bronchiectasis is a multidimensional disease , and hence , its severity or prognosis could not be defined with any precision using a single variable.13 accordingly , some recently published multidimensional scores combine clinical , functional , microbiological , and radiological variables to evaluate the prognosis and severity of bronchiectasis more comprehensively.2,3 one of these scores is the faced score,3 an acronym derived from five dichotomized variables ( f : forced expiratory volume in 1 s [ fev1 ] ; a : age ; c : chronic colonization by pseudomonas aeruginosa [ pa ] , e : radiological extension [ number of pulmonary lobes affected ] , and d : dyspnea ) .
the faced score is easy to remember and to apply in clinical practice ; it presents a range of values of growing severity spanning 07 points , and it has demonstrated an excellent prognostic capacity for mortality ( both all - cause and respiratory ) in both medium and long term , and an excellent capacity to discriminate different degrees of severity.4 it also had a good external validation in europe as well as in various latin american countries in bronchiectasis patients with wide - ranging etiology and severity and with characteristics different from those of the initial cohort.46 nevertheless , some authors have found that the faced score does not predict exacerbations accurately.6 one of the key clinical aspects in the prognosis and severity of airway diseases is exacerbations , particularly serious ones that require hospitalization . in bronchiectasis , exacerbations have been associated with higher mortality2 and a greater impairment of lung function,7 as well as more severe forms of the disease.8,9 furthermore , another important aspect of exacerbations in these patients is the substantial concomitant health costs.10 as exacerbations are potentially preventable , exacerbations and hospitalizations are main outcomes in most of the clinical studies that seek to evaluate the efficacy and , above all , cost - efficiency of different treatments for bronchiectasis.1116 hence , tools that can predict future exacerbations and particularly those patients ar risk of presenting multiple exacerbations ( especially severe ones requiring hospitalizations ) are required . since the faced score was not constructed with the variable 
exacerbations ,  the present study aimed to analyze , in a wide - ranging series of patients different from the original faced cohort , the prognostic capacity for exacerbations ( and more particularly for patients with multiple exacerbations ) of a new score called e - faced and compare it with that of faced . it also sought to evaluate whether e - faced also maintains the faced score s excellent predictive capacity for all - cause and respiratory mortality , as well as its capacity to discriminate different degrees of severity of bronchiectasis .
this is an observational , multicenter study of two large historical cohorts involving seven teaching centers in spain ( construction series ) and six teaching centers in latin america ( validation series ) , all with multidisciplinary and protocolized monographic bronchiectasis outpatient clinics .
the spanish series corresponds with the one used for the construction and internal validation of the faced score .
it comprised a total of 819 patients with bronchiectasis whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile ,
651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score .
all the patients were diagnosed as having bronchiectasis by means of a primary diagnosis with hrct , and they presented a wide range of severity , etiologies , and clinical and functional alterations , following the criteria described by naidich et al.17 patients aged < 18 years were excluded , along with those whose vital state was unknown at the end of the follow - up .
this study was approved by the ethics and research committee of each participating center ( table s1 ) .
the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score .
in other words , the recorded variables corresponded as closely as possible to a radiological diagnosis of bronchiectasis .
the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . since there is no standard definition of an exacerbation , this study defined an exacerbation as a worsening of the respiratory symptoms concomitant with an increase in the volume or purulence of sputum that requires antibiotic treatment .
this enabled identification of the number of exacerbations more objectively , according to the number of antibiotic treatments prescribed for changes in the volume or purulence of sputum or symptoms .
similarly , a severe exacerbation was defined as one that was considered by the physician to require hospitalization .
patients from both cohorts were followed up for 5 years after the radiological diagnosis of bronchiectasis .
the number of deaths and their causes were identified via the hospitals computerized records or the corresponding death certificates .
the main variable was the prognostic capacity for exacerbations in a year , according to different cut - off points as regards number and severity .
the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity .
the spss 20.0 ( ibm corporation , armonk , ny , usa ) statistical package was used for all the calculations .
the quantitative variables were evaluated as mean ( standard deviation ) in cases of normal distribution or as median ( inter - quartile range ) in other cases .
the qualitative or dichotomic variables were evaluated as an absolute value ( percentage of the total ) .
various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score .
the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of  at least one  ,  at least two  , or  more than two  exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of  at least two exacerbations or one hospitalization  in the previous year .
the dependent variable was the number of deaths from any cause in the spanish construction cohort ( 819 patients ) .
the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score ,
only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci .
a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc . all the results were compared with those obtained from the application of the faced score to the same series of patients .
the roc curves obtained from the e - faced and faced scores were compared by means of c - statistics .
finally , the kaplan  meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . in all cases ,
this is an observational , multicenter study of two large historical cohorts involving seven teaching centers in spain ( construction series ) and six teaching centers in latin america ( validation series ) , all with multidisciplinary and protocolized monographic bronchiectasis outpatient clinics .
the spanish series corresponds with the one used for the construction and internal validation of the faced score . it comprised a total of 819 patients with bronchiectasis
whose characteristics have previously been published.3 this series will be used as the construction cohort for the e - faced . meanwhile ,
651 patients with bronchiectasis from six latin american centers ( four in brazil , one in argentina , and one in chile ) who were the subjects for the external validation of the faced score will also be used for the external validation of the e - faced score .
all the patients were diagnosed as having bronchiectasis by means of a primary diagnosis with hrct , and they presented a wide range of severity , etiologies , and clinical and functional alterations , following the criteria described by naidich et al.17 patients aged < 18 years were excluded , along with those whose vital state was unknown at the end of the follow - up .
this study was approved by the ethics and research committee of each participating center ( table s1 ) .
the variables recorded in both the construction cohort ( n=819 ) and the validation cohort ( n=651 ) were the same as those used in the original studies for the construction , internal validation , and external validation of the faced score .
in other words , the recorded variables corresponded as closely as possible to a radiological diagnosis of bronchiectasis .
the exacerbations , hospitalizations , and microbiological data of the construction cohort pertained to the year prior to the patients inclusion in the study , while these data were also available longitudinally for the validation cohort during the 5 years of the follow - up . since there is no standard definition of an exacerbation , this study defined an exacerbation as a worsening of the respiratory symptoms concomitant with an increase in the volume or purulence of sputum that requires antibiotic treatment .
this enabled identification of the number of exacerbations more objectively , according to the number of antibiotic treatments prescribed for changes in the volume or purulence of sputum or symptoms .
similarly , a severe exacerbation was defined as one that was considered by the physician to require hospitalization .
patients from both cohorts were followed up for 5 years after the radiological diagnosis of bronchiectasis .
the number of deaths and their causes were identified via the hospitals computerized records or the corresponding death certificates .
the main variable was the prognostic capacity for exacerbations in a year , according to different cut - off points as regards number and severity .
the secondary outcomes were established as the predictive capacity for death from any cause or from respiratory causes , and the capacity to discriminate different degrees of severity .
the spss 20.0 ( ibm corporation , armonk , ny , usa ) statistical package was used for all the calculations .
the quantitative variables were evaluated as mean ( standard deviation ) in cases of normal distribution or as median ( inter - quartile range ) in other cases .
the qualitative or dichotomic variables were evaluated as an absolute value ( percentage of the total ) .
various logistic regressions were made to determine the optimal cut - off point for the number of exacerbations and hospitalizations that would add the best independent prognostic value to the faced score .
the independent values in the construction series ( n=819 ) , apart from the value of the faced itself , the cut - off points of  at least one  ,  at least two  , or 
more than two  exacerbations or hospitalizations in the year prior to inclusion in the study , including a composite of  at least two exacerbations or one hospitalization  in the previous year .
the dependent variable was the number of deaths from any cause in the spanish construction cohort ( 819 patients ) .
the prognostic capacity of the best score for the number or severity of the exacerbations was quantified by means of the odds ratio ( or ; 95% confidence interval [ 95% ci ] ) , while the value of the or , rounded up to its highest whole value , was used as a relative weight for the variable in the new score that was formed ( e - faced ) . in order to simplify the score ,
only different cut - off points were used for the number or severity of exacerbations , to make the variable dichotomic , as in the original faced score . once the range of the e - faced score had been calculated and obtained , its prognostic capacity for future exacerbations and hospitalizations over the course of a year ( according to different cut - off points for number and severity ) was determined by using the external validation database ( n=651 ) and calculating the corresponding areas under the receiver operating characteristic curve ( auc - roc ) and 95% ci .
a prognostic capacity was considered excellent if the auc - roc is > 80% of the total area.18 the prognostic capacity of the e - faced for all - cause and respiratory mortality was then calculated , again by using the auc - roc .
all the results were compared with those obtained from the application of the faced score to the same series of patients .
the roc curves obtained from the e - faced and faced scores were compared by means of c - statistics .
finally , the kaplan  meier curves and the log - rank test for comparing the curves two by two were used to determine the capacity of the new index ( e - faced ) to discriminate the degree of severity , in order to divide bronchiectasis into mild ( first tertile ) , moderate ( second tertile ) , or severe ( third tertile ) . in all cases ,
the construction cohort comprised 819 patients with bronchiectasis from the initial spanish cohort for the construction and internal validation of the faced , whereas the external validation cohort ( n=651 ) comprised the latin american cohort for the external validation of the faced .
data from 20 patients from the initial spanish cohort ( 2.4% ) and 21 patients from the latin american cohort ( 3.1% ) were not obtained , mainly because of lack of information on their vital status at the end of the study ( figure 1 ) .
the differential characteristics of both cohorts are shown in table 1 . as can be seen , patients in the external validation cohort are younger , and they present greater clinical and functional severity , a greater radiological extension of bronchiectasis , a lower percentage of etiologies of unknown origin , and a significantly lower number of exacerbations .
table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality .
as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was  at least one hospitalization in the previous year ,  with an or of 1.8 , and so this was the variable chosen to be added to the faced .
this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations .
table 3 shows that the e - faced score has a range of values of 09 points .
the three tertiles running from least to greatest severity are therefore 03 points , 46 points , and 79 points .
table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year .
the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) .
figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) .
these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity .
furthermore , in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) .
similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two .
furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) .
it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores .
similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) .
table 2 shows the results of the logistical regressions , which included different cut - off points for the number of exacerbations and hospitalizations in the previous year ( at least one , at least two , or more than two , as well as a composite of two exacerbations or at least one hospitalization ) to evaluate the faced score s independent prognostic capacity for all - cause mortality .
as can be seen , the cut - off point that added the most statistically significant prognostic value to the faced score was  at least one hospitalization in the previous year ,  with an or of 1.8 , and so this was the variable chosen to be added to the faced .
this process therefore added two extra points to the faced score in cases of at least one hospitalization in the previous year , or none in cases of no hospitalizations .
table 3 shows that the e - faced score has a range of values of 09 points .
the three tertiles running from least to greatest severity are therefore 03 points , 46 points , and 79 points .
table 4 shows how the prognostic capacity of the e - faced score was significantly greater than that of the faced , in both the number and severity ( hospitalizations ) of the future exacerbations over the course of a year .
the greatest benefit from e - faced was found in the identification of patients with future multiple exacerbations ( at least two exacerbations per year [ figure 2 ] , or at least two exacerbations or one hospitalization per year ) .
figure 3 shows the auc - roc of both the e - faced and faced scores for all - cause mortality in the initial construction cohort ( n=819 ) .
these are 0.87 ( 0.830.91 ) and 0.87 ( 0.820.91 ) , respectively , with no significant differences between them ; they are always > 0.80 , which means that they both present an excellent prognostic capacity . furthermore ,
in the case of prognostic capacity for respiratory deaths , the results were again similarly excellent : the e - faced score presented an auc - roc of 0.86 ( 0.810.9 ) , whereas the faced score presented one of 0.82 ( 0.780.87 ) .
similarly , when both the e - faced and the faced scores were applied to the series of 651 patients from the latin american cohorts , the auc - rocs were 0.84 ( 0.800.88 ) and 0.81 ( 0.770.86 ) , respectively , which are as excellent as those of the construction cohort , with no statistically significant differences between the two .
furthermore , in the case of the prognostic capacity for respiratory mortality , the e - faced presented an auc - roc of 0.87 ( 0.830.91 ) and the faced one of 0.84 ( 0.800.88 ) ( figure 4 ) .
it was also demonstrated ( figure 5 ) that the three tertiles of both the faced and the e - faced scores accurately differentiated patients into three groups of increasing severity , with no significant differences between the classifications created by the faced and e - faced scores .
similarly , the comparative division of this validation cohort into three tertiles in both the e - faced and the faced scores also presented an excellent discriminatory capacity in three groups of increasing severity , with no significant differences from the construction cohort ( figure 6 ) .
according to the results of this study of two large series of patients with bronchiectasis , the addition to the faced score of a simple dichotomized variable relating to the presence of previous severe exacerbations ( hospitalizations ; e - faced score ) significantly increases the prognostic capacity for future exacerbations and hospitalizations over the course of 1 year , making it a valid score for detecting patients with more exacerbated bronchiectasis .
furthermore , the addition of this variable does not make the score any more complex but does maintain its excellent prognostic capacity for both all - cause and respiratory mortality , while also retaining the capacity of faced score to discriminate different degrees of severity . by adding
a variable that is potentially modifiable or preventable with treatment , the e - faced would broaden its clinical applicability and its potential use in research studies on new treatments for bronchiectasis .
the faced score is a very simple ( and even memorizable ) multilevel system for evaluating ( without any need for a computer ) the initial severity and prognosis of patients with bronchiectasis.3 however , one of its limitations is the fact that it does not cover exacerbations .
and although there is little literature on this topic , some important studies have shown this variable to have a decisive impact , especially in its more severe forms , on both the severity and prognosis of bronchiectasis.2,6,7 in this respect , chalmers et al,2 in a study of the construction and validation of the bsi ( bronchiectasis severity index ) score , observed that both previous exacerbations ( at least two exacerbations ; hr ( hazard ratio ) 2.03 [ 1.024.03 ] ) and , above all , severe exacerbations ( hospitalization ; hr 2.43 [ 1.024.03 ] ) were significantly associated with an increase in all - cause mortality , with a very substantial relative weight ( above all severe exacerbations ) in the final value of the score . although the bsi score is more complex than the faced , it has demonstrated an excellent prognostic capacity for exacerbations / hospitalizations in patients with bronchiectasis.2 moreover , martnez - garca et al have demonstrated that , over and above any chronic bronchial infection or presence of systemic inflammation , severe exacerbations were capable of producing a steep drop in lung function , of up to 120 ml of fev1 in a year 
more than double the mean observed overall in patients with bronchiectasis ( ~50 ml / year).7 this substantial impact of exacerbations on bronchiectasis patients can be explained by the increase in diagnoses,19 related mortality,20 and hospitalizations due to bronchiectasis21,22 in recent years , even in industrialized countries , despite the excellent therapeutic arsenal at disposal .
ringshausen et al,21 for example , found a rise in hospitalization from 9.4 to 39.4/100,000 inhabitants , especially in women and the elderly .
similar findings have been observed in the usa , with an annual increase of 2.5%3% in the period 19932006.22 furthermore , patients who present the greatest number or severity of exacerbations are also the ones who accumulate a higher percentage of health costs . de la rosa et al10 found that the annual cost of treating a bronchiectasis patient in spain in 2012 was 4,671 euros , and ~10,000 euros in cases of severe bronchiectasis ( faced 57 points ) , with exacerbations being one of the variables that explained a higher percentage of cost .
thus , the group of patients with more than two exacerbations ( 21.5% ) was responsible for 34.5% of the total cost of the 456 patients included , while those patients who required two or more hospitalizations ( 5.7% ) were responsible for 20.4% of the total cost .
therefore , even without any definition of an exacerbatory clinical phenotype in bronchiectasis , the impact of exacerbations on these patients calls for a simple and prompt identification of the type of patients who will present a higher number or greater severity of exacerbations in the future . in this study ,
the e - faced score ( a simple score that only adds to the faced score one easily memorized dichotomic variable pertaining to the presence of hospitalizations in the year prior to the patient s diagnosis ) presents advantages over the faced .
while maintaining the substantial prognostic capacity of the latter , for all - cause and respiratory mortality and for discrimination of degrees of severity , the e - faced is capable , without accruing any greater complexity , of significantly improving the early identification of patients who will have at least two exacerbations or one hospitalization over the course of a year , or both , despite receiving treatment .
this means that it is important to establish a prompt and thorough preventive strategy for these patients .
moreover , another advantage of the e - faced over the faced is its greater susceptibility to change after the administration of different treatments .
this may make it more suitable for research studies , both because it incorporates the main variable customarily used in randomized clinical trials ( the number and severity of exacerbations ) and because it presents a great prognostic capacity for these factors .
although the present study has several significant strengths , particularly large numbers of patients being included in both the series ( construction and external validation ) , it also presents a number of noteworthy limitations . on the one hand ,
exacerbations have been defined by the need for antibiotic treatment prescribed for changes in the volume or purulence of sputum or in the symptoms .
they are therefore open to a more objective classification , although it should be emphasized that smaller exacerbations that do not require such treatment have also been recorded .
it is nevertheless believed that the impact of this limitation on the conclusion can not be very significant as most patients with exacerbations of bronchiectasis are treated with antibiotics,3 and furthermore , more severe exacerbations have the greatest impact on patients.2,6 on the other hand , the e - faced was constructed using different cut - off points as regards the number of hospitalizations in the year prior to the patient s inclusion in the study , but no longitudinal data on the hospitalizations in the construction cohort ( n=819 ) in the subsequent years were available .
such longitudinal data on the number of yearly exacerbations and hospitalizations are available , however , for the validation cohort ( n=651 ) , and , thanks to the way the e - faced was constructed , it has demonstrated an excellent prognostic capacity .
moreover , we can not exclude the influence of the chronic treatments used on clinical outcomes , especially in the number or severity of exacerbations .
however , this is a limitation of all studies that include historical cohorts of patients with data recorded longitudinally from the time of diagnosis .
furthermore , no treatment has yet been proved to have any influence on the natural history of the disease . finally ,
data were not obtained from 2.4% of patients from the original faced score cohort and 3.1% of patients from the latin american series , mainly because of lack of information on their vital status , but such a small percentage of missing data can not be expected to change the conclusion of the study .
in conclusion , the e - faced offers a more versatile alternative to the faced that is more susceptible to variations after medication , while maintaining the excellent prognostic capacity of the latter for mortality and its capacity to discriminate degrees of severity , without increasing the complexity of the score .
moreover , it presents a greater prognostic capacity for exacerbations and hospitalizations , especially in patients in which these are more frequent .
although further studies are required to investigate the applicability of the e - faced in research studies , it is considered that the greater capacity of this score for change makes it more suitable than the faced for use in clinical trials to evaluate different treatments for bronchiectasis .
list of ethical committees and approval numbers this approval was obtained from the original database of faced score , published in eur respir j. 2014;43:1357 .
 backgroundalthough the faced score has demonstrated a great prognostic capacity in bronchiectasis , it does not include the number or severity of exacerbations as a separate variable , which is important in the natural history of these patients.objectiveconstruction and external validation of a new index , the e - faced , to evaluate the predictive capacity of exacerbations and mortality.methodsthe new score was constructed on the basis of the complete cohort for the construction of the original faced score , while the external validation was undertaken with six cohorts from three countries ( brazil , argentina , and chile ) . 
 the main outcome was the number of annual exacerbations / hospitalizations , with all - cause and respiratory - related deaths as the secondary outcomes . 
 a statistical evaluation comprised the relative weight and ideal cut - off point for the number or severity of the exacerbations and was incorporated into the faced score ( e - faced ) . 
 the results obtained after the application of faced and e - faced were compared in both the cohorts.resultsa total of 1,470 patients with bronchiectasis ( 819 from the construction cohorts and 651 from the external validation cohorts ) were followed up for 5 years after diagnosis . 
 the best cut - off point was at least two exacerbations in the previous year ( two additional points ) , meaning that the e - faced has nine points of growing severity . 
 e - faced presented an excellent prognostic capacity for exacerbations ( areas under the receiver operating characteristic curve : 0.82 for at least two exacerbations in 1 year and 0.87 for at least one hospitalization in 1 year ) that was statistically better than that of the faced score ( 0.72 and 0.78 , p<0.05 , respectively ) . 
 the predictive capacities for all - cause and respiratory mortality were 0.87 and 0.86 , respectively , with both being similar to those of the faced.conclusione-faced score significantly increases the faced capacity to predict future yearly exacerbations while maintaining the score s simplicity and prognostic capacity for death . 
Introduction
Methods
Design
Patients
Variables
Follow-up and main endpoints
Statistical analysis
Results
Construction of the E-FACED score
Prognostic capacity for exacerbations
All-cause mortality: comparison of E-FACED and FACED  construction and validation cohorts
Capacity to discriminate different degrees of severity
Discussion
Conclusion
Supplementary material