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Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Enteropathogenic Escherichia coli (EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic DeltaespB mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the DeltaespB mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the DeltaespB mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.

Background: Shigella enterotoxin 1 is a novel enterotoxin elaborated by Shigella flexneri 2a that causes fluid accumulation in rabbit ileal loops and a rise in short circuit current in Ussing chambers., Aims: To gain insights into the mechanism of action of shigella enterotoxin 1., Methods: Supernatants from genetically engineered clones either overexpressing shigella enterotoxin 1 or producing deletion mutants of the toxin were tested in rabbit ileum both in vitro and in vivo., Results: In rabbit ileum shigella enterotoxin 1 induced an irreversible rise in short circuit current that was not mediated by any of the recognised intracellular mediators of secretion. Deletion of 90% of the A subunit of the holotoxin ablated its enterotoxicity. In the in vivo perfusion model, the toxin induced a time dependent decrease in water absorption, whereas no changes were detected in the segment perfused with supernatants obtained from the deletion mutant. Finally, partially purified toxin induced a dose dependent increment in short circuit current that reached its plateau at a toxin concentration of 4 x 10(-6) M., Conclusions: Shigella enterotoxin 1 induces a time and dose dependent intestinal secretion in the rabbit animal model, suggesting that it may be responsible for the watery phase of Shigella flexneri 2a infection.

Enteroaggregative Escherichia coli (EAggEC) have been implicated as diarrheal pathogens in several settings. Some EAggEC produce a distinct heat-stable enterotoxin named EAST1. The distribution and prevalence of the EAST1 gene in selected groups of bacterial enteropathogens were determined by colony hybridization. One hundred percent of 75 O157:H7 enterohemorrhagic E. coli (EHEC), 41% of 227 EAggEC, 41% of 149 enterotoxigenic E. coli, 22% of 65 enteropathogenic E. coli (EPEC), and 38% of 47 E. coli stool isolates from asymptomatic children hybridized with an EAST1 DNA probe. None of 55 enteroinvasive E. coli, 12 Yersinia enterocolitica, or 20 Vibrio cholerae non-O1 strains were EAST1 probe-positive. Concordance between EAST1 genotype and enterotoxicity was shown in examined strains of EAggEC, EHEC, and EPEC. The gene encoding EAST1 is more broadly distributed among diarrheogenic E. coli than previously known and may represent an additional determinant in the pathogenesis of E. coli diarrhea.

Human populations throughout the world can be found in diverse conditions. A proportion of the population of developing countries lives in deprived conditions characterized by ramshackle housing, lack of piped water and sanitation, and widespread fecal contamination of the environment. Enteric infections, particularly due to bacterial pathogenes, are readily transmitted under these circumstances. In contrast, the majority of inhabitants of industrialized countries live in a sanitary environment that generally discourages the transmission of enteric pathogenes, particularly bacteria. In both these ecologic niches, changes in human ecology and behavior are leading to the emergence of certain enteric infections. Relevant factors in developing areas include urbanization (leading to periurban slums), diminished breastfeeding, and political upheaval that results in population migrations. In industrialized areas, large-scale food production (e.g., enormous poultry farms), distribution, and retailing (e.g., fast-food chains) create opportunities where widespread and extensive outbreaks of food-borne enteric infection can ensue if a breakdown in food hygiene occurs.

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.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

Enterotoxigenic Escherichia coli (ETEC) was a common cause of traveler's diarrhea in U.S. soldiers in the Middle East in 1989 and 1990. To determine which bacterial components would be useful in a vaccine, potential protective antigens (toxin, colonization factor antigen [CFA], and serotype) from 189 ETEC isolates were examined. Nearly half of the isolates expressed both ETEC toxins, 39% had only heat-stable enterotoxin (ST), and 17% had heat-labile enterotoxin (LT). CFA/I was the least common colonization factor antigen (11%), CFA/II was common (34%), as was CFA/IV (31%), and 24% expressed none of these CFAs. Fifty-seven O:H serotypes were found. Serotype O6:H16 was the most common, occurring in 29% of the ETEC isolates, usually with LT-ST and CFA/II. Generally, CFA/II was associated with expression of both toxins, CFA/IV was associated with expression of ST, and none of the CFAs was routinely found with LT. We conclude that ETEC from soldiers in the Middle East expressed a variety of antigens and that an effective vaccine will require multiple protective antigens.

Enteroaggregative Escherichia coli (EAggEC) has been found to be associated with pediatric diarrhea in developing countries. In order to determine the role of EAggEC as an agent of traveler's diarrhea, we used a sensitive and specific DNA probe for EAggEC to screen bacterial colony blots from 278 volunteers before and after travel. Colonization with EAggEC was infrequent (2.5%) prior to travel but rose to 27 to 33% after travel in volunteers who took either placebo or trimethoprim-sulfamethoxazole. Travelers who took trimethoprimsulfamethoxazole were colonized with organisms that were uniformly resistant to that antimicrobial agent; when volunteers received ciprofloxacin, colonization with EAggEC was prevented (2.0%). Although colonization rates were high in the placebo and trimethoprim-sulfamethoxazole groups, only a minority of travelers who were colonized with EAggEC experienced diarrhea. On the basis of our data, we suggest that colonization with EAggEC alone is not sufficient to cause traveler's diarrhea.

To prepare a field site for evaluating preventive interventions against endemic shigellosis, the authors followed prospectively a cohort of 360 children (90 each of children aged 0-11, 12-23, 24-35, and 36-47 months) in Santa Julia, a low socioeconomic area in Santiago, Chile, from November 1986 through April 1989 with twice weekly household visits for diarrheal disease; infants replaced children who reached 60 months of age. Coprocultures on 2 consecutive days from children with diarrhea and from age-matched controls within the cohort were cultured for Shigella. Bacteriologic surveillance was also maintained in the health center and children's hospital serving Santa Julia. In this community, where all households had access to potable water (68% inside) and all but 3% had access to a toilet, but where there was marked crowding, the overall incidence of diarrheal disease in the cohort was low (2.26 episodes/12 child months of observation in children aged 0-11 months and 2.09 in those aged 12-23 months), yet Shigella infections were common. Shigella accounted for 10% of diarrheal episodes in the cohort (vs. 3.2% isolation rate in controls, p less than 0.0001). The incidence of shigellosis in children aged 12-47 months was 0.16 cases per 12 child months of observation; in the first 5 years of life, a child had a 67% chance of experiencing shigellosis. Shigella sonnei, Shigella flexneri 2a, and S. flexneri 6 caused greater than 79% of the infections. Shigella occurred more often in hospitalized cases of diarrhea than in age-matched cases detected in the health center or by household surveillance (p less than 0.0001). An initial episode of Shigella diarrhea did not diminish overall the risk of subsequent shigellosis but did confer 72% protection (p = 0.05) against illness due to the homologous serotype. The high rate of both S. sonnei and S. flexneri shigellosis in a population with a low background rate of diarrhea makes Santa Julia an appropriate site for assessing the efficacy and effectiveness of measures to reduce Shigella infections.

Considerable progress has been made in the last decade in developing vaccines against the most important enteric infections. Two new, widely licensed vaccines (oral Ty21a and parenteral Vi) are available against typhoid fever, and new attenuated Salmonella typhi strains are ready for testing. An engineered live orally administered cholera vaccine, CVD 103-HgR, is undergoing clinical trials for safety, immunogenicity, and transmissibility in children in areas where cholera is endemic. Multiple candidate vaccines against rotavirus, Shigella, and enterotoxigenic Escherichia coli are in clinical trials. Newly acquired knowledge about pathogenesis and mucosal and cellular immunology, coupled with application of biotechnology, has already resulted in many candidates for vaccines, and more are expected to appear within the next few years.

A simple and economical method was developed for using biotinylated DNA probes to hybridize with bacterial colonies belonging to the various categories of diarrhea-causing Escherichia coli. Simplification and cost containment were achieved by using Whatman no. 541 filter papers instead of nitrocellulose, by minimizing the concentration of proteinase K (an expensive but necessary reagent used to pretreat the colony blots prior to hybridization with biotin-labeled DNA probes) and by reusing hybridization solution containing labeled probe DNA. After exposing the colony blots to lysing solution and steam, followed by lysozyme (1.5 mg/ml), sucrose (25%), and proteinase K (10 micrograms/ml) treatments, biotinylated probes were used to detect enterotoxigenic, enteropathogenic, enterohemorrhagic, diffuse adherence, and enteroinvasive categories of diarrhea-causing E. coli with a high level of sensitivity and specificity. Three independent observers who were experienced in reading DNA blots recorded remarkably similar results, while less satisfactory results were obtained when the blots were read by an inexperienced observer. This technique will be useful in laboratories in which radioactive isotopes are unavailable or impractical and in which budgets are restricted.

Diffusely adherent (DA) Escherichia coli, a newly described category, has been associated with diarrhea in children in some but not all epidemiologic studies. To investigate its diarrheagenic potential, two strains of DA E. coli were fed to adult volunteers in doses of 10(5)-10(10) colony-forming units. None of 22 volunteers who received strain 57-1 or of 21 who received strain C1845 developed symptoms of diarrhea as defined for this study. All volunteers shed the challenge DA E. coli in their stools. Duodenal string cultures were positive in some volunteers only at the highest doses. Only 6 developed IgG or IgA antibodies to whole DA E. coli. Thus, the pathogenic potential of DA E. coli could not be shown in this model. Further studies are needed to characterize potential virulence factors of DA E. coli that might distinguish a pathogenic subset.

To determine whether methodological differences in the HEp-2 adherence assay could explain conflicting results of field studies, 244 strains of Escherichia coli from Mexican children with diarrhea were tested for patterns of adherence by the method used at the Center for Vaccine Development, University of Maryland (CVD), and at the Center for Infectious Diseases, University of Texas Medical School and School of Public Health (UTH). The CVD assay differentiated three phenotypes of adherent E. coli, including localized, diffuse, or aggregative adherence (LA, DA, or AA, respectively). There was agreement on pattern of adherence in 241 of the 244 strains (98.8%) tested by the CVD method in both Baltimore and Houston, and AA+ was the most common phenotype (28.5% of isolates). Among these isolates, the UTH assay detected only two adherent phenotypes (LA and DA), since it did not distinguish the AA pattern. The LA+ strains detected by each assay were compared for positivity with the enteropathogenic E. coli adherence factor (EAF) gene probe. Of the 16 strains LA+ by the CVD method, 100% were EAF+; in contrast, only 11 of 22 strains LA+ by the UTH method were EAF+ (P = 0.00074). These results help explain why in pediatric field studies in Mexico where isolates were tested by the UTH method (J. J. Mathewson, R. A. Oberhelman, H. L. Dupont, F. J. de la Cabada, and E. V. Garibay, J. Clin. Microbiol. 25:1917-1919, 1987) LA+ strains often did not belong to enteropathogenic E. coli O serogroups and why the AA pattern was not observed; the opposite was found in studies of pediatric diarrhea in Chile in which the CVD assay was used (M. M. Levine, V. Prado, R. M. Robins-Browne, H. Lior, J. B. Kaper, S. Moseley, K. Gicquelais, J. P. Nataro, P. Vial, and B. Tall, J. Infect. Dis. 158:224-228, 1988). Since it appears that both assays identify E. coli strains associated with diarrheal illness, the genetic relationships among these strains should be examined in future studies.

A small number of bacterial agents, including enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli (EPEC), Shigella and Vibrio cholerae 01 and one virus, rotavirus, combine to cause a major proportion of the diarrheal illness of public health importance worldwide. Salmonella typhi is by far the major cause of enteric fever. Attempts to develop safe, practical, and effective vaccines against these agents are under way. Examples of vaccine candidates include live oral vaccines against S. typhi, V. cholerae, Shigella, and rotavirus, and inactivated, submit vaccines given parentally or orally against S. typhi, V. cholerae or ETEC. Although the oral attenuated S. typhi vaccine is ready for commercial license, it will be several years before the other vaccines are proven to be practically safe and effective in children and adults.

The experience of Nalin et al. and Patra et al. with a "super oral rehydration solution (ORS)" containing glucose plus glycine to enhance the intestinal absorption of sodium and water prompted us to investigate a similar ORS containing the standard World Health Organization (WHO/ORS) plus either 55 or 110 mmol/L glycine in infants and small children with noncholera diarrhea. We did not find a statistically significant difference between the glycine-fortified ORS and the standard WHO/ORS with respect to the clinical outcome and composition of serum electrolytes.

There are four major categories of diarrheagenic Escherichia coli: enterotoxigenic (a major cause of travelers' diarrhea and infant diarrhea in less-developed countries), enteroinvasive (a cause of dysentery), enteropathogenic (an important cause of infant diarrhea), and enterohemorrhagic (a cause of hemorrhagic colitis and hemolytic uremic syndrome). Besides manifesting distinct clinical patterns, these categories of E. coli differ in their epidemiology and pathogenesis and in their O:H serotypes. Common features (albeit distinct for each category) include plasmid-encoded virulence properties, characteristic interactions with intestinal mucosa, and elaboration of various types of enterotoxins or cytotoxins. A less-well-defined fifth category of diarrheagenic E. coli is that of enteroadherent E. coli, so far identifiable only by their pattern of adherence to Hep-2 cells in tissue culture.

In vitro and animal experiments indicated that lactobacilli might prevent Escherichia coli from colonizing the intestine and may produce substances counteracting enterotoxin. Lactinex, a commercial preparation of dried Lactobacillus acidophilus and L. bulgaricus, is marketed for uncomplicated diarrhea. Preliminary experiments in nonfasting volunteers indicated that lactobacilli in this preparation colonized the small intestine for up to 6 h. To evaluate the protective efficacy of Lactinex, a double-blind randomized study was carried out in which 48 volunteers (23 receiving Lactinex and 25 receiving placebos) were challenged with E. coli strains that produced heat-stable or heat-labile enterotoxins or both. No significant differences between the two groups were noted with respect to attack rate, incubation period, duration of diarrhea, volume and number of liquid stools, and coproculture yields. These data suggest that this lactobacillus preparations does not prevent or alter the course of enterotoxigenic E. coli diarrhea in adults. Lack of efficacy occurred despite efforts to maximize small bowel colonization, including administration of Lactinex in milk and in a 6-hour-interval regimen during 36 h before and 96 h after challenge.

Although most enteropathogenic Escherichia coli strains do not produce recognized enterotoxins, we wished to examine whether they produce any factors like heat-stable enterotoxin b or cholera toxin active subunits that might be missed by conventional assay methods. E. coli strains E851 (O142) and E2348 (O127) that had caused diarrhea in volunteers were negative for heat-labile enterotoxin and heat-stable enterotoxin a in Chinese hamster ovary cell and suckling mouse assays, failed to cause secretion in ligated small bowel loops from 6- to 8-week-old pigs after 4 to 5 h (used to show heat-stable enterotoxin b), and did not activate adenylate cyclase in pigeon erythrocyte lysates (used to demonstrate cholera toxin A subunit). We conclude that crude, unconcentrated culture filtrates and sonicates do not mimic heat-labile or heat-stable enterotoxins or cholera toxin or its A subunit and that enteropathogenic strains of E. coli probably have yet another mechanism or group of mechanisms by which they cause diarrhea.

Controversy exists as to whether all strains of enterotoxigenic Escherichia coli must possess colonization factor antigen pili I or II (CFA/I, CFA/II) in order to be virulent for humans. Failure to detect CFA/I or CFA/II in enterotoxigenic strains from human diarrhea has been explained by some as due to plasmid loss and by others as evidence that CFA/I and CFA/II pili are not prerequisites for human virulence in all enterotoxigenic strains of E. coli. Seven enterotoxigenic and three enteropathogenic strains of E. coli that have been used in volunteer challenge studies were tested for CFA/I, CFA/II, and type 1 somatic pili after culture on solid agar and in broth. Six of the seven enterotoxigenic and two of the three enteropathogenic strains caused diarrhea in humans. Of these eight virulent strains, one produced CFA/I, and one had CFA/II. Threre remained four enterotoxigenic and two enteropathogenic strains that unequivocally caused diarrhea while lacking CFA/I or CFA/II. In such strains other organelles or surface properties must be operative to promote adhesion to and colonization of small intestinal mucosa. CFA/I and CFA/II are not prerequisites of virulence for all E. coli strains that cause diarrhea in humans.

Escherichia coli that exhibit the aggregative pattern of adherence to HEp-2 cells (enteroadherent-aggregative E. coli [EA-AggEC]) have been epidemiologically incriminated as a cause of diarrhea. We undertook a preliminary microbiological and pathogenetic characterization of 42 isolates of this putative pathogen. The strains were negative by tests with DNA probes for enteropathogenic, enterotoxigenic, enteroinvasive, and enterohemorrhagic E. coli and, by serotype, did not fit these categories. Thirty-nine of 42 strains had a 55-65-megadalton plasmid; many shared DNA homology. With one representative strain, plasmid transfer was accompanied by transfer of smooth lipopolysaccharide, fimbriae expression, and the aggregative property. EA-AggEC caused characteristic lesions in rabbit and rat ileal loops. The intestinal lesions and (Shiga-like) limb paralysis and death in rabbits inoculated with live organisms suggest toxin involvement; assays for Shiga-like toxins were negative. These preliminary results support the contention that EA-AggEC may represent a distinct category of diarrheagenic E. coli.

Clinical and microbiologic studies were conducted to evaluate the capability of exogenous lactobacilli to implant in the upper small intestine, the ability of exogenous lactobacilli to prevent enterotoxigenic Escherichia coli (ETEC) diarrhea, and the effect of lactobacillus therapy in reduction of diarrhea due to neomycin. Microbiologic studies of jejunal aspirates indicated that orally ingested Lactobacillus acidophilus and L. bulgaricus in LactinexR and L. acidophilus in Infloran BernaR survived passage through the stomach and remained viable in the proximal small bowel for three to six hours in most, not all, individuals. Challenge studies with ETEC revealed that despite the ability of lactobacilli in LactinexR to persist in the upper small intestine for several hours, the lactobacillus preparation did not prevent or alter the course of ETEC diarrhea in adults. Lactobacillus therapy with one lot of LactinexR reduced the volume and duration of neomycin-associated diarrhea, whereas another lot of LactinexR had no therapeutic effect. Thus, lot to lot variations in lactobacillus preparations may account for some conflicting results observed with lactobacillus therapy for intestinal disorders.

In order to determine the descriptive epidemiology of persistent diarrhoea in rural northern India, a cohort of 963 children aged 0-71 months was followed prospectively for 12 months through weekly household visits. The incidence of persistent diarrhoea was 6.3 per 100 child-years among those aged 0-71 months, and was highest (31 per 100 child-years) among those aged 0-11 months. There were no significant sex-related differences in the incidence of the disease, and the overall seasonal distribution of acute and persistent diarrhoea was similar. The persistence of diarrhoeal symptoms was significantly correlated with a higher initial mean stool frequency (P less than 0.01) and passage of gross blood with stools (P less than 0.001). Persistent diarrhoea was an important problem among children during the first 2 years of life. Established enteric pathogens were isolated during the initial illness in 46.4% of persistent and 55.4% of acute episodes. Pathogens isolated during persistent episodes included enterotoxigenic Escherichia coli (ETEC 9.3%), Salmonella spp. (4.7%), as well as campylobacter (4.7%), Shigella spp. (2.3%), Entamoeba histolytica (2.3%), and rotavirus (2.3%). Similar proportions of these pathogens were isolated also during episodes of acute diarrhoea. Multiple pathogens were isolated in 7% of the persistent and 5% of the acute episodes. E. coli that manifested aggregative adherence (EAEC-A) was more common (34.9% versus 12.3%) in persistent than acute episodes (P less than 0.01), and initial faecal excretion of EAEC-A was significantly associated with the persistence of a diarrhoeal episode.

The development of a solid-phase microtiter radioimmunoassay blocking test to detect serum antibody to Escherichia coli heat-labile enterotoxin is described. The assay is easy to perform and quantitate, and it is sensitive and specific.

Diarrheal diseases remain a leading cause of morbidity and mortality in the developing countries and represent at least a nuisance in the industrialized world. Fluid and electrolyte replacement, particularly via oral rehydration, is the mainstay of therapy for the prevention and treatment of dehydration associated with these illnesses. Antibiotics are not indicated for the majority of enteric infections, and their promiscuous use can contribute to the escalating prevalence of bacterial resistance worldwide. Used judiciously, however, antimicrobial agents can ameliorate illness or curtail pathogen excretion and spread of disease, or both, in some diarrheal infections. Antimicrobial agents are indicated for shigellosis, cholera, traveler's diarrhea, amebiasis, and giardiasis. They are indicated in some specific circumstances to treat infections caused by Campylobacter, some categories of diarrheagenic E. coli, C. difficile, nontyphoidal Salmonella, and certain Vibrionaceae. Few adjunctive treatments provide proven benefit without risk of adverse reactions; most offer no advantage over placebo, and their general use is not encouraged.

Impressive advancements in our understanding of the mechanisms of diarrhea production and of the epidemiologic importance of these "classical" bacterial enteropathogens have been accomplished, but many areas are in need of further exploration. The recent development of gene probes and enzyme-linked immunosorbent assays for the identification of EPEC, ETEC and EIEC will enhance immeasurably the ability to carry out large scale epidemiologic studies which are still needed to clarify the global importance of these pathogens in infantile diarrhea. For some of these organisms pathogenic mechanisms remain incompletely understood and the role of antibiotics is not well-established. The delineation of virulence factors, immunity and the construction of attenuated strains through DNA recombination technology are bringing the worthy goal of prevention through vaccination into view. These advances should not, however, detract attention from the primary reason that these bacteria continue to be a major cause of childhood morbidity and mortality in the developing world, which is lack of adequate sanitation. Efforts to provide clean water, safe waste disposal and hygiene education need to proceed in conjunction with development of vaccines.

In a double-blind study of the treatment of disease caused by enterotoxigenic Escherichia coli (ETEC), diarrhea was induced in volunteers with a trimethoprim (TMP)- and sulfamethoxazole (SMZ)-susceptible strain of E. coli that produces both heat-stable and heat-labile toxin. III volunteers were then treated with TMP, TMP-SMZ, or placebo. Volunteers treated with both TMP alone and the TMP-SMZ combination showed a substantial decrease in the duration and severity of the illness, as compared with the placebo-treated controls. TMP-resistant (MIC, 3.1-12.5 micrograms/ml) ETEC were isolated from stool cultures of five of 10 TMP-treated volunteers and none of 10 TMP-SMZ-treated volunteers after 48 hr of therapy, and in two volunteers the appearance of resistant organisms was associated with a clinical relapse. These data suggest that the TMP-SMZ combination should be evaluated in field trials to determine its usefulness as an adjunct to replacement of fluid and electrolytes in the therapy of ETEC diarrhea.

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.

Enterotoxigenic Escherichia coli (ETEC) are the major aetiological agent of travellers' diarrhoea, usually accounting for 30-60% of cases. Thus a safe and effective vaccine against ETEC could play an important role in prevention of this infection. A successful vaccine must somehow protect against a heterogenous array of ETEC pathogens comprising many O:H serotypes and three enterotoxin phenotypes (LT+/ST-, LT-/ST+, and LT+/ST+). Three major approaches to vaccine development are currently in progress including: 1) LT and ST toxoids that stimulate antitoxic immunity; 2) purified pili vaccines that induce anti-adhesion immunity; 3) attenuated strains (prepared by recombinant DNA techniques) that mimic natural infection by stimulating antibacterial and anti-adhesive immunity as well as antitoxic immunity. Since Shigella represents the second most common cause of travellers' diarrhoea, immunoprophylaxis against this pathogen is also desirable. A recently described attenuated Salmonella typhi strain (Ty21a) expressing Shigella sonnei O antigen on its surface represents an attractive Shigella vaccine prototype.

Acute diarrheal disease may be due to viral, bacterial, or protozoal enteropathogens. In our current state of knowledge and medical practice, specific antiviral agents are not used in the treatment of known or presumed viral diarrhea. In contrast, for a number of the bacterial and protozoal diarrheal infections, therapy with certain antimicrobial agents can significantly ameliorate the severity and duration of illness and curtail the excretion of the pathogen. A recurring theme encountered in reviewing information on the therapy for diarrheal infections is that demonstration of the susceptibility in vitro of a bacterial pathogen to a particular antibiotic by no means assures clinical success. Many antibiotics that show potent activity in vitro have little or no efficacy in vivo. Controlled clinical trials are necessary to assess the clinical and bacteriologic efficacy of an antibiotic in diarrheal infections.

A total of 516 Escherichia coli strains randomly isolated from coprocultures of 154 Chilean children with diarrhea and 66 controls were examined with DNA probes and tested for adherence to HEp-2 cells. Three adherence patterns were distinguished, localized, true diffuse and "aggregative." Enteropathogenic E. coli (EPEC) were detected by EPEC adherence factor probe among 86 of the 372 isolates (23%) from patients with diarrhea vs. 14 of 144 (10%) strains from controls (P less than 0.0002). Of 95 strains that manifested localized adherence, 97% were EPEC adherence factor probe-positive; thus the HEp-2 assay may serve as an alternative to the probe in identifying EPEC adherence factor-positive EPEC. True diffuse adherence was not associated with diarrhea. In contrast the aggregative pattern appears to signify a new, distinct class of diarrheagenic E. coli (enteroadherent-aggregative E. coli). The aggregative pattern was found in only 3 of 27 enterotoxigenic, 0 of 4 enteroinvasive, 0 of 2 enterohemorrhagic and 2 of 86 EPEC strains but in 84 of 253 probe-negative strains (P less than 0.00001) from patients with diarrhea; in comparison only 20 of 134 probe-negative strains from controls were aggregative E. coli (P less than 0.00001 vs. probe-negative strains from diarrhea patients).

Enterotoxigenic Escherichia coli strains represent the most frequent etiological agent of travelers diarrhea. Challenge studies with several of these strains were undertaken in volunteers to evaluate the mechanisms of disease-induced immunity. Seventeen students and other community volunteers were given 10(6) or 10(8) organisms of E. coli B7A (O148:H28), which produces heat-labile and heat-stable enterotoxins. Ten individuals developed diarrheal illness closely resembling natural travelers diarrhea; of these ten, rises in titer of serum antitoxin and anti-O antibody occurred in eight (80%). Eight of the volunteers who developed diarrhea in the first test agreed to undergo rechallenge 9 weeks later with 10(8) B7A organisms. Only one of these eight "veterans" developed diarrhea versus seven of twelve controls given the same challenge (P = 0.05). Despite clinical protection, all "veterans" excreted B7A after rechallenge. Four controls who developed diarrhea during the homologous B7A rechallenge test were rechallenged 9 weeks later with 10(9) organisms of E. coli strain E2528-C1 (O25:H-), which produces only heat-labile enterotoxin and possesses a different O, H, and pili antigen composition than B7A. Three of four "veterans" and two of six controls developed comparable diarrhea. These studies demonstrate that prior disease due to enterotoxigenic E. coli confers homologous immunity against subsequent challenge, and the operative mechanism apparently is not bactericidal and is not mediated by serum anti-O antibodies. Heterologous protection was not conferred where the only common antigen was heat-labile enterotoxin, indicating that serum infection-derived antitoxin to heat-labile enterotoxin also is not protective.

Fimbriae from enteropathogenic Escherichia coli strain E2349/69 (0127:H6) and its plasmid-minus derivative, MAR20, were purified and characterized as type 1-like by their physicochemical and hemagglutination patterns. Sera from adult volunteers challenged with the diarrheagenic parent strain and the attenuated plasmid-minus derivative were examined to detect an immune response, using the purified fimbriae as antigens in an enzyme linked immunosorbent assay (ELISA) and immunoblot assay. An anti-fimbrial response was evident in sera of 7 of 10 volunteers fed the diarrheagenic parent strain E2348 but also in 8 of 9 individuals fed the attenuated, plasmid-cured, derivative MAR20. The antibody response appeared specific in that the sera failed to react in an ELISA and by immunoblot assay with type 1 fimbriae from other E. coli. These findings suggest that the type 1 fimbriae of this representative EPEC strain are antigenically distinct. The results of this investigation provide the first evidence of seroconversion to type 1-like fimbriae in infections caused by diarrheagenic E. coli.

Three enteropathogenic Escherichia coli (E.P.E.C.) strains (O127:K63:H6, O128:K67:H2, and O142:K86:H6) isolated from outbreaks of infantile diarrhoea and one strain from the "normal" colonic flora (E. coli HS) of a healthy adult were fed in doses of 10(6), 10(8), and 10(10) organisms in NaHCO3 to adult volunteers. The strains, which had been stored for 7--9 years, gave negative results in sensitive tests for heat-labile (L.T.) enterotoxin (Y-1 adrenal-cell test), heat-stable (S.T.) enterotoxin (infant mouse assay), invasiveness (guineapig eye test), and gross fluid accumulation (infant rabbit assay). Two strains (O142 and O127) caused diarrhoea. L.T. or S.T. enterotoxins were not found in E. coli stool isolates from individuals with diarrhoea and no one had a rise in L.T. antitoxin titre; the findings suggest that L.T. and S.T. enterotoxins were not involved in pathogenesis of the diarrhoea. Non-invasive E.P.E.C. strains probably induce diarrhoea by a mechanism (presumably an enterotoxin) distinct from L.T. or S.T. enterotoxins.

A cohort of 452 rural children was followed longitudinally for 13 mo to ascertain the role of HEp-2 cell adherent Escherichia coli and other pathogens in causing acute (less than or equal to 14 d) and persistent (greater than 14 d) diarrhea. Aeromonas, Campylobacter jejuni, E. coli manifesting localized adherence to HEp-2 cells and enterotoxigenic E. coli were significantly associated with acute diarrhea. E. coli strains that exhibit aggregative adherence, so-called enteroaggregative E. coli, a newly-described category of diarrheagenic E. coli distinct from enterotoxigenic, enteroinvasive, enterohemorrhagic, and enteropathogenic E. coli, were found significantly more often in patients with persistent diarrhea (29.5%) than with acute diarrhea (12.8%) (P = .0052) or controls (9.9%) (P = .0006).

During studies of diarrhoea due to Escherichia coli in 316 adult volunteers, ABO and Rh blood group determinations were done to look for differences in the occurrence or severity of illness in association with certain blood groups. In studies using heat-labile enterotoxin-producing E. coli, volunteers with O blood group had a significantly higher attack rate for diarrhoea than persons with other blood groups. In contrast, in studies with enteropathogenic or heat-stable enterotoxin-producing E. coli, no association was found between occurrence of diarrhoea and ABO group. These studies, and previous studies finding a similarly increased susceptibility to cholera in persons with O blood group, suggest that the mechanism of increased risk involves an interaction between blood group substances and the similar enterotoxin produced by E. coli and Vibrio cholerae.