Etiology of childhood diarrhea in Korea.

To assess the role of recently recognized enteropathogens in childhood diarrhea in Korea, 231 children with diarrhea admitted to and 104 children without diarrhea seen at the well-baby clinic or the outpatient department of Hanyang University Hospital in Seoul, Korea, were evaluated during a 14-month period. Stools were cultured for bacterial pathogens, including enterotoxigenic (heat-labile and heat-stable enterotoxin-producing) and enteroadherent organisms. Only those stools obtained from patients with diarrhea were examined for rotavirus. All Escherichia coli isolates were screened for Shiga-like toxin (SLT) I, SLT-II, enterohemorrhagic E. coli fimbriae, and enteroinvasiveness by colony hybridization. One or more pathogens were identified in 75.8% of the children with diarrhea. Rotavirus was the most frequently identified pathogen, accounting for 47% of the cases. Other major enteropathogens were enterotoxigenic E. coli (22%), Clostridium difficile (16%), enteroadherent E. coli (15%), and enteropathogenic E. coli (6%). Shigella spp., Campylobacter jejuni, Salmonella spp., SLT-I-and enterohemorrhagic-E. coli-fimbria-probe-positive E. coli serotype O26:H11 and enteroinvasive E. coli were isolated from only a few patients. Aeromonas hydrophila and E. coli O157 were not isolated. Compared with those of the controls, the isolation rates of heat-stable-enterotoxin-producing E. coli (P less than 0.05), C. difficile (P less than 0.025), and enteroadherent E. coli (P less than 0.05) were significantly higher in the patients with diarrhea. The greatest number of rotavirus, enterotoxigenic E. coli, and C. difficile cases were identified during the cool, dry months of October and November.     

Etiology of childhood diarrhea in Beijing, China.

To determine the role of recently recognized enteropathogens in childhood diarrhea in China, 221 children with diarrhea and 108 controls seen at the Beijing Children's Hospital were studied during April and May 1989. Stools were examined for ova, parasites, and rotavirus, cultured for bacterial pathogens, and probed for enterotoxigenic Escherichia coli (ETEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), and enteropathogenic adherence factor-positive (EAF+) E. coli. Pathogens were identified in 56.5% of children with diarrhea and 43.5% of controls (P = 0.04). Detection of enteropathogens was significantly greater in patients examined within 1 week of symptom onset (65%) than in patients examined later (39%; P = 0.01). ETEC was the most frequently detected pathogen in children with diarrhea, accounting for 20% of the cases. Other agents identified in patients included the following: salmonellae, 12%; rotavirus, 7%; EIEC, 7%; EHEC, 7%; members of the Aeromonas hydrophila group, 6%; EAF+ E. coli, 5%; Ascaris lumbricoides, 3%; shigellae, 3%; campylobacters, 2%; and Vibrio spp., 0.5%. The isolation rates of salmonellae (P = 0.02), EAF+ E. coli (P = 0.04), and mixed pathogens (P = 0.05) were significantly greater for diarrhea patients than for controls. Resistance to multiple antimicrobial agents occurred in 39% of the Salmonella isolates, 22% of the Aeromonas isolates, and 17% of the Shigella isolates. Multiresistant salmonellae (P = 0.05) and shigellae were recovered from diarrheal stools only. Ciprofloxacin, cefotaxime, and imipenem were the only agents tested to which all bacterial isolates were susceptible in vitro. These results suggest that both traditional and newly recognized agents are important causes of childhood diarrhea in Beijing and that therapy may be complicated by indigenous antimicrobial resistance.     

Differentiation of pathogenic Escherichia coli strains in Brazilian children by PCR.

A PCR technique to differentiate pathogenic enteric Escherichia coli strains in a field setting was evaluated. Among 76 children with acute diarrhea, this technique identified 12 children (16%) with enterotoxigenic E. coli, 6 (8%) with enteropathogenic E. coli, and 1 (1%) with enteroinvasive E. coli infection. Compared with the conventional assays, the PCR method proved to be simpler, more rapid, and inexpensive and therefore suitable for application in a developing-country field setting.     

Nationwide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand.

Escherichia coli strains isolated from children with diarrhea were collected from 16 hospitals in different districts in Thailand during 1985 and 1986 and submitted to the National Reference Laboratory. Isolates were identified by serogrouping or as enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) adhesin factor (EAF) E. coli, or Shiga-like-toxin (SLT)-producing E. coli by DNA hybridization. EPEC strains of known serogroups were isolated from 10%, ETEC strains were isolated from 6%, EAF E. coli strains were isolated from 4%, EIEC strains were isolated from less than 1%, and SLT-producing E. coli strains were isolated from none of 393 children with diarrhea. Among 278 children whose ages were recorded, the highest rate of isolation of EAF E. coli was 11% (9 of 85) from children less than 6 months old. ETEC was isolated from 5% (4 of 85) of children less than 6 months old, from 10% (12 of 118) of children 6 to 23 months old, and from 1% (1 of 75) of children greater than 23 months old. EPEC strains of known serogroups were isolated from 18% (15 of 85) of children less than 6 months old, from 11% (13 of 118) of children 6 to 23 months old, and from 9% (7 of 75) of children greater than 23 months old. E. coli strains that hybridized with the EIEC probe were isolated from three children who were 20, 36, and 48 months old. Examining E. coli for hybridization with DNA probes for virulence determinants is a practical way of conducting nationwide surveillance of diarrhea-causing E. coli. Since only 33% (13 of 39) of EPEC serogroups hybridized with the EAF probe and none hybridized with the SLT probes, identification of EPEC by serogroups analysis, followed by serotyping, should continue to be used in the identification of EPEC.     

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.     

Typical enteroaggregative Escherichia coli is the most prevalent pathotype among E. coli strains causing diarrhea in Mongolian children

Diarrhea remains one of the main sources of morbidity and mortality in the world, and a large proportion is caused by diarrheagenic Escherichia coli. In Mongolia, the epidemiology of diarrheagenic E. coli has not been well studied. A total of 238 E. coli strains from children with sporadic diarrhea and 278 E. coli strains from healthy children were examined by PCR for 10 virulence genes: enteropathogenic E. coli (EPEC) eae, tir, and bfpA; enterotoxigenic E. coli (ETEC) lt and st; enteroinvasive E. coli (EIEC) ipaH; enterohemorragic E. coli stx1 and stx2; and enteroaggregative E. coli (EAEC) aggR and astA. EAEC strains without AggR were identified by the HEp-2 cell adherence test. The detection of EAEC, ETEC, EPEC, and EIEC was significantly associated with diarrhea. The incidence of EAEC (15.1%), defined by either a molecular or a phenotypic assay, was higher in the diarrheal group than any other category (0 to 6.0%). The incidence of AggR-positive EAEC in the diarrheal group was significantly higher than in the control group (8.0 versus 1.4%; P = 0.0004), while that of AggR-negative EAEC was not (7.1 versus 4.3%). Nineteen AggR-positive EAEC strains harbored other EAEC virulence genes-aggA, 2 (5.5%); aafA, 4 (11.1%); agg-3a, 5 (13.8%); aap, 8 (22.2%); aatA, 11 (30.5%); capU, 9 (25.0%); pet, 6 (16.6%); and set, 3 (8.3%)-and showed 15 genotypes. EAEC may be an important pathogen of sporadic diarrhea in Mongolian children. Genetic analysis showed the heterogeneity of EAEC but illustrated the importance of the AggR regulon (denoting typical EAEC) as a marker for virulent EAEC strains.     

Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H⁻, O111ab:H₂, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea.     

Prevalence of enteroaggregative Escherichia coli among children with and without diarrhea in Switzerland

In a prospective study between July 1999 and September 2000, stool specimens of children below the age of 16 years with (n = 187) and without (n = 137) diarrhea were tested for the presence of enterovirulent bacteria by standard culture methods and by PCR. Targets for the PCR were the plasmid pCVD432 for enteroaggregative Escherichia coli (EAEC), the verotoxin 1 and verotoxin 2 genes for enterohemorrhagic E. coli, ipaH for enteroinvasive E. coli (EIEC) and Shigella spp., genes coding for heat-stable and heat-labile toxins for enterotoxigenic E. coli (ETEC), and the eaeA gene for enteropathogenic E. coli. The following bacteria could be associated with diarrhea: Salmonella enterica (P = 0.001), Campylobacter spp. (P = 0.036), ETEC (P = 0.012), and EAEC (P = 0.006). The detection of EAEC, ETEC, and S. enterica was strongly associated with a history of recent travel outside of Switzerland. EAEC isolates were found in the specimens of 19 (10.2%) of 187 children with diarrhea and in those of 3 (2.2%) of 137 children without diarrhea (P = 0.006) and were the most frequently detected bacteria associated with diarrhea. Among the children below the age of 5 years, the specimens of 18 (11.9%) of 151 with diarrhea were positive for EAEC, while this agent was found in the specimens of 2 (2.2%) of 91 controls (P = 0.007). Enteropathogenic E. coli isolates were found in the specimens of 30 (16.4%) of the patients and in those of 15 (10.9%) of the controls, with similar frequencies in all age groups (P > 0.05). We conclude that EAEC bacteria are involved in a significant proportion of diarrhea cases among children. Children younger than 5 years of age are more often affected by EAEC than older children.     

Molecular epidemiology of Escherichia coli diarrhea in children in Hong Kong.

This pediatric hospital-based study of 388 diarrhea cases and 306 controls analyzed predominant E. coli colonies from primary culture (253 cases and 177 controls) with eight DNA probes for enteropathogenic, enterotoxigenic, enteroaggregative, and diffusely adherent E. coli. Only enteropathogenic E. coli adherence factor was identified significantly more frequently in cases (10) than in controls (0).     

Etiology of children's diarrhea in Montevideo, Uruguay: associated pathogens and unusual isolates

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies. Escherichia coli enteric virotypes, especially enteropathogenic E. coli (EPEC), were shown to be prevalent. Rotavirus, Cryptosporidium, Campylobacter (mainly Campylobacter jejuni), and Shigella flexneri were also often identified. Enterotoxigenic E. coli, Salmonella, and Giardia lamblia were sporadically recognized. Unusual findings included two enteroinvasive E. coli strains, one Shigella dysenteriae 2 isolate, and a non-O:1 Vibrio cholerae culture. EPEC bacteria and S. flexneri (but not Salmonella) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.     

Epidemiologic study of enterotoxigenic and enteropathogenic Escherichia coli isolated from infantile diarrhea cases on the Wallis and Futuna Islands (French Overseas Territory)

We have studied the incidence of enteropathogenic and enterotoxigenic Escherichia coli strains associated with infant diarrhoeal disease in Wallis and Futuna (South Pacific) during a period of 3 months. We have isolated enteropathogenic E. coli in 30,4% of children. The most frequently serotypes isolated were 0119:B14 (32%), 0111:B4 (23%) and 0126:B16 (19%). In this last serotype 6 strains released heat-stable enterotoxin. Enterotoxigenic E. coli were isolated from 8,1% of the children with diarrhea (20 strains), 13 strains released heat-labile toxin, 6 released heat-stable toxin (serotype 0126:B16) and 1 strain produced both.     

Case-Control Study of Enteropathogens Associated with Childhood Diarrhea in Dhaka, Bangladesh

The International Centre for Diarrhoeal Disease Research, Bangladesh, is a major center for research into diarrheal diseases. The center treats more than 100,000 patients a year. To obtain useful information representative of all patients, a surveillance system in which a 4% systematic sample of all patients is studied in detail, including etiological agents of diarrhea, was installed in October 1979. The first paper on etiology for the surveillance patients was published in 1982, which identified a potential enteric pathogen in 66% of patients. In subsequent years, several new agents of diarrhea have been identified. To assess the importance of a broader spectrum of diarrheal agents including the ones identified relatively recently, we studied 814 children with diarrhea. The children were up to 5 years of age and were part of the surveillance system. They were matched with an equal number of community controls without diarrhea. The study was conducted from February 1993 to June 1994. A potential enteric pathogen was isolated from 74.8% of diarrheal children and 43.9% of control children (P = 0.0001). Even though the first study was not a case-control study, it identified rotavirus, Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp. , and Vibrio cholerae O1 as major pathogens. The present study identified these pathogens as being significantly associated with diarrhea. In addition, the study also identified six additional agents, including enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, enterotoxigenic Bacteroides fragilis, Clostridium difficile, and Cryptosporidium parvum, as being significantly associated with diarrhea. Plesiomonas shigelloides, Salmonella spp., diffusely adherent E. coli, enteroaggregative E. coli, Entamoeba histolytica, and Giardia lamblia were not significantly associated with diarrhea. Enteroinvasive E. coli, enterohemorrhagic E. coli, and Cyclospora cayetanensis were not detected in any of the children. The major burden of diseases due to most pathogens occurred in the first year of life. As in the previous study, seasonal patterns were seen for diarrhea associated with rotavirus, V. cholerae, and enterotoxigenic E. coli, and infections with multiple pathogens were common. With a few exceptions, these findings are in agreement with those from other developing countries. This knowledge of a broader spectrum of etiological agents of diarrhea in the surveillance patients will help us plan studies into various aspects of diarrheal diseases in this population.     

Laboratory investigation of enteroaggregative Escherichia coli O untypeable:H10 associated with a massive outbreak of gastrointestinal illness.

A massive outbreak of gastrointestinal illness occurred in Tajimi city, Gifu prefecture, in June of 1993 in which 2,697 children in elementary and junior high schools developed severe diarrhea. Stool specimens from 30 children with severe protracted diarrhea were studied. Twenty-seven strains of enteroaggregative Escherichia coli (EAggEC) isolated from 12 of 30 patients all belonged to the same serotype, O untypeable (OUT):H10, and showed the same biochemical characteristics and antibiotic susceptibility pattern. These strains were negative for the virulence factors of the four standard categories of diarrheagenic E. coli (enterotoxigenic, enteropathogenic, enteroinvasive, and enterohemorrhagic). However, the isolates showed an aggregative pattern of adherence to HEp-2 cells and had a 60-MDa plasmid and an astA gene, which encodes heat-stable enterotoxin-1 production. These data suggested that the EAggEC serotype OUT:H10 was associated with this massive outbreak of gastrointestinal illness.     

Examination of colonies and stool blots for detection of enteropathogens by DNA hybridization with eight DNA probes

We compared three methods for detecting enteropathogens in 416 children with diarrhea: (i) examination of 10 lactose-fermenting and all non-lactose-fermenting Escherichia coli (colony blots); (ii) examination of 300 colonies (replicate blots); and (iii) determination of the total bacterial growth of stools (stool blots). All specimens were spotted onto Whatman 541 filters and hybridized with specific radiolabeled DNA probes. Enterotoxigenic E. coli was detected in 38 patients by examining colony blots, in 52 patients by examining replicate blots, and in 45 patients by examining stool blots. Enteropathogenic E. coli adhesin factor was detected in 12 patients by colony blots, in 25 patients by replicate blots, and in 16 patients by stool blots. E. coli that hybridized with the enterohemorrhagic E. coli probe was detected in 2 patients by colony blots, in 11 patients by replicate blots, and in 0 patients by stool blots. Shiga-like toxin-producing E. coli was detected in 0 patients by colony blots, in 12 patients by replicate blots, and in 0 patients by stool blots. Shigella spp. were identified by standard bacteriological methods in 82 patients, and enteroinvasive E. coli was identified by colony blots in 11 patients (total, 93), by replicate blots in 56 patients, and by stool blots in 35 patients. Of 82 culture-confirmed Shigella infections, 45 were identified by examining replicate blots with the 17-kilobase-pair probe and 36 were identified by examination with the Ipa probe (P less than 0.05). Examining replicate blots with specific probes identified more enterotoxigenic E.coli (P < 0.005), enteropathogenic E.coli adhesion factor-producing E.coli (P < 0.001), and Shiga-like toxin-producing E.coli (P < 0.005) infections than examining colony blots. More Shigella and enteroinvasive E.coli infections were identified by standard bacteriological methods and examining colony blots with a specific probe than by examining replicate and stool blots (P < 0.001).     

Cohort study of Guinean children: incidence,pathogenicity, conferred protection,and attributable risk for enteropathogens during the first 2 years of life

We recruited 200 children shortly after birth and collected stool specimens weekly, irrespective of whether the children had diarrhea, until up to 2 years of age. All children were recruited during the first year of the study and were monitored for a median of 18.4 months. To measure pathogenicity, the odds ratio for diarrhea, adjusted for age, sex, and coinfections with other enteropathogens, was determined by logistic regression. Standard estimation of the population attributable risk indicated that rotavirus, enterotoxigenic Escherichia coli that produced only the heat-stable toxin ST, Isospora spp., Cryptosporidium parvum, Shiga toxin (Stx)-producing E. coli (STEC), and Shigella spp. or enteroinvasive E. coli were the most important contributors to diarrhea in this population. Stx2- but not Stx1-producing STEC strains were pathogenic. Enteroaggregative E. coli, diffusely adherent E. coli, and attaching-and-effacing E. coli strains, which were the most commonly isolated microorganisms, were not associated with diarrhea. For most of the microorganisms, primary infections did not confer protection against reinfection with the same organism, but some conferred protection against diarrhea from reinfection.     

Detection and characterization of diarrheagenic Escherichia coli from young children in Hanoi, Vietnam

Diarrhea continues to be one of the most common causes of morbidity and mortality among infants and children in developing countries. Escherichia coli is an emerging agent among pathogens that cause diarrhea. The development of a highly applicable technique for the detection of different categories of diarrheagenic E. coli is important. We have used multiplex PCR by combining eight primer pairs specific for enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli, enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC). This facilitates the identification of five different categories of diarrheagenic E. coli from stool samples in a single reaction simultaneously. The prevalences of diarrheagenic E. coli were 22.5 and 12% in the diarrhea group and the control group, respectively. Among 587 fecal samples from Vietnamese children under 5 years of age with diarrhea, this technique identified 132 diarrheagenic E. coli strains. This included 68 samples (11.6%) with EAEC, 12 samples (2.0%) with EIEC, 39 samples (6.6%) with EPEC, and 13 samples (2.2%) with ETEC. Among the 249 age-matched controls, 30 samples were positive for diarrheagenic E. coli. The distribution was 18 samples (7.2%) with EAEC, 11 samples (4.4%) with EPEC, and 1 sample (0.4%) with ETEC.     

Diarrhoeagenic Escherichia coli detected by 16-plex PCR in children with and without diarrhoea in Burkina Faso

The importance of diarrhoeagenic Escherichia coli (DEC) in Africa is poorly understood, and is unknown in Burkina Faso. This study investigated the occurrence of five major DEC pathogroups in primary cultures of stool samples from 658 Burkinabe children under 5 years old using 16-plex PCR for virulence-associated genes. At least one DEC pathogroup was detected in 45% of 471 children with diarrhoea and in 29% of 187 children without diarrhoea (p <0.001). More than one DEC pathogroup was detected in 11% of children with and 1% of children without diarrhoea (p <0.001). Enteroaggregative E. coli was the most common pathogroup in both children with diarrhoea (26%) and children without diarrhoea (21%). Enteropathogenic E. coli and enterotoxigenic E. coli were detected significantly more often in children with diarrhoea (16% and 13%) than in children without diarrhoea (5% and 4%; p <0.001 for both pathogroups). Shiga toxin-producing E. coli and enteroinvasive E. coli were detected only in children with diarrhoea (2% and 1%, respectively). Diarrhoeagenic E. coli, especially enteropathogenic and enterotoxigenic, may be important, unrecognized causes of childhood diarrhoea in Burkina Faso.     

Prevalence of diarrheagenic Escherichia coli strains detected by PCR in patients with travelers' diarrhea

Objective: To determine the prevalence of the different categories of diarrheagenic Escherichia coli , enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), verotoxin-producing E. coli (VTEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enteropathogenic E. coli (EPEC), associated with travelers' diarrhea.
Methods: Stool specimens from 350 patients with travelers' diarrhea were collected between 1994 and 1996. The virulence factors of the diarrheagenic E. coli isolated were detected by PCR technique, in subcultures of single colonies of all morphotypes of E. coli observed in culture on MacConkey agar.
Results: ETEC (15.7%), EAggEC (13.4%) and DAEC (9.14%) are significantly more prevalent than EIEC (3.4%), EPEC (2.86%) and VTEC (0.86%) (p < 0.05; z -test). The prevalence of ETEC and EAggEC was similar in all geographic areas visited.
Conclusions: PCR is a rapid and specific technique to use in the identification of the different categories of diarrheagenic E. coli and greatly increases the yield of potential enteropathogens from cases of travelers' diarrhea. Not only ETEC but also EAggEC and DAEC strains play a major role in the etiology of travelers' diarrhea, whereas EIEC, EPEC, and VTEC strains play a minor role, leading to the question of whether it is necessary to search routinely for these three types of E. coli in diagnostic laboratories.     

Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detection of these organisms to be practical. We developed a real-time fluorescence-based multiplex PCR for the detection of all six of the currently recognized classes of diarrheagenic E. coli. The primers were designed to specifically amplify eight different virulence genes in the same reaction: aggR for enteroaggregative E. coli, stIa/stIb and lt for enterotoxigenic E. coli, eaeA for enteropathogenic E. coli and Shiga toxin-producing E. coli (STEC), stx(1) and stx(2) for STEC, ipaH for enteroinvasive E. coli, and daaD for diffusely adherent E. coli (DAEC). Eighty-nine of ninety diarrheagenic E. coli and 36/36 nonpathogenic E. coli strains were correctly identified using this approach (specificity, 1.00; sensitivity, 0.99). The single false negative was a DAEC strain. The total time between preparation of DNA from E. coli colonies on agar plates and completion of PCR and melting-curve analysis was less than 90 min. The cost of materials was low. Melting-point analysis of real-time multiplex PCR is a rapid, sensitive, specific, and inexpensive method for detection of diarrheagenic E. coli.     

Enteroadherent Escherichia coli and diarrhea in children: a prospective case-control study.

The relative contribution of diarrheagenic Escherichia coli was examined during a 1-year prospective study of hospitalized children in Clermont-Ferrand, France, including 220 case patients (with diarrhea) and 211 matched controls. Fecal isolates were characterized by means of their pattern of adherence to HEp-2 cells and by colony hybridization with DNA probes specific for the six categories of diarrheagenic E. coli. No enteroinvasive or enterotoxigenic E. coli isolates were isolated. Twenty-eight (6.5%) eae-positive isolates and 39 (9%) enteroaggregative E. coli isolates characterized with the aggregative adherence probe and/or by their adherence pattern were detected; they were equally distributed among the patients and the controls. Diffusely adhering E. coli was the predominant pathotype: 30.7% were detected by their adherence pattern and 13.7% were detected with the daaC probe. They were isolated with similar frequencies from the patients and the controls, thereby showing no association with diarrhea. However, daaC-positive strains were significantly associated with a past record of urinary tract infections. These results suggest that the diffusely adhering E. coli organisms isolated in the present study are not true intestinal pathogens but may be regarded as resident colonic strains.