In this review, we showed that more than half of the severe diarrhea episodes, most likely to result in death among children under the age of 5 years in 2011, could be attributed to rotavirus, EPEC, calicivirus, and ETEC. Our estimates have been adjusted for age in studies that did not cover all children <5 years old, and to add to 100%, including a fraction of episodes with unknown etiology. Such adjustments have not been done in previously published estimates for single diarrhea etiologies 
We identified a potential selection bias among studies that focus on a single pathogen. For example, the median proportion of diarrheal episodes with rotavirus identified varied from 39% in single-pathogen studies to 20% in studies that sought more than 4 pathogens. It is possible that studies looking for a particular pathogen are more likely to be conducted in a study site with a high prevalence of that pathogen and/or a low prevalence of other pathogens. An urban hospital that treats children of higher socioeconomic status and living in more hygienic conditions than children in rural areas may find a higher proportion of cases with rotavirus. A study of cholera done in a hospital in an endemic area may not be representative of national or regional populations. Because of the low number of studies that sought multiple pathogens, we have not restricted our analysis to only those studies, in an attempt to include as much global data as possible, but it should be recognized that the inclusion of single-etiology studies may result in a biased higher estimate for some pathogens.
By including 13 pathogens in this review we are able to address the problem of mixed infections, an important factor ignored in previously published single-pathogen estimates of deaths. No methodology has been developed to identify the true cause of an episode when more than one pathogen is identified in the stool. Our adjustment of all percentages to fit 100% is done to correct for this problem, assuming that each pathogen is equally likely to cause the illness. This is probably not correct because some organisms are carried in the feces for a relatively long time after infection-causing illness, like norovirus 
, or may not cause illness, especially in older children who have acquired immunity that protects against disease, but not carriage of the organism, like some protozoa 
. This method of including all equally in the constraint to 100% of diarrhea deaths may result in an underestimate of the importance of some pathogens, such as rotavirus in young children, and overestimate the importance of others, such as Giardia. We do not have data on the presence of these pathogens in the stools of asymptomatic children in the studies selected in this review so we cannot determine the attributable fraction related to each pathogen as done in other studies 
. However, controlling for pathogens found in non-ill children does not necessarily eliminate the problem because some pathogens with long excretion periods after illness, like norovirus, may be wrongly classified as not causing diarrhea. Carefully conducted longitudinal studies are needed to separate long-term excretors after illness from asymptomatic infections, to reveal the true pathogenic role of these different organisms in developing countries.
We estimated that the number of diarrhea episodes for which no pathogen can be identified is 34%, which is based on studies that sought at least 8 pathogens, not necessarily all 13 and thus may be an overestimate. These “unknowns” could be due either to the same pathogens not detected because insensitive methods were used to identify them (either the method itself or to using a rectal swab instead of a stool sample) 
, to the use of antibiotics prior to obtaining the stool sample, to other yet undiscovered infections, or to non-infectious causes of diarrhea. The proportion of samples with unknown causes was based on a selected group of 12 studies that searched for 8 or more pathogens. These studies do not represent the world as the rest of the studies did. The recently conducted studies called The Global Enterics Multicenter Study (GEMS) in 7 countries in Africa and Asia were designed to fill this gap 
. However, they studied cases with moderate and severe diarrhea seen in health services (hospitals, emergency rooms and community clinics), not separating those being hospitalized from milder outpatient cases, therefore, those studies would not meet our inclusion criteria. Given that we cannot distinguish among the reasons no pathogen was found during the episode, our estimates may represent an under-estimate, at least for some causes. We could not include some pathogens known to cause diarrhea in our review, such as organisms that cause food-borne outbreaks (i.e. Clostridium perfringens
, or Staphylococcus aureus
producing enterotoxins 
), because there are very little data on their importance in developing countries.
A recent review of rotavirus studies estimated that rotavirus caused 453 000 deaths in children <5 in 2008 
. If we would apply the median proportion of 38% rotavirus isolation found in the 242 inpatient studies that sought it in our review, without any adjustment, to the 1 236 million U5 diarrheal deaths in 2008, we would estimate 472 000 rotavirus deaths in 2008. In 2011 it is estimated that diarrhea deaths have been reduced to 712 000 
. Our estimate of 197 000 deaths due to rotavirus, using our improved methods, still represents an important global public health problem, with 23 children dying due to this condition every hour. This estimate does not account for any recent reduction in rotavirus-specific proportionate mortality due to the introduction of rotavirus vaccine, as seen in some Latin America countries 
, but these countries account for a very small fraction of global diarrhea mortality. Wide scale use of the rotavirus vaccine in high mortality countries will allow a more precise estimate of the true proportion of diarrhea deaths caused by rotavirus.
Our estimate of 28 000 deaths for shigella is much lower than a previous estimate of 667 695 deaths due to shigellosis in children under 5 years in the world in 1995 published by Kotloff et al 
. This initial estimate was not based on a systematic review of the literature; rather, it used a single study in Latin America to estimate the proportion of shigella cases that were hospitalized and a Bangladeshi study to estimate the case-fatality rate of children hospitalized with shigellosis to estimate the global burden due to this organism. Using the same methodology of Kotloff et al but with an updated review of the literature and current case fatality rates observed in Bangladesh, Bardhan P et al 
estimated that only 14 000 children younger than 5 years of age died due to shigellosis in Asia in 2005. Our estimates are compatible with this Asian estimate.
The total number of deaths due to calicivirus of 71 000 deaths has indicated to be the third most common cause of death due to diarrhea in children under 5 years of age. Few studies differentiated between GI and GII norovirus and other types of human caliciviruses, but in those few that did, most of calicivirus isolated in children with severe diarrhea have been due to norovirus GII 
. Patel et al 
estimated 218 000 deaths due to norovirus among children under 5, but this was calculated using very different methods and assumptions: they used an attributable fraction due to norovirus when data on asymptomatic children was available, and applied their mean isolation rate of 12.1% from inpatient studies (not much different from our median isolation rate of 13.8%) to 1.8 million deaths due to diarrhea in the world; they did not adjust for mixed infections or unknowns.
The 79 000 deaths estimated to be caused by EPEC represent different sub-types of this type of pathogenic E. coli
, a group that requires further epidemiological studies in different parts of the world to further characterize them since some sub-types are isolated with the same frequency in diarrhea and control children 
, new “typical” and “atypical” EPEC strains have been identified 
, and in some regions have been identified to cause more persistent than acute diarrhea 
These estimates have several limitations. The studies included in this review were conducted in selected sites and in some cases in populations with increased risk of diarrheal diseases. Thus, they may not be representative of the countries where they were conducted, nor of the world. For several regions, such as Russia and the former Soviet states or Sub-Saharan Africa we have limited or no data (, ). The gap of information from Africa, for pathogens other than rotavirus, is most acute because of the number of diarrhea deaths in this region is very high 
. No study has been conducted to identify pathogens in children who died due to diarrheal diseases, so we assume that children in need of hospitalization are the best proxy of diarrhea deaths in low to middle income countries, but this may not be true for some pathogens. Another limitation is the combination of laboratory methods with different sensitivities to identify a pathogen: from the culture-based identification of salmonella or shigella to the highly sensitive real-time PCR method for norovirus. This may have affected the relative importance of one vs
another pathogen in our estimates. We excluded studies on nosocomial infections, on displaced populations and on diarrhea outbreaks, which may have caused us to under-represent deaths due to some pathogens like V. cholerae
We included in our estimates a total of 13 pathogens (4 viruses, 6 bacteria and 3 parasites) that have been incriminated as causes of severe diarrheal diseases. Some viruses, like adenovirus, and parasites, like G. lamblia
, have not been completely documented as a cause of severe diarrhea in developing countries 
. The subject of causality of diarrheal diseases is still not completely understood in settings where children are heavily exposed to many pathogens early in life. Young infants may be protected by breast milk and trans-placental maternal immunity and very low doses of ingested pathogens early in life may result in subclinical infections and development of immunity. This immunity may not preclude, however, the excretion of these pathogens in the child's feces. Practically all studies done in children who were studied when they were healthy as well as when they developed an acute diarrheal episode have found the same pathogens, although usually with lower frequency, in healthy states. Thus, the assumption that any pathogen identified in a child with diarrhea is the cause of the episode is naive and additional methods are needed to determine the pathogenicity of microbes. With a better understanding of the pathogenicity of key organisms our estimates could be further adjusted. Also, some studies suggest that children ill with a pathogen, as with EPEC, may excrete higher amounts in the stool, as compared with asymptomatic infections 
, so future studies may consider quantifying the amount of each pathogen in the stool to help identifying those ill with it. Finally, the review period covering studies published between 1990 and 2011 (studies were conducted with a median mid-study period of 2005, only 24 (8%) studies were done prior to 1990). We have not identified a significant change of the proportions assigned to each pathogen over time, so this does not seem to affect our estimates, as shown in for rotavirus.
Median proportion of stool samples with rotavirus by mid study period.
The Global Burden of Disease Study recently published cause of death estimates for 187 countries in 2010 
. For children <5 years of age, GBD estimated a total of 666 000 deaths due to diarrheal diseases in 2010 while CHERG estimated 712 000 deaths for 2011. GBD also estimated deaths due to 9 etiologies and produced estimates for 0–6, 7–27, and 28–364 days and 1–4 years of age. CHERG estimates for 2011 in children <5 years of age are slightly higher than GBD estimates for rotavirus (198 000 vs
173 000 rotavirus deaths, respectively), similar for EPEC and ETEC deaths (79 000 vs
73 000, and 43 000 vs
39 000, respectively), and lower for cholera, salmonella, shigella, campylobacter, Entamoeba histolytica
, and Cryptosporidium spp
. (). GBD did not estimate deaths due to norovirus, which was the third leading cause of death in our review. GBD used rates reported in diarrhea studies published between 1975 and 2010 done in outpatients, case-control, and community-based studies as a reference category to adjust the proportions seen in inpatient studies. CHERG only used data from inpatient studies published between 1990 and 2011. Both GBD and CHERG used modeling to obtain the total number of diarrheal deaths for children <5, but unlike GBD, CHERG has not used models for etiology-specific causes of deaths for each age group and for each country to produce its global estimate. Age specific data and modeling may produce spurious results, more so if there are no data. For example, very few studies have been done describing causes of diarrhea in neonates in developing countries, but GBD has estimated deaths caused by each of the 9 pathogens in neonates 0–6 and 7–27 days of age (). GBD only produced estimates for 9 etiologies of diarrhea and by subtracting the total of these estimates from the total of diarrheal deaths; they estimated the proportion of other causes of diarrheal deaths. CHERG estimated the proportion due to unknowns from studies that searched for 9–13 pathogens, which we feel realistically addresses the fact that a causative agent is not identified in every illness. This also explains why we estimated a higher number of deaths in this category (176 000) than GBD for “other causes” which should include unknowns (109 000). GBD and CHERG recognized the problem of mixed infections, but the methods used to adjust for it was different: GBD only used proportions for each etiology from inpatient studies that searched for 2–8 etiologies and used that information to produce weights to adjust their estimates in the models. We choose to constrain all proportions, including unknowns, to 100% to correct for mixed infections, which we feel it is more appropriate until better data and analytical tools are available. We have done an extensive search of the literature to include the 286 inpatient studies used in our estimates. GBD has not published the studies included, their search strategy, or modeling methods. Until these are published we will not be able to completely compare these estimates.
Comparison between Global Burden of Diseases (for 2010) and the Child Epidemiology Reference Group (for 2011) estimates of the number of diarrhea deaths (×1000), by cause and age, in children <5 years of age in the world.
This is the first systematic review attempting to estimate the cause of deaths for these 13 enteric pathogens. Rotavirus, calicivirus, enteropathogenic and enterotoxigenic E. coli cause more than half of all diarrheal deaths in children <5 in the world. We have identified a potential selection bias in studies searching for only one enteropathogen, and the problem when mixed infections (more than one enteropathogen is identified in a stool sample taken from a child with severe diarrhea) are not taken into consideration when estimating causes of diarrheal deaths, factors that has affected previous published estimates. Future studies should be done in hospital services dealing with all types of severe diarrhea, searching for all known enteropathogens, removing the effect of asymptomatic excretes, and establishing a mechanism to attribute to one enteropathogen the cause of a diarrheal episode in cases of mixed infections.