Search

Background Viruses are a leading cause of pediatric diarrheal illness, resulting in over 200,000 childhood deaths annually worldwide. Rotavirus is the most common viral cause of moderate-to-severe diarrhea in low to middle-income countries, but adenovirus 40/41, norovirus, sapovirus, and astrovirus are increasingly recognized as important contributors to the global viral gastroenteritis burden. Comparisons of clinical severity between these five viruses among infants and young children are currently limited. Methods The objective of this analysis was to describe the clinical severity of rotavirus, adenovirus 40/41, norovirus, sapovirus, and astrovirus among children enrolled in the Global Enteric Multicenter Study (GEMS). Stool samples from children younger than 5 years of age with moderate-to-severe diarrhea at four sites in Africa and three in Asia were tested by enzyme immunoassays (rotavirus, adenovirus) and multiplex reverse transcriptase polymerase chain reaction (norovirus, sapovirus, astrovirus). A modified Vesikari score (MVS) to assess clinical severity was calculated based on symptoms and clinical characteristics. Results Among 3,305 viral diarrhea cases, rotavirus had the highest median MVS (9 [interquartile range [IQR: 7,11]) followed by adenovirus 40/41 (8 [IQR]: 6,10]); sapovirus and astrovirus had a similar MVS distribution to norovirus (7 [IQR: 6, 9]). The proportion of patients experiencing > 3 episodes of vomiting was high for those with rotavirus (66.3%) and adenovirus 40/41 (55.7%); astrovirus (40.8%), sapovirus (40.0%), and norovirus (35.9%) patients also experienced substantial episodes of vomiting. A high proportion of cases with adenovirus 40/41 (54.0%) and rotavirus (46.3%) experienced ≥ 7 episodes of diarrhea within 24 hours. While hospitalization or intravenous fluids were required most frequently for patients with rotavirus (42.1%), the proportion was also high in those with adenovirus 40/41 (30.6%). Conclusion These findings demonstrate the clinical severity of viral diarrheal pathogens besides rotavirus, in particular adenovirus 40/41, warranting the continued development of vaccines and therapeutics to prevent morbidity and mortality due to viral enteric pathogens. Disclosures All Authors: No reported disclosures.

Background The association between childhood diarrheal disease and linear growth faltering in developing countries is well described. However, the impact attributed to specific pathogens has not been elucidated, nor has the impact of recommended antibiotic treatment. Methods The Global Enteric Multicenter Study enrolled children with moderate to severe diarrhea (MSD) seeking healthcare at 7 sites in sub-Saharan Africa and South Asia. At enrollment, we collected stool samples to identify enteropathogens. Length/height was measured at enrollment and follow-up, approximately 60 days later, to calculate change in height-for-age z scores (ΔHAZ). The association of pathogens with ΔHAZ was tested using linear mixed effects regression models. Results Among 8077 MSD cases analyzed, the proportion with stunting (HAZ below −1) increased from 59% at enrollment to 65% at follow-up (P < .0001). Pathogens significantly associated with linear growth decline included Cryptosporidium (P < .001), typical enteropathogenic Escherichia coli (P = .01), and untreated Shigella (P = .009) among infants (aged 0–11 months) and enterotoxigenic E. coli encoding heat-stable toxin (P < .001) and Cryptosporidium (P = .03) among toddlers (aged 12–23 months). Shigella-infected toddlers given antibiotics had improved linear growth (P = .02). Conclusions Linear growth faltering among children aged 0–23 months with MSD is associated with specific pathogens and can be mitigated with targeted treatment strategies, as demonstrated for Shigella.

Background Shigella is a leading cause of childhood diarrhea and target for vaccine development. Microbiologic and clinical case definitions are needed for pediatric field vaccine efficacy trials. Methods We compared characteristics of moderate to severe diarrhea (MSD) cases in the Global Enteric Multicenter Study (GEMS) between children with culture positive Shigella to those with culture-negative, quantitative polymerase chain reaction (qPCR)-attributable Shigella (defined by an ipaH gene cycle threshold, Molecular methods identified Shigella more commonly than microbiologic culture in younger and stunted children. A simplified clinical score containing dehydration, hospitalization, and diarrhea duration could be used to stratify vaccine trial endpoints by severity based on its ability to predict death.

(See the editorial commentary by Steiner on pages 1860–2.) Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic Escherichia coli that is a cause of acute and persistent diarrhea in many settings [1–7]. EAEC strains express a heterogeneous array of putative virulence factors [8–12] encoded on the bacterial chromosome or on the EAEC-specific pAA plasmid. EAEC strains often harbor a transcriptional activator of the AraC/XylS class, called “AggR” [13], which controls genes on both the plasmid and the chromosome. Among the genes under AggR control include those that encode the aggregative adherence fimbriae (AAF), of which at least 4 variants exist [14–18]. AAF adhesins have been shown to be essential for EAEC adherence to human intestinal explants and to elicit both cytokine release and opening of epithelial tight junctions in a polarized epithelial model [19, 20]. EAEC strains also often harbor a variable number of serine protease autotransporters of Enterobacteriaceae (SPATEs) that are implicated in immune evasion, mucosal damage, secretogenicity, and colonization [21]. In 2011, an outbreak of foodborne hemorrhagic colitis originated in Germany, spreading to other European countries. Over 4000 individuals were affected, including primary and secondary cases [22]. Hemolytic uremic syndrome (HUS) developed in approximately 22% of the cases [22], and 54 people died [23, 24]. The implicated pathogen was an EAEC strain of the rare serotype O104:H4 [23], which was lysogenized with an Stx2a-converting phage. Genomic analysis [25] demonstrated that the outbreak strain contained the genes required to produce the AAF/I variant and 3 SPATEs (Pic, SigA, and SepA). Although much is known about the pathogenesis of serotype O157:H7 Shiga toxin (Stx)–producing E. coli, it is unclear how an EAEC strain would be able to elicit severe hemorrhagic colitis and HUS, even when harboring an Stx-encoding gene. In this study, we tested the hypothesis that the plasmid-borne virulence factors of EAEC contributed to the high pathogenicity of the German outbreak strain by promoting strong adherence to the epithelium and/or by opening epithelial tight junctions.

Recent evidence suggests that the abundance of enteric pathogens in the stool correlates with the presence of clinical diarrhea. We quantified the fecal pathogen after feeding enterotoxigenic Escherichia coli (ETEC) strain H10407 to 30 adult volunteers. Stools were collected daily and examined using qualitative and quantitative (Q) culture. DNA was isolated, and quantitative (Q) PCR targeting the heat-labile toxin (LT) gene was performed. Nine volunteers developed diarrhea. Among 131 stool specimens with complete data, pathogen abundance by QPCR was strongly correlated with Qculture, ρ = 0.61, P < 0.0001. Receiver operating characteristic curve analysis comparing quantitative data against diarrhea status suggested cut-points, based on a maximum Youden Index, of 2.8 × 10(4) LT gene copies and 1.8 × 10(7) CFU. Based on these cut-points, QPCR had a sensitivity and specificity compared with diarrheal status of 0.75 and 0.87, respectively, and an OR of 20.0 (95% CI 5.7-70.2), whereas Qculture had a sensitivity and specificity of 0.73 and 0.91, respectively, and an OR of 28.6 (95% CI 7.7-106.6). Qculture had a sensitivity and specificity of 0.82 and 0.48, respectively and an OR of 4.4 (95% CI 1.2-16.0). The correlation between Qculture and QPCR was highest in diarrheal specimens, and both quantitative methods demonstrated stronger association with diarrhea than qualitative culture.

To understand the etiology of moderate-to-severe diarrhea among children in high mortality areas of sub-Saharan Africa and South Asia, we performed a comprehensive case/control study of children aged

Background. Enteroaggregative Escherichia coli (EAEC) is a cause of epidemic and sporadic diarrhea, yet its role as an enteric pathogen is not fully understood. Methods. We characterized 121 EAEC strains isolated in 2008 as part of a case-control study of moderate to severe acute diarrhea among children 0–59 months of age in Bamako, Mali. We applied multiplex polymerase chain reaction and comparative genome hybridization to identify potential virulence factors among the EAEC strains, coupled with classification and regression tree modeling to reveal combinations of factors most strongly associated with illness. Results. The gene encoding the autotransporter protease SepA, originally described in Shigella species, was most strongly associated with diarrhea among the EAEC strains tested (odds ratio, 5.6 [95% confidence interval, 1.92–16.17]; P = .0006). In addition, we identified 3 gene combinations correlated with diarrhea: (1) a clonal group positive for sepA and a putative hemolysin; (2) a group harboring the EAST-1 enterotoxin and the flagellar type H33 but no other previously identified EAEC virulence factor; and (3) a group carrying several of the typical EAEC virulence genes. Conclusion. Our data suggest that only a subset of EAEC strains are pathogenic in Mali and suggest that sepA may serve as a valuable marker for the most virulent isolates.

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.

We have previously described a protein termed Shigella enterotoxin 2 (ShET-2), which induces rises in short-circuit current in rabbit ileum mounted in the Ussing chamber. Published reports have postulated that ShET-2 may be secreted by the Shigella type III secretion system (T3SS). In this study, we show that ShET-2 secretion into the extracellular space requires the T3SS in Shigella flexneri 2a strain 2457T and a ShET-2-TEM fusion was translocated into epithelial cells in a T3SS-dependent manner. The ShET-2 gene, sen, is encoded downstream of the ospC1 gene of S. flexneri, and we show that sen is cotranscribed with this T3SS-secreted product. Considering that T3SS effectors have diverse roles in Shigella infection and that vaccine constructs lacking ShET-2 are attenuated in volunteers, we asked whether ShET-2 has a function other than its enterotoxic activity. We constructed a ShET-2 mutant in 2457T and tested its effect on epithelial cell invasion, plaque formation, guinea pig keratoconjunctivitis and interleukin 8 (IL-8) secretion from infected monolayers. Although other phenotypes were not different compared with the wild-type parent, we found that HEp-2 and T84 cells infected with the ShET-2 mutant exhibited significantly reduced IL-8 secretion into the basolateral compartment, suggesting that ShET-2 might participate in the Shigella-induced inflammation of epithelial cells.

Enteroaggregative Escherichia coli (EAEC) causes diarrhea in diverse populations worldwide. The AraC-like regulator AggR is a key virulence regulator in EAEC. AggR-regulated genes include those encoding the Aggregative Adherence Fimbria, the dispersin protein, and a type VI secretion system. This study characterizes the regulation of the aggR promoter (P(aggR)). Using primer extension analysis, the transcriptional start site of the aggR promoter was located 40 nucleotides upstream of the translational start. P(aggR) was found to be autoregulated and DNA footprinting revealed the presence of two AggR-binding sites: one upstream of the transcriptional start site and one downstream. Additionally, P(aggR) was found to be positively regulated by the DNA-binding protein FIS and negatively regulated by the global regulator H-NS. To further understand this complex regulation scheme, a bacterial luciferase reporter system was used with a mouse model of EAEC colonization. This allowed for the in vivo measurement of P(aggR), P(fis), and P(hns) activity. EAEC present in the mouse intestine possessed relatively high levels of P(fis) and P(aggR) activity and a low level of P(hns) when compared with in vitro experiments. The data provide significant insights into the regulation cascade leading to aggR expression in the mammalian intestine during EAEC infection.

Background Imipenem-resistant Pseudomonas aeruginosa (IRPA) is an emerging problem. The causal role of antibiotic selective pressure versus patient-to-patient transmission has not been assessed using a large cohort.

Background. Diarrhea remains a common complaint among US patients who seek medical attention. Methods. We performed a prospective study to determine the etiology of diarrheal illness among patients and control subjects of all ages presenting to the emergency departments and outpatient clinics of 2 large academic hospitals in Baltimore, Maryland, and New Haven, Connecticut. We used molecular methods to detect the presence of diarrheagenic Escherichia coli pathotypes, including enteroaggregative E. coli (EAEC), as well as Shiga toxin-producing, cytodetaching, enterotoxigenic and enteropathogenic E. coli. Results. Of the pathotypes sought, only EAEC was found in an appreciable proportion (4.5%) of case patients, and it was found more frequently among case patients than control subjects (P

A DNA microarray was used to analyze the distribution of plasmid and chromosomal genes among strains of enteroaggregative Escherichia coli (EAEC) isolated from a prospective diarrhoea surveillance study in the United Kingdom. Target genes were extracted from existing databases and from the genome sequence of prototype EAEC strain 042. We found that strains exhibiting the aggregative adherence (AA) phenotype could be broadly divided into two groups depending upon whether they harboured genes from the EAEC virulence plasmid (pAA) and a set of chromosomal genes found in EAEC strain 042. Several chromosomal loci were inherited en bloc, and were more common in strains which we designated Group 1; genes at the pheU locus were particularly conserved. Genes encoded on the pAA plasmid and those under control of the master regulator AggR were also concentrated in the Group 1 EAEC. A gene encoding a type 1 pilin allele was detected more frequently in Group 2 EAEC. Our data suggest that strains previously designated as typical EAEC harbour a large number of conserved plasmid and chromosomal loci, further illuminating a package of virulence genes common to the most important EAEC.

Background We created a live, attenuated, oral Shigella vaccine by constructing a lineage of guanine auxotrophs and conducted a double-blind, placebo-controlled trial to ascertain (1) the attenuation profile of Delta guaBA Shigella flexneri 2a, which harbors deletions in the guanine nucleotide synthesis pathway (CVD 1204); (2) additional attenuation conferred by deletions in set and sen genes encoding Shigella enterotoxins (ShETs) 1 and 2, respectively (CVD 1208); and (3) the relative immunogenicity of these constructs. Methods Inpatient volunteers received a single oral dose of CVD 1204, CVD 1208 (10(7), 10(8), or 10(9) cfu), or placebo. Clinical, immunologic, and microbiologic responses were evaluated. Results Reactogenicity occurred in 8 of 23 recipients of CVD 1204, characterized by diarrhea (30%), fever (22%), and/or dysentery (17%), but in only 1 (5%) of 21 recipients of CVD 1208 (brief fever) (P=.02, Fisher's exact test). Antilipopolysaccharide responses, as measured by antibody-secreting cell, serum, or fecal antibody levels, occurred in 67%, 71%, and 100% of recipients of CVD 1204 and in 86%, 43%, and 100% of recipients of CVD 1208 at doses of 10(7), 10(8), and 10(9) cfu, respectively. Conclusions We conclude that 1 or both ShETs are virulence determinants in humans; their inactivation, in combination with Delta guaBA, leads to a well-tolerated and immunogenic Shigella vaccine candidate.

Enteroaggregative Escherichia coli (EAEC) is distinguished by its characteristic aggregative adherence (AA) pattern to cultured epithelial cells. In this study we investigated the role of type I fimbriae (TIF) in the AA pattern to HEp-2 cells and in biofilm formation. Accentuation of this pattern was observed when the adherence assay was performed in the absence of mannose. This effect was observed in the prototype EAEC strain 042 (O44:H18), O128:H35 strains and for other EAEC serotypes. Antiserum against TIF decreased AA by 70% and 90% for strains 042 and 18 (O128:H35 prototype strain), respectively. A non-polar knockout of fimD, the TIF usher, in strains 042 and 18 resulted in inhibition of the accentuated AA pattern of approximately 80% and 70% respectively, and biofilm formation diminution of 49% for 042::fimD and 76% for 18::fimD. Our data evidence a role for TIF in the AA pattern and in EAEC biofilm formation, demonstrating that these phenotypes are multifactorial.

Enteroaggregative Escherichia coli (EAEC) is associated with diarrhea in Spanish travelers to developing countries. In this study, the polymerase chain reaction was used to test EAEC isolates for genes encoding putative virulence factors, including EAEC adhesins, the plasmid-encoded toxin (Pet), a heat-stable enterotoxin (EAST), and Shigella enterotoxins 1 and 2 (ShET1 and ShET2). Findings included a low prevalence of genes for Pet (4.3%), ShET2 (4.3%), and the adherence factor AAF/II (8.7%). The overlapping genes encoding the ShET1 and the Pic mucinase were present in most EAEC strains tested (56.5%); however, some strains that carried this locus did not produce both proteins, as determined by Western immunoblot. Surprisingly, ShET1 and ShET2 genes were also found in other E. coli pathotypes, as was the EAST toxin locus. These findings underscore the heterogeneity of EAEC strains and suggest that the ShET1 may be an important virulence factor in traveler's diarrhea.

Persistent diarrhea (PD; duration >/=14 days) is a growing part of the global burden of diarrheal diseases. A 45-month prospective cohort study (with illness, nutritional, and microbiologic surveillance) was conducted in a shantytown in northeastern Brazil, to elucidate the epidemiology, nutritional impact, and causes of PD in early childhood (0-3 years of age). A nested case-control design was used to examine children's diarrhea burden and nutritional status before and after a first PD illness. PD illnesses accounted for 8% of episodes and 34% of days of diarrhea. First PD illnesses were preceded by a doubling of acute diarrhea burdens, were followed by further 2.6-3.5-fold increased diarrhea burdens for 18 months, and were associated with acute weight shortfalls. Exclusively breast-fed children had 8-fold lower diarrhea rates than did weaned children. PD-associated etiologic agents included Cryptosporidium, Giardia, enteric adenoviruses, and enterotoxigenic Escherichia coli. PD signals growth shortfalls and increased diarrhea burdens; children with PD merit extended support, and the illness warrants further study to elucidate its prevention, treatment, and impact.

Enterotoxigenic Escherichia coli (ETEC) is the most commonly isolated pathogen responsible for travelers' diarrhea and the cause of up to 650 million cases of pediatric diarrhea per year in the developing world. As a safe alternative to the prophylactic use of antibiotics, a hyperimmune bovine milk antibody product with specific activity against purified colonization factor antigens (CFAs) was developed and evaluated in a human challenge study. Twenty-five healthy adult volunteers were challenged orally with 10(9) cfu of a virulent CFA/I-bearing ETEC. In the randomized double-blind trial, 7 of 10 volunteers receiving a lactose-free placebo developed clinical diarrhea after challenge, compared with only 1 of 15 cases in volunteers receiving active product (Fisher's exact test, P < .0017). It is concluded that antibodies against CFAs alone are sufficient for protection and that prophylaxis with milk-derived immunoglobulin is a feasible alternative to existing drug interventions.

Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 10(6) cfu of a rugose variant of V. cholerae O1 El Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined.

Background. \ud Diarrhea is a leading cause of illness and death among children aged

Vibrio cholerae O139 Bengal has recently emerged as a cause of epidemic cholera in Asia. To evaluate clinical and immunologic responses to infection, V. cholerae O139 Bengal AI1837 was administered to healthy adult North American volunteers. Two of 4 persons ingesting 10(4) cfu became ill (incubation period, 48 h; mean diarrheal stool, 1873 g), as did 7 of 9 persons receiving 10(6) cfu (incubation period, 28 h; mean diarrheal stool, 4548 g). Ill volunteers did not demonstrate a vibriocidal antibody response to the challenge strain or other V. cholerae. Three months later, volunteers were rechallenged with the homologous O139 Bengal strain. Only 1 of 6 persons who had been ill on initial challenge had diarrhea, compared with 11 of 13 controls (P = .01; protective efficacy = 80%). V. cholerae O139 Bengal can cause severe diarrhea typical of cholera, with clinical characteristics and a dose-response similar to those seen with V. cholerae O1 El Tor. A moderately high level of protection against subsequent disease is provided by initial clinical infection.

Enteric infection with adherent bacteria has been seen in a person with chronic diarrhea who was infected with the human immunodeficiency virus. In this study, adherent bacteria were seen in 17% of all patients with AIDS evaluated during a 1-year period. The infection was centered in the cecum and right colon. Three distinct histopathologic patterns of adherence were observed: attaching and effacing lesions, bacteria intercalated between microvilli, and aggregates of bacteria more loosely attached to the damaged epithelium. The infections were associated with weight loss (P < .005) and peripheral blood CD4+ cell of counts < 100/mm3 (P < .05). Eight of 9 patients treated with antibiotics had symptomatic improvement. Bacterial cultures of rectal biopsies frozen at endoscopy yielded Escherichia coli in 12 of 18 cases; aggregative adherence was seen in 6. Isolates from 2 cases hybridized with a DNA probe encoding aggregative properties. These results suggest that chronic infection with adherent bacteria may produce a syndrome of AIDS-associated diarrhea and wasting.

Enteroaggregative Escherichia coli (EAggEC) are diarrheal pathogens defined by aggregative adherence to HEp-2 cells. In an effort to identify pathogenic EAggEC isolates, four groups of 5 volunteers were fed 1 of 4 different EAggEC strains, each at a dose of 10(10) cfu. Strain 042 caused diarrhea in 3 of 5 adults; 3 other EAggEC isolates (17-2, 34b, and JM221) failed to elicit diarrhea. A gene encoding enterotoxin EAST1 was found in strains 042 and 17-2 but not 34b or JM221; a 108-kDa cytotoxin was expressed in all 4 isolates. All 4 isolates showed a modest degree of gentamicin protection in HEp-2 cells. 17-2, 34b, and JM221 expressed the fimbrial antigen AAF/I; 042 did not express this fimbria as determined by immunogold electron microscopy and genetic probe hybridization.

rcsp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal delta aroC locus of attenuated delta aroC, delta aroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 omega (delta aroC1019::tacP-rcsp) was well tolerated by 10 volunteers who were fed two doses of 5 x 10(7) organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: 1 developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8+ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein. Improved foreign gene expression should enhance immunogenicity.

The incidence of diarrhea due to six categories of diarrheogenic Escherichia coli was determined in two pediatric cohorts in a low socioeconomic level community in Santiago, Chile, with access to chlorinated water. An age cross-sectional cohort of 340 children aged birth to 47 months was assembled. A newborn cohort was assembled by enrolling 10-12 newborns monthly for 12 months. Episodes of diarrhea were detected by twice weekly household visits. E. coli from stool cultures of cases and matched controls were hybridized with DNA probes specific for enterotoxigenic, enteroinvasive, enteropathogenic, enterohemorrhagic, enteroaggregative, and diffuse adherence E. coli. Overall, the incidence of diarrhea was low (2.1 episodes/infant/year). Nevertheless, a putative E. coli enteropathogen was found in a large proportion of diarrheal episodes, particularly during the summer. In both cohorts, enterotoxigenic E. coli were important pathogens. Enteropathogenic E. coli were incriminated during the first year of life in the newborn cohort, where they were found significantly more often in cases (p = 0.021) than in controls; beyond this age, isolation rates were similar. In contrast, the relative risk of isolation of diffuse adherence E. coli increased with age in the age cross-sectional cohort, where, overall, the difference in rate of isolation between cases and controls was significant (p = 0.0024). Enteroinvasive and enterohemorrhagic E. coli were isolated infrequently. Enteroaggregative E. coli were encountered equally in cases and controls. Facile transmission of E. coli enteropathogens is occurring in this community despite the availability of potable water.Researchers conducted an age cross sectional cohort analysis of 340 0-47 month old children and newborn cohort analysis of 144 newborns to determine the diarrheogenic Escherichia coli incidence in Santa Julia, a low socioeconomic community in Santiago, Chile. Children in the age cross sectional cohort had age, sex, and sector matched controls. The newborns had sex matched controls. A public health nurse or nurse auxiliary visited the household of each subject 2 times a week to detect diarrhea episodes. Between December 1986 and February 1990, the age cross sectional cohort had 1178 episodes of diarrhea and the newborn cohort had 674 episodes. The overall diarrhea incidence was only 2.1 episodes/child/year. An E. coli enteropathogen was isolated in many of these episodes, especially during the summer (e.g. enterotoxigenic E. coli [ETEC], 2.2 cases/month in summer vs. 0.4 cases/month in winter; p = .00001). Diffuse adherence E. coli (DAEC) and enteropathogenic E. coli (EPEC) infections also peaked in the summer. ETEC contributed greatly to diarrheal episodes in both cohorts. Among newborns, EPEC was isolated significantly more often in cases than controls during the 1st 12 months of life (6.7% vs. 2.5%; p = .021). After 1 year, however, E. coli isolation rates were essentially the same. On the other hand, in the age cross sectional cohort, the relative risk of isolation of DAEC rose with age (e.g., 1.1 for 0.11 months, 1.4 for 36-47 months, and 2.1 for = or 48 months). In the same cohort, DAEC infections were much more common in cases than controls (16.6% vs. 11.9%; p = .0024). Enteroinvasive and enterohemorrhagic E. coli were the most rarely isolated E. coli types. No difference in the isolation rate of enteroaggregative E. coli existed between cases and controls. Since most households in Santa Julia have access to potable water (68%) and an indoor toilet (64%), food contamination were likely the vehicles of E. coli transmission because more than 50% of households do not have a refrigerator.

CVD 103-HgR is a live oral cholera vaccine that, in phase I and II studies to date, has been well tolerated and immunogenic. In challenge studies of US volunteers conducted 4-5 weeks after vaccination, CVD 103-HgR provided significant protection against experimental cholera due to classical and El Tor Vibrio cholerae O1. To determine the onset and duration of protection, two volunteer challenge studies were conducted: the first, 6 months after vaccination and the second, 8 days after vaccination. In both studies, CVD 103-HgR was 100% protective against diarrhea and significantly reduced the rate of shedding of vibrios after challenge with V. cholerae classical Inaba strain 569B, the virulent parent strain of CVD 103-HgR. Previously vaccinated subjects were less likely than naive controls to develop rises in titer of vibriocidal antibodies after challenge (P = .002), and the mean peak titer of vibriocidal antibodies was less than among controls. CVD 103-HgR can provide homologous protective immunity as soon as 8 days after vaccination and protection can persist for at least 6 months.

The volunteer challenge model was used to study the virulence of strains of Vibrio cholerae O1 El Tor recently isolated from cases of cholera in South America. Fifteen of the 24 volunteers (62%) were of blood group O, the group most prevalent in South America and the group at increased risk of more severe cholera. Two El Tor Inaba strains and 2 El Tor Ogawa strains were given to volunteers at a dose of 1–2 × 106 colony-forming units. All 4 strains caused diarrhoea in 67–83% of volunteers. Volunteers with blood group antigen O had an increased attack rate for diarrhoea (P = 0.015) and a marginally increased mean diarrhoeal stool volume (P = 0.08) after challenge. One-third of the volunteers with blood group O, and none of the volunteers with other blood groups, developed severe diarrhoea (>5 L) (P = 0.01). This study established a model of South American cholera that can be used to predict field efficacy of candidate vaccines among populations with a high prevalence of blood group antigen O.

A DNA probe to detect genes conferring localized adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 cells was evaluated by using E. coli isolates from the stools of Peruvian infants with and without diarrhea. The probe was both sensitive and specific and revealed that Hep-2 adherence (because of the EPEC adherence factor [EAF] was more frequent in some O serogroups of EPEC than in others. Those serogroups in which EAF is almost always found have been designated class I EPEC; serogroups in which EAF is rarely found have been designated class II. Both class I (EAF-positive) and class II EPEC are associated with diarrheal disease.

Enterohemorrhagic Escherichia coli (EHEC) cause hemorrhagic colitis and hemolytic uremic syndrome (HUS), make potent cytotoxins (Verotoxins [VT] or Shiga-like toxins), and possess a plasmid (approximately 60 megadaltons) that encodes a new fimbrial antigen and promotes attachment to epithelial cells. We evaluated the use of a DNA probe, prepared from a 3.4-kilobase segment of the EHEC plasmid, to detect EHEC. The probe hybridized with 106 (99%) of 107 O157:H7 and 34 (77%) of 44 O26:H11, VT-positive strains from patients with colitis, HUS, and diarrheal disease and hybridized with 21 (81%) of 26 VT-positive E. coli of serotypes other than O157:H7 or O26:H11 from patients with hemorrhagic colitis and HUS. We examined 601 other strains, including 18 serotype O26 isolates of H types other than H11, 306 enteropathogenic E. coli, 60 enteroinvasive E. coli, 119 enterotoxigenic E. coli, and 20 isolates from the urinary tract and 77 isolates from the normal intestinal flora; only one (O127:H-) was positive (specificity, 99.8%). Serotype O26:H11, previously considered a classic enteropathogenic E. coli serotype, is now shown to be EHEC.

Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers. This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.