kit (Roche, Tucson, AZ) and ultraView Universal Alkaline Phosphatase Red Detection Kit (Roche). The slides were counterstained with Mayer’s hematoxylin. Appropriate positive and negative controls were used. Three grading systems based on the presence of solid components have been recognized: the Perzin/Szanto system, , the Spiro system, and the van Weert system. These systems describe the presence of solid components as negative prognosticators at thresholds of 30%, 50%, and any solid component, respectively. All schemes were applied on our cohort, as depicted in Table . TruSight Oncology 500 Kit (TS500) Mutation analysis and fusion-transcript detection were performed using the TruSight Oncology 500 Kit (Illumina, San Diego, CA). RNA was extracted using the Maxwell RSC DNA FFPE Kit and the Maxwell RSC Instrument (Promega, Madison, WI) according to the manufacturer’s instructions and quantified using the Qubit HS RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). DNA was extracted using the QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany) and quantified using the Qubit BR DNA Assay Kit (Thermo Fisher Scientific). The quality of DNA was assessed using the FFPE QC kit (Illumina), and the quality of RNA was assessed using Agilent RNA ScreenTape Assay (Agilent, Santa Clara, CA). DNA samples with Cq <5 and RNA samples with DV200 ≥20 were used for further analysis. After enzymatic fragmentation of DNA with KAPAFrag Kit (KAPA Biosystems, Wilmington, MA), DNA and RNA libraries were prepared using the TruSight Oncology 500 Kit (Illumina) according to the manufacturer’s protocol. Sequencing was performed on the NovaSeq. 6000 sequencer (Illumina) following manufacturer´s recommendations. Data analysis was conducted using the TruSight Oncology 500 v2.2 Local App (Illumina). Variant annotation and filtering were performed using the Omnomics NGS analysis software (Euformatics, Espoo, Finland). A custom variant filter was set up, including only nonsynonymous variants with coding sequences and a read depth >50, while benign variants according to the ClinVar database were excluded. The remaining subset of variants was checked visually, and suspected artefactual variants were excluded. Fluorescence In Situ Hybridization (FISH) Analysis Before FISH, hematoxylin and eosin-stained slides were examined to determine the areas for cell evaluation. Then, a 4-μm-thick FFPE section was placed onto a positively charged slide. The unstained slide was routinely deparaffinized and incubated in 1× Target Retrieval Solution Citrate pH 6 (Dako, Glostrup, Denmark) for 40 minutes at 95°C, subsequently cooled for 20 minutes at room temperature in the same solution and washed in deionized water for 5 minutes. The slide was digested in protease solution with pepsin (0.5 mg/mL) (Sigma Aldrich, St Louis, MO) in 0.01 mol/L HCl at 37°C for 45 to 60 minutes, depending on sample conditions. The slide was then rinsed in deionized water for 5 minutes, dehydrated in a series of ethanol solutions (70%, 85%, and 96% for 2 min each), and air-dried. The details of EWSR1 , MYB , NFIB break-apart, and MYB : NFIB fusion analysis have been described previously. , For the detection of EWSR1 : MYB fusion, custom designed EWSR1:MYB dual fusion probes comprising the catalogue 22q12.2. EWSR1 DF 498 kb probe and custom MYB probe with chromosomal location: chr6:135,271,382-135,771,382 (Agilent Technologies) were used following similar protocols. Detection of HPV For HPV studies, genomic DNA was isolated from formalin-fixed, paraffin-embedded tissue using QIAsymphony SP and special precautions were taken to prevent HPV DNA microcontamination. Briefly, five 5-μm-thick sections were cut from the blocks. A new microtome blade was used each time a new case was sectioned. DNA was extracted by the QIAsymphony DNA Mini Kit (Qiagen) according to manufacturer’s protocol. The quality of isolated DNA was checked by PCR which amplifies a set of control genes. Detection of HPV DNA was performed using a set of several PCRs with different primers (Table ) to cover a wide range of high-risk and low-risk HPV types. For all samples, the primers’ systems targeting both L1 and E1 region were used: CPSGB, GP5+/GP6+, as previously described. To avoid false negative findings (because of loss of L1 or E1 regions due to HPV integration into host genome) PCR targeting HPV oncogenes E6, and E7 of the 6 most prevalent HR-HPV types including types 16, 18, 31, 33, 35, and 45 was performed. All PCRs were run on the cycler GeneAmp PCR System 9700 (PE/Applied Biosystem, Forster City, CA). Amplicons were analyzed in 2% agarose gel with ethidium bromide. Positive PCR samples were genotyped by hybridizing them to type-specific probes, or sequenced and compared with BLAST databases. Positive and negative controls were included in every run. Mutation analysis and fusion-transcript detection were performed using the TruSight Oncology 500 Kit (Illumina, San Diego, CA). RNA was extracted using the Maxwell RSC DNA FFPE Kit and the Maxwell RSC Instrument (Promega, Madison, WI) according to the manufacturer’s instructions and quantified using the Qubit HS RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA). DNA was extracted using the QIAsymphony DSP DNA Mini Kit (Qiagen, Hilden, Germany) and quantified using the Qubit BR DNA Assay Kit (Thermo Fisher Scientific). The quality of DNA was assessed using the FFPE QC kit (Illumina), and the quality of RNA was assessed using Agilent RNA ScreenTape Assay (Agilent, Santa Clara, CA). DNA samples with Cq <5 and RNA samples with DV200 ≥20 were used for further analysis. After enzymatic fragmentation of DNA with KAPAFrag Kit (KAPA Biosystems, Wilmington, MA), DNA and RNA libraries were prepared using the TruSight Oncology 500 Kit (Illumina) according to the manufacturer’s protocol. Sequencing was performed on the NovaSeq. 6000 sequencer (Illumina) following manufacturer´s recommendations. Data analysis was conducted using the TruSight Oncology 500 v2.2 Local App (Illumina). Variant annotation and filtering were performed using the Omnomics NGS analysis software (Euformatics, Espoo, Finland). A custom variant filter was set up, including only nonsynonymous variants with coding sequences and a read depth >50, while benign variants according to the ClinVar database were excluded. The remaining subset of variants was checked visually, and suspected artefactual variants were excluded. Before FISH, hematoxylin and eosin-stained slides were examined to determine the areas for cell evaluation. Then, a 4-μm-thick FFPE section was placed onto a positively charged slide. The unstained slide was routinely deparaffinized and incubated in 1× Target Retrieval Solution Citrate pH 6 (Dako, Glostrup, Denmark) for 40 minutes at 95°C, subsequently cooled for 20 minutes at room temperature in the same solution and washed in deionized water for 5 minutes. The slide was digested in protease solution with pepsin (0.5 mg/mL) (Sigma Aldrich, St Louis, MO) in 0.01 mol/L HCl at 37°C for 45 to 60 minutes, depending on sample conditions. The slide was then rinsed in deionized water for 5 minutes, dehydrated in a series of ethanol solutions (70%, 85%, and 96% for 2 min each), and air-dried. The details of EWSR1 , MYB , NFIB break-apart, and MYB : NFIB fusion analysis have been described previously. , For the detection of EWSR1 : MYB fusion, custom designed EWSR1:MYB dual fusion probes comprising the catalogue 22q12.2. EWSR1 DF 498 kb probe and custom MYB probe with chromosomal location: chr6:135,271,382-135,771,382 (Agilent Technologies) were used following similar protocols. For HPV studies, genomic DNA was isolated from formalin-fixed, paraffin-embedded tissue using QIAsymphony SP and special precautions were taken to prevent HPV DNA microcontamination. Briefly, five 5-μm-thick sections were cut from the blocks. A new microtome blade was used each time a new case was sectioned. DNA was extracted by the QIAsymphony DNA Mini Kit (Qiagen) according to manufacturer’s protocol. The quality of isolated DNA was checked by PCR which amplifies a set of control genes. Detection of HPV DNA was performed using a set of several PCRs with different primers (Table ) to cover a wide range of high-risk and low-risk HPV types. For all samples, the primers’ systems targeting both L1 and E1 region were used: CPSGB, GP5+/GP6+, as previously described. To avoid false negative findings (because of loss of L1 or E1 regions due to HPV integration into host genome) PCR targeting HPV oncogenes E6, and E7 of the 6 most prevalent HR-HPV types including types 16, 18, 31, 33, 35, and 45 was performed. All PCRs were run on the cycler GeneAmp PCR System 9700 (PE/Applied Biosystem, Forster City, CA). Amplicons were analyzed in 2% agarose gel with ethidium bromide. Positive PCR samples were genotyped by hybridizing them to type-specific probes, or sequenced and compared with BLAST databases. Positive and negative controls were included in every run. Demographic and Clinicopathologic Findings We collected a cohort of 88 cases of sinonasal AdCC. Clinical data, follow-up, and molecular genetic results are summarized in Tables and . The patients comprised 45 men and 41 women (with gender unknown in 2 cases), ranging in age from 20 to 86 years (mean: 58.8 y). The tumors arose in the nasal cavity (49 cases), the maxillary sinus (26 cases), the sphenoid sinus (8 cases), the ethmoid sinus (4 cases), and the nasopharynx (1 case). Treatment consisted of excision or radical surgical resection in 30/47 (64%) cases. Chemotherapy, radiation and/or proton therapy were administered before surgery, after surgery, or as the sole therapeutic approach in 44/49 patients (90%). One patient received targeted therapy with Imatinib. Distant metastatic spread was reported in 14/60 (23%) cases, with one or more metastases in the lung (9 cases), the liver (2 cases), the brain (1 case), bones (pelvis and sacrum, 1 case), or the axilla (1 case). Local recurrence was observed in 26/60 cases (43%) (Table ). Follow-up information was available for 60/88 (68%) patients, with follow-up periods ranging from shortly after diagnosis to 276 months (mean follow-up: 62.7 mo). Eighteen patients (18/60, 30%) were alive without evidence of disease (mean follow-up of 91.9 mo), 9 patients (15%) were alive with disease (mean follow-up of 35.6 mo), 18 patients (30%) died of the disease (mean follow-up of 63 mo), 4 patients (7%) died of unrelated causes (mean follow-up of 46.3 mo), and 11 patients (18%) were lost to follow-up after a follow-up period with regular check-ups (mean follow-up of 33.6 mo) (Table ). Grading All 3 grading systems were applied on our cohort. The presence of solid component is independent on the presence of canonical or alternative gene fusion. In our cohort, 49 cases showed more than 30% solid component, 33 cases showed more than 50%, and 66 cases showed any amount of solid component. The presence of a solid component was independent of the presence of canonical or alternative gene fusions. The total amount and categorization of our cohort into 3 independent grading systems is depicted in Table . High-grade transformation/dedifferentiation was not present. Molecular Findings Sinonasal AdCC was predominantly characterized by canonical MYB:NFIB (49 cases) (Fig. E) and MYBL1:NFIB (9 cases) (Fig. E) fusions. In addition, rearrangements in MYB (8 cases), NFIB (1 case), and EWSR1 (1 case) genes were detected in 9 cases using FISH. NGS analysis revealed novel noncanonical fusion transcripts, including ACTB:MYB; ACTN4:MYB; ESRRG:DNM3 , and EWSR1:MYB , each in 1 case. Mutational analysis was performed by NGS in 31/88 (35%) AdCCs. Mutations in genes with established roles in oncogenesis were identified in 21/31 tumors (68%), including BCOR (4/21; 19%), NOTCH1 (3/21; 14%), EP300 (3/21; 14%), SMARCA4 (2/21; 9%), RUNX1 (2/21; 9%), KDM6A (2/21; 9%), SPEN (2/21; 9%), and RIT1, MGA, RB1, PHF6, PTEN, CREBBP, DDX41, CHD2, ROS1, TAF1, CCD1, NF1, PALB2, AVCR1B, ARID1A, PPM1D, LZTR1, GEN1 , PDGFRA , each in 1 case (1/21; 5%). In addition, 24/31 (77%) cases exhibited a spectrum of gene mutations of uncertain pathogenetic significance. Moreover, no known pathogenic mutations were detected in 9/31 (29%) cases. None of the detected mutations occurred at a high level of recurrence. Even the most frequent mutations of BCOR and NOTCH1 showed a low level of recurrence at 19% and 14%, respectively, in our AdCC cases as a whole. The clinical features of all 23 cases of AdCC harboring noncanonical gene fusions and oncogenic/likely pathogenic mutations are summarized in Table . The clinical outcome of patients with NOTCH and BCOR mutated AdCCs was notably poor. There were 3 patients with NOTCH -mutated AdCCs, and 2 of them experienced multiple recurrences, and died of disease at 20 and 64 months, respectively. One patient is currently alive and receives proton therapy, with brain metastasis at 5 months after primary diagnosis. Two of 4 patients with BCOR mutated AdCC are alive with disease at 53 and 60 months respectively, but both have experienced local recurrences at 53 and 50 months, and 1 is alive with lung metastasis, while 2 patients were lost to follow-up (Table ). Histopathologic and Immunohistochemical Findings Sinonasal Adenoid Cystic Carcinomas With Canonical MYB/MYB1:NFIB Gene Fusions Histologically, there was no morphologic difference between AdCCs with MYBL1:NFIB and those with MYB:NFIB fusion. All these fusion-positive sinonasal AdCCs exhibited an infiltrative biphasic growth pattern consisting of both ductal epithelial and myoepithelial cells. Identification of both pseudocysts and true glandular lumina was required to make the diagnosis. The most common architectural patterns, namely cribriform, tubular, and solid were observed in varying proportions. The cribriform pattern was the most prevalent in both molecular subtypes MYB:NFIB and MYBL1:NFIB , as illustrated in Figures A and A,C, respectively. The cribriform/pseudocystic growth pattern is characterized by nests of tumor cells with microcystic-like spaces filled with hyaline basement membrane-like or basophilic mucoid material. The solid pattern, characterized by tumor sheets composed of basaloid cells lacking tubular or cribriform formations, was also observed in both subtypes of AdCC with canonical fusion transcripts (Fig. B, Fig. D). Combinations of these growth patterns were common (Fig. C). Abundant deposits of hyalinized eosinophilic extracellular matrix and reduplicated basement membrane materials were often seen (Fig. D). The tubular pattern, consisting of well-formed ducts and tubules lined by luminal ductal and abluminal myoepithelial cells, was the least common growth pattern (Fig. B). Sinonasal Adenoid Cystic Carcinomas With Noncanonical Fusion We identified 4 cases of sinonasal AdCC harboring novel noncanonical fusion transcripts, namely ACTB:MYB, ACTN4:MYB , ESRRG:DNM3 , and EWSR1:MYB. Two cases with fusions involving ACTB and ACTN4 genes predominantly exhibited a solid growth pattern of monomorphic basaloid cells without significant nuclear atypia. In the former of those 2 (in-frame ACTBex3:MYBex3 fusion), the tumor fragments were 8 cm in the largest diameter and displayed a macroscopic mucinous appearance. Microscopic examination revealed a polypoid and partly cystic lesion of predominantly myoepithelial appearance with lobulated multinodular architecture, a hypercellular periphery and hypocellular center with large necrotic areas (Fig. A). Tumor cells were basaloid and had monomorphic hyperchromatic nuclei (Fig. B) with high-grade features (Fig. C). Areas of conventional cribriform adenoid cystic carcinoma were present at the periphery and represented 20% of the tumor mass (Fig. D). The stroma was partly hyalinized and partly myxoid. The growth pattern was biphasic with p63 positivity in the cells of the abluminal component. Ki-67 proliferation index was estimated at 30%. The latter case represented an example of metatypical AdCC (in-frame ACTN4ex18:MYBex2 fusion, and a break of the MYB gene). The tumor consisted of a solid tumor mass composed of 2 cell populations. The predominant component was composed of monomorphic basaloid cells without significant nuclear atypia or mitotic activity. A less frequent cell population was represented by groups of cells with pale eosinophilic to vacuolated cytoplasm (Fig. A). These cells were identical both in morphology and immunoprofile with cells described by Altemani et al. A stromal component was minor and characterized by fibrous bands with distended vascular spaces and large areas of dense sclerosis with mucoid material (Fig. B). Basaloid tumor cells were positive for p63 (Fig. C) and CK14. Vacuolated (clear) cells showed CK7 expression (Fig. D). Ki-67 proliferation index was estimated at 2%. The third case featuring a novel ESRRGex3:DNM3ex14 fusion not previously described in AdCC was a polypoid lesion infiltrating the right maxilla. The tumor consisted of a main tumor mass corresponding to conventional AdCC while areas in the periphery of the lesion resembled a seromucinous hamartoma. The AdCC component of the lesion comprised different architectural patterns, including solid, tubular, micropapillary, and cribriform (containing hyaline or basophilic mucoid material) (Fig. A). Areas of stromal edema or fibromyxohyaline change of the interstitium contained small clusters, cords, or isolated tumor cells, that were sometimes bizarre. The AdCC tumor cells were pleomorphic with irregular nuclear borders, nuclear hyperchromasia, or chromatin clearing with prominent nucleoli and nuclear grooves. The cytoplasm was eosinophilic to clear and bubbly (Fig. B). Perineural invasion was prominent (Fig. C). Mitotic activity was high. A transition zone contained atypical sinonasal glands arising from the respiratory adenomatoid hamartoma-like periphery (Fig. D) connected to the AdCC tumor mass. The fourth case was an 82-year-old man with a tumor mass of the sphenoid sinus (in-frame EWSR1ex6:MYBex2 ). This case was reported in a previous cohort of metatypical AdCC. The tumor was composed of hypercellular and hypocellular areas. The hypercellular areas consisted of basaloid cells forming tubular and cribriform patterns with multiple areas of comedo-like necrosis (Fig. A), extensive tubular epithelial hypereosinophilia (Fig. B), and foci of intraluminal clear cells (Fig. C). The islands of tumor cells were circumscribed by basement membrane deposits or basophilic mucoid material. The hypocellular area was composed of fibrosclerotic and hyalinized stroma with focal myxoid change and cords and ducts of tumor cells (Fig. D). Tumor cells were pleomorphic with irregular nuclear borders, nuclear grooves, and multiple nucleoli. The cytoplasm was eosinophilic to clear. Tumor cells showed typical biphasic immunohistochemical pattern with the luminal cells positive for CK7, while the abluminal cells were positive for p63, CK14, and SOX10. Proliferative activity was mostly intermediate, but reached up to 25% in hot spot regions. Sinonasal Adenoid Cystic Carcinomas With NOTCH Gene Family Mutations Three cases of AdCC harbored mutations in NOTCH1 gene. These tumors were characterized by a predominant solid growth pattern, nuclear enlargement, and polymorphism (Fig. A). There were nests forming confluent sheets of tumor cells, and comedonecrosis was pronounced (Fig. B). Mitotic and Ki-67 labeling indices were significantly higher in solid areas as compared with cribriform/tubular pattern in conventional AdCC (Figs. C–D). Two NOTCH -mutated AdCCs exhibited metatypical features, including unusual growth patterns with squamous differentiation (Fig. A), tubular epithelial hypereosinophilia with luminal cell prominence (Fig. B), and focal sebaceous metaplasia (Fig. C). In one metatypical AdCC, multiple foci of foamy vaculated eosinophilic cells were present within the nests composed of basaloid neoplastic cells (Fig. D). Our cohort included 31/88 (35%) cases of AdCC (with canonical or noncanonical fusions) associated with seromucinous hamartoma (SH) and/or atypical sinonasal glands arising in SH (ASGSH) (Fig. D). The nuclei varied in size and shape and were often hyperchromatic. Forty-four of 88 (50%) cases consisted of a pure AdCC tumor mass with no evidence of surface or other structures of normal mucosa. In 11 cases, a normal sinonasal epithelium with no atypia covered the tumor mass. In 4 additional cases, squamous epithelium without dysplasia lined the surface of some tumor fragments. We collected a cohort of 88 cases of sinonasal AdCC. Clinical data, follow-up, and molecular genetic results are summarized in Tables and . The patients comprised 45 men and 41 women (with gender unknown in 2 cases), ranging in age from 20 to 86 years (mean: 58.8 y). The tumors arose in the nasal cavity (49 cases), the maxillary sinus (26 cases), the sphenoid sinus (8 cases), the ethmoid sinus (4 cases), and the nasopharynx (1 case). Treatment consisted of excision or radical surgical resection in 30/47 (64%) cases. Chemotherapy, radiation and/or proton therapy were administered before surgery, after surgery, or as the sole therapeutic approach in 44/49 patients (90%). One patient received targeted therapy with Imatinib. Distant metastatic spread was reported in 14/60 (23%) cases, with one or more metastases in the lung (9 cases), the liver (2 cases), the brain (1 case), bones (pelvis and sacrum, 1 case), or the axilla (1 case). Local recurrence was observed in 26/60 cases (43%) (Table ). Follow-up information was available for 60/88 (68%) patients, with follow-up periods ranging from shortly after diagnosis to 276 months (mean follow-up: 62.7 mo). Eighteen patients (18/60, 30%) were alive without evidence of disease (mean follow-up of 91.9 mo), 9 patients (15%) were alive with disease (mean follow-up of 35.6 mo), 18 patients (30%) died of the disease (mean follow-up of 63 mo), 4 patients (7%) died of unrelated causes (mean follow-up of 46.3 mo), and 11 patients (18%) were lost to follow-up after a follow-up period with regular check-ups (mean follow-up of 33.6 mo) (Table ). All 3 grading systems were applied on our cohort. The presence of solid component is independent on the presence of canonical or alternative gene fusion. In our cohort, 49 cases showed more than 30% solid component, 33 cases showed more than 50%, and 66 cases showed any amount of solid component. The presence of a solid component was independent of the presence of canonical or alternative gene fusions. The total amount and categorization of our cohort into 3 independent grading systems is depicted in Table . High-grade transformation/dedifferentiation was not present. Sinonasal AdCC was predominantly characterized by canonical MYB:NFIB (49 cases) (Fig. E) and MYBL1:NFIB (9 cases) (Fig. E) fusions. In addition, rearrangements in MYB (8 cases), NFIB (1 case), and EWSR1 (1 case) genes were detected in 9 cases using FISH. NGS analysis revealed novel noncanonical fusion transcripts, including ACTB:MYB; ACTN4:MYB; ESRRG:DNM3 , and EWSR1:MYB , each in 1 case. Mutational analysis was performed by NGS in 31/88 (35%) AdCCs. Mutations in genes with established roles in oncogenesis were identified in 21/31 tumors (68%), including BCOR (4/21; 19%), NOTCH1 (3/21; 14%), EP300 (3/21; 14%), SMARCA4 (2/21; 9%), RUNX1 (2/21; 9%), KDM6A (2/21; 9%), SPEN (2/21; 9%), and RIT1, MGA, RB1, PHF6, PTEN, CREBBP, DDX41, CHD2, ROS1, TAF1, CCD1, NF1, PALB2, AVCR1B, ARID1A, PPM1D, LZTR1, GEN1 , PDGFRA , each in 1 case (1/21; 5%). In addition, 24/31 (77%) cases exhibited a spectrum of gene mutations of uncertain pathogenetic significance. Moreover, no known pathogenic mutations were detected in 9/31 (29%) cases. None of the detected mutations occurred at a high level of recurrence. Even the most frequent mutations of BCOR and NOTCH1 showed a low level of recurrence at 19% and 14%, respectively, in our AdCC cases as a whole. The clinical features of all 23 cases of AdCC harboring noncanonical gene fusions and oncogenic/likely pathogenic mutations are summarized in Table . The clinical outcome of patients with NOTCH and BCOR mutated AdCCs was notably poor. There were 3 patients with NOTCH -mutated AdCCs, and 2 of them experienced multiple recurrences, and died of disease at 20 and 64 months, respectively. One patient is currently alive and receives proton therapy, with brain metastasis at 5 months after primary diagnosis. Two of 4 patients with BCOR mutated AdCC are alive with disease at 53 and 60 months respectively, but both have experienced local recurrences at 53 and 50 months, and 1 is alive with lung metastasis, while 2 patients were lost to follow-up (Table ). Sinonasal Adenoid Cystic Carcinomas With Canonical MYB/MYB1:NFIB Gene Fusions Histologically, there was no morphologic difference between AdCCs with MYBL1:NFIB and those with MYB:NFIB fusion. All these fusion-positive sinonasal AdCCs exhibited an infiltrative biphasic growth pattern consisting of both ductal epithelial and myoepithelial cells. Identification of both pseudocysts and true glandular lumina was required to make the diagnosis. The most common architectural patterns, namely cribriform, tubular, and solid were observed in varying proportions. The cribriform pattern was the most prevalent in both molecular subtypes MYB:NFIB and MYBL1:NFIB , as illustrated in Figures A and A,C, respectively. The cribriform/pseudocystic growth pattern is characterized by nests of tumor cells with microcystic-like spaces filled with hyaline basement membrane-like or basophilic mucoid material. The solid pattern, characterized by tumor sheets composed of basaloid cells lacking tubular or cribriform formations, was also observed in both subtypes of AdCC with canonical fusion transcripts (Fig. B, Fig. D). Combinations of these growth patterns were common (Fig. C). Abundant deposits of hyalinized eosinophilic extracellular matrix and reduplicated basement membrane materials were often seen (Fig. D). The tubular pattern, consisting of well-formed ducts and tubules lined by luminal ductal and abluminal myoepithelial cells, was the least common growth pattern (Fig. B). Sinonasal Adenoid Cystic Carcinomas With Noncanonical Fusion We identified 4 cases of sinonasal AdCC harboring novel noncanonical fusion transcripts, namely ACTB:MYB, ACTN4:MYB , ESRRG:DNM3 , and EWSR1:MYB. Two cases with fusions involving ACTB and ACTN4 genes predominantly exhibited a solid growth pattern of monomorphic basaloid cells without significant nuclear atypia. In the former of those 2 (in-frame ACTBex3:MYBex3 fusion), the tumor fragments were 8 cm in the largest diameter and displayed a macroscopic mucinous appearance. Microscopic examination revealed a polypoid and partly cystic lesion of predominantly myoepithelial appearance with lobulated multinodular architecture, a hypercellular periphery and hypocellular center with large necrotic areas (Fig. A). Tumor cells were basaloid and had monomorphic hyperchromatic nuclei (Fig. B) with high-grade features (Fig. C). Areas of conventional cribriform adenoid cystic carcinoma were present at the periphery and represented 20% of the tumor mass (Fig. D). The stroma was partly hyalinized and partly myxoid. The growth pattern was biphasic with p63 positivity in the cells of the abluminal component. Ki-67 proliferation index was estimated at 30%. The latter case represented an example of metatypical AdCC (in-frame ACTN4ex18:MYBex2 fusion, and a break of the MYB gene). The tumor consisted of a solid tumor mass composed of 2 cell populations. The predominant component was composed of monomorphic basaloid cells without significant nuclear atypia or mitotic activity. A less frequent cell population was represented by groups of cells with pale eosinophilic to vacuolated cytoplasm (Fig. A). These cells were identical both in morphology and immunoprofile with cells described by Altemani et al. A stromal component was minor and characterized by fibrous bands with distended vascular spaces and large areas of dense sclerosis with mucoid material (Fig. B). Basaloid tumor cells were positive for p63 (Fig. C) and CK14. Vacuolated (clear) cells showed CK7 expression (Fig. D). Ki-67 proliferation index was estimated at 2%. The third case featuring a novel ESRRGex3:DNM3ex14 fusion not previously described in AdCC was a polypoid lesion infiltrating the right maxilla. The tumor consisted of a main tumor mass corresponding to conventional AdCC while areas in the periphery of the lesion resembled a seromucinous hamartoma. The AdCC component of the lesion comprised different architectural patterns, including solid, tubular, micropapillary, and cribriform (containing hyaline or basophilic mucoid material) (Fig. A). Areas of stromal edema or fibromyxohyaline change of the interstitium contained small clusters, cords, or isolated tumor cells, that were sometimes bizarre. The AdCC tumor cells were pleomorphic with irregular nuclear borders, nuclear hyperchromasia, or chromatin clearing with prominent nucleoli and nuclear grooves. The cytoplasm was eosinophilic to clear and bubbly (Fig. B). Perineural invasion was prominent (Fig. C). Mitotic activity was high. A transition zone contained atypical sinonasal glands arising from the respiratory adenomatoid hamartoma-like periphery (Fig. D) connected to the AdCC tumor mass. The fourth case was an 82-year-old man with a tumor mass of the sphenoid sinus (in-frame EWSR1ex6:MYBex2 ). This case was reported in a previous cohort of metatypical AdCC. The tumor was composed of hypercellular and hypocellular areas. The hypercellular areas consisted of basaloid cells forming tubular and cribriform patterns with multiple areas of comedo-like necrosis (Fig. A), extensive tubular epithelial hypereosinophilia (Fig. B), and foci of intraluminal clear cells (Fig. C). The islands of tumor cells were circumscribed by basement membrane deposits or basophilic mucoid material. The hypocellular area was composed of fibrosclerotic and hyalinized stroma with focal myxoid change and cords and ducts of tumor cells (Fig. D). Tumor cells were pleomorphic with irregular nuclear borders, nuclear grooves, and multiple nucleoli. The cytoplasm was eosinophilic to clear. Tumor cells showed typical biphasic immunohistochemical pattern with the luminal cells positive for CK7, while the abluminal cells were positive for p63, CK14, and SOX10. Proliferative activity was mostly intermediate, but reached up to 25% in hot spot regions. Sinonasal Adenoid Cystic Carcinomas With NOTCH Gene Family Mutations Three cases of AdCC harbored mutations in NOTCH1 gene. These tumors were characterized by a predominant solid growth pattern, nuclear enlargement, and polymorphism (Fig. A). There were nests forming confluent sheets of tumor cells, and comedonecrosis was pronounced (Fig. B). Mitotic and Ki-67 labeling indices were significantly higher in solid areas as compared with cribriform/tubular pattern in conventional AdCC (Figs. C–D). Two NOTCH -mutated AdCCs exhibited metatypical features, including unusual growth patterns with squamous differentiation (Fig. A), tubular epithelial hypereosinophilia with luminal cell prominence (Fig. B), and focal sebaceous metaplasia (Fig. C). In one metatypical AdCC, multiple foci of foamy vaculated eosinophilic cells were present within the nests composed of basaloid neoplastic cells (Fig. D). Our cohort included 31/88 (35%) cases of AdCC (with canonical or noncanonical fusions) associated with seromucinous hamartoma (SH) and/or atypical sinonasal glands arising in SH (ASGSH) (Fig. D). The nuclei varied in size and shape and were often hyperchromatic. Forty-four of 88 (50%) cases consisted of a pure AdCC tumor mass with no evidence of surface or other structures of normal mucosa. In 11 cases, a normal sinonasal epithelium with no atypia covered the tumor mass. In 4 additional cases, squamous epithelium without dysplasia lined the surface of some tumor fragments. MYB/MYB1:NFIB Gene Fusions Histologically, there was no morphologic difference between AdCCs with MYBL1:NFIB and those with MYB:NFIB fusion. All these fusion-positive sinonasal AdCCs exhibited an infiltrative biphasic growth pattern consisting of both ductal epithelial and myoepithelial cells. Identification of both pseudocysts and true glandular lumina was required to make the diagnosis. The most common architectural patterns, namely cribriform, tubular, and solid were observed in varying proportions. The cribriform pattern was the most prevalent in both molecular subtypes MYB:NFIB and MYBL1:NFIB , as illustrated in Figures A and A,C, respectively. The cribriform/pseudocystic growth pattern is characterized by nests of tumor cells with microcystic-like spaces filled with hyaline basement membrane-like or basophilic mucoid material. The solid pattern, characterized by tumor sheets composed of basaloid cells lacking tubular or cribriform formations, was also observed in both subtypes of AdCC with canonical fusion transcripts (Fig. B, Fig. D). Combinations of these growth patterns were common (Fig. C). Abundant deposits of hyalinized eosinophilic extracellular matrix and reduplicated basement membrane materials were often seen (Fig. D). The tubular pattern, consisting of well-formed ducts and tubules lined by luminal ductal and abluminal myoepithelial cells, was the least common growth pattern (Fig. B). We identified 4 cases of sinonasal AdCC harboring novel noncanonical fusion transcripts, namely ACTB:MYB, ACTN4:MYB , ESRRG:DNM3 , and EWSR1:MYB. Two cases with fusions involving ACTB and ACTN4 genes predominantly exhibited a solid growth pattern of monomorphic basaloid cells without significant nuclear atypia. In the former of those 2 (in-frame ACTBex3:MYBex3 fusion), the tumor fragments were 8 cm in the largest diameter and displayed a macroscopic mucinous appearance. Microscopic examination revealed a polypoid and partly cystic lesion of predominantly myoepithelial appearance with lobulated multinodular architecture, a hypercellular periphery and hypocellular center with large necrotic areas (Fig. A). Tumor cells were basaloid and had monomorphic hyperchromatic nuclei (Fig. B) with high-grade features (Fig. C). Areas of conventional cribriform adenoid cystic carcinoma were present at the periphery and represented 20% of the tumor mass (Fig. D). The stroma was partly hyalinized and partly myxoid. The growth pattern was biphasic with p63 positivity in the cells of the abluminal component. Ki-67 proliferation index was estimated at 30%. The latter case represented an example of metatypical AdCC (in-frame ACTN4ex18:MYBex2 fusion, and a break of the MYB gene). The tumor consisted of a solid tumor mass composed of 2 cell populations. The predominant component was composed of monomorphic basaloid cells without significant nuclear atypia or mitotic activity. A less frequent cell population was represented by groups of cells with pale eosinophilic to vacuolated cytoplasm (Fig. A). These cells were identical both in morphology and immunoprofile with cells described by Altemani et al. A stromal component was minor and characterized by fibrous bands with distended vascular spaces and large areas of dense sclerosis with mucoid material (Fig. B). Basaloid tumor cells were positive for p63 (Fig. C) and CK14. Vacuolated (clear) cells showed CK7 expression (Fig. D). Ki-67 proliferation index was estimated at 2%. The third case featuring a novel ESRRGex3:DNM3ex14 fusion not previously described in AdCC was a polypoid lesion infiltrating the right maxilla. The tumor consisted of a main tumor mass corresponding to conventional AdCC while areas in the periphery of the lesion resembled a seromucinous hamartoma. The AdCC component of the lesion comprised different architectural patterns, including solid, tubular, micropapillary, and cribriform (containing hyaline or basophilic mucoid material) (Fig. A). Areas of stromal edema or fibromyxohyaline change of the interstitium contained small clusters, cords, or isolated tumor cells, that were sometimes bizarre. The AdCC tumor cells were pleomorphic with irregular nuclear borders, nuclear hyperchromasia, or chromatin clearing with prominent nucleoli and nuclear grooves. The cytoplasm was eosinophilic to clear and bubbly (Fig. B). Perineural invasion was prominent (Fig. C). Mitotic activity was high. A transition zone contained atypical sinonasal glands arising from the respiratory adenomatoid hamartoma-like periphery (Fig. D) connected to the AdCC tumor mass. The fourth case was an 82-year-old man with a tumor mass of the sphenoid sinus (in-frame EWSR1ex6:MYBex2 ). This case was reported in a previous cohort of metatypical AdCC. The tumor was composed of hypercellular and hypocellular areas. The hypercellular areas consisted of basaloid cells forming tubular and cribriform patterns with multiple areas of comedo-like necrosis (Fig. A), extensive tubular epithelial hypereosinophilia (Fig. B), and foci of intraluminal clear cells (Fig. C). The islands of tumor cells were circumscribed by basement membrane deposits or basophilic mucoid material. The hypocellular area was composed of fibrosclerotic and hyalinized stroma with focal myxoid change and cords and ducts of tumor cells (Fig. D). Tumor cells were pleomorphic with irregular nuclear borders, nuclear grooves, and multiple nucleoli. The cytoplasm was eosinophilic to clear. Tumor cells showed typical biphasic immunohistochemical pattern with the luminal cells positive for CK7, while the abluminal cells were positive for p63, CK14, and SOX10. Proliferative activity was mostly intermediate, but reached up to 25% in hot spot regions. NOTCH Gene Family Mutations Three cases of AdCC harbored mutations in NOTCH1 gene. These tumors were characterized by a predominant solid growth pattern, nuclear enlargement, and polymorphism (Fig. A). There were nests forming confluent sheets of tumor cells, and comedonecrosis was pronounced (Fig. B). Mitotic and Ki-67 labeling indices were significantly higher in solid areas as compared with cribriform/tubular pattern in conventional AdCC (Figs. C–D). Two NOTCH -mutated AdCCs exhibited metatypical features, including unusual growth patterns with squamous differentiation (Fig. A), tubular epithelial hypereosinophilia with luminal cell prominence (Fig. B), and focal sebaceous metaplasia (Fig. C). In one metatypical AdCC, multiple foci of foamy vaculated eosinophilic cells were present within the nests composed of basaloid neoplastic cells (Fig. D). Our cohort included 31/88 (35%) cases of AdCC (with canonical or noncanonical fusions) associated with seromucinous hamartoma (SH) and/or atypical sinonasal glands arising in SH (ASGSH) (Fig. D). The nuclei varied in size and shape and were often hyperchromatic. Forty-four of 88 (50%) cases consisted of a pure AdCC tumor mass with no evidence of surface or other structures of normal mucosa. In 11 cases, a normal sinonasal epithelium with no atypia covered the tumor mass. In 4 additional cases, squamous epithelium without dysplasia lined the surface of some tumor fragments. Adenoid cystic carcinoma (AdCC) is one of the most common salivary gland malignancies, found in both major and minor salivary and seromucous gland sites. Historically, AdCCs of salivary gland origin have been categorized as fusion-defined carcinomas due to the consistent presence of MYB:NFIB fusion genes in over 80% of the cases and MYBL1:NFIB in ∼5%. , MYB/MYBL1 activation through gene fusion or other mechanisms is therefore considered a critical event in the pathogenesis of AdCC. Further exploration has unveiled additional partner genes linked to MYB and NFIB , such as EWSR1:MYB , FUS:MYB , MYB:PDCD1LG2 , MYB:EFR3A , and NFIB:AIG1 , respectively, expanding our understanding of AdCC pathogenesis. Intriguingly, some AdCCs lack these rearrangements, raising questions about possible additional fusion-transcript mechanisms. This is particularly important in small biopsy samples, where AdCC might be misdiagnosed as another entity. In the previous study of our group, NGS analysis revealed noncanonical fusion genes including ACTB:MYB, ACTN4:MYB, ESRRG:DNM3 , each found in 1 tumor case. In the current study, these data are presented in a more comprehensive manner, along with additional details, including morphology and patient outcomes, that were not provided in the prior study. Interestingly, tumors with these alternative fusion genes appear to associate with an unusual variant of morphologic features. Our cases displayed foci of squamous metaplasia, extensive tubular epithelial hypereosinophilia, and clusters of eosinophilic to clear cells with vacuolated or bubbly cytoplasm, leading to the diagnosis of “metatypical AdCC.” This term was introduced by Mathew et al. The authors presented 3 cases of AdCC with unusual features, including extensive squamous differentiation, macrocystic, and trabecular growth patterns, while harboring MYB/MYBL1:NFIB fusion transcripts. Weinreb et al subsequently expanded the spectrum of metatypical morphologies by including AdCC with foci of striking tubular epithelial hypereosinophilia, present in both cases with canonical and novel EWSR1:MYB and FUS:MYB fusions. In our series, 3 of previously published cases with noncanonical fusions ( ACTN4:MYB, ACTB:MYB , and ESRRG:DNM3 ) showed morphologies of metatypical AdCCs. The tumors were composed of different architectural patterns including solid, tubular, micropapillary, and cribriform with predominant component made of monomorphic basaloid cells that showed peripheral palisading of the nuclei. A less frequent cellular population was represented by groups of cells with pale eosinophilic to vacuolated cytoplasm and extensive tubular hypereosinophilia. In addition, we have observed metatypical features, namely sebaceous foci, clusters of eosinophilic and foamy cells, and extensive squamous metaplasia also in AdCCs harboring the canonical MYB:NFIB fusion and variable mutations including those in the NOTCH gene family, BCOR, RUNX1, RIT1 , and other genes. Our cohort of primary sinonasal AdCCs included 31/88 (35%) cases (both with canonical and noncanonical fusions) associated with growth patterns of SH and/or ASGSH. These lesions, first recognized and described by our group, lack the typical lobular arrangement of SH, displaying an irregular and angled overall shape with atypical and disorganised inner secretory epithelial cells and outer myoepithelial cells. These lesions harbor both canonical MYB/MYBL1:NFIB fusions and noncanonical fusions. The possible precursor/neoplastic nature of ASGSH was supported by various mutations revealed by NGS in 5 cases, including BRAF Val600Glu (2 cases), RET Arg912Trp (2 cases), and FAT1 Pro1665Leu (1 case). According to our recent observations, ASGSH structures may also be associated with other sinonasal malignancies, such as low-grade tubulopapillary adenocarcinoma and a subset of sinonasal adenosquamous carcinoma. Sinonasal AdCCs are particularly aggressive salivary gland malignancies, mostly with poor clinical outcome and no effective systemic therapy. , Therefore, identifying potentially targetable genetic alterations in AdCC is crucial. Our findings confirm several observations from previous AdCC whole exome sequencing studies. Somatic mutations in NOTCH1, NOTCH2 and other Notch pathway molecules have been reported previously in AdCC. , – In our cohort, NOTCH1 was one of the most commonly disrupted genes (in 14% of analyzable cases). Notch signaling with its pleiotropic, context-dependent effects on cell differentiation, survival, and growth is disrupted in many human malignancies. , NOTCH1 alterations can result in either tumor suppression or oncogenesis, depending on the tumor entity. , Further characterization of Notch signaling alterations in AdCC is warranted, particularly since it is a potentially targetable pathway. Patients with such aberrations might potentially benefit from anti-NOTCH drugs, such as Brontictuzumab. Morphologically, all 3 NOTCH- mutated AdCCs in our series revealed a prevailing solid/basaloid pattern with a minor component of cribriform and tubular patterns. These tumors were characterized by confluent solid sheets of tumor cells with pronounced nuclear enlargement and polymorphism, and high mitotic and Ki-67 indices, along with perineural, intraneural and bone invasion, and comedonecrosis. In addition to high-grade morphology, metatypical features were noted in 2 AdCCs with NOTCH mutations. They presented with unusual growth patterns, including squamous differentiation, tubular epithelial hypereosinophilia with luminal cell prominence and focal sebaceous metaplasia. The clinical outcome of the 3 patients with NOTCH- mutated AdCCs was particularly poor. In our study, 19 cases displayed atypical morphologic features consistent with metatypical AdCC, including tubular epithelial hypereosinophilia, vacuolated clear cells, and small clusters, cords and isolated tumor cells in hyalinized extracellular matrix. , Multiple foci of foamy vacuolated eosinophilic cells within tumor nests composed of basaloid neoplastic cells were present in 1 case. Similar groups of vacuolated neoplastic cells were previously described by Altemani et al, although their tumors were not termed metatypical AdCC, as they called such cells signet-ring cells. In our study, 3 of the 5 AdCCs with metatypical morphology harbored noncanonical fusions ESWR1:DNM3 , EWSR1:MYB , or ACTN4:MYB , while 1 had the canonical MYB:NFIB fusion, and in 1 case no fusion was detected. In addition, all 5 metatypical AdCCs displayed multiple mutations in genes such as NOTCH3, PTEN, CHD2, ARID1B, RB1, APC, CSF3R, SPEN, NCOR1, KDM6A, BCOR, GATA3 , or MET . The clinical outcome of patients with metatypical AdCCs harboring NOTCH and BCOR gene mutations was notably poor. The clinical significance of mutations in several of the genes identified in this study has not been previously investigated in AdCC. We found mutations in SWI/SNF complex genes, in particular in SMARCA4, ARID1A , and PBRM1 . The latter is known to be required for stability of the SWI/SNF chromatin remodeling complex. The PI3K-Akt pathway was represented by the PTEN gene, the RAS/MAPK pathway by the RIT1 and NF1 genes, and the Wnt signaling pathway by the CDH2 and CCD1 genes. However, none of the mutations observed in this study had a high recurrence rate in our sinonasal AdCCs, and consequently they do not appear to play essential roles in the pathogenetic mechanism of AdCC. In contrast, it is likely that they have pathogenetic importance for those rare patients whose tumors harbor them. In conclusion, this study emphasizes the significance of broad molecular profiling in expanding our understanding of AdCC. No morphologic differences were observed between AdCCs with MYBL1:NFIB and MYB:NFIB fusions. Mutations in the NOTCH genes were associated with high-grade and metatypical phenotypes, and worse clinical outcome. Noncanonical fusions were predominantly associated with metatypical AdCC. These discoveries illustrate how broad molecular profiling enables correlating morphologic changes, genetic alterations, and tumor behavior in AdCCs. |
Establishing Synoptic Cancer Pathology Reporting in Low- and Middle-Income Countries: A Nicaraguan Experience | 730b2234-6dba-442f-905e-7ffa3ee8392c | 8853616 | Pathology[mh] | A cancer pathology report is the final written product of a surgical pathology laboratory with information crucial for patient care and cancer surveillance. Traditional narrative reports contain text that describes information in relevant headings: macroscopic description, microscopic description, and final diagnosis. These descriptive free-text reports, however, show significant variability in style and content, do not contain all clinically important data, and can cause erroneous decisions. CONTEXT Key Objective Is it possible to establish synoptic cancer pathology reporting in a low- or middle-income country? Knowledge Generated In a Nicaraguan public hospital, there were 10,012 histopathologic cases during the study period. Of these, 724 met the criteria for use of organ-specific cancer pathology reporting protocols. Upon review, it was found that every report used the newly introduced synoptic format. Benefits of conversion from limited narrative to structured synoptic cancer pathology reporting include accurate documentation with consistent format, elimination of transcription errors, and concise information available for treatment options and research. Relevance Synoptic cancer reporting is now established in Nicaragua, confirming that it is possible in a low- or middle-income country. This conversion to synoptic from narrative reporting can serve as a model to improve cancer patient care and the quality of data for population-based cancer registries in other countries. Synoptic reporting is a clinical documentation method that has developed over recent decades. In the 1980s, the use of templates, protocols, and checklists became common in a variety of areas of medicine and were associated with improved communication and completeness of tasks. Hutter, in 1984, pointed out that the pathologist's documentation of breast biopsies and mastectomies can provide useful information for the selection of treatment and provide information to estimate prognosis and determine the need for adjunctive therapy. Markel and Hirsch in 1991 were first to report replacing the narrative format with the synoptic style format in their pathology department. Practice protocols for pathology were introduced and standardized in the next few years. , Leslie and Rosai recommended that synoptic templates be used for standardization of pathology reports, with the goal of attaining uniform and consistent data relevant to clinical management of patients. Concise, accurate tumor reporting is accomplished through the consistent use of element-based synoptic protocols. In 1998, the College of American Pathologists (CAP) introduced the first synoptic cancer-reporting guidelines for use by practicing pathologists. Multidisciplinary task forces began to release individual protocols for a variety of types of cancers. One for gastric carcinoma serves as an example. Two years later, CAP published information regarding the process of their development and approval. The protocols were designed for patient care in all types of practice settings and to assist registrars and governing bodies in the uniform collection of pathology data. Protocols include the TNM classification system, used primarily in solid tumors. Synoptic elements, including classified TMN staging, show the extent of disease. Cancer staging with TNM aids prognosis, improves communication between providers, and allows for better information sharing and research across populations. The International Classification of Diseases for Oncology (ICD-O), a standard coding tool for cancer registries, is incorporated. In June 2021, the release of updated CAP Cancer Protocols included 87 revised and three new protocols. Collection of data regarding individual tissue specimens is obtained using protocol templates in a question and answer format. For each template, required data items are followed by a list of possible appropriate responses. Templates may also include optional data items. Each diagnostic parameter pair (data item and response) must appear on a separate line to achieve visual separation. The number of parameters that a pathologist needs to include in a report depends on the organ and specimen type. Table shows English and Spanish versions of an acceptable synoptic pathology report. Table shows an example of a synoptic report that clearly delineates TNM staging. Conversely, Table shows an example of a report that is unacceptable, in part due to the absence of TNM. Key Objective Is it possible to establish synoptic cancer pathology reporting in a low- or middle-income country? Knowledge Generated In a Nicaraguan public hospital, there were 10,012 histopathologic cases during the study period. Of these, 724 met the criteria for use of organ-specific cancer pathology reporting protocols. Upon review, it was found that every report used the newly introduced synoptic format. Benefits of conversion from limited narrative to structured synoptic cancer pathology reporting include accurate documentation with consistent format, elimination of transcription errors, and concise information available for treatment options and research. Relevance Synoptic cancer reporting is now established in Nicaragua, confirming that it is possible in a low- or middle-income country. This conversion to synoptic from narrative reporting can serve as a model to improve cancer patient care and the quality of data for population-based cancer registries in other countries. All pathologists in Nicaragua had traditionally and exclusively used the narrative form of pathology reporting. The adoption of synoptic reporting in pathology practice was contemplated as part of a nation-wide system to capture and report cancer data. Significant milestones of the development process for the entire country, beginning in 2015, are outlined in Table . Initially, the authors explored the concept of synoptic reporting with interested pathologists and oncologists at national meetings. Positive feedback led to discussions with the national Ministry of Health (MINSA). After receiving permission from CAP, translations into Spanish were carried out by 11 pathologists. The translated protocols were approved by a committee of experts of MINSA. In 2017, authorization to proceed with implementation was obtained from MINSA. A successful 3-month trial of reporting breast cancer cases was carried out in one hospital. Training sessions were carried out for all public sector pathologists. Starting in 2018, several hospitals in Nicaragua began using synoptic reporting for some types of cancer cases. By 2019, all pathologists had been trained in the use of protocols, and all hospitals in Nicaragua became qualified to accept them for cancer reporting. The ongoing process includes translating CAP updates, including the most recent TNM staging, and providing continuing education. The Department of Pathology, Hospital Escuela Oscar Danilo Rosales Argüello (HEODRA), León, Nicaragua, decided to establish and implement synoptic reporting for all appropriate cancer cases beginning in January 2018. The process from microscope to signed synoptic report utilized at HEODRA follow. Registering of data begins in the operating or procedure room. The patient is identified, a unique national code is assigned, a procedure is performed, and a surgical specimen is obtained. These initial data are entered into a software program for surveillance and prevention of cancer called Sistema de Vigilancia y Prevención del Cáncer (SIVIPCAN). The second step in registration occurs when the specimen is received in the Department of Pathology where the case is assigned a hospital code. The pathologist receives the case slides along with a paper request form, examines the tissue, and handwrites a report using a synoptic protocol for appropriate cancer cases. When an element of data cannot be responded to because of the type of specimen, the reported response is not applicable or not available. A brief reason is written in parentheses (eg, not possible to specify the state of margins because of fragmented specimen). A secretary or clerk types the pathology report that the pathologist then reviews, corrects as needed, approves, and signs. Though free text is never allowed within the synoptic report, it may appear separately after the formal report. Data elements of the protocol are entered into SIVIPCAN. Copies of the report are printed for the clinical physician and the pathology archives. All 10,012 histopathologic case reports issued by the Department of Pathology, HEODRA, from January 1, 2018, through June 30, 2020, were reviewed. Benign cases, as well as recurrent tumors, endometrial biopsies, endometrial curettages, and primary resection cytologic specimens, were excluded. Since synoptic CAP reports are designed for primary tumors, metastatic tumor cases were excluded when surgery for the primary lesion had been performed in another hospital or when the patient had died without a histopathologic report of a primary tumor. As no cancer biomarkers were available at HEODRA, it was not possible to include that type of protocol. After exclusions, 724 cancer cases met criteria for synoptic style examination and reporting using CAP protocols. All 724 synoptic-style cancer pathology reports issued by the Department of Pathology, HEODRA, from January 1, 2018, through June 30, 2020, were reviewed. Each case report was analyzed to assess completeness of synoptic reporting. All (100%) were found to be in the synoptic format without deficiency of required data items. None of the 724 cases were in the narrative style. In addition, in every report, all elements were listed in correct order for the specific protocol template used. Topographic distribution was based on the anatomical site of origin (Table ). The 10 most common types of cancer seen at HEODRA, in descending order, were as follows: uterine cervix 338 (46.7%), breast 90 (12.4%), skin 82 (11.3%), colon 39 (5.4%), lymph nodes 25 (3.5%), thyroid 24 (3.3%), corpus uteri 20 (2.8%), stomach 13 (1.8%), ovary 11 (1.5%), and kidney 10 (1.4%). Patient age was distributed by age groups (Table ). The 15- to 39-year-old age group had the largest number and percentage of synoptic case reports: 270 (35.9%). Diagnostic and demographic data for all 724 cases were submitted to SIVIPCAN, the national database. By 2030, the United Nations Sustainable Development Goal 3 seeks to reduce premature mortality from noncommunicable diseases, including cancer, by one third. More than 60% of the world's cancer cases occur in Africa, Asia, and Central and South America, and these regions account for about 70% of the cancer deaths. The president of the International Association of Cancer Registries noted that in low- and middle-income countries (LMICs), registry coverage with high-quality data remains well below 10% in Africa, Asia, and Latin America. In addition to accurate demographic information, registries need to obtain detailed case data from clinicians and pathologists. Essential variables include the following: personal identifiers and demographics, tumor biopsy or resection reports, initial therapy, and case follow-up. Registry data can then be used for selection of patient treatment options, epidemiologic studies, and public health planning. In Nicaragua, the desire for a national cancer registry to provide accurate information for clinicians and public health planning was the initial impetus to seek a more advanced level of cancer reporting. The principal benefits of conversion from narrative to structured synoptic cancer pathology reporting include accurate documentation without variability in format, elimination of transcription errors, concise information available for treatment options and research, and robust reliable data for population-based registry and public health planning. Standardized population-based synoptic cancer pathology reports have improved support for clinical decision making, research, and clinician satisfaction. , The value of a national approach to structured reporting is well delineated in a report to the Australian Government Department of Health by the Royal College of Pathologists of Australasia. It recognized that synoptic reporting contributes to cancer control at levels of clinical management, notification and registration, aggregated analyses, and clinical research. In addition to CAP, other national pathology groups, including the Royal College of Pathologists, have released standards and data sets. The International Collaboration on Cancer Reporting has also been developing data sets (in English, with some available in Spanish, French, and Portuguese) that provide an evidence-based approach for reporting many individual cancers. Although synoptic pathology reporting is in common use in high-income countries, the only previous study we could find in a LMIC was an investigation in Nigeria limited to comparing narrative versus synoptic reporting for prostate needle biopsies. Our experience in Nicaragua is unique since synoptic protocols were used to report all cancer cases for which there are designated CAP protocols. Though it would have been interesting to compare completion of synoptic with previous narrative style reporting, permission could not be obtained to carry out a review of previous archived narrative reports. The two styles were never used at the same time. Implementation of synoptic reporting for all cases occurred after approval of the new platform. Evaluation of the cases at HEODRA revealed areas for improvement that could speed the process overall, minimize errors, and maximize the use of enhanced information. These include introducing special checklists for cancers of greatest frequency, entering data directly into the computer instead of handwriting initial reports, moving to electronic reporting with drop-down menus, and promoting the use of data by clinicians and tumor boards. In addition, availability of immunohistochemistry would allow inclusion of biomarkers in reporting. Synoptic reporting is now also being carried out by pathologists in all public hospitals in Nicaragua. For each case, demographic data, synoptic elements (including classified TNM staging), and other diagnostic data (from ICD-O catalogs) are entered into SIVIPCAN, the national database. This information can be reviewed on the network from any hospital in the country. For clinicians, the synoptic protocols used by pathologists are providing more complete data for consideration of patient treatment options. For registrars and researchers, these data provide more reliable information for epidemiologic studies and public health planning. Resulting data, however, are not entered in a formal cancer registry that allows it to be shared and analyzed nationally. Currently, it is not possible to access precise information on the percentage of cases that receive care at individual public or private hospitals. An optimal strategy would be to have reports in a computerized mode that would allow them to be dispersed rapidly for use by consultants in real time for diagnosis, treatment planning, and surveillance. The goal is to be able to provide these data to all clinics, hospitals, and cancer care physicians, as well as the national public social security system. This capacity awaits the establishment of a national cancer registry. In conclusion, conversion from narrative to synoptic reporting, as reported here, has inaugurated a valuable system change in the care of Nicaraguan patients with cancer. Early acceptance by local physicians and health officials led to a heartening spirit for collaboration and easy sharing of information. Even at this early stage in implementation, it provides complete, uniform, and reliable information essential for decision making, especially in regard to further diagnostic evaluation and appropriate treatment. Data from reports are submitted electronically to the national population-based data registry. Accurate reporting flows from the pathologist's desk to the cancer care clinician. We acknowledge that the change from narrative to synoptic reporting can have limitations. LMICs that do not yet have hospitals or centers that care for patients with cancer, or are not public health-oriented, may have to await increased interest or resources. As far as is known, the introduction of synoptic cancer pathology reporting in all public hospitals in Nicaragua is a first for a LMIC. On the basis of our successful experience, establishment of synoptic cancer reporting is recommended for all LMICs at a level that is achievable and affordable. As exemplified by our case, the lack of financial means to install an electronic synoptic software program should not delay basic conversion from narrative to synoptic style. Transition to an electronic format can occur as resources become available. With a dedicated collaborative step-by-step approach, successful establishment of synoptic reporting can significantly improve the quality of cancer patient care and the quality of data in population-based cancer registries. |
Long-term follow-up study of work status among patients with work-related mental disorders referred to departments of occupational medicine in Denmark | cf6ca205-3be3-446e-9500-f5301659b5fe | 10632875 | Preventive Medicine[mh] | Many studies have linked exposure to psychosocial risk factors in the workplace with increased risk of work-related common mental disorders such as depression, anxiety, stress-related diseases and post-traumatic stress disorder (PTSD). In Denmark, when general practitioners (GPs) suspect that working conditions may have contributed to a patient’s common mental disorder, they can refer patients to departments of occupational medicine for further examination. An examination provides a diagnostic assessment and causal evaluation of whether or not the disease occurred due to exposures at work. In addition to diagnostic assessment, patients who are on sick leave at referral receive advice on optimal return to work practices. Psychological treatment for patients with stress disorders has been introduced during the last 10 years at some Danish departments of occupational medicine based on results from randomised clinical studies. Except from a recent cohort profile study, no studies have reported on long-term follow-up of patients with mental disorders referred to Danish departments of occupational medicine. Long-term follow-up studies from other countries are also scarce. The burden and costs associated with mental disorders are high for the affected individuals, their employers and society. This is in agreement with a recent systematic review reporting that mental disorders are a major cause of long-term sick leave and disability expenses. Employees on sick leave due to mental disorders often experience challenges on attempting to return to work, and the risk of recurrent sick leave or job loss is high. A 7–year follow-up study by Koopmans et al found that 19% of employees who had experienced sick leave due to a common mental disorder experienced relapse within 3 years. While some long-term follow-up studies on large samples are available, most research has focused on short follow-up periods. Few studies have assessed work-relatedness of common mental disorders, even though this would be highly relevant for return to work, and the advice given to these patients. In a public healthcare system, where financial resources are often scarce, prediction of which patients are at risk of losing their labour market attachment after having a work-related common mental disorder, is highly valuable. Emphasis should be on identifying which patients might be at particular risk of recurrence or job loss and could profit from treatment and extended consultative interventions. Studies are however, sparse that focus on prognostic factors or prognostic modelling of long-term labour market attachment after a work-related common mental disorder. The primary endpoint of most studies has been time to return to work, often within 1–2 year’s follow-up. Two reviews reported strong evidence that increasing age is associated with continuing disability and longer time to return to work after common mental disorders. A scoping review found consistent evidence that lower symptom severity, no previous absenteeism and positive sick leave duration expectations were associated with earlier return to work. Recent studies find that ‘degree of motivation to return to work’ and higher ‘work ability at baseline’ are positively associated with improved return to work. High socioeconomic position is associated with a greater likelihood of return to work after depression but not after severe stress. The prognostic value of gender and educational level remains unclear. Further research is needed to determine the importance of all the prognostic factors studied so far. The purposes of this paper is to examine long-term prognosis after a work-related common mental disorder among patients in a nationwide occupational medicine cohort and to develop a prognostic model predicting which patients experience a relatively stable return to work process and which patients are at risk of losing their long-term labour market attachment. Study design and participants We present a register-based nation-wide longitudinal study. The study was based on registrations in the Danish National Patient Registry, which records all hospital contacts. We identified all patients referred to departments of occupational medicine in Denmark 2000–2013. Inclusion into the mental health cohort was defined as: first contact to a Department of Occupational Medicine for a range of mental disorders specified by ICD-10 diagnoses F00-F99, Z56, Z63.7, Z73.0-Z73.3 and R45.7. For the descriptive part of the study, patients younger than 18 years and older than 67 years of age at assessment were excluded from the study population as were patients registered as receiving public retirement pension, early public retirement pension or emigrated . For the prognostic model, patients older than 62 years at assessment and patients receiving permanent health-related public benefits at time of assessment were also excluded. Variables and data sources All data were obtained through Statistics Denmark. We retrieved register data from 5 years before and 5 years after patient assessment. Data in these registries were complete from 1995 and updated until the end of 2018, enabling us to include patients seen during 2000–2013. The patient’s unique social security number was linked to nation-wide registries (The Population Register, The Danish National Patient Registry, The Danish Register for Evaluation of Marginalisation (DREAM), The Population Education Register, The Job Register, The Danish National Health Service Register, The Danish Psychiatric Central Research Register, The Cause of Death Register and the Danish Occupational Cohort (DOC*X)) and used to merge all data at the individual level. In Denmark, all citizens are registered by their social security number and entitled to payment in case of unemployment or disease. The DREAM register provided information on all transfer payments provided by the Danish authorities to citizens. The DREAM register comprises numerous codes for transfer payment, emigration and death recorded on a weekly basis. Absence of a weekly code refers to patients not receiving transfer payment, most likely due to an income from working but for a small proportion it can be due to no income at all (ie, spousal support or similar). DREAM codes were grouped as (1) working, (2) education, maternity or parental leave, (3) temporary public benefits due to either sick leave or temporary unemployment, (4) permanent health-related public benefits, (5) retirement due to old age public retirement or voluntary early retirement, (6) emigration and (7) death. The dependent variable in the prognostic analyses was the Work Participation Score (WPS) during the fifth year of follow-up of each patient. A WPS was calculated per follow-up year. The first year comprised the period from the week of the first visit and the following 51 weeks. The time periods for the following years were 52 weeks. WPS was defined as weeks of employment, education, maternity or parental leave relative to the total number of potential weeks of work (ie, weeks of employment, education, maternity, parental leave plus weeks on temporary public benefits or permanent health-related public benefits). Patients were censored from the year of public retirement pension, voluntary early retirement pension, emigration or death. Thus, weeks on public retirement pension and voluntary early retirement as well as death and emigration were not included in the calculation of the WPS. Similar to other studies, a WPS>75% was defined as high work participation. The non-dependent variables in the prognostic analyses were selected if we estimated that the variable was theoretically relevant for the association between suggested work-related common mental disorder and work participation. Besides, the variable had to be available for the clinician in a daily clinical setting and available from Statistics Denmark (as this study is strictly register based). The theoretical relevance of the variables was among others based on a literature search (presented in objectives). Altogether the following non-dependent variables were selected: gender, age, marital status, education, occupational groups, geographical region, diagnostic group, calendar time period, sick leave, comorbidity, geographical region, prior healthcare utilisation, prior work participation and comorbidity. The ICD-10 diagnoses were grouped into the following categories: stress disorder, depression, PTSD, anxiety and other mental disorders. Stress disorder was used as an umbrella diagnosis aggregated by the following diagnoses: psychological strain disease (F43.0, F43.8 or F43.9), adjustment disorder (F43.2), the Z56 diagnoses (problems with employment), Z63.7 traumatising life experiences, Z73.0 exhaustion, Z73.2 lacking relaxation and spare time and Z73.3 psychological stress not otherwise classified. Depression was aggregated by single episode depression (F32), periodic depression (F33), persistent affective disorders (F34) and depression when bipolar (F31.0, F31.3 and F31.4). PTSD (F43.1) was a single group. Anxiety (F40.0–F41.9) was kept as a single group in but was grouped with ‘other mental disorders’ (the remaining F diagnoses) in due to rules stated by Statistics Denmark enforcing the General Data Protection Regulation, which prohibits cells in tables with <5 persons. ‘Other mental disorders’ were the remaining psychiatric ICD-10 F-diagnoses. The number of professional groups (classified in 10 ISCO categories) were reduced to: (1) managers and professionals, (2) technicians and associate professionals, (3) clerical support workers, (4) service and sales workers, (5) craftsmen, agriculture and armed forces, (6) elementary occupations and plant and machine operators and (7) unknown. Sample size Traditionally, multivariable logistic regression analyses require 10 outcome events per prognostic variable. The 12 non-dependent variables in this study require 120 outcome events of the dependent variable. Because more than 15 000 patients were included, no formal sample size calculation was performed. Missing data The non-dependent variables had missing data in the range of 0%–1.4% which we considered low, and no attempt to correct for missing data was performed. There were no missing data for the patients included in the multivariable logistic regressions. Statistical analyses Descriptive show the work status of the patient cohort on a weekly basis. Data are presented for each week from 5 years before to 5 years after first contact to a Department of Occupational medicine. Two different prognostic models were built that analysed probability of high work participation (working >75% of total number of potential work weeks during the fifth year of follow-up). Twelve non-dependent variables were included in the analyses. Correlations between the variables were examined by performing a correlation matrix. Ten variables were analysed as categorical, whereas age and calendar time period were kept on a continuous scale. Statistical analyses were performed using Stata V.16 (STATA Corp, College Station, Texas, USA). A model using multivariable logistic regression analyses was built that included all 12 non-dependent variables. Impact of non-dependent variables was analysed using backwards selection, excluding variables from the model if p-values exceeded 0.20. Performance of the model was tested with Hosmer-Lemeshow goodness-of-fit. Additionally, a regression model using the LASSO technique (least absolute shrinkage and selection operator) was built to apply penalised estimation and investigate variable selection based on the full logistic model. The LASSO technique was originally introduced to improve prediction accuracy and interpretability of regression models by shrinking the number of covariates used in the model. Performance was tested with LASSO goodness-of-fit using deviance and deviance ratios. To assess calibration of the two models, we plotted expected against observed probabilities of outcome in a calibration belt plot with 80% and 95% CIs. Estimates of discrimination were achieved by calculating the area under the receiver operator curve (ROC AUC) and applying the Brier score with Spiegelhalter’s Z-statistic. Patient and public involvement None. We present a register-based nation-wide longitudinal study. The study was based on registrations in the Danish National Patient Registry, which records all hospital contacts. We identified all patients referred to departments of occupational medicine in Denmark 2000–2013. Inclusion into the mental health cohort was defined as: first contact to a Department of Occupational Medicine for a range of mental disorders specified by ICD-10 diagnoses F00-F99, Z56, Z63.7, Z73.0-Z73.3 and R45.7. For the descriptive part of the study, patients younger than 18 years and older than 67 years of age at assessment were excluded from the study population as were patients registered as receiving public retirement pension, early public retirement pension or emigrated . For the prognostic model, patients older than 62 years at assessment and patients receiving permanent health-related public benefits at time of assessment were also excluded. All data were obtained through Statistics Denmark. We retrieved register data from 5 years before and 5 years after patient assessment. Data in these registries were complete from 1995 and updated until the end of 2018, enabling us to include patients seen during 2000–2013. The patient’s unique social security number was linked to nation-wide registries (The Population Register, The Danish National Patient Registry, The Danish Register for Evaluation of Marginalisation (DREAM), The Population Education Register, The Job Register, The Danish National Health Service Register, The Danish Psychiatric Central Research Register, The Cause of Death Register and the Danish Occupational Cohort (DOC*X)) and used to merge all data at the individual level. In Denmark, all citizens are registered by their social security number and entitled to payment in case of unemployment or disease. The DREAM register provided information on all transfer payments provided by the Danish authorities to citizens. The DREAM register comprises numerous codes for transfer payment, emigration and death recorded on a weekly basis. Absence of a weekly code refers to patients not receiving transfer payment, most likely due to an income from working but for a small proportion it can be due to no income at all (ie, spousal support or similar). DREAM codes were grouped as (1) working, (2) education, maternity or parental leave, (3) temporary public benefits due to either sick leave or temporary unemployment, (4) permanent health-related public benefits, (5) retirement due to old age public retirement or voluntary early retirement, (6) emigration and (7) death. The dependent variable in the prognostic analyses was the Work Participation Score (WPS) during the fifth year of follow-up of each patient. A WPS was calculated per follow-up year. The first year comprised the period from the week of the first visit and the following 51 weeks. The time periods for the following years were 52 weeks. WPS was defined as weeks of employment, education, maternity or parental leave relative to the total number of potential weeks of work (ie, weeks of employment, education, maternity, parental leave plus weeks on temporary public benefits or permanent health-related public benefits). Patients were censored from the year of public retirement pension, voluntary early retirement pension, emigration or death. Thus, weeks on public retirement pension and voluntary early retirement as well as death and emigration were not included in the calculation of the WPS. Similar to other studies, a WPS>75% was defined as high work participation. The non-dependent variables in the prognostic analyses were selected if we estimated that the variable was theoretically relevant for the association between suggested work-related common mental disorder and work participation. Besides, the variable had to be available for the clinician in a daily clinical setting and available from Statistics Denmark (as this study is strictly register based). The theoretical relevance of the variables was among others based on a literature search (presented in objectives). Altogether the following non-dependent variables were selected: gender, age, marital status, education, occupational groups, geographical region, diagnostic group, calendar time period, sick leave, comorbidity, geographical region, prior healthcare utilisation, prior work participation and comorbidity. The ICD-10 diagnoses were grouped into the following categories: stress disorder, depression, PTSD, anxiety and other mental disorders. Stress disorder was used as an umbrella diagnosis aggregated by the following diagnoses: psychological strain disease (F43.0, F43.8 or F43.9), adjustment disorder (F43.2), the Z56 diagnoses (problems with employment), Z63.7 traumatising life experiences, Z73.0 exhaustion, Z73.2 lacking relaxation and spare time and Z73.3 psychological stress not otherwise classified. Depression was aggregated by single episode depression (F32), periodic depression (F33), persistent affective disorders (F34) and depression when bipolar (F31.0, F31.3 and F31.4). PTSD (F43.1) was a single group. Anxiety (F40.0–F41.9) was kept as a single group in but was grouped with ‘other mental disorders’ (the remaining F diagnoses) in due to rules stated by Statistics Denmark enforcing the General Data Protection Regulation, which prohibits cells in tables with <5 persons. ‘Other mental disorders’ were the remaining psychiatric ICD-10 F-diagnoses. The number of professional groups (classified in 10 ISCO categories) were reduced to: (1) managers and professionals, (2) technicians and associate professionals, (3) clerical support workers, (4) service and sales workers, (5) craftsmen, agriculture and armed forces, (6) elementary occupations and plant and machine operators and (7) unknown. Traditionally, multivariable logistic regression analyses require 10 outcome events per prognostic variable. The 12 non-dependent variables in this study require 120 outcome events of the dependent variable. Because more than 15 000 patients were included, no formal sample size calculation was performed. The non-dependent variables had missing data in the range of 0%–1.4% which we considered low, and no attempt to correct for missing data was performed. There were no missing data for the patients included in the multivariable logistic regressions. Descriptive show the work status of the patient cohort on a weekly basis. Data are presented for each week from 5 years before to 5 years after first contact to a Department of Occupational medicine. Two different prognostic models were built that analysed probability of high work participation (working >75% of total number of potential work weeks during the fifth year of follow-up). Twelve non-dependent variables were included in the analyses. Correlations between the variables were examined by performing a correlation matrix. Ten variables were analysed as categorical, whereas age and calendar time period were kept on a continuous scale. Statistical analyses were performed using Stata V.16 (STATA Corp, College Station, Texas, USA). A model using multivariable logistic regression analyses was built that included all 12 non-dependent variables. Impact of non-dependent variables was analysed using backwards selection, excluding variables from the model if p-values exceeded 0.20. Performance of the model was tested with Hosmer-Lemeshow goodness-of-fit. Additionally, a regression model using the LASSO technique (least absolute shrinkage and selection operator) was built to apply penalised estimation and investigate variable selection based on the full logistic model. The LASSO technique was originally introduced to improve prediction accuracy and interpretability of regression models by shrinking the number of covariates used in the model. Performance was tested with LASSO goodness-of-fit using deviance and deviance ratios. To assess calibration of the two models, we plotted expected against observed probabilities of outcome in a calibration belt plot with 80% and 95% CIs. Estimates of discrimination were achieved by calculating the area under the receiver operator curve (ROC AUC) and applying the Brier score with Spiegelhalter’s Z-statistic. None. In total, 17 822 patients, 18–67 years of age, were seen for the first time due to mental disorders at a Department of Occupational Medicine in Denmark during 2000–2013 . Most patients were women (73%), median age of 45 at the first contact to a Department of Occupational Medicine. Almost three-fourths of patients had either a short or medium level of further education and 59% were married. More than three-fourths had high work participation prior to study inclusion. Patients were diagnosed with conditions within the following categories: stress disorder 72%, depression 13%, PTSD 6% anxiety 2%, and ‘other mental disorder’ 7%. presents high labour market attachment for all diagnostic subgroups and the combined group of patients with mental disorders in the 5 years preceding a first contact to a department of occupational medicine. Within the subgroup of patients with stress disorder, around 85% of patients diagnosed with stress disorder were working or on leave of absence (categorised as working) 2–5 years before assessment at a department of occupational medicine. At assessment time only around 30% were working, increasing to around 60% after 1 year, after which practically no further increase was seen. The same tendencies with practically no increase in work status after the first year of follow-up were also seen within the other subgroups of mental disorders. Among patients with PTSD, a particularly small increase in work status (28%) was seen after the first contact. Correspondingly, an increase in the proportion of patients receiving permanent health-related public benefits during the 5 year follow-up period was seen in all diagnostic subgroups. In , weekly employment status for patients diagnosed with stress disorder is shown for three different calendar time periods. A slightly higher level of employment (60%) at 5 years’ follow-up was seen in the most recent calendar time period (2010–2013), as compared with approximately 50% in 2000–2004. Prognostic model development The study population available for prognostic model development decreased from 17 092 to 15 910 due to retirement, emigration or death from index date to the beginning of the fifth year of follow-up (see ). The study population was further reduced to 15 547 due to missing information on non-dependent variables. Dichotomised outcome of high work participation in the fifth year of follow-up was used as dependent variable for both models . 10.1136/bmjopen-2023-072217.supp1 Supplementary data 10.1136/bmjopen-2023-072217.supp2 Supplementary data In the model using multivariable logistic regression analyses, the 12 selected non-dependent variables were included in the analysis. Gender had a p-value of 0.9 and was excluded from the analyses according to the predefined steps in the backwards selection procedure. The remaining 11 non-dependent variables had p-values below 0.20 and were kept in the model. Variables with single subcategories with p-values above 0.20, for example, widowed, were kept in the analysis as the other subcategories in the same variable had p-values below 0.20. According to Riley et al p-values and CIs should not be included in prognostic models. We decided, however, that p-values and CIs should be presented in the multivariable logistic regression analysis . Young age, being married or widow/widower, having long further education, being a manager or professional or being diagnosed with stress disorder were all factors statistically significantly associated with high work participation at 5 years’ follow-up. Additionally, not being sick notified at assessment, low level of prior healthcare utilisation, previous high work participation, no comorbid somatic disease and being assessed in most recent calendar years were also statistically significantly associated with high work participation at 5 years’ follow-up. In the second model, the regression model using the LASSO technique, the 12 non-dependent variables were also included. The LASSO model also found that all non-dependent variables except gender were important for the model. The purpose of a penalising model, such as the LASSO model, is to reduce the number of variables and additionally reduce subcategories within the variables as well as the size of the estimates to prevent over-optimism of the regression model. In this population, the LASSO model resulted in reduction of only two subcategories (two of the five geographical regions), as compared with the model using multivariable logistic regression analyses. The estimates in the LASSO model were lower than estimates presented in the multivariable logistic regression model; the tendencies were, however, identical. Hosmer-Lemeshow goodness-of-fit of the multivariable model did not indicate model lack of fit (p=0.38). Comparing the two models, the LASSO goodness-of-fit deviance and deviance ratios were almost identical for the multivariable logistic regression and the LASSO model (deviance 1.19 vs 1.22 and deviance ratio 0.14 vs 0.11, respectively). In terms of discrimination, identical AUCs of the ROC of 0.72 (0.71–0.73) were found for the two models. A Brier score of 0.21 was found for both the models and Spiegelhalter’s Z-scores of 0.54 for the logistic regression analysis and 0.80 for the LASSO model, where Z-scores>0.05 indicate acceptable discrimination. Calibration belt plots were made for both models and showed slim belt plots for both models . The study population available for prognostic model development decreased from 17 092 to 15 910 due to retirement, emigration or death from index date to the beginning of the fifth year of follow-up (see ). The study population was further reduced to 15 547 due to missing information on non-dependent variables. Dichotomised outcome of high work participation in the fifth year of follow-up was used as dependent variable for both models . 10.1136/bmjopen-2023-072217.supp1 Supplementary data 10.1136/bmjopen-2023-072217.supp2 Supplementary data In the model using multivariable logistic regression analyses, the 12 selected non-dependent variables were included in the analysis. Gender had a p-value of 0.9 and was excluded from the analyses according to the predefined steps in the backwards selection procedure. The remaining 11 non-dependent variables had p-values below 0.20 and were kept in the model. Variables with single subcategories with p-values above 0.20, for example, widowed, were kept in the analysis as the other subcategories in the same variable had p-values below 0.20. According to Riley et al p-values and CIs should not be included in prognostic models. We decided, however, that p-values and CIs should be presented in the multivariable logistic regression analysis . Young age, being married or widow/widower, having long further education, being a manager or professional or being diagnosed with stress disorder were all factors statistically significantly associated with high work participation at 5 years’ follow-up. Additionally, not being sick notified at assessment, low level of prior healthcare utilisation, previous high work participation, no comorbid somatic disease and being assessed in most recent calendar years were also statistically significantly associated with high work participation at 5 years’ follow-up. In the second model, the regression model using the LASSO technique, the 12 non-dependent variables were also included. The LASSO model also found that all non-dependent variables except gender were important for the model. The purpose of a penalising model, such as the LASSO model, is to reduce the number of variables and additionally reduce subcategories within the variables as well as the size of the estimates to prevent over-optimism of the regression model. In this population, the LASSO model resulted in reduction of only two subcategories (two of the five geographical regions), as compared with the model using multivariable logistic regression analyses. The estimates in the LASSO model were lower than estimates presented in the multivariable logistic regression model; the tendencies were, however, identical. Hosmer-Lemeshow goodness-of-fit of the multivariable model did not indicate model lack of fit (p=0.38). Comparing the two models, the LASSO goodness-of-fit deviance and deviance ratios were almost identical for the multivariable logistic regression and the LASSO model (deviance 1.19 vs 1.22 and deviance ratio 0.14 vs 0.11, respectively). In terms of discrimination, identical AUCs of the ROC of 0.72 (0.71–0.73) were found for the two models. A Brier score of 0.21 was found for both the models and Spiegelhalter’s Z-scores of 0.54 for the logistic regression analysis and 0.80 for the LASSO model, where Z-scores>0.05 indicate acceptable discrimination. Calibration belt plots were made for both models and showed slim belt plots for both models . In this cohort of 17 822 patients with suspected work-related common mental disorders seen at departments of occupational medicine in Denmark in 2000–2013, we observed high labour market attachment during the years preceding the first contact. We consider this previous high labour market attachment as indicating a relatively healthy population until the patients begin suffering from a common mental disorder. We have not located other similar large-scale studies on this topic covering both the 5 years before and the 5 years after clinical assessment at a Department of Occupational Medicine. During the first year of follow-up, an increase, which was not, however, to the previous level, in the percentage of patients working was seen for all subgroups, after which practically no further increase was observed. This is in agreement with a recent systematic review reporting that common mental disorders are a major cause of long-term sick leave. We also developed a prognostic model of high work participation, however it was not suitable for personal prognostication as ROC AUC estimates do not exceed 0.80 and are barely sufficient for prognostication at a population level, where they should lie between 0.70 and 0.80. Applying a machine-based LASSO model for prognostication of high work participation was not superior to a simple multivariable logistic regression model when comparing performance and calibrations of the two models. When developing prognostic models, emphasis is not on comparing the size of individual ORs with one another. Emphasis is on the performance of the model. Nevertheless, the findings that younger age, not being sick notified at assessment, and high previous work participation were associated with high work participation after a suggested work-related common mental disorder are in agreement with previous studies. Our finding that a high level of further education was significantly associated with high work participation at 5 years’ follow-up was not entirely in agreement with previously published conflicting results. Only limited and sporadic treatment was offered to the patients at the departments of occupational medicine during 2000–2013, primarily restricted to patients with stress disorders. Among GPs, the municipal system and private psychologists, treatment offers may, as well, have varied (eg, therapy, medicine and exercise). A relevant concern is that the most resourceful and most well-educated patients may have received help from private psychologists to a greater extent than others, resulting in a higher degree of recovery. The overall results from our and other relevant studies call for new and better intervention approaches that focus on significant, sustainable and long-term return to work for patients with work-related common mental disorders. Numerous randomised controlled studies (RCT) have investigated the effects of various types of return-to-work interventions and reported at most only modest effect. The interventions included individual cognitive behavioural or problem-focused approaches to enhance individual coping and/or gradual return to work with or without workplace involvement. With regard to stress disorders, a Cochrane review with a limited number of studies, found no substantial effect of cognitive behavioural or problem-focused interventions on return to work. Another review from 2018 found an overall little improvement in return to work among sick listed employees with common mental disorders after interventions, particularly, employees with stress disorders. Strong evidence for including workplace contact and moderate evidence for including graded return to work was reported. The effect of physical exercise on return to work after depression or other common mental disorders has rarely been studied. The results from a large RCT reported no effect of exercise on return to work after depression as compared with treatment as usual. The majority of intervention studies addressing return to work have only employed short follow-up periods of 1 year, and follow-up studies addressing relapse are scarce. Thus, so far, intervention studies have not showed large convincing improvements in return to work after common mental disorders. Additionally, recurrent sick leave or job loss have rarely been studied in a long-term perspective. In a recent position paper, Nielsen et al argued that return to work research, to a large extent focuses primarily on resources enhancing return to work during the absence period, ignoring other resources that may facilitate sustainable return to work. They call for studies that follow returned workers over time to understand their return journey. Our study makes an early contribution to this agenda by providing an overall description of the long-term return to work journey among central diagnostic groups. Although more detailed studies are needed. Managing to separate patients in those with high work participation from those with low work participation after 5 years of follow-up could be a step towards studying resources facilitating sustainable return to work and towards development of a prognostic model that can be implemented in a clinical setting to potentially enhance resources that support long-term return to work among vulnerable patients. Strengths and limitations A strength of this study is the large and nation-wide patient cohort, which was followed for 5 years before and after the index date. The patients serve as their own controls regarding work status. Another strength is the use of nation-wide registries linked with a patient’s social security number. The availability of these registries is unique for Denmark, and provides us with a detailed endpoint to study and a wide range of prognostic variables, involving demographic, work and health characteristics with few missing data. A limitation is the endpoint studied. Labour market attachment is an endpoint exposed to changing legal terms in a politically changing system. Over time several political interventions have been initiated in Denmark to increase the employment rate. The law on permanent health-related public benefits was introduced in January 2013 and may have affected the outcome at 5 years’ follow-up for patients seen in the most recent years. Financial fluctuations may also have impacted the numbers employed in different sectors of the labour market. However, many patients in this cohort had professions within the public sector, which is less frequently affected by financial fluctuations. Furthermore, we present figures for patients with stress disorder in three different calendar time periods and in the prognostic study, we included the continuous variable calendar year to deal with confounding due to calendar time periods. Our findings in the descriptive analyses as well as the prognostic study confirmed that labour market attachment was highest for the most recent calendar years, which could indicate some effect of previous political initiatives to improve labour market attachment. Another limitation of the study is the lack of data from patient questionnaires and hospital records. Thus, we have no information on life style factors such as smoking habits and alcohol consumption, severity of the mental health symptoms, general work environment at the work place, the cognitive demands in performing the work, the triggering psychological exposures at the work place, the persons’ own expectations of sick leave duration and other self-reported information. Besides we have no information on our professional conclusion on the work-relatedness of the common mental disorder. We have decided to use the term work-related mental disorder based on the fact that a GP have suggested the disorder to be triggered by work-exposures and have referred the patient to assessment at a Department of Occupational Medicine. A third limitation of the study is the fact that organising a healthcare system with departments of occupational medicine is primarily seen in northern Europe, limiting the relevance of the study to other countries. Conclusion Most patients with common mental disorders in this previously healthy population, referred to departments of occupational medicine in Denmark, are working 1–5 years after assessment. Some patients are however not working 5 years after assessment especially among patients diagnosed with PTSD or depression. To select which patients are at low risk and which are at high risk of losing their labour market attachment, we tried to develop prognostic models for work participation 5 years after assessment. None of the models were, however, suitable for personal prognostication and are barely suitable for prognostication at a population level and none of the models included data from patient questionnaires or hospital records. We hope to improve the models in the future to a sufficient quality enabling us to implement a prognostic model in our daily clinical practice. A strength of this study is the large and nation-wide patient cohort, which was followed for 5 years before and after the index date. The patients serve as their own controls regarding work status. Another strength is the use of nation-wide registries linked with a patient’s social security number. The availability of these registries is unique for Denmark, and provides us with a detailed endpoint to study and a wide range of prognostic variables, involving demographic, work and health characteristics with few missing data. A limitation is the endpoint studied. Labour market attachment is an endpoint exposed to changing legal terms in a politically changing system. Over time several political interventions have been initiated in Denmark to increase the employment rate. The law on permanent health-related public benefits was introduced in January 2013 and may have affected the outcome at 5 years’ follow-up for patients seen in the most recent years. Financial fluctuations may also have impacted the numbers employed in different sectors of the labour market. However, many patients in this cohort had professions within the public sector, which is less frequently affected by financial fluctuations. Furthermore, we present figures for patients with stress disorder in three different calendar time periods and in the prognostic study, we included the continuous variable calendar year to deal with confounding due to calendar time periods. Our findings in the descriptive analyses as well as the prognostic study confirmed that labour market attachment was highest for the most recent calendar years, which could indicate some effect of previous political initiatives to improve labour market attachment. Another limitation of the study is the lack of data from patient questionnaires and hospital records. Thus, we have no information on life style factors such as smoking habits and alcohol consumption, severity of the mental health symptoms, general work environment at the work place, the cognitive demands in performing the work, the triggering psychological exposures at the work place, the persons’ own expectations of sick leave duration and other self-reported information. Besides we have no information on our professional conclusion on the work-relatedness of the common mental disorder. We have decided to use the term work-related mental disorder based on the fact that a GP have suggested the disorder to be triggered by work-exposures and have referred the patient to assessment at a Department of Occupational Medicine. A third limitation of the study is the fact that organising a healthcare system with departments of occupational medicine is primarily seen in northern Europe, limiting the relevance of the study to other countries. Most patients with common mental disorders in this previously healthy population, referred to departments of occupational medicine in Denmark, are working 1–5 years after assessment. Some patients are however not working 5 years after assessment especially among patients diagnosed with PTSD or depression. To select which patients are at low risk and which are at high risk of losing their labour market attachment, we tried to develop prognostic models for work participation 5 years after assessment. None of the models were, however, suitable for personal prognostication and are barely suitable for prognostication at a population level and none of the models included data from patient questionnaires or hospital records. We hope to improve the models in the future to a sufficient quality enabling us to implement a prognostic model in our daily clinical practice. Reviewer comments Author's manuscript |
Critical congenital heart disease: contemporary prenatal screening performance and outcomes in a multi-centre perinatology service | d2149a77-554c-475f-8779-17f7ece587ec | 10893667 | Pediatrics[mh] | Congenital heart disease (CHD) has a prevalence of 4–13 per 1000 live births and is the most common structural congenital anomaly . Approximately 30% of cases of congenital heart disease are considered critical, requiring urgent intervention (surgical or catheter-based) in the early neonatal period . Survival and neurodevelopmental outcome are dependent on timely prenatal diagnosis and input from fetal, neonatal and cardiology services. Global prenatal detection rates remain variable, with reported screening performance ranging from 13% detection in the Slovak Republic to 87% in Northern France and are influenced by availability of ultrasound expertise and access to fetal medicine and fetal cardiology services . Where national anatomy screening policies are introduced and/or targeted fetal cardiac training provided, studies have demonstrated substantial improvements in prenatal detection, up to 59% nationally and up to 91% in targeted training studies . This study sought to determine contemporary prenatal detection rates and survival for critical congenital heart disease (CCHD) managed in a designated hospital group that includes a large tertiary perinatology centre and two affiliated regional secondary sites. This retrospective cohort study examined all cases of duct-dependent CCHD requiring or expected to require cardiac intervention in the first six weeks of life among pregnancies managed in a single hospital group. This study was approved by ethics boards across all institutions involved. The Rotunda Hospital , Dublin is a tertiary care centre that offers a fetal cardiology service in collaboration with the single National Pediatric Cardiac Centre that provides care to all cases of CCHD delivered in Ireland. Prenatal ultrasound is performed by qualified sonographers who participate in ongoing training in fetal echocardiography. This study examined the care pathways from early diagnosis to neonatal life over a 4-year period among a cohort of infants with critical congenital heart disease. All cases of CCHD that underwent pregnancy termination, died in-utero or in the early neonatal period were also ascertained. The protocol in this tertiary care prenatal screening service and its affiliated sites included a dating ultrasound scan at 12–14 weeks’ gestational age, a fetal anatomy screen at 18–22 weeks and a repeat screening ultrasound for cases of non-acquisition of views sufficient to constitute a normal fetal cardiac examination. The following pre-specified cardiac screening views were required for prenatal clearance of the fetal heart: 4-chamber heart view, left and right outflow tract views, three vessel and three vessel tracheal views. Colour assessment was not mandated for the screening examination. In addition to the standard screening cardiac examination at 18–22 weeks, patients attending any of the study centres who had identified risk factors for CHD were offered targeted fetal echocardiography according to criteria that align with prior ISUOG and updated ISUOG and AIUM practice guidelines. Targeted examination of the fetal heart included the use of colour flow Doppler to evaluate inlet and outlet valves, determination of systemic and pulmonary venous return and extended sagittal cardiac views. Referral was made to the tertiary Fetal Medicine service in the event of non-acquisition of cardiac views on a second attempt by the screening sonographer. All cases of suspected CCHD were referred to the Fetal Medicine service, offered genetic testing where appropriate and underwent detailed fetal echocardiography with a dedicated fetal cardiac team, with Fetal Medicine and Paediatric Cardiology expertise. In the event of multiple congenital anomalies, confirmed genetic abnormality or isolated CCHD associated with significant long-term morbidity, pregnancy termination was discussed. In the setting of an anticipated lethal prognosis, pregnancy termination was offered in accordance with Irish Law. For all cases of prenatally recognised CCHD, a multidisciplinary perinatal plan was determined. All cases of CCHD delivered in Ireland undergo treatment in a single national centre. Data sources included dedicated prenatal ultrasound and pediatric cardiology databases, perinatal, neonatal, and pediatric chart review, and examination of perinatal mortality reports from the tertiary care centre. Individual cases were traced back to delivery records and birth registration where necessary. Data management and data quality checks were performed using SAS V9.2. Basic summary statistics were described for the study population. An exact 95% confidence interval was calculated for the overall prenatal detection rate. From January 2019 to December 2022, among a consecutive cohort of 49,950 pregnancies, 202 (0.4%) cases of CHD were identified, of whom 104 were considered to meet the definition for critical congenital heart disease (CCHD), expected to require intervention within the first six weeks of life, for a population incidence of 2 per 1000 pregnancies (Fig. ). Within the cohort of 104 CCHD cases, 96 were diagnosed in the prenatal period, for a prenatal detection rate of 92% (95% CI: 84%—96%). The 96 prenatal cases represented a prevalence of 1.9 per 1000 births over the study. The prenatal cases are presented in Fig. and are categorized into four groups: Left duct-dependant heart disease (Hypoplastic left heart disease, critical mitral valve stenosis, critical aortic stenosis/atresia, Shone complex, interrupted aortic arch, aortic coarctation) Right duct-dependant lesions (Hypoplastic right heart, tricuspid stenosis, critical pulmonary stenosis/atresia, Tetralogy of Fallot with critical pulmonary stenosis) Transposition of the great arteries (TGA) Complete congenital heart block (CHB) No case of isolated total anomalous pulmonary venous drainage (TAPVD) was observed during the study period, an observation consistent with the rarity of isolated TAPVD diagnosis in the literature . There were three cases of TAPVD identified in association with other complex critical cardiac disease, namely right duct-dependent pathology. In the event of multiple structural cardiac anomalies, cases were classified according to the most critical or duct-dependant anomaly. Comparative prenatal and postnatal detection rates for this CCHD classification are presented in Fig. . Maternal demographic data for all cases of CCHD are presented in Table , which demonstrates a mean gestational age at prenatal detection of 22.2 weeks (range 15 to 34 + 4 weeks). Eight percent of cases were significant for a family history of congenital heart disease with one percent of patients having a pre-pregnancy diagnosis of diabetes. All CCHD cases with pre-existing risk factors for CCHD were identified by the routine anatomy screening programme. 33% of CCHD cases (34/104) received prenatal screening in a regional centre, of whom 15% (5/34) were not detected until the postnatal period. This screening performance compares with 4% (3/70) of tertiary-care cases evading prenatal diagnosis. 63% of CCHD cases in this cohort occurred in isolation, with the remaining cases (37%) complicated by an extracardiac structural or genetic abnormality. Perinatal outcomes for 96 cases following prenatal diagnosis are presented in Table . A decision to terminate the pregnancy was taken by patients in 10 cases, some of whom met criteria for pregnancy termination in Ireland (legally permissible in the setting of a lethal prognosis). Spontaneous in-utero fetal death occurred in 7% (7/96) (Table ). Among 79 live-born infants, 11 patients had a palliative care plan in place prenatally. Among the remaining infants considered for surgery, 62 infants were transferred to the Cardiac centre on day 1–14 of life, with prostaglandin administered where needed for the purposes of maintaining ductal patency in accordance with the pre-specified fetal cardiology plan (Table ). A 94% detection rate (16/17) for TGA was observed with a 92% survival rate at one year. The three TGA cases that did not survive comprised of one case of complex TGA (with a genetic diagnosis) where the patient chose pregnancy termination, one case deemed unsuitable for surgery by virtue of severe inlet valvular dysfunction and one perioperative neonatal death. All three cases of congenital heart block were detected prenatally, one fetus died in utero with the remaining two infants alive at 12 months. At one year of age, an 88% survival rate for left-sided duct-dependant lesions and 78% for right-sided duct-dependant cases was observed. The eight postnatally-diagnosed CCHD cases are described in Fig. . There were two cases of single-ventricle pathology undetected by the prenatal screening programme. One had hypoplastic left heart disease (HLHD) and one had double inlet left ventricle (DILV) with TGA and aortic coarctation. Both cases delivered in secondary, regional centres. 62% (5/8) of CCHD cases that were not identified prenatally received prenatal care in regional sites. 87% (7/8) of cases diagnosed postnatally were left-sided duct-dependant lesions (Fig. ). All cases of single ventricle pathology, TGA and CHB in the tertiary care centre were identified by the screening programme. Prenatal detection of CCHD makes a compelling case for targeted screening in the prenatal period owing to its complexity and the time-sensitive nature of neonatal intervention. A neonatal mortality risk of up to 50% is reported in the setting of neonatal hospital discharge with undetected CCHD . An Irish study in 2017 reported a one-year mortality rate for CCHD that was tenfold higher in babies diagnosed postnatally compared to CCHD cases born with a prenatal diagnosis . A meta-analysis of eight studies reported a survival advantage and lower levels of neonatal morbidity for CCHD diagnosed in the prenatal period . Although the use of pulse oximetry as a postnatal screening tool can help identify CCHD not detected by prenatal screening, many new-borns with CCHD do not develop clinically appreciable hypoxaemia until after discharge from hospital . Some critical lesions such as hypoplastic left heart disease may present with significant cardiovascular compromise without apparent cyanosis . Therefore, optimal timely detection of CCHD remains heavily reliant on prenatal diagnosis. An undeniable paradox of prenatal screening is that cases at the greatest disadvantage from non-detection in the prenatal period, namely those where delivery is planned in a centre remote from the pediatric cardiac service, are those for whom access to optimal screening expertise may be less readily available . The extent to which specialist fetal echocardiography for selected pregnancies considered to be at heightened risk for congenital heart disease may result in an appreciable increase in prenatal detection is unclear. The vast majority of cases of CCHD in this study had no risk factors for CHD. Furthermore, the study period was notable for a 12-month period (March 2020 to February 2021) during which the COVID-19 pandemic-related rationalisation protocols were introduced to limit patient-facing exposure. During that time, all fetal cardiac screening was limited to the mid-trimester fetal anatomy examination, with no supplementary imaging offered to women with identified risk factors for CHD. The examination time was also reduced in the pandemic from 30 to 15 min for routine ultrasound examination as a means of reducing sonographer exposure to viral risk. These constraints did not have a detrimental effect on prenatal detection of CCHD. Indeed, throughout the four-year study period, no case of CCHD was detected by targeted screening in the selected population. This observation suggests that the prenatal detection of CCHD does not rely on the provision of targeted supplementary examinations for pregnancies perceived to be at heightened risk. Rather, excellent prenatal detection rates can be achieved women perceived to be at with a protocolised screening service and an established sonographer training programme. This study was conducted in one of the busiest maternity hospitals in Europe, with two additional affiliated referral centres, over a four-year period. The existence of a single National Pediatric Cardiac Centre in Ireland, participating in an externally validated Pediatric Cardiac data collection system that is submitted in real time, results in complete ascertainment of all cases of CCHD evaluated for surgical and catheter intervention within the first six weeks of life. In addition, all cases of fetal abnormality identified in the two regional sites, including all cases of CCHD, are referred to the tertiary Fetal Medicine unit, which maintains a contemporaneous record of all cases of CCHD identified in all three centres. No case of pregnancy termination within Ireland for CCHD proceeds without referral to the centralised fetal cardiac service. All cases of in-utero fetal death in the context of fetal abnormality are recorded and reported in the Annual Report of the tertiary centre . Postnatally diagnosed cases in the event of infant mortality are reported to the cardiac database by the paediatric pathology service centralised in CHI (Children’s Health Ireland)@Crumlin. Therefore, this study should be considered to reflect complete ascertainment of all cases of CCHD identified in the prenatal period and diagnosed in the neonatal period during the four-year study timeframe across all three contributing centres. The prevalence of CCHD in this population was 1.9 per 1000 live births, which is aligned with the prevalence seen in other series. In 2018, Bakker et al. reported a prevalence of 19.1 per 10,000 births in a retrospective cohort study across 15 international centres in Europe, North America and Asia . In 2016, the Irish National Maternity Strategy 2016–2026 devised an implementation plan of action aimed at improving services across all 19 maternity units in Ireland. This included a plan for provision of a routine first trimester ultrasound examination at 10–14 weeks’ gestation and a mid-trimester anatomy scan (18–22 weeks) to all pregnant women . Prior to the strategy, 7/19 units at that time were providing a mid-trimester anatomy scan, 7/19 units were not providing the service and 5/19 units provided the service to select groups of patients . A national detection rate of 57% was reported in 2019, for CCHD in Ireland with 71% reported in centres with an established universal screening protocol . Retrospective population studies have emphasised the importance of national screening programmes and protocols for improvement in the prenatal detection of CCHD . In the Netherlands, the largest reported population screening programme for CCHD to date resulted in an increase in prenatal detection from 36 to 60% in 2007 . In Sweden, where a national screening programme was introduced between 2014 and 2017, the overall detection rate for CCHD was reported as 59%. While the detection of single ventricle pathologies was reported at 100%, only 9% of TGA cases were detected during pregnancy and the prenatal detection of aortic coarctation was 18% . A National anatomy screening programme in the UK has been in place for 25 years and updated Fetal Anatomy Screening Programme (FASP) guidelines were introduced in 2015 with the aim of increasing detection rates to 50%. The reported national prenatal detection rate for CHD in the UK is 30–50% among infants undergoing catheterisation or surgery . Importantly, published data that do not include fetal deaths, pregnancy terminations, pre-intervention neonatal death or palliative care cases will inevitably reflect an under-representation of both CCHD prevalence and the performance of prenatal screening programmes. Targeted fetal echocardiography training programmes have been shown to yield significant improvements in prenatal detection. Following implementation of a training programme CHD detection rates of 91% (21/23) were reported in a review of 5445 fetal anatomy screening ultrasound examinations over a two-year period in a single unit in the UK . Comparable success was reported by The Rotunda Hospital, Dublin programme, where targeted sonographer training resulted in an increase in the prenatal detection of major CHD from 31% in 2010 to 91% in 2012 . Suard and colleagues recently reported a prenatal detection rate of 71% in southern France across two large centres, with 97% of single ventricle abnormalities and 80% of TGA cases identified prenatally. Evans improved overall CHD detection rates from 36–71% in Nevada through implementation of training and multidisciplinary support from fetal medicine and cardiology teams between 2012 and 2014 . His later paper in 2019 reported a further increase in overall CHD detection rates to 78% . It is worth noting that the tertiary centre performed better at prenatal detection of CCHD during the study period. The achievement of performance standards in regional/ peripheral sites equivalent to those observed in tertiary care university centres is a challenge that prevails in all areas of healthcare . While the regional sites in this study have access to the same standard of equipment and with a comparable sonographer workload, readiness of available on-site MFM expertise in the tertiary centre represents the most glaring service disparity. Enhancement of MFM staffing presence at regional centres, in addition to the sharing of practice protocols and the introduction of educational initiatives in fetal echocardiography are progressing since this study period. A national US study in 2020 highlighted variable levels of education among sonographers as a contributory factor to poor prenatal detection . In Ireland, all practitioners involved in the provision of fetal cardiac ultrasound services are qualified sonographers with a background in either midwifery or radiography and all hold a postgraduate qualification in obstetric ultrasound. In 2021, 86/157 (55%) of sonographers in Ireland held a Master’s degree qualification in ultrasound . The majority of studies reporting on the performance of prenatal screening programmes do not provide information relating to the competencies, experience or qualifications held by sonographers . The provision of on-site Fetal Medicine support was cited as an independent contributor to improved prenatal detection in a Spanish study . Equally, Pinto’s 2020 national report associated the absence of fetal medicine support in some US centres with low rates of prenatal detection of CCHD . Periodic training updates, revision of screening protocols and ongoing sonographer support from fetal medicine and fetal cardiology services have continued since The Rotunda Hospital Cardiac Screening Programme was established in 2010, with extension of the screening protocol to implementation in two regional centres, including on-site fetal medicine support at both regional centres. Among almost 50,000 deliveries in this hospital group during the four-year study period, eight cases of unanticipated CCHD that required urgent intervention were managed at the National Pediatric Children’s Heart Centre. A retrospective review of prenatal imaging was beyond the scope of this audit, but is planned for a future study, subject to Institutional Ethics Board approval. A limitation of the study is that not all perinatal deaths at any participating hospital underwent post-mortem examination, nor did all pregnancy terminations undergo post-mortem confirmation of a prenatal diagnosis. While all cases of perinatal mortality in the tertiary centre were ascertained by way of an institutional perinatal mortality report that describes each perinatal death, and all cases were examined for prenatal or neonatal features consistent with CCHD, it is possible that a perinatal death in one of the regional centres may have featured CCHD and was not ascertained by this review. Post-mortem confirmation of CCHD among cases that underwent termination was not routinely undertaken. However, all cases of CCHD that proceeded to TOP were subjected to review by two MFM specialists, a paediatric cardiologist and with multidisciplinary review of imaging both at MFM and Paediatric Cardiothoracic fora in order to eliminate diagnostic uncertainty. A further limitation is that the low population prevalence of CCHD inevitably results in wide confidence intervals surrounding the detection rate estimates. The termination of pregnancy (TOP) rate in the setting of CCHD was low (10%) in this cohort and the palliative care rate high (26%). Since 2018, pregnancy termination in Ireland is legally permissible in the setting of a diagnosis that is expected to lead to death within the first 28 days of life . However, some parents opt for perinatal palliation, particularly where societal or cultural attitudes to pregnancy termination prevail in spite of the recent removal of legal constraints. The low termination rate had the unintended effect of maximising the opportunity for postnatal confirmation. To our knowledge, the overall 92% prenatal detection rate for CCHD reported in this population, comprising a single large tertiary and two affiliated regional maternity sites, is the highest prenatal detection rate reported in the literature to date in an unselected population of this scale. Our data illustrate that optimal rates of prenatal detection can be achieved by a protocolised approach to mid-trimester fetal anatomy ultrasound in all pregnancies, underpinned by a programme of sonographer education and training. While led by the tertiary centre, this approach should include all teams in referring regional centres. It is notable that all detected cases of CCHD were identified by sonographers performing routine anatomy screening and not in specialist cardiac screening clinics. This is a welcome finding, as CCHD typically occurs in pregnancies that at the outset would not be identified as high risk for CCHD. The review confirms that left heart obstruction lesions remain the Achilles Heel of prenatal cardiac imaging. |
Diary of an ophthalmology resident: Recollection of lessons learned at Dr. R. P. Centre, AIIMS, New Delhi | f6c10771-6501-4f34-992d-76aa15dbcd94 | 10229972 | Ophthalmology[mh] | Nil. There are no conflicts of interest. |
An assessment of excess mortality during the COVID-19 pandemic, a retrospective post-mortem surveillance in 12 districts – Zambia, 2020–2022 | 49bb75bf-0af0-4448-bb80-c678251fabc4 | 11437817 | Forensic Medicine[mh] | Since the first cases of SARS-CoV-2 were reported in Zambia in March 2020 through to October 12, 2023, 349,287 cases and 4,069 COVID-19-related deaths were reported . During this period, several phylogenetically distinct strains of SARS-CoV-2 were identified in Zambia, each associated with varying case fatality rates across different wave periods . The number of cases and deaths reported in Zambia likely underestimates the true impact of the pandemic. A nationwide SARS-CoV-2 prevalence survey, using nasal specimens for PCR testing, revealed that for every reported SARS-CoV-2 infection in Zambia, approximately 92 infections went unreported . A systematic postmortem prevalence study among deaths that occurred at a tertiary hospital in Lusaka showed a high SARS-CoV-2 PCR prevalence among deceased persons from January 2021 to June 2021, with greater per cent positivity during peak COVID-19 transmission periods . However, it should be noted that while SARS-CoV-2 was detected among the deceased individuals in this study, it was not determined whether the virus directly caused their deaths or if its presence was merely incidental. Among those with a positive SARS-CoV-2 diagnosis postmortem, only a minority had a COVID-19 diagnosis antemortem. As such, officially reported COVID-19 deaths in Zambia may underestimate the total number of COVID-19 deaths that occurred during the pandemic. During a public health emergency such as the COVID-19 pandemic, monitoring all-cause mortality trends may help assess the severity and impact of the emergency on the population affected . Deaths among confirmed COVID-19 patients do not capture the full extent of the COVID-19 burden. In contrast, deaths from all causes can be used to estimate excess mortality and provide a more complete picture of the impact of the COVID-19 pandemic . For many countries, reporting statistics on COVID-19 mortality has been a challenge due to variations in access to testing, differential diagnostic capacity, the inconsistent and sub-optimal certification of COVID-19 as the immediate cause of death, and the nonspecific clinical presentation of COVID-19 (including in fatal cases) . Additionally, the pandemic led to an increase in deaths due to other causes, because of disruptions to routine health services and loss of livelihoods due to the stringent non-pharmacological interventions put in place to mitigate the pandemic . Therefore, the World Health Organization (WHO) recommends the surveillance of confirmed COVID-19 mortality and all-cause mortality during the COVID-19 pandemic . A study of mortality trends among community deaths at the University Teaching Hospital (UTH) in Lusaka from April 2020 to December 2020 showed 1,139 excess deaths from all causes during the study period compared to the pre-pandemic baseline (2017–2019) . Whilst excess mortality during the COVID-19 pandemic has been established in Lusaka , the country’s predominantly urban capital, it remains unclear whether other regions within the country with different socio-demographic factors had comparable or worse outcomes. We, therefore, conducted this study to assess excess mortality across 12 districts of Zambia during the COVID-19 pandemic. We conducted a mixed methods retrospective analysis of mortality records in Zambia by triangulating data from two different sources (health facility and community death mortuary records) between January 2017 and December 2022. These records were obtained from the mortuaries of the main district referral health facilities in the selected districts. Typically, the most life-threatening medical cases are referred to these referral health facilities from other health facilities within the district and deaths occurring within the communities outside health facilities are brought to these district hospital morgues. We purposively selected 12 districts of Zambia (Chingola, Kabwe, Kitwe, Livingstone, Luanshya, Lusaka, Kasama, Mansa, Chipata, Mongu, Solwezi and Ndola) primarily based on the known availability of mortuary records over the study period, to ensure an adequate mix of urban and rural districts and to ensure adequate geographic representation from across the country. Combined, the selected districts are home to an estimated 6,882,437 (37.4% of the total projected 2021 population) Zambians . Monthly counts of all health facility deaths and all reported community deaths between 2017 and 2019 were collected to estimate baseline all-cause mortality before the COVID-19 pandemic and monthly counts from 2020 to 2022 to estimate excess deaths during the COVID-19 pandemic. A COVID-19 wave period was defined as a sustained increase in the SARS-CoV-2 national test positivity of greater than five per cent, and between January 2020 and December 2022 days were dichotomized as wave or non-wave days. Additionally, wave days were further classified as the first wave (1 Jun 2020 to 1 Oct 2020), second wave (3rd Jan 2021 to 17th March 2021), third wave (29th May 2021 to 20th Aug 2021), fourth wave (12th Dec 2021 to 9th May 2022) and fifth wave (24th Dec 2022 to 31st Dec 2022), all other dates during the study period were non-wave days. Additionally, from January 2020 through to December 2022, daily individual records of all inpatient deaths and all reported community deaths by age and sex disaggregation were collected to analyse changes in the sex and age distribution of deaths between COVID-19 wave periods and non-COVID-19 wave periods. A categorical variable, age group, was recoded from the continuous variable age. For the all-cause excess mortality analysis, the district-specific monthly counts of all deaths from 2017 to 2019 were used as the COVID-19 pre-pandemic baseline to compare with the period of the COVID-19 pandemic (2020–2022) using a Microsoft Excel-based tool developed by Resolve to Save Lives/Prevent Epidemics to calculate monthly medians and 95% confidence intervals for historic deaths . This tool is recommended by an expert panel for Rapid Mortality Surveillance during COVID-19 . We quantified the availability of death records by calculating the percentage of months with complete monthly count information over the total number of months within the study period (January 2017 to December 2022) for each district. To address missing monthly death counts at the district level, mean imputation was applied to the districts with missing data (Ndola, Solwezi, and Livingstone), as the monthly death counts at the district level were normally distributed. Excess mortality was defined as the difference between the all-cause pandemic monthly death counts (2020–2022) (Observed events) and the median pre-pandemic all-cause monthly death counts and the 95% confidence interval (2017–2019) in the 12 districts (The previous step yields baseline mortality and a “normal” range of variability around that baseline). The 95% confidence interval for the median historical monthly count was calculated separately for each month using the sample standard deviations. Excess mortality was present if the observed count was greater than the 95% confidence limit of the historic number of deaths. District-specific excess mortality was calculated separately among community deaths and health facility deaths and then summed to get monthly and yearly excess mortality counts for each district. To estimate overall excess mortality in Zambia, we assumed that there was sufficient heterogeneity in factors contributing to excess deaths in the 12 selected districts to be representative of the entire population of Zambia and that the excess mortality rate was only a function of the population. Based on these assumptions, we extrapolated the median excess mortality rate from the 12 districts to the entire country to estimate the total excess deaths in Zambia. We then compared this national estimate of excess deaths to the number of officially reported COVID-19 deaths in Zambia to calculate the ratio of reported COVID-19 deaths to excess deaths during the pandemic. The individual-level death information from 2020 to 2022 was used to calculate daily death counts among community deaths and health facility deaths. Decedents with missing age or sex variables were dropped from subsequent analyses involving age or sex variables but included in the time series graphs of daily deaths and daily death count analysis. Shapiro-Wilk test was used to test the normality assumption for the distribution of the daily death counts and age variables. All reported daily deaths were plotted as a 14-day rolling average time series disaggregated by place of death (community versus facility), district and age groups. To detect any difference in the median number of reported daily deaths between wave periods and non-wave period days, we used the Mann-Whitney u test. We then used the Kruskal-Wallis rank sum test to determine whether there was a statistically significant difference between the median daily number of deaths across the six identified wave periods (non-wave period, first wave, second wave, third wave, fourth wave, fifth wave). To identify which median daily death counts varied significantly by wave period, pairwise comparisons of median daily death count by wave period were conducted using the Wilcoxon rank sum test with continuity correction. The Benjamini & Hochberg p -value adjustment method was employed for multiple comparisons. Furthermore, we compared disparities in the distribution of the median age at death, the proportion of individuals above 65 at the time of death, as well as sex, place of death (facility or community), and district of death between wave periods and non-wave periods. These comparisons were conducted using the Wilcoxon rank sum test for median age and Pearson’s Chi-squared test for categorical variables. All statistical analyses were performed using R version 4.2.1 statistical software. Between 2017 and 2022, there were 217 140 deaths reported in the 12 districts (range 32786 [2018] to 41613 [2021]) (Fig. ). In the baseline period (2017–2019), October had the highest average death count (3071.3, 95% CI: 2984.8-3157.8) while May had one of the lowest (2710.0, 95% CI: 2528.1-2891.9) (Table ). Districts contributed from 2.4% (Mongu) to 41.0% (Lusaka) of the reported deaths. A total of 112,768 deaths were reported in the 12 districts between 2020 and 2022 of these 17,111 (15.2%) were excess (Fig. ). The median district excess mortality rate was 237.5 (Interquartile range (IQR) [170.5-282.5]) per 100,000 population (Table ). There was district-by-district variation in the number of excess deaths with the highest excess deaths reported in Chingola (449.5 per 100,000 population) and the lowest excess deaths reported in Mongu (101.1 per 100,000 population) (Fig. ; Table ). Most excess deaths were observed in 2021 ( n = 8992, 52.6%). By extrapolation from these 12 districts, we estimate the median excess deaths across all three years of the COVID-19 pandemic in Zambia to be 43,701 (IQR, 31372-51,982). Compared to officially reported COVID-19 deaths , for every reported COVID-19 death, there was a median of 11 (IQR, 8–13) excess deaths during the COVID-19 pandemic in Zambia. We analysed 112,768 individual death records across the 12 districts between January 2020 to December 2022. Overall, only 2.4% of all deaths had a variable missing (age or sex). Over the entire study period, on average, there were more deaths per day during wave periods than during non-wave periods (median [IQR]: 107 [95–126] versus, 96 [85–107]) respectively, < 0.001) (Table ). The second (median [IQR]: 124 [113–138]), third (median IQR: 140 [116–168]) and fourth (median [IQR]: 102 [92–109]) waves had more deaths per day than non-wave periods (median [IQR]: 96 [85–107], p < 0.001), but no difference was seen between the first and fifth waves (Fig. ; Table ). During wave periods, there was an increase in the daily number of deaths across all districts with a return to baseline after the wave periods (Fig. ). When disaggregated by place of death, more deaths during the third wave were reported as having occurred within the community than in health facilities (Fig. ). During non-wave periods approximately half of all reported deaths across all 12 districts occurred in the community (50.6%), and this proportion increased during wave periods (53.1%, p < 0.001) (Table ). There was district-by-district variation in the baseline number of deaths per day, with Lusaka accounting for the highest proportion of all deaths (33.49%) and Mongu the least (2.96%). During wave periods, there was an increase in the proportion of all deaths from Lusaka (45.39%, p < 0.001) (Table ). In both wave and non-wave periods, more males died (58.7%, non-wave period versus 58.6%), However, the difference in proportions of men who died between the two periods was not statistically significant ( p = 0.860). There was an increase in the median age at death during wave periods with a return to baseline median age at death after the wave period (41.0 years, IQR [22.0–63.0] versus 44.0 years, IQR [25.0–67.0], p < 0.001) (Table , supplementary Figs. and ). There was a corresponding increase in the proportion of individuals aged 65 and over who died during wave periods than those that died during non-wave periods (12886 [23.4%] non-wave period versus 11721 [27.7%] wave periods, p < 0.001) (Table , supplementary Fig. ). When the time series of daily deaths was plotted by age group, the most noticeable change from baseline rates was in the 60 and over age group, which showed an increased number of deaths in this age category across the first, second, third and fourth waves (Fig. ). There was excess mortality in all 12 districts included in the study between January 2020-December 2022, with the most excess deaths (52.6%) in 2021. These results suggest that the impact of the COVID-19 pandemic in Zambia was more widespread than official statistics indicate, and not only restricted to urban centres such as Lusaka . Similar findings of excess mortality in other sub-Saharan countries during the COVID-19 pandemic have been observed . Globally, the World Health Organisation estimates excess mortality is 2.74 times higher than reported COVID-19 deaths . Modelled estimates of excess mortality during the COVID-19 pandemic in Zambia vary widely (74.3 credible interval (CI) [2.5-147.6] vs. 228.2 [165.9-322.8] per 100,000 population ). Due to the unavailability of publicly available population-level mortality data, these current estimates of excess mortality in Zambia used statistical models to directly predict excess mortality for Zambia or used mathematical models to generate historical and current mortality data and then calculated the excess mortality rate . Our national estimate of the excess mortality rate (median 237.5 [(IQR)170.5-282.5] per 100,000 population), which lies within the range of modelled estimates for Zambia, is most likely an underestimate as not all deaths that occur within communities are reported at health facilities, and therefore were not included in this analysis. Our study demonstrates the value of applying mortality surveillance to understand the impact of a major public health event. If done in real-time, it could have also helped inform public health messaging and policymaking in response to the COVID-19 pandemic in Zambia. Further studies need to be conducted to understand the characteristics and explore surveillance strategies to detect and report these otherwise undocumented community deaths. The observed variation in excess mortality rates across districts may be attributed to district-specific differences in geospatial factors influencing COVID-19 transmission dynamics, variations in healthcare quality and its utilization by the community, and the proportion of deaths occurring within the community that are reported to health facilities. Consequently, there is a degree of underreporting of community deaths at health facilities, the extent of which is unknown but tends to be higher in rural areas compared to urban areas. Evidence suggests that rural-urban residence significantly impacts the location of deaths in Zambia . Additionally, differences in geographical factors between different regions have been shown to affect the spread of COVID-19 . Further studies are required to identify these factors and their impact on the spread of COVID-19 in Zambia. Most of the observed excess deaths were likely due to COVID-19 or due to the socioeconomic disruption due to the pandemic. We observed an increase in the daily number of deaths during COVID-19 wave periods compared to non-COVID-19 wave periods. To our knowledge, there were no other reported widespread public health emergencies during the study period that could explain the abrupt increase in the number of deaths during the specific wave periods and across all 12 districts almost simultaneously . Additionally, when we analysed the different wave periods, we noted that the relative increase in daily deaths during wave periods was associated with the relative case fatality of the predominant strain of SARS-CoV-2 associated with that wave. The predominant strain during Zambia’s third wave, which had the highest median daily death count, was the delta variant, a finding consistent with numerous other countries . Further, after an increase in the daily number of deaths during a COVID-19 wave period, we observed a return to baseline pre-pandemic mortality rates between waves and this was consistent across all 12 districts visited and across the different age groups. COVID-19 mortality has been shown to disproportionately affect the elderly . We observed an increase in the overall median age at death (44 years vs. 41 years, p < 0.001) and in the proportion of deceased persons aged 65 years and older (27.7 per cent vs. 23.4 per cent, p < 0.001) during COVID-19 wave periods with a return to baseline between wave periods respectively. However, the exact proportion of these excess deaths that were directly attributable to COVID-19 remains unknown because of limitations in antemortem and postmortem SARS-CoV-2 testing and limited death registration and certification across the country. There were more community deaths than facility deaths in both wave periods and non-wave periods. Community deaths increased during wave periods, suggesting potential gaps in health services brought on by the COVID-19 pandemic. As not all deaths that occur within the community are brought to health facilities before burial, the actual proportion of total deaths that occur within the community may even be higher. An analysis of places of death in Zambia among adults 15–59 years between 2010 and 2012 showed that slightly less than half of the adult deaths occurred in the home, factors associated with dying in a health facility included higher educational attainment, urban versus rural residence, and being of female gender . The observed increase in the proportion of deaths in the community during COVID-19 waves could be due to barriers to access to health facilities as some facilities were repurposed to serve as specialised COVID-19 treatment centres whilst other facilities scaled down services offered by only offering essential health services or attending to only emergencies . This could have compromised the quality of outpatient care that chronically ill patients received. Additionally, the myths and misconceptions around COVID-19 could have prevented those in most need of care from seeking health care . We recommend risk communication and engagement strategies tailored to increasing demand within the community for seeking health services during public health emergencies. Additionally, surge capacity plans should be developed by the Ministry of Health and implemented during public health emergencies. These could help ensure the continued provision of essential health services as well as provide additional capacity to respond to the public health emergency. Our study had several limitations. As we investigated excess deaths, we were unable to quantify the proportion of these excess deaths that were due to COVID-19, however, due to the timing and demographic composition of these deaths and the absence of other explanatory events, we believe that most of these deaths could have been due to COVID-19. Only community deaths that were reported at health facilities were analysed during this investigation, as such our findings underestimate the total number of deaths that occurred within these districts, as not all community deaths are reported at these facilities. While our study employed purposive sampling for site selection, which may have introduced selection bias and impacted the generalizability of our findings, the consistency observed across all selected districts—despite varying sociodemographic factors—suggests that our results are broadly reflective of the entire country. This consistent pattern across diverse districts supports the robustness of our findings and their applicability at a national level. Sampling additional districts was impossible because of resource limitations (data abstraction was time-consuming). Our method of determining excess mortality did not consider potential seasonal variations. However, due to the large size of the data set, the consistency of findings across the different districts, the consistency of our findings with current known epidemiological characteristics of COVID-19 and the consistency of our findings with other similar studies, we believe our findings are credible. There was excess mortality in all 12 districts visited during the COVID-19 pandemic in Zambia with most of these deaths occurring within the community and among the elderly. These findings suggest the impact of the COVID-19 pandemic in Zambia was far greater than implied by reported COVID-19 deaths alone. Strengthening routine and continuous mortality surveillance systems with cause of death ascertainment especially among community deaths could help guide public health decision-making and strengthen risk communication and community engagement during public health emergencies. Below is the link to the electronic supplementary material. Supplementary Figure 1: Population pyramid of deaths by wave period, 12 districts of Zambia, 2020–2022 Supplementary Figure 2: Trendline of median age at death and median daily count of deaths in 12 districts, Zambia 2020–2022 |
PEGDA-based HistoBrick for increasing throughput of cryosectioning and immunohistochemistry in organoid and small tissue studies | 53f1402b-bb5b-400a-8b5d-74556df6d768 | 11696907 | Anatomy[mh] | Histology provides insights into tissue structure and cellular morphology and is the gold standard analytical method for clinicians and researchers. Tissue sectioning along with immunohistochemical staining reveals changes in tissue morphology or physiology in response to different treatments and environments. Histological sections are most commonly prepared via paraffin infiltration or cryosectioning. In terms of antigen accessibility for immunohistochemical stainings, cryosections are considered to be superior to paraffin sections. Paraffin infiltration hides some of the antigens, necessitating long non-standardized antigen retrieval protocols for immunostaining. Cryosectioning generally preserves antigens well and enables their visualization via immunohistochemistry , . Microtissues (organoids, spheroids, tumoroids and related complex 3D cellular models) serve as invaluable in vitro models of tissues, offering insights into organ development, disease phenotypes and drug responses , . While some microtissues can be produced in large numbers, their high-throughput analysis remains a challenge – . Preparing histological sections to analyze microtissues or small tissues is a low-throughput, labor-intensive and operator-dependent process , . Typically, the operator manually embeds a few (less than ten) microtissues into a matrix to form a block (Fig. a). Because individual microtissues in the block cannot be easily traced during conventional embedding and subsequent analysis steps, often one or multiple blocks need to be prepared per experimental condition. The time-consuming histological process is especially a bottleneck in the field of drug discovery, where large numbers of compounds are often validated in multiple sample replicates . Strategies have been deployed to increase the throughput of microtissue histology. We and others have described planar arrays to spatially organize samples within blocks – or to centrifuge samples for arrangement on the same sectioning plane . For cryosectioning, tissue microarray-inspired approaches remain limited either in terms of alignment precision, intensive labor, or process compatibility with various microtissue culture approaches , . An ideal and versatile method to increase throughput of microtissues histological analysis should provide: 1. Spatially organizing microtissues in the sectioning plane to trace individual samples throughout the entire study, allowing the combination of different experimental conditions within one block; 2. Highly increasing the number of microtissues within one block; 3. Aligning microtissues in a narrow horizontal plane in the embedded block to reduce the number of sections required for the analysis; 4. Ease-of-use and standardized procedure involving readily accessible tools and materials with integration potential in automated workflows. We aimed to create a solution adapted to cryosectioning that addresses the previous advantages by improving our previously established HistoBrick tool, in which spheroid arrangement is spatially controlled in a paraffin embedded agarose block with arrayed mini-wells (Fig. b). Because the original agarose-based HistoBrick is not suited for cryosectioning, there is a need to develop a HistoBrick made of a cryosectioning-compatible material. The selection of a suitable embedding matrix for HistoBrick preparation is crucial to ensure the integrity of the frozen block (also called cryoblock) throughout the sectioning process. Fragile samples that lack rigidity (neuronal tissue), contain cavities (cochlea biopsy), or present complex surface topography (retinal organoids) can be structurally distorted during cryosectioning, rendering analysis of their immunostaining less informative – . Optimal Cutting Temperature compound (OCT) is a widely used matrix for embedding samples for cryosectioning. However, OCT melts at room temperature, leaving the histological sections which may disrupt fragile sample structures . Gelatine is preferred by some scientists to embed soft tissues such as brain and cochlea. One advantage of gelatine is the mechanically stable sample-matrix interfaces , , . However, gelatine presents the disadvantage of needing to be kept above 37 °C to stay liquid. At room temperature, the viscosity of gelatine increases, making pipetting difficult. Other hydrogels, such as PEGDA, have a stable viscosity at room temperature and their crosslinking can be precisely controlled using UV light. However, PEG-derived hydrogels alone are not suitable for cryosectioning . Here, we describe a new hydrogel mixture composed of PEGDA, gelatine and sucrose suited for HistoBrick preparation and cryosectioning. The new material composition is tested against different parameters including ease of preparation, integrity of cryosections, vertical alignment of microtissues and preservation of their structure. The impact of the embedding matrix on the structural integrity of the sample is studied using retinal organoids. Retinal organoids, like the adult human retina, are organized in layers containing different cell types , . Along the outer organoid surface, they present particularly fragile, hair-like structures made of light-sensitive photoreceptor outer segments. Cells of the human retina, in particular photoreceptors and their outer segments, are affected by several diseases leading to blindness , . Reliable visualization of retinal layers, photoreceptors and their outer segment structures is thus essential. We confirm that the new embedding material for cryosectioning preserves layering of the organoids, photoreceptor appearance and the fragile outer segments throughout the histological process. Taking advantage of PEGDA-gelatine HistoBricks, we analyze a two-year time-course of retinal organoid development and describe features of human retina degeneration (such as the loss of photoreceptor outer segments) and features of human retina aging (such as displaced photoreceptors in the region of outer segments). Embedding up to 16 retinal organoids from different experimental conditions into one HistoBrick increases the throughput of microtissue histological analysis for cryosectioning, while decreasing cost of the analysis by saving reagents and time. Optimizing hydrogel formulation for HistoBrick preparation and cryosectioning We aimed to identify a hydrogel to adapt the HistoBrick method to cryosectioning. The HistoBrick was prepared by first filling liquid embedding material into a silicone mold, and subsequently crosslinking to obtain a gel well plate. The gel well plate contained an array of mini-wells in which microtissues were transferred. After sedimentation of the microtissues to the well bottom, the wells were filled by liquid embedding material and the resulting HistoBrick was snap frozen and cut into thin Sections (10–30 µm) on a cryostat (Fig. ). In this manuscript, the word HistoBrick refers to both the embedding method and the resulting block containing the gel well plate, the microtissues and the embedding matrix. The gel well plate is the empty arrayed hydrogel and the embedding matrix is the hydrogel in direct contact with the microtissues in the wells. The gel well plate and the embedding matrix are always made of the same material. After snap freezing, the frozen HistoBrick is called cryoblock. We first tested the fabrication and cryosectioning of the HistoBrick with either gelatine or PEGDA, not containing microtissues. OCT was not considered for HistoBrick fabrication because an OCT HistoBrick would need to be loaded while frozen which would not allow sedimentation of small samples that would freeze along the cold wells at random heights. We successfully molded gel well plates with the two materials. While crosslinking of the gel well plate prepared with gelatine required one hour at 4 °C, crosslinking with UV for the PEGDA solution only took a few minutes. The unmolding of the gelatine gel well plate was more challenging than the one prepared of PEGDA, as solidified gelatine tended to stick to the silicone mold. We subsequently investigated the integrity of the sections, and the interface between the gel well plate and the embedding matrix. Sectioning the PEGDA HistoBrick (8 v% and 10 v% PEGDA) resulted in frequent wrinkling and breakage of the sections, highlighting poor mechanical stability. Additionally, filling the PEGDA gel well plate with PEGDA solution resulted in a non-adherent interface, which led to the disassociation of the embedded matrix from the gel well plate (Fig. a left). Consequently, some embedding matrix regions were lost or folded during sectioning. During an analysis experiment containing organoids this would result in the loss of microtissues located inside. The sections of the gelatine HistoBricks were stable without major cracks or folds, and with good cohesion at the interface of the well plate and embedding matrix (Fig. a middle). Due to its poor sectionability, the PEGDA hydrogel was not used for further testing with organoids. To promote a coherent interface between organoids and the embedding matrix, organoids were incubated in the embedding solutions for 15 min prior to the transfer into the gel well plate. The pipetting of organoids, incubated in gelatine solution, into the wells trapped air bubbles at the bottom. The presence of air bubbles at the bottom of the wells prevented the organoids from sedimenting and aligning on a unique plane while reducing the integrity of the sections during cutting. An additional barrier to a planar arrangement of organoids in gelatine HistoBricks was their tendency to attach to the sides of the wells during transfer. Consequently, we had to gently push them down to the bottom of the wells using a pipette tip. To optimize organoid planar embedding and HistoBrick sectioning, we aimed to build on the advantages of both tested materials. We developed a new embedding matrix by mixing 8 v% PEGDA with 2.5 wt% of gelatine (thereafter called PEGDA-gelatine hydrogel). The PEGDA-gelatine HistoBrick combined the fast and easy unmolding of the PEGDA HistoBrick and the structural stability and coherence of embedding matrix-gel well plate interface during cryosectioning of the gelatine HistoBrick (Fig. a right). We did not observe air bubble trapping when pipetting organoids incubated in PEGDA-gelatine solution and organoids efficiently sedimented to the bottom of the wells. We then investigated the interface between the organoids and the embedding matrix in more detail with a hematoxilin and eosin staining, as a coherent interface is important to support the tissue structure. Eosin stained the proteins contained in gelatine and thus enabled embedding matrix visualization. Both gelatine and PEGDA-gelatine matrix presented a stable interface with the organoids, meaning that the embedding matrices did not detach from the organoid surface throughout the whole histological process (Fig. b). The gelatine sections have a structure less homogeneous than the PEGDA-gelatine sections but this was not found to impact the interface with the organoids. Conventional embedding in OCT was also performed, as it is known to present a weak interface . Indeed, during the staining procedure the OCT was washed away, not maintaining an interface with the tissue (Fig. b). Hydrogel rheological analysis We next aimed to understand how viscosity of the hydrogel solutions affected their compatibility with organoid transfer procedures. Using rheology, we measured changes in viscosity of the PEGDA, gelatine, and PEGDA-gelatine solutions over time at temperatures representative of the HistoBrick loading step. To recreate similar conditions, the measurements were started with a temperature ramp going from 37 to 20 °C (5 °C/min), followed by an isotherm at 20 °C (Fig. c). As the solutions underwent temperature-driven crosslinking during the tests, we measured the complex viscosity that considered the elastic and viscous component of crosslinked hydrogels. The complex viscosity of the PEGDA solution stayed stable throughout the whole experiment. The isotherm at 20 °C was shortened as the PEGDA solution showed a stable behavior. The viscosity of the gelatine solution started to increase during the temperature ramp at 22 °C and stabilized at the end of the ramp when the temperature reached 20 °C. The increase of viscosity was caused by the crosslinking of the gelatine polymers inside the solution upon cooling. On the other hand, the viscosity of the PEGDA-gelatine solution started to increase at 20 °C and stabilized 6 min later. The PEGDA polymers do not influence the viscosity as in these experiments no UV crosslinking was performed. Therefore, the difference between the two solutions is interpreted to come from the difference in gelatine concentration. These results support experimental observations that PEGDA-gelatine solution was easier to handle when pipetting into the gel well plate due to its slower increase of viscosity during the process. PEGDA-gelatine preserves fragile organoid substructures To investigate if the embedding process of the HistoBrick has an impact on the structure of fragile organoid substructures, we used human retinal organoids. Like the human retina, retinal organoids are structured into layers with each layer containing specific cell types. Photoreceptor cell bodies are arranged in a layer called outer nuclear layer along the outside of retinal organoids. The inner nuclear layer and ganglion cell layers of retinal organoids, like the human adult retina, contain the other major cell types of the neural retina as described in Cowan et al . (Fig. a). Along the outer surface, retinal organoids display fragile outer segments. Outer segments are the light-sensitive “antennae” of photoreceptor cells. We embedded PFA-fixed, outer-segment-containing retinal organoids from the same organoid differentiation and compared conventionally embedded to HistoBrick embedded samples using both gelatine and PEGDA-gelatine as embedding materials. We evaluated both, overall organoid integrity, and the maintenance of outer segments on cryosections (Fig. , Supplementary Fig. ). As a negative control, we embedded retinal organoids in OCT, as we and others have described that this treatment does not preserve outer segments , . Due to the liquid state of OCT at room temperature and its very high viscosity, we used it only for conventional embedding. To analyze retinal organoid sections obtained using the different embedding materials we performed immunostainings (Fig. b–f). For visualization of cone photoreceptors, we stained them with an antibody against arrestin 3. Rod photoreceptor morphology was visualized using an antibody against rhodopsin labelling rod cell bodies in the outer nuclear layer and rod outer segments as thin appendices and dots surrounding the organoid. For both conventional and HistoBrick embedding using gelatine and PEGDA-gelatine, and for OCT embedding, the outer nuclear layer containing cone (ARR3 positive) and rod (RHO positive) photoreceptor cell bodies was well preserved (Fig. b–f). We stained for various retinal cell types, like cones expressing M- and L-opsin, horizontal cells (ONECUT2 positive), ON bipolar cells (TRPM1 positive), Müller cells (RLBP1 positive), and ribbon synapses (Basson) and observed that organoid sections were well maintained and the cell types, as well as ribbon synapses, could be detected across all conditions (Supplementary Fig. a–e). To observe cone outer segments, we stained organoid cryosections with the lectin Peanut agglutinin (PNA). PNA in retinal organoids labels both inner segments and outer segments of cones (Supplementary Fig. f). Cone inner segments appear bud-like adjacent to the outer nuclear layer, and the outer segments extend significantly farther than the inner segments (Supplementary Fig. f). In both gelatine and PEGDA-gelatine cryoblock preparations (conventional and HistoBrick embedding), RHO and PNA staining confirm the presence of cone and rod outer segments along the outside of retinal organoids (Fig. b–f). Upon OCT embedding, the RHO and PNA positive outersegments were completely lost, and only PNA-positive and RHO positive inner segment buds remained (Fig. d). Our stainings revealed that gelatine in some cases did not preserve outer segments well, leading to stretched outer segments, holes in the outer segment area that overall appeared rather porous, or outer segments ripped off the organoid (Supplementary Fig. g–i). While we observed those artefacts for both conventional and HistoBrick embedding using gelatine, it happened more frequently using the HistoBrick. Interestingly, we observed less damage to outer segments in the form of stretching or holes using PEGDA-gelatine than using gelatine embedding, irrespective of conventional or HistoBrick embedding. To quantify the preservation of outer segments, we estimated the average thickness ϑ of the PNA-positive outer and inner segments (“OS + IS thickness”) under different embedding conditions. We first defined a region-of-interest (ROI) of the retinal organoid by the PNA signal (PNA ROI), which contained outer segments, inner segments and the inner part of the retinal organoid. We then defined a ROI of the retinal organoid by the Hoechst signal (Hoechst ROI), which contained nuclei and the inner part of the retinal organoid but excluded outer and inner segments. The area of the outer and inner segments (OS + IS area) was estimated by subtracting the area of the Hoechst ROI (Hoechst area) from the area of the PNA ROI (PNA area; Fig. g and Supplementary Fig. b–e). The OS + IS thickness ϑ was estimated from the PNA area and the Hoechst area by a circular approximation (Methods) and was independent of the Hoechst area (Supplementary Figs. f, g and i, j), which varied due to variance in organoid size (Supplementary Fig. h,i) or cross-section area. A significant reduction in ϑ was observed comparing samples conventionally embedded in OCT to the gelatine control. There were no significant differences in ϑ for organoids treated with conventional gelatine embedding compared to gelatine HistoBrick, conventional PEGDA-gelatine, or PEGDA-gelatine HistoBrick embedding, respectively. Overall, these results show that PEGDA-gelatine is suitable for HistoBrick preparation and well preserves organoid layering and fragile photoreceptor outer segments. The HistoBrick improves organoid traceability and analysis throughput We optimized the HistoBrick design to contain up to 16 retinal organoids while keeping external dimensions compatible with cryosectioning (Fig. a). Organoid diameters are on average 1.17 mm (+ /− 0.25 mm SD; n = 16,306 organoid images quantified from but can measure up to 2.66 mm (Supplementary Fig. i). We designed the HistoBrick wells to be 2.4 mm in diameter. Up to three sections of the HistoBrick fit on one glass slide. Here, we show an example with two sections enabling the simultaneous analysis of up to 32 organoids (Fig. b). Sometimes, the interface between the embedding matrix and the gel well plate is not continuous. Residual PBS, from the pre-wetting of the wells, is hypothesized to not be fully mixed with the embedding matrix, thereby disrupting the interface. Sedimentation of organoids at the bottom of the wells results in serial cryosections with maximized information content per section. More than 80% of organoids were present in the cryoblock over a thickness of 390 ± 212 μm, which corresponds to 19 ± 10 consecutive sections of 20 μm. This large standard deviation is hypothesized to come from the heterogeneity of organoids size (Supplementary Fig. h,i) and morphology. In addition, the HistoBrick is often cut at a slightly tilted angle on the cryotome. A tilting angle between the HistoBrick and the blade was indicated by the fact that some wells only appeared in later sections (Supplementary Fig. a,b). Due to this misalignment, aligned organoids in the blocks would appear on different sections. The tilting angle inherent to manual sectioning was analyzed in more detail in our previous work and was hypothesized to reduce the number of visible organoids on the Sections . The individualization of organoids in an array facilitates their traceability throughout the analysis process from embedding into the HistoBrick to the sectioned samples on slides. When serial sections are prepared on consecutive glass slides, each organoids position can be tracked within the array. The different slides can be stained with various sets of dyes and antibodies, thus providing multiple datasets for a single organoid. First, the high information content obtained by immunohistochemistry on consecutive sections facilitates the correlation between the organoid structure and immunolocalization (Fig. b, c). Second, consecutive organoid sections can provide some insight into organoid 3D structure (Fig. b, d; Supplementary Fig. b). Finally, increasing the number of organoids embedded within one block makes their analysis cheaper. While in conventional blocks our lab previously embedded on average 4 organoids, the HistoBrick fits 16 organoids. Thus, depending on the scale of a performed experiment, using the HistoBrick can save 91% of sectioning time and 88% of reagents necessary for antibody stainings in an experiment in which 2 organoid replicates per treatment are analyzed (Supplementary Fig. d). Structural changes of human retinal organoids over time Retinal organoids require over six months of development to reach a stage where they exhibit transcriptomic and morphological characteristics closely resembling those of the adult human retina . We have previously shown that at week 46, the transcriptome of rod photoreceptors changes and some rod marker genes are down-regulated . It was not known whether from that age onward, organoids would lose photoreceptors, and until when photoreceptors can be maintained in retinal organoids. We cultured retinal organoids for 98 weeks, fixed organoids at week 30, 38, 46, 52, 70, 81 and 98 (n = 3–8 retinal organoids per time-point), and embedded them in PEGDA-gelatine HistoBricks. Processing the organoids using HistoBrick embedding greatly reduced the number of blocks that needed to be sectioned and stained (3 HistoBricks instead of 11 conventional blocks). We then assessed several quality criteria that are potentially compromised in aging organoids: 1. the presence of layered retina structures, 2. the presence of rod- and cone photoreceptors and 3. the presence of photoreceptor outer segments. We observed organoids that maintained both their outer nuclear layer and inner nuclear layer up to week 98 (Fig. a). Staining for the rod marker rhodopsin (RHO) and cone marker arrestin 3 (ARR3) revealed the presence and maintenance of both photoreceptor types up to week 98 in culture. However, while organoids are surrounded by dense outer segments between weeks 30 and 52, we observed a gradual loss of photoreceptor outer segments past week 52 (Fig. b). To quantify whether outer segments decrease over time, we estimated the OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} . For each individual organoid, we manually selected the section with the most abundant outer segments for quantification. Our measurements revealed that OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} negatively correlates with time (Fig. c, Spearman rank correlation r = − 0.38, P = 0.01). Up to week 52, OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} was significantly larger than the negative control (Fig. d). At week 98, we measured a significant decrease of OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} as compared to the positive control (Fig. d). Although retina structures and photoreceptors could still be detected, some degeneration events very likely occurred during the long culture period. While analyzing the stained images, in older organoids we unexpectedly observed displaced nuclei outside the outer nuclear layer, in the area where outer segments are located (Fig. b, Supplementary Fig. ). To determine the cell type identity of these displaced cells, we performed further stainings. The most common cell types for retinal organoids were examined using the markers SOX9 for Müller cell nuclei, MiTF for retinal pigment epithelium cells, and MAP2 for neuronal cells. The displaced cells were negative for Sox9 and MiTF (Supplementary Fig. a). The nuclei of the displaced cells in the Hoechst staining overlapped with the stained cell bodies in the MAP2 stained images (Supplementary Fig. b–h, left), confirming a neuronal cell identity. Further evaluation of the ARR3 stained images indicated that these cells were cones, (Supplementary Fig. b–h, right). Gartner et al . and Lai et al . described displaced nuclei in the region of outer segments in human retinas upon aging , . We found examples of displaced cells in both young and old organoids. However, while the displaced cells appeared individually in organoids at week 30 and 38, we saw examples of displaced nuclei arranged chain-like near each other in older organoids (Supplementary Fig. b–h). We aimed to identify a hydrogel to adapt the HistoBrick method to cryosectioning. The HistoBrick was prepared by first filling liquid embedding material into a silicone mold, and subsequently crosslinking to obtain a gel well plate. The gel well plate contained an array of mini-wells in which microtissues were transferred. After sedimentation of the microtissues to the well bottom, the wells were filled by liquid embedding material and the resulting HistoBrick was snap frozen and cut into thin Sections (10–30 µm) on a cryostat (Fig. ). In this manuscript, the word HistoBrick refers to both the embedding method and the resulting block containing the gel well plate, the microtissues and the embedding matrix. The gel well plate is the empty arrayed hydrogel and the embedding matrix is the hydrogel in direct contact with the microtissues in the wells. The gel well plate and the embedding matrix are always made of the same material. After snap freezing, the frozen HistoBrick is called cryoblock. We first tested the fabrication and cryosectioning of the HistoBrick with either gelatine or PEGDA, not containing microtissues. OCT was not considered for HistoBrick fabrication because an OCT HistoBrick would need to be loaded while frozen which would not allow sedimentation of small samples that would freeze along the cold wells at random heights. We successfully molded gel well plates with the two materials. While crosslinking of the gel well plate prepared with gelatine required one hour at 4 °C, crosslinking with UV for the PEGDA solution only took a few minutes. The unmolding of the gelatine gel well plate was more challenging than the one prepared of PEGDA, as solidified gelatine tended to stick to the silicone mold. We subsequently investigated the integrity of the sections, and the interface between the gel well plate and the embedding matrix. Sectioning the PEGDA HistoBrick (8 v% and 10 v% PEGDA) resulted in frequent wrinkling and breakage of the sections, highlighting poor mechanical stability. Additionally, filling the PEGDA gel well plate with PEGDA solution resulted in a non-adherent interface, which led to the disassociation of the embedded matrix from the gel well plate (Fig. a left). Consequently, some embedding matrix regions were lost or folded during sectioning. During an analysis experiment containing organoids this would result in the loss of microtissues located inside. The sections of the gelatine HistoBricks were stable without major cracks or folds, and with good cohesion at the interface of the well plate and embedding matrix (Fig. a middle). Due to its poor sectionability, the PEGDA hydrogel was not used for further testing with organoids. To promote a coherent interface between organoids and the embedding matrix, organoids were incubated in the embedding solutions for 15 min prior to the transfer into the gel well plate. The pipetting of organoids, incubated in gelatine solution, into the wells trapped air bubbles at the bottom. The presence of air bubbles at the bottom of the wells prevented the organoids from sedimenting and aligning on a unique plane while reducing the integrity of the sections during cutting. An additional barrier to a planar arrangement of organoids in gelatine HistoBricks was their tendency to attach to the sides of the wells during transfer. Consequently, we had to gently push them down to the bottom of the wells using a pipette tip. To optimize organoid planar embedding and HistoBrick sectioning, we aimed to build on the advantages of both tested materials. We developed a new embedding matrix by mixing 8 v% PEGDA with 2.5 wt% of gelatine (thereafter called PEGDA-gelatine hydrogel). The PEGDA-gelatine HistoBrick combined the fast and easy unmolding of the PEGDA HistoBrick and the structural stability and coherence of embedding matrix-gel well plate interface during cryosectioning of the gelatine HistoBrick (Fig. a right). We did not observe air bubble trapping when pipetting organoids incubated in PEGDA-gelatine solution and organoids efficiently sedimented to the bottom of the wells. We then investigated the interface between the organoids and the embedding matrix in more detail with a hematoxilin and eosin staining, as a coherent interface is important to support the tissue structure. Eosin stained the proteins contained in gelatine and thus enabled embedding matrix visualization. Both gelatine and PEGDA-gelatine matrix presented a stable interface with the organoids, meaning that the embedding matrices did not detach from the organoid surface throughout the whole histological process (Fig. b). The gelatine sections have a structure less homogeneous than the PEGDA-gelatine sections but this was not found to impact the interface with the organoids. Conventional embedding in OCT was also performed, as it is known to present a weak interface . Indeed, during the staining procedure the OCT was washed away, not maintaining an interface with the tissue (Fig. b). We next aimed to understand how viscosity of the hydrogel solutions affected their compatibility with organoid transfer procedures. Using rheology, we measured changes in viscosity of the PEGDA, gelatine, and PEGDA-gelatine solutions over time at temperatures representative of the HistoBrick loading step. To recreate similar conditions, the measurements were started with a temperature ramp going from 37 to 20 °C (5 °C/min), followed by an isotherm at 20 °C (Fig. c). As the solutions underwent temperature-driven crosslinking during the tests, we measured the complex viscosity that considered the elastic and viscous component of crosslinked hydrogels. The complex viscosity of the PEGDA solution stayed stable throughout the whole experiment. The isotherm at 20 °C was shortened as the PEGDA solution showed a stable behavior. The viscosity of the gelatine solution started to increase during the temperature ramp at 22 °C and stabilized at the end of the ramp when the temperature reached 20 °C. The increase of viscosity was caused by the crosslinking of the gelatine polymers inside the solution upon cooling. On the other hand, the viscosity of the PEGDA-gelatine solution started to increase at 20 °C and stabilized 6 min later. The PEGDA polymers do not influence the viscosity as in these experiments no UV crosslinking was performed. Therefore, the difference between the two solutions is interpreted to come from the difference in gelatine concentration. These results support experimental observations that PEGDA-gelatine solution was easier to handle when pipetting into the gel well plate due to its slower increase of viscosity during the process. To investigate if the embedding process of the HistoBrick has an impact on the structure of fragile organoid substructures, we used human retinal organoids. Like the human retina, retinal organoids are structured into layers with each layer containing specific cell types. Photoreceptor cell bodies are arranged in a layer called outer nuclear layer along the outside of retinal organoids. The inner nuclear layer and ganglion cell layers of retinal organoids, like the human adult retina, contain the other major cell types of the neural retina as described in Cowan et al . (Fig. a). Along the outer surface, retinal organoids display fragile outer segments. Outer segments are the light-sensitive “antennae” of photoreceptor cells. We embedded PFA-fixed, outer-segment-containing retinal organoids from the same organoid differentiation and compared conventionally embedded to HistoBrick embedded samples using both gelatine and PEGDA-gelatine as embedding materials. We evaluated both, overall organoid integrity, and the maintenance of outer segments on cryosections (Fig. , Supplementary Fig. ). As a negative control, we embedded retinal organoids in OCT, as we and others have described that this treatment does not preserve outer segments , . Due to the liquid state of OCT at room temperature and its very high viscosity, we used it only for conventional embedding. To analyze retinal organoid sections obtained using the different embedding materials we performed immunostainings (Fig. b–f). For visualization of cone photoreceptors, we stained them with an antibody against arrestin 3. Rod photoreceptor morphology was visualized using an antibody against rhodopsin labelling rod cell bodies in the outer nuclear layer and rod outer segments as thin appendices and dots surrounding the organoid. For both conventional and HistoBrick embedding using gelatine and PEGDA-gelatine, and for OCT embedding, the outer nuclear layer containing cone (ARR3 positive) and rod (RHO positive) photoreceptor cell bodies was well preserved (Fig. b–f). We stained for various retinal cell types, like cones expressing M- and L-opsin, horizontal cells (ONECUT2 positive), ON bipolar cells (TRPM1 positive), Müller cells (RLBP1 positive), and ribbon synapses (Basson) and observed that organoid sections were well maintained and the cell types, as well as ribbon synapses, could be detected across all conditions (Supplementary Fig. a–e). To observe cone outer segments, we stained organoid cryosections with the lectin Peanut agglutinin (PNA). PNA in retinal organoids labels both inner segments and outer segments of cones (Supplementary Fig. f). Cone inner segments appear bud-like adjacent to the outer nuclear layer, and the outer segments extend significantly farther than the inner segments (Supplementary Fig. f). In both gelatine and PEGDA-gelatine cryoblock preparations (conventional and HistoBrick embedding), RHO and PNA staining confirm the presence of cone and rod outer segments along the outside of retinal organoids (Fig. b–f). Upon OCT embedding, the RHO and PNA positive outersegments were completely lost, and only PNA-positive and RHO positive inner segment buds remained (Fig. d). Our stainings revealed that gelatine in some cases did not preserve outer segments well, leading to stretched outer segments, holes in the outer segment area that overall appeared rather porous, or outer segments ripped off the organoid (Supplementary Fig. g–i). While we observed those artefacts for both conventional and HistoBrick embedding using gelatine, it happened more frequently using the HistoBrick. Interestingly, we observed less damage to outer segments in the form of stretching or holes using PEGDA-gelatine than using gelatine embedding, irrespective of conventional or HistoBrick embedding. To quantify the preservation of outer segments, we estimated the average thickness ϑ of the PNA-positive outer and inner segments (“OS + IS thickness”) under different embedding conditions. We first defined a region-of-interest (ROI) of the retinal organoid by the PNA signal (PNA ROI), which contained outer segments, inner segments and the inner part of the retinal organoid. We then defined a ROI of the retinal organoid by the Hoechst signal (Hoechst ROI), which contained nuclei and the inner part of the retinal organoid but excluded outer and inner segments. The area of the outer and inner segments (OS + IS area) was estimated by subtracting the area of the Hoechst ROI (Hoechst area) from the area of the PNA ROI (PNA area; Fig. g and Supplementary Fig. b–e). The OS + IS thickness ϑ was estimated from the PNA area and the Hoechst area by a circular approximation (Methods) and was independent of the Hoechst area (Supplementary Figs. f, g and i, j), which varied due to variance in organoid size (Supplementary Fig. h,i) or cross-section area. A significant reduction in ϑ was observed comparing samples conventionally embedded in OCT to the gelatine control. There were no significant differences in ϑ for organoids treated with conventional gelatine embedding compared to gelatine HistoBrick, conventional PEGDA-gelatine, or PEGDA-gelatine HistoBrick embedding, respectively. Overall, these results show that PEGDA-gelatine is suitable for HistoBrick preparation and well preserves organoid layering and fragile photoreceptor outer segments. We optimized the HistoBrick design to contain up to 16 retinal organoids while keeping external dimensions compatible with cryosectioning (Fig. a). Organoid diameters are on average 1.17 mm (+ /− 0.25 mm SD; n = 16,306 organoid images quantified from but can measure up to 2.66 mm (Supplementary Fig. i). We designed the HistoBrick wells to be 2.4 mm in diameter. Up to three sections of the HistoBrick fit on one glass slide. Here, we show an example with two sections enabling the simultaneous analysis of up to 32 organoids (Fig. b). Sometimes, the interface between the embedding matrix and the gel well plate is not continuous. Residual PBS, from the pre-wetting of the wells, is hypothesized to not be fully mixed with the embedding matrix, thereby disrupting the interface. Sedimentation of organoids at the bottom of the wells results in serial cryosections with maximized information content per section. More than 80% of organoids were present in the cryoblock over a thickness of 390 ± 212 μm, which corresponds to 19 ± 10 consecutive sections of 20 μm. This large standard deviation is hypothesized to come from the heterogeneity of organoids size (Supplementary Fig. h,i) and morphology. In addition, the HistoBrick is often cut at a slightly tilted angle on the cryotome. A tilting angle between the HistoBrick and the blade was indicated by the fact that some wells only appeared in later sections (Supplementary Fig. a,b). Due to this misalignment, aligned organoids in the blocks would appear on different sections. The tilting angle inherent to manual sectioning was analyzed in more detail in our previous work and was hypothesized to reduce the number of visible organoids on the Sections . The individualization of organoids in an array facilitates their traceability throughout the analysis process from embedding into the HistoBrick to the sectioned samples on slides. When serial sections are prepared on consecutive glass slides, each organoids position can be tracked within the array. The different slides can be stained with various sets of dyes and antibodies, thus providing multiple datasets for a single organoid. First, the high information content obtained by immunohistochemistry on consecutive sections facilitates the correlation between the organoid structure and immunolocalization (Fig. b, c). Second, consecutive organoid sections can provide some insight into organoid 3D structure (Fig. b, d; Supplementary Fig. b). Finally, increasing the number of organoids embedded within one block makes their analysis cheaper. While in conventional blocks our lab previously embedded on average 4 organoids, the HistoBrick fits 16 organoids. Thus, depending on the scale of a performed experiment, using the HistoBrick can save 91% of sectioning time and 88% of reagents necessary for antibody stainings in an experiment in which 2 organoid replicates per treatment are analyzed (Supplementary Fig. d). Retinal organoids require over six months of development to reach a stage where they exhibit transcriptomic and morphological characteristics closely resembling those of the adult human retina . We have previously shown that at week 46, the transcriptome of rod photoreceptors changes and some rod marker genes are down-regulated . It was not known whether from that age onward, organoids would lose photoreceptors, and until when photoreceptors can be maintained in retinal organoids. We cultured retinal organoids for 98 weeks, fixed organoids at week 30, 38, 46, 52, 70, 81 and 98 (n = 3–8 retinal organoids per time-point), and embedded them in PEGDA-gelatine HistoBricks. Processing the organoids using HistoBrick embedding greatly reduced the number of blocks that needed to be sectioned and stained (3 HistoBricks instead of 11 conventional blocks). We then assessed several quality criteria that are potentially compromised in aging organoids: 1. the presence of layered retina structures, 2. the presence of rod- and cone photoreceptors and 3. the presence of photoreceptor outer segments. We observed organoids that maintained both their outer nuclear layer and inner nuclear layer up to week 98 (Fig. a). Staining for the rod marker rhodopsin (RHO) and cone marker arrestin 3 (ARR3) revealed the presence and maintenance of both photoreceptor types up to week 98 in culture. However, while organoids are surrounded by dense outer segments between weeks 30 and 52, we observed a gradual loss of photoreceptor outer segments past week 52 (Fig. b). To quantify whether outer segments decrease over time, we estimated the OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} . For each individual organoid, we manually selected the section with the most abundant outer segments for quantification. Our measurements revealed that OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} negatively correlates with time (Fig. c, Spearman rank correlation r = − 0.38, P = 0.01). Up to week 52, OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} was significantly larger than the negative control (Fig. d). At week 98, we measured a significant decrease of OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} as compared to the positive control (Fig. d). Although retina structures and photoreceptors could still be detected, some degeneration events very likely occurred during the long culture period. While analyzing the stained images, in older organoids we unexpectedly observed displaced nuclei outside the outer nuclear layer, in the area where outer segments are located (Fig. b, Supplementary Fig. ). To determine the cell type identity of these displaced cells, we performed further stainings. The most common cell types for retinal organoids were examined using the markers SOX9 for Müller cell nuclei, MiTF for retinal pigment epithelium cells, and MAP2 for neuronal cells. The displaced cells were negative for Sox9 and MiTF (Supplementary Fig. a). The nuclei of the displaced cells in the Hoechst staining overlapped with the stained cell bodies in the MAP2 stained images (Supplementary Fig. b–h, left), confirming a neuronal cell identity. Further evaluation of the ARR3 stained images indicated that these cells were cones, (Supplementary Fig. b–h, right). Gartner et al . and Lai et al . described displaced nuclei in the region of outer segments in human retinas upon aging , . We found examples of displaced cells in both young and old organoids. However, while the displaced cells appeared individually in organoids at week 30 and 38, we saw examples of displaced nuclei arranged chain-like near each other in older organoids (Supplementary Fig. b–h). In this work, we present a novel embedding matrix PEGDA-gelatine hydrogel to prepare an adapted HistoBrick for organoid cryosectioning. Identifying a novel embedding mixture was key to adapt the HistoBrick for cryosectioning of complex 3D microtissues. The embedding material should meet the following criteria. First, it should provide structural stability of the sections during cryosectioning. Importantly, a coherent interface between the gel well plate and the embedding matrix prevents the loss of valuable samples. Second, the embedding matrix should support and preserve the sample’s structural integrity throughout the whole process. A stable interface between the embedding material and the sample is crucial. Third, handling of the HistoBrick should be easy, including gel well plate unmolding from the silicone mold. Finally, the embedding material solution should allow easy transfer of organoids onto the bottom of the gel well plate. Ferguson et al. presented an embryoid body array providing traceability for six experimental conditions using OCT as an embedding matrix . The major limitation of this technique resides in the use of OCT which is a viscous material difficult to pipette, solidifying only below – 10 °C and not maintaining fragile structures such as outer segments of retinal organoids . Gelatine, which provides good stability to soft samples , was used to create an array of six half rodent brains (cm-scale ). The study highlights gelatine as a promising candidate for organoid or tissue arrays in the mm-scale like the HistoBrick. However, gelatine presents the disadvantage of needing to be kept above 37 °C to stay liquid. At room temperature, the viscosity of gelatine increases, making pipetting difficult. Consequently, UV-crosslinkable hydrogels with stable viscosity at room temperature are an interesting embedding matrix for the HistoBrick, facilitating gel pipetting and promoting organoid sedimentation inside the HistoBrick wells. PEGDA is a promising UV-curable candidate because it is widely commercially available, well studied in literature and exhibits biocompatibility properties . A previous study investigated the cryosectioning of a tissue-engineered construct composed of polyethylene glycol hydrogel (PEG) mixed with human mesenchymal stem cells. The untreated PEG-derived hydrogels were incompatible with the cryosectioning process . This behavior is due to the water-rich and polymeric nature of these compounds, which makes them very sensitive to routine tissue histology procedures. The highly ordered tetrahedral structure of water molecules in large ice crystals results in sample brittleness . The authors of the study demonstrated that overnight incubation in OCT or 1% Polyvinyl alcohol (PVA) of the tissue-engineered PEG-derived constructs increases the flexibility of the frozen sample facilitating sectioning . In another study, spheroids cultured in alginate and after fixation embedded in OCT were successfully cryosectioned . Alginate was not considered for the HistoBrick fabrication because its crosslinking relies on the diffusion of ions which is difficult to implement for large volumes. Based on these considerations, we compared gelatine and PEGDA as materials for the HistoBrick. As expected, PEGDA HistoBrick cryoblocks were very brittle and shattered during sectioning. On the other hand, gelatine acts as a plasticizer promoting material flexibility and reducing the sections’ brittleness. A small amount of gelatine (2.5 wt%) added to the PEGDA hydrogel enables the cutting of entire crack-free and fold-free sections while maintaining the advantage of easy handling low-viscous solution at room temperature and UV-crosslinking. Using a mixture of 8 v% PEGDA and 2.5 wt% gelatine, the user can prepare PEGDA-based cryoblocks without the need for prolonged overnight incubation in OCT or PVA solution, as required in a related published protocol . Furthermore, the addition of gelatine promotes the adhesion between the gel well plate and the embedding matrix, which is crucial to avoid the loss of precious samples. During freezing and sectioning, a separation of the tissue-matrix interface may create damage to the tissue. Proper infiltration of the embedding matrix inside the tissue promotes a coherent interface. Analysis of the substructure of human retinal organoids revealed that PEGDA-gelatine mixture preserves the fragile structure of outer segments, as well as the organization of the organoid nuclear layers and cell types. It correlates with the stable interface obtained between the embedding matrix and the organoids. Additionally, the PEGDA-gelatine mixture has a 3.4 times lower viscosity at 37 °C than gelatine 7.5 wt%, which promotes the diffusion of the matrix during incubation supporting the structure of fragile tissue (Supplementary Fig. b,c). Embedding with the gelatine HistoBrick protocol frequently resulted in damaged outer segments. The rapid solidification of gelatine during organoid transfer into the well plate required that organoids were pushed into well bottoms with pipette tips (to prevent their attachment to well walls or displacement by air bubbles), which may have damaged the organoids. PEGDA-gelatine circumvented this problem, as it is less viscous at room temperature and thereby more suited for organoid transfer into wells and retained the substructure of retinal organoid outer segments more efficiently. PEGDA-gelatine is thus the preferred material for fabrication and use of a HistoBrick for cryosectioning. The PEDGA-gelatine HistoBricks easily allowed the analysis of a two-year retinal organoid culture time-course. Instead of preparing and sectioning 11 conventional blocks with different time points and biological replicates, only 3 PEGDA-gelatine HistoBricks were required to analyze all samples. We have previously analyzed the development of retinal organoids in detail by single-cell RNA sequencing and compared them to the adult human retina . At week 46, we have found the cell type diversity of organoids to be decreased and that some cell type marker genes such as rhodopsin were downregulated in rods . One could interpret reduced expression of cell type marker genes as a decrease in cell quality or health. The decrease of outer segments that we visually observed from week 52 supports the indication that photoreceptors degenerate to some degree. In some retinal diseases, outer segments of photoreceptors degenerate, while their cell bodies remain viable . It has been unclear for how long photoreceptors can be maintained in cultured retinal organoids. We were surprised to still find organoids with clearly separated outer nuclear and inner nuclear layers between week 70 and up to week 98. We observed both cone and rod photoreceptors up to week 98. Unexpectedly, in older organoids we found displaced nuclei that we identified as cones, on top of the outer nuclear layer, in the region where outer segments are. It has been described in sections of human retinas that photoreceptor cell bodies can be displaced out of the outer nuclear layer into the region of outer segments during aging , . Further studies are needed to validate whether the gradual loss of photoreceptor outer segments and displacement of cones is a form of degradation in retinal organoids and whether this is similar to degeneration processes happening in human disease or aging. The HistoBrick method faces general challenges associated with the analysis of complex 3D microtissues on two-dimensional sections. First, each section contains only part of an organoid at a random sectioning angle which may or may not contain the tissue region or cell type of interest (Supplementary Fig. c), introducing variability in tissue composition. Second, organoids largely vary in size and shape , and on each given section only a fraction of the entire organoid is visible, introducing variability in size and geometry. Third, retinal organoids contain not only retina, but also regions of non-retinal identity, introducing variability in the ratio of retinal to non-retinal tissues. Fourth, depending on the cutting angle, appearance of retinal organoid layering, outer segment thickness, or distance between layers could be influenced by the sectioning angle. These sources of variability could be reduced by analyzing a larger number of sections per organoid, or optimally their full 3D signals. In addition, the analysis would benefit from automated organoid segmentation based on antibody staining. A future version of the HistoBrick, potentially using yet another material, could be adapted to organoid embedding for wholemount staining, clearing and imaging in 3D. Some aspects of the HistoBrick method can be further optimized to improve its user-friendliness. First, color can be added to the embedding material to help the visualization on the slide after sectioning and staining. Secondly, a marking sign visible on transparent sections could be incorporated into the HistoBrick design to ensure optimal sample traceability and re-orientation. Finally, automated transfer of organoids into the gel well plate and automated staining would further increase the throughput of the HistoBrick approach. We successfully adapted the HistoBrick methodology for an efficient preparation and analysis of organoid frozen sections. We expect the PEGDA-gelatine HistoBrick to provide optimal support to various fragile samples like brain organoids and cochlea biopsies. The PEGDA-gelatine HistoBrick increases the throughput of immunohistochemistry by simultaneously embedding up to 16 human retinal organoids from different experimental conditions in an organized manner. Processing a smaller number of cryoblocks for sectioning and stainings saves time and consumables such as antibodies required for staining the resulting sections (Supplemenatary Fig. d). Depending on the microtissue to analyze, the HistoBrick can easily be adapted to different organoid and tissue sizes, and organoid numbers per block can still be increased when smaller or more wells are used. Simultaneous immunohistochemical staining and imaging of multiple organoid samples on the same section reduces sample-to-sample variation and enables more reliable analysis. The array organization facilitates traceability of individual organoids through different sections and offers high potential for automated organoid transfer and image analysis. Furthermore, the HistoBrick is a versatile labware that can be easily implemented in standard laboratories thanks to its simple methodology and low-cost materials. The ease of HistoBrick re-design enables adoption of the technology across the field of microtissue research. Finally, with this tool in hand, the immunohistochemical validation of organoid high-throughput experiments such as compound-, toxicology-, or gene-therapy screening will be simplified. Gel well plate fabrication The silicone mold was designed similarly to our previous work . The diameter of the wells was increased to 2.4 mm to accommodate large retinal organoids. The outer dimensions of the HistoBrick were set to 20.7× 34.7 mm to contain 16 wells (Supplementary Fig. a). The mold was 3D printed in silicone “TrueSil Shore 50A” by the company Spectoplast. The schematic of the HistoBrick fabrication is depicted in Fig. . The HistoBrick 3D design file is available upon request to the corresponding authors. HistoBrick well-sizes and dimensions can be optimized for different organoid types. Gelatine gel well plate In a first approach, the silicone mold was placed with the pillars facing downward on a petri dish. A gelatine solution (7.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84,100) in 1 × PBS) was warmed at 37 °C and pipetted inside the silicone mold. The gelatine solution was crosslinked at 4 °C for 1 h. Afterwards, the mold was flipped and the gel well plate was released by gently lifting it from below. The obtained gel well plate was stored in a closed box with damp paper at 4 °C until use. PEGDA-based gel well plate In a second approach the silicone mold was placed with the pillars facing downward on a petri dish. The PEGDA-gelatine solution was prepared by mixing 8 v% PEGDA (Mw = 700, Sigma Aldrich, #455008), 2.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84100) and 0.05 wt% LAP (Sigma Aldrich, #900889) in 1 × PBS. The solution was pipetted inside the silicone mold and crosslinked by a 30 s exposure to 365 nm wavelength light with 35 mW/cm power (total illumination dose = 1.05 J/s*cm). The UV light was shined from above the mold. Afterward, the silicone mold was vertically lifted to release the gel well plate. The obtained gel well plate was stored in a closed box with humid paper at 4 °C until use. For the material selection, additional HistoBricks were prepared using a solution of 8 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS and 10 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS with the same crosslinking procedure. Organoid transfer into the gel well plate and HistoBrick freezing When the gelatine embedding material was used, the gel well plate was left to equilibrate at room temperature for 1 h. We refer to the liquid hydrogel solution filling the wells of the gel well plate as liquid embedding matrix. Before organoid transfer into the gel well plate, PFA-fixed, sucrose-cryopreserved retinal organoids were incubated in liquid embedding matrix of the same embedding material as the gel well plate for 15 min at 37 °C on a warming plate to enhance organoid-embedding matrix interaction. Incubation can also be performed in an incubator at 37 °C. The organoids were manually transferred with 20–50 µL of liquid embedding matrix to the gel well plate with a 200 µL tip pre-coated with 2% BSA (Sigma Aldrich, #A2153) in deionized water, to prevent organoids from sticking. The end of the tip was cut with a razor blade to create a larger aperture to avoid damaging the organoids. The gel well plate can be pre-wet by adding 20 µL of PBS in each well to minimize air bubble trapping. After the transfer, the wells were filled up with the same liquid embedding matrix used during the incubation of the organoids. Organoids were aligned onto a unique plane by centrifuging the filled gel well plate for 20 s at 200×g. The filled gel well plate underwent a second crosslinking (as previously described) to fix the position of the organoids at the bottom of the wells. The HistoBrick blocks were trimmed on all four sides so that three sections would later fit on the glass slides during cryosectioning. Finally, the HistoBrick was fixed on a labelled cardboard with a drop of OCT and snap frozen for 2 min by submersion in isopentane (Sigma Aldrich, #277258) cooled down to − 40 °C using dry ice pellets. The frozen HistoBricks were wrapped in aluminum foil to avoid drying and stored at − 80 °C until cryosectioning. Conventional cryoblock preparation Unless specified otherwise, the steps were performed using the same solutions and timings as for the gel well plate fabrication and organoid transfer. Gelatine and PEGDA-gelatine cryoblock The respective solution was pipetted inside a plastic weighing boat or an embedding mold and crosslinked to form a base layer of 2–4 mm thickness. During this time, retinal organoids were incubated in their respective embedding solutions for 15 min at 37 °C. Then, they were transferred one by one and placed on the base layer, followed by a second crosslinking. Finally, liquid embedding material was pipetted on top of the organoids to fill up the weighing boat and crosslinked. The blocks were trimmed with a scalpel to leave 1–2 mm space between the organoids and the block edge. The samples were then fixed on a labelled cardboard with a drop of OCT, snap frozen in isopentane at − 40 °C and stored at − 80 °C. OCT-based cryoblock OCT (Sakura, #4583) was poured inside a plastic weigh boat and crosslinked by freezing on dry ice pellets to form a base layer. The organoids were then transferred from their storage solution (without incubation) and placed on the base layer. Additional OCT was poured on the organoids, the blocks were frozen on dry ice and stored wrapped in aluminum foil at − 80 °C. HistoBrick cryoblock and conventional cryoblock sectioning The HistoBrick cryoblocks and the conventionally embedded cryoblocks were stored at − 20 °C overnight to acclimate to sectioning temperature. They were then mounted on a cryostat holder with OCT. If necessary, HistoBrick cryoblocks were again trimmed to fit 3 sections per slide, and conventional cryoblocks were trimmed to fit 8–12 sections per slide. Then they were sectioned in thin slices of 20 µm or 25 µm and placed on SuperFrost ® /Plus glass slides (Biosystems, #85-0911-00 or Epredia, #K5800AMNZ72). For results shown in Figs. , , Supplementary Figs. , and consecutive Histobrick sections were placed one section per glass slide on 5 slides. Afterwards, a second section was placed on each of the slides, followed by a third section, again starting from the first slide. After filling the first 5 slides, a second slide series of 5 slides was prepared using the same procedure. In this manner each slide contains sections from different organoid cutting depths, increasing the likelihood that there will be at least one usable section per organoid. The slides were stored at − 20 °C or − 80 °C until staining. Hematoxylin and Eosin staining To localize the organoids, slides were stained with hematoxylin and eosin. The slides were equilibrated and dried at room temperature for 1 h, then rehydrated in deionized water for 10 min. The slides were then incubated in Harris Hematoxylin (Harris Hematoxylin: Biosystems, #3873.2500) for 5 min and washed with running tap water. They were then differentiated for a few seconds in 1% acid-alcohol (Absolute alcohol: 7:10, VWR #20820.362, Hydrochloric acid 37%: 0.1:10, Sigma Aldrich, #30721 and H2O MiliQ 2.9:10) and washed under running tap water for 10 min. The slides were incubated in Eosine-Phloxine solution for 1 min (Eosin: 1:100, Sigma Aldrich, #E4382; Phloxine: 1:100, Sigma Aldrich, #P2759) and washed a last time. Finally, the slides were mounted with Eukitt (Sigma Aldrich, #03989) and dried overnight at room temperature. Immunostainings The slides were equilibrated and dried at room temperature for 1 h, then rehydrated in 1 × PBS for 10 min. The sections were blocked with 180 µL of 10% NDS blocking solution (10% Normal Donkey Serum (Sigma Aldrich, S30-M), 1% BSA (Sigma Aldrich, #A2153), 0.5% TritonX (Sigma Aldrich, #T9284), 0.1% sodium azide (Biosciences, #786-299), 1 × PBS) for 1 h. The blocking solution was removed, and the primary antibodies were added (ARR3: 1:400, Sigma Aldrich, #HPA063129; PNA: 1:1200, Sigma Aldrich #L6135; Rhodopsin: 1:400, Sigma Aldrich, #R5403; SOX9: 1:200, R&D systems, #AF3075; MiTF: 1:500, Exalpha, #X2398M; MAP2: 1:400, Sigma Aldrich, #AB5622; OneCut2: 1:100, R&D systems, # AF6294; Cralbp: 1:200, Abcam, #ab15051; Trpm1: 1:200, Sigma Aldrich, #HPA014779; Bassoon: 1:500, Enzo, # SAP7F407, L-M Opsin: 1:100, Merck, # AB5405). The primary antibodies were diluted in a 3% NDS solution (3% Normal Donkey Serum, 1% BSA, 0.5% TritonX, 0.1% sodium azide, 1 × PBS), 180 µL was added to each slide. The sections were covered with parafilm and incubated with the primary antibodies overnight at 4 °C in a humidified chamber. Afterwards, the slides were washed 3 times with PBS-T (1 × PBS, 0.05% TritonX) for 10 min each. The secondary antibodies (Hoechst 33342: 1:2000, Invitrogen, #H3570; anti-Mouse 488: 1:500, Invitrogen, #A32766; Streptavidin 555: 1:400, Invitrogen, #S32355; anti-Rabbit 640: 1:500, Invitrogen, #A32795; anti-Goat 488: 1:500, Invitrogen, #A32814; anti-Mouse 568: 1:500, Invitrogen, #A10037; anti-Sheep 488: 1:500, Invitrogen, # A11015) were diluted in 3% NDS and the sections were incubated for 2 h with 180 µL antibody solution at 4 °C. The slides were washed twice with PBS-T for 10 min each and once with 1 × PBS for 10 min. As a last step, the slides were mounted with Prolong Gold (Invitrogen, #P36934), coverslipped, and dried overnight at room temperature, sealed with nail polish (Electron Microscopy Sciences, #72180) and imaged. Image acquisition Images of the H&E-stained sections in Figs. and were acquired with an Olympus VS200 slide scanner. An overview image of the entire glass slide was acquired at 10x (UplaXapo, NA = 0.40) magnification in brightfield. Immunostained sections were acquired with a confocal spinning disc microscope (Olympus IXplore SpinSR10). Different magnifications were used depending on the desired image (4x: UplanSApo, NA = 0.16; 10x: UplanSApo, NA = 0.40; 20x: UplanSApo, NA = 0.75; 40x: UPlanSApo, NA = 1.05 Sil). For higher throughput of image acquisition and in Supplementary Fig. , a slide scanner was used (Zeiss Axio Scan.Z1) with 10 × magnification (Plan-Apochromate, NA = 0.45). OS + IS area measurements were performed on images obtained with the Zeiss Axio Scan.Z1 slide scanner. For each individual organoid, the section containing the highest quality outer segments (criteria: abundant OS with as little disruption as possible, intact organoid section) was manually selected for analysis. Image analysis Analysis of the vertical alignment of the human retinal organoids was performed on consecutive H&E-stained sections on a slide series by manually counting the number of organoids present on each section. For each HistoBrick, we determined the number of sections in which at least 80% of the organoids were visible. The analysis was conducted on two HistoBricks containing 15, respectively 16, organoids. Although the HistoBrick contains a marking at one edge to localize the top right corner, one well can be left empty for experiments in which the embedding matrix cannot easily be visualized during the microscopy step. This improves the certainty of organoid re-identification, because during sectioning and microscopy the sections and the images thereof can be rotated. FIJI was employed in this study for image modification and the quantification of the OS + IS area within images. For images displayed in the figures, brightness and contrast were adjusted for optimal visualization of the signal. Each channel underwent independent analysis. OS + IS area measurements To obtain single, fully filled regions of interest (ROIs), we applied the following procedure separately for each channel. For area masking, first, the “Fire” lookup table (LUT) for color mapping was applied to enhance visualization (Supplementary Fig. c, 2nd panel). A color threshold was manually set for each channel such that the organoid including the OS was above, and the background below threshold. Thresholds were channel-specific and kept constant within one embedding material in Fig. and across all conditions in Fig. . A binary mask was defined as the pixels above threshold (Supplementary Fig. c, 3rd panel). Image parts of the retinal organoids below threshold were included into the mask (Supplementary Fig. d), while parts outside the retinal organoid with values above threshold, including non-retinal, unstructured or disrupted organoid regions were manually separated from the mask (Supplementary Fig. e) using the “Overlay brush” tool. Subsequently, the “Fill Holes” command was executed to include all pixels inside the retinal organoid into the mask. The “Analyze Particles” function was applied to define a region of interest (ROI) by the binary mask with the largest area, corresponding to the retinal organoid (Supplementary Fig. c, right panel, Supplementary Fig. d,e, right panels). Area measurements of the ROIs were performed using the “Analyze Particles” command. To measure the OS + IS area, first, a single, fully filled ROI for the Peanut agglutinin (PNA) signal was determined (PNA ROI, Supplementary Fig. b,c). Second, a single, fully filled ROI for the Hoechst signal was determined (Hoechst ROI, Supplementary Fig. b,c). Finally, to calculate the OS + IS area, the area of the Hoechst ROI was subtracted from the area of the PNA ROI. The used FIJI macro is available in the Supplementary Information. Quantification of OS + IS thickness To quantify outer segments independent of organoid size and cross-section area, we estimated the average thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} of the outer and inner segments labeled by PNA (“OS + IS thickness”) by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta = \frac{{\sqrt {PNA area} }}{\sqrt \pi } - \frac{{\sqrt {Hoechst area} }}{\sqrt \pi }$$\end{document} where PNA area denotes the area of the PNA ROI and Hoechst area denotes the area of the Hoechst ROI. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} provides the exact solution for circular organoids with uniform OS + IS segments and an average thickness estimate for organoids with less regular shape and OS + IS segments. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} can also be expressed as a ratio between the OS + IS area and the Hoechst area, providing an intuitive interpretation of their relationship: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{OS+IS area }{\sqrt{Hoechst area} \cdot \sqrt{\pi } \cdot 2} = \frac{PNA area - Hoechst area }{\sqrt{Hoechst area} \cdot \sqrt{\pi } \cdot 2}= \frac{\left(2\cdot r\cdot \vartheta +{\vartheta }^{2}\right)\cdot \pi }{r\cdot \sqrt{\pi } \cdot \sqrt{\pi } \cdot 2}=\vartheta + \frac{{\vartheta }^{2}}{2r}\approx \vartheta ,$$\end{document} with radius \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r$$\end{document} of the Hoechst area, radius \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r+\vartheta$$\end{document} of the PNA area, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta \ll r$$\end{document} . Retinal organoid culture and fixation for embedding Retinal organoids were generated from induced pluripotent stem cells as previously described in Cowan et al . and Spirig et al . , . Retinal organoids were generated from the induced pluripotent stem cell line 01F49i-N-B7 (IOBi001-A (RRID:CVCL_C1TR) described in Cowan et al . . Experiments in this study were performed using organoids aged between 30 and 98 weeks. Organoids were fixed using 4% paraformaldehyde (PFA) (Merck, #1.00496) for 4 h at 4 °C. The samples were washed three times for 10 min each in 1 × PBS. Then the organoids were incubated in 30% sucrose (Millipore, #84100) in 1 × PBS until sunken to the bottom of the tube for cryopreservation and stored at − 80 °C until embedding. Statistical analysis Graphpad Prism (Prism10 for MacOS, Version 10.3.1 (464), August 21, 2024) was used for statistical analysis. To test whether the OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} labeled by PNA differed significantly for the results shown in Figs. h and d, Welch’s ANOVA was used to compare the different groups followed by posthoc Dunnett’s test. For the posthoc test in Fig. h, we compared all conditions to the conventional gelatine embedding. For the graph in Fig. d, we included data from Fig. as controls. We used the PEGDA-gelatine HistoBrick data as positive controls and the OCT data as negative controls. For the posthoc test, we compared all against negative control and all against positive control. To determine, whether OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} decreases over time, we plotted \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} values of the organoids at different developmental ages against their age in weeks. We then calculated the nonparametric Spearman rank correlation. The P value and r are represented in the figure. Multiplicity adjusted P values are indicated as asterisks in the figures as follows: P > 0.05 (ns), P ≤ 0.05 , P ≤ 0.001 , P ≤ 0.001 . Only significant P values were indicated on the graphs, the other comparisons were not significant. Each dot on the graphs represents the results of one organoid. Rheological measurements The gelatine solution, PEGDA-gelatine solution and a 8 v% PEGDA + 10 wt% sucrose solution were measured by pipetting 1 ml of the solution into a rheometer (Anton Paar, MCR 702e). For oscillation testing, the geometry used was a PP-40 with a gap of 0.5 mm. The normal force was set at 0 N so that the gap would adjust to maintain this force during the phase transition. A ramp from 37 to 20 °C (5 °C/min) followed by an isotherm at 20 °C for 60 min was performed for the gelatine solution and the PEGDA-gelatine solution. For the PEGDA solution, a ramp from 37 to 20 °C (5 °C/min) followed by an isotherm at 20 °C for 10 min was performed. The isotherm at 20 °C was shortened for the PEGDA solution because it showed stable behavior. During these 2 intervals, the storage Modulus (G’) and the loss Modulus (G’’) as well as the complex viscosity were recorded at a strain of 1% and 1 Hz. Prior to this experiment, an amplitude sweep test was performed (data not shown) to evaluate the Linear viscoelastic range (LVE range). 1% strain was selected within the LVE range. For rotational testing, the geometry used was a CP-50-1 (diameter of 50 mm with a 1° angle and cone truncation of 0.045 mm). Dynamic viscosity was recorded at 37 °C from a shear rate of 0.1–1000 s −1 . The exploitable data range is from 10 to 1000 s −1 . The rotational tests were performed three times for each sample. The dynamic viscosities of the different solutions were calculated as the average of the three performed tests over the whole shear rate range. The silicone mold was designed similarly to our previous work . The diameter of the wells was increased to 2.4 mm to accommodate large retinal organoids. The outer dimensions of the HistoBrick were set to 20.7× 34.7 mm to contain 16 wells (Supplementary Fig. a). The mold was 3D printed in silicone “TrueSil Shore 50A” by the company Spectoplast. The schematic of the HistoBrick fabrication is depicted in Fig. . The HistoBrick 3D design file is available upon request to the corresponding authors. HistoBrick well-sizes and dimensions can be optimized for different organoid types. Gelatine gel well plate In a first approach, the silicone mold was placed with the pillars facing downward on a petri dish. A gelatine solution (7.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84,100) in 1 × PBS) was warmed at 37 °C and pipetted inside the silicone mold. The gelatine solution was crosslinked at 4 °C for 1 h. Afterwards, the mold was flipped and the gel well plate was released by gently lifting it from below. The obtained gel well plate was stored in a closed box with damp paper at 4 °C until use. PEGDA-based gel well plate In a second approach the silicone mold was placed with the pillars facing downward on a petri dish. The PEGDA-gelatine solution was prepared by mixing 8 v% PEGDA (Mw = 700, Sigma Aldrich, #455008), 2.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84100) and 0.05 wt% LAP (Sigma Aldrich, #900889) in 1 × PBS. The solution was pipetted inside the silicone mold and crosslinked by a 30 s exposure to 365 nm wavelength light with 35 mW/cm power (total illumination dose = 1.05 J/s*cm). The UV light was shined from above the mold. Afterward, the silicone mold was vertically lifted to release the gel well plate. The obtained gel well plate was stored in a closed box with humid paper at 4 °C until use. For the material selection, additional HistoBricks were prepared using a solution of 8 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS and 10 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS with the same crosslinking procedure. In a first approach, the silicone mold was placed with the pillars facing downward on a petri dish. A gelatine solution (7.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84,100) in 1 × PBS) was warmed at 37 °C and pipetted inside the silicone mold. The gelatine solution was crosslinked at 4 °C for 1 h. Afterwards, the mold was flipped and the gel well plate was released by gently lifting it from below. The obtained gel well plate was stored in a closed box with damp paper at 4 °C until use. In a second approach the silicone mold was placed with the pillars facing downward on a petri dish. The PEGDA-gelatine solution was prepared by mixing 8 v% PEGDA (Mw = 700, Sigma Aldrich, #455008), 2.5 wt% gelatine (Sigma Aldrich, #G1890), 10 wt% sucrose (Sigma Aldrich, #84100) and 0.05 wt% LAP (Sigma Aldrich, #900889) in 1 × PBS. The solution was pipetted inside the silicone mold and crosslinked by a 30 s exposure to 365 nm wavelength light with 35 mW/cm power (total illumination dose = 1.05 J/s*cm). The UV light was shined from above the mold. Afterward, the silicone mold was vertically lifted to release the gel well plate. The obtained gel well plate was stored in a closed box with humid paper at 4 °C until use. For the material selection, additional HistoBricks were prepared using a solution of 8 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS and 10 v% PEGDA Mw = 700, 10 wt% sucrose, 0.05 wt% LAP in 1 × PBS with the same crosslinking procedure. When the gelatine embedding material was used, the gel well plate was left to equilibrate at room temperature for 1 h. We refer to the liquid hydrogel solution filling the wells of the gel well plate as liquid embedding matrix. Before organoid transfer into the gel well plate, PFA-fixed, sucrose-cryopreserved retinal organoids were incubated in liquid embedding matrix of the same embedding material as the gel well plate for 15 min at 37 °C on a warming plate to enhance organoid-embedding matrix interaction. Incubation can also be performed in an incubator at 37 °C. The organoids were manually transferred with 20–50 µL of liquid embedding matrix to the gel well plate with a 200 µL tip pre-coated with 2% BSA (Sigma Aldrich, #A2153) in deionized water, to prevent organoids from sticking. The end of the tip was cut with a razor blade to create a larger aperture to avoid damaging the organoids. The gel well plate can be pre-wet by adding 20 µL of PBS in each well to minimize air bubble trapping. After the transfer, the wells were filled up with the same liquid embedding matrix used during the incubation of the organoids. Organoids were aligned onto a unique plane by centrifuging the filled gel well plate for 20 s at 200×g. The filled gel well plate underwent a second crosslinking (as previously described) to fix the position of the organoids at the bottom of the wells. The HistoBrick blocks were trimmed on all four sides so that three sections would later fit on the glass slides during cryosectioning. Finally, the HistoBrick was fixed on a labelled cardboard with a drop of OCT and snap frozen for 2 min by submersion in isopentane (Sigma Aldrich, #277258) cooled down to − 40 °C using dry ice pellets. The frozen HistoBricks were wrapped in aluminum foil to avoid drying and stored at − 80 °C until cryosectioning. Unless specified otherwise, the steps were performed using the same solutions and timings as for the gel well plate fabrication and organoid transfer. Gelatine and PEGDA-gelatine cryoblock The respective solution was pipetted inside a plastic weighing boat or an embedding mold and crosslinked to form a base layer of 2–4 mm thickness. During this time, retinal organoids were incubated in their respective embedding solutions for 15 min at 37 °C. Then, they were transferred one by one and placed on the base layer, followed by a second crosslinking. Finally, liquid embedding material was pipetted on top of the organoids to fill up the weighing boat and crosslinked. The blocks were trimmed with a scalpel to leave 1–2 mm space between the organoids and the block edge. The samples were then fixed on a labelled cardboard with a drop of OCT, snap frozen in isopentane at − 40 °C and stored at − 80 °C. OCT-based cryoblock OCT (Sakura, #4583) was poured inside a plastic weigh boat and crosslinked by freezing on dry ice pellets to form a base layer. The organoids were then transferred from their storage solution (without incubation) and placed on the base layer. Additional OCT was poured on the organoids, the blocks were frozen on dry ice and stored wrapped in aluminum foil at − 80 °C. The respective solution was pipetted inside a plastic weighing boat or an embedding mold and crosslinked to form a base layer of 2–4 mm thickness. During this time, retinal organoids were incubated in their respective embedding solutions for 15 min at 37 °C. Then, they were transferred one by one and placed on the base layer, followed by a second crosslinking. Finally, liquid embedding material was pipetted on top of the organoids to fill up the weighing boat and crosslinked. The blocks were trimmed with a scalpel to leave 1–2 mm space between the organoids and the block edge. The samples were then fixed on a labelled cardboard with a drop of OCT, snap frozen in isopentane at − 40 °C and stored at − 80 °C. OCT (Sakura, #4583) was poured inside a plastic weigh boat and crosslinked by freezing on dry ice pellets to form a base layer. The organoids were then transferred from their storage solution (without incubation) and placed on the base layer. Additional OCT was poured on the organoids, the blocks were frozen on dry ice and stored wrapped in aluminum foil at − 80 °C. The HistoBrick cryoblocks and the conventionally embedded cryoblocks were stored at − 20 °C overnight to acclimate to sectioning temperature. They were then mounted on a cryostat holder with OCT. If necessary, HistoBrick cryoblocks were again trimmed to fit 3 sections per slide, and conventional cryoblocks were trimmed to fit 8–12 sections per slide. Then they were sectioned in thin slices of 20 µm or 25 µm and placed on SuperFrost ® /Plus glass slides (Biosystems, #85-0911-00 or Epredia, #K5800AMNZ72). For results shown in Figs. , , Supplementary Figs. , and consecutive Histobrick sections were placed one section per glass slide on 5 slides. Afterwards, a second section was placed on each of the slides, followed by a third section, again starting from the first slide. After filling the first 5 slides, a second slide series of 5 slides was prepared using the same procedure. In this manner each slide contains sections from different organoid cutting depths, increasing the likelihood that there will be at least one usable section per organoid. The slides were stored at − 20 °C or − 80 °C until staining. To localize the organoids, slides were stained with hematoxylin and eosin. The slides were equilibrated and dried at room temperature for 1 h, then rehydrated in deionized water for 10 min. The slides were then incubated in Harris Hematoxylin (Harris Hematoxylin: Biosystems, #3873.2500) for 5 min and washed with running tap water. They were then differentiated for a few seconds in 1% acid-alcohol (Absolute alcohol: 7:10, VWR #20820.362, Hydrochloric acid 37%: 0.1:10, Sigma Aldrich, #30721 and H2O MiliQ 2.9:10) and washed under running tap water for 10 min. The slides were incubated in Eosine-Phloxine solution for 1 min (Eosin: 1:100, Sigma Aldrich, #E4382; Phloxine: 1:100, Sigma Aldrich, #P2759) and washed a last time. Finally, the slides were mounted with Eukitt (Sigma Aldrich, #03989) and dried overnight at room temperature. The slides were equilibrated and dried at room temperature for 1 h, then rehydrated in 1 × PBS for 10 min. The sections were blocked with 180 µL of 10% NDS blocking solution (10% Normal Donkey Serum (Sigma Aldrich, S30-M), 1% BSA (Sigma Aldrich, #A2153), 0.5% TritonX (Sigma Aldrich, #T9284), 0.1% sodium azide (Biosciences, #786-299), 1 × PBS) for 1 h. The blocking solution was removed, and the primary antibodies were added (ARR3: 1:400, Sigma Aldrich, #HPA063129; PNA: 1:1200, Sigma Aldrich #L6135; Rhodopsin: 1:400, Sigma Aldrich, #R5403; SOX9: 1:200, R&D systems, #AF3075; MiTF: 1:500, Exalpha, #X2398M; MAP2: 1:400, Sigma Aldrich, #AB5622; OneCut2: 1:100, R&D systems, # AF6294; Cralbp: 1:200, Abcam, #ab15051; Trpm1: 1:200, Sigma Aldrich, #HPA014779; Bassoon: 1:500, Enzo, # SAP7F407, L-M Opsin: 1:100, Merck, # AB5405). The primary antibodies were diluted in a 3% NDS solution (3% Normal Donkey Serum, 1% BSA, 0.5% TritonX, 0.1% sodium azide, 1 × PBS), 180 µL was added to each slide. The sections were covered with parafilm and incubated with the primary antibodies overnight at 4 °C in a humidified chamber. Afterwards, the slides were washed 3 times with PBS-T (1 × PBS, 0.05% TritonX) for 10 min each. The secondary antibodies (Hoechst 33342: 1:2000, Invitrogen, #H3570; anti-Mouse 488: 1:500, Invitrogen, #A32766; Streptavidin 555: 1:400, Invitrogen, #S32355; anti-Rabbit 640: 1:500, Invitrogen, #A32795; anti-Goat 488: 1:500, Invitrogen, #A32814; anti-Mouse 568: 1:500, Invitrogen, #A10037; anti-Sheep 488: 1:500, Invitrogen, # A11015) were diluted in 3% NDS and the sections were incubated for 2 h with 180 µL antibody solution at 4 °C. The slides were washed twice with PBS-T for 10 min each and once with 1 × PBS for 10 min. As a last step, the slides were mounted with Prolong Gold (Invitrogen, #P36934), coverslipped, and dried overnight at room temperature, sealed with nail polish (Electron Microscopy Sciences, #72180) and imaged. Images of the H&E-stained sections in Figs. and were acquired with an Olympus VS200 slide scanner. An overview image of the entire glass slide was acquired at 10x (UplaXapo, NA = 0.40) magnification in brightfield. Immunostained sections were acquired with a confocal spinning disc microscope (Olympus IXplore SpinSR10). Different magnifications were used depending on the desired image (4x: UplanSApo, NA = 0.16; 10x: UplanSApo, NA = 0.40; 20x: UplanSApo, NA = 0.75; 40x: UPlanSApo, NA = 1.05 Sil). For higher throughput of image acquisition and in Supplementary Fig. , a slide scanner was used (Zeiss Axio Scan.Z1) with 10 × magnification (Plan-Apochromate, NA = 0.45). OS + IS area measurements were performed on images obtained with the Zeiss Axio Scan.Z1 slide scanner. For each individual organoid, the section containing the highest quality outer segments (criteria: abundant OS with as little disruption as possible, intact organoid section) was manually selected for analysis. Analysis of the vertical alignment of the human retinal organoids was performed on consecutive H&E-stained sections on a slide series by manually counting the number of organoids present on each section. For each HistoBrick, we determined the number of sections in which at least 80% of the organoids were visible. The analysis was conducted on two HistoBricks containing 15, respectively 16, organoids. Although the HistoBrick contains a marking at one edge to localize the top right corner, one well can be left empty for experiments in which the embedding matrix cannot easily be visualized during the microscopy step. This improves the certainty of organoid re-identification, because during sectioning and microscopy the sections and the images thereof can be rotated. FIJI was employed in this study for image modification and the quantification of the OS + IS area within images. For images displayed in the figures, brightness and contrast were adjusted for optimal visualization of the signal. Each channel underwent independent analysis. To obtain single, fully filled regions of interest (ROIs), we applied the following procedure separately for each channel. For area masking, first, the “Fire” lookup table (LUT) for color mapping was applied to enhance visualization (Supplementary Fig. c, 2nd panel). A color threshold was manually set for each channel such that the organoid including the OS was above, and the background below threshold. Thresholds were channel-specific and kept constant within one embedding material in Fig. and across all conditions in Fig. . A binary mask was defined as the pixels above threshold (Supplementary Fig. c, 3rd panel). Image parts of the retinal organoids below threshold were included into the mask (Supplementary Fig. d), while parts outside the retinal organoid with values above threshold, including non-retinal, unstructured or disrupted organoid regions were manually separated from the mask (Supplementary Fig. e) using the “Overlay brush” tool. Subsequently, the “Fill Holes” command was executed to include all pixels inside the retinal organoid into the mask. The “Analyze Particles” function was applied to define a region of interest (ROI) by the binary mask with the largest area, corresponding to the retinal organoid (Supplementary Fig. c, right panel, Supplementary Fig. d,e, right panels). Area measurements of the ROIs were performed using the “Analyze Particles” command. To measure the OS + IS area, first, a single, fully filled ROI for the Peanut agglutinin (PNA) signal was determined (PNA ROI, Supplementary Fig. b,c). Second, a single, fully filled ROI for the Hoechst signal was determined (Hoechst ROI, Supplementary Fig. b,c). Finally, to calculate the OS + IS area, the area of the Hoechst ROI was subtracted from the area of the PNA ROI. The used FIJI macro is available in the Supplementary Information. To quantify outer segments independent of organoid size and cross-section area, we estimated the average thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} of the outer and inner segments labeled by PNA (“OS + IS thickness”) by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta = \frac{{\sqrt {PNA area} }}{\sqrt \pi } - \frac{{\sqrt {Hoechst area} }}{\sqrt \pi }$$\end{document} where PNA area denotes the area of the PNA ROI and Hoechst area denotes the area of the Hoechst ROI. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} provides the exact solution for circular organoids with uniform OS + IS segments and an average thickness estimate for organoids with less regular shape and OS + IS segments. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} can also be expressed as a ratio between the OS + IS area and the Hoechst area, providing an intuitive interpretation of their relationship: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\frac{OS+IS area }{\sqrt{Hoechst area} \cdot \sqrt{\pi } \cdot 2} = \frac{PNA area - Hoechst area }{\sqrt{Hoechst area} \cdot \sqrt{\pi } \cdot 2}= \frac{\left(2\cdot r\cdot \vartheta +{\vartheta }^{2}\right)\cdot \pi }{r\cdot \sqrt{\pi } \cdot \sqrt{\pi } \cdot 2}=\vartheta + \frac{{\vartheta }^{2}}{2r}\approx \vartheta ,$$\end{document} with radius \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r$$\end{document} of the Hoechst area, radius \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$r+\vartheta$$\end{document} of the PNA area, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta \ll r$$\end{document} . Retinal organoids were generated from induced pluripotent stem cells as previously described in Cowan et al . and Spirig et al . , . Retinal organoids were generated from the induced pluripotent stem cell line 01F49i-N-B7 (IOBi001-A (RRID:CVCL_C1TR) described in Cowan et al . . Experiments in this study were performed using organoids aged between 30 and 98 weeks. Organoids were fixed using 4% paraformaldehyde (PFA) (Merck, #1.00496) for 4 h at 4 °C. The samples were washed three times for 10 min each in 1 × PBS. Then the organoids were incubated in 30% sucrose (Millipore, #84100) in 1 × PBS until sunken to the bottom of the tube for cryopreservation and stored at − 80 °C until embedding. Graphpad Prism (Prism10 for MacOS, Version 10.3.1 (464), August 21, 2024) was used for statistical analysis. To test whether the OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} labeled by PNA differed significantly for the results shown in Figs. h and d, Welch’s ANOVA was used to compare the different groups followed by posthoc Dunnett’s test. For the posthoc test in Fig. h, we compared all conditions to the conventional gelatine embedding. For the graph in Fig. d, we included data from Fig. as controls. We used the PEGDA-gelatine HistoBrick data as positive controls and the OCT data as negative controls. For the posthoc test, we compared all against negative control and all against positive control. To determine, whether OS + IS thickness \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} decreases over time, we plotted \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\vartheta$$\end{document} values of the organoids at different developmental ages against their age in weeks. We then calculated the nonparametric Spearman rank correlation. The P value and r are represented in the figure. Multiplicity adjusted P values are indicated as asterisks in the figures as follows: P > 0.05 (ns), P ≤ 0.05 , P ≤ 0.001 , P ≤ 0.001 . Only significant P values were indicated on the graphs, the other comparisons were not significant. Each dot on the graphs represents the results of one organoid. The gelatine solution, PEGDA-gelatine solution and a 8 v% PEGDA + 10 wt% sucrose solution were measured by pipetting 1 ml of the solution into a rheometer (Anton Paar, MCR 702e). For oscillation testing, the geometry used was a PP-40 with a gap of 0.5 mm. The normal force was set at 0 N so that the gap would adjust to maintain this force during the phase transition. A ramp from 37 to 20 °C (5 °C/min) followed by an isotherm at 20 °C for 60 min was performed for the gelatine solution and the PEGDA-gelatine solution. For the PEGDA solution, a ramp from 37 to 20 °C (5 °C/min) followed by an isotherm at 20 °C for 10 min was performed. The isotherm at 20 °C was shortened for the PEGDA solution because it showed stable behavior. During these 2 intervals, the storage Modulus (G’) and the loss Modulus (G’’) as well as the complex viscosity were recorded at a strain of 1% and 1 Hz. Prior to this experiment, an amplitude sweep test was performed (data not shown) to evaluate the Linear viscoelastic range (LVE range). 1% strain was selected within the LVE range. For rotational testing, the geometry used was a CP-50-1 (diameter of 50 mm with a 1° angle and cone truncation of 0.045 mm). Dynamic viscosity was recorded at 37 °C from a shear rate of 0.1–1000 s −1 . The exploitable data range is from 10 to 1000 s −1 . The rotational tests were performed three times for each sample. The dynamic viscosities of the different solutions were calculated as the average of the three performed tests over the whole shear rate range. Supplementary Information. |
Regenerative fibroblasts derived from autologous skin tissue for the treatment of Sjögren’s syndrome: a case report | 0ee5fa01-e95d-4222-81bc-952b95f02d04 | 11808821 | Surgical Procedures, Operative[mh] | Introduction Sjögren’s syndrome (SS) is a systemic autoimmune disease that is typically characterized by symptoms such as dry mouth, dry eyes, fatigue, joint pain, muscle pain, and dental caries, among others . In severe cases, SS can cause systemic complications, including rash, kidney damage, lung damage, and neuropathy . One of the most severe and life-threatening complications of SS is cryoglobulinemic vasculitis . Mesenchymal stem cells (MSCs), known for their immune regulatory and tissue repair functions, can directly influence T-cell differentiation and function by secreting immune regulatory factors. These cells particularly enhance the production of regulatory T cells (Tregs), thereby suppressing inflammation . Additionally, MSCs inhibit the activity of Th1 and Th17 cells, which are critical in the autoimmune response of SS . MSCs can also inhibit the proliferation of B cells and the production of antibodies, including autoantibodies, by directly interacting with B cells and modulating their signaling pathways, thereby reducing the autoimmune response . Additionally, MSCs are effective in repairing damaged tissues . However, allogeneic MSC treatment for SS presents several challenges. These include the risk of immune rejection following transplantation, which can lead to inflammation, as well as the variability in outcomes depending on different cell donors . Therefore, autologous MSCs are an ideal choice for SS treatment. The patented technology developed by the laboratory of the first author (who is also a clinical trial volunteer in this paper) reprograms chemically induced skin fibroblasts into MSC-like cells, referred to as regenerative fibroblasts (rFib). This process preserves the cells’ genetic sequence and ensures their functional activity is unaffected by the health status of the volunteer . Safety evaluations have been completed for both single and multiple intravenous administrations of autologous rFib in monkeys. In this paper, despite treatment with UCMSC therapy, the volunteer’s condition continued to deteriorate, significantly impacting her work, life, and emotional well-being. Following a review by the medical ethics committee at the first author’s institution, the case was treated as that of a volunteer. Autologous rFib was used for SS treatment research in a compassionate use context. The results are reported as follows. Case report 2.1 Volunteer information The volunteer was pregnant in 2017 and frequently noticed small red spots on her feet. Since 2018, she has experienced dry mouth, dry eyes, and occasional red spots on her lower limbs. After speaking for 5 min without water, she struggles to pronounce words clearly. In March 2021, she sought medical attention due to widespread bruising in both lower limbs following prolonged standing. Tests revealed a positive anti-SSB antibody. A biopsy of the labial gland revealed focal infiltration of lymphocytes, with more than 50 cells/4 mm 2 . Dry eye indicators include a decreased lacrimal lake height, reduced tear film rupture time, and absence of active meibomian glands. These findings confirm a diagnosis of SS. 2.2 Therapeutic process MSCs are known for their immunomodulatory functions and have been reported to alleviate SS to some extent . With the volunteer’s consent, we followed the method by Liang et al. to prepare and test the quality of UCMSCs. Between May 2021 and November 2022, UCMSCs (5 × 10 7 cells per injection) were administered intravenously 10 times. Unfortunately, the volunteer’s symptoms, including purpura, dry mouth, and fatigue, did not improve, significantly affecting her work, daily life, and mental health. After a review by the Medical Ethics Committee at Kunming University and obtaining informed consent, skin fibroblasts were collected from the volunteer in May 2023 and reprogrammed into rFib using small molecule compounds, following the patented technology (US 11,674,122 B2) by Hu et al. . rFib can differentiate into osteoblasts, adipocytes, and chondrocytes . It secretes vascular endothelial growth factor (VEGF) and platelet-derived growth factor A (PDGFA) in vitro and exhibits immune regulatory functions . Following quality testing, the volunteer received 12 intravenous infusions of autologous rFib between October 2023 and June 2024. The first four times were administered weekly, while the last eight were scheduled every 4 weeks. For the initial seven infusions, the number of cells was 5 × 10 7 . Given the absence of adverse reactions during the clinical trial, the cell count was increased to 7.5 × 10 7 for the final five infusions. illustrates the treatment progression for this volunteer, including UCMSC and rFib. 2.3 Change of body condition The volunteer was diagnosed with SS in March 2021, but her condition did not improve after UCMSC treatment. She experienced dry mouth and had to wake up in the middle of the night to drink water. She also had reduced tear production and general weakness, particularly in her lower limbs, which felt heavy. Climbing the stairs of the 10th floor was difficult, requiring two to three rest breaks during the journey, and she frequently felt tired. Purpura frequently appeared on both lower limbs following fatigue, prolonged standing, or extended sitting . After four infusions of rFib, the volunteer experienced relief from dry mouth and dry eyes, and large areas of purpura rarely appeared on her lower limbs . After 12 transfusions of rFib, the volunteer experienced an increase in saliva production, no longer needed to get up during the night to drink water, and had tears. She felt stronger overall, with relaxed legs, and was able to hike 5 km up a mountain without feeling tired afterward. Additionally, the purpura marks on her lower limbs had faded . By November 2024, the purpura had completely disappeared from her lower limbs . 2.4 Changes in serological indexes Cytokines play an important role in the occurrence and development of SS . Therefore, we measured 12 related cytokines in serum before rFib therapy, as well as again after four and 12 treatments. Before the use of autologous rFib, levels of all cytokines, except interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α) were elevated, particularly the proinflammatory cytokines IL-1β, IL-6, and IL-17α, as well as the chemokine IL-8, which were more than 10 times higher than normal values. After four transfusions of rFib, the levels of IL-1β, IL-17α, and IL-8 decreased, while the levels of other cytokines increased compared to their levels before infusion. After 12 infusions of rFib, the values of seven cytokines (IFN-α, IFN-γ, IL-10, IL-1β, IL-5, IL-8, TNF-α) returned to normal levels, while the other five cytokines (IL-12p70, IL-17α, IL-2, IL-4, IL-6) remained elevated. However, the levels of the remaining cytokines were close to the normal range; notably, the level of IL-17α decreased from 199.63 to 9.76 pg/ml, which is only 0.45 pg/ml below the normal range . SS is an autoimmune inflammatory disease characterized by the infiltration of lymphocytes into the salivary and lacrimal glands, which ultimately leads to gland destruction. The activation of T lymphocytes and B cells triggers the infiltration of inflammatory cells, resulting in glandular tissue damage and metabolic changes associated with SS . We analyzed peripheral blood lymphocyte subsets before the infusion of rFib and after four and 12 infusions. Before the infusion of rFib, the proportion of NK cells and CD3+CD4+CD45+ T lymphocytes was below the reference value, while the proportion of B lymphocytes was above the reference value. After four transfusions of rFib, the proportion of NK cells increased and returned to the normal reference range. The proportion of B lymphocytes decreased after 12 transfusions of rFib, while the proportion of NK cells increased further, and the proportion of CD3+CD4+CD45+ T lymphocytes increased, returning to the normal reference range . Patients with SS typically show elevated levels of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in their serum . Consequently, we measured ESR and CRP levels before and after the injection of rFib. The results indicated that CRP remained within the normal range both before and after the infusion. In contrast, the initial ESR was 36 mm/h before the infusion of rFib, decreased to 31 mm/h after four infusions, and further decreased to 25 mm/h after 12 infusions of rFib . SS is an autoimmune disease characterized by the excessive production of autoantibodies, including rheumatoid factors (RF) . We similarly tested immunoglobulin and RF levels before and after the infusion of rFib. Before the infusion, RF A was 305.055 U/ml, and RF M was 331.472 U/ml. After four transfusions of rFib, RF A increased to 375.829 U/ml, while RF M decreased to 191.032 U/ml. After 12 transfusions of rFib, RF A decreased to 95.402 U/ml, while RF M decreased to 179.775 U/ml. These results are summarized in . One of the most severe organ and life-threatening complications of SS is cryoglobulinemic vasculitis . To determine whether the purpura on the volunteer’s legs was related to the immunoglobulin and complement levels, we measured the serum levels of immunoglobulins A, G, and M, as well as complement levels 3 and 4, before and after rFib injection. The results indicated that, before the rFib injection, immunoglobulins A and M, as well as complement levels 3 and 4, were normal, while immunoglobulin G levels were elevated. However, after the rFib injection, immunoglobulin G levels remained unchanged, even as the purpura disappeared . 2.5 Other indicators remain unchanged We conducted several examinations, including antinuclear antibody detection, echocardiogram, and chest CT, both before and after the infusion of rFib. However, since these measurements did not show any changes, they are not included in the results. Volunteer information The volunteer was pregnant in 2017 and frequently noticed small red spots on her feet. Since 2018, she has experienced dry mouth, dry eyes, and occasional red spots on her lower limbs. After speaking for 5 min without water, she struggles to pronounce words clearly. In March 2021, she sought medical attention due to widespread bruising in both lower limbs following prolonged standing. Tests revealed a positive anti-SSB antibody. A biopsy of the labial gland revealed focal infiltration of lymphocytes, with more than 50 cells/4 mm 2 . Dry eye indicators include a decreased lacrimal lake height, reduced tear film rupture time, and absence of active meibomian glands. These findings confirm a diagnosis of SS. Therapeutic process MSCs are known for their immunomodulatory functions and have been reported to alleviate SS to some extent . With the volunteer’s consent, we followed the method by Liang et al. to prepare and test the quality of UCMSCs. Between May 2021 and November 2022, UCMSCs (5 × 10 7 cells per injection) were administered intravenously 10 times. Unfortunately, the volunteer’s symptoms, including purpura, dry mouth, and fatigue, did not improve, significantly affecting her work, daily life, and mental health. After a review by the Medical Ethics Committee at Kunming University and obtaining informed consent, skin fibroblasts were collected from the volunteer in May 2023 and reprogrammed into rFib using small molecule compounds, following the patented technology (US 11,674,122 B2) by Hu et al. . rFib can differentiate into osteoblasts, adipocytes, and chondrocytes . It secretes vascular endothelial growth factor (VEGF) and platelet-derived growth factor A (PDGFA) in vitro and exhibits immune regulatory functions . Following quality testing, the volunteer received 12 intravenous infusions of autologous rFib between October 2023 and June 2024. The first four times were administered weekly, while the last eight were scheduled every 4 weeks. For the initial seven infusions, the number of cells was 5 × 10 7 . Given the absence of adverse reactions during the clinical trial, the cell count was increased to 7.5 × 10 7 for the final five infusions. illustrates the treatment progression for this volunteer, including UCMSC and rFib. Change of body condition The volunteer was diagnosed with SS in March 2021, but her condition did not improve after UCMSC treatment. She experienced dry mouth and had to wake up in the middle of the night to drink water. She also had reduced tear production and general weakness, particularly in her lower limbs, which felt heavy. Climbing the stairs of the 10th floor was difficult, requiring two to three rest breaks during the journey, and she frequently felt tired. Purpura frequently appeared on both lower limbs following fatigue, prolonged standing, or extended sitting . After four infusions of rFib, the volunteer experienced relief from dry mouth and dry eyes, and large areas of purpura rarely appeared on her lower limbs . After 12 transfusions of rFib, the volunteer experienced an increase in saliva production, no longer needed to get up during the night to drink water, and had tears. She felt stronger overall, with relaxed legs, and was able to hike 5 km up a mountain without feeling tired afterward. Additionally, the purpura marks on her lower limbs had faded . By November 2024, the purpura had completely disappeared from her lower limbs . Changes in serological indexes Cytokines play an important role in the occurrence and development of SS . Therefore, we measured 12 related cytokines in serum before rFib therapy, as well as again after four and 12 treatments. Before the use of autologous rFib, levels of all cytokines, except interferon-α (IFN-α) and tumor necrosis factor-α (TNF-α) were elevated, particularly the proinflammatory cytokines IL-1β, IL-6, and IL-17α, as well as the chemokine IL-8, which were more than 10 times higher than normal values. After four transfusions of rFib, the levels of IL-1β, IL-17α, and IL-8 decreased, while the levels of other cytokines increased compared to their levels before infusion. After 12 infusions of rFib, the values of seven cytokines (IFN-α, IFN-γ, IL-10, IL-1β, IL-5, IL-8, TNF-α) returned to normal levels, while the other five cytokines (IL-12p70, IL-17α, IL-2, IL-4, IL-6) remained elevated. However, the levels of the remaining cytokines were close to the normal range; notably, the level of IL-17α decreased from 199.63 to 9.76 pg/ml, which is only 0.45 pg/ml below the normal range . SS is an autoimmune inflammatory disease characterized by the infiltration of lymphocytes into the salivary and lacrimal glands, which ultimately leads to gland destruction. The activation of T lymphocytes and B cells triggers the infiltration of inflammatory cells, resulting in glandular tissue damage and metabolic changes associated with SS . We analyzed peripheral blood lymphocyte subsets before the infusion of rFib and after four and 12 infusions. Before the infusion of rFib, the proportion of NK cells and CD3+CD4+CD45+ T lymphocytes was below the reference value, while the proportion of B lymphocytes was above the reference value. After four transfusions of rFib, the proportion of NK cells increased and returned to the normal reference range. The proportion of B lymphocytes decreased after 12 transfusions of rFib, while the proportion of NK cells increased further, and the proportion of CD3+CD4+CD45+ T lymphocytes increased, returning to the normal reference range . Patients with SS typically show elevated levels of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) in their serum . Consequently, we measured ESR and CRP levels before and after the injection of rFib. The results indicated that CRP remained within the normal range both before and after the infusion. In contrast, the initial ESR was 36 mm/h before the infusion of rFib, decreased to 31 mm/h after four infusions, and further decreased to 25 mm/h after 12 infusions of rFib . SS is an autoimmune disease characterized by the excessive production of autoantibodies, including rheumatoid factors (RF) . We similarly tested immunoglobulin and RF levels before and after the infusion of rFib. Before the infusion, RF A was 305.055 U/ml, and RF M was 331.472 U/ml. After four transfusions of rFib, RF A increased to 375.829 U/ml, while RF M decreased to 191.032 U/ml. After 12 transfusions of rFib, RF A decreased to 95.402 U/ml, while RF M decreased to 179.775 U/ml. These results are summarized in . One of the most severe organ and life-threatening complications of SS is cryoglobulinemic vasculitis . To determine whether the purpura on the volunteer’s legs was related to the immunoglobulin and complement levels, we measured the serum levels of immunoglobulins A, G, and M, as well as complement levels 3 and 4, before and after rFib injection. The results indicated that, before the rFib injection, immunoglobulins A and M, as well as complement levels 3 and 4, were normal, while immunoglobulin G levels were elevated. However, after the rFib injection, immunoglobulin G levels remained unchanged, even as the purpura disappeared . Other indicators remain unchanged We conducted several examinations, including antinuclear antibody detection, echocardiogram, and chest CT, both before and after the infusion of rFib. However, since these measurements did not show any changes, they are not included in the results. Discussion From 2018 to 2023, the volunteer experienced dry mouth, purpura in both lower limbs, and fatigue, which significantly affected her daily life, work, and mood. Since rFib is derived from the volunteer’s own body and has undergone GLP safety evaluation in preclinical studies, intravenous infusion of autologous rFib was conducted under compassionate use conditions. Following autologous rFib treatment, a significant improvement was observed in the absence of widespread purpura. Symptoms such as dry mouth, fatigue, and difficulty walking briskly also improved greatly. Unfortunately, no ultrasound or lower limb skin biopsy was performed prior to the rFib cell infusion, nor was the saliva flow rate assessed. Consequently, the impact of these laboratory findings remains uncertain. Laboratory serology results showed significant changes in cytokines, lymphocyte subsets, and erythrocyte sedimentation rate before and after rFib infusion. Abnormal cytokine secretion plays a significant role in the pathogenesis of SS, including the overexpression of proinflammatory factors such as TNF-α, IFN-γ, IL-1β, IL-6, and IL-17, alongside reduced expression of the anti-inflammatory factor TGF-β . Prior to the infusion of autologous rFib, the expression levels of proinflammatory cytokines IL-1β, IL-6, IL-17α, and the chemokine IL-8 were over 10 times higher than normal, consistent with previous reports . After the infusion, the levels of highly expressed proinflammatory cytokines decreased to normal or near-normal levels. The histological features of SS include immune cell infiltration in the lacrimal and salivary glands, involving CD4+ Th cells, CD8+ cytotoxic T (Tc) cells, B cells, and plasma cells. CD4+ T cells predominate in the early stage of SS, whereas B cells or plasma cells become more dominant as the disease progresses . Before the infusion of rFib, the volunteer’s serum exhibited a reduced proportion of NK cells and an increased proportion of B lymphocytes. Following the infusion, NK cell levels returned to normal, while B lymphocyte proportions decreased . The ESR is a key inflammatory marker in SS volunteers . In this case, the ESR was 36 mm/h before the infusion of autogenic rFib, decreased to 31 mm/h after four transfusions, and further declined to 25 mm/h after 12 transfusions , indicating that rFib cells can effectively reduce ESR. The mechanism by which autologous rFib treats SS involves significant inhibition of PHA-stimulated lymphocyte proliferation, as demonstrated by our in vitro experimental results . Following autologous rFib therapy, changes in cytokines and lymphoid subsets in vivo may be related to the immunomodulatory functions of rFib. The levels of PDGFA and VEGF increased in the rFib culture medium . PDGF primarily facilitates angiogenesis and cell proliferation , while VEGF promotes angiogenesis and enhances tissue blood supply, potentially aiding in the functional recovery of glands. Moreover, VEGF plays a crucial role in regulating the inflammatory response and may influence the onset and progression of inflammation by modulating immune cell activity in the microenvironment . In a clinical study, Pan et al. used an anti-VEGF antibody to treat a volunteer with bilateral cystoid macular edema complicated by anaphylactoid purpura . We speculate that the disappearance of purpura in both lower limbs of the volunteer may be related to these two cytokines rather than serum immunoglobulin and complement levels . Previous studies indicate that allogeneic UCMSCs can alleviate dryness symptoms in both mouse models and SS patients . However, the volunteer with Sjögren’s syndrome in this study did not respond to allogeneic UCMSCs. We speculate that several factors may contribute to this outcome. First, UCMSCs are derived from various umbilical cord donors, each with a unique biological backgrounds. As a result, UCMSCs from different donors may produce varying therapeutic effects on SS. Secondly, while UCMSCs have low immunogenicity, they are eventually cleared after allotransplantation due to immune rejection by the host (SS patients), leading to short-lived and limited efficacy. The survival duration of UCMSCs in the host (SS patient) depends on both the immunogenicity of the UCMSCs and the immune status of the host. Our findings suggest that autologous rFib may help treat SS by modulating the immune system, reducing inflammatory cytokine production, and lowering the erythrocyte sedimentation rate. Although the volunteer’s physical condition has significantly improved, the limited treatment duration has resulted in few studies on the mechanism of autologous rFib in treating SS. In the future, we will continue to monitor the volunteer’s health changes and use mouse models to investigate the mechanism of autologous rFib in treating SS. Conclusion This clinical trial demonstrates that intravenous injection of autologous rFib is safe and effective for treating SS, potentially due to its immunomodulatory function. This is the first case in which autologous rFib cells have been used to treat SS. Our data are limited to purpura and serological indicators from the lower limbs, with no involvement of lung tissue, joints, or other lesions in this case. If future volunteers present with more severe symptoms, we can consider treating them with autologous rFib as well. |
Do summaries of evidence enable informed decision-making about COVID-19 and influenza vaccination equitably across more and less disadvantaged groups? Study protocol for a multi-centre cluster randomised controlled trial of ‘fact boxes’ in health and social care in Germany | 25daf338-7776-4a97-abce-2f617e9884d1 | 11529688 | Health Communication[mh] | Background Ensuring informed consent as an ethical principle for medical research involving human subjects is enshrined in the Declaration of Helsinki. This consent relies on informed choices for or against medical interventions and requires evidence-based health information (EBHI) being comprehensible and transparent about the benefits and harms of all treatment options. However, information often fails evidence-based and risk communication criteria, which means that people facing a health decision are not enabled to make a decision based on evidence-based knowledge and in line with their personal preferences (informed choice). One example are package leaflets that are written at a higher reading level and therefore difficult to access, particularly for people with low health literacy or other influencing constructs such as low education —making informed decisions more difficult. As a result, people are more likely to experience regret, reduced ability to use medicines appropriately and poorer health, and are less likely to opt for follow-up treatment (eg, booster vaccinations). In particular, there are disadvantaged groups in society who make less informed decisions (eg, less educated, non-native speakers) —leading to inequities. To reduce inequities and achieve high-quality healthcare, the structures in the healthcare sector and approaches (eg, communication formats and channels for patient education) must enable disadvantaged people to access and navigate the health system for effectively managing their health and care. Patient-oriented information such as EBHI and decision aids (DAs) are key elements in improving health communication and thus healthcare by enabling more people to make informed and shared decisions. The benefits of such information over standard care (no evidence-based information or no intervention) are already clear. A Cochrane review found that DAs have a positive effect on knowledge, information and perception of values, and that decision makers are likely to take a more active role in the decision-making process and have a better perception of risk. However, there is a risk that only certain groups of the population will benefit from EBHI and DAs. Even if materials are ‘target group-oriented’, the factors that lead to inequity in terms of shared and informed decision-making have not yet been sufficiently taken into account in their development process. For example, there are many patient-oriented materials written at an advanced level, which makes them less accessible to people with reading difficulties, lower education or health literacy. Overall, only few studies have looked specifically at the impact of shared decision-making (SDM) or informed decision-making in groups that are more likely to have communication barriers due to their racial or ethnic background and/or level of education. Objectives The aim of our study is to assess whether evidence summaries using the fact box format can facilitate informed decisions for laypeople from different social backgrounds. Further, we explore whether these fact boxes contribute to reducing inequity in healthcare—with the help of shared decision-making. Fact boxes provide a tabular or graphical overview of the benefits and harms of medical options through transparent risk communication. They are used to inform a range of health decisions, such as medical treatments, cancer screening and vaccinations (see https://hardingcenter.de/en/transfer-and-impact/fact-boxes ). In contrast to DAs, fact boxes do not contain preference elicitation. By reducing complexity and balancing evidence, they aim to promote informed decisions instead of predetermined behavioural change (eg, having a vaccine). Consequently, they are in line with those Western health standards and ethical principles that differ from persuasion or nudging, which can be at odds with informed choice and pose the risk of undermining the trustworthiness and credibility of the communicator and vaccinator. Fact boxes are developed according to the Medical Research Council guideline Development and Evaluation of Complex Interventions. Several experiments have shown that fact boxes support informed decisions by improving, for example, risk perception, short-term knowledge and knowledge recall, and comprehension. At present, it is unclear whether fact boxes can be an effective format outside (online) laboratory conditions. Further, fact boxes have been tested with the lay public, but only three studies analysed its effect by vulnerability factors. Additionally, our study should reveal to what extent evidence-based summaries using the fact box format can be integrated into the communication of health educators (eg, doctors) in standard care and outreach settings, and what added value they have in supporting SDM compared with conventional information or no information at all. Although there is evidence for the benefits of SDM interventions (eg, EBHI) in improving people’s knowledge of risks and benefits, feeling informed and clarity about their values in a variety of decision-making contexts, and thus for their use in clinical practice, the real-world effects of implementing EBHI as an SDM intervention are still unclear. What are the prerequisites, facilitators and barriers for people to benefit from them in practice? What are the prerequisites and barriers for clinicians to implement them? Already in 2013, the authors of a review called for more research into the extent to which EBHI and DAs are effective in medical care, as they are not yet widely used in medical practice. Moreover, in 2019 only 21% of DAs were introduced after trials and a further 7% were part of implementation studies. Barriers included outdated evidence in DAs and disagreement among clinicians about the use of DAs. This paper reports on the study protocol of an implementation study and equity evaluation of evidence-based summaries on COVID-19 and influenza vaccination in different health education settings in Germany using fact box formats. The trial was prospectively registered on clinicaltrials.gov (NCT06076421). Acknowledging that public health initiatives generally aim to promote vaccination to protect the population from vaccine-preventable death and disease (societal perspective), individual autonomy and informed choice are ensured in many health systems (eg, ‘the 2013 German Patients' Rights Act’ ). Therefore, the aim of health campaigns is also to provide comprehensive information about the benefits and harms of vaccination, to consider the needs of citizens and patients, and to increase transparency and individual understanding of the social benefits of vaccination through education (see also Goal 3 of the U.S. Vaccines National Strategic Plan ). Improving public understanding of vaccination policy in order to strengthen trust and compliance is crucial for future positive vaccination behaviour—particularly so in the case of seasonal vaccinations. Thus, the study will provide insights into the extent to which non-directed evidence summaries, with a focus on supporting informed decision-making, can contribute to achieving public health goals. The study will therefore be of interest to other organisations that produce health information and is thus relevant to both producers and disseminators of health-related information, as well as to decision-makers at various levels who develop strategies to achieve societal goals for maintaining and promoting public health. Ensuring informed consent as an ethical principle for medical research involving human subjects is enshrined in the Declaration of Helsinki. This consent relies on informed choices for or against medical interventions and requires evidence-based health information (EBHI) being comprehensible and transparent about the benefits and harms of all treatment options. However, information often fails evidence-based and risk communication criteria, which means that people facing a health decision are not enabled to make a decision based on evidence-based knowledge and in line with their personal preferences (informed choice). One example are package leaflets that are written at a higher reading level and therefore difficult to access, particularly for people with low health literacy or other influencing constructs such as low education —making informed decisions more difficult. As a result, people are more likely to experience regret, reduced ability to use medicines appropriately and poorer health, and are less likely to opt for follow-up treatment (eg, booster vaccinations). In particular, there are disadvantaged groups in society who make less informed decisions (eg, less educated, non-native speakers) —leading to inequities. To reduce inequities and achieve high-quality healthcare, the structures in the healthcare sector and approaches (eg, communication formats and channels for patient education) must enable disadvantaged people to access and navigate the health system for effectively managing their health and care. Patient-oriented information such as EBHI and decision aids (DAs) are key elements in improving health communication and thus healthcare by enabling more people to make informed and shared decisions. The benefits of such information over standard care (no evidence-based information or no intervention) are already clear. A Cochrane review found that DAs have a positive effect on knowledge, information and perception of values, and that decision makers are likely to take a more active role in the decision-making process and have a better perception of risk. However, there is a risk that only certain groups of the population will benefit from EBHI and DAs. Even if materials are ‘target group-oriented’, the factors that lead to inequity in terms of shared and informed decision-making have not yet been sufficiently taken into account in their development process. For example, there are many patient-oriented materials written at an advanced level, which makes them less accessible to people with reading difficulties, lower education or health literacy. Overall, only few studies have looked specifically at the impact of shared decision-making (SDM) or informed decision-making in groups that are more likely to have communication barriers due to their racial or ethnic background and/or level of education. The aim of our study is to assess whether evidence summaries using the fact box format can facilitate informed decisions for laypeople from different social backgrounds. Further, we explore whether these fact boxes contribute to reducing inequity in healthcare—with the help of shared decision-making. Fact boxes provide a tabular or graphical overview of the benefits and harms of medical options through transparent risk communication. They are used to inform a range of health decisions, such as medical treatments, cancer screening and vaccinations (see https://hardingcenter.de/en/transfer-and-impact/fact-boxes ). In contrast to DAs, fact boxes do not contain preference elicitation. By reducing complexity and balancing evidence, they aim to promote informed decisions instead of predetermined behavioural change (eg, having a vaccine). Consequently, they are in line with those Western health standards and ethical principles that differ from persuasion or nudging, which can be at odds with informed choice and pose the risk of undermining the trustworthiness and credibility of the communicator and vaccinator. Fact boxes are developed according to the Medical Research Council guideline Development and Evaluation of Complex Interventions. Several experiments have shown that fact boxes support informed decisions by improving, for example, risk perception, short-term knowledge and knowledge recall, and comprehension. At present, it is unclear whether fact boxes can be an effective format outside (online) laboratory conditions. Further, fact boxes have been tested with the lay public, but only three studies analysed its effect by vulnerability factors. Additionally, our study should reveal to what extent evidence-based summaries using the fact box format can be integrated into the communication of health educators (eg, doctors) in standard care and outreach settings, and what added value they have in supporting SDM compared with conventional information or no information at all. Although there is evidence for the benefits of SDM interventions (eg, EBHI) in improving people’s knowledge of risks and benefits, feeling informed and clarity about their values in a variety of decision-making contexts, and thus for their use in clinical practice, the real-world effects of implementing EBHI as an SDM intervention are still unclear. What are the prerequisites, facilitators and barriers for people to benefit from them in practice? What are the prerequisites and barriers for clinicians to implement them? Already in 2013, the authors of a review called for more research into the extent to which EBHI and DAs are effective in medical care, as they are not yet widely used in medical practice. Moreover, in 2019 only 21% of DAs were introduced after trials and a further 7% were part of implementation studies. Barriers included outdated evidence in DAs and disagreement among clinicians about the use of DAs. This paper reports on the study protocol of an implementation study and equity evaluation of evidence-based summaries on COVID-19 and influenza vaccination in different health education settings in Germany using fact box formats. The trial was prospectively registered on clinicaltrials.gov (NCT06076421). Acknowledging that public health initiatives generally aim to promote vaccination to protect the population from vaccine-preventable death and disease (societal perspective), individual autonomy and informed choice are ensured in many health systems (eg, ‘the 2013 German Patients' Rights Act’ ). Therefore, the aim of health campaigns is also to provide comprehensive information about the benefits and harms of vaccination, to consider the needs of citizens and patients, and to increase transparency and individual understanding of the social benefits of vaccination through education (see also Goal 3 of the U.S. Vaccines National Strategic Plan ). Improving public understanding of vaccination policy in order to strengthen trust and compliance is crucial for future positive vaccination behaviour—particularly so in the case of seasonal vaccinations. Thus, the study will provide insights into the extent to which non-directed evidence summaries, with a focus on supporting informed decision-making, can contribute to achieving public health goals. The study will therefore be of interest to other organisations that produce health information and is thus relevant to both producers and disseminators of health-related information, as well as to decision-makers at various levels who develop strategies to achieve societal goals for maintaining and promoting public health. The protocol follows the SPIRIT 2013 guideline and CONSORT Equity Reporting Guideline 2017. Design and setting We will conduct a cluster randomised controlled trial with a 2×2-between-subjects design: format (usual health communication plus fact box vs usual health communication) and health educator (cluster unit medical practice vs cluster unit outreach work settings) . Health professionals in particular are still the first-contact for people seeking health information in Germany. Therefore, the cluster units will include all those who communicate about health issues (eg, doctors and other health professionals, community health workers) in Germany, as well as settings where traditional communication about disease prevention takes place (eg, doctors’ offices) and where communication about health issues is carried out in the context of outreach work (mothers’ and fathers’ cafés, intercultural meeting places) if more than one person per unit participates. We will refer to these units as health educators. The study started in November 2023, and data collection is expected to be completed towards the likely end of the influenza season in Germany (Spring 2025). Research questions Do disadvantaged people benefit to the same extent as non-disadvantaged people in terms of informed and shared decision-making from receiving COVID-19 and influenza vaccination fact boxes as opposed to standard vaccination communication in medical practices and outreach work (field settings)? Disadvantaged populations In order to reduce inequities in healthcare, it should be recognised that certain individual and sociocultural factors affect health opportunities and outcomes and lead to disadvantages in the impact of interventions. These factors are defined within the PROGRESS framework as place of residence, race/ethnicity/culture/language, occupation, gender/sex, religion, socioeconomic status and social capital. Ignoring them can exacerbate health inequalities, even if public health interventions (eg, education, communication and information) are generally effective. For examining the impact of our intervention, we take the following factors and concepts into account : education, subjective socioeconomic status (SSS), health literacy as well as mother tongue (German, Arabic, Turkish, Russian, other) and language (subjective native reading literacy, perception of own German language skills). Research questions (RQ) and main hypotheses (HYP) RQ1 Is the use of fact boxes more effective than standard communication (control condition) in the field? Primary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Higher vaccination knowledge: A number of studies have shown that fact boxes improve short-term knowledge and recall and comprehension, enabling people to weigh the benefits and harms of an intervention. More vaccination intentions that are in line with attitudes and vaccination knowledge (informed vaccination intentions): Because knowledge is a component of informed choice and attitudes about vaccines’ benefit–harm ratios are not biased by the presentation of a vaccine fact box. Secondary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Improved risk perception: One study found that studying fact boxes can improve accuracy of COVID-19 risk perceptions without affecting vaccination intentions. Increased patient involvement in medical decision-making: As based on data from a Cochrane Review evidence-based tools aimed at enabling informed decision-making have the potential to increase patient involvement. Decreased decisional conflict: A study found that, compared with controls, participants who received fact boxes experienced less decisional conflict about antibiotic use (non-standardised beta (β) = −8.35, 95% CI, −12.43, −4.28) and artificial hydration (β = −6.02, 95% CI, −9.84, –2.20) at 1 month compared with baseline. RQ2 Are fact boxes as effective for people with disadvantaging factors as for those without those factors? HYP. We will analyse the effect of fact boxes compared with usual care among people with more and less advantaging factors (eg, in terms of education), taking into account known baseline differences between advantaged and disadvantaged people. The added value of evidence-based tools (eg, DAs) over traditional (non-evidence-based or usual) healthcare and interventions tailored to specific target groups (eg, people with low levels of education) has already been demonstrated. We therefore expect that the gap will narrow between more and less advantaged people in terms of outcomes relevant for informed vaccination decisions. We hypothesise that the implementation of fact boxes compared with standard vaccination communication will lead to a greater alignment of knowledge, informed vaccination intentions and accuracy of risk perception between: People with low and high education. People with problematic or inadequate and excellent or sufficient level of health literacy. Non-native (Arabic, Turkish and Russian speaking participants, only with low German skills) and native German speakers (including non-native speakers with high German skills), because we not only provided information in the native language but also tested it with these target groups. People with low and high reading literacy in different languages, because fact boxes are a complexity-reduced format of health information and we tried to address accessibility through pilot testing with different groups. RQ3 Does the use of fact boxes in outreach work promote more shared and informed decision-making than in regular healthcare settings? HYP. We assume that fact boxes more likely lead to more informed vaccination intentions and shared decisions in outreach work than in regular healthcare settings. In order to respond to the needs of especially people with disadvantaging factors and to reach them in a more effective way, community-based health services are becoming more and more important. The use of non-primary care professionals (eg, social workers, community nurses or community health workers) who can build trust and provide psychoeducational interventions has been shown to be effective in providing health knowledge, improving healthcare, changing health behaviour and improving health status. Explorative analyses We will analyse the effect of fact boxes on knowledge, vaccination intentions and accuracy of risk perception between people with low and medium or high SSS through fact boxes compared with usual vaccination communication. We will analyse the effect of fact boxes on vaccination intentions, knowledge, risk perception, patient involvement and decisional conflict among people with migration-related indicators (eg, residential status, length of stay in Germany) compared with usual care, controlling for other factors describing the social situation (eg, education, health literacy). This may be crucial because people with immigrant history are less receptive to information from authorities or health providers in Germany, the host country (even if it is translated), if they have less trust in them. Also campaigns or formats that work for the majority of the host population may not be the right channel for communicating with immigrant groups. An increased length of stay is associated with a greater orientation towards the host society and thus a change in lifestyle habits, health perceptions and health behaviour. On the other hand, a shorter duration of stay, as well as not having German citizenship or a residence permit, may be associated with higher barriers to access to the German healthcare system (eg, due to lack of insurance or more difficult access to (multilingual) health information). Eligibility and recruitment strategy Study participants in general The trial is open to people who are currently facing a decision about vaccination for themselves or a family member (eg, caring relatives). Adults of legal age with current residence in Germany and who speak German, Russian, Turkish or Arabic will be included. All participants must confirm their informed consent online to take part in our study. Patients who do not speak any of the four languages and who do not give informed consent will be excluded. Health educators will be asked to inform all potential participants about the study. The health educators will then distribute the study flyer as part of the health communication or afterwards. To ensure sufficient variability of a potential disadvantage status, the recruitment of health educators is focused on facilities and practices that serve a very heterogeneous audience. In particular, involving outreach organisations will ensure that disadvantaged people who are underserved are reached. In addition, health educators will be sensitised to approach these groups but will also be selected on the basis of their geographical area, for example, in districts with higher socioeconomic deprivation (socioeconomic differences that lead to differences in health outcomes). In addition, health authorities will be asked to help identify facilities and practices that primarily serve a disadvantaged population. In order to make the study as accessible as possible for the target group, information about the study and about data protection will be provided in their preferred and in plain language. Those who decide to take part in our study will complete an online survey. There will also be a read-aloud function for the questionnaire in all languages. After completing the initial online survey (T1), participants will have the opportunity to provide their email address for a follow-up survey 4 weeks later (T2). At the end of this second survey, participants will be rewarded with a voucher worth 15 Euro. Health educators Key requirement for involving health educators is that they provide information on health-related topics and have access to potential study participants. Furthermore, informed written consent is a prerequisite. To reach health educators, we will contact German public health authorities and ask them to distribute the study call to all kinds of health educators in their respective areas of responsibility (districts). We will also directly contact potential health educators by email or post. All health educators will first be given general information about the study and asked for their consent. Health educators will not be remunerated. Our study will be conducted in close cooperation with the health departments in Germany, which provides access to the planned cluster settings and the target groups. Intervention Health educators in the intervention group will receive a flyer with a brief description of the study, a QR code and a link to an online survey, including on the reverse side a fact box about COVID-19 or the influenza vaccine in Arabic, German, Russian or Turkish. In several prestudies, the intervention underwent a comprehensive development process. This included feedback from various public health stakeholders on a COVID-19 fact box implemented in January 2021 by the Robert Koch Institute, the German national health authority. Further, information needs and requirements of the population in Germany were incorporated in updates with the help of secondary data analyses. Simplified COVID-19 and influenza vaccination fact boxes were tested for comprehensibility in 52 cognitive interviews between July 2021 and September 2022. First, a tabular COVID-19 fact box was tested with German-speaking participants and translated into a visualised fact box, which was then tested with Arabic-, Turkish- and Russian-speaking participants from disadvantaged districts of Berlin. Cognitive interviewing methods were used to ask whether the information in the fact boxes was understood and how it was understood. This method is usually used to pretest questionnaires. In individual interviews by telephone (during the COVID-19 period) and in person, a partially standardised questionnaire was used to ask respondents to reproduce the content of the fact boxes and to identify any comprehension problems in terms of content and wording. A visualised influenza fact box was piloted by the University of Erfurt with German-speaking laypeople with low numeracy. The non-directional vaccination fact boxes will be available either in tabular form (for influenza) or as a visualisation (for COVID-19), each in all languages and for both age groups. COVID-19 and influenza vaccine fact boxes are available for two different age groups each: COVID-19 vaccination for people aged 18–59 and over 60; influenza vaccination for people aged 16–64 and over 65. Accordingly, the study neither compares fact box formats (tabular, visual) nor examines disease-specific effects. Health educators are free to decide if and how to use the fact boxes, whether during, before or after vaccination education and whom to address. Therefore, there will be no training, as we cannot expect a one-size-fits-all solution for all health educators in different settings and especially in outreach settings (which are very heterogeneous). By asking health educators about their use of the fact boxes as part of a telephone assessment at the end of the study, we want to find out if and how they can be implemented in usual care. Control condition Health educators in the control condition will also receive the flyer with a brief description of the study, a QR code and a link to the online study to distribute to potential study participants, but without the fact box on the back. Patient and public involvement As described in the Intervention section, the COVID-19 and influenza vaccination fact boxes were developed through a comprehensive process involving various stakeholders and in particular the target groups, at all stages: (1) Information needs and requirements of the population in Germany were incorporated in updates with the help of secondary data analyse, (2) Simplified COVID-19 and influenza tabular vaccination fact boxes were tested for comprehensibility in 52 cognitive interviews with German-, Arabic-, Turkish- and Russian-speaking participants from disadvantaged districts of Berlin and (3) A visualised influenza fact box was piloted with German-speaking laypeople with low numeracy. The study design, outcomes, strategies for reaching vulnerable target groups and the questionnaire were discussed with various expert groups (eg, scientists from different disciplines, representatives of health authorities and outreach work). Randomisation and allocation concealment First, health educators will be randomised, either at the individual level or at the institutional level if there is more than one health educator per setting. Health educators who gave written informed consent will be assigned a study ID number and randomly assigned to one of the two study conditions: (1) usual health communication plus fact box (intervention) or (2) no information/usual health communication (control) (see ) by an independent researcher using computer-generated random numbers in a 1:1 ratio. Health educators will then distribute their assigned flyer format to individuals who are about to make a vaccination decision about the COVID-19 or the influenza vaccine, taking into account their reference group (age group). Blinding Health educators and study participants will not know which study condition they belong to. Health educators will be assigned by a person independent of the study, which means that the assignment will be blinded. Health educators in the control group will also have the opportunity to receive the fact boxes for their health communication post-trial. The data analyst will also be blinded. Outcomes Health educators Preintervention the type of health education setting (doctor’s office or outreach work (alone or in a community setting/group practice)) is registered. Health educators will be asked about perceived changes in vaccination behaviour from the start of the intervention, once a cluster is no longer recruiting. Health educators will be asked about their demographics (age, gender, mother tongue) and, in the intervention group, how they used the fact boxes (during, before or after vaccination communication) and about involving patients from their perspective (SDM-Q-Doc). Participants Sociodemographic information and outcomes will be collected after people receive the flyer from their health educator and access the study link or QR code. As the study is voluntary and those who approach people do not know who among those approached will participate in the study, we will not be able to control who participates in the study and we will not be able to follow-up with those approached unless they complete the first survey. All information will be collected at the time of the first survey (T1), as participation in the second survey (T2) 4 weeks after the initial survey is also voluntary. The primary outcomes are knowledge and informed vaccination intention. For exploratory purposes, we will analyse on knowledge differences on the item level. The secondary outcomes are risk perception, decisional conflict and patient involvement . Furthermore, we will collect sociodemographic information, SSS, migration history and health literacy . Study participants will be also asked whether the fact box was used as part of the counselling process and how they judged it. Sample size We aim to demonstrate moderate main effects (equal to Cohen’s d=0.50) of the study intervention on informed intentions (10% expected in usual care) and a knowledge sum score (equal to the proportion of correct responses; with an expected variance of 0.20). Taking into account a cluster correlation coefficient (ICC=0.10) common for primary care studies with patient endpoints, we will target three health educators (eg, doctors’ offices) per study arm with 40 patients/clients per health educator. So, 240 participants in each subgroup undergoing the same analysis are required . Furthermore, to demonstrate small-to-moderate interaction effects (equal to Cohen’s d=0.375) of the study intervention in respective settings on informed intentions and knowledge, we have to target instead seven health educators per study arm with 40 patients/clients per health educator (n=560). In total, we aim at 800 participants because the proportion of subgroup members cannot be predicted, and we cannot rule out that interaction effects in the field are smaller than expected from experimental evidence (eg, d=0.20 would call for up to 20 health educators per study arm). Further details on the assumptions underlying the sample size calculations in the power calculation with respect to the critical hypotheses and outcomes are provided in in the supplementary section. Data collection and management After giving their online consent, participants will complete an online survey via SoSciSurvey. The survey (T1) is completely anonymous for the study participants. Personal data (email address) will be only collected if there is interest in participating in the follow-up survey (T2) and in the remuneration, for which also first name and surname will be recorded. For this purpose, a computer-generated random ID will be generated by someone independent of the study, which is stored with the unblinding list separately from the collected personal and survey data at the Harding Center (HC) on password-protected computers and is only accessible to researchers of the HC until the end of the data collection. The survey of the health educators will be conducted under pseudonym. The random generated ID, personal and survey data will be separated from the contact information immediately after completion of the postsurvey, and the de-blinding list is also kept until the end of the data collection. All relevant data will be provided in the manuscript and supporting information or made available in a public repository after the completion of the study. Statistical analysis Data analysis will initially be based on descriptive statistics as well as frequency distributions and histograms to identify outliers and missing data. Baseline data of the study arms will be compared to see if the distribution is balanced between study sites/clusters. SPSS will be used to conduct all analyses. All participants will be asked to indicate at T1 whether they received the intervention to allow for intention-to-treat and as-treat analyses. Statistical methods for analysing primary and secondary outcomes We use linear mixed models to analyse the influence of both individual (eg, education status, health literacy) and cluster level factors (eg, setting) and account for the expected cluster variability in realising usual care or the intervention (details are outlined in ). Missing data We will record the number of flyers distributed per cluster. Because participation is voluntary, anonymous and not aware to the health educators, we will not be able to send a reminder and record the response rate for those who were approached. We will record the numbers of the total randomised sample for each group as well as the reasons for exclusion. It is compulsory for participants to answer all questions within the surveys. As participation in the follow-up survey is voluntary, we will record the number of drop-outs from the first to the second survey. We will conduct a cluster randomised controlled trial with a 2×2-between-subjects design: format (usual health communication plus fact box vs usual health communication) and health educator (cluster unit medical practice vs cluster unit outreach work settings) . Health professionals in particular are still the first-contact for people seeking health information in Germany. Therefore, the cluster units will include all those who communicate about health issues (eg, doctors and other health professionals, community health workers) in Germany, as well as settings where traditional communication about disease prevention takes place (eg, doctors’ offices) and where communication about health issues is carried out in the context of outreach work (mothers’ and fathers’ cafés, intercultural meeting places) if more than one person per unit participates. We will refer to these units as health educators. The study started in November 2023, and data collection is expected to be completed towards the likely end of the influenza season in Germany (Spring 2025). Do disadvantaged people benefit to the same extent as non-disadvantaged people in terms of informed and shared decision-making from receiving COVID-19 and influenza vaccination fact boxes as opposed to standard vaccination communication in medical practices and outreach work (field settings)? Disadvantaged populations In order to reduce inequities in healthcare, it should be recognised that certain individual and sociocultural factors affect health opportunities and outcomes and lead to disadvantages in the impact of interventions. These factors are defined within the PROGRESS framework as place of residence, race/ethnicity/culture/language, occupation, gender/sex, religion, socioeconomic status and social capital. Ignoring them can exacerbate health inequalities, even if public health interventions (eg, education, communication and information) are generally effective. For examining the impact of our intervention, we take the following factors and concepts into account : education, subjective socioeconomic status (SSS), health literacy as well as mother tongue (German, Arabic, Turkish, Russian, other) and language (subjective native reading literacy, perception of own German language skills). Research questions (RQ) and main hypotheses (HYP) RQ1 Is the use of fact boxes more effective than standard communication (control condition) in the field? Primary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Higher vaccination knowledge: A number of studies have shown that fact boxes improve short-term knowledge and recall and comprehension, enabling people to weigh the benefits and harms of an intervention. More vaccination intentions that are in line with attitudes and vaccination knowledge (informed vaccination intentions): Because knowledge is a component of informed choice and attitudes about vaccines’ benefit–harm ratios are not biased by the presentation of a vaccine fact box. Secondary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Improved risk perception: One study found that studying fact boxes can improve accuracy of COVID-19 risk perceptions without affecting vaccination intentions. Increased patient involvement in medical decision-making: As based on data from a Cochrane Review evidence-based tools aimed at enabling informed decision-making have the potential to increase patient involvement. Decreased decisional conflict: A study found that, compared with controls, participants who received fact boxes experienced less decisional conflict about antibiotic use (non-standardised beta (β) = −8.35, 95% CI, −12.43, −4.28) and artificial hydration (β = −6.02, 95% CI, −9.84, –2.20) at 1 month compared with baseline. RQ2 Are fact boxes as effective for people with disadvantaging factors as for those without those factors? HYP. We will analyse the effect of fact boxes compared with usual care among people with more and less advantaging factors (eg, in terms of education), taking into account known baseline differences between advantaged and disadvantaged people. The added value of evidence-based tools (eg, DAs) over traditional (non-evidence-based or usual) healthcare and interventions tailored to specific target groups (eg, people with low levels of education) has already been demonstrated. We therefore expect that the gap will narrow between more and less advantaged people in terms of outcomes relevant for informed vaccination decisions. We hypothesise that the implementation of fact boxes compared with standard vaccination communication will lead to a greater alignment of knowledge, informed vaccination intentions and accuracy of risk perception between: People with low and high education. People with problematic or inadequate and excellent or sufficient level of health literacy. Non-native (Arabic, Turkish and Russian speaking participants, only with low German skills) and native German speakers (including non-native speakers with high German skills), because we not only provided information in the native language but also tested it with these target groups. People with low and high reading literacy in different languages, because fact boxes are a complexity-reduced format of health information and we tried to address accessibility through pilot testing with different groups. RQ3 Does the use of fact boxes in outreach work promote more shared and informed decision-making than in regular healthcare settings? HYP. We assume that fact boxes more likely lead to more informed vaccination intentions and shared decisions in outreach work than in regular healthcare settings. In order to respond to the needs of especially people with disadvantaging factors and to reach them in a more effective way, community-based health services are becoming more and more important. The use of non-primary care professionals (eg, social workers, community nurses or community health workers) who can build trust and provide psychoeducational interventions has been shown to be effective in providing health knowledge, improving healthcare, changing health behaviour and improving health status. In order to reduce inequities in healthcare, it should be recognised that certain individual and sociocultural factors affect health opportunities and outcomes and lead to disadvantages in the impact of interventions. These factors are defined within the PROGRESS framework as place of residence, race/ethnicity/culture/language, occupation, gender/sex, religion, socioeconomic status and social capital. Ignoring them can exacerbate health inequalities, even if public health interventions (eg, education, communication and information) are generally effective. For examining the impact of our intervention, we take the following factors and concepts into account : education, subjective socioeconomic status (SSS), health literacy as well as mother tongue (German, Arabic, Turkish, Russian, other) and language (subjective native reading literacy, perception of own German language skills). RQ1 Is the use of fact boxes more effective than standard communication (control condition) in the field? Primary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Higher vaccination knowledge: A number of studies have shown that fact boxes improve short-term knowledge and recall and comprehension, enabling people to weigh the benefits and harms of an intervention. More vaccination intentions that are in line with attitudes and vaccination knowledge (informed vaccination intentions): Because knowledge is a component of informed choice and attitudes about vaccines’ benefit–harm ratios are not biased by the presentation of a vaccine fact box. Secondary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Improved risk perception: One study found that studying fact boxes can improve accuracy of COVID-19 risk perceptions without affecting vaccination intentions. Increased patient involvement in medical decision-making: As based on data from a Cochrane Review evidence-based tools aimed at enabling informed decision-making have the potential to increase patient involvement. Decreased decisional conflict: A study found that, compared with controls, participants who received fact boxes experienced less decisional conflict about antibiotic use (non-standardised beta (β) = −8.35, 95% CI, −12.43, −4.28) and artificial hydration (β = −6.02, 95% CI, −9.84, –2.20) at 1 month compared with baseline. RQ2 Are fact boxes as effective for people with disadvantaging factors as for those without those factors? HYP. We will analyse the effect of fact boxes compared with usual care among people with more and less advantaging factors (eg, in terms of education), taking into account known baseline differences between advantaged and disadvantaged people. The added value of evidence-based tools (eg, DAs) over traditional (non-evidence-based or usual) healthcare and interventions tailored to specific target groups (eg, people with low levels of education) has already been demonstrated. We therefore expect that the gap will narrow between more and less advantaged people in terms of outcomes relevant for informed vaccination decisions. We hypothesise that the implementation of fact boxes compared with standard vaccination communication will lead to a greater alignment of knowledge, informed vaccination intentions and accuracy of risk perception between: People with low and high education. People with problematic or inadequate and excellent or sufficient level of health literacy. Non-native (Arabic, Turkish and Russian speaking participants, only with low German skills) and native German speakers (including non-native speakers with high German skills), because we not only provided information in the native language but also tested it with these target groups. People with low and high reading literacy in different languages, because fact boxes are a complexity-reduced format of health information and we tried to address accessibility through pilot testing with different groups. RQ3 Does the use of fact boxes in outreach work promote more shared and informed decision-making than in regular healthcare settings? HYP. We assume that fact boxes more likely lead to more informed vaccination intentions and shared decisions in outreach work than in regular healthcare settings. In order to respond to the needs of especially people with disadvantaging factors and to reach them in a more effective way, community-based health services are becoming more and more important. The use of non-primary care professionals (eg, social workers, community nurses or community health workers) who can build trust and provide psychoeducational interventions has been shown to be effective in providing health knowledge, improving healthcare, changing health behaviour and improving health status. Is the use of fact boxes more effective than standard communication (control condition) in the field? Primary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Higher vaccination knowledge: A number of studies have shown that fact boxes improve short-term knowledge and recall and comprehension, enabling people to weigh the benefits and harms of an intervention. More vaccination intentions that are in line with attitudes and vaccination knowledge (informed vaccination intentions): Because knowledge is a component of informed choice and attitudes about vaccines’ benefit–harm ratios are not biased by the presentation of a vaccine fact box. Secondary HYP. We hypothesise that compared with standard vaccination communication implementation of fact boxes in medical practices and outreach work will lead to: Improved risk perception: One study found that studying fact boxes can improve accuracy of COVID-19 risk perceptions without affecting vaccination intentions. Increased patient involvement in medical decision-making: As based on data from a Cochrane Review evidence-based tools aimed at enabling informed decision-making have the potential to increase patient involvement. Decreased decisional conflict: A study found that, compared with controls, participants who received fact boxes experienced less decisional conflict about antibiotic use (non-standardised beta (β) = −8.35, 95% CI, −12.43, −4.28) and artificial hydration (β = −6.02, 95% CI, −9.84, –2.20) at 1 month compared with baseline. Are fact boxes as effective for people with disadvantaging factors as for those without those factors? HYP. We will analyse the effect of fact boxes compared with usual care among people with more and less advantaging factors (eg, in terms of education), taking into account known baseline differences between advantaged and disadvantaged people. The added value of evidence-based tools (eg, DAs) over traditional (non-evidence-based or usual) healthcare and interventions tailored to specific target groups (eg, people with low levels of education) has already been demonstrated. We therefore expect that the gap will narrow between more and less advantaged people in terms of outcomes relevant for informed vaccination decisions. We hypothesise that the implementation of fact boxes compared with standard vaccination communication will lead to a greater alignment of knowledge, informed vaccination intentions and accuracy of risk perception between: People with low and high education. People with problematic or inadequate and excellent or sufficient level of health literacy. Non-native (Arabic, Turkish and Russian speaking participants, only with low German skills) and native German speakers (including non-native speakers with high German skills), because we not only provided information in the native language but also tested it with these target groups. People with low and high reading literacy in different languages, because fact boxes are a complexity-reduced format of health information and we tried to address accessibility through pilot testing with different groups. Does the use of fact boxes in outreach work promote more shared and informed decision-making than in regular healthcare settings? HYP. We assume that fact boxes more likely lead to more informed vaccination intentions and shared decisions in outreach work than in regular healthcare settings. In order to respond to the needs of especially people with disadvantaging factors and to reach them in a more effective way, community-based health services are becoming more and more important. The use of non-primary care professionals (eg, social workers, community nurses or community health workers) who can build trust and provide psychoeducational interventions has been shown to be effective in providing health knowledge, improving healthcare, changing health behaviour and improving health status. We will analyse the effect of fact boxes on knowledge, vaccination intentions and accuracy of risk perception between people with low and medium or high SSS through fact boxes compared with usual vaccination communication. We will analyse the effect of fact boxes on vaccination intentions, knowledge, risk perception, patient involvement and decisional conflict among people with migration-related indicators (eg, residential status, length of stay in Germany) compared with usual care, controlling for other factors describing the social situation (eg, education, health literacy). This may be crucial because people with immigrant history are less receptive to information from authorities or health providers in Germany, the host country (even if it is translated), if they have less trust in them. Also campaigns or formats that work for the majority of the host population may not be the right channel for communicating with immigrant groups. An increased length of stay is associated with a greater orientation towards the host society and thus a change in lifestyle habits, health perceptions and health behaviour. On the other hand, a shorter duration of stay, as well as not having German citizenship or a residence permit, may be associated with higher barriers to access to the German healthcare system (eg, due to lack of insurance or more difficult access to (multilingual) health information). Study participants in general The trial is open to people who are currently facing a decision about vaccination for themselves or a family member (eg, caring relatives). Adults of legal age with current residence in Germany and who speak German, Russian, Turkish or Arabic will be included. All participants must confirm their informed consent online to take part in our study. Patients who do not speak any of the four languages and who do not give informed consent will be excluded. Health educators will be asked to inform all potential participants about the study. The health educators will then distribute the study flyer as part of the health communication or afterwards. To ensure sufficient variability of a potential disadvantage status, the recruitment of health educators is focused on facilities and practices that serve a very heterogeneous audience. In particular, involving outreach organisations will ensure that disadvantaged people who are underserved are reached. In addition, health educators will be sensitised to approach these groups but will also be selected on the basis of their geographical area, for example, in districts with higher socioeconomic deprivation (socioeconomic differences that lead to differences in health outcomes). In addition, health authorities will be asked to help identify facilities and practices that primarily serve a disadvantaged population. In order to make the study as accessible as possible for the target group, information about the study and about data protection will be provided in their preferred and in plain language. Those who decide to take part in our study will complete an online survey. There will also be a read-aloud function for the questionnaire in all languages. After completing the initial online survey (T1), participants will have the opportunity to provide their email address for a follow-up survey 4 weeks later (T2). At the end of this second survey, participants will be rewarded with a voucher worth 15 Euro. Health educators Key requirement for involving health educators is that they provide information on health-related topics and have access to potential study participants. Furthermore, informed written consent is a prerequisite. To reach health educators, we will contact German public health authorities and ask them to distribute the study call to all kinds of health educators in their respective areas of responsibility (districts). We will also directly contact potential health educators by email or post. All health educators will first be given general information about the study and asked for their consent. Health educators will not be remunerated. Our study will be conducted in close cooperation with the health departments in Germany, which provides access to the planned cluster settings and the target groups. The trial is open to people who are currently facing a decision about vaccination for themselves or a family member (eg, caring relatives). Adults of legal age with current residence in Germany and who speak German, Russian, Turkish or Arabic will be included. All participants must confirm their informed consent online to take part in our study. Patients who do not speak any of the four languages and who do not give informed consent will be excluded. Health educators will be asked to inform all potential participants about the study. The health educators will then distribute the study flyer as part of the health communication or afterwards. To ensure sufficient variability of a potential disadvantage status, the recruitment of health educators is focused on facilities and practices that serve a very heterogeneous audience. In particular, involving outreach organisations will ensure that disadvantaged people who are underserved are reached. In addition, health educators will be sensitised to approach these groups but will also be selected on the basis of their geographical area, for example, in districts with higher socioeconomic deprivation (socioeconomic differences that lead to differences in health outcomes). In addition, health authorities will be asked to help identify facilities and practices that primarily serve a disadvantaged population. In order to make the study as accessible as possible for the target group, information about the study and about data protection will be provided in their preferred and in plain language. Those who decide to take part in our study will complete an online survey. There will also be a read-aloud function for the questionnaire in all languages. After completing the initial online survey (T1), participants will have the opportunity to provide their email address for a follow-up survey 4 weeks later (T2). At the end of this second survey, participants will be rewarded with a voucher worth 15 Euro. Key requirement for involving health educators is that they provide information on health-related topics and have access to potential study participants. Furthermore, informed written consent is a prerequisite. To reach health educators, we will contact German public health authorities and ask them to distribute the study call to all kinds of health educators in their respective areas of responsibility (districts). We will also directly contact potential health educators by email or post. All health educators will first be given general information about the study and asked for their consent. Health educators will not be remunerated. Our study will be conducted in close cooperation with the health departments in Germany, which provides access to the planned cluster settings and the target groups. Health educators in the intervention group will receive a flyer with a brief description of the study, a QR code and a link to an online survey, including on the reverse side a fact box about COVID-19 or the influenza vaccine in Arabic, German, Russian or Turkish. In several prestudies, the intervention underwent a comprehensive development process. This included feedback from various public health stakeholders on a COVID-19 fact box implemented in January 2021 by the Robert Koch Institute, the German national health authority. Further, information needs and requirements of the population in Germany were incorporated in updates with the help of secondary data analyses. Simplified COVID-19 and influenza vaccination fact boxes were tested for comprehensibility in 52 cognitive interviews between July 2021 and September 2022. First, a tabular COVID-19 fact box was tested with German-speaking participants and translated into a visualised fact box, which was then tested with Arabic-, Turkish- and Russian-speaking participants from disadvantaged districts of Berlin. Cognitive interviewing methods were used to ask whether the information in the fact boxes was understood and how it was understood. This method is usually used to pretest questionnaires. In individual interviews by telephone (during the COVID-19 period) and in person, a partially standardised questionnaire was used to ask respondents to reproduce the content of the fact boxes and to identify any comprehension problems in terms of content and wording. A visualised influenza fact box was piloted by the University of Erfurt with German-speaking laypeople with low numeracy. The non-directional vaccination fact boxes will be available either in tabular form (for influenza) or as a visualisation (for COVID-19), each in all languages and for both age groups. COVID-19 and influenza vaccine fact boxes are available for two different age groups each: COVID-19 vaccination for people aged 18–59 and over 60; influenza vaccination for people aged 16–64 and over 65. Accordingly, the study neither compares fact box formats (tabular, visual) nor examines disease-specific effects. Health educators are free to decide if and how to use the fact boxes, whether during, before or after vaccination education and whom to address. Therefore, there will be no training, as we cannot expect a one-size-fits-all solution for all health educators in different settings and especially in outreach settings (which are very heterogeneous). By asking health educators about their use of the fact boxes as part of a telephone assessment at the end of the study, we want to find out if and how they can be implemented in usual care. Health educators in the control condition will also receive the flyer with a brief description of the study, a QR code and a link to the online study to distribute to potential study participants, but without the fact box on the back. As described in the Intervention section, the COVID-19 and influenza vaccination fact boxes were developed through a comprehensive process involving various stakeholders and in particular the target groups, at all stages: (1) Information needs and requirements of the population in Germany were incorporated in updates with the help of secondary data analyse, (2) Simplified COVID-19 and influenza tabular vaccination fact boxes were tested for comprehensibility in 52 cognitive interviews with German-, Arabic-, Turkish- and Russian-speaking participants from disadvantaged districts of Berlin and (3) A visualised influenza fact box was piloted with German-speaking laypeople with low numeracy. The study design, outcomes, strategies for reaching vulnerable target groups and the questionnaire were discussed with various expert groups (eg, scientists from different disciplines, representatives of health authorities and outreach work). First, health educators will be randomised, either at the individual level or at the institutional level if there is more than one health educator per setting. Health educators who gave written informed consent will be assigned a study ID number and randomly assigned to one of the two study conditions: (1) usual health communication plus fact box (intervention) or (2) no information/usual health communication (control) (see ) by an independent researcher using computer-generated random numbers in a 1:1 ratio. Health educators will then distribute their assigned flyer format to individuals who are about to make a vaccination decision about the COVID-19 or the influenza vaccine, taking into account their reference group (age group). Health educators and study participants will not know which study condition they belong to. Health educators will be assigned by a person independent of the study, which means that the assignment will be blinded. Health educators in the control group will also have the opportunity to receive the fact boxes for their health communication post-trial. The data analyst will also be blinded. Health educators Preintervention the type of health education setting (doctor’s office or outreach work (alone or in a community setting/group practice)) is registered. Health educators will be asked about perceived changes in vaccination behaviour from the start of the intervention, once a cluster is no longer recruiting. Health educators will be asked about their demographics (age, gender, mother tongue) and, in the intervention group, how they used the fact boxes (during, before or after vaccination communication) and about involving patients from their perspective (SDM-Q-Doc). Participants Sociodemographic information and outcomes will be collected after people receive the flyer from their health educator and access the study link or QR code. As the study is voluntary and those who approach people do not know who among those approached will participate in the study, we will not be able to control who participates in the study and we will not be able to follow-up with those approached unless they complete the first survey. All information will be collected at the time of the first survey (T1), as participation in the second survey (T2) 4 weeks after the initial survey is also voluntary. The primary outcomes are knowledge and informed vaccination intention. For exploratory purposes, we will analyse on knowledge differences on the item level. The secondary outcomes are risk perception, decisional conflict and patient involvement . Furthermore, we will collect sociodemographic information, SSS, migration history and health literacy . Study participants will be also asked whether the fact box was used as part of the counselling process and how they judged it. Preintervention the type of health education setting (doctor’s office or outreach work (alone or in a community setting/group practice)) is registered. Health educators will be asked about perceived changes in vaccination behaviour from the start of the intervention, once a cluster is no longer recruiting. Health educators will be asked about their demographics (age, gender, mother tongue) and, in the intervention group, how they used the fact boxes (during, before or after vaccination communication) and about involving patients from their perspective (SDM-Q-Doc). Sociodemographic information and outcomes will be collected after people receive the flyer from their health educator and access the study link or QR code. As the study is voluntary and those who approach people do not know who among those approached will participate in the study, we will not be able to control who participates in the study and we will not be able to follow-up with those approached unless they complete the first survey. All information will be collected at the time of the first survey (T1), as participation in the second survey (T2) 4 weeks after the initial survey is also voluntary. The primary outcomes are knowledge and informed vaccination intention. For exploratory purposes, we will analyse on knowledge differences on the item level. The secondary outcomes are risk perception, decisional conflict and patient involvement . Furthermore, we will collect sociodemographic information, SSS, migration history and health literacy . Study participants will be also asked whether the fact box was used as part of the counselling process and how they judged it. We aim to demonstrate moderate main effects (equal to Cohen’s d=0.50) of the study intervention on informed intentions (10% expected in usual care) and a knowledge sum score (equal to the proportion of correct responses; with an expected variance of 0.20). Taking into account a cluster correlation coefficient (ICC=0.10) common for primary care studies with patient endpoints, we will target three health educators (eg, doctors’ offices) per study arm with 40 patients/clients per health educator. So, 240 participants in each subgroup undergoing the same analysis are required . Furthermore, to demonstrate small-to-moderate interaction effects (equal to Cohen’s d=0.375) of the study intervention in respective settings on informed intentions and knowledge, we have to target instead seven health educators per study arm with 40 patients/clients per health educator (n=560). In total, we aim at 800 participants because the proportion of subgroup members cannot be predicted, and we cannot rule out that interaction effects in the field are smaller than expected from experimental evidence (eg, d=0.20 would call for up to 20 health educators per study arm). Further details on the assumptions underlying the sample size calculations in the power calculation with respect to the critical hypotheses and outcomes are provided in in the supplementary section. After giving their online consent, participants will complete an online survey via SoSciSurvey. The survey (T1) is completely anonymous for the study participants. Personal data (email address) will be only collected if there is interest in participating in the follow-up survey (T2) and in the remuneration, for which also first name and surname will be recorded. For this purpose, a computer-generated random ID will be generated by someone independent of the study, which is stored with the unblinding list separately from the collected personal and survey data at the Harding Center (HC) on password-protected computers and is only accessible to researchers of the HC until the end of the data collection. The survey of the health educators will be conducted under pseudonym. The random generated ID, personal and survey data will be separated from the contact information immediately after completion of the postsurvey, and the de-blinding list is also kept until the end of the data collection. All relevant data will be provided in the manuscript and supporting information or made available in a public repository after the completion of the study. Data analysis will initially be based on descriptive statistics as well as frequency distributions and histograms to identify outliers and missing data. Baseline data of the study arms will be compared to see if the distribution is balanced between study sites/clusters. SPSS will be used to conduct all analyses. All participants will be asked to indicate at T1 whether they received the intervention to allow for intention-to-treat and as-treat analyses. Statistical methods for analysing primary and secondary outcomes We use linear mixed models to analyse the influence of both individual (eg, education status, health literacy) and cluster level factors (eg, setting) and account for the expected cluster variability in realising usual care or the intervention (details are outlined in ). We use linear mixed models to analyse the influence of both individual (eg, education status, health literacy) and cluster level factors (eg, setting) and account for the expected cluster variability in realising usual care or the intervention (details are outlined in ). We will record the number of flyers distributed per cluster. Because participation is voluntary, anonymous and not aware to the health educators, we will not be able to send a reminder and record the response rate for those who were approached. We will record the numbers of the total randomised sample for each group as well as the reasons for exclusion. It is compulsory for participants to answer all questions within the surveys. As participation in the follow-up survey is voluntary, we will record the number of drop-outs from the first to the second survey. The trial has been approved by the Ethics Committee of the University of Potsdam, Germany (application numbers: 34/2021 and 57/2022). Written informed consent will be obtained by the health educators prior to randomisation. Informed consent will be obtained online from study participants and/or their legal guardians after opening the QR code/link to the study on the flyer, and all study and ethical information has been provided (see for an example). Any changes to the protocol that affect the conduct of the trial, as well as changes to the study design, patient population, sample size, primary outcomes or statistical analysis plan, will be made transparent in the trial registration and final report. We plan to disseminate our findings through publications in peer-reviewed journals, national and international conferences, and relevant working groups and networks (eg, German Network for Evidence-Based Medicine, German Society for Public Health and Population Medicine), also targeting relevant community stakeholders. 10.1136/bmjopen-2023-083515 online supplemental file 1 10.1136/bmjopen-2023-083515 online supplemental file 2 |
Evaluation of effectiveness of bacteriophage purification methods | 67df9afc-4e81-4360-b8a8-add233906855 | 11656862 | Microbiology[mh] | Bacteriophages, independently discovered in 1915 and 1917 by Frederick Twort and Felix d’Herelle, respectively, are viruses that specifically infect bacteria . Their use in modern medicine has been propelled by increased antibiotic resistance and they are considered frontrunner alternatives to antibiotics , especially after the declaration of the antibiotic resistance pandemic . Developing bacteriophage therapeutics is considered more affordable than designing new chemical antimicrobial compounds . Further, the capacity for bacteriophages to overcome bacterial resistance through their co-evolutionary capacity is a useful tool for evolving therapeutics . Accordingly, western medicine has seen a significant increase in clinical trials evaluating the safety and efficacy of these viruses . Because bacteriophages are diverse biological entities, procedures to test the safety of each virus need to be employed prior to clinical use. Despite reports of bacteriophage safety in therapy , retrospective observational studies have suggested that bacteriophage therapy may have resulted in non-serious adverse events in up to 8% of patients, life-threatening reactions in 1% of patients, and contributed to mortality in 6% of patients . Although bacteriophages and conventional antibiotics are mechanistically different, many experts agree that bacteriophage therapy needs to be proven in the context of randomised controlled trials (RCTs), similar to antibiotics. However, unlike antibiotics, bacteriophages are dynamic and evolve quickly, and may require an outside-the-box regulatory framework for their application . Underestimating RCT and regulation framework requirements for bacteriophages can be costly, as evidenced by the failed ‘Phagoburn’ RCT where inadequate care in preparation of bacteriophages resulted in inactive viruses and ineffective therapy . Presently, it is unclear how bacteriophages may be adapted for use, but examples may be taken from other biologics such as faecal microbiota transplant and how they are regulated . As regulatory discussions progress, bacteriophage therapy is finding its way to patients through compassionate use in many Western hospitals and as magistral preparations in Belgium . In both circumstances, specific bacteria (usually multi-resistant to antibiotics) are isolated from the patient and several bacteriophage biobanks are used to screen for active bacteriophages. If no active bacteriophages are found, bacteriophage hunting is commenced in clinical and research laboratories. The host bacteria strain is cultured for this hunting process and for the production of more bacteriophage particles. Once an active bacteriophage is found, several considerations are taken into account before administration to the patient. Firstly, their genomes are characterised for presence of toxins and antibiotic resistance genes, and then assessed for obligatory lytic capacity. To ensure safety, bacteriophages are purified to remove endotoxins using purification methods in clinical and research laboratories that are often without good manufacturing practice certification . It is therefore reasonable to suggest that safety assurance protocols are required before compassionate and/or magistral applications of bacteriophages in therapy. The United States Food and Drug Administration (FDA), along with other major regulatory bodies including the Australian Therapeutic Goods Administration and the European Medicines Agency, provide guidelines for maximal endotoxin concentration of parenteral medicines. They place the upper limit at 5 Endotoxin Units (EU) per kg of body weight per hour for non-intrathecal administration, and 2 EU per kg of body weight per hour for intrathecal administration . This is measured by a Limulus Amebocyte Lysate (LAL) test as the gold standard assay. However, as this assay is based on horseshoe crab plasma response , it may not reflect or give an indication of expected safety when administered to a patient. Phase I safety trials and case studies have shown variable outcomes in some patients. The adverse events observed can be non-specific and range from local responses such as redness and pain at the application site; mild events such as abdominal discomfort, coughing; life-threatening events such as heart failure; fatal events including septic shock, and bacteriophage therapy associated tumour progression . Further, some methods for removing endotoxins may require researchers to dilute their bacteriophage preparations in order not to exceed recommended FDA limits , which may limit bioavailability and efficacy. There is a paucity of data on the evaluation of endotoxin concentration in bacteriophage preparations. Two studies have compared different bacteriophage purification methods, and used the amoebocyte lysate-based assay to determine endotoxin concentration. One of these compared several sequential bacteriophage purifications by using EndoTrap ® HD affinity column followed by CsCl ultracentrifugation or Triton X-100, 1-octanol extraction, enzymatic inactivation of endotoxins and anion-exchange chromatography. Sequential steps of EndoTrap ® HD affinity column followed by CsCl ultracentrifugation was found to be the most effective in reducing endotoxin concentration . However, a lack of experimental replicates made it difficult to validate the findings. The other research compared several methods including PEG precipitation/Triton X-100, octanol extraction, anion exchange, and two endotoxin removal columns (EndoTrap ® HD affinity column and Pierce™ High-Capacity Endotoxin Removal Resin spin columns). This study observed that using EndoTrap ® affinity column in combination with Vivaspin ultrafiltration columns with 100,000 MWCO polyethersulfone membrane was the most effective in removing endotoxin . While the study included data from experimental replicates, diverse bacteriophage morphologies were not directly compared. In this study, we employed a novel human immune cell-based system for determining concentration of endotoxins in bacteriophage preparations and assessed three common purification techniques: Triton X-100 exposure, Pierce™ High-Capacity Endotoxin Removal Resin spin columns and CsCl density gradient ultracentrifugation . We also tested bacteriophages of three different morphotypes to understand whether the viral morphology may affect endotoxin purification. Bacteriophage preparations and titre assessment Three bacteriophages were used in this study to represent the three most common morphotypes of the Caudoviricetes. These included Latrobevirus FNU1 (morphotype: siphovirus, genome size 130 kb, GenBank accession number: MK554696), unclassified Ahphunavirus LAh5 (morphotype: podovirus, genome size 42 kb, GenBank accession number: MK838111), and Ludhianavirus LAh10 (morphotype: myovirus genome size 260 kb, GenBank accession number: MK838116). The capsid diameters of these bacteriophages were 82 nm (LAh5), 116 nm (LAh10), and 88 nm (FNU1). The bacteriophages were propagated on their host strains, Fusobacterium nucleatum ATCC 10953 for FNU1 , and Aeromonas hydrophila strains AHB0147 and AHB0116 for LAh5 and LAh10, respectively . F. nucleatum was cultured anaerobically at 37 o C using anaerobic generating packs (AnaeroGen™, Oxoid, Australia) in Heart Infusion broth or agar (0.8% w/v) media supplemented with 0.5% cysteine (Sigma, Australia) and 0.5% haemin (Sigma, Australia) while A. hydrophila was cultured aerobically at 37 o C in nutrient broth or agar (1% w/v) as previously described . A bacteriophage stock was prepared using the spread technique to increase the volume and concentration. Briefly, 200 µL of bacteriophage stock was spread on a fresh lawn of host bacteria and incubated over 24–48 h, for A. hydrophila and F. nucleatum , respectively. Bacteriophages were harvested by washing and filtering using 0.2 μm filters (Microanalytix, Australia) and titres calculated using an established method . Filtered bacteriophage stock obtained at this point were considered a crude preparation for this study. Bacteriophage purification Three independently prepared 0.2 μm filtered bacteriophage stocks (normalised to 1 × 10 9 PFU mL − 1 ) of each bacteriophage type were used in three purification methods. These included the (i) Polyethylene Glycol 8000 (Sigma-Aldrich, Australia) precipitation and Triton X-100 (Sigma-Aldrich, Australia) purification , (ii) Caesium chloride (CsCl; Sigma-Aldrich, Australia) density gradient ultracentrifugation , and (iii) Pierce™ High-Capacity Endotoxin Removal Resin spin columns (ThermoFisher, Australia) . Bacteriophage titres were assayed before and after purification and stored at -80 o C before endotoxin assessment. PEG precipitation and Triton X-100 purification Five mL of 0.2 μm filtered bacteriophages were purified as previously described . Briefly, 5 mmol L − 1 of MgCl 2 and 1.0 µL each of RNase A (Promega, Australia) and DNase I (Promega, Australia) to a final concentration of 10 µg mL − 1 were added before incubation at room temperature for 30 min to digest extraneous DNA and RNA. Polyethylene glycol 8000 (PEG) at 10% [w/v] and NaCl at 1 g L − 1 were added and dissolved by gentle shaking on an orbital mixer (Ratek, Australia) for 5 min. To this mixture, 2% v/v Triton X-100 was added at room temperature, and the material shaken gently for 5 min before a 15 min incubation at 4 o C. After centrifugation (4000 × g ; 15 min), the supernatant was discarded and the pellet of precipitated bacteriophages resuspended in 5 mL sterile PBS (phosphate buffered saline, pH 7.4). The PEG/NaCl/Triton X-100 treatment was repeated two more times, following which the bacteriophages were centrifuged (4000 × g; 15 min) and resuspended with fresh sterile 5 mL PBS three times then filtered with a 0.2 μm filter, a precautionary step to ensure that no precipitates are carried forward in the purified bacteriophage solution. Caesium chloride gradient ultracentrifugation Purification using CsCl was adapted from the method described by Luong and colleagues . Protocol transfer was achieved using the Beckman protocol transfer assistant ‘Intellifuge’ . Briefly, CsCl densities of 1.30 g mL − 1 , 1.50 g mL − 1 and 1.60 g mL − 1 were carefully layered in 13.2 mL open-top thin wall ultra-clear centrifuge tubes (Beckman, Australia) in 0.2 μm sterile filtered Tris-sodium chloride buffer, pH 7.0 (10 mM Tris (pH 7.0) and 150 mM NaCl). Adjusting the weight of the centrifuge holders, approximately 4 mL of crude bacteriophage preparation was added. The gradients were centrifuged in a Beckman SW40 Ti rotor at 28,000 × g , 4 °C, for 258 min (Beckman Coulter, Optima L-100 XP Ultracentrifuge). Purified bacteriophages were extracted using a 26-gauge needle and 3 mL syringe from the base of a visible whitish/grey band. Pierce™ high-capacity endotoxin removal resin spin columns One mL spin columns and endotoxin removal resins were purchased from ThermoFisher, Australia. Before use, resins were equilibrated overnight at room temperature with 8 mL of 0.2 N NaOH to remove storage solution as per manufacturer’s instructions. All solutions are removed from resins by 500 × g centrifugation for 1 min. The NaOH was then replaced with 8 mL of 2 M NaCl, which was replaced with 8 mL of endotoxin-free water and then endotoxin-free buffer. The resins were further washed twice in 8 mL endotoxin-free buffer. After removing endotoxin-free buffer, 5 mL of crude bacteriophage preparations were added to the resin and incubated while mixing at room temperature for 2 h. Purified bacteriophages were eluted by 500 × g centrifugation for 1 min. Resins were then treated with 8 mL of 0.2 N NaOH overnight and stored in 20% ethanol at 4 o C for subsequent re-use. Cytokine detection assay Interleukin Receptor Activated Kinase (IRAK) 3 knockout THP-1 monocytes were obtained from the La Trobe University collection . The IRAK3 knockout monocytes were cultured in 10% Foetal Bovine Serum (Sigma-Aldrich, Australia) supplemented Roswell Park Memorial Institute (RPMI) 1640 media (Sigma-Aldrich, Australia) at 5% CO 2 and 37 o C. Approximately 2 × 10 5 cells per well were seeded in 12-well plates. The cells were exposed to either 300 µL of crude or purified bacteriophage adjusted to 3.0 × 10 8 PFU mL − 1 , or PBS, or lipopolysaccharide (LPS) ( Escherichia coli O55:B5, Sigma; at a concentration of 1 µg/mL) before incubating for 24 h. The supernatant was then collected after centrifugation (300 × g , 5 min, 37 °C) and analysed using BD OptEIA™ Set Human IL-6 (Interleukin 6) and TNF-α (Tumour necrosis factor – α) commercial sandwich Enzyme-Linked Immunosorbent Assays (ELISA; BD Biosciences), according to manufacturer’s instructions. Endotoxin correlation For correlation of cytokine production to endotoxin levels, IRAK3 knockout monocytes were exposed to the 10,000 United States Pharmacopeia Endotoxin Unit Reference Standard diluted from 50 EU mL − 1 . Endotoxin free water was used as a 0 EU mL − 1 (Blank) standard. Cytokine production was analysed as above and standard curves generated by plotting cytokine levels against endotoxin concentrations. Estimated concentration of endotoxin present in the crude and purified bacteriophage samples were calculated by linear regression with cytokine production as the independent variable. Statistical analysis All data were collected into Microsoft Excel spreadsheets before importing to R/R studio (R version 4.3.1 (2024.04.2 + 764)). The Shapiro–Wilk test was used to determine whether the data were normally distributed. A paired t -test was used for quantitative data that were normally distributed while Wilcoxon signed-rank test was used for non-parametric statistical analysis. A p value less than 0.05 was considered statistically significant. Standard curves of endotoxin assay were plotted using the ggscatter function. The correlation factor ( R ) and equation of the linear regression line were calculated using the stat_cor and stat_regline_equation functions. Three bacteriophages were used in this study to represent the three most common morphotypes of the Caudoviricetes. These included Latrobevirus FNU1 (morphotype: siphovirus, genome size 130 kb, GenBank accession number: MK554696), unclassified Ahphunavirus LAh5 (morphotype: podovirus, genome size 42 kb, GenBank accession number: MK838111), and Ludhianavirus LAh10 (morphotype: myovirus genome size 260 kb, GenBank accession number: MK838116). The capsid diameters of these bacteriophages were 82 nm (LAh5), 116 nm (LAh10), and 88 nm (FNU1). The bacteriophages were propagated on their host strains, Fusobacterium nucleatum ATCC 10953 for FNU1 , and Aeromonas hydrophila strains AHB0147 and AHB0116 for LAh5 and LAh10, respectively . F. nucleatum was cultured anaerobically at 37 o C using anaerobic generating packs (AnaeroGen™, Oxoid, Australia) in Heart Infusion broth or agar (0.8% w/v) media supplemented with 0.5% cysteine (Sigma, Australia) and 0.5% haemin (Sigma, Australia) while A. hydrophila was cultured aerobically at 37 o C in nutrient broth or agar (1% w/v) as previously described . A bacteriophage stock was prepared using the spread technique to increase the volume and concentration. Briefly, 200 µL of bacteriophage stock was spread on a fresh lawn of host bacteria and incubated over 24–48 h, for A. hydrophila and F. nucleatum , respectively. Bacteriophages were harvested by washing and filtering using 0.2 μm filters (Microanalytix, Australia) and titres calculated using an established method . Filtered bacteriophage stock obtained at this point were considered a crude preparation for this study. Three independently prepared 0.2 μm filtered bacteriophage stocks (normalised to 1 × 10 9 PFU mL − 1 ) of each bacteriophage type were used in three purification methods. These included the (i) Polyethylene Glycol 8000 (Sigma-Aldrich, Australia) precipitation and Triton X-100 (Sigma-Aldrich, Australia) purification , (ii) Caesium chloride (CsCl; Sigma-Aldrich, Australia) density gradient ultracentrifugation , and (iii) Pierce™ High-Capacity Endotoxin Removal Resin spin columns (ThermoFisher, Australia) . Bacteriophage titres were assayed before and after purification and stored at -80 o C before endotoxin assessment. PEG precipitation and Triton X-100 purification Five mL of 0.2 μm filtered bacteriophages were purified as previously described . Briefly, 5 mmol L − 1 of MgCl 2 and 1.0 µL each of RNase A (Promega, Australia) and DNase I (Promega, Australia) to a final concentration of 10 µg mL − 1 were added before incubation at room temperature for 30 min to digest extraneous DNA and RNA. Polyethylene glycol 8000 (PEG) at 10% [w/v] and NaCl at 1 g L − 1 were added and dissolved by gentle shaking on an orbital mixer (Ratek, Australia) for 5 min. To this mixture, 2% v/v Triton X-100 was added at room temperature, and the material shaken gently for 5 min before a 15 min incubation at 4 o C. After centrifugation (4000 × g ; 15 min), the supernatant was discarded and the pellet of precipitated bacteriophages resuspended in 5 mL sterile PBS (phosphate buffered saline, pH 7.4). The PEG/NaCl/Triton X-100 treatment was repeated two more times, following which the bacteriophages were centrifuged (4000 × g; 15 min) and resuspended with fresh sterile 5 mL PBS three times then filtered with a 0.2 μm filter, a precautionary step to ensure that no precipitates are carried forward in the purified bacteriophage solution. Caesium chloride gradient ultracentrifugation Purification using CsCl was adapted from the method described by Luong and colleagues . Protocol transfer was achieved using the Beckman protocol transfer assistant ‘Intellifuge’ . Briefly, CsCl densities of 1.30 g mL − 1 , 1.50 g mL − 1 and 1.60 g mL − 1 were carefully layered in 13.2 mL open-top thin wall ultra-clear centrifuge tubes (Beckman, Australia) in 0.2 μm sterile filtered Tris-sodium chloride buffer, pH 7.0 (10 mM Tris (pH 7.0) and 150 mM NaCl). Adjusting the weight of the centrifuge holders, approximately 4 mL of crude bacteriophage preparation was added. The gradients were centrifuged in a Beckman SW40 Ti rotor at 28,000 × g , 4 °C, for 258 min (Beckman Coulter, Optima L-100 XP Ultracentrifuge). Purified bacteriophages were extracted using a 26-gauge needle and 3 mL syringe from the base of a visible whitish/grey band. Pierce™ high-capacity endotoxin removal resin spin columns One mL spin columns and endotoxin removal resins were purchased from ThermoFisher, Australia. Before use, resins were equilibrated overnight at room temperature with 8 mL of 0.2 N NaOH to remove storage solution as per manufacturer’s instructions. All solutions are removed from resins by 500 × g centrifugation for 1 min. The NaOH was then replaced with 8 mL of 2 M NaCl, which was replaced with 8 mL of endotoxin-free water and then endotoxin-free buffer. The resins were further washed twice in 8 mL endotoxin-free buffer. After removing endotoxin-free buffer, 5 mL of crude bacteriophage preparations were added to the resin and incubated while mixing at room temperature for 2 h. Purified bacteriophages were eluted by 500 × g centrifugation for 1 min. Resins were then treated with 8 mL of 0.2 N NaOH overnight and stored in 20% ethanol at 4 o C for subsequent re-use. Five mL of 0.2 μm filtered bacteriophages were purified as previously described . Briefly, 5 mmol L − 1 of MgCl 2 and 1.0 µL each of RNase A (Promega, Australia) and DNase I (Promega, Australia) to a final concentration of 10 µg mL − 1 were added before incubation at room temperature for 30 min to digest extraneous DNA and RNA. Polyethylene glycol 8000 (PEG) at 10% [w/v] and NaCl at 1 g L − 1 were added and dissolved by gentle shaking on an orbital mixer (Ratek, Australia) for 5 min. To this mixture, 2% v/v Triton X-100 was added at room temperature, and the material shaken gently for 5 min before a 15 min incubation at 4 o C. After centrifugation (4000 × g ; 15 min), the supernatant was discarded and the pellet of precipitated bacteriophages resuspended in 5 mL sterile PBS (phosphate buffered saline, pH 7.4). The PEG/NaCl/Triton X-100 treatment was repeated two more times, following which the bacteriophages were centrifuged (4000 × g; 15 min) and resuspended with fresh sterile 5 mL PBS three times then filtered with a 0.2 μm filter, a precautionary step to ensure that no precipitates are carried forward in the purified bacteriophage solution. Purification using CsCl was adapted from the method described by Luong and colleagues . Protocol transfer was achieved using the Beckman protocol transfer assistant ‘Intellifuge’ . Briefly, CsCl densities of 1.30 g mL − 1 , 1.50 g mL − 1 and 1.60 g mL − 1 were carefully layered in 13.2 mL open-top thin wall ultra-clear centrifuge tubes (Beckman, Australia) in 0.2 μm sterile filtered Tris-sodium chloride buffer, pH 7.0 (10 mM Tris (pH 7.0) and 150 mM NaCl). Adjusting the weight of the centrifuge holders, approximately 4 mL of crude bacteriophage preparation was added. The gradients were centrifuged in a Beckman SW40 Ti rotor at 28,000 × g , 4 °C, for 258 min (Beckman Coulter, Optima L-100 XP Ultracentrifuge). Purified bacteriophages were extracted using a 26-gauge needle and 3 mL syringe from the base of a visible whitish/grey band. One mL spin columns and endotoxin removal resins were purchased from ThermoFisher, Australia. Before use, resins were equilibrated overnight at room temperature with 8 mL of 0.2 N NaOH to remove storage solution as per manufacturer’s instructions. All solutions are removed from resins by 500 × g centrifugation for 1 min. The NaOH was then replaced with 8 mL of 2 M NaCl, which was replaced with 8 mL of endotoxin-free water and then endotoxin-free buffer. The resins were further washed twice in 8 mL endotoxin-free buffer. After removing endotoxin-free buffer, 5 mL of crude bacteriophage preparations were added to the resin and incubated while mixing at room temperature for 2 h. Purified bacteriophages were eluted by 500 × g centrifugation for 1 min. Resins were then treated with 8 mL of 0.2 N NaOH overnight and stored in 20% ethanol at 4 o C for subsequent re-use. Interleukin Receptor Activated Kinase (IRAK) 3 knockout THP-1 monocytes were obtained from the La Trobe University collection . The IRAK3 knockout monocytes were cultured in 10% Foetal Bovine Serum (Sigma-Aldrich, Australia) supplemented Roswell Park Memorial Institute (RPMI) 1640 media (Sigma-Aldrich, Australia) at 5% CO 2 and 37 o C. Approximately 2 × 10 5 cells per well were seeded in 12-well plates. The cells were exposed to either 300 µL of crude or purified bacteriophage adjusted to 3.0 × 10 8 PFU mL − 1 , or PBS, or lipopolysaccharide (LPS) ( Escherichia coli O55:B5, Sigma; at a concentration of 1 µg/mL) before incubating for 24 h. The supernatant was then collected after centrifugation (300 × g , 5 min, 37 °C) and analysed using BD OptEIA™ Set Human IL-6 (Interleukin 6) and TNF-α (Tumour necrosis factor – α) commercial sandwich Enzyme-Linked Immunosorbent Assays (ELISA; BD Biosciences), according to manufacturer’s instructions. For correlation of cytokine production to endotoxin levels, IRAK3 knockout monocytes were exposed to the 10,000 United States Pharmacopeia Endotoxin Unit Reference Standard diluted from 50 EU mL − 1 . Endotoxin free water was used as a 0 EU mL − 1 (Blank) standard. Cytokine production was analysed as above and standard curves generated by plotting cytokine levels against endotoxin concentrations. Estimated concentration of endotoxin present in the crude and purified bacteriophage samples were calculated by linear regression with cytokine production as the independent variable. All data were collected into Microsoft Excel spreadsheets before importing to R/R studio (R version 4.3.1 (2024.04.2 + 764)). The Shapiro–Wilk test was used to determine whether the data were normally distributed. A paired t -test was used for quantitative data that were normally distributed while Wilcoxon signed-rank test was used for non-parametric statistical analysis. A p value less than 0.05 was considered statistically significant. Standard curves of endotoxin assay were plotted using the ggscatter function. The correlation factor ( R ) and equation of the linear regression line were calculated using the stat_cor and stat_regline_equation functions. Pro-inflammatory cytokine production in IRAK3 knockout THP-1 cells Bacteriophages purified via CsCl ultracentrifugation or Pierce™ High-Capacity Endotoxin Removal Resin spin columns produced significantly higher levels of TNF-α and IL-6 ( p < 0.001) in IRAK3 knockout monocytes (Table ), compared to those purified using Triton X-100. The production of these pro-inflammatory cytokines by IRAK3 knockout monocytes treated with crude bacteriophage preparations was significantly higher ( p < 0.001) than those when treated with any of the purified preparations (Fig. ). Assay standardisation Standard curves were derived from TNF-α and IL-6 production by the IRAK3 knockout THP-1 cells when exposed to known endotoxin standards. Both the standard curves of TNF-α and IL-6 showed a bi-phasic linear correlation between cytokine production and endotoxin standards (Fig. ). For the TNF-α production, the lower and higher endotoxin standards showed a correlation of R = 0.9, p < 0.0001 and R = 0.98, p < 0.0001 respectively. The correlation for the lower and higher endotoxin standards with production of IL-6 was R = 0.95, p < 0.0001, and R = 0.96, p < 0.0001, respectively. Comparison of the bacteriophage purification methods We found significantly reduced endotoxin concentrations in purified bacteriophages using all three methods, compared to crude preparations (Table ). The estimated endotoxin concentrations of purified bacteriophage preparations using CsCl ultracentrifugation and Pierce™ High-Capacity Endotoxin Removal Resin spin columns were significantly higher ( p = 0.001, p < 0.001, respectively) than the Triton X-100 bacteriophage preparations, when assessed by measuring TNF-α production in the IRAK3 knockout monocytes (Fig. ). When assessed by measuring IL-6 cytokine production, the CsCl ultracentrifugation and Triton-X methods of purification were not significantly different, while the Pierce™ High-Capacity Endotoxin Removal Resin spin columns yielded significantly higher endotoxin concentrations ( p < 0.001) (Fig. ). Effect of bacteriophage characteristics on efficacy of purification methods To assess whether there was a difference in purification efficacy between the bacteriophages, levels of endotoxins measured from standard curves derived from responses of both of the pro-inflammatory cytokines were employed. There was no significant difference between the different bacteriophage types, p > 0.05 (Table ). Effectiveness of subsequent use of Pierce™ high-capacity endotoxin removal Resin spin columns Pierce™ High-Capacity Endotoxin Removal Resin spin columns are considered multi-use products . In this study, the columns were used three times, each time with a new crude sample, to determine whether these columns were still as effective in removing endotoxins. Estimated endotoxin concentration of purified bacteriophage preparations using the columns was lowest when they were used the first time, p < 0.001 (Table ). When columns were used for the third time, the endotoxin concentration was significantly higher than the second use, p < 0.001 (Fig. ), although this is still an improvement of nearly a 3,000-fold decrease in endotoxin concentration compared to the crude preparations (Table ). Bacteriophage viability following purification The recovered bacteriophages were assayed following each of the purification techniques to determine any loss in bacteriophage titre. Bacteriophages purified using CsCl ultracentrifugation or the Pierce™ High-Capacity Endotoxin Removal Resin spin columns lost approximately 90% of their titre, a significantly higher loss than bacteriophage preparations purified using Triton X-100 (average of 71% loss of titre) (Fig. ). Final concentrations of bacteriophages after purifications are listed in the supplementary Table . Bacteriophages purified via CsCl ultracentrifugation or Pierce™ High-Capacity Endotoxin Removal Resin spin columns produced significantly higher levels of TNF-α and IL-6 ( p < 0.001) in IRAK3 knockout monocytes (Table ), compared to those purified using Triton X-100. The production of these pro-inflammatory cytokines by IRAK3 knockout monocytes treated with crude bacteriophage preparations was significantly higher ( p < 0.001) than those when treated with any of the purified preparations (Fig. ). Standard curves were derived from TNF-α and IL-6 production by the IRAK3 knockout THP-1 cells when exposed to known endotoxin standards. Both the standard curves of TNF-α and IL-6 showed a bi-phasic linear correlation between cytokine production and endotoxin standards (Fig. ). For the TNF-α production, the lower and higher endotoxin standards showed a correlation of R = 0.9, p < 0.0001 and R = 0.98, p < 0.0001 respectively. The correlation for the lower and higher endotoxin standards with production of IL-6 was R = 0.95, p < 0.0001, and R = 0.96, p < 0.0001, respectively. We found significantly reduced endotoxin concentrations in purified bacteriophages using all three methods, compared to crude preparations (Table ). The estimated endotoxin concentrations of purified bacteriophage preparations using CsCl ultracentrifugation and Pierce™ High-Capacity Endotoxin Removal Resin spin columns were significantly higher ( p = 0.001, p < 0.001, respectively) than the Triton X-100 bacteriophage preparations, when assessed by measuring TNF-α production in the IRAK3 knockout monocytes (Fig. ). When assessed by measuring IL-6 cytokine production, the CsCl ultracentrifugation and Triton-X methods of purification were not significantly different, while the Pierce™ High-Capacity Endotoxin Removal Resin spin columns yielded significantly higher endotoxin concentrations ( p < 0.001) (Fig. ). To assess whether there was a difference in purification efficacy between the bacteriophages, levels of endotoxins measured from standard curves derived from responses of both of the pro-inflammatory cytokines were employed. There was no significant difference between the different bacteriophage types, p > 0.05 (Table ). Pierce™ High-Capacity Endotoxin Removal Resin spin columns are considered multi-use products . In this study, the columns were used three times, each time with a new crude sample, to determine whether these columns were still as effective in removing endotoxins. Estimated endotoxin concentration of purified bacteriophage preparations using the columns was lowest when they were used the first time, p < 0.001 (Table ). When columns were used for the third time, the endotoxin concentration was significantly higher than the second use, p < 0.001 (Fig. ), although this is still an improvement of nearly a 3,000-fold decrease in endotoxin concentration compared to the crude preparations (Table ). The recovered bacteriophages were assayed following each of the purification techniques to determine any loss in bacteriophage titre. Bacteriophages purified using CsCl ultracentrifugation or the Pierce™ High-Capacity Endotoxin Removal Resin spin columns lost approximately 90% of their titre, a significantly higher loss than bacteriophage preparations purified using Triton X-100 (average of 71% loss of titre) (Fig. ). Final concentrations of bacteriophages after purifications are listed in the supplementary Table . Three bacteriophage morphotypes were purified using three common techniques to determine the efficacy of purification methods in reducing monocyte response to bacteriophage preparations. All three purification methods used in this study reduced endotoxin levels regardless of morphology and size of the virus. Triton X-100 was optimal at removing endotoxins and retaining bacteriophage titre. CsCl ultracentrifugation had comparable efficacy in reducing endotoxin but with approximately 20% lower recovery rate of bacteriophages. The Pierce™ High-Capacity Endotoxin Removal Resin spin columns were the least effective in removing endotoxin, and resulted in similar loss of bacteriophage titre as CsCl ultracentrifugation. In measuring endotoxin concentrations, commercially available amoebocyte lysate assays and wild-type monocyte assays are limited to detecting less than 1 EU mL − 1 of endotoxin. In comparison, the IRAK3 knockout monocyte assay we developed and employed here was able to detect a broader range of endotoxin concentrations. This may limit measurement errors that are possibly introduced if samples need to be serially diluted for measurement with commercial kits. When we tested a broad range of concentrations of endotoxin standards, the IRAK3 knockout monocytes produced a biphasic cytokine response. The biphasic response revealed strong correlation between the production of IL-6 or TNF-α and endotoxin concentration. It is unclear why there was a biphasic response. Other mediators of cytokine release impact responses to endotoxin as IRAK3 deletion in human primary monocytes or THP-1 cells results in upregulation of multiple families of cytokines . Of the two cytokines, IL-6 had a better correlation for the low concentration standards, while TNF-α had a better correlation for the high concentration standards. Both cytokines are therefore key in establishing accurate endotoxin concentrations in bacteriophage preparations using our assay. The production of pro-inflammatory cytokines TNF-α and IL-6 was significantly higher in the crude bacteriophage preparations compared to the purified preparations for all methods tested. Endotoxin concentrations were significantly lower for Triton X-100 purification, compared to CsCl ultracentrifugation, when measured via TNF-α production (which has a lower correlation coefficient) from the standard curve using low concentrations. When using IL-6 production as a measure, bacteriophage preparations purified with Triton X-100 did not differ to those purified with CsCl ultracentrifugation. These results suggest that bacteriophage purification with Triton X-100 was at least as effective as CsCl ultracentrifugation without the need for expensive equipment such as ultracentrifuges. Previous studies have used at least two sequential methods to effectively remove endotoxins from bacteriophage preparations. We found that single-step purifications with Triton X-100 or CsCl ultracentrifugation reduced endotoxin concentration to levels comparable to those of the sequential techniques . The efficacy of the purification techniques was similar for the three different bacteriophages tested. These differed in capsid size, genome size and morphology (representing the three common morphotypes of siphovirus, myovirus and podovirus). Although some chemicals are known to affect bacteriophages of different morphology differently , we did not see any variances in purification efficacy here. There have been suggestions that purification methods such as Triton X-100 and CsCl, result in remnant chemicals rendering bacteriophage preparations not safe for clinical use . Previous studies have shown that CsCl may suppress the growth of HeLa cells and has been associated with chromosomal aberrations in mice . Similarly, while some Triton X detergents are well tolerated , low concentrations of Triton X-100 may lead to cell death, raising concerns about use in preparation of therapeutics . On occasion, clinical studies have included additional steps such as dialysis in PBS are employed to remove these harmful chemicals before administering to patients . In our study, three PBS wash steps were employed to minimise remnant Triton X-100 in purified bacteriophage preparations. Using this method, we have shown that the resulting bacteriophage preparations do not adversely affect growth of human epithelial cells . It is also important to note that bacteriophage purification using these methods is not new, and that endotoxin purifications using CsCl have been applied for many years and considered generally safe . Bacteriophage preparations purified using Pierce™ High-Capacity Endotoxin Removal Resin spin columns reduced endotoxin levels significantly compared to crude preparations, but the levels were significantly higher than both CsCl ultracentrifugation and Triton X-100 preparations. Although promoted as a reusable technique, the spin column was most effective in removing endotoxin in bacteriophage preparations during the first use. Each subsequent reuse of the spin columns was less effective in removing endotoxins. This is similar to findings observed by Hietala et al. . It is possible that high endotoxin concentrations in crude bacteriophage preparations caused resin saturation after each use and the equilibration steps did not allow dissociation of bound endotoxin. The bacteriophage titre loss in resin purified bacteriophages was similar to CsCl ultracentrifugation, at approximately 90%. We and others have previously shown that purified bacteriophages induce low levels of cytokine production. This minimal immune response induced by the purified bacteriophages illustrates that they may be a safe option when administered in therapy. However, studies have also reported that bacteriophage specific antibodies are present after exposure , and it remains unclear whether this is a result of inadequately purified bacteriophages that activate the innate and subsequent adaptive immunity. Another complicating factor is that innate responses to bacteria can affect responses to bacteriophages . In this study, Triton X-100 was the most effective method for bacteriophage recovery. Although we found lower retention of viable bacteriophages when using CsCl, endotoxin removal efficacy was comparable to Triton X-100. Both these methods were significantly more effective than resins in removing endotoxins. Further, differences in the morphology, capsid size or genomic size of the bacteriophages used in this study did not influence the efficacy of bacteriophage purification. Below is the link to the electronic supplementary material. Supplementary Material 1 |
Pilot implementation of a sonography simulator in gynecological medical training and continuing education: A practical report | bb4f2812-ff1e-40bc-990e-16f03d0c859e | 11656176 | Gynaecology[mh] | Sonography is firmly integrated into preventive, routine, emergency, and follow-up diagnostics. Standardized and structured training of all medical professionals is therefore essential, while at the same time conserving human, spatial and financial resources . At the Medical Interprofessional Training Centre (MITZ) of the Faculty of Medicine at TU Dresden (MFD), students of medicine, dentistry and midwifery train basic practical skills and communication skills. The MITZ also offers opportunities for further and advanced training. Since 2015, ultrasound teaching at the MITZ has been successively implemented, expanded and professionalized. During the collaboration with the Clinic and Polyclinic for Gynecology and Obstetrics at Dresden University Hospital (GYN) in the midwifery degree program, points of intersection became clear regarding efforts to ensure resource-conserving, needs-oriented teaching and the bundling of didactic skills. Limitations in current sonography training due to limited space and personnel capacities became apparent in both areas, as well as the need for patient-friendly teaching. In 2022, the MITZ purchased a fully equipped sonography simulator (SoSim) with the support of the MFD: the SCANTRAINER 7A of Skills Med Germany. In the pilot phase, the SoSim was tested in cooperation with the GYN. Under the technical and didactic supervision of the authors, five young professionals from GYN trained two defined modules on the SoSim over a period of five months and were asked about their subjective appraisal after the training (see figure 1 ). The aim was to use the pilot phase to identify positive effects in terms of competence gain and to create a spatial and organizational basis for a sustainable and comprehensive use of the SoSim for entire University Medicine Dresden. Initially the authors were familiarized with the SoSim, and the necessary structural and organizational conditions were created to adequately support the users learning progress (see figure 1 ). As part of the pilot program, all new recruits at the GYN at this point of time trained in transvaginal and transabdominal ultrasound with SoSim at determined and individually agreed training times. Two specific learning modules were defined to be completed during the course (see figure 1 ). All modules contain a structured learning program with text and video-based learning aids, automatic error correction and audiovisual feedback. The five learners were monitored by means of cloud-based evaluation of the learning outcomes and accompanying self-assessment at a measurement point via online-based evaluation using the EvaSys software [ https://www.electricpaper.biz/ /]. The survey included questions on demographics, previous experience, attitude, self-assessment of competences before and after training on the simulator, questions on general use of the simulator and transfer of learning to the clinic. The 14 competency questions were divided into technical (compliance with hygiene, probe handling, orientation in the B-scan, technical terms, setting standard sections, differentiation between normal and pathological findings), social (positioning, communication) and personal aspects (own limits, stress experience). The questions were answered using a five-point Likert scale from “applies” to “does not apply” (see figure 2 ). The piloting processes were analyzed and reflected in a circular manner by the authors by means of an exploratory survey of the perspectives of the participating authors and employees at MITZ in individual interviews. For this purpose, observation protocols and text documents were evaluated in the categories organizational-spatial, didactic-content, structural-personal. The process analysis confirmed research findings on self-directed learning to the effect that learners require learning assistance to reflect on their own learning process despite the availability of a comprehensive and transparent learning program with integrated learning management and feedback . As part of the pilot, this was realized by preselecting and controlling the sequence of the learning modules to be completed with additional feedback from the supervising senior physician to enable prompt clinical applicability. Furthermore, the greatest administrative effort for all those involved was required to coordinate individual appointments. The response rate for the evaluation of SoSim utilization was 100%. The results for the competency questions in the evaluation of the five GYN career starters are shown in figure 2 . The agreement values for the items increased for 11 of 14 questions in comparison before/after the training, while the values remained identical for three questions. The results influence the further organization of structural framework conditions, the transfer of the project to other areas as well as the didactic support and education research to be established. Structural For an acceptable use by learners, the SoSim-training as part of the overall training concept needs to be integrated into medical working hours, existing curricula, and further education programs . Departments need to provide appropriate resources. In addition to standards for instruction and training specifications, the transfer of the learnt to the patient must be ensured. To reduce the organizational effort, individual appointment booking for users was established using automated processes and digital booking tools. Transfer Due to the positive changes in the approval ratings in the evaluation of SoSim use, the simulator will soon be accessible to medical staff in further training in the specialist areas of internal medicine and anesthesia. The learning modules to be completed will be determined according to learning requirements and clinical applicability. The SoSim will be successively integrated into student training and accompanied by educational research. Didactic support Despite the integrated SoSim learning program, a learning assistant is required to pre-select and manage the learning modules. The results of the one-off evaluation indicate a subjective increase in learning. A larger sample with a pre-post design is aimed for significance tests and clear causal relationships. For an acceptable use by learners, the SoSim-training as part of the overall training concept needs to be integrated into medical working hours, existing curricula, and further education programs . Departments need to provide appropriate resources. In addition to standards for instruction and training specifications, the transfer of the learnt to the patient must be ensured. To reduce the organizational effort, individual appointment booking for users was established using automated processes and digital booking tools. Due to the positive changes in the approval ratings in the evaluation of SoSim use, the simulator will soon be accessible to medical staff in further training in the specialist areas of internal medicine and anesthesia. The learning modules to be completed will be determined according to learning requirements and clinical applicability. The SoSim will be successively integrated into student training and accompanied by educational research. Despite the integrated SoSim learning program, a learning assistant is required to pre-select and manage the learning modules. The results of the one-off evaluation indicate a subjective increase in learning. A larger sample with a pre-post design is aimed for significance tests and clear causal relationships. A transfer to further areas of education and the gradual implementation of SoSim training for student tutors and students is in planning and will be monitored via a separate study. Anne Röhle: [ 0009-0001-6691-5401 ] Marie-Christin Willemer: [ 0009-0000-7950-5922 ] The authors declare that they have no competing interests. |
An untargeted metabolomic analysis of acute AFB1 treatment in liver, breast, and lung cells | 948a5e81-c704-40e3-b3ac-82185d06bd34 | 11781672 | Biochemistry[mh] | Aflatoxin B1 (AFB1) is the most toxic member of the aflatoxins, a class of mycotoxins produced by the fungus Aspergillus flavus . AFB1 has been widely established as a causative agent in hepatocellular carcinoma (HCC) and is officially deemed a group 1 human carcinogen by the International Agency for Research on Cancer . AFB1 has been found to contribute to 4.6–28.2% of HCC cases globally, partly due to a synergistic interaction with hepatitis B virus infection . In addition, AFB1 has been shown to play a role in other detrimental health effects such as growth suppression, immune system modulation, and malnutrition which may be linked to liver or intestinal damage induced by AFB1 . A . flavus contaminates essential food supplies (rice, corn, groundnuts, etc.) with AFB1, especially under high humidity and heat . Due to their inability to be destroyed by standard cooking or industrial processing practices, strict monitoring of AFB1 in food sources is essential to prevent exposure to this toxin . AFB1 levels are restricted in variable amounts depending on the country. The maximum allowed level (MAL) ranges from 2 ug/kg in Romania to 20 ug/kg in the United States, and countries in regions with high humidity and heat or inadequate climate-controlled storage are especially vulnerable to AFB1 contamination . Thus, contamination and subsequent exposure to AFB1 is inevitable for certain populations. Therefore, an understanding of the mechanisms in which AFB1 causes its negative health effects is imperative to uncovering interventions to protect these populations. The main mechanism of action of AFB1 that contributes to its carcinogenicity is triggered when it is metabolized by cytochrome P450 enzymes, particularly CYP3A4 and CYP1A2, to form aflatoxin B1-8,9-epoxide (AFBO), a highly reactive metabolite that forms AFB1-N7-guanine adducts when reacting with cellular DNA. AFB1-N7-guanine can spontaneously be converted to a decyclized AFB1-formamidopyridine adduct (AFB1-FAPy) or the guanine residue undergoes depurination and leaves an apurinic (AP) site. Both outcomes result in forms of DNA mutations . A common form of mutation due to AFB1 exposure found to be implicated in HCC formation is a G→T transversion causing the mutation R249S in the p53 protein, a widely known tumor suppressor protein . AFBO can also spontaneously form AFB1 dihydrodiol which readily interconverts with its open ring dialdehyde form, AFB1 dialdehyde. The latter form can react with primary amine groups on proteins and is presumed to contribute to a pro-inflammatory and pro-proliferative state in tissues associated with carcinogenesis . Furthermore, AFBO can directly bind to RNA and protein and disrupt their normal functions . Other metabolites or derivatives of AFB1 can form spontaneous adducts with cellular structures, notably aflatoxin M1 (AFM1) and aflatoxin B2a (AFB2a). AFM1 was found to have a similar mechanism as AFB1 in forming adducts with DNA after epoxidation following biotransformation by cytochrome P450s. However, AFM1 forms epoxides much less readily as compared to AFB1 and has been shown to be less mutagenic . AFB2a binds to primary amines on amino acids, which has been shown to inhibit the activity of proteins such as deoxyribonucleases. AFB2a is also unique in its ability to form an adduct with phospholipids by binding to phosphoethanolamine head groups . In addition to HCC, AFB1 exposure has been illustrated to participate in the development of other detrimental health disorders such as malnutrition, growth impairment, and immune modulation . Multiple studies have found higher levels of aflatoxin biomarkers in kwashiorkor, marasmus, and marasmic kwashiorkor patients . It has been hypothesized that AFB1 interferes with metal bioavailability, lipogenesis, renal function, and parathyroid metabolism . Downregulation of the vitamin D receptor by AFB1 has been supported by in vitro studies . Enteropathy, including impaired intestinal function and absorption, has been observed to correlate with childhood malnutrition and AFB1 exposure . Taken together, this suggests a mechanism in which AFB1 causes malnutrition as well as growth impairment, possibly by affecting metabolic functions. Finally, impairment of immune function has been seen following AFB1 exposure by a reduction in immunoglobulins, anti-inflammatory cytokines, lymphocyte activation, and general suppressed humoral immunity in animal models . Some studies do indicate that this effect may only be true for lower doses of AFB1 and higher doses cause greater activation of immune functions . Altogether, these studies indicate that the toxic effects of AFB1 exposure are widespread and not all effects have a clearly defined mechanism. Moreover, the effects of AFB1 are likely to be different by cell type due to differences in the expression of enzymes that metabolize AFB1. While the mechanism of all AFB1-related toxicities is not clear, evidence suggests that metabolic disruptions may play a role, particularly in growth impairment and malnutrition. A comprehensive investigation into the metabolic effects of AFB1 exposure could provide valuable insight into understanding potential mechanisms that underlie AFB1 toxicity, and may point towards intervention strategies to prevent or mitigate those effects. Metabolomics enables the elucidation of changes in metabolism that occur in response to exposures which can be used to elucidate mechanisms and potential interventions to mitigate their negative effects . Identification of affected pathways and metabolites could better inform the understanding of how AFB1 adversely affects the body, how it contributes to various health issues, and how targeted pharmacological or nutritional therapies can be developed to combat the effects of AFB1 exposure. We present herein an untargeted metabolomic analysis of acute AFB1 treatment in HepG2, MDA-MB-231, and A549 cell lines in order to investigate metabolic pathways that are affected by AFB1 in liver, breast, and lung cells. Chemical reagents Optima grade solvents (water with 0.1% formic acid and methanol with 0.1% formic acid) and fetal bovine serum (FBS) were purchased from Fisher Scientific (Waltham, MA, USA). Phosphate-buffered saline (PBS) and Dubelcco’s Modified Eagle Medium (DMEM) with high glucose were purchased from Gibco (Grand Island, NY, USA). AFB1 was purchased from Cayman Chemical (Ann Arbor, MI, USA). A549, HepG2, and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). Cell culture A549, HepG2, and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells were plated in 6 well plates and grown to 80% confluency and were treated with AFB1 at doses of 0, 2.5, 5, and 10 μM. Dimethyl sulfoxide (DMSO) was used as the vehicle with a concentration of 0.1% for all treatments. All treatments were performed for 24 h (n = 4 per treatment). The range of treatment concentrations were based off of our previous work which showed bioactivity of AFB1 in liver cells at similar doses . Metabolite extraction After treatment, metabolites were extracted from cell samples as described previously . Briefly, treatment media was aspirated, and cells were washed with 2 mL of ice-cold PBS followed by the addition of 1 mL of an ice-cold solution of 80% methanol and 20% water. Cells were detached using cell scrapers and vortexed at 5000 rpm for 10 min. Protein concentration was measured by a bicinchoninic acid (BCA) assay, and additional 80% methanol was added to each tube to normalize for protein concentration. Samples were centrifuged at 16,000× g at 4°C for 10 min and supernatants were transferred to autosampler vials for analysis by ultra-high-pressure liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS). Quality control study pools (QCSP) were created by combining 50 μL of each sample into a single mixture. Three separate QCSPs were made for each cell line. Method blanks were created by adding 500 μL of 80% methanol to empty tubes and were processed in an identical manner as the study samples. All samples for a given cell line were derived from the same cryovial. UHPLC-HRMS metabolomics data acquisition, preprocessing, and multivariate analysis The metabolomics data in this study were generated using established methods as previously described . In brief, the data were collected using a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved with an HSS T3 C18 column (2.1 × 100 mm, 1.7 μm, Waters Corporation) held at a constant temperature of 50°C. The mobile phases consisted of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). The mobile phase gradient was initiated with 2% B, increased to 100% B over 16 minutes, and then maintained at 100% for 4 minutes, all at a flow rate of 400 μL/min. Mass spectral data were acquired using a data-dependent acquisition mode in positive polarity within a mass range of 70–1050 m/z. Data-dependent acquisition mode was used to fragment the top 20 ions in each spectra. To monitor instrument stability and data quality, quality control samples (QCSP) and blank injections were interspersed throughout the sample set during data acquisition, and each sample was analyzed with a 5 μL injection volume. The acquired UHPLC-HRMS raw data were subsequently processed using Progenesis QI (version 2.1, Waters Corporation, MA, USA) for alignment, peak picking, and deconvolution. Background noise was eliminated by filtering out peaks with higher average abundances in the blank injections compared to the QCSP injections. Data normalization was performed utilizing a reference sample from the QCSP, employing the "normalize to all" function within the Progenesis software. Compound identification/annotation Peaks were matched to an in-house library of reference standards or public mass spectral databases from the National Institute of Standards and Technology (NIST), the Human Metabolome Database (HMDB), and METLIN. The matching criteria encompassed multiple parameters, including retention time (RT, within ±0.5 minutes for in-house library matches), exact mass (MS, within <5 ppm), and fragmentation pattern (MS/MS, with a similarity score > 30). An ontology system was used to denote the evidence basis for each metabolite assignment, with the following designations: OL1 indicated a match to the in-house library for retention time, exact mass, and MS/MS; OL2a indicated an in-house match to the in-house library for retention time and exact mass; OL2b referred to a match to the in-house library for exact mass and MS/MS; PDa indicated a match to public databases for exact mass and MS/MS; PDb represented a public database match for exact mass and theoretical MS/MS (e.g., HMDB); PDc was used when there was a match to public databases for exact mass and isotopic similarity, and PDd signified a match to public databases for exact mass only. Multivariate and Univariate Statistical Analysis Following normalization and filtration, data were imported into SIMCA 16 (Sartorius Stedim Data Analytics AB, Umeå, Sweden). Pareto scaling was applied to all peaks for multivariate analysis. Principal component analysis (PCA) plots were built to evaluate data quality and unsupervised sample grouping, while orthogonal partial least squares-discriminant analysis (OPLS-DA) plots were employed as a supervised method to assess the differentiation of metabolomes between vehicle-treated cells and the experimental groups. Variable importance to projection (VIP) scores were calculated for each detected peak using the OPLS-DA models. All models were built using a 7-fold cross validation to assess the predictive ability of the model (Q2). Fold changes and p-values (using Students’ t-test) were calculated for each peak. P-values were not adjusted for multiple testing due to the exploratory, rather than confirmatory, nature of this study . Pathway analysis In-house matched peaks were uploaded to MetaboAnalyst 5.0 for pathway analysis. Correlations were performed using the “Statistical Analysis [metadata table]” module. Concentration of AFB1 treatment (0 uM, 2.5 μM, 5 μM, 10 μM) was categorized as a continuous variable under the covariate study design. A correlation and partial correlation analysis was performed using the correlation measure Pearson r. Metabolites with a p-value < 0.05 for each cell type were used for Pathway Analysis. Fold changes and two-sided Student’s t-test were calculated between each individual treatment dosage group with its control in all cell types using the volcano plot function in the “Statistical Analysis [one factor]” module in MetaboAnalyst. Metabolites with a p-value < 0.1 were used for Pathway Analysis. Significant compounds from each analysis were imported into MetaboAnalyst 5.0’s “Pathway Analysis” module to identify significantly perturbed metabolic pathways. The Small Molecule Pathway Database (SMPDB) pathway library was used as the pathway database and pathways with a p<0.1 were considered significant. Optima grade solvents (water with 0.1% formic acid and methanol with 0.1% formic acid) and fetal bovine serum (FBS) were purchased from Fisher Scientific (Waltham, MA, USA). Phosphate-buffered saline (PBS) and Dubelcco’s Modified Eagle Medium (DMEM) with high glucose were purchased from Gibco (Grand Island, NY, USA). AFB1 was purchased from Cayman Chemical (Ann Arbor, MI, USA). A549, HepG2, and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas, VA, USA). A549, HepG2, and MDA-MB-231 cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 50 U/mL penicillin, and 50 μg/mL streptomycin. Cells were plated in 6 well plates and grown to 80% confluency and were treated with AFB1 at doses of 0, 2.5, 5, and 10 μM. Dimethyl sulfoxide (DMSO) was used as the vehicle with a concentration of 0.1% for all treatments. All treatments were performed for 24 h (n = 4 per treatment). The range of treatment concentrations were based off of our previous work which showed bioactivity of AFB1 in liver cells at similar doses . After treatment, metabolites were extracted from cell samples as described previously . Briefly, treatment media was aspirated, and cells were washed with 2 mL of ice-cold PBS followed by the addition of 1 mL of an ice-cold solution of 80% methanol and 20% water. Cells were detached using cell scrapers and vortexed at 5000 rpm for 10 min. Protein concentration was measured by a bicinchoninic acid (BCA) assay, and additional 80% methanol was added to each tube to normalize for protein concentration. Samples were centrifuged at 16,000× g at 4°C for 10 min and supernatants were transferred to autosampler vials for analysis by ultra-high-pressure liquid chromatography–high-resolution mass spectrometry (UHPLC-HRMS). Quality control study pools (QCSP) were created by combining 50 μL of each sample into a single mixture. Three separate QCSPs were made for each cell line. Method blanks were created by adding 500 μL of 80% methanol to empty tubes and were processed in an identical manner as the study samples. All samples for a given cell line were derived from the same cryovial. The metabolomics data in this study were generated using established methods as previously described . In brief, the data were collected using a Vanquish UHPLC system coupled to a Q Exactive™ HF-X Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Chromatographic separation was achieved with an HSS T3 C18 column (2.1 × 100 mm, 1.7 μm, Waters Corporation) held at a constant temperature of 50°C. The mobile phases consisted of water with 0.1% formic acid (A) and methanol with 0.1% formic acid (B). The mobile phase gradient was initiated with 2% B, increased to 100% B over 16 minutes, and then maintained at 100% for 4 minutes, all at a flow rate of 400 μL/min. Mass spectral data were acquired using a data-dependent acquisition mode in positive polarity within a mass range of 70–1050 m/z. Data-dependent acquisition mode was used to fragment the top 20 ions in each spectra. To monitor instrument stability and data quality, quality control samples (QCSP) and blank injections were interspersed throughout the sample set during data acquisition, and each sample was analyzed with a 5 μL injection volume. The acquired UHPLC-HRMS raw data were subsequently processed using Progenesis QI (version 2.1, Waters Corporation, MA, USA) for alignment, peak picking, and deconvolution. Background noise was eliminated by filtering out peaks with higher average abundances in the blank injections compared to the QCSP injections. Data normalization was performed utilizing a reference sample from the QCSP, employing the "normalize to all" function within the Progenesis software. Peaks were matched to an in-house library of reference standards or public mass spectral databases from the National Institute of Standards and Technology (NIST), the Human Metabolome Database (HMDB), and METLIN. The matching criteria encompassed multiple parameters, including retention time (RT, within ±0.5 minutes for in-house library matches), exact mass (MS, within <5 ppm), and fragmentation pattern (MS/MS, with a similarity score > 30). An ontology system was used to denote the evidence basis for each metabolite assignment, with the following designations: OL1 indicated a match to the in-house library for retention time, exact mass, and MS/MS; OL2a indicated an in-house match to the in-house library for retention time and exact mass; OL2b referred to a match to the in-house library for exact mass and MS/MS; PDa indicated a match to public databases for exact mass and MS/MS; PDb represented a public database match for exact mass and theoretical MS/MS (e.g., HMDB); PDc was used when there was a match to public databases for exact mass and isotopic similarity, and PDd signified a match to public databases for exact mass only. Following normalization and filtration, data were imported into SIMCA 16 (Sartorius Stedim Data Analytics AB, Umeå, Sweden). Pareto scaling was applied to all peaks for multivariate analysis. Principal component analysis (PCA) plots were built to evaluate data quality and unsupervised sample grouping, while orthogonal partial least squares-discriminant analysis (OPLS-DA) plots were employed as a supervised method to assess the differentiation of metabolomes between vehicle-treated cells and the experimental groups. Variable importance to projection (VIP) scores were calculated for each detected peak using the OPLS-DA models. All models were built using a 7-fold cross validation to assess the predictive ability of the model (Q2). Fold changes and p-values (using Students’ t-test) were calculated for each peak. P-values were not adjusted for multiple testing due to the exploratory, rather than confirmatory, nature of this study . In-house matched peaks were uploaded to MetaboAnalyst 5.0 for pathway analysis. Correlations were performed using the “Statistical Analysis [metadata table]” module. Concentration of AFB1 treatment (0 uM, 2.5 μM, 5 μM, 10 μM) was categorized as a continuous variable under the covariate study design. A correlation and partial correlation analysis was performed using the correlation measure Pearson r. Metabolites with a p-value < 0.05 for each cell type were used for Pathway Analysis. Fold changes and two-sided Student’s t-test were calculated between each individual treatment dosage group with its control in all cell types using the volcano plot function in the “Statistical Analysis [one factor]” module in MetaboAnalyst. Metabolites with a p-value < 0.1 were used for Pathway Analysis. Significant compounds from each analysis were imported into MetaboAnalyst 5.0’s “Pathway Analysis” module to identify significantly perturbed metabolic pathways. The Small Molecule Pathway Database (SMPDB) pathway library was used as the pathway database and pathways with a p<0.1 were considered significant. After data preprocessing, 20,315 features remained in the untargeted dataset. PCA of all peaks showed tight clustering of each cell line’s QCSPs in the center of the study samples, a common benchmark of metabolomics data quality . Additionally, PCA showed a moderate separation of dosage levels in each cell line . Supervised multivariate analysis using OPLS-DA showed strong and reproducible separation of each dose level from the vehicle control in each cell line (R2X, Q2 > 0.7) . OPLS-DA models were used to calculate VIP values for each peak. Fold changes and p-values were also calculated for each peak using normalized peak intensities for each dose-vehicle comparison for each cell line . Matching peaks to compounds revealed 566 peaks matched to the in-house library and 14,898 peaks matched to public databases. Correlation analysis between peak intensities and AFB1 dose levels was then performed using only in-house matched metabolites. For A549 samples, this correlation analysis yielded 99 in-house matched metabolites that significantly correlated with AFB1 dose levels (p<0.05). These significant metabolites were subsequently imported into MetaboAnalyst’s pathway analysis, identifying nine significant pathways (p<0.1) which included catecholamine biosynthesis, carnitine biosynthesis, glycine and serine metabolism, methionine metabolism, arginine and proline metabolism, oxidation of branched chain fatty acids, ammonia recycling, beta-alanine metabolism, and beta oxidation of very long chain fatty acids ( and ). Pairwise pathway comparisons between the vehicle and each dose level were also performed using metabolites with p<0.1. Comparison of the vehicle with the 2.5 μM group produced 82 significant in-house matched metabolites which produced one significant pathway ( and ). Comparison of the vehicle and 5 μM groups showed 147 significant in-house matched metabolites which led to six significant pathways ( and ). The 10 μM treatment versus vehicle comparison found 160 significant in-house matched metabolites, which produced five significant pathways ( and ). Catecholamine biosynthesis, carnitine synthesis, beta oxidation of very long chain fatty acids, arginine and proline metabolism, and ammonia recycling were found to be significant in higher dosage treatment group analyses (5 μM and 10 μM) as well as in the correlation analysis. The only significant pathway yielded by the 2.5 μM treated cells was the tricarboxylic acid cycle. For HepG2 cells, correlation of metabolite intensities with AFB1 dose levels revealed 93 significant metabolites (p<0.05) which revealed six significant pathways (p<0.1) in pathway analysis ( and ). Pairwise comparisons of the 2.5 μM treatment with vehicle-treated HepG2 cells revealed 193 significant matched metabolites which corresponded to seven significant pathways ( and ). The analysis of the 5 μM treatment group found 218 significant matched metabolites which revealed five significant pathways ( and ). The 10 μM treatment group analysis generated 189 significant matched metabolites and three significant pathways ( and ). Major pathways found across multiple analyses of treated HepG2 cells include spermidine and spermine biosynthesis, alpha linolenic acid and linoleic acid metabolism, purine metabolism, and catecholamine biosynthesis. Lastly, for MDA-MB-231 cells, correlation analyses of the concentrations of matched features and AFB1 treatment level found 146 significant matched metabolites (p<0.05) which corresponded to ten pathways with p<0.1 ( and ). When comparing metabolite intensities between vehicle and 2.5 μM AFB1 treated MDA-MB-231 cells, 141 matched metabolites with p<0.1 were identified. Inputting these metabolites into pathway analysis, seven significant pathways (p<0.1) were revealed ( and ). The same statistical and pathway analysis with MDA-MB-231 cells treated with 5 μM AFB1 generated 194 significant in-house matched metabolites which yielded eight significant pathways ( and ). Lastly, the 10 μM AFB1 treatment resulted in 208 significant matched metabolites and 11 pathways ( and ). Some notable pathways that were affected aligned with those found in A549 and HepG2, such as spermidine and spermine biosynthesis, carnitine synthesis, and catecholamine biosynthesis. Oxidation of branched chain fatty acids were seen to be significant in some analyses of every cell type examined. Methionine metabolism, phosphatidylcholine biosynthesis, glycine and serine metabolism were consistently and uniquely found to be significant in MDA-MB-231 cells following AFB1 treatment. AFB1 has been identified as an international hazard to human health due to its demonstrated role in HCC as well as its potential role in malnutrition, growth impairment, and immune modulation. Yet, its precise mechanism of action in some of these effects is still not completely understood. Using untargeted metabolomics, we sought to further investigate the effect of acute AFB1 treatment on cellular metabolism and how this differs between lung (A549), liver (HepG2), and breast (MDA-MB-231) cells. A549 and HepG2 cells were selected for our investigation due to AFB1’s established carcinogenic effect on liver and lung tissue, while MDA-MB-231 cells were evaluated as breast milk is a common transmission method of AFB1 toxicity to infants. Our data revealed numerous lipid-related pathways related as altered by AFB1 across the studied cell lines. Treatment of HepG2 cells with AFB1 caused perturbations in alpha linolenic and linoleic acid metabolism, oxidation of branched chain fatty acids, carnitine synthesis, and beta oxidation of very long chain fatty acids. With the exception of alpha linolenic and linoleic acid metabolism, all of these pathways were also altered in A549 cells, and all except for beta oxidation of very long chain fatty acids were altered in MDA-MB-231 cells. Notably, catecholamine biosynthesis was also perturbed in all cell lines by AFB1 treatment. In addition to similarities between cell lines, our data also show that AFB1 effects are different for different cell lines. For example, purine metabolism as well as spermine and spermidine biosynthesis were altered in both HepG2 and MDA-MB-231 cells, but not in A549. This data supports that AFB1 can have significantly different metabolic effects depending on the tissue type, but lipid and catecholamine metabolism are most commonly affected. There have been relatively few metabolomic studies examining the effects of AFB1. One study using HepB3 cells found that pathways associated with lipids in the cell membrane such as arachidonic acid metabolism and glycerophospholipid metabolism were altered following AFB1 treatment . Upregulation of glycerophospholipid metabolism and choline metabolism were observed in goat plasma after AFB1 administration . Induced mammary gland toxicity in pregnant and lactating rats also identified glycerophospholipid metabolism as an altered pathway from AFB1, as did an analysis of NCM460 intestinal cells and a lipidomics study of zebrafish brains which also found alterations in sphingolipid and glycerolipid metabolism . These findings align with our data that identified alpha-linolenic acid and linoleic acid metabolism as altered pathways in HepG2 cells as well as phosphatidylcholine metabolism in MDA-MB-231 cells. Our data also revealed changes in various amino acid pathways following AFB1 treatment which was consistent with other metabolomic studies . Finally, our study found that purine metabolism was altered in HepG2 cells following AFB1 treatment, a finding that was also shown previously in HepB3 cells . Lipid metabolism dysregulation has been a known consequence of cancer, and treatments that target it have been a topic of research. Cancer cells need an abundance of lipids to form membranes for daughter cells, generate energy, and act as signaling molecules . Metabolic studies that evaluated the effects of AFB1 commonly implicated lipid metabolism dysregulation in its mechanism to induce toxicity in the liver . Lipid biosynthesis and desaturation has been found to be a necessary component of HCC proliferation and has suppressed cancer growth and/ or induced apoptosis when inhibited. This is evident by upregulation of fatty acid synthase, acetyl CoA carboxylase, stearoyl CoA desaturase-1, and ATP citrate lyase in HCC . Carnitine metabolism was also identified in our study as a perturbed pathway. Upregulation of CPT1c has been seen in many cancers, likely as a mechanism to generate ATP and NADPH in response to glucose deprivation . CPT1 has also been seen to have a role in cancer cell gene expression, growth, metastasis, and apoptosis in certain cell types . Taken together, our data suggests that alterations in lipid metabolism may be a potential mechanism that supports AFB1-induced carcinogenesis in liver and lung cells. Purine metabolism was an additional pathway that was significantly affected in HepG2 cells when treated with AFB1 in multiple analyses. As highlighted previously, it has been established that a mechanism in which AFB1 causes cancer is by forming AFB1-N7-guanine adducts which then form AFB1-FAPy or AP sites, causing DNA mutations. Furthermore, N6-methyladenosine (m6A) dysregulation has been observed to have a role in liver carcinogenesis, possibly indicating another mechanism in which AFB1 causes the development of HCC via altered levels of purine metabolism. Given that purines and their associated enzymes are necessary to provide the building blocks for the uncontrolled cell growth seen in cancer, it is plausible that altered purine metabolism may contribute to AFB1-induced carcinogenesis . AFB1 and its metabolites have been established to be transmitted in the breast milk to impart toxicity on infants and sometimes cause growth impairment. The altered pathways that we observed in MDA-MB-231 cells could be reflective of the altered nutrient profile of the breast milk and contribute to the growth abnormalities associated with consuming AFB1-contaminated breast milk. Spermine and spermidine biosynthesis, carnitine synthesis, phospholipid/unsaturated fatty acid metabolism, and amino acid pathways were among the major metabolic effects of AFB1 treatment in MDA-MB-231 cells. Many of these pathways play a significant role in fatty acid beta oxidation and energy production which may affect development. Preterm infants have reduced capacity to store and synthesize carnitine and a diet containing carnitine is necessary for adequate growth . Furthermore, breast milk is commonly the source of carnitine for infants and carnitine intake in preterm infants also has been shown to have a positive correlation with brain size, height, weight, and head circumference z-score change with very preterm infants . If this pathway is altered in a mother’s breast cells and affects the nutritional composition of breast milk, this could play a role in growth stunting associated with AFB1 toxicity in infants. Pathways related to plasma membrane components were also identified in many MDA-MB-231 analyses. Phosphatidylcholine biosynthesis was a very prominent pathway, but alpha linolenic acid and linoleic acid metabolism were also implicated in the analyses of MDA-MB-231 cells when treated with 5 μM of AFB1. In Malawi children with environmental enteric dysfunction, a common source of growth stunting in children of low-income countries, phospholipids such as phosphatidylcholine levels were lowered . A study of undernourished rural Pakistani infants found that they had a deficiency in essential fatty acids (linoleic acid and total n-6 polyunsaturated fatty acids). Because of this, the body would counteract this effect by having high levels of oleic acid and elongase and desaturase activity used in n-3 and n-6 synthesis. This deficiency was found to be greatly associated with acute and chronic growth faltering in children with environmental enteric dysfunction . AFB1 exposure is known to trigger dysfunction in the intestinal barrier as well as intestinal immune function . Thus, changes in phosphatidylcholine and essential polyunsaturated fatty acid synthesis in the breast may participate in its deficiency in the infant, contributing to the growth stunting associated with AFB1 toxicity. In conclusion, this study evaluated the effects of varying concentrations of AFB1 treatment (2.5 μM, 5 μM, 10 μM) on HepG2 liver cells, A549 lung cells, and MDA-MB-231 breast cells. Our analyses yielded various significantly affected metabolic pathways in each cell type–some pathways were commonly affected across all cell lines whereas other pathways were affected in a cell type-dependent manner. While the metabolic effects of AFB1 were broad, pathways related to lipid metabolism (fatty acid beta oxidation, phoshoplipid metabolism) and tyrosine metabolism (catecholamine biosynthesis) emerged as major metabolic pathways affected in all cell lines. As liver and lung cancers are known to be associated with AFB1 exposure, understanding which pathways are changed may be essential to reveal possible therapeutic targets to combat AFB1-induced carcinogenesis. Given that breast milk is a common vehicle for AFB1 exposure to infants and subsequently causes growth stunting, we analyzed the treatment of MDA-MBA-231 cells to consider how its affected pathways may affect the nutrient composition of breast milk and contribute to AFB1-related developmental toxicity. Similarly, a proper understanding of what metabolic pathways contribute to AFB1 toxicity may enable the development of targeted treatments to prevent or mitigate its adverse effects. Future research in this area includes conducting a similar investigation using animal models where the metabotypes of liver, lung, and breast tissue are evaluated. This has several advantages over in vitro approaches which can be influenced from experimental conditions (e.g., media conditions, types of plastics used) that can alter results. Our findings will provide a foundation for these future investigations with larger sample sizes and in vivo models. In addition, other key organs could also be examined such as the brain or gut epithelium to further understand tissue-specific effects of AFB1. Studies involving measuring the exometabolome of treated cells as well as circulating metabolites in breast milk or blood in AFB1-exposed individuals would also be valuable follow up studies. Additionally, future experiments should also investigate whether targeting these metabolic processes alters the cytotoxicity or genotoxicity of AFB1 in various tissues, which could potentially identify new interventions to protect against AFB1 toxicity. S1 Fig Quality control plot of metabolomics data. PCA of all study samples and quality control study pools (QCSPs) for (A) all cell lines used, (B) A549 cells only, (C) HepG2 cells only, and (D) MDA-MB-231 cells only using all metabolomics peaks. (TIF) S1 Table Peak statistics for all cell lines and treatment groups. (XLSX) |
Cryoablation-induced modulation of Treg cells and the TGF-β pathway in lung adenocarcinoma: implications for increased antitumor immunity | d8fd6c7b-fcfa-4f09-a5a9-8e92dba9cb46 | 11827211 | Surgical Procedures, Operative[mh] | As is well documented, lung cancer is one of the most prevalent malignant tumors worldwide and threatens the health and life of humans. According to the Global Cancer Data 2022 report, lung cancer is currently the leading cause of cancer deaths globally, accounting for 18.7% of all cancer-related deaths . Lung adenocarcinoma (LUAD) is the primary pathological type of lung cancer . While radical surgery can yield positive outcomes in early-stage patients with a favorable prognosis, a considerable number of patients are diagnosed only at an advanced stage with a dismal prognosis . Cryoablation, a minimally invasive treatment for tumors, has long been used for the treatment of inoperable or unresectable LUAD patients. It offers distinct advantages over other treatment modalities, including a lower incidence of side effects and trauma, as well as improved visualization and targeting . Cryoablation-mediated tumor cell death involves a multifaceted mechanism comprising rapid freezing and slow rewarming processes at ultra-low temperatures, leading to tumor cell demise and the induction of apoptosis . Furthermore, ablated tumors that persist within the patient’s body, being no longer viable, continually release tumor-associated antigens (TAAs) that are subsequently engulfed by antigen-presenting cells (APCs), thereby priming specific immune responses against residual tumor cells . Notably, some studies have concluded that cryoablation can induce the regression of distant metastatic tumors, a phenomenon referred to as the abscopal effect . The tumor microenvironment (TME) plays a pivotal role in the immune evasion of lung cancer. Comprised of tumor cells, immune cells, endothelial cells, fibroblasts, and extracellular matrix , the TME is a complex interplay that collectively fosters conditions conducive to the growth and metastasis of lung cancer. Tregs, which characterized by the surface markers CD4 and CD25, are instrumental in maintaining immune suppression. High frequency of Tregs indicates worse outcome of NSCLC patients . The differentiation and function of Tregs are crucially regulated by the TGF-β pathway, which promotes the expression of Forkhead box protein 3 (Foxp3). Foxp3, essential for the development and immunosuppressive function of Tregs, plays a pivotal role in their inhibitory activities. In addition to its role in Treg differentiation, TGF-β1 also contributes to the immunosuppressive tumor microenvironment by being secreted by Tregs themselves. This secretion of TGF-β1 by Tregs further suppresses antitumor immunity, which creates a negative feedback loop that allows the tumor to evade immune surveillance . In Foxp3-deficient mice, CD4 + CD25 + T cells have been shown to lack immune regulatory function. High frequency of Tregs indicates worse outcome of NSCLC patients . Cryoablation of LUAD has demonstrated its effectiveness in not only killing cancer cells but also destroying the TME. Previous studies have shown that cryoablation can effectively reduce the number of Tregs in different types of solid tumors, such as kidney cancer, liver cancer, and prostate cancer . However, there is currently no research investigating the alterations in Treg content and the underlying mechanisms following cryoablation, specifically in LUAD. In this study, multiomics approaches were employed to investigate the relationship between cryoablation and the local immune microenvironment in LUAD. Briefly, a cell cryoablation model and a mouse subcutaneous transplanted tumor model were established. In vitro and in vivo experiments validated that cryoablation inhibits Tregs and enhances antitumor immune responses by modulating the TGF-β pathway. Taken together, our findings highlight the dual role of cryoablation in eliminating lung adenocarcinoma cells and concomitantly stimulating antitumor immunity, offering potential benefits in preventing tumor progression and recurrence among LUAD patients. scRNA-seq analysis Tissue preparation and scRNA-seq library construction Samples were obtained from the Dongfang Hospital, Beijing University of Chinese Medicine. Samples of carcinoma tissues were derived from three lung adenocarcinoma cases carrying non-metastasis, major, non-treated carcinomas receiving lung adenocarcinoma resecting. The adjacent normal tissues received the taking from over 5 mm in cancerous tissues’ negative margins. Tissues were preserved in sCelLive™ and processed within 30 min. Single-cell suspensions were prepared and scRNA-seq libraries were constructed using the GEXSCOPE® Single-cell RNA Library Kits protocol . Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate gene expression profiles. Cell cluster analysis Quality control was performed to exclude low-quality cells. Normalized and log-transformed data were subjected to variable gene selection and principal component analysis (PCA). The Louvain algorithm was used for cell clustering, and UMAP was used for visualization. Differential expression analysis identified DEGs, and pathway enrichment analysis was performed from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to explore the functions of Treg subclusters. Cell type identification was determined based on the expression of canonical markers from the SynEcoSysTM database, and major cell types were further subtyped through reclustering. Trajectory analysis To explore cell differentiation and developmental potential, trajectory analysis was conducted using CytoTRACE and Monocle2. CytoTRACE v0.3.3 was used to predict the differentiation state of cell subpopulations based on gene counts and expression derived from scRNA-seq data. Monocle2 v2.22.0 was employed to reconstruct the cell differentiation trajectory of monocyte subtypes by selecting top highly variable genes using FindVairableFeatures in Seurat, performing dimension reduction using DDRTree, and visualizing the trajectory using the plot_cell_trajectory function of Monocle2. These analyses provided insights into cellular differentiation pathways and potential. Sample collection and processing Blood samples were collected from a group of stage IIIb/IV LUAD patients undergoing cryoablation at Dongfang Hospital, Beijing University of Chinese Medicine, to evaluate changes in TGF-β and Tregs levels before and after cryoablation. The cryoablation system was used for percutaneous probe insertion under CT guidance, with probe size and quantity tailored to the tumor’s characteristics. The procedure involved a dual freeze–thaw cycle: 20 min of ice ball formation, 10 min of thawing, and 15 min of refreezing, achieving cryoprobe temperatures of − 196 °C within 5 min (registration number: ChiCTR2000038580). Peripheral blood samples were collected from patients 1 day before, 3 days after, and 1 month after the procedure, and flow cytometry was performed to analyze the content of Tregs, whilst ELISA was used to analyze TGF-β1 levels. This prospective study was approved by the Ethics Committee of Dongfang Hospital, Beijing University of Chinese Medicine. Mouse models and cryoablation The animal experiments in this study were approved by the Laboratory Animal Ethics Committee of Dongfang Hospital, Beijing University of Chinese Medicine. Six to 8 weeks C57BL/6 male wild-type mice ( n = 20) were purchased from Sibeifu Inc. (Beijing, China). A tumor‐bearing mouse model was constructed by subcutaneously injecting of Lewis lung adenocarcinoma cells (2 × 10 6 cells/mouse) into the right flank of each mouse. Next, the mice were randomly divided into four groups, namely the control group (Con), TGF-β1 inhibitor group (SB431542), cryoablation group (Cry), and cryoablation + TGF-β1 inhibitor group (Cry + SB431542). After 7 days, the tumor was subjected to cryoablation using a cryoablation system for two cycles. The temperature of each cycle was reduced to − 120 °C for 10 s, followed by rewarming to 10 °C . Mice in the control group underwent only surgical incision, and suturing under strict aseptic conditions. Mice in the TGF-β1 inhibitor group were only intraperitoneally injected with SB431542 (APExBIO, Houston, TX, USA) once daily. On the other hand, mice in the cryoablation + TGF-β1 inhibitor group were intraperitoneally injected with 1 μM SB431542 in DMSO once daily from postoperative day 0 to postoperative day 14. After the intervention, the surgical site was disinfected once daily to prevent infection. The size and volume of the tumors were recorded after the operation, and a growth curve was plotted for 14 days. Histology and immunostaining Tumor tissues from mice in each group were fixed and embedded in paraffin, and sections were prepared. Hematoxylin–eosin (HE) staining, Masson staining, and immunohistochemistry for Ki67 (1:200) (ab15580, Abcam, Cambridge, UK) were performed according to standard protocols . Enzyme‐linked immunosorbent assay TGF-β1 and IFN-γ levels were measured in human and mouse serum samples using a commercial ELISA kit (Proteintech, Chicago, USA). ELISA was performed according to the manufacturer’s protocol. Bulk RNA-seq and data analysis Three tumor tissue samples from mice in both the cryoablation group and the control group were selected for total RNA was extraction using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA). Next, the RNA samples were sequenced by Gene Denovo Biotechnology Co. (Guangzhou, China). Strand-specific libraries were constructed from the extracted RNA, followed by sequencing using the Illumina HiSeq 4000 platform. Differential expression analysis was performed using DESeq2 , comparing samples across different groups. Genes with a false discovery rate (FDR) < 0.05 and an absolute fold change ≥ 2 were considered differentially expressed genes and were further subjected to Gene Ontology (GO) and KEGG pathway enrichment analyses. Real‐time quantitative polymerase chain reaction Three tumor tissue samples were obtained from each group of mice. Total RNA was extracted using RNA extraction solution (Servicebio, Wuhan, China), while reverse transcription was performed using the Reveraid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA), and PCR was performed using SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China). β-Actin served as an internal control for mRNA. The 2 −ΔΔCT method was used for relative quantification. The primers (5′–3′) used for gene amplification are listed in Additional file 1: Table S1. Western blot (WB) analysis WB analysis was performed as described previously . The following primary antibodies were used: TGF-β1 (21,898–1-AP, Proteintech), SMAD2 (12,570–1-AP, Proteintech), p-SMAD2 (ab188334, Abcam), SMAD3 (66,516–1-Ig, Proteintech), p-SMAD3 (ab52903, Abcam), FOXP3 (ab215206, Abcam), Mouse anti-GAPDH (60,004–1-Ig, Proteintech), and Rabbit anti-GAPDH (10,494–1-AP, Proteintech). Cell culture and treatment Human lung adenocarcinoma cell lines A549 and H1299 were procured from Wuhan Pu-nuo-sai Life Technology Co. Ltd. (Wuhan, China); mouse lung adenocarcinoma cells (Lewis lung carcinoma cells, LLC) were sourced from Jiangsu Kaiji Bio-Technology Co. Ltd (Nanjing, China). Both A549 and LLC cells were incubated in high-glucose DMEM (HyClone, USA) medium supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA), whereas H1299 cells were incubated in RPMI-1640 medium (HyClone, USA). All cells were incubated in an incubator at 37 °C and 5% CO 2 . To activate T cells within PBMCs obtained from healthy volunteers, a 24-well plate was coated with anti-CD3 antibody (clone: UCHT1, BioLegend, CA, USA) diluted to 5 μg/mL in PBS. A volume of 400 μL of the diluted antibody was added to each well and incubated overnight at 4 °C. Following incubation, the wells were aspirated and washed three times with PBS to remove any unbound antibody. The PBMCs were then added to these wells. Construction of the cryoablation cell model LUAD cells (5 × 10 5 cells/mL, 5 mL) were sequentially collected from cryogenic tubes, frozen in a − 80 °C refrigerator for 7 min, and rewarmed in a 37 °C water bath. These cells remained partially viable and were characterized as sublethal cells outside the cryoprobe. The other cryogenic tube was frozen in liquid nitrogen for 5 min, resulting in cell lysis and necrosis. The supernatant was collected by centrifugation. The necrotic medium was prepared by combining the complete medium and supernatant at a ratio of 2:1 . Experimental groups: Group A: healthy cells + complete medium (unfrozen area), Group B: healthy cells + necrotic medium (healthy cells located near the frozen area), Group C: sublethal cells + complete medium (away from the necrotic sublethal area), Group D: sublethal cells + necrotic medium (close to the necrotic sublethal area). After incubation for 24 h, the ensuing experiments were performed. Co-culture assay Co-culture experiments were performed in 6-well plates with inserts with a pore size of 0.4 μm (Corning, NY, USA). The LUAD cells (5 × 10 5 cells, 1 ml), including the A549 and H1299 cell lines, were seeded and incubated in the outer wells of a 6-well plate. PBMCs (1 × 10 6 cells, 2 mL) pre-treated with anti-CD3 antibody as described in section “ ” were added to the inner wells of a Transwell system. All cells were incubated in a complete medium containing 5 µg/mL anti-CD28 (Clone: CD28.2, BioLegend, CA, USA) and 0.4 µL/mL IL-2 (Catalog No.: GMP-CD66, Novoprotein, Shanghai, China). After 72 h, cells in the inner wells were harvested to quantify the Tregs via flow cytometry. Treatment of samples and flow cytometric analysis The serum samples of patients, co-cultured cells in the inner wells, and spleen tissue of mice were collected to detect the content of Tregs. Splenic tissues were ground, filtered, and centrifuged into a single-cell suspension, respectively. The staining protocol was adapted based on the samples. Tregs were detected as either CD4, CD25 bright and Foxp3 staining, or CD4, CD25 bright , and CD127 low staining. Intracellular staining for Foxp3 was performed using a Foxp3 Staining Buffer Set (eBioscience, CA, USA), according to the manufacturer’s instructions. The antibodies are summarized in Additional file 1: Table S2. Statistical analysis Statistical analyses were performed using GraphPad Prism 8 (GraphPad, USA) and SPSS 20 (IBM, USA). P -value < 0.05 was considered statistically significant. Paired t -tests were employed for clinical patient samples, while independent t -tests were utilized for between-group comparisons. One-way ANOVA was conducted for comparisons across multiple groups. Tissue preparation and scRNA-seq library construction Samples were obtained from the Dongfang Hospital, Beijing University of Chinese Medicine. Samples of carcinoma tissues were derived from three lung adenocarcinoma cases carrying non-metastasis, major, non-treated carcinomas receiving lung adenocarcinoma resecting. The adjacent normal tissues received the taking from over 5 mm in cancerous tissues’ negative margins. Tissues were preserved in sCelLive™ and processed within 30 min. Single-cell suspensions were prepared and scRNA-seq libraries were constructed using the GEXSCOPE® Single-cell RNA Library Kits protocol . Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate gene expression profiles. Cell cluster analysis Quality control was performed to exclude low-quality cells. Normalized and log-transformed data were subjected to variable gene selection and principal component analysis (PCA). The Louvain algorithm was used for cell clustering, and UMAP was used for visualization. Differential expression analysis identified DEGs, and pathway enrichment analysis was performed from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to explore the functions of Treg subclusters. Cell type identification was determined based on the expression of canonical markers from the SynEcoSysTM database, and major cell types were further subtyped through reclustering. Trajectory analysis To explore cell differentiation and developmental potential, trajectory analysis was conducted using CytoTRACE and Monocle2. CytoTRACE v0.3.3 was used to predict the differentiation state of cell subpopulations based on gene counts and expression derived from scRNA-seq data. Monocle2 v2.22.0 was employed to reconstruct the cell differentiation trajectory of monocyte subtypes by selecting top highly variable genes using FindVairableFeatures in Seurat, performing dimension reduction using DDRTree, and visualizing the trajectory using the plot_cell_trajectory function of Monocle2. These analyses provided insights into cellular differentiation pathways and potential. Samples were obtained from the Dongfang Hospital, Beijing University of Chinese Medicine. Samples of carcinoma tissues were derived from three lung adenocarcinoma cases carrying non-metastasis, major, non-treated carcinomas receiving lung adenocarcinoma resecting. The adjacent normal tissues received the taking from over 5 mm in cancerous tissues’ negative margins. Tissues were preserved in sCelLive™ and processed within 30 min. Single-cell suspensions were prepared and scRNA-seq libraries were constructed using the GEXSCOPE® Single-cell RNA Library Kits protocol . Libraries were sequenced on an Illumina NovaSeq 6000 platform to generate gene expression profiles. Quality control was performed to exclude low-quality cells. Normalized and log-transformed data were subjected to variable gene selection and principal component analysis (PCA). The Louvain algorithm was used for cell clustering, and UMAP was used for visualization. Differential expression analysis identified DEGs, and pathway enrichment analysis was performed from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to explore the functions of Treg subclusters. Cell type identification was determined based on the expression of canonical markers from the SynEcoSysTM database, and major cell types were further subtyped through reclustering. To explore cell differentiation and developmental potential, trajectory analysis was conducted using CytoTRACE and Monocle2. CytoTRACE v0.3.3 was used to predict the differentiation state of cell subpopulations based on gene counts and expression derived from scRNA-seq data. Monocle2 v2.22.0 was employed to reconstruct the cell differentiation trajectory of monocyte subtypes by selecting top highly variable genes using FindVairableFeatures in Seurat, performing dimension reduction using DDRTree, and visualizing the trajectory using the plot_cell_trajectory function of Monocle2. These analyses provided insights into cellular differentiation pathways and potential. Blood samples were collected from a group of stage IIIb/IV LUAD patients undergoing cryoablation at Dongfang Hospital, Beijing University of Chinese Medicine, to evaluate changes in TGF-β and Tregs levels before and after cryoablation. The cryoablation system was used for percutaneous probe insertion under CT guidance, with probe size and quantity tailored to the tumor’s characteristics. The procedure involved a dual freeze–thaw cycle: 20 min of ice ball formation, 10 min of thawing, and 15 min of refreezing, achieving cryoprobe temperatures of − 196 °C within 5 min (registration number: ChiCTR2000038580). Peripheral blood samples were collected from patients 1 day before, 3 days after, and 1 month after the procedure, and flow cytometry was performed to analyze the content of Tregs, whilst ELISA was used to analyze TGF-β1 levels. This prospective study was approved by the Ethics Committee of Dongfang Hospital, Beijing University of Chinese Medicine. The animal experiments in this study were approved by the Laboratory Animal Ethics Committee of Dongfang Hospital, Beijing University of Chinese Medicine. Six to 8 weeks C57BL/6 male wild-type mice ( n = 20) were purchased from Sibeifu Inc. (Beijing, China). A tumor‐bearing mouse model was constructed by subcutaneously injecting of Lewis lung adenocarcinoma cells (2 × 10 6 cells/mouse) into the right flank of each mouse. Next, the mice were randomly divided into four groups, namely the control group (Con), TGF-β1 inhibitor group (SB431542), cryoablation group (Cry), and cryoablation + TGF-β1 inhibitor group (Cry + SB431542). After 7 days, the tumor was subjected to cryoablation using a cryoablation system for two cycles. The temperature of each cycle was reduced to − 120 °C for 10 s, followed by rewarming to 10 °C . Mice in the control group underwent only surgical incision, and suturing under strict aseptic conditions. Mice in the TGF-β1 inhibitor group were only intraperitoneally injected with SB431542 (APExBIO, Houston, TX, USA) once daily. On the other hand, mice in the cryoablation + TGF-β1 inhibitor group were intraperitoneally injected with 1 μM SB431542 in DMSO once daily from postoperative day 0 to postoperative day 14. After the intervention, the surgical site was disinfected once daily to prevent infection. The size and volume of the tumors were recorded after the operation, and a growth curve was plotted for 14 days. Tumor tissues from mice in each group were fixed and embedded in paraffin, and sections were prepared. Hematoxylin–eosin (HE) staining, Masson staining, and immunohistochemistry for Ki67 (1:200) (ab15580, Abcam, Cambridge, UK) were performed according to standard protocols . TGF-β1 and IFN-γ levels were measured in human and mouse serum samples using a commercial ELISA kit (Proteintech, Chicago, USA). ELISA was performed according to the manufacturer’s protocol. Three tumor tissue samples from mice in both the cryoablation group and the control group were selected for total RNA was extraction using the Trizol reagent kit (Invitrogen, Carlsbad, CA, USA). Next, the RNA samples were sequenced by Gene Denovo Biotechnology Co. (Guangzhou, China). Strand-specific libraries were constructed from the extracted RNA, followed by sequencing using the Illumina HiSeq 4000 platform. Differential expression analysis was performed using DESeq2 , comparing samples across different groups. Genes with a false discovery rate (FDR) < 0.05 and an absolute fold change ≥ 2 were considered differentially expressed genes and were further subjected to Gene Ontology (GO) and KEGG pathway enrichment analyses. Three tumor tissue samples were obtained from each group of mice. Total RNA was extracted using RNA extraction solution (Servicebio, Wuhan, China), while reverse transcription was performed using the Reveraid First Strand cDNA Synthesis Kit (Thermo Scientific, MA, USA), and PCR was performed using SYBR qPCR SuperMix Plus (Novoprotein, Shanghai, China). β-Actin served as an internal control for mRNA. The 2 −ΔΔCT method was used for relative quantification. The primers (5′–3′) used for gene amplification are listed in Additional file 1: Table S1. WB analysis was performed as described previously . The following primary antibodies were used: TGF-β1 (21,898–1-AP, Proteintech), SMAD2 (12,570–1-AP, Proteintech), p-SMAD2 (ab188334, Abcam), SMAD3 (66,516–1-Ig, Proteintech), p-SMAD3 (ab52903, Abcam), FOXP3 (ab215206, Abcam), Mouse anti-GAPDH (60,004–1-Ig, Proteintech), and Rabbit anti-GAPDH (10,494–1-AP, Proteintech). Human lung adenocarcinoma cell lines A549 and H1299 were procured from Wuhan Pu-nuo-sai Life Technology Co. Ltd. (Wuhan, China); mouse lung adenocarcinoma cells (Lewis lung carcinoma cells, LLC) were sourced from Jiangsu Kaiji Bio-Technology Co. Ltd (Nanjing, China). Both A549 and LLC cells were incubated in high-glucose DMEM (HyClone, USA) medium supplemented with 10% FBS (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA), whereas H1299 cells were incubated in RPMI-1640 medium (HyClone, USA). All cells were incubated in an incubator at 37 °C and 5% CO 2 . To activate T cells within PBMCs obtained from healthy volunteers, a 24-well plate was coated with anti-CD3 antibody (clone: UCHT1, BioLegend, CA, USA) diluted to 5 μg/mL in PBS. A volume of 400 μL of the diluted antibody was added to each well and incubated overnight at 4 °C. Following incubation, the wells were aspirated and washed three times with PBS to remove any unbound antibody. The PBMCs were then added to these wells. LUAD cells (5 × 10 5 cells/mL, 5 mL) were sequentially collected from cryogenic tubes, frozen in a − 80 °C refrigerator for 7 min, and rewarmed in a 37 °C water bath. These cells remained partially viable and were characterized as sublethal cells outside the cryoprobe. The other cryogenic tube was frozen in liquid nitrogen for 5 min, resulting in cell lysis and necrosis. The supernatant was collected by centrifugation. The necrotic medium was prepared by combining the complete medium and supernatant at a ratio of 2:1 . Experimental groups: Group A: healthy cells + complete medium (unfrozen area), Group B: healthy cells + necrotic medium (healthy cells located near the frozen area), Group C: sublethal cells + complete medium (away from the necrotic sublethal area), Group D: sublethal cells + necrotic medium (close to the necrotic sublethal area). After incubation for 24 h, the ensuing experiments were performed. Co-culture experiments were performed in 6-well plates with inserts with a pore size of 0.4 μm (Corning, NY, USA). The LUAD cells (5 × 10 5 cells, 1 ml), including the A549 and H1299 cell lines, were seeded and incubated in the outer wells of a 6-well plate. PBMCs (1 × 10 6 cells, 2 mL) pre-treated with anti-CD3 antibody as described in section “ ” were added to the inner wells of a Transwell system. All cells were incubated in a complete medium containing 5 µg/mL anti-CD28 (Clone: CD28.2, BioLegend, CA, USA) and 0.4 µL/mL IL-2 (Catalog No.: GMP-CD66, Novoprotein, Shanghai, China). After 72 h, cells in the inner wells were harvested to quantify the Tregs via flow cytometry. The serum samples of patients, co-cultured cells in the inner wells, and spleen tissue of mice were collected to detect the content of Tregs. Splenic tissues were ground, filtered, and centrifuged into a single-cell suspension, respectively. The staining protocol was adapted based on the samples. Tregs were detected as either CD4, CD25 bright and Foxp3 staining, or CD4, CD25 bright , and CD127 low staining. Intracellular staining for Foxp3 was performed using a Foxp3 Staining Buffer Set (eBioscience, CA, USA), according to the manufacturer’s instructions. The antibodies are summarized in Additional file 1: Table S2. Statistical analyses were performed using GraphPad Prism 8 (GraphPad, USA) and SPSS 20 (IBM, USA). P -value < 0.05 was considered statistically significant. Paired t -tests were employed for clinical patient samples, while independent t -tests were utilized for between-group comparisons. One-way ANOVA was conducted for comparisons across multiple groups. Human single-cell sequencing analysis reveals cellular heterogeneity in lung adenocarcinoma microenvironment To gain a comprehensive understanding of the cellular diversity within the lung adenocarcinoma microenvironment, single-cell RNA sequencing (scRNA-Seq) was performed on six samples collected from three patients with lung adenocarcinoma. The samples comprised three cases of lung adenocarcinoma tissues and three adjacent nontumor tissues. Following stringent quality control measures, single-cell transcriptomes were acquired for 95,318 cells. As depicted in the UMAP plot, seven major cell clusters were identified based on marker gene expression profiles: epithelial cells, stromal cells, proliferating cells, B cells, T cells, NK cells, mast cells, and mononuclear phagocytes (Fig. A). Histograms illustrating the specificity of the expression of each cell cluster across different samples revealed that epithelial cells and stromal cells were significantly overexpressed in lung adenocarcinoma tissues than in adjacent nontumor tissues (Fig. B). Conversely, T and NK cells and mast cells exhibited higher expression levels in adjacent nontumor tissues. To further identify immune cells associated with lung adenocarcinoma, a sub-clustering analysis of T and NK cells was conducted according to marker identification and subsequent merger of the clusters, yielding 11 distinct subtypes (Fig. C, E). According to the histograms representing these immune cell subgroups, the expression levels of Treg-IL2RA, NKT-HSPA6, and NaïveT-RPL34 were higher in tumor tissues compared to adjacent nontumor tissues (Fig. D). Detailed classification and developmental trajectory of Tregs in the human lung adenocarcinoma microenvironment The UMAP plot revealed seven cell clusters identified which were labeled as follows: Treg-LTB, Treg-PELI1, Treg-CCL5, Treg-HSPA6, Treg-CXCL13, Treg-CCL20, and Treg-IFI27 (Fig. A). For instance, the Treg-CCL5 cell cluster primarily expressed CCL5, GZMB, and CCL4, while the Treg-CXCL13 cell cluster predominantly expressed CXCL13, DRAIC, and C19orf70. The Treg-HSPA6 cell cluster was characterized by overexpression of HSPE1, DNAJB1, and HSPA6 (Additional file 1: Fig. S1). To elucidate the differentiation and developmental process of Tregs, the monocle and cytoTRACE R packages were used for pseudotime trajectory analysis of Treg subpopulations. According to the pseudotime analysis, during the early branch-point stage, Treg-IFI27 cells dominated, possessing greater differentiation potential and gradually transitioning towards intermediate and late-stage Treg differentiation. In the late stages of differentiation, Tregs primarily expressed HSPA6 and PELI1. Conversely, Treg-CXCL13, Treg-CCL5, Treg-CCL20, and Treg-LTB cells were predominantly concentrated in the intermediate stages of differentiation (Fig. B–D). Interestingly, both early and late Treg cell types were primarily localized within cancer-adjacent tissues, whereas Tregs within lung cancer tissues exhibited a more mature phenotype (Additional file 1: Fig. S2). The gene clustering heatmap illustrated the temporal trends of critical genes throughout the pseudotime analysis (Fig. E). The present study specifically focused on TGF-β and simulated the expression trajectory of TGFB1, noting that its expression level was increased during Treg differentiation (Additional file 1: Fig. S3). Pathway analysis suggested that genes within the Treg population regulated PD-L1 expression, Th17 cell differentiation, and the TGF-β pathway, indicating close relationships with tumor immune responses (Fig. F). Cryoablation-mediated regulation of Tregs in clinical and experimental models of LUAD To investigate the impact of cryoablation on Tregs and its potential to enhance immune responses in LUAD, a total of 22 patients were recruited at the Dongfang Hospital, Beijing University of Chinese Medicine, between August 2021 and February 2022, with 17 completing the trial. CD4 + CD25 high CD127 low/− was employed as a marker for Treg expression. To assess Treg content, aggregates and debris were initially excluded based on cell size and granularity, then lymphocyte populations were selected based on CD45 expression, and CD4 + T lymphocytes were identified by CD3 + and CD4 + expression. Finally, Tregs were determined through CD25 + CD127 low/− expression. As anticipated, the proportion of CD4 + CD25 high CD127 low/− in the majority of lung adenocarcinoma patients initially increased and subsequently decreased after cryoablation (Treg frequencies were 7.893 ± 2.0744% pre-surgery, 8.624 ± 2.6687% 3 days post-surgery, and 6.612 ± 1.0948% 30 days post-surgery). Statistical analysis revealed a significant decrease in Treg content 30 days post-surgery compared to both pre-surgery levels and 3 days post-surgery ( t = 2.793, p = 0.013; t = 2.923, p = 0.01) (Fig. A, B). We speculate that the transient increase in Tregs 3 days post-surgery may be linked to treatment response. Furthermore, cellular and murine cryoablation models were constructed to examine the effects of cryoablation on Tregs both in vitro and in vivo. Lung adenocarcinoma A549 and H1299 cells were subjected to liquid nitrogen to simulate cell lysis during cryoablation. Considering the temperature gradient from the center to the periphery of the ice ball, an environment of − 80 °C was designed to simulate the freezing conditions of peripheral ice balls. To evaluate the influence of cryoablation on Treg differentiation in vitro, the cellular cryoablation model was co-cultured with PBMCs (previously activated by CD3/CD28 and IL2). The results demonstrated that groups B, C, and D influenced Treg differentiation to varying degrees compared to the control group A (Fig. A, B). Notably, after accounting for the influence of tumor cell heterogeneity, lung adenocarcinoma cell lines cultured in a necrotic medium were found to more effectively inhibit Treg differentiation, with minimal association with cryogenic interventions. Additionally, flow cytometry experiments on mouse spleens revealed a reduction in the number of Tregs 14 days post-cryoablation compared to the control group. Compared to the Cry + SB431542 group, the Cry group showed a trend towards higher Treg numbers, though not statistically significant ( p > 0.05). This may result from the complex interactions between TGF-β signaling inhibition and the tumor microenvironment’s immune regulation, potentially masking the effects of cryoablation and SB431542 on Treg levels (Fig. C, D). Cryoablation enhances antitumor capacity in lung cancer mouse model To comprehensively investigate the antitumorigenic effects of cryoablation, a mouse model of lung cancer treated with cryoablation was established. After treatment, a significant reduction in tumor volume was observed in the cryoablation group compared to the control group 14 days post-surgery (Fig. A, B). Moreover, cryoablation altered the size, morphology, and structure of lung cancer cells (Fig. C). Meanwhile, immunohistochemistry assays also that the expression level of Ki-67 was significantly lower in the cryoablation group compared to the control group (Fig. D, E). These findings collectively indicated that cryoablation effectively suppressed the proliferation of residual tumor cells after ablation, thereby inhibiting tumor progression. Interestingly, cryoablation induced changes in collagen fiber structure within the tumor tissue, rendering the post-ablation tissue loose and predominantly exhibiting curved structures surrounding the tumor nests. Moreover, the Cry + SB431542 group showed a more pronounced change in this regard when compared with the Cry group (Fig. A, B). Upon collagen fibers transitioning from a curled to a linear configuration traversing through the tumor foci, tumor cells rapidly migrated along these reconstructed linear collagen fibers, a phenomenon referred to as “highways” for tumor invasion by researchers . In the current study, cryoablation suppressed changes in collagen fibers in Lewis lung adenocarcinoma mouse xenografts, thereby inhibiting tumor metastasis. Additionally, a lower number of lung metastases were noted in mice in the cryoablation group (60% vs. 0%, Con vs. Cry) (Fig. C). Bulk RNA-seq results indicate that cryoablation reverses the immunosuppressive microenvironment in mouse models To further explore the molecular mechanisms underlying cryoablation-enhanced antitumor immunity, RNA seq was performed on tumors collected from mice 14 days after cryoablation. Tumors were snap-frozen in liquid nitrogen for 15 min and stored at − 80 °C until processing. DESeq analysis was carried out to identify DEGs, which were visualized using volcano plots (Fig. A). Our results identified 236 upregulated genes and 153 downregulation genes in the cryoablation group compared to the control group. GO and KEGG enrichment analyses were performed on the DEGs using ClusterProfiler to determine their primary biological functions. Of note, the DEGs were significantly enriched in biological processes (BP) terms related to the regulation of lymphocyte proliferation, regulation of mononuclear cell proliferation, regulation of inflammatory response, and leukocyte migration. They were significantly enriched in cellular components (CC) terms associated with the MHC protein complex and the receptor complex and in molecular functions (MF) terms linked to receptor ligand activity, immune receptor activity, and immunoglobulin binding (Fig. B). Moreover, KEGG enrichment analysis identified several signaling pathways associated with the differential gene expression profile resulting from cryoablation, including Th17 cell differentiation, HIF-1 signaling pathway, p53 signaling pathway, VEGF signaling pathway, TGF-beta signaling pathway, and JAK − STAT signaling pathway (Fig. C). These findings suggest the involvement of multiple signaling cascades in mediating the effects of cryoablation on immune regulation and antitumor responses, providing valuable insights into potential therapeutic targets for enhancing cryoablation efficacy in cancer treatment. Further research is warranted to unravel the intricate interplay between these pathways and their contributions to cryoablation-induced immune modulation. Cryoablation inhibits the TGF-β/SMAD/FOXP3 pathway,suppressing immune evasion in LUAD Our previous bulk RNA-seq unveiled that lung adenocarcinoma cryoablation influences the TGF-β signaling pathway. scRNA-seq demonstrated a strong correlation between the TGF-β pathway and Tregs. Concurrently, flow cytometry corroborated that cryoablation suppressed Treg differentiation. To further investigate the underlying mechanisms, TGF-β1 expression was detected in the serum of patients, revealing a post-treatment pattern of initial increase followed by a decrease, which paralleled the trend in Treg differentiation (Fig. A). Both in vivo mouse models and in vitro cell experiments substantiated the inhibitory effect of cryoablation on TGF-β1 expression (Fig. B, C). qPCR and Western blot analyses determined that cryoablation downregulates TGF-β expression, thereby impairing the phosphorylation of Smad2 and Smad3 and subsequently suppressing downstream Foxp3 expression. The inhibitor SB431542 markedly suppressed the transcription levels of Smad2, Smad3, and Foxp3. Nevertheless, its effect on protein expression was less pronounced (Fig. D–I). These findings signaled that cryoablation inhibits the TGF-β/SMAD/FOXP3 pathway, hindering Treg differentiation and reshaping the immune microenvironment surrounding lung adenocarcinoma. This mechanistic understanding enhances our understanding of the mechanism by which cryoablation enhances antitumor immunity and provides potential therapeutic targets for optimizing cryoablation-based interventions in LUAD treatment. Cryoablation enhances antitumor capacity via IFN - γ To further investigate the potential of cryoablation to augment the killing of LUAD in addition to its effects on suppressing Tregs differentiation, we conducted a series of experiments. Our results demonstrated that the expression of IFN-γ progressively increased, whereas that of IL-2 decreased in lung cancer patients after cryoablation (Fig. A, D). Subsequently, ELISA was carried out to detect the levels of IFN-γ and IL-2 in mouse serum 2 weeks after cryoablation. Cryoablation was observed to produce a trend towards increased levels of IFN-γ, albeit non-statistically significant, which was higher when combined with a TGF-β1 inhibitor. In contrast, IL-2 expression was significantly lower in the treated group than in the control group (Fig. B, E). Moreover, a cell cryoablation model co-cultured with PBMCs was constructed, and ELISA was performed on the supernatant. Our results showed that, compared to the control group, the expression level of IFN-γ and IL-2 was increased and decreased in sublethal cells + necrotic medium ( D ), respectively, with a more prominent effect observed in the A549 cell line (Fig. C, F). These findings collectively suggest that cryoablation enhanced antitumor capacity through the modulation of IFN-γ and IL-2 expression. The upregulation of IFN-γ and concurrent downregulation of IL-2 may contribute to an improved immune response against lung adenocarcinoma cells, highlighting the potential of cryoablation as a complementary strategy in cancer immunotherapy. To gain a comprehensive understanding of the cellular diversity within the lung adenocarcinoma microenvironment, single-cell RNA sequencing (scRNA-Seq) was performed on six samples collected from three patients with lung adenocarcinoma. The samples comprised three cases of lung adenocarcinoma tissues and three adjacent nontumor tissues. Following stringent quality control measures, single-cell transcriptomes were acquired for 95,318 cells. As depicted in the UMAP plot, seven major cell clusters were identified based on marker gene expression profiles: epithelial cells, stromal cells, proliferating cells, B cells, T cells, NK cells, mast cells, and mononuclear phagocytes (Fig. A). Histograms illustrating the specificity of the expression of each cell cluster across different samples revealed that epithelial cells and stromal cells were significantly overexpressed in lung adenocarcinoma tissues than in adjacent nontumor tissues (Fig. B). Conversely, T and NK cells and mast cells exhibited higher expression levels in adjacent nontumor tissues. To further identify immune cells associated with lung adenocarcinoma, a sub-clustering analysis of T and NK cells was conducted according to marker identification and subsequent merger of the clusters, yielding 11 distinct subtypes (Fig. C, E). According to the histograms representing these immune cell subgroups, the expression levels of Treg-IL2RA, NKT-HSPA6, and NaïveT-RPL34 were higher in tumor tissues compared to adjacent nontumor tissues (Fig. D). The UMAP plot revealed seven cell clusters identified which were labeled as follows: Treg-LTB, Treg-PELI1, Treg-CCL5, Treg-HSPA6, Treg-CXCL13, Treg-CCL20, and Treg-IFI27 (Fig. A). For instance, the Treg-CCL5 cell cluster primarily expressed CCL5, GZMB, and CCL4, while the Treg-CXCL13 cell cluster predominantly expressed CXCL13, DRAIC, and C19orf70. The Treg-HSPA6 cell cluster was characterized by overexpression of HSPE1, DNAJB1, and HSPA6 (Additional file 1: Fig. S1). To elucidate the differentiation and developmental process of Tregs, the monocle and cytoTRACE R packages were used for pseudotime trajectory analysis of Treg subpopulations. According to the pseudotime analysis, during the early branch-point stage, Treg-IFI27 cells dominated, possessing greater differentiation potential and gradually transitioning towards intermediate and late-stage Treg differentiation. In the late stages of differentiation, Tregs primarily expressed HSPA6 and PELI1. Conversely, Treg-CXCL13, Treg-CCL5, Treg-CCL20, and Treg-LTB cells were predominantly concentrated in the intermediate stages of differentiation (Fig. B–D). Interestingly, both early and late Treg cell types were primarily localized within cancer-adjacent tissues, whereas Tregs within lung cancer tissues exhibited a more mature phenotype (Additional file 1: Fig. S2). The gene clustering heatmap illustrated the temporal trends of critical genes throughout the pseudotime analysis (Fig. E). The present study specifically focused on TGF-β and simulated the expression trajectory of TGFB1, noting that its expression level was increased during Treg differentiation (Additional file 1: Fig. S3). Pathway analysis suggested that genes within the Treg population regulated PD-L1 expression, Th17 cell differentiation, and the TGF-β pathway, indicating close relationships with tumor immune responses (Fig. F). To investigate the impact of cryoablation on Tregs and its potential to enhance immune responses in LUAD, a total of 22 patients were recruited at the Dongfang Hospital, Beijing University of Chinese Medicine, between August 2021 and February 2022, with 17 completing the trial. CD4 + CD25 high CD127 low/− was employed as a marker for Treg expression. To assess Treg content, aggregates and debris were initially excluded based on cell size and granularity, then lymphocyte populations were selected based on CD45 expression, and CD4 + T lymphocytes were identified by CD3 + and CD4 + expression. Finally, Tregs were determined through CD25 + CD127 low/− expression. As anticipated, the proportion of CD4 + CD25 high CD127 low/− in the majority of lung adenocarcinoma patients initially increased and subsequently decreased after cryoablation (Treg frequencies were 7.893 ± 2.0744% pre-surgery, 8.624 ± 2.6687% 3 days post-surgery, and 6.612 ± 1.0948% 30 days post-surgery). Statistical analysis revealed a significant decrease in Treg content 30 days post-surgery compared to both pre-surgery levels and 3 days post-surgery ( t = 2.793, p = 0.013; t = 2.923, p = 0.01) (Fig. A, B). We speculate that the transient increase in Tregs 3 days post-surgery may be linked to treatment response. Furthermore, cellular and murine cryoablation models were constructed to examine the effects of cryoablation on Tregs both in vitro and in vivo. Lung adenocarcinoma A549 and H1299 cells were subjected to liquid nitrogen to simulate cell lysis during cryoablation. Considering the temperature gradient from the center to the periphery of the ice ball, an environment of − 80 °C was designed to simulate the freezing conditions of peripheral ice balls. To evaluate the influence of cryoablation on Treg differentiation in vitro, the cellular cryoablation model was co-cultured with PBMCs (previously activated by CD3/CD28 and IL2). The results demonstrated that groups B, C, and D influenced Treg differentiation to varying degrees compared to the control group A (Fig. A, B). Notably, after accounting for the influence of tumor cell heterogeneity, lung adenocarcinoma cell lines cultured in a necrotic medium were found to more effectively inhibit Treg differentiation, with minimal association with cryogenic interventions. Additionally, flow cytometry experiments on mouse spleens revealed a reduction in the number of Tregs 14 days post-cryoablation compared to the control group. Compared to the Cry + SB431542 group, the Cry group showed a trend towards higher Treg numbers, though not statistically significant ( p > 0.05). This may result from the complex interactions between TGF-β signaling inhibition and the tumor microenvironment’s immune regulation, potentially masking the effects of cryoablation and SB431542 on Treg levels (Fig. C, D). To comprehensively investigate the antitumorigenic effects of cryoablation, a mouse model of lung cancer treated with cryoablation was established. After treatment, a significant reduction in tumor volume was observed in the cryoablation group compared to the control group 14 days post-surgery (Fig. A, B). Moreover, cryoablation altered the size, morphology, and structure of lung cancer cells (Fig. C). Meanwhile, immunohistochemistry assays also that the expression level of Ki-67 was significantly lower in the cryoablation group compared to the control group (Fig. D, E). These findings collectively indicated that cryoablation effectively suppressed the proliferation of residual tumor cells after ablation, thereby inhibiting tumor progression. Interestingly, cryoablation induced changes in collagen fiber structure within the tumor tissue, rendering the post-ablation tissue loose and predominantly exhibiting curved structures surrounding the tumor nests. Moreover, the Cry + SB431542 group showed a more pronounced change in this regard when compared with the Cry group (Fig. A, B). Upon collagen fibers transitioning from a curled to a linear configuration traversing through the tumor foci, tumor cells rapidly migrated along these reconstructed linear collagen fibers, a phenomenon referred to as “highways” for tumor invasion by researchers . In the current study, cryoablation suppressed changes in collagen fibers in Lewis lung adenocarcinoma mouse xenografts, thereby inhibiting tumor metastasis. Additionally, a lower number of lung metastases were noted in mice in the cryoablation group (60% vs. 0%, Con vs. Cry) (Fig. C). To further explore the molecular mechanisms underlying cryoablation-enhanced antitumor immunity, RNA seq was performed on tumors collected from mice 14 days after cryoablation. Tumors were snap-frozen in liquid nitrogen for 15 min and stored at − 80 °C until processing. DESeq analysis was carried out to identify DEGs, which were visualized using volcano plots (Fig. A). Our results identified 236 upregulated genes and 153 downregulation genes in the cryoablation group compared to the control group. GO and KEGG enrichment analyses were performed on the DEGs using ClusterProfiler to determine their primary biological functions. Of note, the DEGs were significantly enriched in biological processes (BP) terms related to the regulation of lymphocyte proliferation, regulation of mononuclear cell proliferation, regulation of inflammatory response, and leukocyte migration. They were significantly enriched in cellular components (CC) terms associated with the MHC protein complex and the receptor complex and in molecular functions (MF) terms linked to receptor ligand activity, immune receptor activity, and immunoglobulin binding (Fig. B). Moreover, KEGG enrichment analysis identified several signaling pathways associated with the differential gene expression profile resulting from cryoablation, including Th17 cell differentiation, HIF-1 signaling pathway, p53 signaling pathway, VEGF signaling pathway, TGF-beta signaling pathway, and JAK − STAT signaling pathway (Fig. C). These findings suggest the involvement of multiple signaling cascades in mediating the effects of cryoablation on immune regulation and antitumor responses, providing valuable insights into potential therapeutic targets for enhancing cryoablation efficacy in cancer treatment. Further research is warranted to unravel the intricate interplay between these pathways and their contributions to cryoablation-induced immune modulation. Our previous bulk RNA-seq unveiled that lung adenocarcinoma cryoablation influences the TGF-β signaling pathway. scRNA-seq demonstrated a strong correlation between the TGF-β pathway and Tregs. Concurrently, flow cytometry corroborated that cryoablation suppressed Treg differentiation. To further investigate the underlying mechanisms, TGF-β1 expression was detected in the serum of patients, revealing a post-treatment pattern of initial increase followed by a decrease, which paralleled the trend in Treg differentiation (Fig. A). Both in vivo mouse models and in vitro cell experiments substantiated the inhibitory effect of cryoablation on TGF-β1 expression (Fig. B, C). qPCR and Western blot analyses determined that cryoablation downregulates TGF-β expression, thereby impairing the phosphorylation of Smad2 and Smad3 and subsequently suppressing downstream Foxp3 expression. The inhibitor SB431542 markedly suppressed the transcription levels of Smad2, Smad3, and Foxp3. Nevertheless, its effect on protein expression was less pronounced (Fig. D–I). These findings signaled that cryoablation inhibits the TGF-β/SMAD/FOXP3 pathway, hindering Treg differentiation and reshaping the immune microenvironment surrounding lung adenocarcinoma. This mechanistic understanding enhances our understanding of the mechanism by which cryoablation enhances antitumor immunity and provides potential therapeutic targets for optimizing cryoablation-based interventions in LUAD treatment. - γ To further investigate the potential of cryoablation to augment the killing of LUAD in addition to its effects on suppressing Tregs differentiation, we conducted a series of experiments. Our results demonstrated that the expression of IFN-γ progressively increased, whereas that of IL-2 decreased in lung cancer patients after cryoablation (Fig. A, D). Subsequently, ELISA was carried out to detect the levels of IFN-γ and IL-2 in mouse serum 2 weeks after cryoablation. Cryoablation was observed to produce a trend towards increased levels of IFN-γ, albeit non-statistically significant, which was higher when combined with a TGF-β1 inhibitor. In contrast, IL-2 expression was significantly lower in the treated group than in the control group (Fig. B, E). Moreover, a cell cryoablation model co-cultured with PBMCs was constructed, and ELISA was performed on the supernatant. Our results showed that, compared to the control group, the expression level of IFN-γ and IL-2 was increased and decreased in sublethal cells + necrotic medium ( D ), respectively, with a more prominent effect observed in the A549 cell line (Fig. C, F). These findings collectively suggest that cryoablation enhanced antitumor capacity through the modulation of IFN-γ and IL-2 expression. The upregulation of IFN-γ and concurrent downregulation of IL-2 may contribute to an improved immune response against lung adenocarcinoma cells, highlighting the potential of cryoablation as a complementary strategy in cancer immunotherapy. Cryoablation is a well-established treatment modality for various solid tumors, including prostate cancer, breast cancer, kidney cancer, and lung cancer . Using imaging techniques such as CT, cryogenic media (argon or liquid nitrogen) are delivered through probes into tumor tissue, achieving freezing of the tumor at ultra-low temperatures of − 175 °C, leading to cellular damage through repeated freeze–thaw cycles . With the widespread clinical application of cryoablation, the post-procedure immune response has garnered increasing attention. Reports dating back to the 1970s have documented the regression of distant metastases following cryoablation . Current research posits that cryoablation, by lysing tumor cells, exposes previously shielded tumor antigens to the immune system, offering therapeutic opportunities for immune cells . Tumor cells can evade immune cell attack through multiple mechanisms, one of which involves the activation of Tregs to suppress immune responses. In this study, scRNA-seq was conducted on cells isolated from cancer-adjacent and lung adenocarcinoma tissues, yielding a total of 95,318 cells. Besides, distinct cell types were identified in the peripheral regions of LUAD, and differences in immune cell subtypes were examined by isolating T and NK cells. Particularly, we focused on Treg populations, elucidating their developmental trajectories and enriching pathways associated with DEGs. Seven Tregs subtypes were prominently enriched in the tumor microenvironment, namely Treg-LTB, Treg-PELI1, Treg-CCL5, Treg-HSPA6, Treg-CXCL13, Treg-CCL20, and Treg-IFI27. Pseudo-chronological analysis revealed the presence of both early-stage differentiating Treg-IFI27 cells and late-stage Treg-PELI1 cells in adjacent non-cancerous tissues, while mature Treg-LTB cells predominated in lung adenocarcinoma tissue. The immunosuppressive role of Tregs in the lung adenocarcinoma microenvironment is well-established . Notably, prior investigations have concluded that Tregs can adapt to a hypoglycemic, high-lactate environment, allowing them to utilize energy sources inaccessible to other immune cells, thereby facilitating immune escape within the distinctive microenvironment of lung adenocarcinoma . Modulating the lung adenocarcinoma microenvironment holds the potential to impact the survival status of Tregs. Importantly, scRNA-seq identified a correlation between TGF-β1 and the developmental trajectory of Tregs. The TGF-β family comprises three isoforms, namely TGF-β1, TGF-β2, and TGF-β3, existing as homodimers within the biological system . Noteworthily, TGF-β1, highly expressed in tumor tissues, plays a pivotal role in the tumor microenvironment (TME) . Indeed, the TGF-β1 signaling pathway directly influences various cellular components within the TME, including cancer cells, immune cells, and other cell types, playing a decisive role in cancer progression. Consequently, inhibiting TGF-β signal transduction can potentially attenuate immune suppression, angiogenesis, and the activation of cancer-associated fibroblasts (CAFs) , thereby enhancing the effectiveness of alternative therapeutic modalities. Considering the elevated TGF-β activity in the tumor vicinity, therapeutic strategies must concurrently target cancer cells and the immune and stromal components within the TME. This dual approach ensures sustained inhibition of TGF-β signal transduction, facilitating enduring regression of cancer. Overall, this research outlined the distinctive characteristics of cryoablation, emphasizing its targeted ablation capabilities. Our study explored the overarching impact of cryoablation, not only in completely eliminating of lung adenocarcinoma but also in altering the Tregs landscape within the tumor microenvironment. This alteration is speculated to enhance the host’s anti-tumor immune response. Clinical blood tests of patients undergoing cryoablation revealed a reduction in Treg frequency. Subsequent in vivo and in vitro experiments further corroborated this phenomenon. Importantly, the decrease in Tregs was correlated with changes in TGF-β1 levels. Pathological staining revealed cryoablation-induced alterations in the lung adenocarcinoma microenvironment in the murine lung cancer model. These alterations manifested as observable changes in tumor tissue structure, reduced invasiveness, and limited proliferation in LUAD. Naïve precursor CD4 + T cells are guided by TGF-β1 to differentiate into Tregs, which suppress antitumor immunity. DEGs were analyzed in Treg subsets, and KEGG enrichment confirmed the association between Tregs and the TGF-β/Smad signaling pathway in LUAD. Furthermore, our transcriptomic study of mouse cryoablation revealed a correlation between the TGF-β signaling pathway and cryoablation. It is worthwhile emphasizing that the TGF-β signaling pathway promotes the progression of late-stage lung cancer. Upon activation, TGF-β forms a dimeric complex, phosphorylates Smad2 and Smad3, and binds to Smad4 to form the Smad complex. This complex subsequently translocates to the cell nucleus, activates Foxp3, and generates Tregs by promoting Foxp3 CNS1 site activation . Our hypothesis was validated in the animal experiments, wherein increased TGF-β1 expression was observed in serum after cryoablation. Interestingly, variations in TGF-β1 expression were less pronounced in lung adenocarcinoma tissues. Observing the impact of TGF-β1 expression, downstream Smad2 and Smad3 exhibited increased phosphorylation, thereby activating FOXP3 and promoting the transformation of naïve precursor CD4 T cell precursors into Tregs. Furthermore, our study uncovered an additional facet of cryoablation, demonstrating its capability to upregulate the expression of IFN-γ, thereby amplifying its anti-tumorigenic effects. These findings not only shed light on the intricate interplay between the TGF-β/Smad signaling and cryoablation but also highlight the multifaceted impact of cryoablation on the immune landscape within the lung adenocarcinoma microenvironment. Despite its proven clinical efficacy, cryoablation has limitations in achieving complete ablation due to factors such as tumor size and proximity to major blood vessels in lung adenocarcinoma cases. Leveraging the principle that cryoablation can activate the host’s antitumor immune response, we aimed to enhance the immunotherapeutic effects by combining cryoablation with Treg inhibitors. Regrettably, our experimental findings indicated that the use of Tregs inhibitors in isolation did not exert a significant effect. The combination drug regimen demonstrated only marginal improvements over the cryoablation-alone group. This suggests that the complex interplay between TGF-β signaling inhibition and immune modulation within the tumor microenvironment may have mitigated the direct effects of cryoablation and SB431542 on Treg levels. Such complexity highlights the need for a more detailed understanding of the interactions among cryoablation, Treg inhibitors, and the tumor microenvironment, to better elucidate these interactions and develop more effective strategies to enhance the immunotherapeutic benefits of cryoablation. Nevertheless, some limitations of this study cannot be overlooked. To begin, while changes in the levels of IFN-γ and IL-2 were monitored post-cryoablation, our investigation did not detect the levels of serum cytokines or chemokines. Including these factors could provide a more holistic perspective on cryoablation-induced immunological changes. Secondly, the lack of data on tumor-infiltrating lymphocytes (TILs), additional chemokines, and the temporal evolution of immune cell populations following cryoablation restricted our understanding of the sustainability of the immune response. Thirdly, technical difficulties associated with acquiring post-cryoablation tissue samples impeded a spatial analysis of immune cells within the tumor microenvironment, which is crucial for elucidating the spatial dynamics of the immune response. To address these limitations, future research should aim to encompass a more exhaustive cytokine profile and conduct longitudinal analyses of immune cell populations. Such an approach will not only enhance our comprehension of the immunological effects of cryoablation but also contribute to the formulation of more precise and effective targeted cancer therapeutics. Lung adenocarcinoma cryoablation emerges as a potent intervention capable of eradicating lung adenocarcinoma cells and reshaping the tumor microenvironment. Its influence on Treg differentiation mediated by the TGF-β/Smad signaling pathway represents a pivotal mechanism that fosters antitumor immune responses. Additional file 1. Table S1—Quantitative real-time PCR primers; Table S2—Information on antibodies used in flow cytometric analysis; Fig. S1—Dot plots depicting markers of different Tregs populations; Fig. S2—The trajectory distribution of each Treg cell population over time in different samples. At the top is the starting point of development, and at the bottom is the developmental endpoint; Fig S3—The trajectory of TGF-β1. |
Total Arch Replacement is Safe as Hemiarch Repair in Acute Type A
Aortic Dissection in a Middle-Income Country Setting | fa112977-1fc0-4798-919d-b6529732c261 | 11817151 | Surgical Procedures, Operative[mh] | Acute type A aortic dissection (ATAAD) is a challenging surgical emergency that requires early diagnosis and optimal surgical decision making. It is the most frequent fatal aortic syndrome with an estimated incidence of 3-5 cases per 100.000 people every year and it might be underestimated due to deaths before admission . The incidence is higher in men with the peak within the 6 th decade of life . In large scale series, authors report an in-hospital mortality ranging from 17 to 26% remaining as a challenge for cardiothoracic surgeons . Treatment of choice is the open surgery for most patients. The goal is the resection of the primary entry tear through a graft interposition in the involved aortic segment, thus re-establishing blood flow to the true lumen . This procedure is effective in preventing fatal complications and death and it may be achieved either by hemiarch or total arch replacement (TAR) depending on the location of the primary entry tear, surgeon preference, and centre experience in open aortic surgery . The hemiarch repair is the preferred surgical approach among most groups because it is less technically demanding. TAR, on the other hand, is a more complex procedure that requires longer periods of circulatory arrest, cardiopulmonary bypass (CPB), and greater surgical expertise . Nevertheless, evidence is beginning to prove effectiveness of TAR by improving complete false lumen thrombosis and distal aortic remodelling, thus preventing long-term adverse outcomes such as aortic dilation, rupture, or new distal entry tears. The rationale for a more aggressive approach (specially in younger patients and those with connective tissue disorders) is to induce complete false lumen thrombosis and aortic remodelling in the long term . Besides the increasing of performing TAR procedures, last decade trend is toward the use of the newer hybrid techniques. The frozen elephant trunk (FET), the B-SAFER procedure, and the warm stented techniques have become popular as extended arch techniques with potentially improved long-term outcomes . However, the best surgical procedure to treat patients with ATAAD is still unclear. While some large-scale registries show no difference in terms of mortality, stroke, or paraplegia rates between hemiarch, TAR, and FET, there is also opposing evidence showing worse outcomes with TAR as compared to hemiarch repair . Therefore, this study aimed to compare the early clinical outcomes of patients with ATAAD who underwent proximal aortic repair or extended arch repair in a reference centre in Colombia. This single-centre retrospective cohort study was conducted in a high-complexity hospital in Bogotá, Colombia. Using the electronic registry of the Cardiovascular Surgery Department that follows the guidelines of the Society of Thoracic Surgeons (STS) Adult Cardiac Surgical Database, we identified all aortic dissections with arch compromise between January 2002 and November 2022. Then we included all patients diagnosed with ATAAD who underwent surgical aortic repair within 14 days after symptom onset in this period. Patient flowchart selection is shown in . Patients who underwent aortic debranching were excluded from this study, and no other exclusion criteria were considered. We included data from pre, intra, and postoperative variables. Groups of analysis were defined by the extent of the repair that was performed (hemiarch repair vs. TAR). Approval was obtained from our Institutional Review Board (CEIC-0395-2022), and the need for individual informed consent was waived. The diagnosis of ATAAD was made by computerized tomography in all patients.The decision of the extension of the graft was determined by surgeon’s choice on an individualized evaluation considering the location and extension of the entry tear, the patient’s clinical and hemodynamic status, and comorbidities. All repairs were performed through a median sternotomy using CPB, anterograde cardioplegia, hypothermic circulatory arrest, and anterograde cerebral perfusion. Baseline characteristics included age, sex, comorbidities, history of previous cardiac surgery, left ventricular ejection fraction, risk of in-hospital mortality (estimated by the European System for Cardiac Operative Risk Evaluation II), and functional class (assessed by New York Heart Association [NYHA] functional class classification). Definition Chronicity of aortic dissection was defined following the 2022 American College of Cardiology/American Heart Association guidelines for the diagnosis and management of aortic disease that uses the definition for acute and hyperacute aortic dissection proposed by the Society for Vascular Surgery/STS as following: hyperacute occurring in < 24 hours, and acute developing in a one day- 14 days interval from the onset of symptoms . TAR was defined as an aortic repair involving the whole aortic arch including its branches using the conventional elephant trunk or the hybrid FET techniques. The hemiarch repair was defined as an aortic repair limited to the ascending aorta including the lesser curvature of the aortic arch and with no involvement of arch branches. Surgical priority was divided into three categories following the STS database coding: urgent priority as a surgery performed within the same in-hospital stay, emergency as critical refractory ill with or without hemodynamic impairment patients who needs immediate cardiac surgery, and emergent salvage as patients requiring extracorporeal membrane oxygenation for keeping alive or under cardiac arrest who requires cardiopulmonary resuscitation on the way to the operating room or before the anesthesia induction. Outcomes The primary outcomes of this study were stroke rate, spinal cord injury (SCI), and in-hospital mortality. Stroke rate was defined as any acute focal brain function impairment with imaging evidence of recent brain ischemia occurring during total in-hospital stay after the surgery. SCI was defined as an acute neurological deficit of the spinal cord of new onset after the surgery. Death during hospital or intensive care unit (ICU) stay after surgery was defined as in-hospital mortality. The secondary endpoints included mediastinum or sternum infection classified as surgical site infection, and acute kidney injury (AKI) defined as any kidney function impairment that accomplished the Kidney Disease Improving Global Outcomes criteria for AKI after surgery. Bleeding reoperation was determined as the needing of a second intervention due to clear signs of internal bleeding after the surgery. All outcomes were measured during the total in-hospital stay follow-up. Statistical Analysis Standard statistical analysis was performed for between-group continuous variables and was presented using median with the 25 th to 75 th percentile interval, and categorical data were summarized as frequency and percentages. The bivariate analyses were done using the Mann-Whitney U test, and for categorical variables the Chi-square test or Fisher’s exact test according to the nature and distribution of the variable. Statistical significance was established at alpha level of 0.05. All statistical analyses were performed using IBM Corp. Released 2021, IBM SPSS Statistics for Windows, Version 28.0, Armonk, NY: IBM Corp. and STATA 15 (Stata Corp. 2017. Stata Statistical Software: Release 15. College Station, TX: Stata Corp LLC) for Windows. Chronicity of aortic dissection was defined following the 2022 American College of Cardiology/American Heart Association guidelines for the diagnosis and management of aortic disease that uses the definition for acute and hyperacute aortic dissection proposed by the Society for Vascular Surgery/STS as following: hyperacute occurring in < 24 hours, and acute developing in a one day- 14 days interval from the onset of symptoms . TAR was defined as an aortic repair involving the whole aortic arch including its branches using the conventional elephant trunk or the hybrid FET techniques. The hemiarch repair was defined as an aortic repair limited to the ascending aorta including the lesser curvature of the aortic arch and with no involvement of arch branches. Surgical priority was divided into three categories following the STS database coding: urgent priority as a surgery performed within the same in-hospital stay, emergency as critical refractory ill with or without hemodynamic impairment patients who needs immediate cardiac surgery, and emergent salvage as patients requiring extracorporeal membrane oxygenation for keeping alive or under cardiac arrest who requires cardiopulmonary resuscitation on the way to the operating room or before the anesthesia induction. The primary outcomes of this study were stroke rate, spinal cord injury (SCI), and in-hospital mortality. Stroke rate was defined as any acute focal brain function impairment with imaging evidence of recent brain ischemia occurring during total in-hospital stay after the surgery. SCI was defined as an acute neurological deficit of the spinal cord of new onset after the surgery. Death during hospital or intensive care unit (ICU) stay after surgery was defined as in-hospital mortality. The secondary endpoints included mediastinum or sternum infection classified as surgical site infection, and acute kidney injury (AKI) defined as any kidney function impairment that accomplished the Kidney Disease Improving Global Outcomes criteria for AKI after surgery. Bleeding reoperation was determined as the needing of a second intervention due to clear signs of internal bleeding after the surgery. All outcomes were measured during the total in-hospital stay follow-up. Standard statistical analysis was performed for between-group continuous variables and was presented using median with the 25 th to 75 th percentile interval, and categorical data were summarized as frequency and percentages. The bivariate analyses were done using the Mann-Whitney U test, and for categorical variables the Chi-square test or Fisher’s exact test according to the nature and distribution of the variable. Statistical significance was established at alpha level of 0.05. All statistical analyses were performed using IBM Corp. Released 2021, IBM SPSS Statistics for Windows, Version 28.0, Armonk, NY: IBM Corp. and STATA 15 (Stata Corp. 2017. Stata Statistical Software: Release 15. College Station, TX: Stata Corp LLC) for Windows. A total of 107 patients diagnosed with ATAAD were identified, 69 underwent hemiarch repair, and 38 underwent TAR (26 FET), as shown in . Patient’s baseline characteristics are displayed in . Preoperative Both groups had comparable preoperative characteristics. The most frequent comorbidity was hypertension, followed by dyslipidaemia in both groups. There were no differences in functional class between groups, and most patients had NYHA functional class II. The median left ventricular ejection fraction in both groups was 51%. Comparing the timing of dissection, 7.5% of the patients presented in the first 24 hours from the onset of symptoms, and 92.5%o consulted in a > 24 hours-14 days interval after the onset. There were no differences in the median preoperative mortality risk between the two groups. Intraoperative Intraoperative results are displayed on . Most patients underwent surgery as an emergency surgical priority (n=78, 72.9%o), 26 (24.3%) as urgent, and three (2.8%) as emergent salvage. The mean duration of CPB was significantly longer ( P =0.01 ) in TAR (242 min [210 min-290 min]) when compared to the hemiarch repair group (217 min [168 min-253 min]). The median aortic crossclamping time was similar for both groups (129 min vs. 132 min, P =0.83).The most frequent cannulation site was the axillary artery as shown in . From the 38 patients who underwent TAR, 26 patients underwent surgery by hybrid approach with FET, and 12 cases by the conventional elephant trunk technique. Proximal extension of the repair included ascending aortic replacement in 57%o of patients, aortic root replacement in 38%o, Bentall procedure in 30%o, and valve-sparing aortic root replacement in 9%o of cases. Aortic valve interventions were repairs in 13% of cases, and replacements in 3%. Postoperative Results of postoperative outcomes are shown in . There were no significant differences in terms of mortality ( P =0.45), stroke rate ( P =0.06), and SCI. There were no differences in secondary postoperative outcomes such as surgical site infection or AKI. Bleeding reoperation was higher in the hemiarch group, but no statistically significant difference was found ( P =0.05) when compared to the total arch group. There was a total of 18 (16.8%) in-hospital deaths, six (5.6%) cases of stroke, and two cases of SCI were identified. In-hospital mortality rate was 18.8% for the hemiarch repair group and 13.2%o for the total replacement group. Median total length of stay was significantly longer ( P =0.03) in TAR (16 days [11 days-27 days]) than hemiarch repair (12 days [8 days-20 days]) with no significant differences in ICU stay. Both groups had comparable preoperative characteristics. The most frequent comorbidity was hypertension, followed by dyslipidaemia in both groups. There were no differences in functional class between groups, and most patients had NYHA functional class II. The median left ventricular ejection fraction in both groups was 51%. Comparing the timing of dissection, 7.5% of the patients presented in the first 24 hours from the onset of symptoms, and 92.5%o consulted in a > 24 hours-14 days interval after the onset. There were no differences in the median preoperative mortality risk between the two groups. Intraoperative results are displayed on . Most patients underwent surgery as an emergency surgical priority (n=78, 72.9%o), 26 (24.3%) as urgent, and three (2.8%) as emergent salvage. The mean duration of CPB was significantly longer ( P =0.01 ) in TAR (242 min [210 min-290 min]) when compared to the hemiarch repair group (217 min [168 min-253 min]). The median aortic crossclamping time was similar for both groups (129 min vs. 132 min, P =0.83).The most frequent cannulation site was the axillary artery as shown in . From the 38 patients who underwent TAR, 26 patients underwent surgery by hybrid approach with FET, and 12 cases by the conventional elephant trunk technique. Proximal extension of the repair included ascending aortic replacement in 57%o of patients, aortic root replacement in 38%o, Bentall procedure in 30%o, and valve-sparing aortic root replacement in 9%o of cases. Aortic valve interventions were repairs in 13% of cases, and replacements in 3%. Results of postoperative outcomes are shown in . There were no significant differences in terms of mortality ( P =0.45), stroke rate ( P =0.06), and SCI. There were no differences in secondary postoperative outcomes such as surgical site infection or AKI. Bleeding reoperation was higher in the hemiarch group, but no statistically significant difference was found ( P =0.05) when compared to the total arch group. There was a total of 18 (16.8%) in-hospital deaths, six (5.6%) cases of stroke, and two cases of SCI were identified. In-hospital mortality rate was 18.8% for the hemiarch repair group and 13.2%o for the total replacement group. Median total length of stay was significantly longer ( P =0.03) in TAR (16 days [11 days-27 days]) than hemiarch repair (12 days [8 days-20 days]) with no significant differences in ICU stay. ATAAD is a life-threatening emergency that requires immediate surgical treatment. The main goal of surgical repair for ATAAD is preventing death and its complications in the short term by the resection of the initial dissection tear. The management of ATAAD has been evolving throughout the years, however, there is still no consensus regarding the most adequate extension of the repair. The available evidence shows contradictory information about the early-, mid-, and long-term outcomes when comparing hemiarch repair versus TAR. Recent studies have started to show better long-term outcomes with an extended replacement in specific populations . Some series show better outcomes with the hemiarch repair. Kim J et al. reported a retrospective cohort study including 188 patients with DeBakey I aortic dissection that showed greater late death rate ( P ≤0.05) for the total arch group 22.7% (n=10) as compared to the hemiarch group 9.7% (n=14) . Likewise, a systematic review and meta-analysis conducted by Ma et al. concluded that hemiarch replacement provides better early mortality rate, lower permanent neurological dysfunction, renal failure, and dialysis in this type of patients. On the other hand, other contemporary series have reported no differences in terms of mortality rate with the TAR as the studies by Ok et al. , Vendramin et al. , and Hayashi et al. show. Patel et al. and Omura et al. also reported similar outcomes in terms of early mortality. Summarized data of the studies presented above are displayed on . A systematic review and metaanalysis conducted by Poon et al. including 14 retrospective cohort studies for a total of 2,221 patients reported no statistically significant differences of in-hospital mortality between hemiarch and TAR with a mortality rate of 3.60-24.1% and 3.85-28.57% ( P =0.20), respectively. Our study compared the outcomes of 107 patients diagnosed with ATAAD who underwent aortic repair surgery in a 20-year period. We reviewed the impact of the graft extension on major early outcomes. Even in high volume centres, mortality remains high, ranging from 17% to 26% . However, in coherence with the aforementioned studies, our results showed no significant difference in terms of early mortality, stroke rate, or SCI. The hemiarch repair has been associated with an increased risk of residual dissection as it demands a longer suture line for distal anastomosis. Is has been well established that residual dissection in the early phase causes an unfavourable distal remodelling and a greater risk of complicated aortic dilatation . Whereas a TAR performed by either conventional or FET technique provides a safer anastomosis and distal remodelling , therefore providing a greater long-term survival and lesser risk of reoperation in patients with ATAAD . Even if the TAR is a more complex and technically demanding procedure, the surgical strategy for ATAAD has been migrating towards favouring extended repairs as these have begun to show improved long-term outcomes with comparable short- and mid-term results if performed in experienced centres. While in the early phase of our study we performed the hemiarch repair more frequently, with the appearance of newer techniques the number of extended, and hybrid repairs has been gradually increasing over time. Surgeons in our study preferred the FET technique over the classic elephant trunk forTAR. ATAAD treatment is a technically challenging procedure. Thus, in some patients, it is still reasonabletolimittherepairtotheascending aorta. However, evidence seems to show that an extended arch replacement is preferred in the following situations : A. When the tear is located within the arch B. In malperfusion syndrome C. In younger patients D. In patients with connective tissue disorders or hereditary aortic syndromes with an aortic aneurysm > 45 mm Moreover, replacement with FET has shown to favour and facilitate distal aortic remodelling, future descending aortic surgery, and might reduce the number of future operations. However, performing an extended repair requires experience in aortic arch surgery . Limitations The limitations of our study are inherent to the retrospective observational design. Additionally, this was a single-centre experience in a referral centre in Colombia with a homogeneous and limited sample. Bias related to surgeon’s preference on the extension of the surgery was present as well because each surgeon had an approach based on patient’s condition and experience. Long-term follow-up was available in a very low percentage of patients, and hence long-term outcomes were not measured. The limitations of our study are inherent to the retrospective observational design. Additionally, this was a single-centre experience in a referral centre in Colombia with a homogeneous and limited sample. Bias related to surgeon’s preference on the extension of the surgery was present as well because each surgeon had an approach based on patient’s condition and experience. Long-term follow-up was available in a very low percentage of patients, and hence long-term outcomes were not measured. ATAAD management remains as a challenge for cardiothoracic surgeons. Our results show thatTAR does not seem to increase the risk of major early postoperative complications when compared to hemiarch repair and it would even provide some benefits such as a lesser risk of reoperation. Therefore, we believe that TAR should be performed when feasible, in selected population, to improve long-term outcomes. |
Genomics in nephrology: identifying informatics opportunities to improve diagnosis of genetic kidney disorders using a human-centered design approach | 6f3405e6-016e-4bed-9896-7e7d5934365d | 11105128 | Internal Medicine[mh] | As recognition of the contribution of genetics to disease grows, improvements to equitable access to genetic testing in clinical practice are necessary. The rapidly evolving field of genomic precision medicine presents significant challenges for clinicians, particularly those without formal training in genetics, as they are often first to encounter patients with genetic conditions. Genetic conditions are individually rare, though collectively common. Most clinicians have not encountered them in training, which causes them not to be considered during initial differential diagnosis. While many research studies have examined integration of genetic testing into the management of cancer, , little has been done in other areas such as chronic kidney disease (CKD), which affects 1 in 7 US adults. The diagnosis of CKD may benefit from improved integration of genetic testing as a monogenic cause may affect as many as 10% of patients with CKD. Kidney Disease Improving Global Outcomes (KDIGO) 2012 guidelines recommend referral to nephrologists (physicians who specialize in diagnosing and treating kidney conditions) for those with more advanced CKD, extensive/recurrent kidney stones, or hereditary kidney disease. , Genetic testing may help when there are monogenic subtypes in a clinical category (eg, congenital/cystic nephropathies, steroid-resistant nephrotic syndrome), positive family history, early age of onset, syndromic features, possibility of identifying a condition in which a targeted treatment may be available. Genetic testing is also important for potential kidney donors with family history of kidney disease, and to inform family planning. Even when clinical diagnosis is easy, as in the case of polycystic kidney disease (PKD), genetic information (such as the differences between PKD1 truncating, PKD2 truncating, PKD1/PKD2 missense variants, other genes, or negative results) can aid in predicting disease severity and prognosis. Without a genetic diagnosis, other kidney diseases, such as Autosomal Dominant Alport Syndrome, are underdiagnosed and misdiagnosed. Earlier initiation of condition-specific management based on genetic diagnosis may improve outcomes. Challenges to genetic testing include insufficient experience and knowledge among nephrologists, cost and access barriers, and lack of electronic health record integration. Our systematic review highlighted the potential for clinical decision support (CDS) tools to improve the uptake of genetic services and the challenges in effectively implementing them, such as the reliance on alerts and reminders, lack of standards for genomic data integration, and underuse of implementation frameworks. The review also demonstrated genetic CDS tools primarily focus on cancer and pharmacogenomics, indicating a knowledge gap in applying genotype and family history data for other specialties, such as nephrology. Scant attention has been paid to clinician needs and workflow, which has led to low adoption of genetic diagnosis. Use of implementation frameworks to objectively evaluate CDS systems in practice is uncommon, which may contribute to the poor uptake of genetic CDS tools in practice. Understanding nephrologists’ perspectives and experience on genetic diagnosis in their clinical workflow and how genetics should be incorporated into it enables the development of tailored CDS tools addressing the specific challenges faced by nephrologists and those who treat kidney conditions. Human-centered design (HCD), also known as user-centered design, is a collection of methodologies that include the user or recipient of a service throughout the design and implementation process. HCD methodologies include qualitative research to understand and empathize with the user’s current experience, use that deep understanding of the current state (such as how genetic diagnosis is implemented in clinical care at the present time) to identify innovative solutions, and iteratively design and test increasingly sophisticated prototypes with end users engaged in every stage. Objective This project aimed to use qualitative and HCD research to understand the current state of genetic diagnosis in kidney disease. We addressed the following research questions: What is the current state of diagnosis and treatment of genetic kidney conditions at multiple institutions? What is the experience, from the perspective of nephrologists and those who treat kidney conditions, of diagnosing and treating patients with complex kidney conditions that may have a genetic cause? What pain points, barriers, and facilitators exist in the process of diagnosing and treating patients with complex kidney conditions that may have a genetic cause? This project aimed to use qualitative and HCD research to understand the current state of genetic diagnosis in kidney disease. We addressed the following research questions: What is the current state of diagnosis and treatment of genetic kidney conditions at multiple institutions? What is the experience, from the perspective of nephrologists and those who treat kidney conditions, of diagnosing and treating patients with complex kidney conditions that may have a genetic cause? What pain points, barriers, and facilitators exist in the process of diagnosing and treating patients with complex kidney conditions that may have a genetic cause? Design and setting Interview process We used semistructured interviews to understand the experiences of nephrologists and internal medicine doctors who diagnose and treat genetic conditions in nephrology. An interview guide was developed by the study team using an experiential phenomenological approach. Interview questions were informed by the literature on the barriers experienced by clinicians to conducting genetic testing. Interview topics included experience(s) with genetic kidney diagnosis and experience(s) with genetic testing in general. The interview guide included a demographic survey and open-ended questions exploring interviewee experience diagnosing and treating genetic conditions, their most complicated and most simple experiences diagnosing a genetic condition, and asking them to share what their ideal experience diagnosing genetic conditions would be. Each interview was scheduled for 45 minutes and was conducted by a single investigator (DKJ) in the presence of an experienced medical geneticist (MSW) who was available for clarification and follow-up questions. Participant selection and sampling We used a purposive sampling strategy to elicit diverse experiences and reactions to making genetic diagnoses. The population of interest in this study consisted of nephrologists and other clinicians with experience in diagnosing and treating kidney conditions with genetic causes. Additionally, while the study focuses on the current state of genetic diagnosis at Geisinger, we also wanted to capture the current state of genetic diagnosis in the nephrology community beyond Geisinger. This led us to recruit both academic and nonacademic clinicians, from large and small institutions to identify common characteristics that could be applied to Geisinger. Nephrologists external to Geisinger were recruited via convenience sampling at Geisinger, from non-Geisinger study team members’ organizations (University of Utah) and via Twitter—a tweet inviting US nephrologists using #nephtwitter. Finally, opportunistic snowball sampling from the participants ensured a diverse and broadly representative sample. Eligible participants were contacted via email, inviting them to participate in an interview. Follow-up emails were sent to schedule the interviews. Due to low participant numbers at individual sites, interviewers were unable to reach thematic saturation within any subgroup. As a result, interviews were conducted until thematic saturation was reached across all participants, meaning new concepts were not being identified in additional interviews. Data analysis Interviewers completed episodic summaries for each interview within 24 hours of interview completion. The notes captured the context and summary of the interview conversation. Interviews were audio-recorded and transcribed verbatim by medical transcriptionists at Geisinger. Study data were collected and managed using a framework based on the interview guide, in an Excel spreadsheet. Thematic analysis Emergent themes were analyzed using a rapid, thematic approach (RADaR: Rapid Data Analysis and Reporting) by 2 independent reviewers (DKJ and HMR). RADaR is a method of organizing and analyzing qualitative data in a rigorous and systematic way that is faster than other methods, and is particularly appropriate for applied qualitative research which informs the design and implementation of practical applications. It uses widely available software (any word processing and spreadsheet software) to organize, reduce, code, and summarize data iteratively into tables. RADaR does not use any form of inter-rater reliability score, rather is an iterative approach that involves reducing data into concise actionable data tables. The reviewers iteratively coded and reviewed their coding together until they reached consensus. RADaR was used to summarize and identify themes in the answers to the interview questions. Summaries and exemplar quotes were entered into the study database (Excel). Themes (barriers, facilitators, and opportunities for innovation) were summarized from the study database. Capturing variability across different users is important as diagnostic processes evolve to incorporate genetics. To generate complementary visualizations of how genetic diagnosis is implemented, we used visualizations of qualitative data to synthesize findings and build empathy with the end user. We created service blueprints (visual diagrams representing the service being performed, mapping roles, tools, and tasks) and process maps (visual diagrams detailing the sequence of actions) to help stakeholders visualize and understand processes. Service blueprints illustrate the overall service design and delivery inclusive of context, as no tool or resource exists in a vacuum, and identify barriers to success and opportunities for improvement and innovation. They represent the most common processes, and while they illustrate complexity and can be quite detailed, they tend to have a bird’s eye view of the larger process. In contrast, workflow process mapping depicts the variability seen within heterogenous groups of users. A workflow process map details a sequence of actions to help relevant stakeholders visualize and understand processes. Historically, process maps have been applied to health services research and quality improvement studies to help visualize those steps and pinpoint sites of intervention. , Service blueprinting Using the rapid analysis data set, the interview data were iteratively synthesized into 2 service blueprints: one representing the process of diagnosis by primary care clinicians from the perspective of nephrologists, and one representing the process of diagnosis by nephrologists from their own perspective. , For the purposes of this study, we are focusing on the nephrology service blueprint. Information about roles, actions (front stage, or those actions conducted within the view of the subject of the service blueprint; and back stage, those actions conducted out of the view of the subject of the service blueprint) and tools or resources used to support those actions were captured and summarized. This information was used to draft an increasingly sophisticated service blueprint representing the current state of genetic diagnosis of kidney disease. To triangulate the findings, the draft maps were presented to the larger study team which included nephrologists, medical geneticists, and informaticians for their feedback, which informed updates to the maps. The maps were designed using Miro, an online visualization and collaboration tool. Process mapping Concurrent to service-blueprinting, using data from the rapid analysis following the interviews with clinicians from 7 health care systems, ZMS listed process and contextual differences for ordering germline genetic testing and/or appropriately referring to a genetic counselor. These data were then iteratively adapted to workflow process maps, representing each pathway a nephrologist or other clinician may take to diagnose genetic kidney disease. These workflow process maps were then presented to the study team of content experts, both from within and outside the health care organizations, to communicate the current state, verify pathway validity, and update maps accordingly. This approach was adapted from the process mapping methodological approach from Salvati et al. Interview process We used semistructured interviews to understand the experiences of nephrologists and internal medicine doctors who diagnose and treat genetic conditions in nephrology. An interview guide was developed by the study team using an experiential phenomenological approach. Interview questions were informed by the literature on the barriers experienced by clinicians to conducting genetic testing. Interview topics included experience(s) with genetic kidney diagnosis and experience(s) with genetic testing in general. The interview guide included a demographic survey and open-ended questions exploring interviewee experience diagnosing and treating genetic conditions, their most complicated and most simple experiences diagnosing a genetic condition, and asking them to share what their ideal experience diagnosing genetic conditions would be. Each interview was scheduled for 45 minutes and was conducted by a single investigator (DKJ) in the presence of an experienced medical geneticist (MSW) who was available for clarification and follow-up questions. Participant selection and sampling We used a purposive sampling strategy to elicit diverse experiences and reactions to making genetic diagnoses. The population of interest in this study consisted of nephrologists and other clinicians with experience in diagnosing and treating kidney conditions with genetic causes. Additionally, while the study focuses on the current state of genetic diagnosis at Geisinger, we also wanted to capture the current state of genetic diagnosis in the nephrology community beyond Geisinger. This led us to recruit both academic and nonacademic clinicians, from large and small institutions to identify common characteristics that could be applied to Geisinger. Nephrologists external to Geisinger were recruited via convenience sampling at Geisinger, from non-Geisinger study team members’ organizations (University of Utah) and via Twitter—a tweet inviting US nephrologists using #nephtwitter. Finally, opportunistic snowball sampling from the participants ensured a diverse and broadly representative sample. Eligible participants were contacted via email, inviting them to participate in an interview. Follow-up emails were sent to schedule the interviews. Due to low participant numbers at individual sites, interviewers were unable to reach thematic saturation within any subgroup. As a result, interviews were conducted until thematic saturation was reached across all participants, meaning new concepts were not being identified in additional interviews. Data analysis Interviewers completed episodic summaries for each interview within 24 hours of interview completion. The notes captured the context and summary of the interview conversation. Interviews were audio-recorded and transcribed verbatim by medical transcriptionists at Geisinger. Study data were collected and managed using a framework based on the interview guide, in an Excel spreadsheet. Thematic analysis Emergent themes were analyzed using a rapid, thematic approach (RADaR: Rapid Data Analysis and Reporting) by 2 independent reviewers (DKJ and HMR). RADaR is a method of organizing and analyzing qualitative data in a rigorous and systematic way that is faster than other methods, and is particularly appropriate for applied qualitative research which informs the design and implementation of practical applications. It uses widely available software (any word processing and spreadsheet software) to organize, reduce, code, and summarize data iteratively into tables. RADaR does not use any form of inter-rater reliability score, rather is an iterative approach that involves reducing data into concise actionable data tables. The reviewers iteratively coded and reviewed their coding together until they reached consensus. RADaR was used to summarize and identify themes in the answers to the interview questions. Summaries and exemplar quotes were entered into the study database (Excel). Themes (barriers, facilitators, and opportunities for innovation) were summarized from the study database. Capturing variability across different users is important as diagnostic processes evolve to incorporate genetics. To generate complementary visualizations of how genetic diagnosis is implemented, we used visualizations of qualitative data to synthesize findings and build empathy with the end user. We created service blueprints (visual diagrams representing the service being performed, mapping roles, tools, and tasks) and process maps (visual diagrams detailing the sequence of actions) to help stakeholders visualize and understand processes. Service blueprints illustrate the overall service design and delivery inclusive of context, as no tool or resource exists in a vacuum, and identify barriers to success and opportunities for improvement and innovation. They represent the most common processes, and while they illustrate complexity and can be quite detailed, they tend to have a bird’s eye view of the larger process. In contrast, workflow process mapping depicts the variability seen within heterogenous groups of users. A workflow process map details a sequence of actions to help relevant stakeholders visualize and understand processes. Historically, process maps have been applied to health services research and quality improvement studies to help visualize those steps and pinpoint sites of intervention. , Service blueprinting Using the rapid analysis data set, the interview data were iteratively synthesized into 2 service blueprints: one representing the process of diagnosis by primary care clinicians from the perspective of nephrologists, and one representing the process of diagnosis by nephrologists from their own perspective. , For the purposes of this study, we are focusing on the nephrology service blueprint. Information about roles, actions (front stage, or those actions conducted within the view of the subject of the service blueprint; and back stage, those actions conducted out of the view of the subject of the service blueprint) and tools or resources used to support those actions were captured and summarized. This information was used to draft an increasingly sophisticated service blueprint representing the current state of genetic diagnosis of kidney disease. To triangulate the findings, the draft maps were presented to the larger study team which included nephrologists, medical geneticists, and informaticians for their feedback, which informed updates to the maps. The maps were designed using Miro, an online visualization and collaboration tool. Process mapping Concurrent to service-blueprinting, using data from the rapid analysis following the interviews with clinicians from 7 health care systems, ZMS listed process and contextual differences for ordering germline genetic testing and/or appropriately referring to a genetic counselor. These data were then iteratively adapted to workflow process maps, representing each pathway a nephrologist or other clinician may take to diagnose genetic kidney disease. These workflow process maps were then presented to the study team of content experts, both from within and outside the health care organizations, to communicate the current state, verify pathway validity, and update maps accordingly. This approach was adapted from the process mapping methodological approach from Salvati et al. We used semistructured interviews to understand the experiences of nephrologists and internal medicine doctors who diagnose and treat genetic conditions in nephrology. An interview guide was developed by the study team using an experiential phenomenological approach. Interview questions were informed by the literature on the barriers experienced by clinicians to conducting genetic testing. Interview topics included experience(s) with genetic kidney diagnosis and experience(s) with genetic testing in general. The interview guide included a demographic survey and open-ended questions exploring interviewee experience diagnosing and treating genetic conditions, their most complicated and most simple experiences diagnosing a genetic condition, and asking them to share what their ideal experience diagnosing genetic conditions would be. Each interview was scheduled for 45 minutes and was conducted by a single investigator (DKJ) in the presence of an experienced medical geneticist (MSW) who was available for clarification and follow-up questions. We used a purposive sampling strategy to elicit diverse experiences and reactions to making genetic diagnoses. The population of interest in this study consisted of nephrologists and other clinicians with experience in diagnosing and treating kidney conditions with genetic causes. Additionally, while the study focuses on the current state of genetic diagnosis at Geisinger, we also wanted to capture the current state of genetic diagnosis in the nephrology community beyond Geisinger. This led us to recruit both academic and nonacademic clinicians, from large and small institutions to identify common characteristics that could be applied to Geisinger. Nephrologists external to Geisinger were recruited via convenience sampling at Geisinger, from non-Geisinger study team members’ organizations (University of Utah) and via Twitter—a tweet inviting US nephrologists using #nephtwitter. Finally, opportunistic snowball sampling from the participants ensured a diverse and broadly representative sample. Eligible participants were contacted via email, inviting them to participate in an interview. Follow-up emails were sent to schedule the interviews. Due to low participant numbers at individual sites, interviewers were unable to reach thematic saturation within any subgroup. As a result, interviews were conducted until thematic saturation was reached across all participants, meaning new concepts were not being identified in additional interviews. Interviewers completed episodic summaries for each interview within 24 hours of interview completion. The notes captured the context and summary of the interview conversation. Interviews were audio-recorded and transcribed verbatim by medical transcriptionists at Geisinger. Study data were collected and managed using a framework based on the interview guide, in an Excel spreadsheet. Emergent themes were analyzed using a rapid, thematic approach (RADaR: Rapid Data Analysis and Reporting) by 2 independent reviewers (DKJ and HMR). RADaR is a method of organizing and analyzing qualitative data in a rigorous and systematic way that is faster than other methods, and is particularly appropriate for applied qualitative research which informs the design and implementation of practical applications. It uses widely available software (any word processing and spreadsheet software) to organize, reduce, code, and summarize data iteratively into tables. RADaR does not use any form of inter-rater reliability score, rather is an iterative approach that involves reducing data into concise actionable data tables. The reviewers iteratively coded and reviewed their coding together until they reached consensus. RADaR was used to summarize and identify themes in the answers to the interview questions. Summaries and exemplar quotes were entered into the study database (Excel). Themes (barriers, facilitators, and opportunities for innovation) were summarized from the study database. Capturing variability across different users is important as diagnostic processes evolve to incorporate genetics. To generate complementary visualizations of how genetic diagnosis is implemented, we used visualizations of qualitative data to synthesize findings and build empathy with the end user. We created service blueprints (visual diagrams representing the service being performed, mapping roles, tools, and tasks) and process maps (visual diagrams detailing the sequence of actions) to help stakeholders visualize and understand processes. Service blueprints illustrate the overall service design and delivery inclusive of context, as no tool or resource exists in a vacuum, and identify barriers to success and opportunities for improvement and innovation. They represent the most common processes, and while they illustrate complexity and can be quite detailed, they tend to have a bird’s eye view of the larger process. In contrast, workflow process mapping depicts the variability seen within heterogenous groups of users. A workflow process map details a sequence of actions to help relevant stakeholders visualize and understand processes. Historically, process maps have been applied to health services research and quality improvement studies to help visualize those steps and pinpoint sites of intervention. , Using the rapid analysis data set, the interview data were iteratively synthesized into 2 service blueprints: one representing the process of diagnosis by primary care clinicians from the perspective of nephrologists, and one representing the process of diagnosis by nephrologists from their own perspective. , For the purposes of this study, we are focusing on the nephrology service blueprint. Information about roles, actions (front stage, or those actions conducted within the view of the subject of the service blueprint; and back stage, those actions conducted out of the view of the subject of the service blueprint) and tools or resources used to support those actions were captured and summarized. This information was used to draft an increasingly sophisticated service blueprint representing the current state of genetic diagnosis of kidney disease. To triangulate the findings, the draft maps were presented to the larger study team which included nephrologists, medical geneticists, and informaticians for their feedback, which informed updates to the maps. The maps were designed using Miro, an online visualization and collaboration tool. Concurrent to service-blueprinting, using data from the rapid analysis following the interviews with clinicians from 7 health care systems, ZMS listed process and contextual differences for ordering germline genetic testing and/or appropriately referring to a genetic counselor. These data were then iteratively adapted to workflow process maps, representing each pathway a nephrologist or other clinician may take to diagnose genetic kidney disease. These workflow process maps were then presented to the study team of content experts, both from within and outside the health care organizations, to communicate the current state, verify pathway validity, and update maps accordingly. This approach was adapted from the process mapping methodological approach from Salvati et al. Sixteen clinicians (14 nephrologists, 2 internists) from 7 different healthcare systems were interviewed (Geisinger, Hattiesburg Clinic, University of Cincinnati, University of Utah, Johns Hopkins Medicine, Georgetown University Hospital, and Marshfield Clinic). Fifty percent of participants currently practice at Geisinger, which has a robust genomic medicine focus. One participant was in the process of relocating from one health care system to another; their responses reflected both organizations. Demographic information is included in . All completed the full interview. More participants use genetic testing in general (12 out of 16), many of whom (10 out of 12) use a broad next-generation sequencing-based kidney disease gene panel from external genetic testing vendors. Neither of the 2 internists reported used genetic testing for kidney disease. Genetic diagnosis information needs Nine themes in 3 categories were identified. Participants experience barriers to integrating genetics into clinical practice, including: a limited understanding of genetics and its application in clinical care; difficulty accessing genetic testing resources, and difficulty understanding individual cases and diseases. Participants reported lack of genetics training during their medical education and rely on the expertise of medical geneticists and genetic counselors to address gaps in their genetics knowledge. Participants also shared facilitators to integration of genetics into their clinical practice. Certain commercially available genetic testing services offer online, easy-to-use portals for ordering genetic tests, which participants consider particularly helpful. Clinical scenarios with clear, diagnostic genetic test results make the experience straightforward. Participants reported that is very helpful to have access to genetics specialists or nephrology colleagues with genetic expertise to help guide them through the process. Participants identified 3 information needs they experience related to genetic testing: trustworthy genetics resources, how to order tests, and how to interpret results. They are unfamiliar with where to find reliable, easy to understand information to guide them. When they recognize the need for genetic testing, they are often unsure of the optimal test to order, insurance coverage, out-of-pocket patient cost, and how to place the order correctly. When they receive results, they are often unsure how to interpret results, especially variants of unknown significance, as well as the clinical implications of the genetic findings. The thematic findings are summarized with exemplar quotations in . Service blueprints A service blueprint depicting the current state of genetic diagnosis of kidney disease by nephrologists was drafted and iteratively updated with feedback from the larger research group, including nephrologists . While the left side of the service blueprints, depicting usual practice, shows a streamlined process with minimal confusion or barriers, the right side , depicting the genetic diagnosis portion of the journey, is rife with complexity and barriers (red diamonds). The genetic test experience facilitated by external genetic testing vendors offers a smoother experience (yellow stars). Workflow process maps Ten workflow process maps were created and synthesized to represent 3 primary processes of the current state, using nephrologists’ perspectives of ordering genetic testing across 7 different health care organizations. Two organizations were found to have multiple processes nephrologists used to identify and care for patients with genetic conditions. However, it was noted from provider-stakeholder interviews these were not formal processes; rather, these steps were stakeholder-dependent due to a lack of process standardization. The primary processes to identify and care for patients with suspected genetic conditions, found from stakeholder interviews, are represented by 3 workflow process maps from Organization 1 . Organization 2 had 2 workflow process maps and Organizations 3 through 7 all had a single map representing each site. These workflows were slight variants of the 3 primary processes which can be viewed in . All 3 workflow process maps showed similar approaches to why a nephrologist might order or refer a patient to receive genetic testing and how a patient who receives said testing typically presents in clinic. Genetic testing was thought to be an option for suspected rare conditions, syndromic phenotypes, genotyping PKD to inform prognosis, unexplained kidney disease, or in specific situations such as establishing a cause of kidney disease in potential kidney transplant recipients with CKD of unknown cause and for risk assessment of potential kidney donors. These patients will often be referred to Adult Nephrology for evaluation of CKD of unknown cause or when transitioning from pediatric to adult care. The workflows deviate with the process leading up to ordering genetic testing. One workflow is described by nephrologists prioritizing a clinical diagnosis, then ordering genetic testing if necessary. This can be characterized by taking a complete medical history and family history, conducting a biopsy, imaging procedures, and/or serology. Subsequent genetic testing is warranted if a clinical diagnosis has not been made, but one nephrologist described a patient care barrier involving the return of results: The second primary workflow process map shows taking the medical and family history first and referring to a genetic counselor if the nephrologist suspects a genetic condition. If no genetic condition is suspected, then the typical workup of biopsy, imaging procedures, and/or serology are conducted to identify a clinical diagnosis. Patient care barriers were identified related to ordering genetic testing, The last process involved ordering genetic testing themselves or referring to genetic counselors internally. However, this process was variable. Multiple nephrologists endorsed referring to genetic counselors internally, but others had positive experiences ordering genetic testing directly without referring to a genetic counselor first. Nine themes in 3 categories were identified. Participants experience barriers to integrating genetics into clinical practice, including: a limited understanding of genetics and its application in clinical care; difficulty accessing genetic testing resources, and difficulty understanding individual cases and diseases. Participants reported lack of genetics training during their medical education and rely on the expertise of medical geneticists and genetic counselors to address gaps in their genetics knowledge. Participants also shared facilitators to integration of genetics into their clinical practice. Certain commercially available genetic testing services offer online, easy-to-use portals for ordering genetic tests, which participants consider particularly helpful. Clinical scenarios with clear, diagnostic genetic test results make the experience straightforward. Participants reported that is very helpful to have access to genetics specialists or nephrology colleagues with genetic expertise to help guide them through the process. Participants identified 3 information needs they experience related to genetic testing: trustworthy genetics resources, how to order tests, and how to interpret results. They are unfamiliar with where to find reliable, easy to understand information to guide them. When they recognize the need for genetic testing, they are often unsure of the optimal test to order, insurance coverage, out-of-pocket patient cost, and how to place the order correctly. When they receive results, they are often unsure how to interpret results, especially variants of unknown significance, as well as the clinical implications of the genetic findings. The thematic findings are summarized with exemplar quotations in . A service blueprint depicting the current state of genetic diagnosis of kidney disease by nephrologists was drafted and iteratively updated with feedback from the larger research group, including nephrologists . While the left side of the service blueprints, depicting usual practice, shows a streamlined process with minimal confusion or barriers, the right side , depicting the genetic diagnosis portion of the journey, is rife with complexity and barriers (red diamonds). The genetic test experience facilitated by external genetic testing vendors offers a smoother experience (yellow stars). Ten workflow process maps were created and synthesized to represent 3 primary processes of the current state, using nephrologists’ perspectives of ordering genetic testing across 7 different health care organizations. Two organizations were found to have multiple processes nephrologists used to identify and care for patients with genetic conditions. However, it was noted from provider-stakeholder interviews these were not formal processes; rather, these steps were stakeholder-dependent due to a lack of process standardization. The primary processes to identify and care for patients with suspected genetic conditions, found from stakeholder interviews, are represented by 3 workflow process maps from Organization 1 . Organization 2 had 2 workflow process maps and Organizations 3 through 7 all had a single map representing each site. These workflows were slight variants of the 3 primary processes which can be viewed in . All 3 workflow process maps showed similar approaches to why a nephrologist might order or refer a patient to receive genetic testing and how a patient who receives said testing typically presents in clinic. Genetic testing was thought to be an option for suspected rare conditions, syndromic phenotypes, genotyping PKD to inform prognosis, unexplained kidney disease, or in specific situations such as establishing a cause of kidney disease in potential kidney transplant recipients with CKD of unknown cause and for risk assessment of potential kidney donors. These patients will often be referred to Adult Nephrology for evaluation of CKD of unknown cause or when transitioning from pediatric to adult care. The workflows deviate with the process leading up to ordering genetic testing. One workflow is described by nephrologists prioritizing a clinical diagnosis, then ordering genetic testing if necessary. This can be characterized by taking a complete medical history and family history, conducting a biopsy, imaging procedures, and/or serology. Subsequent genetic testing is warranted if a clinical diagnosis has not been made, but one nephrologist described a patient care barrier involving the return of results: The second primary workflow process map shows taking the medical and family history first and referring to a genetic counselor if the nephrologist suspects a genetic condition. If no genetic condition is suspected, then the typical workup of biopsy, imaging procedures, and/or serology are conducted to identify a clinical diagnosis. Patient care barriers were identified related to ordering genetic testing, The last process involved ordering genetic testing themselves or referring to genetic counselors internally. However, this process was variable. Multiple nephrologists endorsed referring to genetic counselors internally, but others had positive experiences ordering genetic testing directly without referring to a genetic counselor first. To understand the current state of genetic diagnosis of complex conditions in nephrology, we conducted qualitative interviews with nephrologists and internal medicine doctors who diagnose and treat kidney genetic conditions. We identified barriers (lack of knowledge, lack of access, and complexity surrounding the case and disease) and facilitators (good user experience, straightforward diagnoses, and support from colleagues) to timely diagnosis of genetic conditions in nephrology. Similar barriers have been identified by other research exploring why genetic testing for other medical conditions has been poorly implemented, such as minimal genetics knowledge among clinicians, cost and access barriers, and lack of electronic health record integration causing poor user experience. , Notably, participants in our study emphasized their lack of knowledge about what costs might be for their patients—they assume there would be costs, but do not know what they would be because do not know what any individual’s insurance plan covers. This is an interesting facet to the well-known cost barriers surrounding genetic testing: the clinician’s lack of insight into what the cost might be in the first place. To identify areas of opportunity to improve genetic diagnosis, we created a suite of visual artifacts depicting the current state of genetic diagnosis of kidney conditions by nephrologists, which capture both the interaction with the larger context of healthcare and the overall service design of genetic diagnosis (service blueprints) as well as the variability of processes across different nephrologists and healthcare systems (process maps). More participants than expected indicated they have ordered and used genetic information in their practice. This may be because nephrologists interested in genetic testing and diagnosis self-selected to participate in a qualitative research study about genetic diagnosis in nephrology at a higher rate than nephrologists who were less interested in genetic kidney disease. The nephrology field may be more developed in including genomics in their practice compared to other clinical areas, though this work cannot be generalized to all nephrologists. Additionally, Geisinger Clinic has a robust research interest in genomic medicine, increasing the likelihood of recruiting participants here who share that interest. Some external testing vendors have developed nephrology-specific genetic testing products, marketing them to nephrologists directly. These services include support for ordering and result interpretation, which addresses some of the barriers identified in this work. However, external stand-alone services do not address other important aspects of real time genetic diagnosis design identified by participants, such as support from colleagues and management of complexity in individual cases. The large number of genes on the panel, while intending to be helpful by providing more information, were difficult for participants to interpret due to receiving results which seem to be unrelated or having unknown significance to the indication for testing. Furthermore, genetic testing results from external services must be manually added to the patients’ health record, limiting the ability to use informational resources in the EHR which could be triggered by structured data in reports. Having to use external, commercial products introduces other impediments to successful use of genetic information, even as it solves some problems. A systematic review of clinicians’ genetic testing practices found most studies focused on clinicians’ knowledge, attitudes, or beliefs about genetic testing, and none evaluated the experience or process of obtaining or receiving a genetic diagnosis. Within nephrology, recent articles review the current state of evidence for the genetic diagnosis of diseases, and review the indications for pursuing genetic testing. However, these do not include information on the current experience of clinicians in conducting genetic testing within any clinical area, nor specifically nephrology. The results of this study offer opportunities to address the unmet information needs and barriers experienced in implementing genetic diagnosis in clinical care, which have previously not been addressed in the literature. In an area where no guidelines or standard practices exist, we expected to see substantial variability in the steps participants take when diagnosing genetic conditions, and this was confirmed. We also wanted to identify when and where participants experience roadblocks to achieving their goals, because those are opportunities for improvement and innovation. Neither service blueprints nor process workflow maps accomplish those goals alone—by pairing them in the analysis and synthesis of the qualitative data, we illustrate the experience with both a low-power view and a high-power view, akin to the options on a microscope. The low power view, process workflow maps, has a wider field of vision with multiple distinct experiences, less detail, and more information about variation. The high-power view, service blueprints, has a narrower field of vision, more detail, and more information about the context and structures in which the experience of obtaining a genetic diagnosis exists. Using the same data but generated independently, these visualizations provide different perspectives and information that neither would provide alone. The service blueprints and workflow process maps developed in this work offer a novel, complementary, and visual approach to communicating qualitative findings in a compelling, actionable way. Together they allow the HCD researcher to explore both the variability and the problems—and identify opportunities for improvement in genetic diagnosis in nephrology. Limitations This study had limitations related to sampling and data collection. First, data collection was limited to participants from 7 healthcare organizations, and participant sampling ranged from 1 to 6 stakeholder perspectives representing each site. Ultimately, a cross-case comparison by site would not have achieved thematic saturation. Rather, thematic saturation was achieved by looking at all stakeholder perspectives, with Organization 1 representing the 3 primary processes for ordering germline genetic testing in Nephrology. Future studies are needed to further characterize processes across multiple institutions to ensure all possible processes are accounted for. While participant recruitment included healthcare systems outside our own local institutions, common themes at multiple institutions likely represent generalizable knowledge which will be important to address as real time genetic diagnosis systems are built, even as differences are identified. Future work Future work includes conducting a design thinking workshop with nephrologists, medical geneticists, informaticians, and other experts. The workshop will use the findings from the qualitative work, including the data visualizations, to build empathy and shared understanding of the current state of genetic diagnosis in nephrology among the participants. The output of the workshop will be a first draft prototype of the future state of genetic diagnosis in nephrology, using real-time genetic diagnosis innovations. Testing of such a prototype across a diverse group of nephrologists can facilitate the need to characterize processes as noted in the limitations. This study had limitations related to sampling and data collection. First, data collection was limited to participants from 7 healthcare organizations, and participant sampling ranged from 1 to 6 stakeholder perspectives representing each site. Ultimately, a cross-case comparison by site would not have achieved thematic saturation. Rather, thematic saturation was achieved by looking at all stakeholder perspectives, with Organization 1 representing the 3 primary processes for ordering germline genetic testing in Nephrology. Future studies are needed to further characterize processes across multiple institutions to ensure all possible processes are accounted for. While participant recruitment included healthcare systems outside our own local institutions, common themes at multiple institutions likely represent generalizable knowledge which will be important to address as real time genetic diagnosis systems are built, even as differences are identified. Future work includes conducting a design thinking workshop with nephrologists, medical geneticists, informaticians, and other experts. The workshop will use the findings from the qualitative work, including the data visualizations, to build empathy and shared understanding of the current state of genetic diagnosis in nephrology among the participants. The output of the workshop will be a first draft prototype of the future state of genetic diagnosis in nephrology, using real-time genetic diagnosis innovations. Testing of such a prototype across a diverse group of nephrologists can facilitate the need to characterize processes as noted in the limitations. The current state of genetic diagnosis in nephrology is suboptimal for timely diagnosis of kidney diseases with genetic etiology. We have identified opportunities to improve and innovate this experience with the HCD of a real-time genetic diagnosis tool. ocae053_Supplementary_Data |
Safety of the Herbal Medicine SH003 in Patients With Solid Cancer: A Multi-Center, Single-Arm, Open-Label, Dose-Escalation Phase I Study | ab5a2c81-aef2-418f-8556-d9a1f0a6086b | 11528795 | Pharmacology[mh] | Cancer, a group of diseases characterized by the uncontrolled growth and spread of abnormal cells, is a leading cause of death globally, accounting for nearly 10 million deaths in 2020. By 2040, the global cancer burden is projected to reach 28.4 million cases, marking a 47% increase from 2020. Despite extensive efforts and substantial resources devoted to cancer research and treatment, cancer continues to be a pressing health issue worldwide. As the cancer treatment market expands due to rising global prevalence and medical advancements, a corresponding increase in interest toward natural products has emerged. These products, rich in bioactive compounds, are increasingly explored not only as potential sources of novel anticancer agents but also for their roles in alleviating symptoms associated with cancer and its treatment. , Despite significant advancements in cancer therapies, the overall rate remains high, underscoring the necessity for innovative approaches. Herbal medicine and natural products are increasingly explored for their potential to offer new therapeutic options in cancer treatment. , SH003, a new herbal medicine, combines Huang-Qi ( Astragalus membranaceus ), Dang-Gui ( Angelica gigas ), and Gua-Lou-Gen ( Trichosanthes kirilowii ). All of which have historical usage in traditional medicine and have shown anticancer properties in various preclinical studies, both in vitro and in vivo. - Studies into the efficacy and mechanism of SH003 on cancer reveal its multiple therapeutic effects. It suppresses breast cancer growth and metastasis by inducing autophagy and obstructing STAT3-IL-6 signaling, and it represses tumor angiogenesis by inhibiting VEGF-induced VEGFR2 activation. - Additionally, SH003 triggers apoptotic cell death in prostate cancer cells by inhibiting ERK2-mediated signaling, and it also restrains HeLa cervical cancer cell growth via G1 phase cell cycle arrest. , In vivo experiments have demonstrated potent anticancer effects of SH003 across various models. SH003 not only suppresses tumor growth and metastasis in MDA-MB-231 breast cancer cells but also, when combined with doxorubicin, exhibits a synergistic effect that significantly enhances therapeutic efficacy in triple-negative breast cancer models. , , Furthermore, SH003 effectively represses tumor angiogenesis in pancreatic cancer. In studies focusing on lung cancer, the combination of SH003 with docetaxel leads to synergistic antitumor activity, and it further enhances the anticancer effect of dabrafenib, showcasing broad efficacy in lung cancer treatment. , Recent studies highlight that Astragalus polysaccharide, a key component derived from Huang-Qi, inhibits tumor growth and enhances apoptosis, showing therapeutic potential in various cancers including prostate, liver, and non-small-cell lung cancer. Decursin, a bioactive compound from Dang-Gui, has been shown to effectively inhibit both tumor progression and autophagy in cancer cells, reducing CXCR7 expression to curtail cell proliferation, migration, and invasion, while also blocking cathepsin C-mediated autophagic flux, thereby demonstrating significant therapeutic potential. , Trichosanthin, an active component of Gua-Lou-Gen, has demonstrated significant anticancer efficacy in inhibiting the proliferation of cancer cells by inducing autophagy, a process dependent on the generation of reactive oxygen species and the activation of the NF-κB/p53 pathway. In our investigation into SH003, a blend of Huang-Qi, Dang-Gui, Gua-Lou-Gen, we observed that individual extracts influenced cell viability, but their combination at a 1:1:1 ratio appeared to enhance the inhibition of cell proliferation and induce apoptosis in cancer cells more effectively. Preliminary mechanistic studies suggest that this synergy could be effective in modulating critical signaling pathways; the SH003 combination seemed to reduce the phosphorylation of EGFR, SRC, and STAT3—pathways crucial for cancer cell survival and proliferation—more effectively than any single component alone. These findings indicate that the 1:1:1 ratio may not only amplify the anticancer properties of the individual components but also potentially plays a key role in selectively blocking STAT3 phosphorylation, warranting further investigation. Previous research has established the safety of SH003 at doses up to 4800 mg per day in a Phase I clinical trial. The previous Phase I trial administered SH003 up to a dosage of 4800 mg per day. This dosage was the highest tested that did not reach a conventional maximum tolerated dose (MTD), defined as the dose immediately below that at which dose-limiting toxicities (DLTs) of grade 3 or higher are observed in more than one participant. Given the absence of such DLTs at and below this dosage in the earlier study, our current trial aimed to explore higher dosage to rigorously establish the MTD of SH003, seeking to ascertain if higher dosage could be tolerated without exceeding these toxicity thresholds. Building on this foundation, the present study is designed to evaluate the safety of even higher doses of SH003, up to 9600 mg per day. The goal is to expand the permissible dosage range for SH003 in future efficacy trials. Study Design This multi-center, single-arm, open-label, dose-escalation phase I study was conducted at Gachon University Gil Medical Center in Incheon and Ajou University Hospital in Suwon, Republic of Korea from October 2020 to December 2022. This study was approved by the institutional review board (IRB) of the Gachon University Gil Medical Center (reference GFIRB2020-385) and the Ajou University Hospital (reference AJIRB-MED-CT1-20-264). All participants voluntarily signed an informed consent form before enrollment in the clinical trial and the study followed the guidelines of the Declaration of Helsinki and Tokyo for humans. Participants Inclusion criteria were as follows: age 19 years and older; histologically or cytologically confirmed solid cancers for which standard curative measures do not exist or are no longer effective; previous treatment should have been terminated at least 4 weeks ago and no residual toxicity related to previous treatment has been appeared; Eastern Cooperative Oncology Group (ECOG) Performance Status ≤2; estimated life expectancy of at least 12 weeks; participants who can swallow tablets; those whose organ function test results are as follows: hemoglobin at ≥8 g/dL, absolute neutrophil count ≥1500/μL, platelet count ≥75 000/μL, which is above the threshold for clinically significant chemotherapy-induced thrombocytopenia, bilirubin ≤ 2.5 times the upper limit of normal, AST/ALT ≤ 2.5 times the upper limit of normal, and serum creatinine ≤ 1.5 times the upper limit of normal or calculated CCr (Cockroft) ≥ 60 mL/min; female participants must not be capable of becoming pregnant (women who are 60 years or older and have not had a menstrual period for at least a year or who have undergone a hysterectomy or bilateral oophorectomy), if there was any chance of pregnancy, it had to be ruled out by conducting a pregnancy test prior to study participation; both male and female participants of childbearing potential were required to adhere to effective contraception methods throughout the of investigational product administration period and for at least 8 weeks thereafter; participants had to be able to understand the study and be willing to sign a written informed consent document. Exclusion criteria were as follows: patients who were undergoing systemic drug therapy or any local anticancer therapy, including radiation therapy, for the treatment of cancer; participants with known or suspected hypersensitivity reactions or serious adverse reactions to the study materials or materials of the same class, namely A. membranaceus, A. gigas and T. Kirilowii; participants presenting with evidence of active infection such as HBV, HCV, HIV, TB, etc., requiring treatment; patients with a known history of a positive test for HIV infection; patients with uncontrolled cardiovascular disease, including symptomatic unstable angina, heart failure, myocardial infarction, or uncontrolled hypertension, defined as blood pressure higher than 140/90 despite medication; patients with active CMV disease or infection within the last 4 weeks; patients with cerebrovascular disease, including acute coronary syndrome or stroke, or who had major surgery necessitating mechanical ventilation within the last year; pregnant or breastfeeding individuals; patients with symptomatic metastatic brain lesions, including dural metastases (those asymptomatic following previous surgery or radiotherapy, with no recent evidence of progression, were allowed); individuals who had participated in blood donation or other drug and medical device clinical trials within 1 month prior to study entry; organ transplant patients, including recipients of allogeneic stem cell transplants; substance abusers, or individuals with neurological, medical, psychiatric, or social illnesses that may interfere with study participation or the interpretation of study results; patients with a recent (within 1 year) serious medical condition that, in the opinion of the investigator, may increase the risk associated with the receipt of the investigational product or study participation, or may interfere with the interpretation of the study results, and patients deemed incapable of providing consent due to comorbidities such as dementia. Interventions Each SH003 tablet contains 800 mg of total material, consisting of 400 mg of blended solid extract from 3 herbs— A. membranaceus , A. gigas , and T. kirilowii —in a 1:1:1 ratio, obtained through a 30% ethanol extraction process, and 400 mg of excipients. The production of SH003 was undertaken by the pharmaceutical company Hanpoong Pharm and Foods Co. Ltd. (Jeonju, Republic of Korea,), in accordance with Korea Good Manufacturing Practice (KGMP) standards. The chemical constituents of SH003 are diverse. A. membranaceus contributes compounds including Astragaloside I, II, IV, isomucronulatol 7-O-glucoside, calycosin-7-O-β-D-glucoside, pinitol, daucosterol 6’-palmitate, β-sitosterol, sucrose, formononetin. A. gigas offers decursin, decursinol, decursinol angelate, demethylsuberosine, isoimperatorin, umbelliferone, nodakenin. Finally, T. kirilowii contributes trichosanthin, α-hydroxymethylserine, aspartic acid, threonine, serine, glutamic acid, citrulline, glycine, valine, tyrosine, glucose, galactose. Among the major chemical compounds of SH003, formononetin and astragaloside IV are attributed to A. membranaceus , decursin and nodakenin to A. gigas , and trichosanthin to T. kirilowii . , Of these, decursin and formononetin are designated as the marker compounds based on the Korean Herbal Pharmacopoeia, and the content of these 2 compounds were used to validate the SH003 formulation. We used traditional 3 + 3 design, which follows specific dose-escalation rules. This approach begins by recruiting 3 participants into a single cohort to evaluate toxicity, after which a decision is made on whether to proceed to the next cohort. If dose-limiting toxicities (DLTs) develop in more than 1 of 6 participants at a specific dose, this indicates that the maximum tolerated dose (MTD) has been exceeded. According to the protocol, this necessitates the cessation of further dose escalation. The dose increments were guided by a modified Fibonacci sequence. Upon establishing the safety of the preceding dose, it was elevated by 1.5 times for cohort 2, and by 1.33 times for cohort 3. The study participants were administered SH003 for a duration of 3 weeks. They ingested 4 to 8 tablets orally, 3 times a day post meals, with the dosage tailored according to their dose level. Participants consuming 4800, 7200, or 9600 mg/day were classified into Cohorts 1, 2, and 3, respectively. A previous phase I clinical trial determined the MTD of SH003 to be 4800 mg. Consequently, this dosage was designated as the starting dose in this study. The second and third cohort doses were subsequently escalated to 7200 and 9600 mg per day, respectively. Throughout the study duration, participants were instructed to abstain from any other cancer treatments, which included chemotherapy and radiotherapy. Outcome Measurements This study aimed to establish the MTD by evaluating the occurrence DLTs. The DLTs were defined as Grade 3 or higher adverse events, based on the Common Terminology Criteria for Adverse Events (CTCAE) ver. 5.0, outlined by the National Cancer Institute (NCI; Bethesda, MD USA). The MTD was identified as the highest dose at which no more than 1 out of 6 participants experienced a DLT. Furthermore, adverse events of all grades were recorded throughout the study period using the CTCAE. For safety assessments, vital signs, physical examination outcomes, hematologic and biochemical profiles, and urine tests were regularly measured. The present study conducted an exploratory evaluation of anticancer efficacy. This efficacy assessment was based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, enabling a standardized and uniform assessment of tumor response across the diverse solid tumors treated with SH003. Outcome Analysis The MTD was defined as the dose just below the level at which more than 1 of the 6 participants exhibited DLTs during the 4-week trial period. In this study, the MTD of SH003 was determined as the highest dose among the 3 dose groups (4800, 7200, or 9,600 mg per day) that resulted in a DLT in one participant or fewer. All data analyses conducted in this study were descriptive, as the research did not involve inferential analysis or generalized hypothesis testing. Continuous variables were presented as medians and range, while categorical variables were presented in terms of absolute and relative frequencies. This multi-center, single-arm, open-label, dose-escalation phase I study was conducted at Gachon University Gil Medical Center in Incheon and Ajou University Hospital in Suwon, Republic of Korea from October 2020 to December 2022. This study was approved by the institutional review board (IRB) of the Gachon University Gil Medical Center (reference GFIRB2020-385) and the Ajou University Hospital (reference AJIRB-MED-CT1-20-264). All participants voluntarily signed an informed consent form before enrollment in the clinical trial and the study followed the guidelines of the Declaration of Helsinki and Tokyo for humans. Inclusion criteria were as follows: age 19 years and older; histologically or cytologically confirmed solid cancers for which standard curative measures do not exist or are no longer effective; previous treatment should have been terminated at least 4 weeks ago and no residual toxicity related to previous treatment has been appeared; Eastern Cooperative Oncology Group (ECOG) Performance Status ≤2; estimated life expectancy of at least 12 weeks; participants who can swallow tablets; those whose organ function test results are as follows: hemoglobin at ≥8 g/dL, absolute neutrophil count ≥1500/μL, platelet count ≥75 000/μL, which is above the threshold for clinically significant chemotherapy-induced thrombocytopenia, bilirubin ≤ 2.5 times the upper limit of normal, AST/ALT ≤ 2.5 times the upper limit of normal, and serum creatinine ≤ 1.5 times the upper limit of normal or calculated CCr (Cockroft) ≥ 60 mL/min; female participants must not be capable of becoming pregnant (women who are 60 years or older and have not had a menstrual period for at least a year or who have undergone a hysterectomy or bilateral oophorectomy), if there was any chance of pregnancy, it had to be ruled out by conducting a pregnancy test prior to study participation; both male and female participants of childbearing potential were required to adhere to effective contraception methods throughout the of investigational product administration period and for at least 8 weeks thereafter; participants had to be able to understand the study and be willing to sign a written informed consent document. Exclusion criteria were as follows: patients who were undergoing systemic drug therapy or any local anticancer therapy, including radiation therapy, for the treatment of cancer; participants with known or suspected hypersensitivity reactions or serious adverse reactions to the study materials or materials of the same class, namely A. membranaceus, A. gigas and T. Kirilowii; participants presenting with evidence of active infection such as HBV, HCV, HIV, TB, etc., requiring treatment; patients with a known history of a positive test for HIV infection; patients with uncontrolled cardiovascular disease, including symptomatic unstable angina, heart failure, myocardial infarction, or uncontrolled hypertension, defined as blood pressure higher than 140/90 despite medication; patients with active CMV disease or infection within the last 4 weeks; patients with cerebrovascular disease, including acute coronary syndrome or stroke, or who had major surgery necessitating mechanical ventilation within the last year; pregnant or breastfeeding individuals; patients with symptomatic metastatic brain lesions, including dural metastases (those asymptomatic following previous surgery or radiotherapy, with no recent evidence of progression, were allowed); individuals who had participated in blood donation or other drug and medical device clinical trials within 1 month prior to study entry; organ transplant patients, including recipients of allogeneic stem cell transplants; substance abusers, or individuals with neurological, medical, psychiatric, or social illnesses that may interfere with study participation or the interpretation of study results; patients with a recent (within 1 year) serious medical condition that, in the opinion of the investigator, may increase the risk associated with the receipt of the investigational product or study participation, or may interfere with the interpretation of the study results, and patients deemed incapable of providing consent due to comorbidities such as dementia. Each SH003 tablet contains 800 mg of total material, consisting of 400 mg of blended solid extract from 3 herbs— A. membranaceus , A. gigas , and T. kirilowii —in a 1:1:1 ratio, obtained through a 30% ethanol extraction process, and 400 mg of excipients. The production of SH003 was undertaken by the pharmaceutical company Hanpoong Pharm and Foods Co. Ltd. (Jeonju, Republic of Korea,), in accordance with Korea Good Manufacturing Practice (KGMP) standards. The chemical constituents of SH003 are diverse. A. membranaceus contributes compounds including Astragaloside I, II, IV, isomucronulatol 7-O-glucoside, calycosin-7-O-β-D-glucoside, pinitol, daucosterol 6’-palmitate, β-sitosterol, sucrose, formononetin. A. gigas offers decursin, decursinol, decursinol angelate, demethylsuberosine, isoimperatorin, umbelliferone, nodakenin. Finally, T. kirilowii contributes trichosanthin, α-hydroxymethylserine, aspartic acid, threonine, serine, glutamic acid, citrulline, glycine, valine, tyrosine, glucose, galactose. Among the major chemical compounds of SH003, formononetin and astragaloside IV are attributed to A. membranaceus , decursin and nodakenin to A. gigas , and trichosanthin to T. kirilowii . , Of these, decursin and formononetin are designated as the marker compounds based on the Korean Herbal Pharmacopoeia, and the content of these 2 compounds were used to validate the SH003 formulation. We used traditional 3 + 3 design, which follows specific dose-escalation rules. This approach begins by recruiting 3 participants into a single cohort to evaluate toxicity, after which a decision is made on whether to proceed to the next cohort. If dose-limiting toxicities (DLTs) develop in more than 1 of 6 participants at a specific dose, this indicates that the maximum tolerated dose (MTD) has been exceeded. According to the protocol, this necessitates the cessation of further dose escalation. The dose increments were guided by a modified Fibonacci sequence. Upon establishing the safety of the preceding dose, it was elevated by 1.5 times for cohort 2, and by 1.33 times for cohort 3. The study participants were administered SH003 for a duration of 3 weeks. They ingested 4 to 8 tablets orally, 3 times a day post meals, with the dosage tailored according to their dose level. Participants consuming 4800, 7200, or 9600 mg/day were classified into Cohorts 1, 2, and 3, respectively. A previous phase I clinical trial determined the MTD of SH003 to be 4800 mg. Consequently, this dosage was designated as the starting dose in this study. The second and third cohort doses were subsequently escalated to 7200 and 9600 mg per day, respectively. Throughout the study duration, participants were instructed to abstain from any other cancer treatments, which included chemotherapy and radiotherapy. This study aimed to establish the MTD by evaluating the occurrence DLTs. The DLTs were defined as Grade 3 or higher adverse events, based on the Common Terminology Criteria for Adverse Events (CTCAE) ver. 5.0, outlined by the National Cancer Institute (NCI; Bethesda, MD USA). The MTD was identified as the highest dose at which no more than 1 out of 6 participants experienced a DLT. Furthermore, adverse events of all grades were recorded throughout the study period using the CTCAE. For safety assessments, vital signs, physical examination outcomes, hematologic and biochemical profiles, and urine tests were regularly measured. The present study conducted an exploratory evaluation of anticancer efficacy. This efficacy assessment was based on the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, enabling a standardized and uniform assessment of tumor response across the diverse solid tumors treated with SH003. The MTD was defined as the dose just below the level at which more than 1 of the 6 participants exhibited DLTs during the 4-week trial period. In this study, the MTD of SH003 was determined as the highest dose among the 3 dose groups (4800, 7200, or 9,600 mg per day) that resulted in a DLT in one participant or fewer. All data analyses conducted in this study were descriptive, as the research did not involve inferential analysis or generalized hypothesis testing. Continuous variables were presented as medians and range, while categorical variables were presented in terms of absolute and relative frequencies. In our phase I trial using the traditional 3 + 3 design, we closely monitored for any DLTs to determine the MTD. Initially, 3 participants were administered SH003 at 4800 mg/day in Cohort 1. A DLT was observed in one of these initial participants, prompting the enrollment of an additional 3 participants to ensure a robust assessment of this dose level. Ultimately, the cohort expanded to 6 participants, and with only one DLT occurrence, the trial was able to proceed. The dosage was then escalated to 7200 mg/day for the 3 participants in Cohort 2, and eventually to 9600 mg/day for the 4 participants in Cohort 3, following the predefined escalation rules without further DLTs. presents a flow chart of the study, illustrating the participant enrollment process, dose escalation steps, and overall study design. The participant characteristics are detailed in . The median age of participants was 62 years, with a predominance of male participants (9 males and 4 females). The most common cancer types among the participants were colon cancer (6 patients), followed by gastric (2 patients), and pancreatic cancer (2 patients). All participants had an ECOG PS of 2. Laboratory parameters, including WBC, RBC, Hemoglobin, Hematocrit, Platelets, AST, ALT, Creatinine, and BUN were within normal ranges or, if outside normal ranges, deviations were clinically non-significant. The occurrence of adverse events during the study is summarized in . A total of 12 kinds of adverse events were observed, totaling 15 occurrences across all cohorts. Cohort 1 reported 5 adverse events, Cohort 2 had 3, and Cohort 3 experienced 5. Dizziness was the most common adverse event with 3 cases, followed by nausea with 2 cases. All other adverse events were reported only once. In Cohort I, a DLT was observed as the GGT increased corresponded to a Grade 3. Cohorts II and III experienced Grade 2 or lower adverse events, with no Grade 3 or higher toxicities reported. In Cohort I, initially 3 participants were enrolled, but due to the occurrence of a DLT in one participant, the protocol necessitated the recruitment of an additional 3 participants, maintaining the same dosage of 4800 mg/day. For Cohort III, one of the initially recruited participants was excluded due to low medication compliance. As per the protocol, this participant was replaced by enrolling an additional participant, resulting in a total of 4 participants in this cohort receiving 9600 mg/day. We conducted a clinical trial to evaluate the safety of SH003 at doses up to 9600 mg per day. Previously, the safety of SH003 had been established for doses up to 4800 mg per day. This study successfully extended the safety profile of SH003, confirming its tolerability at a dose level of 9600 mg per day. This outcome enables us to explore higher dosages in future clinical trials investigating the efficacy of SH003. With the established safety profile up to 9600 mg per day, it opens new possibilities for upcoming research in various combination treatment settings. In addition to safety assessments, tumor response rates in this study, as well as in a previous study assessing the safety of SH003, were evaluated using RECIST 1.1. This standardized approach was selected due to its broad acceptance for assessing tumor size changes across diverse types of solid tumors. In the current study, one participant in the 4800 mg/day cohort and one in the 9600 mg/day cohort showed stable disease. For the remaining participants in both studies, the response was classified as progressive disease. In our study, stable disease was observed in participants with gallbladder and colon cancer. However, due to the diverse range of solid tumors included and the limited sample size, it remains premature to draw definitive conclusions about the tumor-specific efficacy of SH003. Continued research in larger, more focused trials is essential to determine the therapeutic potential of SH003 across different cancer subtypes. In the Republic of Korea, there exists a regulatory provision that allows the exemption of Phase I safety evaluations for new drug development using natural substances, based on traditional usage experience. This enables the commencement of clinical trials directly from Phase 2. Consequently, there have been very few Phase I clinical trials conducted in Korea on natural substances. SH003 represents the first Phase I clinical trial in Korea focused on a natural substance. In this trial, we have additionally confirmed the safety of SH003 at higher dosages. While many studies on natural products in cancer research have focused on symptom relief in cancer patients, our study with SH003 is distinctly aimed at developing it as a therapeutic anticancer agent. The current phase I trial is crucial for establishing a safe dosing regimen, which lays the groundwork for future rigorous evaluation of its potential anticancer efficacy. Historically, the use of natural products in oncology has been largely limited to supportive care, aiming to alleviate the side effects of conventional cancer treatments or improve the quality of life for patients. However, with SH003, we are exploring the direct antitumor effects of an herbal medicine. We highlighted the diverse therapeutic effects of SH003, such as its capacity for inducing autophagy, obstructing key signaling pathways, , and arresting cell cycle progression in various cancer cells. The diverse range of actions of SH003, attributed to its multi-component nature, marks a departure from traditional single-target cancer therapies. This multi-targeted approach, utilizing the synergy of various bioactive compounds, is designed to concurrently address different aspects of tumor biology. Such an approach aligns with the growing interest in multi-component natural compounds for cancer treatment, as evidenced by recent researches. , The results of our study suggest that SH003 is safe up to a dosage of 9600 mg/day, as significant adverse reactions were not frequently observed. Among the adverse events, dizziness, occurring in 3 instances, was the most common, followed by nausea, reported in 2 instances. Although these were not considered major concerns, the absence of a control group in this study limits our ability to make definitive comparison. Nevertheless, given the common occurrence of such symptoms in patients with advanced cancer, , coupled with clinical judgment of the attending physician, it is considered unlikely that these symptoms are directly related to SH003. Furthermore, in Cohort 1, a case of Grade 3 GGT increase was observed. This event is particularly noteworthy as it occurred in a patient with advanced-stage cancer and liver metastases, conditions often associated with elevated GGT levels. - These factors suggest that the increase in GGT may not be directly attributable to SH003, but rather could be a consequence of the underlying advanced cancer condition of the patient, especially the liver involvement. The evaluating physician supported this view, indicating a likely association of the increased GGT level with the advanced disease state of the patient, rather than with SH003 administration. We plan to keep a close watch on such symptoms in future clinical trials to further investigate their association with SH003. The absence of frequently occurring or severe adverse reactions, even at the highest dosage tested, is a promising indicator of the safety profile of SH003. This finding is particularly significant in cancer treatment, where the utmost importance is placed on patient tolerance and safety, especially considering the often compromised health of many patients. The potential for lower associated risks with SH003 may provide a safer alternative or adjunct to existing treatments, which are often accompanied by substantial side-effect burdens. This study, a single-arm trial involving a small cohort of patients with various solid cancers, represents an early phase in the development of the potential new drug SH003. Despite the safety profile demonstrated in this phase I trial, the limitations posed by the small sample size and the diversity of cancer types must be acknowledged. The limited number of participants and heterogeneity of cancer types studied restrict the generalizability of the findings and the statistical power required for definitive efficacy conclusions. As an initial step in drug development, this study aimed to establish a safety baseline and identify the maximum tolerated dose of SH003. These constraints underscore the need for more focused research to ascertain the effectiveness of the drug across specific cancer subtypes. Future studies are planned to include larger patient cohorts and concentrate on particular types of cancer, enhancing statistical robustness and enabling more precise evaluations of efficacy. Such an approach will provide stronger evidence for the therapeutic potential of SH003 and allow for a more accurate assessment of its clinical relevance to specific cancer subtypes. By increasing both sample size and study population homogeneity, these studies aim to deliver a more definitive understanding of the efficacy and safety profile of SH003, thereby meeting the rigorous requirements for statistical and clinical validation in subsequent clinical trials. Furthermore, the predominant involvement of end-stage cancer patients complicates the clear identification of adverse reactions specifically attributable to SH003, due to symptom overlap commonly seen in advanced cancer stages. The decision to include end-stage cancer patients was driven by ethical considerations, as this was the first human study of SH003. Established guidelines in oncology clinical trials recommend reserving experimental treatments for individuals who lack curative options. This approach ensures that patients with potentially curative cancers are not deprived of effective treatments in favor of experimental therapies. As the safety profile of SH003 becomes better established and if partial efficacy is observed, it may become feasible and ethical to consider including earlier-stage cancer patients in future trials. This would be particularly pertinent in combination therapy trials, where SH003 could potentially be evaluated alongside standard treatments. Therefore, it is essential to conduct future studies with larger and more diverse patient cohorts to better understand unique adverse effect profile of SH003 and validate its safety and efficacy across various cancer stages and types. While special attention should continue to be given to the occurrence of dizziness, reported in 3 instances, and nausea in 2 instances, it is also crucial to carefully monitor and analyze other observed adverse events, such as increased levels of GGT. In this study, a notable limitation is the absence of human pharmacokinetic data for SH003. While pharmacokinetic parameters have been evaluated in animal models, these findings are unpublished and cannot be directly extrapolated to human subjects. The primary challenge in conducting human PK studies lies in the low bioavailability of index compounds in SH003, requiring large blood sample volumes for analysis—a procedure not feasible in patients with cancer without compromising ethical standards. As our research progresses, we will consider the possibility of integrating pharmacokinetic studies into the development process of SH003 under appropriate conditions, potentially including trials with healthy volunteers, ensuring ethical standards are met. Another limitation is that no marker compound was set for T. kirilowii due to the low concentration of its components. We plan to establish a standardization method through techniques such as chemical fingerprinting for SH003, to ensure consistent quality across all components. An important consideration for future research is that although we have established the safety of administering up to 9600 mg/day, the current formulation requires the consumption of a considerable number of tablets. This could potentially reduce patient compliance. Moving forward, it will be crucial to explore optimal dosing that balances efficacy with patient adherence and to consider the development of alternative formulations that could reduce the tablet burden. This study provides initial insights for future clinical trials assessing the efficacy of SH003 in cancer treatment. Separately, another clinical trial is currently underway to evaluate the safety of combining SH003 with docetaxel. This separate study reflects the common practice of combination therapy in oncology and is an important step in understanding the broader potential of SH003 when used in conjunction with established treatments. The initial findings regarding SH003 are encouraging, yet they represent just the initial steps toward a deeper understanding of its potential in cancer therapy. The ongoing exploration of SH003, both as an independent treatment and in combination with other drugs such as docetaxel, is anticipated to significantly contribute to the field of oncology. Furthermore, recent reports have highlighted the potential of SH003 in symptom management for cancer patients. Notably, SH003 has been observed to alleviate pain induced by chemotherapy agents like paclitaxel and docetaxel, , and to enhance immune responses. These properties suggest that SH003 could be a valuable adjunct in cancer therapy, not only for its direct anticancer effects but also for improving the quality of life of patients by mitigating treatment-related symptoms. The prospect of combining SH003 with conventional chemotherapy agents is noteworthy, as it may enhance the overall therapeutic efficacy and potentially reduce adverse effects. This research not only has the potential to lead to the development of new, less toxic cancer treatments but also sets a precedent for future studies on other natural products. The progress made in understanding and utilizing SH003 could serve as a valuable foundation for broader research into natural products in cancer therapy, potentially ushering in a new era of innovative and effective treatment options. In conclusion, this study has established that SH003 is safe for use in cancer patients at doses up to 9600 mg/day, expanding its known safety threshold. This advancement underscores the importance of patient tolerance and safety in cancer treatments, particularly given the often compromised health status in this patient group. While the results are promising, they also highlight the necessity for further, more extensive research to fully determine the efficacy and broader applicability of SH003 in various cancer types and stages. |
Entwicklung der Katheterablation supraventrikulärer Tachykardien unter besonderer Berücksichtigung der Beiträge deutscher Ingenieure und Elektrophysiologen | 1f61bc53-b74f-4965-9aac-15137fb58933 | 10923970 | Internal Medicine[mh] | Wie in der Einleitung bereits kurz dargestellt, wurde zur Katheterablation von Herzrhythmusstörungen zunächst die Gleichstromablation (auch DC-Ablation) eingesetzt. Hierzu wurde die im Bereich des vermuteten Zielgewebes im Herzen platzierte Katheterelektrode über einen Adapter mit der Elektrode eines handelsüblichen Defibrillators verbunden. Als Gegenelektrode wurde eine großflächige Hautelektrode am Patienten fixiert und die Energieabgabe unipolar durchgeführt. Im Moment der Stromabgabe wurden innerhalb von Millisekunden Spannungen von mehreren 1000 V über die Katheterelektrode dem Myokard zugeführt. Durch die Energieabgabe entstehen im Bereich der Katheterelektrode erhebliche Druckwellen, die zu sogenannten barotraumatischen Gewebeeffekten führen. Einerseits konnte so zwar das Zielgewebe erfolgreich zerstört werden, als Folge des Barotraumas stellten sich aber auch gefährliche Komplikationen wie Myokardperforationen und Perikardtamponaden ein. Die relativ hohe Komplikationsrate der Eingriffe war somit eine erhebliche Limitierung der Nutzbarkeit. Als alternative Energiequelle wurde 1985 von dem Ingenieur Dr. Peter Osypka die Hochfrequenzstromenergie zur Katheterablation mit der Vorstellung des Hochfrequenzstrom-Generators HAT 100 eingeführt. Mit dem Generator konnte sinusförmiger nichtmodulierter Hochfrequenzstrom in 10 Leistungsstufen abgegeben werden. Die Energieabgabe erfolgte analog zur Gleichstromablation in unipolarer Elektrodenkonfiguration über die endständige Elektrode eines handelsüblichen diagnostischen Elektrodenkatheters. Durch die Stromabgabe wurde im Übergangsbereich zwischen der Katheterelektrode und dem Herzmuskelgewebe Widerstandswärme erzeugt, die zur Ausbildung einer Koagulationsnekrose in der Umgebung des Elektrodenkatheters führte. Die Stromabgabe war mit dem einfachen Generator schwierig zu kontrollieren, und es stellten sich immer wieder Überhitzungen der Katheterelektrode ein, die teilweise zu Verkohlungen (Karbonisation) mit zum Teil erheblichen Isolationsdefekten des Elektrodenkatheters führten. Um die Überhitzung der Katheterelektrode zu verhindern, wurden spezielle Elektrodenkatheter entwickelt, die mit Thermistoren in der Katheterspitze ausgestattet waren, um die Stromabgabe bei zu starker Temperaturentwicklung zu limitieren (Abb. ). Die erste erfolgreiche klinische Anwendung der temperaturkontrollierten Ablation wurde 1988 von der Arbeitsgruppe in Münster durchgeführt. Die sogenannte temperaturgesteuerte Hochfrequenzstromablation erlaubte eine effektivere und sichere Applikationsbehandlung und setzte sich als Standard der Hochfrequenzstromablation in den nächsten Jahren durch (; Abb. und ). Alternativ wurden auch andere Energiequellen wie Laserenergie und später auch fokussierter Ultraschall hinsichtlich der Möglichkeiten zur Ablationsbehandlung untersucht. Letztlich konnte sich keine der alternativen Energiequellen durchsetzen, und die Hochfrequenzstrom-Katheterablation setzte den technologischen Standard für die Behandlung von Herzrhythmusstörungen. Die in Deutschland entwickelte Technologie und auch die ersten klinischen Anwendungen wurde insbesondere von den Arbeitsgruppen um Borggrefe und Breithardt so wie Kuck und Mitarbeiter befördert . Wesentliche Beiträge zur technologischen Verbesserung der Applikationsmethode wurden aber auch von den amerikanischen Elektrophysiologen David Haines und Hiroshi Nakagawa geleistet . Die Entwicklung der Katheterablation wäre jedoch nicht möglich gewesen ohne die sich parallel entwickelnde Verfügbarkeit von sogenannten elektroanatomischen Mappingsystemen, die eine Verbindung zwischen Elektrophysiologie und Anatomie in bis dahin nicht vorstellbarer Genauigkeit möglich machten. Der wesentlichste Beitrag in diesem Bereich ist sicherlich Shlomo Ben-Haim durch die Entwicklung des Carto-Systems zuzuschreiben. Neben der hohen Präzision dieses ersten nichtfluoroskopischen Mappingsystems konnte so die Strahlenbelastung für die behandelten Patienten und auch für die Behandler substanziell reduziert werden . Nach den oben bereits beschriebenen ersten experimentellen und klinischen Untersuchungen zur Gleichstromablation durch Scheinman und Gallagher wurde die erste Anwendung in Deutschland 1983 durch Lüderitz und Manz beschrieben . Die erste erfolgreiche Anwendung der Hochfrequenzstrom-Ablation zur Durchtrennung der atrioventrikulären Überleitung erfolgte 1985 von Budde und Mitarbeitern . Erste erfolgreiche Anwendungen der Katheterablation zur Modulation des AV-Knotens bei AVNRT wurden 1998 von Haissaguerre und Mitarbeitern vorgestellt. Die Untersuchungen wurden zunächst durch Gleichstromablation der retrograden Leitung des AV-Knotens durchgeführt. Die Methode erwies sich als durchaus effektiv zur kurativen Behandlung der Tachykardien, ging aber mit einer relativ hohen Häufigkeit kompletter AV-Blockierung und nachfolgender Schrittmacherpflichtigkeit einher . 1992 stellten Jackman und Mitarbeiter die Methode der AV-Knotenmodulation durch selektive Ablation der langsam leitenden Anteile des AV-Knotens vor. Die Methode hatte den wesentlichen Vorteil dadurch, dass die Häufigkeit höhergradiger AV-Blockierungen als Folge der Behandlung im Vergleich zur Ablation der schnell leitenden Anteile des AV-Knotens erheblich geringer war . Diese Methode hat sich dann als Standard für die Modulation des AV-Knotens erfolgreich durchgesetzt. Mittlerweile konnten viele 10.000 Patienten durch diesen sicheren und kurativen Eingriff von ihren hochsymptomatischen Herzrhythmusstörungen geheilt werden. Die Modulation des AV-Knotens kann heute in geübter Hand in nahezu allen Fällen erfolgreich und sicher durchgeführt werden. Die Entwicklung der Katheterablation zur erfolgreichen Behandlung von rechtsatrialem, sog. typischem Vorhofflattern ist ohne die pathophysiologischen Untersuchungen von Waldo und Mitarbeitern nicht denkbar . Diese Arbeitsgruppe legte grundlegende Ergebnisse zur Beschreibung des Reentry-Kreises bei typischem Vorhofflattern vor und schuf damit die Grundlage für die Entwicklung einer Strategie zur erfolgreichen Behandlung mittels Katheterablation. Diese wurde von Saoudi und Mitarbeitern aufgegriffen und in ein Ablationskonzept durch die Applikation linearer Läsionen zwischen dem inferioren Bereich des Trikuspidalklappenanulus und der Mündung der Vena cava inferior entwickelt . Saudi und Mitarbeiter konnten zeigen, dass durch eine Läsion entlang dem rechtsatrialen Isthmus der Reentry-Kreis erfolgreich unterbrochen und das Wiederauftreten von typischem Vorhofflattern effektiv verhindert werden konnte. Vergleichbare Behandlungskonzepte wurden später dann auch für die erfolgreiche Behandlung anderer rechts- und linksatrialer Reentry-Tachykardien entwickelt und etabliert. Mit der Entwicklung einer chirurgischen Technik zur Behandlung von Vorhofflimmern leitete der Herzchirurg Jimmy Cox eine neue Ära in der Behandlung von Vorhofflimmern ein. Cox entwickelte nach umfangreichen experimentellen Vorarbeiten die sog. Maze-Prozedur. Im Rahmen der Operation wurden der rechte und der linke Vorhof durch multiple Inzisionen in isolierte Kompartimente unterteilt und dadurch das Auftreten von Vorhofflimmern verhindert. Teil der Maze-Prozedur war auch die komplette Isolation der Pulmonalvenen . Der amerikanische Elektrophysiologie John Schwarz war der Erste, der in klinischen Studien versuchte, das Therapiekonzept der chirurgischen Maze-Operation in eine Katheterprozedur zu übertragen. Die Ergebnisse dieser Studien wurden 1986 im Rahmen der Jahrestagung der Amerikanischen Gesellschaft für Kardiologie vorgestellt, aber leider nie als volles Manuskript publiziert. Inspiriert durch die Untersuchungen und Befunde von John Schwarz arbeitete der französische Elektrophysiologe Michelle Haissaguerre an Ablationskonzepten für die Therapie von Vorhofflimmern . In einer ersten Mitteilung wurde die Beobachtung von sog. fokalem rechtsatrialem Vorhofflimmern und einer erfolgreichen Ablation bei 3 Patienten beschrieben . Der Durchbruch sowohl im pathophysiologischen Verständnis des Vorhofflimmerns wie auch hinsichtlich der Entwicklung von Therapiestrategien gelang Haissaguerre und Mitarbeitern durch die Identifizierung von Pulmonalvenenfoci und deren Bedeutung für das Auftreten von Vorhofflimmern. In einer wirklichen Meilenstein-Arbeit publizierte Haissaguerre diese Befunde 1993 im New England Journal of Medicine . Damit war die Entwicklung des Therapiekonzeptes der Pulmonalvenenisolation zur Behandlung von Vorhofflimmern vorgestellt. Parallel hierzu wurden Elektrodenkatheter entwickelt, durch welche die Elektrophysiologie der Pulmonalvenen abgebildet werden konnte (sog. Lasso-Katheter). Herausragende Beiträge zum besseren Verständnis der Elektrophysiologie der Pulmonalvenen und der Entwicklung effektiver Ablationsstrategien wurden von der Arbeitsgruppe um Karl Heinz Kuck aus Hamburg geleistet. Carlo Pappone stellte als Erster das Konzept der Pulmonalvenenisolation durch zirkumferenzielle Ablation auf Vorhofebene vor . Dieses Konzept wurde durch die Nutzung elektroanatomischer Mappingtechnologie zur klinischen Anwendungsreife geführt. Das Prinzip der kompletten Isolation der Pulmonalvenen hat sich als effektives Therapiekonzept für die Katheterablation von Vorhofflimmern mittlerweile gut etabliert. Von unterschiedlichen Arbeitsgruppen wurden alternative Ablationskonzepte wie die gezielte Ablation fraktionierter arterieller Potenziale oder auch durch Ablation sog. Rotoren klinisch untersucht. Beide Methoden konnten sich aufgrund geringer Effektivität nicht durchsetzen. Auch die Ergänzung der Pulmonalvenenisolation durch sog. lineare Läsionen, beispielsweise entlang des Mitralklappenanulus oder als sog. linksatriale Dachlinie, konnte sich nicht durchsetzen. Die einzige Technik, die in einigen Studien Vorteile gegenüber der alleinigen Pulmonalvenenisolation zeigen konnte, war die sog. Substratmodifikation durch gezielte Ablation atrialer Myokardbezirke, die sich durch sog. Low-Voltage-Areale auszeichnen. Das Konzept der atrialen Kardiomyopathie wurde u. a. von Hans Kottkamp entwickelt und klinisch etabliert . Piorkowski und Mitarbeiter konnten im Rahmen einer randomisierten Studie zeigen, dass die gezielte Ablation dieser Areale zu einer signifikanten Verbesserung der Ablationsergebnisse führt . Die Entwicklung der Kryoablation in den Jahren 2000 bis 2010 wurde wesentlich von der deutschen Elektrophysiologie mitbestimmt. Erste größere Untersuchungsserien wurden von Ellen Hoffmann und ihrer Arbeitsgruppe aus München vorgestellt . Die erste große randomisierte Studie, die eine Gleichwertigkeit der Kryoablation zur Katheterablation mit Hochfrequenzstrom zeigte, wurde von Karl-Heinz Kuck geleitet und durchgeführt: Fire and Ice . Die wesentliche aktuelle technologische Entwicklung im Bereich der Katheterablation von Vorhofflimmern ist sicherlich der Einsatz der sog. „pulsed-field ablation“ zur linksatrialen Ablation . Im Gegensatz zur Katheterablation mit Hochfrequenzstrom oder zur Kryoablation wird die Läsionsinduktion bei Einsatz der PFA-Energie fast ausschließlich durch eine direkte elektrische Schädigung der Zellmembranen ohne wesentliche thermische Effekte induziert. Vorteil der PFA-Technologie ist darüber hinaus, dass eine Schädigung des Ösophagus, der Pulmonalvenen (Pulmonalvenenstenose) oder auch des Nervus phrenicus im Vergleich zu den thermischen Ablationsmethoden deutlich niedriger zu sein scheint. Gleichzeitig kann die Ablationsbehandlung schneller durchgeführt werden. Im Moment laufen erste größere klinische Studien, die vergleichend die Wirksamkeit thermischer und elektrischer Ablationsmethoden untersuchen. Außerdem wurden in den letzten Jahren Studien zur Ablationsbehandlung komplexer Herzrhythmusstörungen durch gezielte Strahlenapplikation mittels Linearbeschleunigern, die ansonsten in der Krebsbehandlung eingesetzt werden, durchgeführt: die transkutane Ablation von Herzrhythmusstörungen . Es geht also immer weiter mit den technischen und technologischen Entwicklungen in der Rhythmologie. Wir sind gespannt! |
Freiburg Neuropathology Case Conference | f2605a3d-a36b-4e5d-9723-53294e893b8a | 8463354 | Pathology[mh] | A 6-year-old girl presented with recurrent daily morning vomiting starting 1 week ago. The clinical examination showed a mild right-sided facial paresis and bilateral congestive papillae. Magnetic resonance imaging (MRI) of the brain showed an extensive heterogeneous temporoparietal tumor formation of the left side with intraventricular infiltration (Figs. , and ). Urgent surgical decompression was indicated. Temporofrontal craniotomy was performed with the patient under general anesthesia in the supine position under neuromonitoring. The brain stem was decompressed by partial temporal resection of the tumor. The tumor appeared soft, bled heavily upon resection and was difficult to separate from the surrounding brain tissue, finally we only performed a partial resection. After surgery, the child promptly woke up and was discharged home without focal neurological deficit on postoperative day 7. Postoperative computed tomography (CT) revealed the extent of the tumor resection, with tumor remnants located in the midline, along the lateral ventricles and in the course of the fornix and septum pellucidum (not shown). An MRI control 4 weeks later showed tumor progression (not shown). After discussion of the case in our multidisciplinary tumor board, chemotherapy with carboplatin-etoposide was initiated. The MRI of the brain revealed a massive space-occupying lesion affecting the mesial temporal lobe, and the underlying white matter of the temporal lobe, the parahippocampal gyrus, as well as the left thalamus. In addition, subependymal growth of the lesion expanding into the ventricular system and subsequent ventricular distension is present at the level of the left temporal horn of the lateral ventricle, the occipital horn of the left lateral ventricle, and the cella media of the left lateral ventricle. Exophytic tumor expansion into the subarachnoidal space causing marked compression of midbrain structures are present at the level of the mesial temporal lobe and the parahippocampal gyrus (Figs. , and ). On fluid attenuated inversion recovery (FLAIR) images (Fig. a), and T2 weighted images (Fig. a and a) the lesions appear relatively homogeneously hyperintense. In addition, cystic components are present indicating regressive changes of the tumor matrix. On T1 weighted images obtained after administration of gadolinium the lesion shows a heterogeneous pattern of enhancement (Fig. b, b and b). The lesion presents with a large number of well-delineated portions showing homogeneous enhancement at the level of the thalamus (Fig. b), the parahippocampal gyrus, and subependymal tumor growth along the temporal horn (Fig. b). On the other hand, there are tumor portions that show no enhancement of contrast. These include the mesiotemporal as well the exophytic tumor portions extending into the subarachnoidal space (Fig. b), but also subependymal tumor portions of the cella media (Fig. c). On diffusion-weighted images (b-value: 1000) the tumor shows restricted diffusion suggesting a hypercellular tumor (Fig. c and c). The diffusion restriction is unrelated to the variable degree of contrast enhancement. On CT images obtained immediately after tumor resection the remaining tumor parts did not show any apparent calcification (not shown). Pilocytic Astrocytoma Pilocytic astrocytoma (PA) is the most common primary brain tumor of childhood accounting for 15% of all pediatric brain tumors with a peak incidence between 5 and 15 years. Pilocytic astrocytomas are low grade World Health Organization (WHO) type I tumors showing a strong association with neurofibromatosis type 1 (NF1) without any gender predisposition . Pilocytic astrocytomas typically arise from midline structures. The most common location in non-NF1 patients is the cerebellum in 60%, whereas PA of the optic pathway are particularly common in NF1 patients. Less common locations include the hypothalamus, brainstem and cerebral hemispheres, cerebral ventricles, velum interpositum and the spinal cord . Pilocytic astrocytoma have a wide range of imaging appearances. On MR imaging four predominant imaging patterns have been described: (a) mass with a nonenhancing cyst and an intensely enhancing mural nodule (21% of cases), (b) mass with an enhancing cyst wall and an intensely enhancing mural nodule (46%), (c) necrotic mass with a central nonenhancing zone (16%), and (d) predominantly solid mass with minimal to no cyst-like component (17%). Calcifications are present in 20% of cases . Given the localization and the extent of the lesion pilocytic astrocytoma was considered a less likely differential diagnosis. Supratentorial Ependymoma Ependymoma is the number three of the most common intracranial neoplasm in children. They arise from ependymal cells of the central nervous system (CNS) and can occur anywhere within the CNS . The incidence is bimodal with one peak between 1–5 years and a smaller peak during the 2–3 decade of life with a male predisposition. Most common symptoms are seizures, focal motor or sensory deficits and headache, but also when large, signs of raised intracranial pressure and obstructive hydrocephalus especially in infratentorial location may occur. Supratentorial location accounts for only one third of ependymomas. They typically arise from the ependymal lining of the ventricular system or fetal ependymal rests. The majority of supratentorial ependymomas are located within the white matter or cortex of the frontal lobe. Ventricular localization is less common. In the WHO classification from 2021, supratentorial ependymomas are classified based on molecular features into two subgroups. RELA fusion-positive supratentorial ependymomas seem to affect older children and have a poor prognosis, whereas supratentorial ependymomas displaying YAP‑1 fusions affect children below 3 years of age . At MRI ependymomas are isointense to slightly hypointense on T1-weighted images and isointense to hyperintense on T2 weighted images. Heterogeneous enhancement is seen on contrast-enhanced images. Restricted diffusion is regularly present, most likely reflecting high cellularity . On CT images, the soft tissue component is commonly hypoattenuating to isoattenuating. Ependymomas frequently demonstrate cystic components, and areas of small chunky calcification. Occasionally intratumoral hemorrhage may be present . Because of ependymal location of the lesion, as well as the diffusion restriction, supratentorial anaplastic ependymoma was considered a valid differential diagnosis in our case. Atypical Teratoid/Rhabdoid Tumor (AT/RT) Atypical teratoid/rhabdoid tumors (AT/RT) are rare, WHO grade IV tumors of the CNS. In > 80% of cases children less than 3 years of age are affected. There is no gender predisposition. They are among the fastest growing CNS tumors with a short interval to symptom onset. By the time of diagnosis, they often reach sizes > 3 cm. Most common symptoms are rapidly increasing lethargy, vomiting and increased head circumference. Smaller supratentorial lesions may cause seizures . The AT/RT may occur supratentorially or infratentorially. Infratentorially they appear off-midline and may resemble medulloblastomas, 12% of AT/RT are found bihemispherically and 15–20% are disseminated at the time of initial diagnosis. Histologically, AT/RTs are embryonal tumors that contain a rhabdoid component as well as areas with primitive neuroectodermal, mesenchymal, and epithelial features. The current WHO classification of 2021 places AT/RT under embryonal tumors. This group of tumors includes medulloblastoma, atypical teratoid/rhabdoid tumor, embryonal tumor with multilayered rosettes, C19MC-altered and embryonal tumor with multilayered rosettes, pineoblastoma, pituitary blastoma, CNS neuroblastoma, and ganglioneuroblastoma . Diagnosis of AT/RT requires confirmation of specific genetic aberration, such as loss of INI1 tumor suppressor gene on chromosome 22 or BRG1 gene . The use of CT imaging often shows a hyperattenuated mass lesion commonly containing cystic structures and/or hemorrhage. Some tumors contain fine calcifications and signs of obstructive hydrocephalus may be present . On T2-weighted images, AT/RTs appear heterogeneous with hyperintense cystic foci, which are hyperintense on T1-weighted images. In addition, restricted diffusion may be present in solid parts of the tumor. Contrast enhancement on T1 weighted images is mostly heterogeneous and can be nodular . Considering the patient’s age and the imaging findings, AT/RT seemed to be a likely differential diagnosis. Choroid Plexus Carcinoma Choroid plexus carcinomas (CPC) are rare entities. They are rapidly growing CNS neoplasms, originating from the epithelium of the choroid plexus and are classified as WHO grade III tumors. They occur predominantly in children, typically younger than 5 years old, most of them (70%) are affected before the age of 2 years with male predisposition . Choroid plexus carcinoma typically occurs in the ventricular system with the lateral (50%) and the 4th ventricle (40%) being the most common locations. Extraventricular manifestation of CPC has been reported in the literature . Clinically CPC presents with unspecific symptoms, such as nausea, vomiting, headache, obtundation as well as focal neurologic deficits. On MR imaging CPC present on T1-weighted and T2-weighted images as heterogeneous isointense to hypointense intraventricular mass lesions with lobulated or irregularly marginated, papillary appearance. In addition, degenerative changes with cystic transformation, necrosis, and hemorrhage can regularly be found. On diffusion-weighted images CPC can show restricted diffusion, a finding correlated with a poor prognosis . Because of its relation to the ventricular system, CPC was considered a likely diagnosis in the present case. Primary Central Nervous System Neuroblastoma (PCNSN) Primary central nervous system neuroblastoma (PCNSN) is a newly defined intracranial embryonal WHO grade IV tumor. These rare tumors have a primitive neuronal architecture and occasional neurocytic differentiation. They mainly affect children under 5 years of age with a female prevalence. The PCNSNs are in most cases located supratentorially and present with a wide spectrum of symptoms due to their variety of possible locations and sizes. Infants may present with few or no signs or symptoms, even when the tumor has reached a considerable size. This is most likely due to compensatory and adaption mechanisms of the surrounding brain structures . In most cases, PCNSN appear as heterogeneous mass lesions with solid and cystic components. The solid tumor parts appear hypointense to isointense on T1-weighted images, and hyperintense on T2-weighted images. Perifocal edema is inconsistent, whereas vascular structures crossing the tumor are not uncommon. PCNSN show markedly heterogeneous enhancement of gadolinium. On diffusion-weighted images the tumor often shows restricted diffusion, whereas the relative cerebral blood volume is reportedly elevated . Although PCNSN are rare entities, we included this tumor in our differential diagnoses. Pilocytic astrocytoma (PA) is the most common primary brain tumor of childhood accounting for 15% of all pediatric brain tumors with a peak incidence between 5 and 15 years. Pilocytic astrocytomas are low grade World Health Organization (WHO) type I tumors showing a strong association with neurofibromatosis type 1 (NF1) without any gender predisposition . Pilocytic astrocytomas typically arise from midline structures. The most common location in non-NF1 patients is the cerebellum in 60%, whereas PA of the optic pathway are particularly common in NF1 patients. Less common locations include the hypothalamus, brainstem and cerebral hemispheres, cerebral ventricles, velum interpositum and the spinal cord . Pilocytic astrocytoma have a wide range of imaging appearances. On MR imaging four predominant imaging patterns have been described: (a) mass with a nonenhancing cyst and an intensely enhancing mural nodule (21% of cases), (b) mass with an enhancing cyst wall and an intensely enhancing mural nodule (46%), (c) necrotic mass with a central nonenhancing zone (16%), and (d) predominantly solid mass with minimal to no cyst-like component (17%). Calcifications are present in 20% of cases . Given the localization and the extent of the lesion pilocytic astrocytoma was considered a less likely differential diagnosis. Ependymoma is the number three of the most common intracranial neoplasm in children. They arise from ependymal cells of the central nervous system (CNS) and can occur anywhere within the CNS . The incidence is bimodal with one peak between 1–5 years and a smaller peak during the 2–3 decade of life with a male predisposition. Most common symptoms are seizures, focal motor or sensory deficits and headache, but also when large, signs of raised intracranial pressure and obstructive hydrocephalus especially in infratentorial location may occur. Supratentorial location accounts for only one third of ependymomas. They typically arise from the ependymal lining of the ventricular system or fetal ependymal rests. The majority of supratentorial ependymomas are located within the white matter or cortex of the frontal lobe. Ventricular localization is less common. In the WHO classification from 2021, supratentorial ependymomas are classified based on molecular features into two subgroups. RELA fusion-positive supratentorial ependymomas seem to affect older children and have a poor prognosis, whereas supratentorial ependymomas displaying YAP‑1 fusions affect children below 3 years of age . At MRI ependymomas are isointense to slightly hypointense on T1-weighted images and isointense to hyperintense on T2 weighted images. Heterogeneous enhancement is seen on contrast-enhanced images. Restricted diffusion is regularly present, most likely reflecting high cellularity . On CT images, the soft tissue component is commonly hypoattenuating to isoattenuating. Ependymomas frequently demonstrate cystic components, and areas of small chunky calcification. Occasionally intratumoral hemorrhage may be present . Because of ependymal location of the lesion, as well as the diffusion restriction, supratentorial anaplastic ependymoma was considered a valid differential diagnosis in our case. Atypical teratoid/rhabdoid tumors (AT/RT) are rare, WHO grade IV tumors of the CNS. In > 80% of cases children less than 3 years of age are affected. There is no gender predisposition. They are among the fastest growing CNS tumors with a short interval to symptom onset. By the time of diagnosis, they often reach sizes > 3 cm. Most common symptoms are rapidly increasing lethargy, vomiting and increased head circumference. Smaller supratentorial lesions may cause seizures . The AT/RT may occur supratentorially or infratentorially. Infratentorially they appear off-midline and may resemble medulloblastomas, 12% of AT/RT are found bihemispherically and 15–20% are disseminated at the time of initial diagnosis. Histologically, AT/RTs are embryonal tumors that contain a rhabdoid component as well as areas with primitive neuroectodermal, mesenchymal, and epithelial features. The current WHO classification of 2021 places AT/RT under embryonal tumors. This group of tumors includes medulloblastoma, atypical teratoid/rhabdoid tumor, embryonal tumor with multilayered rosettes, C19MC-altered and embryonal tumor with multilayered rosettes, pineoblastoma, pituitary blastoma, CNS neuroblastoma, and ganglioneuroblastoma . Diagnosis of AT/RT requires confirmation of specific genetic aberration, such as loss of INI1 tumor suppressor gene on chromosome 22 or BRG1 gene . The use of CT imaging often shows a hyperattenuated mass lesion commonly containing cystic structures and/or hemorrhage. Some tumors contain fine calcifications and signs of obstructive hydrocephalus may be present . On T2-weighted images, AT/RTs appear heterogeneous with hyperintense cystic foci, which are hyperintense on T1-weighted images. In addition, restricted diffusion may be present in solid parts of the tumor. Contrast enhancement on T1 weighted images is mostly heterogeneous and can be nodular . Considering the patient’s age and the imaging findings, AT/RT seemed to be a likely differential diagnosis. Choroid plexus carcinomas (CPC) are rare entities. They are rapidly growing CNS neoplasms, originating from the epithelium of the choroid plexus and are classified as WHO grade III tumors. They occur predominantly in children, typically younger than 5 years old, most of them (70%) are affected before the age of 2 years with male predisposition . Choroid plexus carcinoma typically occurs in the ventricular system with the lateral (50%) and the 4th ventricle (40%) being the most common locations. Extraventricular manifestation of CPC has been reported in the literature . Clinically CPC presents with unspecific symptoms, such as nausea, vomiting, headache, obtundation as well as focal neurologic deficits. On MR imaging CPC present on T1-weighted and T2-weighted images as heterogeneous isointense to hypointense intraventricular mass lesions with lobulated or irregularly marginated, papillary appearance. In addition, degenerative changes with cystic transformation, necrosis, and hemorrhage can regularly be found. On diffusion-weighted images CPC can show restricted diffusion, a finding correlated with a poor prognosis . Because of its relation to the ventricular system, CPC was considered a likely diagnosis in the present case. Primary central nervous system neuroblastoma (PCNSN) is a newly defined intracranial embryonal WHO grade IV tumor. These rare tumors have a primitive neuronal architecture and occasional neurocytic differentiation. They mainly affect children under 5 years of age with a female prevalence. The PCNSNs are in most cases located supratentorially and present with a wide spectrum of symptoms due to their variety of possible locations and sizes. Infants may present with few or no signs or symptoms, even when the tumor has reached a considerable size. This is most likely due to compensatory and adaption mechanisms of the surrounding brain structures . In most cases, PCNSN appear as heterogeneous mass lesions with solid and cystic components. The solid tumor parts appear hypointense to isointense on T1-weighted images, and hyperintense on T2-weighted images. Perifocal edema is inconsistent, whereas vascular structures crossing the tumor are not uncommon. PCNSN show markedly heterogeneous enhancement of gadolinium. On diffusion-weighted images the tumor often shows restricted diffusion, whereas the relative cerebral blood volume is reportedly elevated . Although PCNSN are rare entities, we included this tumor in our differential diagnoses. The initial hematoxylin and eosin (H&E) stained section of the formaldehyde-fixed and paraffin-embedded intraoperative biopsy material revealed a dense and pleomorphic neoplasm with small to maximally medium sized, mainly round nuclei. Regionally, tumor cells clustered in groups surrounded by a neuropil-like matrix (Fig. a). Brisk mitotic activity and apoptotic cells (Fig. b) suggested a malignant entity. Complementing biopsy material further enriched the palette of morphological features of this tumor, ranging from densely packed areas characterized by small, round, blue cells with sparse cytoplasm and an occasional palisading pattern, to regions with moderate cell density and a fibrillar matrix (Fig. c). In the latter regions, sporadic small calcifications (Fig. c, arrowheads) attributed to an oligoid impression. Only relatively small fractions within the tumor tissue were necrotic, infrequently collocated with larger, granular calcifications (Fig. d). Tumor growth appeared to partially follow meningeal structures and diffusely infiltrate the surrounding parenchyma. Immunohistochemistry with antibodies against the synaptic vesicle protein synaptophysin (Fig. a) and against the neural antigen MAP2 (Fig. b) showed strong positive reactions in tumor areas of both high and medium cell density. While the oligodendrocyte transcription factor OLIG2 was expressed in a subset of tumor cells, the glial fibrillary acid protein (GFAP) was limited to relatively few cells showing only local moderate astrogliosis (Fig. c). The tumor cells showed a negative reaction for vimentin and CD99, a marker typical for Ewing’s sarcoma. The nuclear expressions of the transcriptional regulator ATP-dependent X‑linked helicase (ATRX) and the tumor suppressor gene integrase interactor 1 (INI) were maintained in the tumor cells. Immunohistochemistry with a mutation-specific (R132H) antibody against isocitrate dehydrogenase 1 (IDH1) and mutation-specific (G34V, G34R, K27M) antibodies against histone 3‑point mutations revealed no specific reaction in the tumor cells. The expression of CD34 was limited to vascular endothelial cells. Staining against epithelial membrane antigen (EMA) did not reveal dot-like or ring-like structures in any tumor cells. Along with numerous hyperchromatic nuclei, mitotic figures were a frequent characteristic in small round cells and the proliferation index was increased. In Ki67/MIB1 immunohistochemical staining, a total of approximately 20%, locally—mostly in dense areas—even up to 40–50% of tumor cells were stained (Fig. d). In summary, the histomorphological appearance of a poorly differentiated tumor with high proliferation rate and immunohistochemical characterization of neuroectodermal origin categorize this case as an embryonal tumor of the CNS. The co-existence of dense areas of small, round, blue embryonal cells and areas of pleomorphic, neurocytic cells in a neuropil-rich stroma leads to the diagnosis of a central nervous system (CNS) neuroblastoma (WHO grade IV). Key features of this entity visible in this case include co-expression of synaptophysin and OLIG2 and necrotic regions with granular calcifications . In addition, a molecular analysis of the biopsy tissue assessing the tumor cell DNA methylation profile was performed. The identified copy number variation profile revealed increases on chromosomes 1q, 3q and 17q as well as losses on chromosomes 3p and 6q. To discover common patterns with a library of established tumor profiles, the results of this methylation analysis were assessed using the Heidelberg molecular neuropathology classifier version v11b4 . This classifier showed the highest accordance with the methylation class of CNS neuroblastomas with forkhead box R2 (FOXR2) activation (calibrated score 0.98). Assessment by the brain tumor reference center in Bonn, Germany, independently confirmed the diagnosis. Initial differential diagnostic considerations of the tumor at hand comprised other CNS embryonal tumors, like CNS ganglioneuroblastoma, medulloepithelioma, and ependymoblastoma (all WHO grade IV), and mixed neuronal-glial tumors like the diffuse leptomeningeal glioneural tumor . A malignant glioma was also originally considered, but overruled by the relative sparsity of glial marker expression. In comparison to mixed neuronal-glial tumors, the high proliferation rate, abundance of primitive cells and preponderance of neural markers favored a CNS embryonal tumor. Among embryonal tumors, the above described histological key features in combination with the absence of ganglion cells (ganglioneuroblastoma), pseudostratified neuroepithelial structures (medulloepithelioma) and prominent rosettes (ependymoblastoma) led to the diagnosis of a CNS neuroblastoma (WHO grade IV). Central Nervous System (CNS) Neuroblastoma (WHO-grade IV) Historically, this former group of so-called primitive neuroectodermal tumors (PNETs) of the CNS originated from an concept analogous to the related and more frequent medulloblastomas the PNET of the cerebellum . They were first subdivided into four distinct groups in the 2007 WHO classification of CNS tumors, based on histomorphological criteria . In up to 61% of cases, these histologically classified CNS-PNETs could be reclassified to established reference CNS tumors (e.g. high-grade gliomas), based on their molecular profiles . Sturm et al. assessed the remaining tumors with a genuinely different genetic signature, clustering them into four new entities. Among these, CNS neuroblastoma with FOXR2 activation shares the methylation profile and histological features of the tumor described here. Recently, the cIMPACT-NOW consortium suggested that this new entity be integrated in the next WHO classification of CNS tumors . A CNS neuroblastoma is a rare entity. Several small case series suggest a mean age of 5–9 years and a female:male ratio of approximately 2:1 . Detailed epidemiological information is limited due to the low frequency and relatively new and evolving diagnostic criteria of CNS neuroblastomas, specifically with respect to associated mutations. Besides the frequent FOXR2 activation, singular cases may also be characterized by focal MYC amplification . In comparison to other CNS embryonal tumors, a slightly less unfavorable prognosis has been proposed for this entity . Yet the prognostic value of either of these alterations (FOXR2, MYC) in CNS neuroblastomas remains to be determined . Overall, the diagnosis of these heterogeneous and poorly differentiated CNS embryonal tumors remains challenging based on histomorphological features alone and profited from recent advances in molecular pathological assessment. Historically, this former group of so-called primitive neuroectodermal tumors (PNETs) of the CNS originated from an concept analogous to the related and more frequent medulloblastomas the PNET of the cerebellum . They were first subdivided into four distinct groups in the 2007 WHO classification of CNS tumors, based on histomorphological criteria . In up to 61% of cases, these histologically classified CNS-PNETs could be reclassified to established reference CNS tumors (e.g. high-grade gliomas), based on their molecular profiles . Sturm et al. assessed the remaining tumors with a genuinely different genetic signature, clustering them into four new entities. Among these, CNS neuroblastoma with FOXR2 activation shares the methylation profile and histological features of the tumor described here. Recently, the cIMPACT-NOW consortium suggested that this new entity be integrated in the next WHO classification of CNS tumors . A CNS neuroblastoma is a rare entity. Several small case series suggest a mean age of 5–9 years and a female:male ratio of approximately 2:1 . Detailed epidemiological information is limited due to the low frequency and relatively new and evolving diagnostic criteria of CNS neuroblastomas, specifically with respect to associated mutations. Besides the frequent FOXR2 activation, singular cases may also be characterized by focal MYC amplification . In comparison to other CNS embryonal tumors, a slightly less unfavorable prognosis has been proposed for this entity . Yet the prognostic value of either of these alterations (FOXR2, MYC) in CNS neuroblastomas remains to be determined . Overall, the diagnosis of these heterogeneous and poorly differentiated CNS embryonal tumors remains challenging based on histomorphological features alone and profited from recent advances in molecular pathological assessment. |
Neurophysiological signatures of cortical micro-architecture | 6920337d-b22e-4f37-8b85-c4e5625e846b | 10522715 | Physiology[mh] | Signals, in the form of electrical impulses, are perpetually generated, propagated, and integrated via multiple types of neurons and neuronal populations , . The wiring of the brain guides the propagation of signals through networks of nested polyfunctional neural circuits , . The resulting fluctuations in membrane potentials and firing rates ultimately manifest as patterned neurophysiological activity – . A rich literature demonstrates links between cortical micro-architecture and dynamics. Numerous studies have investigated the cellular and laminar origins of cortical rhythms – . For instance, electro- and magneto-encephalography (EEG/MEG) signals appear to be more sensitive to dipoles originating from pyramidal cells of cortical layers II-III and V , . Moreover, specific time-series features of neuronal electrophysiology depend on neuron type, morphology and local gene transcription, particularly genes associated with ion channel regulation – . However, previous studies have mostly focused on single or small sets of features-of-interest, often mapping single micro-architectural features to single dynamical features. Starting with the discovery of 8–12 Hz alpha rhythm in the electroencephalogram , conventional time-series analysis in neuroscience has typically focused on canonical electrophysiological rhythms – . More recently, there has also been a growing interest in studying the intrinsic timescales that display a hierarchy of temporal processing from fast fluctuating activity in unimodal cortex to slower encoding of contextual information in transmodal cortex – . How ongoing neurophysiological dynamics arise from specific features of neural circuit micro-architecture remains a key question in neuroscience , , . Recent analytic advances have opened new opportunities to perform neurophysiological time-series phenotyping by computing comprehensive feature sets that go beyond power spectral measures, including measures of signal amplitude distribution, entropy, fractal scaling and autocorrelation – . Concomitant advances in imaging technologies and data sharing offer new ways to measure brain structure with unprecedented detail and depth – , including gene expression , myelination , , neurotransmitter receptors – , cytoarchitecture – , laminar differentiation , , cell type composition , , , metabolism , and evolutionary expansion , . Here we comprehensively characterize the dynamical signature of neurophysiological activity and relate it to the underlying micro-architecture by integrating multiple, multimodal maps of human cortex. Instead of manually selecting a small number of features-of-interest, we use extensive sets of dynamical and micro-architectural features using data-driven approaches. Specifically, we first derive cortical spontaneous neurophysiological activity using source-resolved magnetoencephalography (MEG) from the Human Connectome Project (HCP; see ref. ). We then apply highly comparative time-series analysis (hctsa; see refs. , ) to estimate a comprehensive set of time-series features for each brain region (Fig. ). At the same time, we construct a micro-architectural atlas of the cortex that includes maps of microstructure, metabolism, neurotransmitter receptors and transporters, laminar differentiation and cell types (Fig. ). Finally, we map these extensive micro-architectural and dynamical atlases to one another using multivariate statistical analysis. Regional neurophysiological time-series were estimated by applying linearly constrained minimum variance (LCMV) beamforming to resting state MEG data from the Human Connectome Project (HCP; see ref. ) using Brainstorm software (see Methods for details). Highly comparative time-series analysis (hctsa; see refs. , ) was then used to perform massive time-series feature extraction from regional MEG recordings. This procedure provides a feature-based representation of time-series, where given time-series are represented by time-series feature vectors , . This time-series phenotyping analysis is a data-driven method that quantifies dynamic repertoire of neural activity using interdisciplinary metrics of temporal structure of the signal and yields a comprehensive ‘fingerprint’ of dynamical properties of each brain region. Applying time-series phenotyping to regional MEG time-series, we estimated 6880 time-series features for 100 cortical regions from the Schaefer-100 atlas . The hctsa library contains a vast and interdisciplinary set of features with potentially correlated values that span various conceptual time-series characteristics. The list of time-series features includes, but is not limited to, statistics derived from the autocorrelation function, power spectrum, amplitude distribution, and entropy estimates (Fig. . To estimate a comprehensive set of multimodal micro-architectural features, we used the recently-developed neuromaps toolbox as well as the BigBrainWarp toolbox , the Allen Human Brain Atlas (AHBA ) and the abagen toolbox to transform and compile a set of 45 features, including measures of microstructure, metabolism, cortical expansion, receptors and transporters, layer thickness and cell type-specific gene expression (Fig. . Note that the microstructure maps include principal gradients of gene expression and neurotransmitter profiles as they each represent proxy measures of certain molecular properties. Specifically, the principal component of gene expression (gene expression PC1) provides a potential proxy for cell type distribution across the cortex , , and the principal component of neurotransmitter receptors and transporters (neurotransmitter PC1) provides a summary measure of protein densities of 18 neurotransmitter receptors and transporters , . We also included individual neurotransmitter receptor and transporter maps as well as cell type-specific gene expression maps to assess their effects separately. In subsequent analyses, we first assess the topographic organization of neurophysiological dynamics by quantifying the dominant patterns of variations in resting-state MEG time-series properties. We then characterize the signature of neurophysiological dynamics with respect to micro-architectural attributes across the cortex. Finally, we perform sensitivity analyses to investigate potential effects of confounding factors on the findings, such as signal-to-noise ratio and parcellation resolution (see Sensitivity analysis for details). Topographic distribution of neurophysiological dynamics The hctsa time-series phenotyping procedure generated 6880 time-series features per brain region. Since hctsa contains multiple algorithmic variants for quantifying any given time-series property, the identified time-series features potentially capture related dynamical behaviour and include groups of correlated properties. Hence, we first sought to identify dominant macroscopic patterns or gradients of neurophysiological dynamics using principal component analysis (PCA) . Applying PCA to the group-average region × feature matrix, we find evidence of a single dominant component that captures 48.7% of the variance in regional time-series features (Fig. a). The dominant component or “gradient” of neurophysiological dynamics (PC1) mainly spans the posterior parietal cortex and sensory-motor cortices on one end and the anterior temporal, orbitofrontal and ventromedial cortices on the other end (Fig. a). Focusing on intrinsic functional networks, we find that the topographic organization of the dominant neurophysiological dynamics varies along a sensory–fugal axis from dorsal attention, somatomotor and visual networks to limbic and default mode networks (Fig. a). We next investigated the top-loading time-series features on the first component, using the univariate correlations between each of the original feature maps and the PC1 map (i.e., PCA loadings). All correlations were statistically assessed using spatial autocorrelation-preserving null models ("spin tests” , ; see Methods for details). Figure b shows that numerous features are positively and negatively correlated with PC1; the full list of features, their correlation coefficients and p -values are available in the online Supplementary Dataset . Inspection of the top loading features reveals that the majority are statistics derived from the structure of the power spectrum or closely related measures. Examples include power in different frequency bands, parameters of various model fits to the power spectrum, and related measures, such as the shape of the autocorrelation function and measures of fluctuation analysis. Figure b shows how the power spectrum varies across the cortex, with each line representing a brain region. Regions are coloured by their position in the putative unimodal–transmodal hierarchy ; the variation visually suggests that unimodal regions display more prominent alpha (8–12 Hz) and beta (15–29 Hz) power peaks. Collectively, these results demonstrate that the traditional focus of electrophysiological time-series analysis on statistics of the power spectrum is consistent with the dominant variations in MEG dynamics captured by the diverse library of hctsa time-series features. Given that the topographic organization of PC1 was closely related to power spectral features, we directly tested the link between PC1 and conventional band-limited power spectral measures – , as well as intrinsic timescale (Supplementary Figure . Figure c shows the correlations between PC1 and delta (2–4 Hz), theta (5–7 Hz), alpha (8–12 Hz), beta (15–29 Hz), lo-gamma (30–59 Hz) and hi-gamma (60–90 Hz) power maps, and intrinsic neural timescale , – , . We find that PC1 is significantly correlated with intrinsic timescale ( r s = 0.84, p spin = 0.038; FDR-corrected) and hi-gamma ( r s = 0.87, p spin = 0.006; FDR-corrected). The results were consistent when we used band-limited power maps that were adjusted for the aperiodic component of the power spectrum as opposed to the total power (Supplementary Fig. . The fact that PC1 correlates with intrinsic timescale is consistent with the notion that both capture broad variations in the power spectrum. Given that the intrinsic timescale reflects characteristics of the aperiodic component of the power spectrum (these measures are mathematically related; see Methods for details), we also directly assessed the association between PC1 and the exponent and offset of the aperiodic component. The exponent describes the “curve” or the overall “line” or the slope of the aperiodic component and the offset describes the overall vertical shift (up and down translation) of the whole power spectrum . PC1 was significantly correlated with both measures, suggesting that time-series features captured by PC1 also reflect properties of the aperiodic component of the power spectrum (Supplementary Fig. . In addition, previous reports suggest that broadband gamma activity also partly reflects the aperiodic neurophysiological activity and broadband shifts in the power spectrum – . This is consistent with our findings that PC1 is associated with gamma power and intrinsic timescale, mainly capturing broad variations in power spectrum and characteristics of the aperiodic activity. Note that we focused on PC1 because the other components (PC2 and above) accounted for 10% or less of the variance in time-series features and were not significantly associated with hctsa time-series features. Moreover, to verify that the apparent low-dimensionality of the data and the identified PC patterns were not driven by the smaller number of samples (i.e., brain regions) than features (i.e., time-series features), we performed a sensitivity analysis where we randomly selected 100 time-series features (from the original list of 6 880 features) and re-ran PCA (1 000 repetitions). The identified PC patterns and their corresponding amount of variance explained were consistent with the original analysis using the full set of time-series features (Supplementary Fig. . Neurophysiological signatures of micro-architecture How do the regional neurophysiological time-series features map onto multimodal micro-architectural features? To address this question, we implemented a multivariate partial least squares analysis (PLS; see refs. , ) that integrates multiple multimodal brain maps into the analysis and seeks to identify linear combinations of time-series features and linear combinations of micro-architectural features that optimally covary with one another. Figure a shows that the analysis identifies multiple such combinations, termed latent variables (similar results were obtained using sparse canonical correlation analysis (sCCA); Supplementary Fig. . Statistical significance of each latent variable was assessed using spatial autocorrelation-preserving permutation tests , . The first latent variable was statistically significant, capturing the greatest covariance between time-series and micro-architectural features (covariance explained = 75.4%, p spin = 0.011). Figure b shows the spatial topography of time-series features and micro-architectural scores for the first latent variable. These are the weighted sums of the original input features according to the weighting identified by the latent variable. The correlation between the score maps is maximized by the analysis ( r s = 0.73, p spin = 0.0059). We therefore sought to estimate whether the same mapping between time-series and micro-architectural features can be observed out-of-sample. We adopted a distance-dependent cross-validation procedure where “seed” regions were randomly chosen and the 75% most physically proximal regions were selected as the training set, while the remaining 25% most physically distal regions were selected as the test set (see Methods for more details). For each train-test split, we fit a PLS model to the train set and project the test set onto the weights (i.e., singular vectors) derived from the train set. The resulting test set scores are then correlated to estimate an out-of-sample correlation coefficient. Figure b shows that micro-architecture and time-series feature scores are correlated in training set (mean r s = 0.75) and test set (mean r s = 0.5), demonstrating consistent findings in out-of-sample analysis. We next examined the corresponding time-series and micro-architecture feature loadings and identified the most contributing features to the spatial patterns captured by the first latent variable (Fig. c, d). The top loading time-series features were mainly related to measures of self-correlation or predictability of the MEG signal. The self-correlation measures mostly reflect the linear correlation structure of neurophysiological time-series, particularly long-lag autocorrelations (at lags > 15 time steps, or > 30 ms). A wide range of other highly weighted time-series features captured other aspects of signal predictability, including measures of the shape of the autocorrelation function (e.g., the time lag at which the autocorrelation function crosses zero), how the autocorrelation structure changes after removing low-order local trends (e.g., residuals from fitting linear models to rolling 5-time-step, or 10 ms, windows), scaling properties assessed using fluctuation analysis (e.g., scaling of signal variance across timescales), and measures derived from a wavelet decomposition (e.g., wavelet coefficients at different timescales). The full list of time-series feature loadings for the first latent variable is available in the online Supplementary Dataset . To illustrate the spatial distribution of highly contributing time-series features, Fig. c shows three top-loading features that mirror the spatial variation of the first PLS latent variable. For example, Figure c, left depicts the distribution of the group-average first zero-crossing point of the autocorrelation function. The autocorrelation function of the unimodal cortex (marked with a pink circle) crosses zero autocorrelation at a lower lag than the transmodal cortex (marked with a purple circle), suggesting faster autocorrelation decay and longer correlation length in transmodal cortex than in unimodal cortex. Another example is the linear autocorrelation of the MEG signal at longer time lags. Figure c, left shows autocorrelation at a lag of 48 ms (24 time steps), demonstrating lower autocorrelation in unimodal cortex and higher autocorrelation in transmodal cortex. Note that the list of top-loading features includes linear autocorrelation at other time lags and autocorrelation at a lag of 48 ms was only selected as an illustrative example (Supplementary Fig. depicts the full range of loadings for linear autocorrelation at all time lags included in hctsa). Finally, we examined the scaling exponent, α , estimated using detrended fluctuation analysis as the slope of a linear fit to the log-log plot of the fluctuations of the detrended signal across timescales , . Figure c, right depicts this scaling exponent across the cortex, which exhibits a similar spatial pattern as the previous two examples, indicating lower self-correlation in unimodal cortex (pink circle) compared to transmodal cortex (purple circle). Other variations of fluctuation analysis also featured heavily in the list of top-loading features, including goodness of fit of the linear fit, fitting of multiple scaling regimes, and different types of detrending and mathematical formulation of fluctuation size. Figure d shows the corresponding micro-architectural loadings. The most contributing micro-architectural features to the spatial patterns captured by the first latent variable are the principal component of gene expression (gene expression PC1; a potential proxy for cell type distribution , , ), T1w/T2w ratio (a proxy for intracortical myelin ), principal component of neurotransmitter receptors and transporters (neurotransmitter PC1), and oxygen and glucose metabolism (strong positive loadings). We also find high contributions (strong negative loadings) from specific neurotransmitter receptor and transporters, in particular metabotropic serotonergic and dopaminergic receptors, as well as from cell type-specific gene expression of oligodendrocyte precursors (opc), which are involved in myelinogenesis – . Consistent findings were obtained when we used univariate analysis to relate regional time-series features and the top loading micro-architectural maps, in particular principal component of gene expression and T1w/T2w ratio, which have previously been extensively studied as archetypical micro-architectural gradients , , , , (Supplementary Fig. . Altogether, this analysis provides a comprehensive chart or ‘lookup table’ of how micro-architectural and time-series feature maps are associated with one another. These results demonstrate that cortical variation in multiple micro-architectural attributes manifests as a gradient of time-series properties of neurophysiological activity, particularly the properties that reflect the long-range self-correlation structure of the signal. Sensitivity analysis To assess the extent to which the results are affected by potential confounding factors and methodological choices, we repeated the analyses using alternative approaches. First, to ensure that the findings are not influenced by MEG signal-to-noise ratio (SNR), we calculated SNR at each source location using a noise model that estimates how sensitive the source-level MEG signal is to source location and orientation , . We performed two follow-up analyses using the SNR map (Supplementary Fig. : (1) SNR was first compared with the full set of MEG time-series features using mass univariate Pearson correlations. Time-series features that were significantly correlated with SNR were removed from the feature set without correcting for multiple comparisons ( p spin < 0.05; 10,000 spatial autocorrelation-preserving permutation tests , ). Note that this is a more conservative feature selection procedure compared to conventional multiple comparisons correction, because fewer features would be removed if correction for multiple comparisons was applied. PCA was applied to the remaining set of features (Supplementary Figure . The principal component of the retained 3819 features (i.e., PC1 - feature subset) explained 31.6% of the variance and was significantly correlated with the original PC1 of the full set of features ( r s = 0.93, p spin = 0.0001), reflecting similar spatial pattern as the original analysis. (2) SNR was regressed out from the full set of time-series features using linear regression analysis. PCA was then applied to the resulting feature residuals (Supplementary Fig. . The principal component of SNR-regressed features (i.e., PC1 - SNR regressed) explained 41.4% of the variance and reflected the same spatial pattern as the original analysis ( r s = 0.70, p spin = 0.0004). Moreover, we assessed the effects of environmental and instrumental noise on the findings, where we applied principal component analysis to the hctsa features obtained from pre-processed empty-room MEG recordings (see Methods for more details). PCA weights of the time-series features of the empty-room MEG recordings were aligned with the PCA weights of the time-series features of the resting-state MEG recordings using the Procrustes method (see ref. ; https://github.com/satra/mapalign ). The principal component of neurophysiological dynamics was then compared with the principal component of time-series features obtained from empty-room recordings, where no significant associations were identified (Supplementary Fig. ; r s = − 0.17, p spin = 0.69). These analyses demonstrate that the time-series features captured by the dominant axis of variation in neurophysiological dynamics are independent from measures of MEG signal-to-noise ratio. Finally, to ensure that the findings are independent from the parcellation resolution, we repeated the analyses using a higher resolution parcellation (Schaefer-400 atlas with 400 cortical regions ). The results were consistent with the original analysis (Supplementary Figs. , . In particular, the first principal component (PC1) accounted for 48.6% of the variance and displayed a similar spatial organization as the one originally obtained for the Schaefer-100 atlas (Supplementary Fig. . As before, the top loading time-series features were mainly related to the characteristics of the power spectral density (Supplementary Fig. . The full list of features, their loadings and p -values are available in the online Supplementary Dataset . Moreover, PLS analysis identified a single significant latent variable ( p spin = 0.0083) that accounted for 75.7% of the covariance (Supplementary Fig. . Micro-architecture and time-series feature scores displayed similar spatial patterns to the ones obtained for the Schaefer-100 atlas (Supplementary Fig. . The corresponding feature loadings were also consistent with the original findings (micro-architectural loadings in Supplementary Fig. and time-series feature loadings in the online Supplementary Dataset .) The hctsa time-series phenotyping procedure generated 6880 time-series features per brain region. Since hctsa contains multiple algorithmic variants for quantifying any given time-series property, the identified time-series features potentially capture related dynamical behaviour and include groups of correlated properties. Hence, we first sought to identify dominant macroscopic patterns or gradients of neurophysiological dynamics using principal component analysis (PCA) . Applying PCA to the group-average region × feature matrix, we find evidence of a single dominant component that captures 48.7% of the variance in regional time-series features (Fig. a). The dominant component or “gradient” of neurophysiological dynamics (PC1) mainly spans the posterior parietal cortex and sensory-motor cortices on one end and the anterior temporal, orbitofrontal and ventromedial cortices on the other end (Fig. a). Focusing on intrinsic functional networks, we find that the topographic organization of the dominant neurophysiological dynamics varies along a sensory–fugal axis from dorsal attention, somatomotor and visual networks to limbic and default mode networks (Fig. a). We next investigated the top-loading time-series features on the first component, using the univariate correlations between each of the original feature maps and the PC1 map (i.e., PCA loadings). All correlations were statistically assessed using spatial autocorrelation-preserving null models ("spin tests” , ; see Methods for details). Figure b shows that numerous features are positively and negatively correlated with PC1; the full list of features, their correlation coefficients and p -values are available in the online Supplementary Dataset . Inspection of the top loading features reveals that the majority are statistics derived from the structure of the power spectrum or closely related measures. Examples include power in different frequency bands, parameters of various model fits to the power spectrum, and related measures, such as the shape of the autocorrelation function and measures of fluctuation analysis. Figure b shows how the power spectrum varies across the cortex, with each line representing a brain region. Regions are coloured by their position in the putative unimodal–transmodal hierarchy ; the variation visually suggests that unimodal regions display more prominent alpha (8–12 Hz) and beta (15–29 Hz) power peaks. Collectively, these results demonstrate that the traditional focus of electrophysiological time-series analysis on statistics of the power spectrum is consistent with the dominant variations in MEG dynamics captured by the diverse library of hctsa time-series features. Given that the topographic organization of PC1 was closely related to power spectral features, we directly tested the link between PC1 and conventional band-limited power spectral measures – , as well as intrinsic timescale (Supplementary Figure . Figure c shows the correlations between PC1 and delta (2–4 Hz), theta (5–7 Hz), alpha (8–12 Hz), beta (15–29 Hz), lo-gamma (30–59 Hz) and hi-gamma (60–90 Hz) power maps, and intrinsic neural timescale , – , . We find that PC1 is significantly correlated with intrinsic timescale ( r s = 0.84, p spin = 0.038; FDR-corrected) and hi-gamma ( r s = 0.87, p spin = 0.006; FDR-corrected). The results were consistent when we used band-limited power maps that were adjusted for the aperiodic component of the power spectrum as opposed to the total power (Supplementary Fig. . The fact that PC1 correlates with intrinsic timescale is consistent with the notion that both capture broad variations in the power spectrum. Given that the intrinsic timescale reflects characteristics of the aperiodic component of the power spectrum (these measures are mathematically related; see Methods for details), we also directly assessed the association between PC1 and the exponent and offset of the aperiodic component. The exponent describes the “curve” or the overall “line” or the slope of the aperiodic component and the offset describes the overall vertical shift (up and down translation) of the whole power spectrum . PC1 was significantly correlated with both measures, suggesting that time-series features captured by PC1 also reflect properties of the aperiodic component of the power spectrum (Supplementary Fig. . In addition, previous reports suggest that broadband gamma activity also partly reflects the aperiodic neurophysiological activity and broadband shifts in the power spectrum – . This is consistent with our findings that PC1 is associated with gamma power and intrinsic timescale, mainly capturing broad variations in power spectrum and characteristics of the aperiodic activity. Note that we focused on PC1 because the other components (PC2 and above) accounted for 10% or less of the variance in time-series features and were not significantly associated with hctsa time-series features. Moreover, to verify that the apparent low-dimensionality of the data and the identified PC patterns were not driven by the smaller number of samples (i.e., brain regions) than features (i.e., time-series features), we performed a sensitivity analysis where we randomly selected 100 time-series features (from the original list of 6 880 features) and re-ran PCA (1 000 repetitions). The identified PC patterns and their corresponding amount of variance explained were consistent with the original analysis using the full set of time-series features (Supplementary Fig. . How do the regional neurophysiological time-series features map onto multimodal micro-architectural features? To address this question, we implemented a multivariate partial least squares analysis (PLS; see refs. , ) that integrates multiple multimodal brain maps into the analysis and seeks to identify linear combinations of time-series features and linear combinations of micro-architectural features that optimally covary with one another. Figure a shows that the analysis identifies multiple such combinations, termed latent variables (similar results were obtained using sparse canonical correlation analysis (sCCA); Supplementary Fig. . Statistical significance of each latent variable was assessed using spatial autocorrelation-preserving permutation tests , . The first latent variable was statistically significant, capturing the greatest covariance between time-series and micro-architectural features (covariance explained = 75.4%, p spin = 0.011). Figure b shows the spatial topography of time-series features and micro-architectural scores for the first latent variable. These are the weighted sums of the original input features according to the weighting identified by the latent variable. The correlation between the score maps is maximized by the analysis ( r s = 0.73, p spin = 0.0059). We therefore sought to estimate whether the same mapping between time-series and micro-architectural features can be observed out-of-sample. We adopted a distance-dependent cross-validation procedure where “seed” regions were randomly chosen and the 75% most physically proximal regions were selected as the training set, while the remaining 25% most physically distal regions were selected as the test set (see Methods for more details). For each train-test split, we fit a PLS model to the train set and project the test set onto the weights (i.e., singular vectors) derived from the train set. The resulting test set scores are then correlated to estimate an out-of-sample correlation coefficient. Figure b shows that micro-architecture and time-series feature scores are correlated in training set (mean r s = 0.75) and test set (mean r s = 0.5), demonstrating consistent findings in out-of-sample analysis. We next examined the corresponding time-series and micro-architecture feature loadings and identified the most contributing features to the spatial patterns captured by the first latent variable (Fig. c, d). The top loading time-series features were mainly related to measures of self-correlation or predictability of the MEG signal. The self-correlation measures mostly reflect the linear correlation structure of neurophysiological time-series, particularly long-lag autocorrelations (at lags > 15 time steps, or > 30 ms). A wide range of other highly weighted time-series features captured other aspects of signal predictability, including measures of the shape of the autocorrelation function (e.g., the time lag at which the autocorrelation function crosses zero), how the autocorrelation structure changes after removing low-order local trends (e.g., residuals from fitting linear models to rolling 5-time-step, or 10 ms, windows), scaling properties assessed using fluctuation analysis (e.g., scaling of signal variance across timescales), and measures derived from a wavelet decomposition (e.g., wavelet coefficients at different timescales). The full list of time-series feature loadings for the first latent variable is available in the online Supplementary Dataset . To illustrate the spatial distribution of highly contributing time-series features, Fig. c shows three top-loading features that mirror the spatial variation of the first PLS latent variable. For example, Figure c, left depicts the distribution of the group-average first zero-crossing point of the autocorrelation function. The autocorrelation function of the unimodal cortex (marked with a pink circle) crosses zero autocorrelation at a lower lag than the transmodal cortex (marked with a purple circle), suggesting faster autocorrelation decay and longer correlation length in transmodal cortex than in unimodal cortex. Another example is the linear autocorrelation of the MEG signal at longer time lags. Figure c, left shows autocorrelation at a lag of 48 ms (24 time steps), demonstrating lower autocorrelation in unimodal cortex and higher autocorrelation in transmodal cortex. Note that the list of top-loading features includes linear autocorrelation at other time lags and autocorrelation at a lag of 48 ms was only selected as an illustrative example (Supplementary Fig. depicts the full range of loadings for linear autocorrelation at all time lags included in hctsa). Finally, we examined the scaling exponent, α , estimated using detrended fluctuation analysis as the slope of a linear fit to the log-log plot of the fluctuations of the detrended signal across timescales , . Figure c, right depicts this scaling exponent across the cortex, which exhibits a similar spatial pattern as the previous two examples, indicating lower self-correlation in unimodal cortex (pink circle) compared to transmodal cortex (purple circle). Other variations of fluctuation analysis also featured heavily in the list of top-loading features, including goodness of fit of the linear fit, fitting of multiple scaling regimes, and different types of detrending and mathematical formulation of fluctuation size. Figure d shows the corresponding micro-architectural loadings. The most contributing micro-architectural features to the spatial patterns captured by the first latent variable are the principal component of gene expression (gene expression PC1; a potential proxy for cell type distribution , , ), T1w/T2w ratio (a proxy for intracortical myelin ), principal component of neurotransmitter receptors and transporters (neurotransmitter PC1), and oxygen and glucose metabolism (strong positive loadings). We also find high contributions (strong negative loadings) from specific neurotransmitter receptor and transporters, in particular metabotropic serotonergic and dopaminergic receptors, as well as from cell type-specific gene expression of oligodendrocyte precursors (opc), which are involved in myelinogenesis – . Consistent findings were obtained when we used univariate analysis to relate regional time-series features and the top loading micro-architectural maps, in particular principal component of gene expression and T1w/T2w ratio, which have previously been extensively studied as archetypical micro-architectural gradients , , , , (Supplementary Fig. . Altogether, this analysis provides a comprehensive chart or ‘lookup table’ of how micro-architectural and time-series feature maps are associated with one another. These results demonstrate that cortical variation in multiple micro-architectural attributes manifests as a gradient of time-series properties of neurophysiological activity, particularly the properties that reflect the long-range self-correlation structure of the signal. To assess the extent to which the results are affected by potential confounding factors and methodological choices, we repeated the analyses using alternative approaches. First, to ensure that the findings are not influenced by MEG signal-to-noise ratio (SNR), we calculated SNR at each source location using a noise model that estimates how sensitive the source-level MEG signal is to source location and orientation , . We performed two follow-up analyses using the SNR map (Supplementary Fig. : (1) SNR was first compared with the full set of MEG time-series features using mass univariate Pearson correlations. Time-series features that were significantly correlated with SNR were removed from the feature set without correcting for multiple comparisons ( p spin < 0.05; 10,000 spatial autocorrelation-preserving permutation tests , ). Note that this is a more conservative feature selection procedure compared to conventional multiple comparisons correction, because fewer features would be removed if correction for multiple comparisons was applied. PCA was applied to the remaining set of features (Supplementary Figure . The principal component of the retained 3819 features (i.e., PC1 - feature subset) explained 31.6% of the variance and was significantly correlated with the original PC1 of the full set of features ( r s = 0.93, p spin = 0.0001), reflecting similar spatial pattern as the original analysis. (2) SNR was regressed out from the full set of time-series features using linear regression analysis. PCA was then applied to the resulting feature residuals (Supplementary Fig. . The principal component of SNR-regressed features (i.e., PC1 - SNR regressed) explained 41.4% of the variance and reflected the same spatial pattern as the original analysis ( r s = 0.70, p spin = 0.0004). Moreover, we assessed the effects of environmental and instrumental noise on the findings, where we applied principal component analysis to the hctsa features obtained from pre-processed empty-room MEG recordings (see Methods for more details). PCA weights of the time-series features of the empty-room MEG recordings were aligned with the PCA weights of the time-series features of the resting-state MEG recordings using the Procrustes method (see ref. ; https://github.com/satra/mapalign ). The principal component of neurophysiological dynamics was then compared with the principal component of time-series features obtained from empty-room recordings, where no significant associations were identified (Supplementary Fig. ; r s = − 0.17, p spin = 0.69). These analyses demonstrate that the time-series features captured by the dominant axis of variation in neurophysiological dynamics are independent from measures of MEG signal-to-noise ratio. Finally, to ensure that the findings are independent from the parcellation resolution, we repeated the analyses using a higher resolution parcellation (Schaefer-400 atlas with 400 cortical regions ). The results were consistent with the original analysis (Supplementary Figs. , . In particular, the first principal component (PC1) accounted for 48.6% of the variance and displayed a similar spatial organization as the one originally obtained for the Schaefer-100 atlas (Supplementary Fig. . As before, the top loading time-series features were mainly related to the characteristics of the power spectral density (Supplementary Fig. . The full list of features, their loadings and p -values are available in the online Supplementary Dataset . Moreover, PLS analysis identified a single significant latent variable ( p spin = 0.0083) that accounted for 75.7% of the covariance (Supplementary Fig. . Micro-architecture and time-series feature scores displayed similar spatial patterns to the ones obtained for the Schaefer-100 atlas (Supplementary Fig. . The corresponding feature loadings were also consistent with the original findings (micro-architectural loadings in Supplementary Fig. and time-series feature loadings in the online Supplementary Dataset .) In the present study, we use time-series phenotyping analysis to comprehensively chart the dynamic fingerprint of neurophysiological activity from the resting-state MEG signal. We then map the resulting dynamical atlas to a multimodal micro-architectural atlas to identify the neurophysiological signatures of cortical micro-architecture. We demonstrate that cortical variation in neurophysiological time-series properties mainly reflects power spectral density and is closely associated with intrinsic timescale and self-correlation structure of the signal. Moreover, the spatial organization of neurophysiological dynamics follows gradients of micro-architecture, such as neurotransmitter receptor and transporters, gene expression and T1w/T2w ratios, and reflects cortical metabolic demands. Numerous studies have previously investigated neural oscillations and their relationship with neural communication patterns in the brain , , , . Previous reports also suggest that neural oscillations influence behaviour and cognition – and are involved in multiple neurological diseases and disorders , . Neural oscillations manifest as the variations of power amplitude of neurophysiological signal in the frequency domain , , , . Power spectral characteristics of the neurophysiological signal, such as mean power amplitude in canonical frequency bands, have previously been used to investigate the underlying mechanisms of large-scale brain activity and to better understand the individual differences in brain function , , , , , . Other time-series properties that are related to the power spectral density have also been used to study neural dynamics, including measures of intrinsic timescale and self-affinity or self-similarity of the signal (e.g., autocorrelation and fluctuation analysis) , , , , – . Applying a data-driven time-series feature extraction analysis, we find that the topographic organization of neurophysiological time-series signature follows a sensory–fugal axis, separating somatomotor, occipital and parietal cortices from anterior temporal, orbitofrontal and ventromedial cortices. This dynamic fingerprint of neurophysiological activity is mainly characterized by linear correlation structure of MEG signal captured by hctsa time-series features. The linear correlation structure manifests in both power spectral properties and the autocorrelation function. This dominant spatial variation of time-series features also resembles the spatial distribution of intrinsic timescale, another measure related to the characteristics of power spectral density , , . Altogether, while the findings highlight under-represented time-series features, they emphasize the importance of conventional methods in characterizing neurophysiological activity and the key role of linear correlation structure in MEG dynamics. Earlier reports found that regional neural dynamics, including measures of power spectrum and intrinsic timescale, reflect the underlying circuit properties and cortical micro-architecture , , . The relationship between neural dynamics and cortical micro-architecture is often examined using a single, or a few microstructural features. Recent advances in data collection and integration and the increasing number of data sharing initiatives have provided a unique opportunity to comprehensively study cortical circuit properties and micro-architecture using a wide range of multimodal datasets , , , , , , . Here we use such datasets and compile multiple micro-architectural maps, including measures of microstructure, metabolism, cortical expansion, receptors and transporters, layer thickness and cell type-specific gene expressions, to chart the multivariate associations between neurophysiological dynamics and cortical micro-architecture. Our findings build on previous reports by showing that neurophysiological dynamics follow the underlying cytoarchitectonic and microstructural gradients. In particular, our findings confirm that MEG intrinsic dynamics are associated with the heterogeneous distribution of gene expression and intracortical myelin , , , and neurotransmitter receptors and transporters . In addition, we link the dynamic signature of ongoing neurophysiological activity with multiple metabolic attributes , ; for instance, we find that regions with greater oxygen and glucose metabolism tend to display lower temporal autocorrelation and therefore more variable moment-to-moment intrinsic activity. This is consistent with previously reported high metabolic rates of oxygen and glucose consumption in the sensory cortex . We also find a prominent association with cell type-specific gene expression of oligodendrocyte precursors (opc), potentially reflecting the contribution of these cells to myelin generation by giving rise to myelinating oligodendrocytes during development – and to myelin regulation and metabolic support of myelinated axons in the adult neural circuits , , . Finally, we find that the dominant dynamic signature of neural activity covaries with the granular cortical layer IV, consistent with the idea that layer IV receives prominent subcortical (including thalamic) feedforward projections , . Collectively, our findings build on the emerging literature on how heterogeneous micro-architectural properties along with macroscale network embedding (e.g., cortico-cortical connectivity and subcortical projections) jointly shape regional neural dynamics – , – . The present findings must be interpreted with respect to several methodological considerations. First, we used MEG data from a subset of individuals with no familial relationships from the HCP dataset. Although all the presented analyses are performed using the group-level data, future work with larger sample sizes can provide more generalizable outcomes , . Larger sample sizes will also help go beyond associative analysis and allow for predictive analysis of neural dynamics and micro-architecture in unseen datasets. Second, MEG is susceptible to low SNR and has variable sensitivity to neural activity from different regions (i.e., sources). Thus, electrophysiological recordings with higher spatial resolution, such as intracranial electroencephalography (iEEG and ECoG), may provide more precise measures of neural dynamics that can be examined with respect to cortical micro-architecture. However, a major caveat with iEEG and ECoG is that they lack whole brain coverage, limiting their practical usage in such analysis. An alternative non-invasive modality is on-scalp MEG, which offers both high SNR and spatial resolution – . Third, we note that the included micro-architectural maps are by no means direct measurements of the underlying neurobiological features. For example, the “myelin” map is estimated based on the ratio of T1-weighted to T2-weighted MRI scans, which is only sensitive to intracortical myelin and is not a true measure of tissue myelin content , . The “cortical layer thickness” maps are from a deep-learning based layer segmentation of the BigBrain histological atlas and are not precise measurements of laminar differentiation of the brain – . Although we aimed to select non-invasive modalities that are most sensitive to microstructure, cytoarchitecture, and cellular and molecular features, the included maps can only provide proxy, indirect assessments of such biological properties. Finally, despite the fact that we attempt to use a comprehensive list of time-series properties and multiple micro-architectural features, neither the time-series features nor the micro-architectural maps are exhaustive sets of measures. Moreover, micro-architectural features are group-average maps that are compiled from different datasets. Multimodal datasets from the same individuals are required to perform individual-level comparisons between the dynamical and micro-architectural atlases. Altogether, using a data-driven approach, the present findings show that neurophysiological signatures of cortical micro-architecture are hierarchically organized across the cortex, reflecting the underlying circuit properties. These findings highlight the importance of conventional measures for studying the characteristics of neurophysiological activity, while also identifying less-commonly used time-series features that covary with cortical micro-architecture. Collectively, this work opens new avenues for studying the anatomical basis of neurophysiological activity. Dataset: human connectome project (HCP) Resting state magnetoencephalography (MEG) data from a sample of healthy young adults ( n = 33; age range 22–35 years; 16 female and 17 male) with no familial relationships were obtained from Human Connectome Project (HCP; S900 release ; informed consent obtained). The WU-Minn HCP Consortium (consortium of US and European institutions led by Washington University and the University of Minnesota) approved the study protocol. The obtained data includes resting state scans of approximately 6 minutes long (sampling rate = 2034.5 Hz; anti-aliasing low-pass filter at 400 Hz) and empty-room recordings for all participants. 3T structural magnetic resonance imaging (MRI) data and MEG anatomical data (i.e., cortical sheet with 8004 vertices and transformation matrix required for co-registration of MEG sensors and MRI scans) of all participants were also obtained for MEG pre-processing. Resting state magnetoencephalography (MEG) Resting state MEG data was analyzed using Brainstorm software, which is documented and freely available for download online under the GNU general public license (see ref. ; http://neuroimage.usc.edu/brainstorm ). For each individual, MEG sensor recordings were registered to their structural MRI scan using the anatomical transformation matrix provided by HCP for co-registration, following the procedure described in Brainstorm online tutorials for the HCP dataset ( https://neuroimage.usc.edu/brainstorm/Tutorials/HCP-MEG ). The data were downsampled to 1/4 of the original sampling rate (i.e., 509 Hz) to facilitate processing. The pre-processing was performed by applying notch filters at 60, 120, 180, 240, and 300 Hz, and was followed by a high-pass filter at 0.3 Hz to remove slow-wave and DC-offset artifacts. Bad channels were marked based on the information obtained through the data management platform of HCP (ConnectomeDB; https://db.humanconnectome.org/ ). The artifacts (including heartbeats, eye blinks, saccades, muscle movements, and noisy segments) were then removed from the recordings using automatic procedures as proposed by Brainstorm. More specifically, electrocardiogram (ECG) and electrooculogram (EOG) recordings were used to detect heartbeats and blinks, respectively. We then used Signal-Space Projections (SSP) to automatically remove the detected artifacts. We also used SSP to remove saccades and muscle activity as low-frequency (1–7 Hz) and high-frequency (40–240 Hz) components, respectively. The pre-processed sensor-level data was then used to obtain a source estimation on HCP’s fsLR4k cortical surface for each participant (i.e., 8004 vertices). Head models were computed using overlapping spheres and the data and noise covariance matrices were estimated from the resting state MEG and noise recordings. Linearly constrained minimum variance (LCMV) beamforming from Brainstorm was then used to obtain the source activity for each participant. We performed data covariance regularization to avoid the instability of data covariance matrix inversion due to the smallest eigenvalues of its eigenspectrum. Data covariance regularization was performed using the “median eigenvalue” method from Brainstorm , such that the eigenvalues of the eigenspectrum of data covariance matrix that were smaller than the median eigenvalue were replaced with the median eigenvalue itself. The estimated source variance was also normalized by the noise covariance matrix to reduce the effect of variable source depth. Source orientations were constrained to be normal to the cortical surface at each of the 8004 vertex locations on the fsLR4k surface. Source-level time-series were parcellated into 100 regions using the Schaefer-100 atlas for each participant, such that a given parcel’s time series was estimated as the first principal component of its constituting sources’ time series. Finally, we estimated source-level signal-to-noise ratio (SNR) as follows , : 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\rm{SNR}}}}}}}}=10{\log }_{10}\left(\frac{{a}^{2}}{N}\mathop{\sum }\limits_{k=1}^{N}\frac{{b}_{k}^{2}}{{s}_{k}^{2}}\right),$$\end{document} SNR = 10 log 10 a 2 N ∑ k = 1 N b k 2 s k 2 , where a is the source amplitude (i.e., typical strength of a dipole, which is 10 nAm ), N is the number of sensors, b k is the signal at sensor k estimated by the forward model for a source with unit amplitude, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{k}^{2}$$\end{document} s k 2 is the noise variance at sensor k . Group-average source-level SNR was parcellated using the Schaefer-100 atlas. To estimate a measure of environmental and instrumental noise, empty-room MEG recordings of all individuals were obtained from HCP and were pre-processed using an identical procedure to the resting-state recordings. The pre-processed source-level time-series obtained from empty-room recordings were parcellated and subjected to time-series feature extraction analysis to estimate time-series features from noise data for each participant (see Time-series feature extraction using hctsa ). Power spectral analysis Welch’s method was used to estimate power spectrum density (PSD) from the source-level time-series for each individual, using overlapping windows of length 4 seconds with 50% overlap. Average power at each frequency band was then calculated for each vertex (i.e., source) as the mean power across the frequency range of a given frequency band. Source-level power data were parcellated into 100 regions using the Schaefer-100 atlas at six canonical electrophysiological bands (i.e., delta ( δ : 2–4 Hz), theta ( θ : 5–7 Hz), alpha ( α : 8–12 Hz), beta ( β : 15–29 Hz), low gamma (lo- γ : 30–59 Hz), and high gamma (hi- γ : 60–90 Hz)). We contributed the group-average vertex-level power maps on the fsLR4k surface to the publicly available neuromaps toolbox . Intrinsic timescale The regional intrinsic timescale was estimated using spectral parameterization with the FOOOF (fitting oscillations & one over f) toolbox . Specifically, the source-level power spectral density were used to extract the neural timescale at each vertex and for each individual using the procedure described in ref. . The FOOOF algorithm decomposes the power spectra into periodic (oscillatory) and aperiodic (1/ f -like) components by fitting the power spectral density in the log-log space (Supplementary Fig. . The algorithm identifies the oscillatory peaks (periodic component), the “knee parameter” k that controls for the bend in the aperiodic component and the aperiodic “exponent” χ , . The knee parameter k is then used to calculate the “knee frequency” as f k = k 1/ χ , which is the frequency where a knee or a bend occurs in the power spectrum density . Finally, the intrinsic timescale τ is estimated as : 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau=\frac{1}{2\pi {f}_{k}}.$$\end{document} τ = 1 2 π f k . We used the FOOOF algorithm to fit the power spectral density with “knee” aperiodic mode over the frequency range of 1–60 Hz. Note that since the first notch filter was applied at 60 Hz during the pre-processing analysis, we did not fit the model above 60 Hz. Following the guidelines from the FOOOF algorithm and Donoghue et al. , the rest of the parameters were defined as: peak width limits (peak_width_limits) = 1–6 Hz; maximum number of peaks (max_n_peaks) = 6; minimum peak height (min_peak_height) = 0.1; and peak threshold (peak_threshold) = 2. Intrinsic timescale τ was estimated at each vertex for each individual and was parcellated using the Schaefer-100 atlas . The performance of model fits by the FOOOF algorithm was quantified as the “goodness of fit” or R 2 for each model fitted to a given power spectrum (Supplementary Fig. ; range of R 2 = [0.95, 0.98]). We contributed the group-average vertex-level intrinsic timescale map on the fsLR4k surface to the publicly available neuromaps toolbox . In addition to the aperiodic component used to calculate the intrinsic timescale, the FOOOF spectral parameterization algorithm also provides the extracted peak parameters of the periodic component at each vertex for each participant. We used the oscillatory peak parameters to estimate band-limited power maps that were adjusted for the aperiodic component as opposed to the total power maps estimated above (see Power spectral analysis ). We defined the power band limits as delta (2–4 Hz), theta (5–7 Hz), alpha (8–14 Hz), and beta (15–30 Hz), based on the distribution of peak center frequencies across all vertices and participants (Supplementary Fig. . Given the lack of clear oscillatory peaks in high frequencies (above 40 Hz), the FOOOF algorithm struggles with detecting consistent peaks in gamma frequencies and above , . Thus, we did not analyze band-limited power in gamma frequencies using spectral parameterization. For each of the 4 predefined power bands, we estimated an “oscillation score” following the procedure described by Donoghue et al. . Specifically, for each participant and frequency band, we identified the extracted peak at each vertex. If more than one peak was detected at a given vertex, the peak with maximum power was selected. The average peak power was then calculated at each vertex and frequency band across participants. The group-average peak power map was then normalized for each frequency band, such that the average power at each vertex was divided by the maximum average power across all vertices. Separately, we calculated a vertex-level probability map for each frequency band as the percentage of participants with at least one detected peak at a given vertex at that frequency band. Finally, the band-limited “oscillation score” maps were obtained by multiplying the normalized group-average power maps with their corresponding probability maps for each frequency band. The oscillation score maps were parcellated using the Schaefer-100 atlas (Supplementary Fig. . Time-series feature extraction using hctsa We used the highly comparative time-series analysis toolbox, hctsa , , to perform a massive feature extraction of the pre-processed time-series for each brain region for each participant. The hctsa package extracted over 7000 local time-series features using a wide range of time-series analysis methods , . The extracted features include, but are not limited to, measures of data distribution, temporal dependency and correlation properties, entropy and variability, parameters of time-series model fit, and nonlinear properties of a given time-series , . The hctsa feature extraction analysis was performed on the parcellated MEG time-series for each participant. Given that applying hctsa on the full time-series is computationally expensive, we used 80 seconds of data for feature extraction after dropping the first 30 seconds. Previous reports suggest that relatively short segments of about 30 to 120 seconds of resting-state data are sufficient to estimate robust properties of intrinsic brain activity . Nevertheless, to ensure that we can robustly estimate time-series features from 80 seconds of data, we calculated a subset of hctsa features using the catch-22 toolbox on subsequent segments of time-series with varying length for each participant. Specifically, we extracted time-series features from short segments of data ranging from 5 to 125 seconds in increments of 5 s. To identify the time-series length required to estimate robust and stable features, we calculated the Pearson correlation coefficient between features of two subsequent segments (e.g., features estimated from 10 and 5 seconds of data). The correlation coefficient between the estimated features started to stabilize at time-series segments of around 30 s, consistent with previous reports (Supplementary Fig. . Following the feature extraction procedure from time-series segments of 80 s, the outputs of the operations that produced errors were removed and the remaining features (6880 features) were normalized across nodes using an outlier-robust sigmoidal transform for each participant separately. A group-average region × feature matrix was generated from the normalized individual-level features. We also applied hctsa analysis to the parcellated empty-room recordings (80 seconds) to estimate time-series features from noise data using an identical procedure to resting-state data, identifying 6148 features per region per participant. The time-series features were normalized across brain regions for each participant. A group-average empty-room feature set was obtained and used for further analysis. Micro-architectural features from neuromaps We used the neuromaps toolbox ( https://github.com/netneurolab/neuromaps ) to obtain micro-architectural and neurotransmitter receptor and transporter maps in the maps’ native spaces. Details about all maps and their data sources are available in . Briefly, all data that were originally available in any surface space were transformed to the fsLR32k surface space using linear interpolation to resample data and were parcellated into 100 cortical regions using the Schaefer-100 atlas in fsLR32k space . All volumetric data were retained in their native MNI152 volumetric space and were parcellated into 100 cortical regions using the volumetric Schaefer atlas in MNI152 space . Micro-architectural maps included T1w/T2w as a proxy measure of cortical myelin , , cortical thickness , principal component of gene expression , , principal component of neurotransmitter receptors and transporters , synapse density (using [ 11 C ]UCB-J PET tracer that binds to the synaptic vesicle glycoprotein 2A (SV2A)) , – , metabolism (i.e., cerebral blood flow and volume, oxygen and glucose metabolism, glycolytic index) , evolutionary and developmental expansion , allometric scaling from Philadelphia Neurodevelopmental Cohort (PNC) and National Institutes of Health (NIH) . Neurotransmitter maps included 18 different neurotransmitter receptors and transporters across 9 different neurotransmitter systems, namely serotonin (5-HT1a, 5-HT1b, 5-HT2a, 5-HT4, 5-HT6, 5-HTT), histamine (H3), dopamine (D1, D2, DAT), norepinephrine (NET), acetylcholine ( α 4 β 2, M1, VAChT), cannabinoid (CB1), opioid (MOR), glutamate (mGluR5), and GABA (GABAa/bz) . BigBrain histological data Layer thickness data for the 6 cortical layers (I-VI) were obtained from the BigBrain atlas, which is a volumetric, high-resolution (20 × 20 × 20 μm) histological atlas of a post-mortem human brain (65-year-old male) – . In the BigBrain atlas, sections of the post mortem brain are stained for cell bodies using Merker staining technique . These sections are then imaged and used to reconstruct a volumetric histological atlas of the human brain that reflects neuronal density and soma size and captures the regional differentiation of cytoarchitecture – , , . The approximate cortical layer thickness data obtained from the BigBrainWarp toolbox , were originally generated using a convolutional neural network that automatically segments the cortical layers from the pial to white surfaces . Full description of how the cortical layer thickness was approximated is available elsewhere . The cortical layer thickness data for the 6 cortical layers were obtained on the fsaverage surface (164k vertices) from the BigBrainWarp toolbox and were parcellated into 100 cortical regions using the Schaefer-100 atlas . Cell type-specific gene expression Regional microarray expression data were obtained from 6 post-mortem brains (1 female, ages 24–57, 42.5 ± 13.4) provided by the Allen Human Brain Atlas (AHBA, https://human.brain-map.org ; see ref. ). Data were processed with the abagen toolbox (version 0.1.3-doc; https://github.com/rmarkello/abagen ; see ref. ) using the Schaefer-100 volumetric atlas in MNI space . First, microarray probes were reannotated using data provided by ; probes not matched to a valid Entrez ID were discarded. Next, probes were filtered based on their expression intensity relative to background noise , such that probes with intensity less than the background in ≥ 50.00% of samples across donors were discarded. When multiple probes indexed the expression of the same gene, we selected and used the probe with the most consistent pattern of regional variation across donors (i.e., differential stability; see ref. ), calculated with: 3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{\Delta }}}_{S}(p)=\frac{1}{\left({{N}\atop{2}}\right)}\,\mathop{\sum }\limits_{i=1}^{N-1}\mathop{\sum }\limits_{j=i+1}^{N}\rho [{B}_{i}(p),\, {B}_{j}(p)],$$\end{document} Δ S ( p ) = 1 N 2 ∑ i = 1 N − 1 ∑ j = i + 1 N ρ [ B i ( p ) , B j ( p ) ] , where ρ is Spearman’s rank correlation of the expression of a single probe, p , across regions in two donors B i and B j , and N is the total number of donors. Here, regions correspond to the structural designations provided in the ontology from the AHBA. The MNI coordinates of tissue samples were updated to those generated via non-linear registration using the Advanced Normalization Tools (ANTs; https://github.com/chrisfilo/alleninf ). To increase spatial coverage, tissue samples were mirrored bilaterally across the left and right hemispheres . Samples were assigned to brain regions in the provided atlas if their MNI coordinates were within 2 mm of a given parcel. If a brain region was not assigned a tissue sample based on the above procedure, every voxel in the region was mapped to the nearest tissue sample from the donor in order to generate a dense, interpolated expression map. The average of these expression values was taken across all voxels in the region, weighted by the distance between each voxel and the sample mapped to it, in order to obtain an estimate of the parcellated expression values for the missing region. All tissue samples not assigned to a brain region in the provided atlas were discarded. Inter-subject variation was addressed by normalizing tissue sample expression values across genes using a robust sigmoid function : 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${x}_{{{{{{{{\rm{norm}}}}}}}}}=\frac{1}{1+\exp (-\frac{(x-\langle x\rangle )}{{{{\mbox{IQR}}}}_{x}})},$$\end{document} x norm = 1 1 + exp ( − ( x − ⟨ x ⟩ ) IQR x ) , where 〈 x 〉 is the median and IQR x is the normalized interquartile range of the expression of a single tissue sample across genes. Normalized expression values were then rescaled to the unit interval: 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${x}_{{{{{{{{\rm{scaled}}}}}}}}}=\frac{{x}_{{{{{{{{\rm{norm}}}}}}}}}-\min ({x}_{{{{{{{{\rm{norm}}}}}}}}})}{\max ({x}_{{{{{{{{\rm{norm}}}}}}}}})-\min ({x}_{{{{{{{{\rm{norm}}}}}}}}})}.$$\end{document} x scaled = x norm − min ( x norm ) max ( x norm ) − min ( x norm ) . Gene expression values were then normalized across tissue samples using an identical procedure. Samples assigned to the same brain region were averaged separately for each donor and then across donors, yielding a regional expression matrix of 15,633 genes. Finally, cell type-specific gene expression maps were calculated using gene sets identified by a cell type deconvolution analysis , , . Detailed description of the analysis is available at . Briefly, cell-specific gene sets were compiled across 5 single-cell and single-nucleus RNA sequencing studies of adult human post-mortem cortical samples – . Gene expression maps of the compiled study-specific cell types were obtained from AHBA. Unsupervised hierarchical clustering analysis was used to identify 7 canonical cell classes that included astrocytes (astro), endothelial cells (endo), microglia (micro), excitatory neurons (neuron-ex), inhibitory neurons (neuron-in), oligodendrocytes (oligo) and oligodendrocyte precursors (opc) . We used the resulting gene sets to obtain average cell type-specific expression maps for each of these 7 cell classes from the regional expression matrix of 15,633 genes. Partial Least Squares (PLS) Partial least squares (PLS) analysis was used to investigate the relationship between resting-state MEG time-series features and micro-architecture maps. PLS is a multivariate statistical technique that identifies mutually orthogonal, weighted linear combinations of the original variables in the two datasets that maximally covary with each other, namely the latent variables , . In the present analysis, one dataset is the hctsa feature matrix (i.e., X n × t ) with n = 100 rows as brain regions and t = 6880 columns as time-series features. The other dataset is the compiled set of micro-architectural maps (i.e., Y n × m ) with n = 100 rows (brain regions) and m = 45 columns (micro-architecture maps). To identify the latent variables, both data matrices were normalized column-wise (i.e., z -scored) and a singular value decomposition was applied to the correlation matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\bf{R}}}}}}}}={{{{{{{{\bf{X}}}}}}}}}^{{\prime} }{{{{{{{\bf{Y}}}}}}}}$$\end{document} R = X ′ Y as follows: 6 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\bf{R}}}}}}}}={{{{{{{{\bf{X}}}}}}}}}^{{\prime} }{{{{{{{\bf{Y}}}}}}}}={{{{{{{{\bf{USV}}}}}}}}}^{{\prime} },$$\end{document} R = X ′ Y = USV ′ , where U t × m and V m × m are orthonormal matrices of left and right singular vectors and S m × m is the diagonal matrix of singular values. Each column of U and V matrices corresponds to a latent variable. Each element of the diagonal of S is the corresponding singular value. The singular values are proportional to the covariance explained by latent variable and can be used to calculate effect sizes as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\eta }_{i}={s}_{i}^{2}/\mathop{\sum }\nolimits_{j=1}^{J}{s}_{j}^{2}$$\end{document} η i = s i 2 / ∑ j = 1 J s j 2 where η i is the effect size for the i -th latent variable (LV i ), s i is the corresponding singular value, and J is the total number of singular values (here J = m ). The left and right singular vectors U and V demonstrate the extent to which the time-series features and micro-architectural maps contribute to latent variables, respectively. Time-series features with positive weights covary with micro-architectural maps with positive weights, while negatively weighted time-series features and micro-architectural maps covary together. Singular vectors can be used to estimate brain scores that demonstrate the extent to which each brain region expresses the weighted patterns identified by latent variables. Brain scores for time-series features and micro-architectural maps are calculated by projecting the original data onto the PLS-derived weights (i.e., U and V ): \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\rm{Brain}}}}}}}}\,{{{{{{{\rm{scores}}}}}}}}\,{{{{{{{\rm{for}}}}}}}}\,{{{{{{{\rm{time}}}}}}}}-{{{{{{{\rm{series}}}}}}}}\,{{{{{{{\rm{features}}}}}}}}={{{{{{{\bf{XU}}}}}}}}\\ {{{{{{{\rm{Brain}}}}}}}}\,{{{{{{{\rm{scores}}}}}}}}\,{{{{{{{\rm{for}}}}}}}}\,{{{{{{{\rm{micro}}}}}}}}-{{{{{{{\rm{architecture}}}}}}}}={{{{{{{\bf{YV}}}}}}}}$$\end{document} Brain scores for time − series features = XU Brain scores for micro − architecture = YV Loadings for time-series features and micro-architectural maps are then computed as the Pearson correlation coefficient between the original data matrices and their corresponding brain scores. For example, time-series feature loadings are the correlation coefficients between the original hctsa time-series feature vectors and PLS-derived brain scores for time-series features. The statistical significance of latent variables was assessed using 10,000 permutation tests, where the original data was randomized using spatial autocorrelation-preserving nulls (see “Null model” for more details). The PLS analysis was repeated for each permutation, resulting in a null distribution of singular values. The significance of the original singular values were then assessed against the permuted null distributions (Fig. a). The reliability of PLS loadings was estimated using bootstrap resampling , where rows of the original data matrices X and Y are randomly resampled with replacement 10,000 times. The PLS analysis was then repeated for each resampled data, generating a sampling distribution for each time-series feature and micro-architectural map (i.e., generating 10,000 bootstrap-resampled loadings). The bootstrap-resampled loading distributions are then used to estimate 95% confidence intervals for loadings (e.g., see Fig. d). Given that PLS-derived brain scores are by design highly correlated, we used a distance-dependent cross-validation analysis to assess the out-of-sample correlations between brain scores . Specifically, 75% of the closest brain regions in Euclidean distance to a random “seed” region were selected as training set, while the 25% remaining distant regions were selected as test set. We then re-ran the PLS analysis on the training set (i.e., 75% of regions) and used the resulting weights (i.e., singular values) to estimated brain scores for test set. The out-of-sample correlation was then calculated as the Spearman’s rank correlation coefficient between test set brain scores of time-series features and micro-architectural maps. We repeated this analysis 99 times, such that each time a random brain region was selected as the seed region, yielding distributions of training set brain scores correlations and test set (out-of-sample) correlations (Fig. b). Note that 99 is the maximum number of train-test splits here given that brain maps consist of 100 regions. Finally, we used sparse canonical correlation analysis (sCCA; see ref. ) as an alternative multivariate analysis technique to assess whether using a different method with sparsity affects the results , . Similar to PLS, CCA is another reduced-rank regression analysis that is used to identify multivariate linear relationships between two sets of data matrices , , – . The main difference between CCA and PLS is that in CCA the correlation matrix between the input sets is corrected for within-set correlations, ensuring that the identified link between the two input data matrices is not driven by the correlation structure within one of them . Moreover, sparse CCA (sCCA) adds a regularization parameter to the analysis to impose sparsity and avoid overfitting . The regularization parameter ranges between 0 and 1, where 0 corresponds to highest possible sparsity and 1 corresponds to lowest possibility sparsity. We used sCCA (regularization parameter = 0.7) to identify multivariate associations between neurophysiological time-series features and micro-architectural features and found similar results to the original PLS analysis (Supplementary Fig. . Null model To make inferences about the topographic correlations between any two brain maps, we implement a null model that systematically disrupts the relationship between two topographic maps but preserves their spatial autocorrelation , , . We used the Schaefer-100 atlas in the HCP’s fsLR32k grayordinate space , . The spherical projection of the fsLR32k surface was used to define spatial coordinates for each parcel by selecting the vertex closest to the center-of-mass of each parcel – . The resulting spatial coordinates were used to generate null models by applying randomly-sampled rotations and reassigning node values based on the closest resulting parcel (10,000 repetitions). The rotation was applied to one hemisphere and then mirrored to the other hemisphere. Where appropriate, the results were corrected for multiple comparisons by controlling the false discovery rate (FDR correction ). Reporting summary Further information on research design is available in the linked to this article. Resting state magnetoencephalography (MEG) data from a sample of healthy young adults ( n = 33; age range 22–35 years; 16 female and 17 male) with no familial relationships were obtained from Human Connectome Project (HCP; S900 release ; informed consent obtained). The WU-Minn HCP Consortium (consortium of US and European institutions led by Washington University and the University of Minnesota) approved the study protocol. The obtained data includes resting state scans of approximately 6 minutes long (sampling rate = 2034.5 Hz; anti-aliasing low-pass filter at 400 Hz) and empty-room recordings for all participants. 3T structural magnetic resonance imaging (MRI) data and MEG anatomical data (i.e., cortical sheet with 8004 vertices and transformation matrix required for co-registration of MEG sensors and MRI scans) of all participants were also obtained for MEG pre-processing. Resting state MEG data was analyzed using Brainstorm software, which is documented and freely available for download online under the GNU general public license (see ref. ; http://neuroimage.usc.edu/brainstorm ). For each individual, MEG sensor recordings were registered to their structural MRI scan using the anatomical transformation matrix provided by HCP for co-registration, following the procedure described in Brainstorm online tutorials for the HCP dataset ( https://neuroimage.usc.edu/brainstorm/Tutorials/HCP-MEG ). The data were downsampled to 1/4 of the original sampling rate (i.e., 509 Hz) to facilitate processing. The pre-processing was performed by applying notch filters at 60, 120, 180, 240, and 300 Hz, and was followed by a high-pass filter at 0.3 Hz to remove slow-wave and DC-offset artifacts. Bad channels were marked based on the information obtained through the data management platform of HCP (ConnectomeDB; https://db.humanconnectome.org/ ). The artifacts (including heartbeats, eye blinks, saccades, muscle movements, and noisy segments) were then removed from the recordings using automatic procedures as proposed by Brainstorm. More specifically, electrocardiogram (ECG) and electrooculogram (EOG) recordings were used to detect heartbeats and blinks, respectively. We then used Signal-Space Projections (SSP) to automatically remove the detected artifacts. We also used SSP to remove saccades and muscle activity as low-frequency (1–7 Hz) and high-frequency (40–240 Hz) components, respectively. The pre-processed sensor-level data was then used to obtain a source estimation on HCP’s fsLR4k cortical surface for each participant (i.e., 8004 vertices). Head models were computed using overlapping spheres and the data and noise covariance matrices were estimated from the resting state MEG and noise recordings. Linearly constrained minimum variance (LCMV) beamforming from Brainstorm was then used to obtain the source activity for each participant. We performed data covariance regularization to avoid the instability of data covariance matrix inversion due to the smallest eigenvalues of its eigenspectrum. Data covariance regularization was performed using the “median eigenvalue” method from Brainstorm , such that the eigenvalues of the eigenspectrum of data covariance matrix that were smaller than the median eigenvalue were replaced with the median eigenvalue itself. The estimated source variance was also normalized by the noise covariance matrix to reduce the effect of variable source depth. Source orientations were constrained to be normal to the cortical surface at each of the 8004 vertex locations on the fsLR4k surface. Source-level time-series were parcellated into 100 regions using the Schaefer-100 atlas for each participant, such that a given parcel’s time series was estimated as the first principal component of its constituting sources’ time series. Finally, we estimated source-level signal-to-noise ratio (SNR) as follows , : 1 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\rm{SNR}}}}}}}}=10{\log }_{10}\left(\frac{{a}^{2}}{N}\mathop{\sum }\limits_{k=1}^{N}\frac{{b}_{k}^{2}}{{s}_{k}^{2}}\right),$$\end{document} SNR = 10 log 10 a 2 N ∑ k = 1 N b k 2 s k 2 , where a is the source amplitude (i.e., typical strength of a dipole, which is 10 nAm ), N is the number of sensors, b k is the signal at sensor k estimated by the forward model for a source with unit amplitude, and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${s}_{k}^{2}$$\end{document} s k 2 is the noise variance at sensor k . Group-average source-level SNR was parcellated using the Schaefer-100 atlas. To estimate a measure of environmental and instrumental noise, empty-room MEG recordings of all individuals were obtained from HCP and were pre-processed using an identical procedure to the resting-state recordings. The pre-processed source-level time-series obtained from empty-room recordings were parcellated and subjected to time-series feature extraction analysis to estimate time-series features from noise data for each participant (see Time-series feature extraction using hctsa ). Welch’s method was used to estimate power spectrum density (PSD) from the source-level time-series for each individual, using overlapping windows of length 4 seconds with 50% overlap. Average power at each frequency band was then calculated for each vertex (i.e., source) as the mean power across the frequency range of a given frequency band. Source-level power data were parcellated into 100 regions using the Schaefer-100 atlas at six canonical electrophysiological bands (i.e., delta ( δ : 2–4 Hz), theta ( θ : 5–7 Hz), alpha ( α : 8–12 Hz), beta ( β : 15–29 Hz), low gamma (lo- γ : 30–59 Hz), and high gamma (hi- γ : 60–90 Hz)). We contributed the group-average vertex-level power maps on the fsLR4k surface to the publicly available neuromaps toolbox . The regional intrinsic timescale was estimated using spectral parameterization with the FOOOF (fitting oscillations & one over f) toolbox . Specifically, the source-level power spectral density were used to extract the neural timescale at each vertex and for each individual using the procedure described in ref. . The FOOOF algorithm decomposes the power spectra into periodic (oscillatory) and aperiodic (1/ f -like) components by fitting the power spectral density in the log-log space (Supplementary Fig. . The algorithm identifies the oscillatory peaks (periodic component), the “knee parameter” k that controls for the bend in the aperiodic component and the aperiodic “exponent” χ , . The knee parameter k is then used to calculate the “knee frequency” as f k = k 1/ χ , which is the frequency where a knee or a bend occurs in the power spectrum density . Finally, the intrinsic timescale τ is estimated as : 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\tau=\frac{1}{2\pi {f}_{k}}.$$\end{document} τ = 1 2 π f k . We used the FOOOF algorithm to fit the power spectral density with “knee” aperiodic mode over the frequency range of 1–60 Hz. Note that since the first notch filter was applied at 60 Hz during the pre-processing analysis, we did not fit the model above 60 Hz. Following the guidelines from the FOOOF algorithm and Donoghue et al. , the rest of the parameters were defined as: peak width limits (peak_width_limits) = 1–6 Hz; maximum number of peaks (max_n_peaks) = 6; minimum peak height (min_peak_height) = 0.1; and peak threshold (peak_threshold) = 2. Intrinsic timescale τ was estimated at each vertex for each individual and was parcellated using the Schaefer-100 atlas . The performance of model fits by the FOOOF algorithm was quantified as the “goodness of fit” or R 2 for each model fitted to a given power spectrum (Supplementary Fig. ; range of R 2 = [0.95, 0.98]). We contributed the group-average vertex-level intrinsic timescale map on the fsLR4k surface to the publicly available neuromaps toolbox . In addition to the aperiodic component used to calculate the intrinsic timescale, the FOOOF spectral parameterization algorithm also provides the extracted peak parameters of the periodic component at each vertex for each participant. We used the oscillatory peak parameters to estimate band-limited power maps that were adjusted for the aperiodic component as opposed to the total power maps estimated above (see Power spectral analysis ). We defined the power band limits as delta (2–4 Hz), theta (5–7 Hz), alpha (8–14 Hz), and beta (15–30 Hz), based on the distribution of peak center frequencies across all vertices and participants (Supplementary Fig. . Given the lack of clear oscillatory peaks in high frequencies (above 40 Hz), the FOOOF algorithm struggles with detecting consistent peaks in gamma frequencies and above , . Thus, we did not analyze band-limited power in gamma frequencies using spectral parameterization. For each of the 4 predefined power bands, we estimated an “oscillation score” following the procedure described by Donoghue et al. . Specifically, for each participant and frequency band, we identified the extracted peak at each vertex. If more than one peak was detected at a given vertex, the peak with maximum power was selected. The average peak power was then calculated at each vertex and frequency band across participants. The group-average peak power map was then normalized for each frequency band, such that the average power at each vertex was divided by the maximum average power across all vertices. Separately, we calculated a vertex-level probability map for each frequency band as the percentage of participants with at least one detected peak at a given vertex at that frequency band. Finally, the band-limited “oscillation score” maps were obtained by multiplying the normalized group-average power maps with their corresponding probability maps for each frequency band. The oscillation score maps were parcellated using the Schaefer-100 atlas (Supplementary Fig. . We used the highly comparative time-series analysis toolbox, hctsa , , to perform a massive feature extraction of the pre-processed time-series for each brain region for each participant. The hctsa package extracted over 7000 local time-series features using a wide range of time-series analysis methods , . The extracted features include, but are not limited to, measures of data distribution, temporal dependency and correlation properties, entropy and variability, parameters of time-series model fit, and nonlinear properties of a given time-series , . The hctsa feature extraction analysis was performed on the parcellated MEG time-series for each participant. Given that applying hctsa on the full time-series is computationally expensive, we used 80 seconds of data for feature extraction after dropping the first 30 seconds. Previous reports suggest that relatively short segments of about 30 to 120 seconds of resting-state data are sufficient to estimate robust properties of intrinsic brain activity . Nevertheless, to ensure that we can robustly estimate time-series features from 80 seconds of data, we calculated a subset of hctsa features using the catch-22 toolbox on subsequent segments of time-series with varying length for each participant. Specifically, we extracted time-series features from short segments of data ranging from 5 to 125 seconds in increments of 5 s. To identify the time-series length required to estimate robust and stable features, we calculated the Pearson correlation coefficient between features of two subsequent segments (e.g., features estimated from 10 and 5 seconds of data). The correlation coefficient between the estimated features started to stabilize at time-series segments of around 30 s, consistent with previous reports (Supplementary Fig. . Following the feature extraction procedure from time-series segments of 80 s, the outputs of the operations that produced errors were removed and the remaining features (6880 features) were normalized across nodes using an outlier-robust sigmoidal transform for each participant separately. A group-average region × feature matrix was generated from the normalized individual-level features. We also applied hctsa analysis to the parcellated empty-room recordings (80 seconds) to estimate time-series features from noise data using an identical procedure to resting-state data, identifying 6148 features per region per participant. The time-series features were normalized across brain regions for each participant. A group-average empty-room feature set was obtained and used for further analysis. We used the neuromaps toolbox ( https://github.com/netneurolab/neuromaps ) to obtain micro-architectural and neurotransmitter receptor and transporter maps in the maps’ native spaces. Details about all maps and their data sources are available in . Briefly, all data that were originally available in any surface space were transformed to the fsLR32k surface space using linear interpolation to resample data and were parcellated into 100 cortical regions using the Schaefer-100 atlas in fsLR32k space . All volumetric data were retained in their native MNI152 volumetric space and were parcellated into 100 cortical regions using the volumetric Schaefer atlas in MNI152 space . Micro-architectural maps included T1w/T2w as a proxy measure of cortical myelin , , cortical thickness , principal component of gene expression , , principal component of neurotransmitter receptors and transporters , synapse density (using [ 11 C ]UCB-J PET tracer that binds to the synaptic vesicle glycoprotein 2A (SV2A)) , – , metabolism (i.e., cerebral blood flow and volume, oxygen and glucose metabolism, glycolytic index) , evolutionary and developmental expansion , allometric scaling from Philadelphia Neurodevelopmental Cohort (PNC) and National Institutes of Health (NIH) . Neurotransmitter maps included 18 different neurotransmitter receptors and transporters across 9 different neurotransmitter systems, namely serotonin (5-HT1a, 5-HT1b, 5-HT2a, 5-HT4, 5-HT6, 5-HTT), histamine (H3), dopamine (D1, D2, DAT), norepinephrine (NET), acetylcholine ( α 4 β 2, M1, VAChT), cannabinoid (CB1), opioid (MOR), glutamate (mGluR5), and GABA (GABAa/bz) . Layer thickness data for the 6 cortical layers (I-VI) were obtained from the BigBrain atlas, which is a volumetric, high-resolution (20 × 20 × 20 μm) histological atlas of a post-mortem human brain (65-year-old male) – . In the BigBrain atlas, sections of the post mortem brain are stained for cell bodies using Merker staining technique . These sections are then imaged and used to reconstruct a volumetric histological atlas of the human brain that reflects neuronal density and soma size and captures the regional differentiation of cytoarchitecture – , , . The approximate cortical layer thickness data obtained from the BigBrainWarp toolbox , were originally generated using a convolutional neural network that automatically segments the cortical layers from the pial to white surfaces . Full description of how the cortical layer thickness was approximated is available elsewhere . The cortical layer thickness data for the 6 cortical layers were obtained on the fsaverage surface (164k vertices) from the BigBrainWarp toolbox and were parcellated into 100 cortical regions using the Schaefer-100 atlas . Regional microarray expression data were obtained from 6 post-mortem brains (1 female, ages 24–57, 42.5 ± 13.4) provided by the Allen Human Brain Atlas (AHBA, https://human.brain-map.org ; see ref. ). Data were processed with the abagen toolbox (version 0.1.3-doc; https://github.com/rmarkello/abagen ; see ref. ) using the Schaefer-100 volumetric atlas in MNI space . First, microarray probes were reannotated using data provided by ; probes not matched to a valid Entrez ID were discarded. Next, probes were filtered based on their expression intensity relative to background noise , such that probes with intensity less than the background in ≥ 50.00% of samples across donors were discarded. When multiple probes indexed the expression of the same gene, we selected and used the probe with the most consistent pattern of regional variation across donors (i.e., differential stability; see ref. ), calculated with: 3 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{\Delta }}}_{S}(p)=\frac{1}{\left({{N}\atop{2}}\right)}\,\mathop{\sum }\limits_{i=1}^{N-1}\mathop{\sum }\limits_{j=i+1}^{N}\rho [{B}_{i}(p),\, {B}_{j}(p)],$$\end{document} Δ S ( p ) = 1 N 2 ∑ i = 1 N − 1 ∑ j = i + 1 N ρ [ B i ( p ) , B j ( p ) ] , where ρ is Spearman’s rank correlation of the expression of a single probe, p , across regions in two donors B i and B j , and N is the total number of donors. Here, regions correspond to the structural designations provided in the ontology from the AHBA. The MNI coordinates of tissue samples were updated to those generated via non-linear registration using the Advanced Normalization Tools (ANTs; https://github.com/chrisfilo/alleninf ). To increase spatial coverage, tissue samples were mirrored bilaterally across the left and right hemispheres . Samples were assigned to brain regions in the provided atlas if their MNI coordinates were within 2 mm of a given parcel. If a brain region was not assigned a tissue sample based on the above procedure, every voxel in the region was mapped to the nearest tissue sample from the donor in order to generate a dense, interpolated expression map. The average of these expression values was taken across all voxels in the region, weighted by the distance between each voxel and the sample mapped to it, in order to obtain an estimate of the parcellated expression values for the missing region. All tissue samples not assigned to a brain region in the provided atlas were discarded. Inter-subject variation was addressed by normalizing tissue sample expression values across genes using a robust sigmoid function : 4 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${x}_{{{{{{{{\rm{norm}}}}}}}}}=\frac{1}{1+\exp (-\frac{(x-\langle x\rangle )}{{{{\mbox{IQR}}}}_{x}})},$$\end{document} x norm = 1 1 + exp ( − ( x − ⟨ x ⟩ ) IQR x ) , where 〈 x 〉 is the median and IQR x is the normalized interquartile range of the expression of a single tissue sample across genes. Normalized expression values were then rescaled to the unit interval: 5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${x}_{{{{{{{{\rm{scaled}}}}}}}}}=\frac{{x}_{{{{{{{{\rm{norm}}}}}}}}}-\min ({x}_{{{{{{{{\rm{norm}}}}}}}}})}{\max ({x}_{{{{{{{{\rm{norm}}}}}}}}})-\min ({x}_{{{{{{{{\rm{norm}}}}}}}}})}.$$\end{document} x scaled = x norm − min ( x norm ) max ( x norm ) − min ( x norm ) . Gene expression values were then normalized across tissue samples using an identical procedure. Samples assigned to the same brain region were averaged separately for each donor and then across donors, yielding a regional expression matrix of 15,633 genes. Finally, cell type-specific gene expression maps were calculated using gene sets identified by a cell type deconvolution analysis , , . Detailed description of the analysis is available at . Briefly, cell-specific gene sets were compiled across 5 single-cell and single-nucleus RNA sequencing studies of adult human post-mortem cortical samples – . Gene expression maps of the compiled study-specific cell types were obtained from AHBA. Unsupervised hierarchical clustering analysis was used to identify 7 canonical cell classes that included astrocytes (astro), endothelial cells (endo), microglia (micro), excitatory neurons (neuron-ex), inhibitory neurons (neuron-in), oligodendrocytes (oligo) and oligodendrocyte precursors (opc) . We used the resulting gene sets to obtain average cell type-specific expression maps for each of these 7 cell classes from the regional expression matrix of 15,633 genes. Partial least squares (PLS) analysis was used to investigate the relationship between resting-state MEG time-series features and micro-architecture maps. PLS is a multivariate statistical technique that identifies mutually orthogonal, weighted linear combinations of the original variables in the two datasets that maximally covary with each other, namely the latent variables , . In the present analysis, one dataset is the hctsa feature matrix (i.e., X n × t ) with n = 100 rows as brain regions and t = 6880 columns as time-series features. The other dataset is the compiled set of micro-architectural maps (i.e., Y n × m ) with n = 100 rows (brain regions) and m = 45 columns (micro-architecture maps). To identify the latent variables, both data matrices were normalized column-wise (i.e., z -scored) and a singular value decomposition was applied to the correlation matrix \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\bf{R}}}}}}}}={{{{{{{{\bf{X}}}}}}}}}^{{\prime} }{{{{{{{\bf{Y}}}}}}}}$$\end{document} R = X ′ Y as follows: 6 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\bf{R}}}}}}}}={{{{{{{{\bf{X}}}}}}}}}^{{\prime} }{{{{{{{\bf{Y}}}}}}}}={{{{{{{{\bf{USV}}}}}}}}}^{{\prime} },$$\end{document} R = X ′ Y = USV ′ , where U t × m and V m × m are orthonormal matrices of left and right singular vectors and S m × m is the diagonal matrix of singular values. Each column of U and V matrices corresponds to a latent variable. Each element of the diagonal of S is the corresponding singular value. The singular values are proportional to the covariance explained by latent variable and can be used to calculate effect sizes as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\eta }_{i}={s}_{i}^{2}/\mathop{\sum }\nolimits_{j=1}^{J}{s}_{j}^{2}$$\end{document} η i = s i 2 / ∑ j = 1 J s j 2 where η i is the effect size for the i -th latent variable (LV i ), s i is the corresponding singular value, and J is the total number of singular values (here J = m ). The left and right singular vectors U and V demonstrate the extent to which the time-series features and micro-architectural maps contribute to latent variables, respectively. Time-series features with positive weights covary with micro-architectural maps with positive weights, while negatively weighted time-series features and micro-architectural maps covary together. Singular vectors can be used to estimate brain scores that demonstrate the extent to which each brain region expresses the weighted patterns identified by latent variables. Brain scores for time-series features and micro-architectural maps are calculated by projecting the original data onto the PLS-derived weights (i.e., U and V ): \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{{{{{{\rm{Brain}}}}}}}}\,{{{{{{{\rm{scores}}}}}}}}\,{{{{{{{\rm{for}}}}}}}}\,{{{{{{{\rm{time}}}}}}}}-{{{{{{{\rm{series}}}}}}}}\,{{{{{{{\rm{features}}}}}}}}={{{{{{{\bf{XU}}}}}}}}\\ {{{{{{{\rm{Brain}}}}}}}}\,{{{{{{{\rm{scores}}}}}}}}\,{{{{{{{\rm{for}}}}}}}}\,{{{{{{{\rm{micro}}}}}}}}-{{{{{{{\rm{architecture}}}}}}}}={{{{{{{\bf{YV}}}}}}}}$$\end{document} Brain scores for time − series features = XU Brain scores for micro − architecture = YV Loadings for time-series features and micro-architectural maps are then computed as the Pearson correlation coefficient between the original data matrices and their corresponding brain scores. For example, time-series feature loadings are the correlation coefficients between the original hctsa time-series feature vectors and PLS-derived brain scores for time-series features. The statistical significance of latent variables was assessed using 10,000 permutation tests, where the original data was randomized using spatial autocorrelation-preserving nulls (see “Null model” for more details). The PLS analysis was repeated for each permutation, resulting in a null distribution of singular values. The significance of the original singular values were then assessed against the permuted null distributions (Fig. a). The reliability of PLS loadings was estimated using bootstrap resampling , where rows of the original data matrices X and Y are randomly resampled with replacement 10,000 times. The PLS analysis was then repeated for each resampled data, generating a sampling distribution for each time-series feature and micro-architectural map (i.e., generating 10,000 bootstrap-resampled loadings). The bootstrap-resampled loading distributions are then used to estimate 95% confidence intervals for loadings (e.g., see Fig. d). Given that PLS-derived brain scores are by design highly correlated, we used a distance-dependent cross-validation analysis to assess the out-of-sample correlations between brain scores . Specifically, 75% of the closest brain regions in Euclidean distance to a random “seed” region were selected as training set, while the 25% remaining distant regions were selected as test set. We then re-ran the PLS analysis on the training set (i.e., 75% of regions) and used the resulting weights (i.e., singular values) to estimated brain scores for test set. The out-of-sample correlation was then calculated as the Spearman’s rank correlation coefficient between test set brain scores of time-series features and micro-architectural maps. We repeated this analysis 99 times, such that each time a random brain region was selected as the seed region, yielding distributions of training set brain scores correlations and test set (out-of-sample) correlations (Fig. b). Note that 99 is the maximum number of train-test splits here given that brain maps consist of 100 regions. Finally, we used sparse canonical correlation analysis (sCCA; see ref. ) as an alternative multivariate analysis technique to assess whether using a different method with sparsity affects the results , . Similar to PLS, CCA is another reduced-rank regression analysis that is used to identify multivariate linear relationships between two sets of data matrices , , – . The main difference between CCA and PLS is that in CCA the correlation matrix between the input sets is corrected for within-set correlations, ensuring that the identified link between the two input data matrices is not driven by the correlation structure within one of them . Moreover, sparse CCA (sCCA) adds a regularization parameter to the analysis to impose sparsity and avoid overfitting . The regularization parameter ranges between 0 and 1, where 0 corresponds to highest possible sparsity and 1 corresponds to lowest possibility sparsity. We used sCCA (regularization parameter = 0.7) to identify multivariate associations between neurophysiological time-series features and micro-architectural features and found similar results to the original PLS analysis (Supplementary Fig. . To make inferences about the topographic correlations between any two brain maps, we implement a null model that systematically disrupts the relationship between two topographic maps but preserves their spatial autocorrelation , , . We used the Schaefer-100 atlas in the HCP’s fsLR32k grayordinate space , . The spherical projection of the fsLR32k surface was used to define spatial coordinates for each parcel by selecting the vertex closest to the center-of-mass of each parcel – . The resulting spatial coordinates were used to generate null models by applying randomly-sampled rotations and reassigning node values based on the closest resulting parcel (10,000 repetitions). The rotation was applied to one hemisphere and then mirrored to the other hemisphere. Where appropriate, the results were corrected for multiple comparisons by controlling the false discovery rate (FDR correction ). Further information on research design is available in the linked to this article. Supplementary Information Peer Review File Description of Additional Supplementary Files Supplementary Dataset 1 Supplementary Dataset 2 Supplementary Dataset 3 Supplementary Dataset 4 Supplementary Dataset 5 Supplementary Dataset 6 Reporting Summary Source Data |
Genomic characterization and histologic analysis of uterine leiomyosarcoma arising from leiomyoma with bizarre nuclei | adfe581b-b9e1-42ea-b028-063977f4b0d3 | 11717496 | Anatomy[mh] | Uterine leiomyoma with bizarre nuclei (LM‐BN) is a rare variant of leiomyoma, and histologic evaluation remains the key method in the diagnosis of LM‐BN , and no reliable biomarkers can be used to clearly separate it from uterine leiomyosarcoma (LMS) . LM‐BN harbors recurrent genomic events that are commonly seen in LMS . LM‐BN is diagnosed at a mean age of 42.5 years, 10–15 years younger than patients with LMS . LM‐BN is a benign variant of leiomyoma with occasional local recurrence, without disease‐related deaths . However, LM‐BN with diffuse nuclear atypia and five to nine mitoses/10 high power fields (HPFs) poses a diagnostic concern, treated as a smooth muscle tumor of uncertain malignant potential (STUMP) . Uterine LMS is a high‐grade malignant neoplasm and histologically heterogeneous with three subtypes: epithelioid, myxoid, and spindle. While rare reports have detailed chemotherapeutic agents or radiation contributing to LMS development, the tumorigenesis of LMS has long been debated . Due to limited histological or molecular characterizations of LMS, the development of uterine LMS is considered a de novo event . Few studies have suggested the presence of possible precursor lesions, including ‘myometrial dysplasia’, symplastic, or leiomyoma‐like areas within LMS . Studies have shown that both uterine LM‐BN and LMS are genomically unstable with DNA copy number alterations (CNAs) in common tumor suppressor genes including TP53 , CDKN2A/B , PTEN , RB1 , or genes involved in AKT pathways . While these shared molecular aberrations have been well characterized, none can be utilized in isolation to aid in diagnostic purposes or pinpoint etiologies or predict tumor progression, and therefore the identification of tumor origin of these two entities remains largely unknown . Many LMS cases can show mixed areas of benign appearing symplastic or leiomyoma‐like areas, with cases showing no clear‐cut boundary between benign and malignant components . In this study, we aimed to examine the relationship and tumorigenesis of uterine LMS in relation to an adjacent LM‐BN counterpart and to identify similar and different molecular changes. We performed histologic, immunohistochemical, digital, and molecular profiling of both LMS and LM‐BN components from the same patients. We demonstrated that these cases possessed two distinct tumor components with unique histology, regional distributions, shared immunohistochemistry (IHC), and overlapping genomic alterations, particularly DNA CNAs. A spatial transcriptome analysis revealed cell‐specific and distinct expression profiles between LM‐BN and LMS regions, highlighting that these are distinct, rather than admixed, phenotypes. Both LM‐BN and LMS harbored many oncogenic mutations, with LMS gaining more mutations and CNAs than LM‐BN. These results suggest that significantly increased genomic instabilities in LM‐BN are highly associated with adjacent LMS. Case review (clinical, gross, and histologic features) The study received approval by the Institutional Review Board. Cases were selected by retrospectively reviewing our LMS file, and cases with two distinct components of LM‐BN and LMS were included. Clinical information was summarized in supplementary material, Table . Cases with ill‐defined ‘leiomyoma area’ within LMS were excluded in this study. All available slides for each case (range: three to 69 slides, mean slides 30.1/case) were reviewed by gynecologic pathologists. Histologic assessment included tumor type, cellularity, nuclear atypia, mitotic count, necrosis, infiltration, and stage. Unique areas of distinct LMS and LM‐BN regions were determined based on WHO 2020 and Blaustein's Pathology of the Female Genital Tract . The histologic criteria for LM‐BN were well‐ demarcated tumor regions with high‐ or low‐density nuclear atypia in a diffuse or patchy pattern with a low mitotic index (all except one were <5/10 HPFs) and no tumor necrosis. One case (case 2) with six mitoses/10 HPFs at time of diagnosis was classified as LM‐BN, and now STUMP per new WHO criteria. The histologic criteria for spindle cell LMS included at least two of the following: hypercellular tumor regions with marked cytologic atypia, high mitotic index (≥10 mitoses/10 HPF), or tumor coagulative necrosis. HPF (high power field): 40× objective, field diameter = 0.55 mm, 10 HPFs = 2.4 mm 2 . Demographic and clinical characteristics recorded included age, history, radiology, surgical procedure, and outcomes. Immunohistochemical analysis Sections of selected blocks, including both LMS and LM‐BN components, were subjected to IHC analysis with a panel of IHC stains: p16 (E6H4 clone), p53 (Bp53.11 clone), ER (SP1 clone), PR (1E2 clone), and Ki‐67 (30–9 clone). Detailed antibody information for this panel and other relevant markers was summarized in supplementary material, Table . IHC was performed using a Ventana Nexus automated system, as described previously . Intensity was scored as absent (0), weak (1+), moderate (2+), or strong (3+), and the percentage of positive tumor cells was scored from 0% to 100%. p53 interpretation was either diffuse mutant (>75% of tumor), null mutant, or wild type (WT). p16 expression was assessed as patchy or diffuse (cytoplasmic and nuclear strong staining in >80% of tumor). Digital AI image analysis of growth pattern and relationship Select slides were scanned using a Leica Aperio GT450 slide scanner. Whole‐slide images were saved as SVS files. Whole‐slide images of H&E slides were analyzed utilizing QuPath open‐source software . Tumor cells were identified using the cell detection analysis algorithm. Five regions for each of the LMS, LM‐BN, and myometrium/leiomyoma (MM/LM) components were annotated at 40× magnification, corresponding to ~1% of total tumor volume/slide. Object classifier analysis was performed on the slides to generate spatial mapping of distinct regions (semantic segmentation). The regions of interest were classified as follows: red, LMS; yellow, LM‐BN; and green, MM/LM. Regions 1 × 1 mm square were analyzed for each component in quintuplet to assess nuclear and cytoplasmic features. Genomic DNA copy number profiling Tumor sections from LM‐BN and LMS were marked by pathologists and dissected by technicians under the supervision of molecular pathologists. DNA from each region was extracted independently. DNA was analyzed using Affymetrix OncoScan CNA arrays (Thermo Fisher Scientific, Santa Clara, CA, USA). The data were analyzed with normal pool genomic DNA reference using Affymetrix Chromosome Analysis Suite (ChAS) software and reviewed by cytogeneticists for CNAs, including copy number gains or losses, and copy neutral loss of heterozygosity (CN‐LOH) (for details see Supplementary and methods). Spatial transcriptomic RNA sequencing and data analysis Cases with the best distributions of both LMS and LM‐BN back to back within 6 × 6 mm regions were selected for spatial transcriptomic RNA sequencing (RNA‐seq). RNA quality was examined using an Agilent Bioanalyzer RNA pico‐chip (Santa Clara, CA, USA) to confirm that DV200 > 30%. Tissue morphology was examined on H&E‐stained slides to confirm the presence of both LMS and LM‐BN components. Deparaffinization, H&E staining and imaging, and decrosslinking of tissue sections were performed following 10× Genomics protocol (CG000520 Rev B) specific to the Visium CytAssist spatial gene expression (Pleasanton, CA, USA) for FFPE kit. Then the slides subjected to human probe (version 2) hybridization and ligation using 10× Genomics Visium CytAssist spatial gene expression FFPE, human transcriptome, 6.5 mm kit (10× Genomics, PN‐1000520). The probes were released from the tissue slide and transferred to a barcoded Visium slide using a Visium CytAssist instrument followed by probe extension. Sequencing libraries were prepared following the manufacturer's protocol. Multiplexed libraries were pooled and sequenced using a Novaseq6000 at the Northwestern University NUseq core facility. Method details are summarized in Supplementary and methods. Raw sequencing data, in base call format (.bcl), were demultiplexed using Space Ranger (version 2.0.0) from 10× Genomics, converting the raw data into FASTQ format. The images were processed and manually aligned using the Loupe Browser (version 5.1.0). Space Ranger was also used for the alignment of the FASTQ files to the human probe set version 2 and to count the number of reads from each cell that aligned to each probe. The resulting matrix files, which summarize the alignment results, were imported into Seurat (Satija Lab, New York, NY, USA) for further analysis. In Seurat, each individual sample was preprocessed, normalized, and scaled. All samples were then combined into a single dataset using the ‘IntegrateData’ function in Seurat, adding metadata with the original sample information. All UMAPs, violin plots, spatial feature expression plots, and heatmaps were generated using Seurat tools. Cell types were automatically assigned using the R package scType. Next‐generation sequencing ( NGS ) For exon sequencing, DNA libraries were prepared and performed using the BGISEQ‐500 platform (BGI, Cambridge, MA, USA). Rolling circle amplification (RCA) was performed, qualified captured libraries were loaded onto the BGISEQ‐500 platform, and high‐throughput sequencing was performed. Raw data were filtered by removing error sequences. The clean sequence data from each sample were mapped to the human reference genome (GRCh37/HG19) using Burrows–Wheeler Aligner (BWA) software ( http://bio-bwa.sourceforge.net ) with a data analysis quality control (QC) system. The 700 customized oncogene panel sequencing was performed at the Northwestern Diagnostic Molecular Biology Laboratory. The DNA library was hybridized to bridge probes targeting 700 genes. Bridge probes were captured with an anchor probe labeled with biotin and magnetic streptavidin‐coated beads. The library was purified, quantified, and normalized into a sequencing pool and sequenced using a NovaSeq® 6000 and a S1 flowcell (Illumina, San Diego, CA, USA). Sequence data were aligned using BWA1 (version 0.7.17) to human genome version hg38 (Human genome build 38). Variants were called using Vardict2 (version 1.8.3) and Pindel3 (version 0.2.5b). Variants were annotated using wAnnovar4 . Additional details are provided in Supplementary and methods. The data were analyzed as follows: reads from cleaned sequence data were mapped to the hg19 (UCSC) and DNA sequences from normal myometrium using BWA (version 0.7.5) with parameters ‘mem–t 8 –P –M’. Generated files were sorted, and PCR duplicates were removed using Picard (version 1.105) ( http://broadinstitute.github.io/picard ). Subsequently, the BAM files were indexed using samtools (version 0.1.19). The WGS had a quality score of 20 (Q20), and 95–96% of reads were successfully aligned (supplementary material, Figure ). Statistical analysis Statistical analysis was performed in GraphPad Prism (GraphPad Software, San Diego, CA, USA) utilizing unpaired t ‐tests and one‐way ANOVA. P values less than 0.05 were considered statistically significant, and p values were defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. The study received approval by the Institutional Review Board. Cases were selected by retrospectively reviewing our LMS file, and cases with two distinct components of LM‐BN and LMS were included. Clinical information was summarized in supplementary material, Table . Cases with ill‐defined ‘leiomyoma area’ within LMS were excluded in this study. All available slides for each case (range: three to 69 slides, mean slides 30.1/case) were reviewed by gynecologic pathologists. Histologic assessment included tumor type, cellularity, nuclear atypia, mitotic count, necrosis, infiltration, and stage. Unique areas of distinct LMS and LM‐BN regions were determined based on WHO 2020 and Blaustein's Pathology of the Female Genital Tract . The histologic criteria for LM‐BN were well‐ demarcated tumor regions with high‐ or low‐density nuclear atypia in a diffuse or patchy pattern with a low mitotic index (all except one were <5/10 HPFs) and no tumor necrosis. One case (case 2) with six mitoses/10 HPFs at time of diagnosis was classified as LM‐BN, and now STUMP per new WHO criteria. The histologic criteria for spindle cell LMS included at least two of the following: hypercellular tumor regions with marked cytologic atypia, high mitotic index (≥10 mitoses/10 HPF), or tumor coagulative necrosis. HPF (high power field): 40× objective, field diameter = 0.55 mm, 10 HPFs = 2.4 mm 2 . Demographic and clinical characteristics recorded included age, history, radiology, surgical procedure, and outcomes. Sections of selected blocks, including both LMS and LM‐BN components, were subjected to IHC analysis with a panel of IHC stains: p16 (E6H4 clone), p53 (Bp53.11 clone), ER (SP1 clone), PR (1E2 clone), and Ki‐67 (30–9 clone). Detailed antibody information for this panel and other relevant markers was summarized in supplementary material, Table . IHC was performed using a Ventana Nexus automated system, as described previously . Intensity was scored as absent (0), weak (1+), moderate (2+), or strong (3+), and the percentage of positive tumor cells was scored from 0% to 100%. p53 interpretation was either diffuse mutant (>75% of tumor), null mutant, or wild type (WT). p16 expression was assessed as patchy or diffuse (cytoplasmic and nuclear strong staining in >80% of tumor). AI image analysis of growth pattern and relationship Select slides were scanned using a Leica Aperio GT450 slide scanner. Whole‐slide images were saved as SVS files. Whole‐slide images of H&E slides were analyzed utilizing QuPath open‐source software . Tumor cells were identified using the cell detection analysis algorithm. Five regions for each of the LMS, LM‐BN, and myometrium/leiomyoma (MM/LM) components were annotated at 40× magnification, corresponding to ~1% of total tumor volume/slide. Object classifier analysis was performed on the slides to generate spatial mapping of distinct regions (semantic segmentation). The regions of interest were classified as follows: red, LMS; yellow, LM‐BN; and green, MM/LM. Regions 1 × 1 mm square were analyzed for each component in quintuplet to assess nuclear and cytoplasmic features. DNA copy number profiling Tumor sections from LM‐BN and LMS were marked by pathologists and dissected by technicians under the supervision of molecular pathologists. DNA from each region was extracted independently. DNA was analyzed using Affymetrix OncoScan CNA arrays (Thermo Fisher Scientific, Santa Clara, CA, USA). The data were analyzed with normal pool genomic DNA reference using Affymetrix Chromosome Analysis Suite (ChAS) software and reviewed by cytogeneticists for CNAs, including copy number gains or losses, and copy neutral loss of heterozygosity (CN‐LOH) (for details see Supplementary and methods). RNA sequencing and data analysis Cases with the best distributions of both LMS and LM‐BN back to back within 6 × 6 mm regions were selected for spatial transcriptomic RNA sequencing (RNA‐seq). RNA quality was examined using an Agilent Bioanalyzer RNA pico‐chip (Santa Clara, CA, USA) to confirm that DV200 > 30%. Tissue morphology was examined on H&E‐stained slides to confirm the presence of both LMS and LM‐BN components. Deparaffinization, H&E staining and imaging, and decrosslinking of tissue sections were performed following 10× Genomics protocol (CG000520 Rev B) specific to the Visium CytAssist spatial gene expression (Pleasanton, CA, USA) for FFPE kit. Then the slides subjected to human probe (version 2) hybridization and ligation using 10× Genomics Visium CytAssist spatial gene expression FFPE, human transcriptome, 6.5 mm kit (10× Genomics, PN‐1000520). The probes were released from the tissue slide and transferred to a barcoded Visium slide using a Visium CytAssist instrument followed by probe extension. Sequencing libraries were prepared following the manufacturer's protocol. Multiplexed libraries were pooled and sequenced using a Novaseq6000 at the Northwestern University NUseq core facility. Method details are summarized in Supplementary and methods. Raw sequencing data, in base call format (.bcl), were demultiplexed using Space Ranger (version 2.0.0) from 10× Genomics, converting the raw data into FASTQ format. The images were processed and manually aligned using the Loupe Browser (version 5.1.0). Space Ranger was also used for the alignment of the FASTQ files to the human probe set version 2 and to count the number of reads from each cell that aligned to each probe. The resulting matrix files, which summarize the alignment results, were imported into Seurat (Satija Lab, New York, NY, USA) for further analysis. In Seurat, each individual sample was preprocessed, normalized, and scaled. All samples were then combined into a single dataset using the ‘IntegrateData’ function in Seurat, adding metadata with the original sample information. All UMAPs, violin plots, spatial feature expression plots, and heatmaps were generated using Seurat tools. Cell types were automatically assigned using the R package scType. NGS ) For exon sequencing, DNA libraries were prepared and performed using the BGISEQ‐500 platform (BGI, Cambridge, MA, USA). Rolling circle amplification (RCA) was performed, qualified captured libraries were loaded onto the BGISEQ‐500 platform, and high‐throughput sequencing was performed. Raw data were filtered by removing error sequences. The clean sequence data from each sample were mapped to the human reference genome (GRCh37/HG19) using Burrows–Wheeler Aligner (BWA) software ( http://bio-bwa.sourceforge.net ) with a data analysis quality control (QC) system. The 700 customized oncogene panel sequencing was performed at the Northwestern Diagnostic Molecular Biology Laboratory. The DNA library was hybridized to bridge probes targeting 700 genes. Bridge probes were captured with an anchor probe labeled with biotin and magnetic streptavidin‐coated beads. The library was purified, quantified, and normalized into a sequencing pool and sequenced using a NovaSeq® 6000 and a S1 flowcell (Illumina, San Diego, CA, USA). Sequence data were aligned using BWA1 (version 0.7.17) to human genome version hg38 (Human genome build 38). Variants were called using Vardict2 (version 1.8.3) and Pindel3 (version 0.2.5b). Variants were annotated using wAnnovar4 . Additional details are provided in Supplementary and methods. The data were analyzed as follows: reads from cleaned sequence data were mapped to the hg19 (UCSC) and DNA sequences from normal myometrium using BWA (version 0.7.5) with parameters ‘mem–t 8 –P –M’. Generated files were sorted, and PCR duplicates were removed using Picard (version 1.105) ( http://broadinstitute.github.io/picard ). Subsequently, the BAM files were indexed using samtools (version 0.1.19). The WGS had a quality score of 20 (Q20), and 95–96% of reads were successfully aligned (supplementary material, Figure ). Statistical analysis was performed in GraphPad Prism (GraphPad Software, San Diego, CA, USA) utilizing unpaired t ‐tests and one‐way ANOVA. P values less than 0.05 were considered statistically significant, and p values were defined as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Clinical and gross findings Ten spindle cell LMS with adjacent LM‐BN and one LMS with adjacent STUMP were included in this study. The median age at the time of diagnosis was 47 (34–55). The median clinical follow‐up was 33.5 months. The mean tumor size was 8.9 ± 2.1 cm, and three cases (3/11, 27.3%) presented with metastatic disease. Six cases were at FIGO stage I, two stage II, one stage III, and two stage IV. Two patients died of disease (18.2%), and among nine patients alive, one had disease recurrence while the remainder are disease free with median follow‐up of 27 months (5–107 months). Six (54.5%) cases had separate leiomyoma(s). The demographic and clinical characteristics are summarized in Table and supplementary material, Table . Microscopic and histologic findings Microscopic analysis demonstrated the presence of distinct, well‐demarcated LMS and LM‐BN regions in all cases (Figure ). Available imaging and gross pathology are illustrated in supplementary material, Figure . The spatial relationships of distinct regions of LM‐BN and LMS were most easily observed through side‐by‐side comparison of H&E‐ and Ki‐67‐stained slides, showing the unique distributions of LM‐BN and LMS regions (Figure ). The LMS regions showed hypercellular tumor cells with atypia, frequent mitoses, necrosis, and higher Ki‐67 index in comparison to LM‐BN regions. LM‐BN was characterized by symplastic‐like nuclear atypia, few mitoses, and absence of necrosis. All cases demonstrated the distinct spatial distribution of LM‐BN and LMS components, highlighted by H&E‐ and Ki‐67‐stained slides (Figure ). A closer view of histologic features in all 11 cases were summarized in supplementary material, Figure . The average tumor volume of LM‐BN components was 23% and 77% for LMS components. The density and distribution of nuclear atypia varied within LM‐BN regions, and mean mitotic counts were lower in LM‐BN (mean 1.7 ± 0.52) than in LMS (24.3 ± 2.78) ( p <0.0001). Tumor necrosis was identified solely in the LMS components. The specific histological findings of each component are summarized in Table . Immunohistochemistry findings All tumors underwent routine IHC to confirm the LMS diagnosis utilizing smooth muscle markers (SMA, desmin, or H‐caldesmon) and melanoma markers (HMB45, Melan A) to exclude PEComa. IHC for ER, PR, p16, and p53 performed in all available cases demonstrated similar expression patterns to previously published data for sporadic LMS and LM‐BN (Figure and supplementary material, Table ) . Mean Ki‐67 expression was significantly higher in LMS regions (44.5%) than LM‐BN regions (5.9%) ( p < 0.001, Figure ). ER and PR expression was higher in LM‐BN (medium 80% and 79%) than LMS (37% and 35%) ( p = 0.0029 and p = 0.0086, Figure ). Diffuse immunoreactivities for p16 and p53 were slightly higher in LMS (90.9%, 72.7%) than LM‐BN (81.8%, 54.5%) without statistical significance (Figure ). FH expression was present in all tumors. Digital image and AI analysis To further solidify the spatial relationships between LMS and LM‐BN, QuPath cell detection and image classifier algorithms were applied to present whole‐slide images with regional annotation assistance. The cell classifier software defined cellular parameters and confirmed the presence of the spatial distribution of different histologic patterns. Based on the cytologic features, AI defined the distinct distribution patterns of LMS (red), LM‐BN (yellow), and LM/myometrium (green), and a color‐coded map was prepared from each case (Figure ). Small overlaps were identified, likely secondary to mild morphologic overlap that exists in cellular details. Cell detection analysis revealed subtle nuclear feature differences between LMS and LM‐BN (Figure and supplementary material, Table ). There were statistically significant differences in nuclear area between LMS and myometrium ( p = 0.0486) and between LM‐BN and myometrium ( p = 0.0017), with no significance between LMS and LM‐BN ( p = 0.1237). LM‐BN had more substantial nuclear hyperchromasia compared to LMS ( p = 0.0177) and myometrium ( p < 0.0001), with LMS having more nuclear hyperchromasia compared to myometrium ( p = 0.0004) Nuclear‐to‐cytoplasmic ratio was most increased in LMS compared to LM‐BN ( p < 0.0001) and myometrium ( p < 0.0001), as well as LM‐BN compared to myometrium ( p = 0.0016). Assessment of DNA CNAs Genome‐wide CNAs were analyzed by chromosomal microarray analysis in nine of 11 cases. DNA from annotated and macrodissected LM‐BN and LMS regions were prepared. CNAs and CN‐LOH plots between paired LMS and LM‐BN lesions were compared and analyzed (Table ). The presence of shared CNA loci between LM‐BN and LMS was identified in all cases (Figure ), strongly suggesting the same origin of two tumor components. The mean site number of CNA is higher in LMS (27.11 ± 3.15) than in LM‐BN (12.22 ± 2.90) ( p = 0.0031), (Figure ) and the mean fraction of genome alteration in LMS was 29.11% of genomic DNA (range 9.39–56.96%) in comparison to 5.79% (range 0.87–10.82%) in LM‐BN, indicating a higher level of DNA instability in LMS (Figure , p = 0.0028). There was no difference in CN‐LOH (Figure ). The sites of total CNAs were 332, including 71 copy number gains, 214 copy number losses, and 47 CN‐LOH. The site and fraction of CNA was summarized in Figure and supplementary material, Table . The common CN losses in LM‐BN component were 13q ( RB1 , 88.9%), 17p ( TP53 , 66.7%), 6p (55.5%), and 14q (44.4%), and in LMS component were 13q ( RB1 , 100%), 10q ( PTEN , 77.8%), 17p ( TP53 , 88.9%), 14q (77.8%), 2q (66.7%), 1p, 4q, 5q, 6q, 7, 9p, and 16q (55.5%), and the common CN gains in LMS were 1q, 2q, 8q, 9q, 13q, 17p, and 17q (33.3%). Of all CNAs observed, the average percentage of shared CNAs between LM‐BN and LMS regions was 29.4% (11.1–76.3%), suggesting LM‐BN and LMS lesions in examined cases had same tumoral origin. The most common shared loci between LMS and LM‐BN were losses in 13q (88.9%), 17p (66.7%), 6p (55.5%), 14q (44.4%), 6q (33.3%), 10q (33.3%), and 16q (33.3%). CN losses accounted for the vast majority of CNAs, accounting for 68.1% of LM‐BN, 65.4% of LMS, and 66.1% of overall CNAs. Notably, these shared CN losses showed several specific genomic alterations in chromosomal loci encoding major genes in tumorigenesis, including PTEN (10q23.31, 11.1% in LN‐BN versus 44.4% in LMS), RB1 (13q14.2) (77.8% in LN‐BN versus 100% in LMS), and TP53 (17p13.1, 66.7% in LN‐BN versus 88.9% in LMS). CN loss of CDKN2A/2B (9p21.3) was only observed in LMS (11.1%) (Table ). Additional CNAs only identified within LMS included alterations in 3p, 5p, 9q, 10p, 12p, 17q, and 21p. Spatial transcriptome analysis ( RNA ‐seq) Sections from four cases of LMS with adjacent LM‐BN that passed RNA quality testing were utilized for spatial transcriptome analysis (supplementary material, Figure ). The depth of reads with spots in tissue is summarized in supplementary material, Table . Spatial transcriptome analysis highlighted distinctly defined geographical patterns of transcription in LM‐BN, LMS, and MM/LM. Importantly, the spatial transcriptome map matched the tumor region and type with the corresponding histology (Figure ). Unsupervised analysis identified a total of 21 clusters across the four cases (Figure ), highlighting distinct regional variances based on RNA signatures. Two top ranking genes, E2F1 (checkpoint regulator) and PGR (progesterone receptor), in all four cases were selected as examples for electronic in situ analysis that revealed distinct expression patterns between LMS and LM‐BN regions, demonstrating their unique geographical transcription differences (Figure ). Quantitative analysis showed significant differences of these two genes between LMS and LM‐BN regions ( p < 0.001, Figure ). To define the zonal difference of spatial transcriptome between LM‐BN and LMS, case 3 was subjected to heightened analysis of cell‐type‐specific transcription . When the gene signatures were defined in 10 specific cell types, the regional distribution of the cell types was highly correlated with histologic cell types (Figure ). As expected, LMS cells showed more heterogeneity in cluster analysis (Figure ) and presented four gene signatures (Figure ). Of note, LM‐BN regions showed two gene signatures (Figure ). The LMS region contained fibroblast, stemness, undifferentiated, or unknown cell natures, suggesting a complex nature of tumor differentiation (Figure ). This might be highly relevant to the complex CNA in LMS (Figure ). Pathway analysis revealed different functional pathways between LMS. MM and LM‐BN (Figure ), and the six top‐ranking pathways in LMS correlated with aggressiveness and progression of tumor nature (Figure ). To further analyze the gene expression, some top‐ranking genes from LM‐BN (Figure ), LM (Figure ), and LMS (Figure ) were selected for spatial geographic expression analysis, and distinct gene expression patterns were highly correlated with the histologically defined tumor natures. The global and spatial transcriptome demonstrated the different gene expression profiles among LM, LM‐BN, and LMS, indicative of the different stages of tumorigenesis. Mutational profiling on oncogene panel To evaluate the connection and tumor progression of LMS from existing LM‐BN, gene mutation analysis from 700 oncogene panels or exon sequencing was performed in six cases (supplementary material, Table ). Variant calling was subjected to analysis and referred to established variant libraries (ClinVar, OncoKB, ClinGen). The top 18 mutated genes were selected for further analysis. As illustrated in Figure , this subset of oncogenes and tumor suppressors was frequently mutated in both LM‐BN and LMS, with significant overlap. While mutant genes were very different in each case, there were trends of shared oncogenic mutations between LM‐BN and LMS components, defined as identical nucleotide changes. Some genes were found to be mutated in either LM‐BN or LMS, but not both, suggesting subclonal mutation events (supplementary material, Table ). Of note, LMS accumulated more oncogenic mutations than LM‐BN. The high rate of RB1 and TP53 alterations, by either CNA or mutation, in both LM‐BN and LMS is consistent with previous studies . Pathway analysis showed that the major oncogenic genes were involved in DNA damage response and cell cycle alteration (Figure ). Several oncogenes frequently mutated in endometrial and ovarian cancer ( ARID1B , ATM , BRCA1 , CHD2 , POLE , and PMS2 ) were also present in LMS. PMS2 was frequently mutated in four of six cases (Figure ). NTRK1 missense mutations were found in three of six LMS components; however, they were not identified within LM‐BN components. The patterns of mutations in these two components suggest (1) there is a wide spectrum of gene mutations in both LM‐BN and LMS; (2) most gene mutations are likely secondary events; and (3) many shared mutations between LM‐BN and LMS support their clonal connection in these tumor components. Ten spindle cell LMS with adjacent LM‐BN and one LMS with adjacent STUMP were included in this study. The median age at the time of diagnosis was 47 (34–55). The median clinical follow‐up was 33.5 months. The mean tumor size was 8.9 ± 2.1 cm, and three cases (3/11, 27.3%) presented with metastatic disease. Six cases were at FIGO stage I, two stage II, one stage III, and two stage IV. Two patients died of disease (18.2%), and among nine patients alive, one had disease recurrence while the remainder are disease free with median follow‐up of 27 months (5–107 months). Six (54.5%) cases had separate leiomyoma(s). The demographic and clinical characteristics are summarized in Table and supplementary material, Table . Microscopic analysis demonstrated the presence of distinct, well‐demarcated LMS and LM‐BN regions in all cases (Figure ). Available imaging and gross pathology are illustrated in supplementary material, Figure . The spatial relationships of distinct regions of LM‐BN and LMS were most easily observed through side‐by‐side comparison of H&E‐ and Ki‐67‐stained slides, showing the unique distributions of LM‐BN and LMS regions (Figure ). The LMS regions showed hypercellular tumor cells with atypia, frequent mitoses, necrosis, and higher Ki‐67 index in comparison to LM‐BN regions. LM‐BN was characterized by symplastic‐like nuclear atypia, few mitoses, and absence of necrosis. All cases demonstrated the distinct spatial distribution of LM‐BN and LMS components, highlighted by H&E‐ and Ki‐67‐stained slides (Figure ). A closer view of histologic features in all 11 cases were summarized in supplementary material, Figure . The average tumor volume of LM‐BN components was 23% and 77% for LMS components. The density and distribution of nuclear atypia varied within LM‐BN regions, and mean mitotic counts were lower in LM‐BN (mean 1.7 ± 0.52) than in LMS (24.3 ± 2.78) ( p <0.0001). Tumor necrosis was identified solely in the LMS components. The specific histological findings of each component are summarized in Table . All tumors underwent routine IHC to confirm the LMS diagnosis utilizing smooth muscle markers (SMA, desmin, or H‐caldesmon) and melanoma markers (HMB45, Melan A) to exclude PEComa. IHC for ER, PR, p16, and p53 performed in all available cases demonstrated similar expression patterns to previously published data for sporadic LMS and LM‐BN (Figure and supplementary material, Table ) . Mean Ki‐67 expression was significantly higher in LMS regions (44.5%) than LM‐BN regions (5.9%) ( p < 0.001, Figure ). ER and PR expression was higher in LM‐BN (medium 80% and 79%) than LMS (37% and 35%) ( p = 0.0029 and p = 0.0086, Figure ). Diffuse immunoreactivities for p16 and p53 were slightly higher in LMS (90.9%, 72.7%) than LM‐BN (81.8%, 54.5%) without statistical significance (Figure ). FH expression was present in all tumors. AI analysis To further solidify the spatial relationships between LMS and LM‐BN, QuPath cell detection and image classifier algorithms were applied to present whole‐slide images with regional annotation assistance. The cell classifier software defined cellular parameters and confirmed the presence of the spatial distribution of different histologic patterns. Based on the cytologic features, AI defined the distinct distribution patterns of LMS (red), LM‐BN (yellow), and LM/myometrium (green), and a color‐coded map was prepared from each case (Figure ). Small overlaps were identified, likely secondary to mild morphologic overlap that exists in cellular details. Cell detection analysis revealed subtle nuclear feature differences between LMS and LM‐BN (Figure and supplementary material, Table ). There were statistically significant differences in nuclear area between LMS and myometrium ( p = 0.0486) and between LM‐BN and myometrium ( p = 0.0017), with no significance between LMS and LM‐BN ( p = 0.1237). LM‐BN had more substantial nuclear hyperchromasia compared to LMS ( p = 0.0177) and myometrium ( p < 0.0001), with LMS having more nuclear hyperchromasia compared to myometrium ( p = 0.0004) Nuclear‐to‐cytoplasmic ratio was most increased in LMS compared to LM‐BN ( p < 0.0001) and myometrium ( p < 0.0001), as well as LM‐BN compared to myometrium ( p = 0.0016). DNA CNAs Genome‐wide CNAs were analyzed by chromosomal microarray analysis in nine of 11 cases. DNA from annotated and macrodissected LM‐BN and LMS regions were prepared. CNAs and CN‐LOH plots between paired LMS and LM‐BN lesions were compared and analyzed (Table ). The presence of shared CNA loci between LM‐BN and LMS was identified in all cases (Figure ), strongly suggesting the same origin of two tumor components. The mean site number of CNA is higher in LMS (27.11 ± 3.15) than in LM‐BN (12.22 ± 2.90) ( p = 0.0031), (Figure ) and the mean fraction of genome alteration in LMS was 29.11% of genomic DNA (range 9.39–56.96%) in comparison to 5.79% (range 0.87–10.82%) in LM‐BN, indicating a higher level of DNA instability in LMS (Figure , p = 0.0028). There was no difference in CN‐LOH (Figure ). The sites of total CNAs were 332, including 71 copy number gains, 214 copy number losses, and 47 CN‐LOH. The site and fraction of CNA was summarized in Figure and supplementary material, Table . The common CN losses in LM‐BN component were 13q ( RB1 , 88.9%), 17p ( TP53 , 66.7%), 6p (55.5%), and 14q (44.4%), and in LMS component were 13q ( RB1 , 100%), 10q ( PTEN , 77.8%), 17p ( TP53 , 88.9%), 14q (77.8%), 2q (66.7%), 1p, 4q, 5q, 6q, 7, 9p, and 16q (55.5%), and the common CN gains in LMS were 1q, 2q, 8q, 9q, 13q, 17p, and 17q (33.3%). Of all CNAs observed, the average percentage of shared CNAs between LM‐BN and LMS regions was 29.4% (11.1–76.3%), suggesting LM‐BN and LMS lesions in examined cases had same tumoral origin. The most common shared loci between LMS and LM‐BN were losses in 13q (88.9%), 17p (66.7%), 6p (55.5%), 14q (44.4%), 6q (33.3%), 10q (33.3%), and 16q (33.3%). CN losses accounted for the vast majority of CNAs, accounting for 68.1% of LM‐BN, 65.4% of LMS, and 66.1% of overall CNAs. Notably, these shared CN losses showed several specific genomic alterations in chromosomal loci encoding major genes in tumorigenesis, including PTEN (10q23.31, 11.1% in LN‐BN versus 44.4% in LMS), RB1 (13q14.2) (77.8% in LN‐BN versus 100% in LMS), and TP53 (17p13.1, 66.7% in LN‐BN versus 88.9% in LMS). CN loss of CDKN2A/2B (9p21.3) was only observed in LMS (11.1%) (Table ). Additional CNAs only identified within LMS included alterations in 3p, 5p, 9q, 10p, 12p, 17q, and 21p. RNA ‐seq) Sections from four cases of LMS with adjacent LM‐BN that passed RNA quality testing were utilized for spatial transcriptome analysis (supplementary material, Figure ). The depth of reads with spots in tissue is summarized in supplementary material, Table . Spatial transcriptome analysis highlighted distinctly defined geographical patterns of transcription in LM‐BN, LMS, and MM/LM. Importantly, the spatial transcriptome map matched the tumor region and type with the corresponding histology (Figure ). Unsupervised analysis identified a total of 21 clusters across the four cases (Figure ), highlighting distinct regional variances based on RNA signatures. Two top ranking genes, E2F1 (checkpoint regulator) and PGR (progesterone receptor), in all four cases were selected as examples for electronic in situ analysis that revealed distinct expression patterns between LMS and LM‐BN regions, demonstrating their unique geographical transcription differences (Figure ). Quantitative analysis showed significant differences of these two genes between LMS and LM‐BN regions ( p < 0.001, Figure ). To define the zonal difference of spatial transcriptome between LM‐BN and LMS, case 3 was subjected to heightened analysis of cell‐type‐specific transcription . When the gene signatures were defined in 10 specific cell types, the regional distribution of the cell types was highly correlated with histologic cell types (Figure ). As expected, LMS cells showed more heterogeneity in cluster analysis (Figure ) and presented four gene signatures (Figure ). Of note, LM‐BN regions showed two gene signatures (Figure ). The LMS region contained fibroblast, stemness, undifferentiated, or unknown cell natures, suggesting a complex nature of tumor differentiation (Figure ). This might be highly relevant to the complex CNA in LMS (Figure ). Pathway analysis revealed different functional pathways between LMS. MM and LM‐BN (Figure ), and the six top‐ranking pathways in LMS correlated with aggressiveness and progression of tumor nature (Figure ). To further analyze the gene expression, some top‐ranking genes from LM‐BN (Figure ), LM (Figure ), and LMS (Figure ) were selected for spatial geographic expression analysis, and distinct gene expression patterns were highly correlated with the histologically defined tumor natures. The global and spatial transcriptome demonstrated the different gene expression profiles among LM, LM‐BN, and LMS, indicative of the different stages of tumorigenesis. To evaluate the connection and tumor progression of LMS from existing LM‐BN, gene mutation analysis from 700 oncogene panels or exon sequencing was performed in six cases (supplementary material, Table ). Variant calling was subjected to analysis and referred to established variant libraries (ClinVar, OncoKB, ClinGen). The top 18 mutated genes were selected for further analysis. As illustrated in Figure , this subset of oncogenes and tumor suppressors was frequently mutated in both LM‐BN and LMS, with significant overlap. While mutant genes were very different in each case, there were trends of shared oncogenic mutations between LM‐BN and LMS components, defined as identical nucleotide changes. Some genes were found to be mutated in either LM‐BN or LMS, but not both, suggesting subclonal mutation events (supplementary material, Table ). Of note, LMS accumulated more oncogenic mutations than LM‐BN. The high rate of RB1 and TP53 alterations, by either CNA or mutation, in both LM‐BN and LMS is consistent with previous studies . Pathway analysis showed that the major oncogenic genes were involved in DNA damage response and cell cycle alteration (Figure ). Several oncogenes frequently mutated in endometrial and ovarian cancer ( ARID1B , ATM , BRCA1 , CHD2 , POLE , and PMS2 ) were also present in LMS. PMS2 was frequently mutated in four of six cases (Figure ). NTRK1 missense mutations were found in three of six LMS components; however, they were not identified within LM‐BN components. The patterns of mutations in these two components suggest (1) there is a wide spectrum of gene mutations in both LM‐BN and LMS; (2) most gene mutations are likely secondary events; and (3) many shared mutations between LM‐BN and LMS support their clonal connection in these tumor components. The tumorigenesis of LMS has long been a topic of debate. While different inciting agents have been implicated in the development of LMS, these etiologies are few and far between . Patients with germline tumor suppressor mutations, notably RB1 and TP53 in hereditary retinoblastoma and Li–Fraumeni syndrome, respectively, are at increased risk for the development of LMS . LMS coexisting with benign leiomyoma has been occasionally reported . The theorized nature of myometrial dysplasia, as well as intertumoral areas of LM or LM‐BN, has also been suggested as a harbinger of LMS development . While these studies advanced our knowledge of LMS, only one case series by Mittal et al observed intertumoral ‘leiomyoma‐like’ or ‘symplastic leiomyoma‐like’ regions within 26 cases of LMS and demonstrated that these cases sharing genomic alterations of leiomyoma‐like components with LMS . Uterine LM‐BN is a rare variant of leiomyoma . Of the 150 cases reported, LM‐BN shows low recurrence rates and no disease‐related deaths, supporting a benign course . However, LM‐BN is a genomically unstable tumor with increased CNAs, of which many are shared with LMS . TP53 and RB1 alterations are common in the cases of LMS with LM‐BN, which has patterns similar to those seen in sporadic LM‐BN and LMS . The molecular relation between LM‐BN and LMS remains unknown. Herein, we present a case series detailing uterine LMS arising against a background of an associated LM‐BN (Figure ). These cases had clear‐cut boundaries between the different histologic components, supported by IHC and digital analysis (Figure ). Of the nine tumors submitted for CNA analysis, all showed regions of shared CNAs in LM‐BN and LMS components, suggesting a similar tumor origin (Figure ). Increased CNAs were noted in LMS regions, demonstrating high genomic instability toward malignancy. The most shared loci of CNA between the two tumor components were losses in 13q (88.9%) and 17p (66.7%), notably encompassing RB1 and TP53 , respectively, while LMS only demonstrated alterations of CDKN2A/2B . RNA‐seq spatial transcriptome analysis showed striking geographical expression profile differences between LM‐BN and LMS regions (Figures and ). These clear‐cut expression differences were further analyzed with cell‐specific cluster analysis, which showcased significant differences in cell expression between LMS and LM‐BN, proving the regional difference of gene expression led to two distinct histologic components. Further assessment of the shared nature of uterine LMS and LM‐BN was achieved by oncogene mutation analysis (Figure ). A significant overlap in mutations within each case was identified, further demonstrating the connection between these lesions. The frequently mutated oncogenes in this cohort included ARID1B , BLM , PMS2 , POLE, TP53 , and RB , which are major players in numerous pathways associated with tumorigenesis that are involved in DNA repair/recombination and cell cycle regulation. Additionally, missense NTRK1 mutations were identified in 50% (3/6) of LMS cases; however, these were not within the kinase domain. NTRK1 rearrangements by gene fusion were previously found in a subset of cervical/uterine sarcomas, but not in LMS . The utilization of AI through cell detection software allowed for the identification of the spatial relationship between LMS, LM‐BN, LM, and myometrium, which could become a supplemental tool for pathologists. These cellular differences and distribution of each tumor component may not always be readily apparent in slide review. Additionally, according to WHO2020, smooth muscle neoplasms with a high density of nuclear atypia and five to nine mitoses/10 HPFs in younger women could be considered to be STUMP, and recent reports detailing IHC workflows have shown the power in using surrogate IHC markers for genetic alterations for challenging cases . Until such next‐generation IHC markers are available, deep learning models may assist with ascertaining selected lesional components that are well established in different cancer studies . Overall, this study provides a comprehensive multimodal histologic and molecular approach to defining the relationship between LMS and LM‐BN in a single mass (Figure ) and provides reasonable observations of the histogenesis in a subset of uterine LMS. These findings were also corroborated in our previous study comparing de novo LM‐BN and LMS . This study reports the spatially separate regions of LM‐BN and LMS in different tumor development stages characterized by their different transcriptomic and gene mutation patterns, such as CDKN2A/B loss in LMS. This is the first study with a comprehensive analysis of the relationship of LMS with adjacent LM‐BN utilizing a multi‐omics approach. Our findings suggest that some LM‐BN can be molecularly related to LMS. The limitation of this study includes its small sample size and further molecular profiling of all cases was not feasible given the nature of cases. While nine of our 11 cases are alive, the relative short‐term follow‐up in these cases precludes a meaningful survival analysis . In a study of 11 cases, we elucidated the histologic, immunohistochemical, and molecular findings in LMS cases arising from LM‐BN. The molecular analysis identified conserved molecular changes between LMS and LM‐BN, with further progressive genomic alterations identified in LMS components. Transcriptome analysis proved the distinct RNA signatures of the LM‐BN and LMS regions. We argue that a subset of LMS arises from an existing LM‐BN, and highly complex genomic alterations could potentially be a high risk associated with disease progression in LM‐BN. We propose a genomic profiling analysis for all LM‐BN for risk assessment and to potentially improve clinical management. J‐JW and SEB developed the study concept and design. CF, J‐JW, YF, ZC‐F, MJS and PY developed the methodology. CF, XL, LJJ, MJS and J‐JW acquired, analyzed and interpreted data and performed statistical analyses. J‐JW, CF and YF provided technical and material support. J‐JW, JK and SEB provided funding support. CF and J‐JW contributed to writing, review and revision of the manuscript. All authors read and approved the final submitted version. Supplementary materials and methods Figure S1. Representative radiographic and macroscopic gross images for selected cases Figure S2. Histologic and Ki‐67 comparison between LM‐BN and LMS components Figure S3. Representative QuPath digital AI analysis Figure S4. Spatial transcriptome analysis stepwise algorithmic analysis followed a detailed step‐by‐step process to identity spatial expression profiling within LM‐BN and LMS components Table S1. Clinical information for cases Table S2A. Antibody details Table S2B. Immunohistochemical staining patterns in LM‐BN and LMS components Table S3. AI‐based nuclear features in LM‐BN and LMS Table S4A. Chromosomal CNA and LOH in nine cases Table S4B. CNA raw data Table S5A. Summary metrics Table S5B. Top 10 ranking genes and differentially expressed genes between LMS, LM‐BN, and LM detected by spatial transcriptomic analysis Table S5C. Differentially expressed genes in each component Table S6A. Gene mutation types detected using 700 oncogene panel Table S6B. The 700 gene panel |
A rare dental avulsion case report highlighting the importance of rapid replantation for long-term survival | c2034dcf-73e8-435c-bf58-5aca268ab7cb | 11849884 | Surgical Procedures, Operative[mh] | Dental avulsion, the complete displacement of a tooth from its socket, is a traumatic injury that frequently occurs in children. It poses a significant challenge in pediatric dentistry due to its potential long-term consequences on oral health, dental function, and quality of life. Prompt and appropriate management of dental avulsion is crucial for preserving the vitality of the avulsed tooth and preventing complications. This case report provides clinical information showing that teeth replanted in accordance with established protocols-when avulsed teeth are delivered to a pediatric dentist within the recommended time frame and in an appropriate transport medium-can maintain their function for years post-treatment. Additionally, it is demonstrated that fixed orthodontic treatment applied six months after replantation does not cause any radiological or clinical pathology in the teeth. A healthy 11-year-old male patient presented to the pediatric dentistry clinic immediately after sustaining a traumatic injury during a sports activity. Upon examination, it was observed that the patient had suffered an avulsion injury to the maxillary central and lateral incisors (teeth #11, #12, #21, and #22). The patient was brought to the clinic by the school nurse and parents within 60 minutes of the injury. Informed written consent was obtained before any examination and treatment were performed. Following the acquisition of consent, the patient was promptly evaluated by the pediatric dentist and consulted with an oral and maxillofacial surgeon for soft tissue injuries. A decision was made to attempt replantation of the avulsed tooth. Intraoral findings showed no carious lesions and previous restorative treatments. No periodontal pathology was observed. The patient had been undergoing treatment with a fixed orthodontic appliance, and the avulsed teeth were still present inside the mouth but outside the socket due to the appliance wires . Recognizing the urgency of the situation, the patient’s orthodontist was informed via phone call, and a second written consent to postpone orthodontic treatment was obtained from the parents. Immediate replantation was initiated by the pediatric dentist. Infiltrative local anesthesia was administered to control bleeding and pain. The portion of the fixed orthodontic appliance encompassing the avulsed teeth was cut away. Since no debris or dirt were observed on the teeth, rinsing was not performed. The teeth were repositioned into their sockets with gentle digital pressure, and bleeding control was achieved. After reposining and controlling the bleeding, a flexible splint was applied, and the interdental papillae were sutured to stabilize the repositioned teeth . A follow-up periapical X-ray was taken to confirm that the repositioned teeth were properly seated in their sockets. The necessary prescriptions were provided, and the patient was scheduled for regular follow-up appointments to monitor the healing process of the replanted teeth. Since reattachment of the gingival connective tissue and periodontal membrane of the root occurs within 2 to 7 days, the sutures of the healed interdental papillae were removed using a scalpel. Two weeks after replantation, calcium hydroxide was placed into the root canals. The root canal treatments were completed in three weeks , and the splint was removed during the same appointment. Since no clinical or radiological complications were detected at the 6-month follow-up after replantation, the patient’s orthodontic treatment was resumed . At the 12-month follow-up appointment, the patient’s maxillary incisors showed complete healing, with no evidence of root resorption or ankylosis on clinical and radiological examinations. The healing process included the reattachment of periodontal ligament fibers and bone remodeling around the root . The teeth continued to function properly, and periodontal health was maintained 4 years after the incident, indicating the high success of the rapid replantation procedure . The primary causes of dentoalveolar injuries commonly involve accidents such as falls, particularly in children, cycling incidents, participation in full-contact sports, traffic collisions, and instances of assault. Dental trauma frequently occurs in domestic environments, educational institutions, and sports facilities. The highest occurrence of dental injuries is observed in the 7-11 age group, with males experiencing incidents at twice the rate of females. Permanent teeth are more susceptible to injury compared to primary teeth, accounting for 60% versus 40%, respectively. A study involving 800 children aged 11-13 revealed that slightly more than half had experienced dental trauma affecting their permanent front teeth.9 Furthermore, it has been documented that at least 32% of athletes engaged in full-contact sports have suffered some form of dental injury. When a dental avulsion occurs, immediate action is essential to maximize the chances of successful replantation and minimize the risk of complications. Emergency precautions play a crucial role in ensuring the optimal outcome of avulsed tooth management. Key emergency precautions include rapid response, careful handling, avoiding dehydration, minimizing additional trauma, and proper storage. Time is critical in dental avulsion cases. Immediate action should be taken to locate the avulsed tooth and administer appropriate first aid measures. The tooth should be handled with extreme care to avoid damaging the delicate periodontal ligament (PDL) fibers attached to its root surface. Touching or scrubbing the root surface should be avoided to prevent further injury. In addition to careful handling, keeping the avulsed tooth moist is vital for preserving the vitality of the PDL cells. If possible, the tooth should be repositioned in its socket immediately. If replantation is not feasible, the tooth should be stored in an appropriate medium to prevent dehydration. The choice of storage medium for an avulsed tooth significantly influences the chances of successful replantation and long-term prognosis. Ideally, the medium should maintain the vitality of the PDL cells and prevent desiccation. Commonly recommended storage media include milk, saline solution, saliva, and cell culture media. In this case, since the avulsed teeth were extra-alveolar but remained in the oral cavity due to the fixed orthodontic appliance, the teeth were promptly delivered to the clinic without the need for any storage medium. As the avulsed teeth were within the patient’s mouth, there was no additional trauma to the PDL. Replantation was attempted as soon as possible, approximately within 30 minutes of the patient arriving at the dental chair. In addition to managing the avulsed teeth, any associated soft tissue injuries should be promptly assessed and treated to minimize trauma and facilitate healing. In the presented case, the soft tissue injuries were sutured, and their healing was supported with appropriate splinting techniques. Stabilization of the replanted tooth is essential to promote healing and prevent displacement. Splinting with a flexible splint or composite material is typically performed for a period of 7 to 21 days to immobilize the tooth and facilitate reattachment of the PDL fibers, as was applied in this case. Additional key management strategies following immediate replantation and splinting include antibiotic therapy, pain management, clinical and radiographic evaluations, pulp treatments, and orthodontic interventions when necessary. Prophylactic antibiotic therapy may be prescribed to reduce the risk of infection after dental avulsion. Broad-spectrum antibiotics, such as amoxicillin or penicillin, are commonly used, especially in cases with soft tissue injuries or open fractures. Analgesic medication may also be prescribed to alleviate pain and discomfort associated with the dental avulsion injury and subsequent treatment. Nonsteroidal anti-inflammatory drugs (NSAIDs) are often recommended for pain relief, with appropriate dosage adjustments based on the child’s age and weight. In this case, following the completion of replantation, the patient was prescribed amoxicillin and ibuprofen, with the dosages tailored to the patient’s weight and age. The use of a sodium bicarbonate-based mouthwash was also recommended to maintain oral hygiene. Long-term follow-up care is essential for monitoring the healing process, assessing the vitality of the replanted tooth, and detecting any signs of complications. Follow-up appointments should be scheduled at regular intervals, initially on a weekly basis during the first month, and then gradually extending to monthly intervals over the following months. Regular clinical examinations are necessary to evaluate the stability of the replanted tooth, assess periodontal health, and detect any signs of root resorption or ankylosis. Periodic radiographic assessments, using periapical or panoramic radiographs, are performed to evaluate the integrity of the root structure, detect signs of root resorption, and monitor the healing process. In this case, the patient was monitored according to current dental avulsion protocols, with weekly dental check-ups during the first month and both clinical and radiographic examinations conducted in the second week following replantation. The patient was subsequently evaluated clinically and radiologically at 6 and 12 months post-replantation, and again at 2 and 4 years. No clinical or radiological pathology was detected in the replanted maxillary incisors during all follow-up visits. Endodontic treatment may be indicated if signs of pulp necrosis or periapical pathology develop in the replanted tooth. Root canal therapy can help preserve the tooth’s functionality and prevent further complications. In a mature (closed-apex) tooth, endodontic treatment begins before splint removal (1 to 2 weeks after replantation). A calcium hydroxide preparation is used for the initial filling and to monitor periodontal healing. In an immature (open-apex) tooth, it is recommended to wait until pulp necrosis is confirmed, as the pulp tissue may revascularize. If inflammatory root resorption is detected, endodontic treatment must be performed immediately. In this case, since the apices of the avulsed maxillary incisors were completely closed, root canal dressing was performed in the second week after replantation according to protocol, and root canal treatment was completed in the third week, after which the splint was removed. Orthodontic treatment may be required to address any occlusal disturbances or malalignment resulting from dental avulsion. Early intervention can help optimize the aesthetic and functional outcomes of treatment. In this case, the patient was undergoing fixed orthodontic treatment due to malocclusion prior to the trauma, and a fixed orthodontic appliance was present on the maxillary teeth at the time of injury. Due to the avulsion trauma, orthodontic treatment was interrupted for 6 months and then resumed, with the treatment being completed in the second year after replantation. Throughout the orthodontic treatment period, no clinical and/or radiological pathology was detected in the replanted teeth. In conclusion, this case highlights the paramount importance of prompt and effective management in achieving successful outcomes following dental avulsion. Rapid replantation, supported by immediate emergency care, is crucial for optimizing the prognosis of avulsed teeth and reducing the risk of complications. Key interventions include careful handling of the avulsed tooth, appropriate storage in a suitable medium if immediate replantation is not possible, and comprehensive follow-up care. The involvement of a multidisciplinary dental team, with expertise in emergency response, endodontics, orthodontics, and ongoing monitoring, is vital to ensuring the long-term success of avulsed tooth treatment. Additionally, raising awareness among emergency departments and emergency physicians about the critical nature of dental avulsion and its management can significantly improve patient outcomes. Effective coordination between dental professionals and emergency healthcare providers is essential for the timely and efficient management of such injuries. This case illustrates that adherence to established protocols, swift action, and cross-disciplinary collaboration can greatly enhance the preservation of oral health and functionality in patients with dental avulsion. |
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