Summary-Enteropathogenic Escherichia coli (EPEC) are among the most important pathogens infecting children worldwide and are one of the main causes of persistent diarrhea. EPEC were originally serogroup-defined E. coli associated with infantile diarrhea. As various mechanisms of pathogenesis have been discovered, EPEC classification has come to be based on the presence of specific genes. The eae (intimin) and bfpA (bundle-forming pilus) genes have both been used for identification of EPEC and for subdivision of this group of bacteria into typical and atypical strains. For many years typical EPEC have been considered to be the leading cause of infantile diarrhea in developing countries and were considered rare in industrialized countries. However, current data suggests that atypical EPEC are more prevalent than typical EPEC in both developing and developed countries. Moreover, the duration of diarrhea in patients infected with atypical EPEC is significantly longer than that caused by other pathogens. When comparing the isolation rates of EPEC among children with diarrhea and healthy controls without diarrhea, in general, there is a higher isolation rate in diarrhea, although not significantly higher in all studies. These inconsistencies probably are related to the study patient populations, reflecting a possible age-related susceptibility to infection.

EPEC load measured by qRT-PCR is higher in diarrheal than in healthy children. qRT-PCR may be useful to study the relationship between disease and colonization in settings of endemicity.

Enterotoxigenic Escherichia coli (ETEC) is a major cause of childhood diarrhea. The present study sought to determine the prevalence and distribution of toxin types, colonization factors (CFs), and antimicrobial susceptibility of ETEC strains isolated from Peruvian children. We analyzed ETEC strains isolated from Peruvian children between 2 and 24 months of age in a passive surveillance study. Five E. coli colonies per patient were studied by multiplex real-time PCR to identify ETEC virulence factors. ETEC-associated toxins were confirmed using a GM1-based enzyme-linked immunosorbent assay. Confirmed strains were tested for CFs by dot blot assay using 21 monoclonal antibodies. We analyzed 1,129 samples from children with diarrhea and 744 control children and found ETEC in 5.3% and 4.3%, respectively. ETEC was more frequently isolated from children >12 months of age than from children <12 months of age (P < 0.001). Fifty-two percent of ETEC isolates from children with diarrhea and 72% of isolates from controls were heat-labile enterotoxin (LT) positive and heat-stable enterotoxin (ST) negative; 25% and 19%, respectively, were LT negative and ST positive; and 23% and 9%, respectively, were LT positive and ST positive. CFs were identified in 64% of diarrheal samples and 37% of control samples (P < 0.05). The most common CFs were CS6 (14% and 7%, respectively), CS12 (12% and 4%, respectively), and CS1 (9% and 4%, respectively). ST-producing ETEC strains caused more severe diarrhea than non-ST-producing ETEC strains. The strains were most frequently resistant to ampicillin (71%) and co-trimoxazole (61%). ETEC was thus found to be more prevalent in older infants. LT was the most common toxin type; 64% of strains had an identified CF. These data are relevant in estimating the burden of disease due to ETEC and the potential coverage of children in Peru by investigational vaccines.

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.

Infectious diarrhea can be classified based on its clinical presentation as noninflammatory or inflammatory disease. In developing countries, among inflammatory diarrhea cases, Shigella is the most common cause, followed by Campylobacter and Salmonella. Because the time frame in which treatment choices must be made is short and conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. The purpose of our study was to develop a multiplex real-time PCR procedure to simultaneously identify Campylobacter spp., Salmonella spp., and Shigella spp. Primers were designed to amplify the invA, ipaH, and 16S rRNA genes simultaneously in a single reaction to detect Salmonella, Shigella, and Campylobacter, respectively. Using this approach, we correctly identified 102 of 103 strains of the targeted enteropathogens and 34 of 34 other pathogens. The melting temperatures were 82.96 ؎ 0.05°C for invA, 85.56 ؎ 0.28°C for ipaH, and 89.21 ؎ 0.24°C for 16S rRNA. The limit of accurate quantification for the assay in stool samples was 10 4 CFU g ؊1 ; however, the limit of detection was 10 3 CFU g ؊1 . This assay is a simple, rapid, inexpensive, and reliable system for the practical detection of these three enteropathogens in clinical specimens. Infectious diarrhea is a global health problem that is still responsible for thousands of deaths worldwide, especially in children (1). It can be classified based on its clinical presentation as one of two syndromes-noninflammatory or inflammatory diarrhea (2). Among cases of inflammatory diarrhea, Shigella is the most common cause, followed by Campylobacter and Salmonella (3). These invasive organisms primarily target the lower bowel; they invade the intestinal mucosa to induce an acute inflammatory reaction and activate cytokines and inflammatory mediators (4).Because the time frame in which treatment choices must be made is short and the conventional stool cultures lack good sensitivity, there is a need for a rapid, sensitive, and inexpensive detection technique. We searched for DNA sequences that were highly conserved between the different species of each genera, and we selected the following genes as targets, invA (invasion A gene) for Salmonella spp., ipaH (invasion plasmid antigen H) for Shigella spp., and 16S rRNA for Campylobacter spp., to develop a fluorescence-based real-time PCR procedure to simultaneously identify these enteropathogens. In this method the post-PCR products are identified based on melting-point curve analysis. We have also standardized the technique to quantify these bacteria directly from stool samples. MATERIALS AND METHODSBacterial strains. A total of 147 enteropathogenic strains (Table 1) were analyzed, including clinical isolates representative of Salmonella spp., Shigella spp., and Campylobacter spp., as well as other enteropathogens. These clinical strains had previously been identified based on serology, biochemical assays, and real-time PCR for the diarrheagenic Escherichia coli (5). In add...