Single multiplex assay to identify simultaneously enteropathogenic, enteroaggregative, enterotoxigenic, enteroinvasive and Shiga toxin-producing Escherichia coli strains in Brazilian children

A multiplex PCR to differentiate typical and atypical enteropathogenic Escherichia coli (EPEC), enteroaggregative E. coli (EAEC), enterotoxigenic (ETEC), enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) strains was developed and evaluated. The targets selected for each group were eae and bfpA for EPEC, aggR for EAEC, elt and est for ETEC, ipaH for EIEC and stx for STEC isolates. This PCR was specific and sensitive for rapid detection of target isolates in stools. Among 79 children with acute diarrhea, this technique identified 13 (16.4%) with atypical EPEC, four (5%) with EAEC, three (3.8%) with typical EPEC, one (1.3%) with ETEC and one (1.3%) with EIEC.     

Prevalence of diarrheagenic Escherichia coli in children with diarrhea in Salvador, Bahia, Brazil

We report the frequency of the different diarrheagenic Escherichia coli (DEC) categories isolated from children with acute endemic diarrhea in Salvador, Bahia. The E. coli isolates were investigated by colony blot hybridization with the following genes probes: eae, EAF, bfpA, Stx1, Stx2, ST-Ih, ST-Ip, LT-I, LT-II, INV, and EAEC, as virulence markers to distinguish typical and atypical EPEC, EHEC/STEC, ETEC, EIEC, and EAEC. Seven of the eight categories of DEC were detected. The most frequently isolated was atypical EPEC (10.1%) followed by ETEC (7.5%), and EAEC (4.2%). EHEC, STEC, EIEC, and typical EPEC were each detected once. The strains of ETEC, EAEC, and atypical EPEC belonged to a wide variety of serotypes. The serotypes of the others categories were O26:H11 (EHEC), O21:H21 (STEC), O142:H34 (typical EPEC), and O:H55 (EIEC). We also present the clinical manifestations and other pathogenic species observed in children with DEC. This is the first report of EHEC and STEC in Salvador, and one of the first in Brazil.     

occurrence of mannose- resistant adhesins MRHA in E.Coli strains isolated from children with diarrhea]

The study was aimed at determination of the frequency of occurrence of mannose-resistant adhesins in E. coli strains isolated from children with diarrhoea. It was also of interest whether their presence is associated with the serological type or other virulence factors. The material used in this study consisted of 1022 strains of E. coli (EPEC, ETEC and EIEC) and 3431 isolates from sick children and 960 from healthy children (non-EPEC-ETEC-EIEC). Enterotoxigenicity and entero-invasiveness of strains was evaluated by biological tests performed on animals and in tissue culture. Production of MRHA adhesins was determined by the test of mannose-resistant active hemagglutination, and of colonization factors antigens CFA by application of agglutination and agar gel immunodiffusion tests. Most frequently MRHA adhesins were produced by ETEC strains-80% of strains. All of them appeared to be a colonization factor antigen CFA/I. EPEC strains produced various MRHA adhesins only by 12.6% of strains. Production of MRHA adhesins by EIEC strains was not detected. Frequency of occurrence of MRHA adhesins in E. coli strains which were non-EPEC-ETEC-EIEC was dependent from the isolation source. MRHA adhesins were most frequently found in strains isolated from sporadic cases of light diarrhoea in ambulatory treated children (49%), much less among isolates from children hospitalized because of severe diarrhoea (33%), and from healthy children in 9% of isolates only. These results may indicate the potential role of MRHA adhesins in pathogenesis of diarrhoea in children.     

Epidemiological study on Jordanian patients suffering from diarrhoea

Stool specimens from 1400 Diarrhoeal patients from the Jordanian population were examined for bacterial pathogens and Rotavirus during a four- year period (1997-2000). Pathogenic bacteria were identified in 343 patients (24.5%), most often from children. Salmonella spp. was the most frequent isolated organism in 10.7% of the patient's cultures, followed by enteropathogenic Escherichia coli (EPEC) in 3.9%, Shigella spp. in 0.8% and Campylobacter spp. in 0.9%. Vibrio spp. was not identified in the stools tested. Resistance to ampicillin was observed in 42.2% of the Salmonella, 77.0% of the Shigella, and 31.0% of the EPEC isolates. Cotrimoxazole resistance was observed in 34.0% of the Shigella and 13.0% of the EPEC isolates and 77.0% of Campylobacter isolates. Rotavirus was identified in 373 samples (26.6%) of the patients     

Pathogenic enteric Escherichia coli in children with and without diarrhea in Maputo, Mozambique

A study was conducted on the circulation of potentially diarrheagenic Escherichia coli in two groups of children, both under the age of seven. The first group (548 children) suffered from mild diarrhea and attended the Xipamanine Health Center of Maputo, in Mozambique. The second group (380 children) included randomly chosen, asymptomatic, children from the same population. A total of 503 E. coli strains were isolated from the two groups of children (n=375 and 128, respectively). All E. coli strains were genotypically and phenotypically screened. The presence of virulence-associated genes was assessed by a set of multiplex PCR specific for st and lt genes of enterotoxic Escherichia coli (ETEC), eae and bfpA genes of enteropathogenic E. coli (EPEC), stx(1) and stx(2) of enterohemorrhagic E. coli (EHEC), ial of enteroinvasive E. coli (EIEC) and the species-specific gene uidA. Adhesion and citotoxicity of isolated E. coli were evaluated in vitro on different cell cultures. A total of 37 isolates harbored virulence-associated genes: 18 were classified as ETEC, (15 from symptomatic, and three from asymptomatic children), 16 as EPEC (respectively, 13 and 3) and three EIEC in the symptomatic group. No stx(1) or stx(2) genes, associated with enterohemorrhagic E. coli were found. On the basis of the adhesion pattern on HeLa cells, 167 E. coli were classified as diffusely adhering, (125 in patients and 42 in controls) and 67 as enteroaggregative, (50 and 17, respectively). To the best of our knowledge, this is the first report in the literature on the circulation of potentially diarrheagenic E. coli in Mozambique.     

Studies on diarrheagenic Escherichia coli isolated from children with diarrhea in Myanmar

Escherichia coli isolates from 217 children in Myanmar with diarrhea were investigated for the presence of virulence genes related to diarrhea by colony hybridization and PCR. The genes examined were lt, stI, stII, stx1, stx2, eae, bfp, pCVD (which is the representative gene of plasmid of pCVD of EAEC), and ial (which is invasion-associated locus of the invasion plasmid found in EIEC). Isolates from 47 of 217 children (21.7%) possessed virulence genes characteristic of diarrheagenic E. coli. No instance was found of co-existence of different E. coli strains with different virulence genes in the same patient. Diarrheagenic E. coli are currently classified into five categories based on their virulence markers: ETEC, EHEC, EPEC, EAEC, and EIEC. Of the 47 isolates examined, 30 were EAEC, 12 were EPEC and 5 were ETEC. Susceptibility tests for antimicrobial agents showed that almost all diarrheagenic isolates were resistant to penicillin, tetracycline and streptomycin. However, the majority of strains were sensitive to cephalexin, nalidixic acid and norfloxacin. In particular, 42 of the 47 isolates were sensitive to norfloxacin, which is a fluoroquinolone. This study shows EAEC and EPEC are responsible for sporadic diarrhea in Myanmar and fluoroquinolones appear to be effective in the treatment of these patients.     

Enteropathogens associated with diarrheal disease in infants of poor urban areas of Porto Velho, Rondônia: a preliminary study

One hundred and thirty cases of diarrhea and 43 age-matched controls, 0 to 5 years old, were studied in a pediatric outpatient unit from a poor peri urban area of Porto Velho, Rond?nia. Eighty percent of diarrheal cases were observed in the groups under 2 years of age. Rotavirus (19.2%) was the most frequent enteropathogen associated with diarrhea, followed by Shigella flexneri (6.15%) and S. sonnei (1.5%) and Salmonella sp. (6.9%). Four cases of E. coli enterotoxigenic infections (3.1%), E. coli enteropathogenic (EPEC)(2.3%) one case of E. coli enteroinvasive infection (0.8%) and one case of Yersinia enterocolitica (0.8%) were also identified. Mixed infections were frequent, associating rotavirus, EPEC and Salmonella sp. with Entamoeba histolytica and Giardia lamblia.     

多重PCR方法快速检测4种主要致腹泻性大肠埃希菌

肠毒素性大肠埃希菌(ETEC)、肠致病性大肠埃希菌(EPEC)、肠出血性大肠埃希菌(EHEC)和侵袭性大肠埃希菌(EIEC)是引起腹泻的主要大肠埃希菌,威胁着食品安全和人类健康,建立同时检测4种致腹泻性大肠埃希菌的方法具有重要意义。基于ETEC LT肠毒素基因、EPEC bfpA基因、EHECO抗原基因和EIEC侵袭性质粒特异性基因,设计了4对特异性引物,通过对单一PCR反应条件的优化建立了快速检测4种主要致腹泻性大肠埃希菌的多重PCR方法,彼此之间无交叉反应。该多重PCR方法具有良好的特异性和灵敏性,对24株致病菌进行检测,所试4株致腹泻性大肠埃希菌均为PCR阳性,其他菌株则为阴性。实践证明,利用所建立的多重PCR方法对124份肉类、奶类制品及人工污染样品等进行检测,检出15份阳性,与国标(GB4789.6-1994)检测结果相同。结果表明本文建立的多重PCR方法可用于ETEC、EPEC、EHEC和EIEC的单一或混合感染的鉴别诊断及食品安全风险评估,具有良好的实用性。     

A comparison of HEp-2 cell invasion by enteropathogenic and enteroinvasive Escherichia coli

In order to further characterize cellular invasion by enteropathogenic Escherichia coli (EPEC), we compared invasion of HEp-2 cells by EPEC and enteroinvasive E. coli (EIEC). We used a gentamicin HEp-2 cell assay and measured bacterial recovery under conditions of varying incubation time and temperature, and in the presence or absence of inhibitors of cellular microfilaments and microtubules. We found that, unlike EIEC, EPEC did not rapidly multiply within HEp-2 cell but invaded well at 32 degrees C. While microfilament inhibitors reduced invasion by both EIEC and EPEC, microtubule inhibitors reduced invasion by EPEC only. These results suggest that EPEC and EIEC differ in their mechanisms of epithelial cell invasion.     

Diarrheagenic Escherichia coli in acute gastroenteritis in infants in North-West Italy

Enteropathogenic Escherichia coli may cause diarrhoea in infancy, but it is not routinely detected and regarded as a major causative agent. The aim of the present study was to estimate the incidence of enteropathogenic E. coli infection and to investigate its epidemiology and pathogenesis from faecal specimens in infants hospitalized for acute gastroenteritis. Between March 2008 and June 2009, faecal samples were collected and examined to recognize diarrhoeal aetiology, especially for E. coli, by cultural identification and multiplex-PCR. E. coli were isolated in 75 of 160 collected samples (46,88%); 10 samples of which (6,3%) had been positively recognised for pathogenic genes. Data showed that the presence of diarrheagenic E. coli infection was 6.3%, but it becomes 5% considering E. coli as a unique agent responsible for diarrhoea. The datum is not statistically meaningful because of the small sample (p>0,05). Bacterial pathogens were also isolated in 60 samples (37,5% of the total collected samples): 15 Salmonella spp., 8 Klebsiella pneumoniae, 9 Klebsiella oxytoca, 11 Citrobacter freundii, 5 Pseudomonas aeruginosa, 2 Serratia spp., 7 Enterobacter cloacae, 1 Shigella spp., 2 Campylobacter spp. Rotavirus was the predominant pathogenic single etiologic agent identified. It was found in 35 samples (21.88% of the overall collected samples), while Adenovirus, serotypes 40 or 41, was isolated in 2 samples (1.3%). Rotavirus infection was found predominantly in winter with respect to autumn. Data provide an interesting epidemiologic survey of enteropathogenic E. coli, which is not usually detected, although it may have potential clinical implications. Abbreviations: CDEC, detaching E. coli; DAEC, diffusely adherent E. coli; EAggEC, enteroaggregative E. coli; EHEC, enterohaemorrhagic E. coli; EIEC, enteroinvasive E. coli; EPEC, enteropathogenic E. coli; ETEC, enterotoxigenic E. coli.     

Occurrence of P.fimbrii in strains from selected genera of Enterobacteriaceae]

This study was aimed at recognition of frequency of occurrence of P fimbriae in strains of Escherichia coli isolated from samples of feces of children with symptoms of diarrhoea and at search of these adhesions in strains representing other than Escherichia genera of Enterobacteriaceae strains. One hundred forty laboratory strains were investigated. They belonged to genus Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Salmonella, Shigella, and Yersinia. Also were tested 1277 colonies of enteric rods isolated from the MacConkey's medium inoculated with samples of feces from 163 children with symptoms of diarrhoea. Mannose-resistant active hemagglutination test was performed with human group O erythrocytes and guinea pig erythrocytes stabilized with glutaraldehyde. Presence of P fimbriae was detected by the slide latex test with latex covered by P1 glycoprotein. Among 140 laboratory strains of Enterobacteriaceae in 21 strains (3-E. cloaceae, 2-Hafnia, 13-K. pneumoniae, 2-P. rettgeri and in one strains of Providencia) presence of MRHA adhesins was demonstrated. Nine of these strains (2-Hafnia, 5-K. pneumoniae and 2-P. rettgeri) reacted specifically in the latex test. Among 1142 colonies of E. coli isolated from children with symptoms of diarrhoea, 326 colonies belonged to 13 EPEC serotypes. With 118 (36.2%) of EPEC colonies a positive result of MRHA reaction was found with human erythrocytes and 34 (10.4%) with guinea pig erythrocytes. Positive latex test was obtained with 77 (23.6%) colonies. All these colonies possessed MRHA adhesins. Remaining 816 colonies of E. coli strains did not represent microorganisms belonging to serotypes accepted as enteropathogenic. From 112 (13.7%) colonies out of 816 not belonging to EPEC, positive results was obtained in the MRHA test with human erythrocytes and this was the case also with 41 (5.0%) colonies in MRHA reaction with application of guinea pig erythrocytes. The latex test was positive in 65 (7.9%) colonies of E. coli. From remaining 135 colonies other than E. coli, positive result of latex test of presence of P fimbriae was obtained with 54 (40.0%) colonies, including 14 colonies of E. cloacae, 23-K. pneumoniae and 17-K. oxytoca. In all these strains presence of MRHA adhesins was demonstrated. These investigations demonstrated that among EPEC strains significantly more frequently, than not belonging to these serotypes of E. coli, MRHA adhesins, including P fimbriae was observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Octaplex PCR and fluorescence-based capillary electrophoresis for identification of human diarrheagenic Escherichia coli and Shigella spp

A multiplex PCR assay, amplifying seven specific virulence genes and one internal control gene in a single reaction, was developed to identify the five main pathotypes of diarrheagenic Escherichia coli and Shigella spp. The virulence genes selected for each category were Stx1, Stx2, and eaeA for enterohemorrhagic E. coli (EHEC), eaeA for enteropathogenic E. coli (EPEC), STIb and LTI for enterotoxigenic E. coli (ETEC), ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., and aggR for enteroaggregative E. coli (EAEC). Each forward primer was labelled with a fluorochrome and the PCR products were separated by multicolour capillary electrophoresis on an ABI PRISM310 Genetic Analyzer (Applied Biosystems). If present, several gene variants of each virulence gene were identified. The internal control gene rrs, encoding 16S rRNA, was amplified in all 110 clinical strains analyzed. Virulence genes were demonstrated in 103 (94%) of these strains. In the majority of the cases (98/103, 95%), classification obtained by the novel multiplex PCR assay was in agreement with that previously determined by phenotypic assays combined with other molecular genetic approaches. Numerous multiplex PCR assays have been published, but only a few of them detect all five E. coli pathotypes within a single reaction, and none of them has used multicolour capillary electrophoresis to separate the PCR products. The octaplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the five main pathotypes of E. coli, and Shigella spp. This assay will replace the previous molecular genetic methods used in our laboratory and work as an important supplement to the more time-consuming phenotypic assays.     

Relationship between O-serogroup and presence of pathogenic factor genes in Escherichia coli

A total of 383 isolates of serogroup-based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes. The serogroup-based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers. The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%). The difference was not significant. The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA-positive EPEC strains from patients but was not detected in the 9 strains from carriers. In serogroup-based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers. The 4 ipaH-positive strains possessed the invasive plasmid. These results suggested that the serogroup-based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene.     

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.     

Development of a set of multiplex PCR assays for the simultaneous identification of enterotoxigenic, enteropathogenic, enterohemorrhagic and enteroinvasive Escherichia coli

Diarrheagenic E. coli comprise a diverse group of microorganisms responsible for gastrointestinal diseases in humans. On the basis of their virulence traits they are distinguished from the non-pathogenic E. coli and classified in several categories. Molecular methods represent the most reliable techniques for distinguishing pathogenic from non-pathogenic E. coli and characterising their pathogenic features. In this paper we report the development of a set of three multiplex PCR assays for the simultaneous and rapid identification of diarrheagenic E. coli belonging to ETEC, EPEC, EHEC and EIEC groups. Assay 1 utilizes primer pairs specific for genes coding for ST and LT toxins of ETEC, and for the E. coli beta-glucuronidase (uidA); assay 2 detects the presence of the eae and bfpA genes of EPEC, and assay 3 recognizes stx1 and stx2 of EHEC, and ial of EIEC. This technique has been validated on 190 E. coli isolated in Angola, Italy and Mozambique from feces of children with diarrhea. Results obtained with the set of multiplex PCR demonstrated 100% accordance with those obtained for the same isolates by PCR on single target genes. The proposed set of multiplex PCRs is the first reported assay that allows the simultaneous characterization of the four categories of diarrheagenic E. coli.     

Diagnostic and epidemiological significance of F-donor activity of Enterobacteria strains

The study of the circulating strains of enteropathogenic Escherichia coli (EPEC), shigellae and salmonellae for the presence of F-factor (the conjunction factor) was carried out. The study revealed that 85% of Shigella flexneri strains 2a had conjugative (F)-factor. The proportion of such strains was 63% among EPEC of different serovars and 15% among Salmonella enteritidis. As 63% EPEC strains had hemolytic activity and were found to carry F-conjugative plasmid it should be taken into consideration in the identification of microorganisms. The presence of conjugation plasmids is an important epidemiological marker.     

Study of polymorphic variable-number of tandem repeats loci in the ECOR collection and in a set of pathogenic Escherichia coli and Shigella isolates for use in a genotyping assay

The Escherichia coli (E. coli) reference collection, ECOR, consists of 72 strains that are representative of the genotypic diversity, as indexed by multilocus enzyme electrophoresis (MLEE), in the species as a whole. MLEE revealed 4 main phylogenetic groups designated A, B1, B2 and D. We present a study of the relationship between the ECOR strains as determined by polymorphisms in seven variable-number of tandem repeats (VNTR) loci. Seven tandem repeats that were present in more than one of the fully sequenced E. coli strains were selected, and primers were constructed in order to amplify the targets in all species where the loci were present. The combined result for all VNTR loci was adapted as a multiple-locus variable-number tandem repeats analysis (MLVA) and showed that the ECOR collection was divided into 63 distinct genotypes. The ECOR phylogenetic groups defined by MLEE were not well conserved by MLVA. A set of 61 pathogenic isolates of both E. coli and Shigella spp. was then tested with the same set of VNTR loci, and revealed 54 distinct genotypes. In addition, the MLVA method was used to genotype isolates from patients and suspected sources in a recent outbreak of E. coli O103 in Norway. The pathogenic E. coli isolates contained the diarrhea causing categories EIEC, EAEC, STEC, ETEC and EPEC. Shigella isolates were of species S. flexneri, S. boydii, S. sonnei and S. dysenteriae. The MLVA method rapidly genotyped all isolates in the study at a Simpson's index of diversity of D=0.98.     

Rapid detection of diarrheagenic E. coli by real-time PCR

Enterovirulent Escherichia coli are among the most important causes of acute diarrhea in developing as well as in developed countries. We have adapted classical PCR to detect these organisms in stool specimens to real-time PCR using the LightCycler (LC) SYBR Green format followed by melting curve analysis. With only two different cycling protocols we could detect enteropathogenic E. coli (EPEC) and verocytotoxin-producing E. coli (VTEC) (duplex assay for both Verotoxin 1 (VT1) and Verotoxin 2 (VT2)) in one run and enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC) (duplex assay detecting both heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT)) in another run. Using serial dilutions of control strains, the LC proved to be clearly more sensitive than conventional PCR for five out of seven investigated targets: VTEC (VT1 and VT2), ETEC (ST and LT) and EIEC. For EPEC and EAEC, LC and conventional PCR had identical sensitivities. With stool samples, we found an optimal agreement between LC-PCR and the conventional PCR when samples were tested in a 1:10 dilution. Only one specimen was discrepant, being repetitively positive for VT by LightCycler but not by conventional PCR. Given the significantly higher sensitivity of the LC-PCR for the VT target (up to a 10(-4) dilution factor by melting curve analysis and up to a 10(-6) dilution factor following gel electrophoresis), this is probably a false negative result by conventional PCR. We conclude that LightCycler PCR is more rapid, easier than and at least as sensitive as our conventional PCR for the detection of enterovirulent E. coli in stool specimens after culture on MacConkey.     

Bacterial, viral and parasitic enteric pathogens associated with acute diarrhea in hospitalized children from northern Jordan

To determine the etiology of acute diarrhea in Jordanian children under 5 years of age, we examined stool samples from 265 children admitted to the pediatric ward at Princess Rahma Hospital for Children, Irbid, Jordan, for parasites, rotavirus and enteric bacteria. Using both traditional and molecular diagnostic techniques, we detected enteropathogens in 66.4% of patients with diarrhea. A single enteric pathogen was detected in 50.9% of the children, and multiple pathogens were detected in 15.5%. The prevalence of enteropathogens identified was as follows: rotavirus (32.5%), enteropathogenic Escherichia coli (12.8%), enteroaggregative E. coli (10.2), enterotoxigenic E. coli (5.7%), Shigella spp. (4.9%), Entamoeba histolytica (4.9%), Salmonella spp. (4.5%), Campylobacter jejuni/coli (1.5%), Cryptosporidium spp. (1.5%), enteroinvasive E. coli (1.5%), eae-, Ehly-positive E. coli (0.8%), Giardia lamblia (0. 8%) and Yersinia enterocolitica (0.4%). No Vibrio cholerae, Shiga toxin-producing E. coli, microsporidia, adenovirus or small round virus were detected. Findings from this study demonstrate that rotavirus and several types of diarrheagenic E. coli, which are not screened for during routine examinations of stool samples in public health laboratories, were the most frequently detected enteropathogens in these children. Our findings highlight the value of using a combination of traditional and molecular techniques in the diagnosis of diarrheal disease in this population.