this retrospective , cohort study includes patient data collected at 5 etus operated by international medical corps ( imc ) in liberia and sierra leone from 15 september 2014 to 15 september 2015 as part of its comprehensive response to the west african evd epidemic .
all patients who presented to the study etus with symptoms concerning for evd were triaged to ensure that they met the suspect case definition , which was created based on world health organization ( who )   and ministry of health guidelines from each country ( supplementary appendix 1 ) .
after triage , patients who met the suspect case definition were admitted to the etu suspect ward and had a blood sample drawn within 24 hours for evd and malaria diagnostic testing .
patients with an initial negative evd test remained as inpatients for repeated testing after 2 days . patients with a second negative evd result were discharged or transferred to another healthcare facility .
all patients aged < 18 years who were admitted at any of imc s 5 etus with a positive evd test and outcome data were included for analysis .
all patients were treated according to standard treatment protocols based on guidelines developed by the who , mdecins sans frontires , and local ministries of health , as permitted by local resources .
empiric treatment included antimalarial medications , broad - spectrum antibiotics , and nutritional supplementation , as well as focused supportive treatment ( appendix 1 ) .
patients were cared for by trained medical staff who recorded clinical data on paper forms .
these data were digitized at each etu and unified into a database , as described previously .
laboratory data , including evd real - time polymerase chain reaction ( rt - pcr ) and malaria testing , were linked to clinical data for analyses .
rt - pcr testing was performed by the us naval medical research center for etus in liberia , by public health england laboratories for lunsar and makeni etus in sierra leone , and by the nigerian laboratory for the kambia etu in sierra leone .
cycle threshold ( ct ) values , which are based on rt - pcr of the same ebola virus ( ebov ) zaire locus , are presented in this study as surrogates of viral load .
a ct > 40 was considered negative in all cases , though rna extraction procedures differed slightly between laboratories .
malaria testing ( conducted in sierra leone only ) was performed at each laboratory site using the commercially available binaxnow rapid diagnostic test , which identifies 4 plasmodium species : falciparum , malariae , vivax , and ovale . throughout hospitalization ,
patients were cared for by trained local or international nurses , physician assistants , or physicians , who rounded at least daily and recorded vital signs , clinical data , and laboratory results on paper forms .
patient temperatures were measured with infrared or oral thermometers . for children who were unable to communicate history and who had no accompanying guardians ,
a random sampling audit concluded that > 99% of the data in imc s unified database was accurate .
the variables of interest included demographic and clinical characteristics , malaria status , initial evd rt - pcr ct values , and mortality .
length of stay was calculated as the number of days from date of admission to date of discharge .
age was categorized into the following groups : < 2 years , 24 years , 59 years , 1014 years , and 1517 years . for descriptive statistics , median values with interquartile ranges ( iqrs )
were calculated for nonnormally distributed continuous data , and proportions were calculated for categorical data .
bivariate analyses were performed to determine the associations between mortality , the primary outcome of interest , and independent covariates using the mann - whitney u test for continuous variables and  or fisher exact test for categorical variables where appropriate .
covariates that had a p value < 0.1 from bivariate analyses were entered in a backward stepwise logistic regression model to determine which variables independently predicted mortality .
we used kaplan - meier curves and the log - rank test to compare survival among children by age group , hemorrhagic features , and viral loads estimated by ct values .
the sierra leone ethics and scientific review committee , the university of liberia  pacific institute for research & evaluation institutional review board , and the lifespan ( rhode island hospital ) institutional review board provided ethics approval for this study and exemption from informed consent .
this retrospective , cohort study includes patient data collected at 5 etus operated by international medical corps ( imc ) in liberia and sierra leone from 15 september 2014 to 15 september 2015 as part of its comprehensive response to the west african evd epidemic .
all patients who presented to the study etus with symptoms concerning for evd were triaged to ensure that they met the suspect case definition , which was created based on world health organization ( who )   and ministry of health guidelines from each country ( supplementary appendix 1 ) .
after triage , patients who met the suspect case definition were admitted to the etu suspect ward and had a blood sample drawn within 24 hours for evd and malaria diagnostic testing .
patients with an initial negative evd test remained as inpatients for repeated testing after 2 days . patients with a second negative evd result were discharged or transferred to another healthcare facility .
all patients aged < 18 years who were admitted at any of imc s 5 etus with a positive evd test and outcome data were included for analysis .
all patients were treated according to standard treatment protocols based on guidelines developed by the who , mdecins sans frontires , and local ministries of health , as permitted by local resources .
empiric treatment included antimalarial medications , broad - spectrum antibiotics , and nutritional supplementation , as well as focused supportive treatment ( appendix 1 ) .
patients were cared for by trained medical staff who recorded clinical data on paper forms .
these data were digitized at each etu and unified into a database , as described previously .
laboratory data , including evd real - time polymerase chain reaction ( rt - pcr ) and malaria testing , were linked to clinical data for analyses .
rt - pcr testing was performed by the us naval medical research center for etus in liberia , by public health england laboratories for lunsar and makeni etus in sierra leone , and by the nigerian laboratory for the kambia etu in sierra leone .
cycle threshold ( ct ) values , which are based on rt - pcr of the same ebola virus ( ebov ) zaire locus , are presented in this study as surrogates of viral load .
a ct > 40 was considered negative in all cases , though rna extraction procedures differed slightly between laboratories .
malaria testing ( conducted in sierra leone only ) was performed at each laboratory site using the commercially available binaxnow rapid diagnostic test , which identifies 4 plasmodium species : falciparum , malariae , vivax , and ovale .
throughout hospitalization , patients were cared for by trained local or international nurses , physician assistants , or physicians , who rounded at least daily and recorded vital signs , clinical data , and laboratory results on paper forms .
patient temperatures were measured with infrared or oral thermometers . for children who were unable to communicate history and who had no accompanying guardians ,
a random sampling audit concluded that > 99% of the data in imc s unified database was accurate .
the variables of interest included demographic and clinical characteristics , malaria status , initial evd rt - pcr ct values , and mortality .
length of stay was calculated as the number of days from date of admission to date of discharge .
age was categorized into the following groups : < 2 years , 24 years , 59 years , 1014 years , and 1517 years . for descriptive statistics , median values with interquartile ranges ( iqrs )
were calculated for nonnormally distributed continuous data , and proportions were calculated for categorical data .
bivariate analyses were performed to determine the associations between mortality , the primary outcome of interest , and independent covariates using the mann - whitney u test for continuous variables and  or fisher exact test for categorical variables where appropriate .
covariates that had a p value < 0.1 from bivariate analyses were entered in a backward stepwise logistic regression model to determine which variables independently predicted mortality .
we used kaplan - meier curves and the log - rank test to compare survival among children by age group , hemorrhagic features , and viral loads estimated by ct values .
the sierra leone ethics and scientific review committee , the university of liberia  pacific institute for research & evaluation institutional review board , and the lifespan ( rhode island hospital ) institutional review board provided ethics approval for this study and exemption from informed consent .
among the 547 pediatric cases admitted to imc etus in liberia and sierra leone during the study period , 123 had pcr - confirmed evd .
one patient , whose outcome was unknown , was transferred to another facility , leaving 122 patients available for analysis . included in this number were 7 patients who were previously admitted briefly with suspected evd but whose diagnosis was confirmed during second admissions .
children were divided into 5 age categories : younger than 2 years ( 15% ) , 24 years ( 21% ) , 59 years ( 25% ) , 1014 years ( 26% ) , and 1517 years ( 13% ) .
the median length of stay was 9 days ( iqr , 615 ; range , 131 ) .
almost all children ( 94% ) had a history of contact with documented cases of evd . at triage ,
more than 50% of children had fever , anorexia , and weakness ( table 1 ) . the most commonly observed signs and symptoms during hospitalization were weakness , fever , diarrhea , and anorexia ( table 1 ) .
clinical characteristics of ebola virus disease patients observed at triage and during hospitalization at ebola treatment units in liberia and sierra leone abbreviation : nd , no data available . 
 
self - reported at triage ; measured during hospitalization at least daily ( range 16 times per person per day ) .
malaria testing was available at the sierra leone sites only ; 68 of 84 ( 81% ) children were tested , of whom 27 ( 40% ) were positive for plasmodium parasitemia .
the differences in incidence by age group were significant ( p = .024 ) , with the most cases reported among children aged < 5 years ( 56% ) compared with 59 years ( 50% ) , 1014 years ( 25% ) , and 1517 years ( 9% ) .
there was no difference in rates of plasmodium parasitemia in children with or without fever ( 38% vs 43% , respectively ; p = 1.0 ) .
as shown in table 2 , median length of stay was significantly longer for those who survived compared with those who died ( 16 vs 6 days ; p < .001 ) . the median age of patients who died was significantly younger than of patients who survived ( 4 vs 11 years ; p < .001 )
. stratified by age , the cfr was highest ( 89% ) for children aged < 5 years compared with other age groups ( 59 years , 43% ; 1014 years , 41% ; 1517 years , 25% ; p < .001 ; figure 1 ) .
in addition to age , other significant predictors of mortality included evidence of bleeding at any time during hospitalization ( 58% in children who died vs 27% in survivors ; p = .003 ; figure 2 ) and median initial pcr ct values ( 19.2 in children who died vs 25.0 in survivors ; p < .001 ) .
overall , a pcr ct cutoff value of 20 predicted mortality across age groups better than a cutoff value of 25 , except for children aged < 2 years ( figure 3 , supplementary figure 1 ) .
a diagnosis of plasmodium parasitemia did not influence mortality in aggregate ( p = .76 ) or by age stratified analyses ( data not shown ) .
comparison of demographic and clinical characteristics in children who survived vs those who died 
  number ( % ) unless otherwise stated . 
 
differences remained significant when stratified by age ( < 5 , 59 , 1014 , 1517 years ; p < .02 for each comparison of outcomes ) .
kaplan - meier curve for overall survival among 122 ebola virus disease patients stratified by age .
children aged < 5 years had significantly greater mortality than older children ( log rank test , p < .001 ) .
kaplan - meier curve for overall survival among 112 ebola virus disease patients stratified by bleeding status .
children with evidence of bleeding at any time during their hospitalization had significantly greater mortality ( log rank test , p = .003 ) .
kaplan - meier curve for overall survival among 95 ebola virus disease ( evd ) patients based on initial evd real - time polymerase chain reaction ( rt - pcr ) cycle threshold ( ct ) cutoff of 20 , stratified by age .
overall , mortality of children with initial rt - pcr ct values 20 was significantly greater than for those with higher values ( log rank test , p < .001 ) .
the youngest and oldest groups of children accounted for most of the difference in outcome .
the difference in death rates among afebrile hospitalized children stratified by age ( < 5 years , 2 ; 59 years , 2 ; 1014 years , 2 ) was not significant ( p = .6 )
. fifty percent of the afebrile children died , but no distinguishing clinical features or rt - pcr ct values predicted these deaths .
significant variables from the bivariate analyses ( table 2 ) were entered in backward stepwise logistic regression models . in the first model that excluded pcr tests ( n = 112 ; goodness of fit , 0.68 ) , the odds of mortality were 20.1-fold higher for patients aged < 5 years compared with older children ( 95% confidence interval [ ci ] , 5.969.0 ) and 5.9-fold higher if there was evidence of bleeding at any time during hospitalization ( 95% ci , 2.215.6 ) . when evd rt - pcr ct values were added to the model ( n = 86 ; goodness of fit , 0.53 ) , the odds of mortality were 14.8-fold higher for patients aged < 5 years compared with older children ( 95% ci , 3.562.1 ) , 5.2-fold higher if the rt - pcr ct value was 20 ( 95% ci , 1.518.3 ) , and 5.0-fold higher if there was evidence of bleeding at any time during hospitalization ( 95% ci , 1.615.8 ) .
this is one of the largest cohort studies of children and adolescents with evd published to date .
the odds of death were 20-fold higher in evd patients aged < 5 years compared with older children .
this finding is consistent with other data reported from the west african evd epidemic [ 4 , 21 , 27 , 28 ] .
days of hospitalization before death for half of those aged < 2 years ranged from 6 to 12 days .
this range is comparable to that reported by shah et al [ 21 , 28 ] .
hemorrhagic features developed in fewer than half of pediatric patients ; when present , they were associated with a 5-fold higher odds of mortality .
this observation is in line with other reports that bleeding was not a prominent finding among all evd patients during this epidemic [ 19 , 21 , 22 , 29 ] .
it is also noteworthy that 1 in 5 children presented without fever and , of these , 27% never developed fever , which is similar to findings from one study   but not another .
our findings support the mounting evidence that fever is not a sensitive screening tool for evd [ 17 , 28 ] .
in fact , the low frequency of hemorrhagic features and the conspicuous absence of fever at presentation in up to 20% of patients during the west african epidemic questions the utility of referring to evd as a disease characterized primarily as  viral hemorrhagic fever ,  which may mislead clinicians .
we confirmed that a high viral load estimated by a low rt - pcr ct value ( < 20 ) at the time of presentation reliably predicted mortality in infected children who had 5-fold higher odds of mortality .
the fact that pcr ct values did not differ between survivors and fatal cases in those aged < 2 years ( unlike the older age groups ) suggests that factors other than viral load , such as immunological or physiological dysregulation , contributed to death in the youngest age group .
therefore , semiquantitative molecular diagnostic testing at the time of initial evaluation is a simple and effective method for screening for evd as well as for prognosticating outcomes ( especially in children aged > 2 years ) if ct values are below a predefined threshold and should be used routinely .
a diagnosis of plasmodium parasitemia did not impact the risk of mortality or presence of fever in a subpopulation of children who had testing performed , which differs from a recent report in which a diagnosis of malaria reduced the risk of evd mortality .
considering that children s body surface area to volume ratio is higher than that of adults and some children are malnourished , children are particularly susceptible to life - threatening dehydration and nutritional decompensation .
chertow et al made the observation that large volumes of diarrhea caused by evd were  not unlike that of cholera  .
treatment protocols for future evd outbreaks could potentially improve outcomes by instituting early and aggressive rehydration strategies .
this along with serum electrolyte balance and nutrition could be the most important treatments to reduce mortality in those aged < 5 years in a low - resource setting .
furthermore , prompt , accurate diagnosis and isolation precautions are critically important to reduce transmission both in the community and in healthcare facilities .
the first and historically most often associated reason is multiorgan failure secondary to coagulopathy associated with a cytokine storm and immune dysregulation .
the odds of mortality in our cohort were significantly higher in children with the  classic  presentation of viral hemorrhagic fever .
the large volumes of diarrhea often accompanied by vomiting and exacerbated by weakness and inability to drink fluids quickly depletes the younger children . in the low - resource settings of west africa ,
the scarcity of healthcare providers to administer oral fluids frequently or appropriate continuous intravenous ( iv ) fluids probably resulted in numerous deaths that could have been prevented with more resources .
the third reason for death is cardiac arrest from electrolyte disturbances . in patients treated in high - resource settings , abnormal serum levels of potassium and magnesium
were observed , along with cardiac arrhythmias ; similar electrolyte findings were noted in children in west africa [ 3436 ] .
the abnormal electrolyte levels may result from profuse diarrhea , prerenal kidney dysfunction from dehydration , or direct acute kidney injury ( aki ) from evd . beyond the direct effects of the virus on the kidney ,
evd has been associated with rhabdomyolysis , which can cause aki and subsequent electrolyte abnormalities .
this study had limitations in common with others conducted during the west african evd epidemic .
environmental conditions made it challenging for clinicians to collect patient data , given that they were working in full evd personal protective equipment in extreme heat , which limited the time with each patient .
patients temperatures were measured with an infrared digital thermometer gun or oral thermometer , which may be less accurate than the gold standard rectal thermometer .
frequently , clinicians were unable to collect dependable medical histories because of patients severity of illness , absence of accompanying guardians , varying skills of clinicians , or language and cultural barriers . because accurate nutritional assessments were not performed , the additional impact of malnutrition was not taken into account . without clinicians ability to confirm the length of illness
prior to arrival at etus , we were limited in determining the total duration of illness and in assessing accurately patients clinical courses .
a few of the patients included in this analysis received an experimental antiviral treatment as part of a separate trial , which may have impacted their outcomes .
in addition , it is quite likely many patients either died or recovered from evd without seeking medical attention , thereby potentially introducing ascertainment bias .
two important interventions that were not accounted for in the analyses could have impacted mortality rates .
first was whether or not patients received iv fluids , which typically was inconsistent among etus because of varying practices relating to the use of iv catheters .
the second intervention that was not taken into consideration was whether young children had constant bedside caregivers , either family members or healthcare workers , who administered and encouraged oral rehydration and nutrition and provided critical emotional support .
practices varied among etus because some parents were incapacitated or did not want to assume the risk of nosocomial infection and caregivers were not always continuously available . in conclusion
, we found that 3 clinical and virologic factors , after adjustment , were significant poor prognostic factors for children with evd : aged < 5 years , bleeding at any time during hospitalization , and high viral loads as estimated by rt - pcr ct values .
there is an urgent need to investigate the mechanisms of disease to understand how these risk factors as well as malnutrition , dehydration , and electrolyte imbalance contribute to severe outcomes .
the goal is to identify simple , scalable , and relatively inexpensive measures that can improve health outcomes substantially in children in resource - limited settings .
these fundamental interventions will effectively complement vaccines and antiviral agents that are currently undergoing human trials .
consisting of data provided by the author to benefit the reader , the posted materials are not copyedited and are the sole responsibility of the author , so questions or comments should be addressed to the author .
acknowledgements . the authors thank the governments of liberia , sierra leone , and guinea for contributing to international medical corps(icm) humanitarian response .
we also thank all of our generous institutional , corporate , foundation , and individual donors who placed their confidence and trust in international medical corps ( icm ) and made our work during the ebola epidemic possible .
we thank the us naval medical research center , public health england , the european union mobile laboratory , and the nigerian laboratory for providing laboratory data to our ebola treatment units ( etus ) .
we further acknowledge the many contributions made by all members of our research review committee and other technical teams that contributed to this research , including rabih torbay , ann canavan , dennis walto , dan rodman , yoav rappaport , samuel grindley , syed hassan , erin shedd , ryan burbach , saikrishna madhireddy , benedict adjogah , melody xie , nadezda sekularac , inka weissbecker , sean casey , farrah zughni , natalie sarles , and august felix .
we also thank medical directors vanessa wolfman and kassahun gebrehiwot and monitoring and evaluation staff including annie abbate , razia laghari , allison stewart , alex tran , matthew siakor , david mansaray , lamin bangura , sorie sesay , and joseph fangawa as well as all other data collection officers at our etus .
finally , we thank the medical and support staff , both local and ex - patriot , who set up and operated international medical corps ( icm) etus and cared for the patients during the ebola epidemic in west africa . financial support .
icm was supported by grants from the national institutes of health , national institute of allergy and infectious diseases ( k08 ai100997 ) and national institute of general medical sciences ( p20gm 104317 ) .  
conflicts that the editors consider relevant to the content of the manuscript have been disclosed .
 summary.in a large , retrospective cohort of ebola virus  infected children aged < 18 years , the odds of death were 5-fold higher if patients were aged < 5 years , had evidence of bleeding , or real - time polymerase chain reaction cycle threshold value 20 . 