infectious diarrhea continues to be a health burden worldwide , especially in children living in developing countries . it is estimated that in these regions it is responsible for 2.5 million infant deaths annually , with a mortality rate of 4.9 per 1,000 children and an annual incidence of 3 episodes per child among children under 5 years of age [ 1 , 2 ] . in developed countries the mortality from diarrhea is relatively low but morbidity is not so different from that seen in developing countries . due to the relatively high incidence , the social impact and health costs are similar in both situations [ 3 , 4 ] . salmonella ; shigella ; yersinia enterocolitica ; campylobacter ; vibrio cholerae ; diarrheagenic escherichia coli pathotypes ( deps ) , and viruses represent leading causes of infantile acute diarrhea in both developing and developed countries . deps comprise several categories : enteropathogenic e. coli ( epec ) , that can be further classified into typical epec ( tepec ) and atypical epec ( aepec ) depending on the presence or absence of the eaf plasmid ( e. coli adherence factor ) ; shiga - toxin - producing e. coli ( stec ) ; enterotoxigenic e. coli ( etec ) ; enteroaggregative e. coli ( eaec ) ; and enteroinvasive e. coli ( eiec ) . however , its relative importance is different in high- and low - income populations [ 510 ] . deps , campylobacter , and most viruses are not routinely investigated as enteric pathogens in most clinical laboratories worldwide ; thus , their importance in infant community - acquired diarrhea is generally underestimated . in our country , most public or private microbiology laboratories only perform the detection of salmonella , shigella , and some viruses in feces from children with diarrhea [ 11 , 12 ] . however , those studies have been mainly performed in children from households with low or very low socioeconomic level [ 4 , 13 , 14 ] . this study aimed to evaluate the association of deps , salmonella , shigella , yersinia enterocolitica , campylobacter , and viruses with cases of acute diarrhea in a subset the children living in households with high socioeconomic level . the clinical characteristics of the illness related to the most common agents were also analyzed . between january 2010 and march 2011 we conducted a descriptive , microbiological and clinical study involving children 5 years of age , with acute community - acquired diarrhea living in households with high socioeconomic level . diarrhea was defined as the passing of three or more liquid or loose stools ( which take the shape of the container ) within a 24-hour period . children with persistent diarrhea ( more than 15 days of duration ) ; those who were receiving antibiotics or had been hospitalized in the 30-days period before the episode of acute diarrhea ; or children enduring gastrointestinal disorders as celiac disease , allergy to cow 's milk , or inflammatory bowel disease were not included . we studied children  5 years of age seeking medical care in clinics or emergency room of a private health institution , british hospital , a 120-bed tertiary hospital with medical and surgical pediatric specialties . children receiving medical care at the british hospital often come from high - income households . we included only those children in whom the stool sample was available during the visit . the study was approved by the technical commission of the british hospital and by the school of medicine ethical committee ( universidad de la repblica ; file number 071140 - 000323 ) . verbal agreement to participate in the study we performed macroscopic observation to trace the presence of blood , mucus , pus , or other abnormal element and searched for fecal leukocytes ( fl ) in smears stained with methylene - blue and then microscopically examined under high - dry power ( 400 ) . the assays were performed at the laboratory of the british hospital by using commercially available qualitative immunochromatographic lateral flow rapid tests for norovirus , rotavirus , and adenovirus ( rida quick norovirus and rida quick rotavirus / adenovirus combi - biopharm ag , damstadt , germany ) , respectively . stool samples were placed in cary - blair medium ( difco , becton , dickinson ) and chilled and transported to the laboratory of the bacteriology and virology department , for detecting salmonella , shigella , campylobacter , yersinia enterocolitica , and deps according to previously described procedures [ 4 , 1416 ] . briefly , selective and differential culture media ( salmonella - shigella agar , macconkey agar , sorbitol macconkey agar , and yersinia selective agar ) ( difco , becton , dickinson ) were used for isolating salmonella , shigella , deps , and yersinia species . tetrathionate broth ( difco , becton , dickinson ) incubated at 42c , peptone - sorbitol bile broth ( psb ) incubated at 4c , and cefixime - tellurite / tryptic soy broth ( ct - tsb ) ( oxoid , basingstoke , uk and difco , becton , dickinson , resp . ) incubated at 37c were used as enrichment media for salmonella , yersinia , and stec , respectively . for campylobacter detection , feces were cultured at 42c in a microaerophilic environment on selective medium prepared with brucella agar base ( difco , becton , dickinson ) , hemin , sodium metabisulfite - ferrous sulfate - sodium pyruvate ( sigma - aldrich ) , sheep blood , and the campylosel mixture ( biomreieux , marcy l'etoile , france ) : cefoperazone , colistin , vancomycin , and amphotericin b . after incubation for 24  h at 37c , up to 40 colonies with typical e. coli morphology were subjected to pcr using the primers shown in the table 1 to detect the following genes : stx1 and stx2 of stec ; eae of enteropathogenic e. coli ( epec ) and some stec strains ; ipah of enteroinvasive e. coli ( eiec ) and shigella ; elt and esta of enterotoxigenic e. coli ( etec ) ; and pcvd432 of eaec [ 4 , 15 ] . bacterial colonies were pooled for dna extraction in 150  l of sterile water , boiled for 10  min , and then centrifuged at 13,000  rpm for 10  min . a 2,5  l aliquot of this supernatant was added to 22,5  l of the pcr mixture reaction ( 50  mm kcl , 10  mm tris - hcl [ ph 8.3 ] , 1.5  mm mgcl2 , and 2  mm of each deoxynucleoside triphosphate ) , 1.25  u of taq dna polymerase ( hybripol bioline , uk ) , and each primer pair ( sbs genetech co. , ltd ) shown in table 1 . the reactions were run in a thermal cycler ( gene amp pcr system 2700 applied biosystem ) with the following cycling conditions : 94c for 5  min ; 35 cycles of denaturation at 95c for 1  min ; and annealing ( see table 1 for annealing temperature ) for 1  min and extension at 72c for 1  min , followed by a final extension at 72c for 10  min . pcr products were visualized after electrophoresis in 2% agarose gels in 0.5x tbe buffer and ethidium bromide staining . stec isolates were further characterized by the presence of the ehxa and eae genes , according to previously described pcr reactions . variants of eae genes present both in epec and stec recovered strains were also determined by pcr assays [ 4 , 17 , 18 ] . in epec isolates ( eae positive and stx1/2 negative ) , the presence of the bfp gene was detected by pcr as we described previously , using the primer pairs shown in the table 1 . the deps strains detected were confirmed as being e. coli by using the api 20 e system ( biomreieux , marcy l'etoile , france ) and conventional biochemical assays . colonies suspected to be of salmonella , shigella , campylobacter , or yersinia genera were studied according to classical procedures described previously [ 1315 ] . salmonella serotyping was done at the centro nacional de salmonella ( cns , department of bacteriology and virology , institute of hygiene ) . antimicrobial susceptibility to amikacin , ampicillin , ceftriaxone , cefuroxime , ciprofloxacin , cloramphenicol , colistin , gentamicin , nalidixic acid , nitrofurantoin , streptomycin , tetracycline , and trimethoprim - sulfamethoxazole ( oxoid ltd . , basingstoke , hampshire , uk ) was established according to clinical laboratory standards institute ( clsi ) guidelines . their parents were enquired about signs , symptoms , and other relevant clinical or epidemiological information by using a standard questionnaire when the child was enrolled in the study . the data collected were age , gender , macroscopic characteristics of stools , presence of fever and/or vomiting , number of stools , and duration of diarrhea in hours from the beginning to the time of the visit . information on housing , educational level , and occupation of the parents and income was collected . aggregate scores were classified into five social class categories ranging from very high i ( 46 points ) to very low v ( 1720 points ) . we considered the children from households within categories i or ii as having high socioeconomic conditions . height and weight were determined according to standard anthropometric methods . in order to analyze the nutritional status , the weight and height of each child were compared with those of same aged boys and girls measured in the national health and nutritional examination survey in the usa ( nhanes ) . the degree of dehydration was estimated clinically by analyzing general appearance , eyes , mucous membranes , and tears . ninety - eight children 5 years of age were taken to british hospital for diarrhea during the study period . however , only 59 ( 60% ) met all inclusion criteria . most excluded children that had acute diarrhea ( 85% ) were not considered because stool samples were not available during the medical visit . the remaining 15% presented associated or underlying intestinal disease ( e.g. , allergy to cow 's milk , lactose intolerance , and malformations ) . the average age was 11 months with a range from 1 month to 5 years . all the analyzed children were from households belonging to categories i or ii of the modified graffar scale . in 30 of 59 enrolled children ( 51% ) the 32 agents identified were the following : rotavirus 3 ; adenovirus 3 ; norovirus 2 ; deps , 16 isolates ; 5 isolates of campylobacter ; and 3 isolates of salmonella . viruses were thus found in 8 ( 13% ) cases and bacteria in 24 ( 40% ) ( see figure 1 ) . campylobacter was recovered as single pathogen in 5 children , while viruses were detected as single agents in 6 of them . four out of the five campylobacter isolates were identified as c. jejuni and the remaining corresponded to c. coli . the distribution of eae subtypes in aepec isolates was as follows : o26 ,  ; o28ac , 1 ; o34 ,  ; o103 , 1 ; o125 , nontypable ; and ont , . the 3 tepec recovered strains showed bfp gene type . serogroup o86 isolates ( 2 strains ) showed eae gene subtype  , while the o26 strain carried subtype . all stec isolates were eae and ehxa positive . both o26 strains carried eae gene subtype 1 , but we were unable to establish the eae subtype of stec strain o153 . one o26 stec strain carried stx1 gene , and the other one carried both stx1 and stx2 genes . two children showed mixed infections ; one was infected with both aepec and rotavirus and the other one with adenovirus and eaec . one aepec serogroup o103 strain showed resistance to nalidixic acid ; one tepec ( serogroup o86 ) was resistant to ampicillin ; and one eaec ( ont ) isolate was resistant to ampicillin , tetracycline , and trimethoprim - sulfamethoxazole . the remaining recovered deps were susceptible to the all tested antibiotics and so were salmonella isolates . selected clinical data of the children with diarrhea due to epec ( both typical and atypical ) , campylobacter , and viruses are given in table 3 . only the clinical findings of children in which a single enteropathogen was detected are shown . overall , the viral infections were characterized by vomiting . in 9 of the 59 studied children we detected fecal leukocytes . in 6 of these children we identified a potential enteropathogen ( one child infected with stec ( serogroup o26 ; genotype : sxt1/2 , eae subtype 1 , and ehxa positive ) had bloody diarrhea and after 20 days developed  complete  hus with microangiopathic hemolytic anemia , thrombocytopenia , and acute renal involvement , requiring dialysis in the acute stage . none of the 59 children showed severe dehydration at the time of pediatric consultation , and none required hospitalization for intravenous fluid reposition . infectious gastroenteritis is one of the most common diseases in humans , with particularly high morbidity and mortality in children younger than 5 years of age . knowledge of the prevalent agents is important to design specific control measures , vaccination strategies , and treatment regimens [ 1 , 2 ] . in our country and in the region there are no published studies about the etiology of acute community diarrhea in children from households with high socioeconomic level . in this first local study , investigating 12 agents ( epec , etec , stec , eaec , eiec , salmonella , shigella , y. enterocolitica , campylobacter , rotavirus , adenovirus , and norovirus ) allowed to recover a potential enteric pathogen in 52% of the children , a similar figure to that reported previously by olesen et al . in denmark . 5 years of age brought to british hospital for diarrhea during the mentioned period were studied . this private health institution attends 4.000 children and is quite representative of the focused on social group . several studies show that viruses are the leading cause of infantile acute diarrhea , both in developed and developing countries [ 21 , 22 ] . the role of viruses could be underestimated here , since agents such as astrovirus and sapovirus , well - known diarrheagenic pathogens , were not investigated in this study [ 23 , 24 ] . with regard to bacterial causes , our findings are in agreement with results of previous studies involving children of high socioeconomic level . our data suggest that campylobacter jejuni and salmonella are common bacterial gastrointestinal pathogens in this group of children [ 25 , 26 ] . neither shigella nor yersinia seems to play an important role as diarrhea agents in these patients . however , a previous study conducted by us that included 95 children  5 years of age from low socioeconomic - level households suffering from acute diarrhea showed the involvement of these agents in that situation . using an identical methodology , we recovered one or more potential enteric pathogens in 74% of these children . the identified agents were rotavirus in 42 cases ; deps , 40 isolates ; shigella , 18 strains ; seven isolates of campylobacter jejuni ; and 3 yersinia enterocolitica strains . within the set of deps , the most frequently found pathotype was epec ( 26 isolates : 15 corresponded to typical and 11 to atypical ones ) followed by etec ( 8 strains ) and eaec ( 6 strains ) . similar results were obtained in other studies conducted by our group [ 13 , 14 ] . atypical epec was the most commonly isolated category of diarrheagenic e. coli in the present study . on the other hand , tepec strains were recovered less frequently in this infantile subpopulation ( see table 2 ) . this finding is consistent with previous observations indicating that infections with aepec exceed those with tepec in both developing and developed countries [ 27 , 28 ] . our results suggest that this trend is also present in uruguay , especially in children from high - income households . the reasons for these changes are not well known but could be related to the increased exposure of these children to the animal reservoir of these agents , to changes in eating habits , or in the way of cooking certain foods , among other causes . stec strains recovered carried genes associated with severe diseases ( stx , eae , and ehxa ) ; and one child infected with o26 stec developed hus . this is interesting because we have previously isolated stec strains belonging to non - o157 serogroups from children with hus from low - income households and also from bovine feces . these findings reinforce our suggestion posed in previous communications about the local participation of non - o157 stec strains in severe infant diseases and also addresses the importance of performing active surveillance of all forms of hus [ 16 , 30 ] . the third stec recovered strain which belonged to a serogroup not previously isolated in our country or in the region . this fact highlights the value of stec screening based on the detection of stx genes . intimin ( encoded by eae gene ) is involved in the binding of epec and stec to enterocytes and provides information about the association of epec and stec with severe diseases . we found a variety of eae subtypes , both in epec and stec isolates . this superficial structure could become a target for local and regional vaccine development , so eaec has been identified as a common cause of acute diarrheal illness in children and adults of inpatient and emergency units throughout the united states . eaec as a pathogen is not preferentially included in clinical diagnostic tests , and the molecular mechanisms underlying pathogenesis are poorly understood . in the absence of a defined virulence marker and considering that all eaec recovered strains were lysine - decarboxylase positive , its role as enteropathogen for this group of children is debatable . despite the high number of colonies with typical e. coli morphology studied per child etec and eiec are well - known pathogens , but they are common in children from households with low socioeconomic level . we recovered etec and eiec isolates in previous studies that included children with acute diarrhea from low - income families admitted to the public pediatric hospital  hospital peditrico - centro hospitalario pereira rossell  in montevideo [ 13 , 14 ] . as expected , only 2 ( 7% ) out of 30 children showed mixed infections in the present study . this figure is lower than that we found previously in children with diarrhea from low - income households . in that subpopulation of children this can be due to the proper application of the guidelines currently recommended for empirical treatment of children with diarrhea ; it may also result from a lower use of antibiotics as a feed supplement for slaughter animals , especially cattle and chickens . with regard to the clinical findings , our data suggest that children suffering from viral or campylobacter diarrhea were brought to the hospital ( emergency room or clinics ) earlier than those infected with epec . this may have occurred because most children with viral infection presented profuse vomits from illness onset and most children infected with campylobacter had blood and/or mucus in the stools . vomits and blood or mucus in the stools could act as alarm signs for the caregivers of these patients , leading them to quickly seek medical attention [ 4 , 26 ] . diarrhea was also frequent , although some of these children had bloody diarrhea ( see table 3 ) . however , the existence of inflammatory elements in the stools of children infected by epec has been demonstrated by using sensitive methods for detecting leukocyte lactoferrin . almost 90% of children infected by campylobacter showed polymorphonuclear leukocytes in feces , while only 20% of those infected by epec showed a positive result . viruses , campylobacter , and salmonella were found as common pathogens in this subpopulation of high - socioeconomic level children suffering acute diarrhea . the role of diarrheagenic e. coli was also confirmed for epec ( aepec and tepec ) and non - o157 stec strains . o26 stec was found responsible for a case of hus , a severe childhood disease . obtained data suggest that children with viral or campylobacter diarrhea were taken to the hospital earlier than those infected with epec . our results highlight the importance of zoonotic bacteria as pathogens associated with acute diarrhea in this subpopulation of high - socioeconomic level children . the examination of stool cultures from children  5 years of age suffering from acute diarrhea and living in households with high socioeconomic level should include tests for viruses , campylobacter , salmonella , epec , and stec . ideally , identification of epec and stec should be based on detection of the eae and stx genes , respectively .
infectious diarrhea , a common disease of children , deserves permanent monitoring in all social groups . 
 to know the etiology and clinical manifestations of acute diarrhea in children up to 5 years of age from high socioeconomic level households , we conducted a descriptive , microbiological , and clinical study . 
 stools from 59 children with acute community - acquired diarrhea were examined , and their parents were interviewed concerning symptoms and signs . 
 rotavirus , adenovirus , and norovirus were detected by commercially available qualitative immunochromatographic lateral flow rapid tests . 
 salmonella , campylobacter , yersinia , and shigella were investigated by standard bacteriological methods and diarrheagenic e. coli by pcr assays . 
 we identified a potential enteric pathogen in 30 children . 
 the most frequent causes of diarrhea were enteropathogenic e. coli ( epec ) , viruses , campylobacter , salmonella , and shiga - toxin - producing e. coli ( stec ) . 
 only 2 patients showed mixed infections . 
 our data suggest that children with viral or campylobacter diarrhea were taken to the hospital earlier than those infected with epec . 
 one child infected with stec o26 developed 
 complete  hus . 
 the microbiological results highlight the importance of zoonotic bacteria such as atypical epec , campylobacter , stec , and salmonella as pathogens associated with acute diarrhea in these children . 
 the findings also reinforce our previous communications about the regional importance of non - o157 stec strains in severe infant food - borne diseases .