We have developed two multiplex PCR assays that detect typical and atypical enteropathogenic Escherichia coli (EPEC) isolates, enteroaggregative E. coli (EAEC) isolates, enterotoxigenic E. coli (ETEC) isolates, enteroinvasive E. coli (EIEC) isolates, Shiga toxin-producing E. coli (STEC) isolates, and Shigella spp. The targets selected for each group were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the genes encoding heat-labile and heat-stable toxins for ETEC isolates, stx 1 and stx 2 for STEC isolates, and ipaH for EIEC isolates and Shigella spp. These PCRs were specific and sensitive for rapid detection of target isolates in stools. Among 150 stool specimens from the acute diarrhea tested, 9 samples (6%) had atypical EPEC, 9 (6%) had typical EPEC, 7 (4.7%) had EAEC, 3 (2%) had EIEC, 3 (2%) had Shigella spp., and 1 (0.7%) had an O26 STEC strain; we also detected mixed infections, 2 (1.3%) with EAEC and Shigella spp., 1 (0.7%) with atypical and typical EPEC strains, and another with atypical EPEC and EAEC strains. One of the multiplex PCRs directly applied to 36 stool specimens correctly identified 100% of EPEC and EAEC isolates.Five categories of Escherichia coli have been well associated with diarrhea in several epidemiological studies (9)and Shiga toxin-producing E. coli (STEC). The virulence mechanisms that characterize these categories of E. coli are genetically encoded by chromosomal, plasmid, and bacteriophage DNAs and are represented by the following genes: eae (attaching and effacing lesions), bfpA (localized adherence), the gene encoding enteroaggregative adherence, ipaH (enteroinvasive mechanism), the genes encoding heat-labile toxin (LT) and heat-stable toxin (ST), and stx 1 and stx 2 (Shiga toxins). To correctly identify diarrheagenic E. coli strains, these organisms must be differentiated from nonpathogenic members of the normal flora. Serotypic markers correlate, sometimes very closely, with specific categories of diarrheagenic E. coli; however, these markers are rarely sufficient in and of themselves to reliably identify a strain as diarrheagenic. Thus, the detection of diarrheagenic E. coli has focused increasingly on the identification of certain characteristics which themselves determine the virulence of these organisms. This identification process may include HEp-2 cell adherence, DNA hybridization, and PCR assays to detect the presence of specific virulence traits or the genes encoding these traits. The first two types of assays require special expertise, employ cell culture and radioactive material, and are time-consuming.We developed two multiplex PCR assays to detect the five categories of diarrheagenic E. coli organisms and Shigella spp.and assessed the direct application of those assays to human diarrheal stool samples.The targets selected for each category were eae and bfpA for EPEC isolates, the target of probe CVD432 for EAEC isolates, the LT and ST genes for ETEC isolates, stx 1 and stx 2 for STEC isolates, and ipaH for EIEC isolates and Shigella sp...

A 1-year prospective study was carried out in two large urban centers of São Paulo State, Brazil, to determine the prevalences and roles of the different Escherichia coli pathotypes in children less than 5 years of age with diarrhea presenting to the emergency rooms of public hospitals or visiting private pediatricians' offices. Of the pathotypes sought, typical enteroaggregative and atypical enteropathogenic types of E. coli were isolated for 8.9% and 5.4% of 774 diarrhea cases, respectively, and were found to be dominant and significantly associated with diarrhea.

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.

The correlation of the different adherence patterns with DNA probes and PCR primers for the identification of Escherichia coli was analyzed in isolates from children, less than 2 years of age with or without diarrhea, from different regions of Brazil. A total of 1,428 isolates obtained from 338 patients and 322 control children were studied. The enteropathogenic E. coli (EPEC) adherence factor (EAF) probe was shown to be as good as the HEp-2 adhesion assay for the detection of typical EPEC strains. The DNA probes used to detect diffusely adhering E. coli and enteroaggregative E. coli (EAEC) showed low sensitivities (64 and 50%, respectively), and the best method of identifying these organisms in clinical research remains the HEp-2 adherence assay. The "bundle-forming pilus" (BFP) and the EAEC PCR assays could be used instead of the DNA probes as a screening method for typical EPEC and EAEC carrying the EAEC probe sequence in the clinical laboratory. In our study, only typical EPEC strains that carried EAF and BFP were associated with acute diarrhea. The LA exhibited by typical enteropathogenic E. coli (typical EPEC) is mediated by an inducible bundle-forming pilus (BFP), whose expression correlates with the presence of a plasmid designated the EPEC adherence factor (EAF) plasmid (1, 17). EPEC strains also cause attaching and effacing lesions on eukaryotic cells that involve a 94-kDa protein encoded by the chromosomal eae gene (25, 28). The pathogenicity of EPEC strains has been demonstrated in human volunteers, and their role in childhood diarrhea was confirmed in epidemiological studies (9,10,11,15,18,26). Atypical EPEC strains do not carry the EAF plasmid, were found to exhibit LAL, and have been isolated from acute infantile diarrhea in São Paulo (35).The adherence of many enteroaggregative E. coli strains requires the presence of a plasmid that contains genes encoding the AA (38). Epidemiological studies have implicated EAEC as a cause of diarrhea in children in developing countries, and the pathogenic potential of EAEC in human infections was substantiated by challenge studies (5,10,23,27,39).Two factors, F1845 and AIDA-I were found to encode DA in diffusely adhering E. coli (DAEC) (4, 6). Several recent studies have implicated DAEC strains as agents of diarrhea, while other studies have not recovered DAEC strains more frequently from diarrheal patients than from asymptomatic controls (2,15,16,21,24).DNA probes derived from the adherence-related sequences have been constructed (3,4,6,17,25,30) and used in hybridization assays for the detection of the different putative categories of diarrheagenic E. coli in many epidemiologial studies. PCR primers have been also developed for several of the categories of diarrheagenic E. coli (8,14,20,36).In order to optimize screening methods for putative pathogenic enteroadherent E. coli in the clinical laboratory we analyzed the correlation of the different HEp-2 adherence patterns with DNA probes and PCR primers in E. coli isolates from different urban centers of Brazil...

We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea.Enteropathogenic Escherichia coli (EPEC), one of the six E. coli diarrheagenic pathotypes, produces an adherence factor chromosomally encoded by the eae (EPEC attaching and effacing) gene located within the locus for enterocyte effacement (LEE) pathogenicity island (15,16,18)."Typical" EPEC strains contain, in addition to eae, the EPEC adherence factor (EAF) plasmid (4), which encodes the bundle-forming pili that mediate localized adherence to epithelial cells (9,26). EPEC strains lacking the EAF plasmid have been designated "atypical" EPEC (aEPEC) (12). Whereas typical EPEC strains express only the virulence factors encoded by the LEE region and the EAF plasmid, aEPEC strains have additional virulence properties (11,32).Recently Afset et al. (2) described several virulence genes associated with diarrhea in aEPEC isolates from Norwegian children. In their study, genes belonging to the pathogenicity island OI-122 (efa1/lifA, nleB, nleE, and sen), present in the enterohemorrhagic E. coli (EHEC) reference strain EDL933 (17), and the gene for long polar fimbriae (lpfA) (10), found in EHEC O113 strains, were particularly frequent. Other genes, such as the porcine A/E-associated gene (paa) (5) and the EHEC hemolysin gene (ehxA) (27), were also found to be associated with diarrheal disease.Other E. coli adhesion factors recently described include the protein ToxB, required for full adherence expression in EHEC O157:H7 (30); Iha, an adherence-conferring protein similar to Vibrio cholerae IrgA (29); Saa, an autoagglutinating adhesin identified in LEE-negative strains (22); and Spf, a sorbitolfermenting EHEC O157 fimbria (8). In addition, we recently identified a diffuse adherence (lda) locus in an aEPEC strain of the O26 serogroup that codes for adherence to .In this report, we describe the prevalence and virulence profile of aEPEC strains isolated from diarrhea patients and control subjects in several cities of Brazil from 1999 through 2004.Stool specimens from 1,102 children under 2 years of age with diarrhea, presenting to the emergency room of public hospitals in seven cities representing different regions of Brazil, and 647 randomly selected children without any gastrointestinal symptoms from the same hospitals were studied. All specimens were investigated for the presence of enteric pathogens, such as diarrheagenic E. coli, Shigella species, Salmonella species, Yersinia enterocolitica, Campylobacter species, and rotavirus (24).Atypical EPEC strains were isolated, identified, and serotyped as described elsewhere (11). One isolate per subject was stored at Ϫ70°C. aEPEC strains were screened by colony blot hybridization wit...