Search

The treatment regimen for diarrhea depends greatly on correct diagnosis of its etiology. Recent diarrhea outbreaks in Bangladesh showed Vibrio cholerae to be the predominant cause, although more than 40% of the suspected cases failed to show cholera etiology by conventional culture methods (CMs). In the present study, suspected cholera stools collected from every 50th patient during an acute diarrheal outbreak were analyzed extensively using different microbiological and molecular tools to determine their etiology. Of 135 stools tested, 86 (64%) produced V. cholerae O1 by CMs, while 119 (88%) tested positive for V. cholerae O1 by rapid cholera dipstick (DS) assay; all but three samples positive for V. cholerae O1 by CMs were also positive for V. cholerae O1 by DS assay. Of 49 stools that lacked CM-based cholera etiology despite most being positive for V. cholerae O1 by DS assay, 25 (51%) had coccoid V. cholerae O1 cells as confirmed by direct fluorescent antibody (DFA) assay, 36 (73%) amplified primers for the genes wbe O1 and ctxA by multiplex-PCR (M-PCR), and 31 (63%) showed El Tor-specific lytic phage on plaque assay (PA). Each of these methods allowed the cholera etiology to be confirmed for 97% of the stool samples. The results suggest that suspected cholera stools that fail to show etiology by CMs during acute diarrhea outbreaks may be due to the inactivation of V. cholerae by in vivo vibriolytic action of the phage and/or nonculturability induced as a host response.

Helicobacter pylori is chiefly acquired in childhood, but the exact timing of acquisition is not well understood. The main goal of this study was to assess H. pylori acquisition in a pediatric population. We studied two cohorts of Native American children: a birth cohort of 50 children and 58 older children (mean age, 53 months). We measured serum immunoglobulin G (IgG), IgM, and IgA antibodies to H. pylori whole-cell antigen and IgG antibodies to CagA. Among 44 birth cohort children monitored for more than 12 months, 24 (54.5%) had seroconversions, 7 (15.9%) were transient, and 17 (38.6%) were persistent. Among the older children, 49 (84.5%) of the 58 children were monitored for 1 year; 34 (69.4%) had H. pylori antibodies at study entry. During the next year, 7 (20.6%) children seroreverted, and of 15 initially negative children, 5 (33.3%) seroconverted. In both groups, evaluation of CagA antibodies increased the sensitivity of H. pylori detection. Serum pepsinogen I (PGI) levels in H. pylori-negative children rose significantly until age 6 months and remained constant for the next 19 months. At the time of H. pylori seroconversion, PGI peaked to levels significantly higher than in the never-seroconverted (P = 0.02) and the pre-seroconverted (P = 0.03) children, but then declined to levels paralleling those of H. pylori-negative children. Thus, H. pylori acquisition, accompanied by a transient PGI increase, was frequent in this population, especially in the second and third years of life, but often was brief.

Aeromonads are causative agents of a number of human infections. Even though aeromonads have been isolated from patients suffering from diarrhea, their etiological role in gastroenteritis is unclear. In spite of a number of virulence factors produced by Aeromonas species, their association with diarrhea has not been clearly linked. Recently, we have characterized a heat-labile cytotonic enterotoxin (Alt), a heat-stable cytotonic enterotoxin (Ast), and a cytotoxic enterotoxin (Act) from a diarrheal isolate of Aeromonas hydrophila. Alt and Ast are novel enterotoxins which are not related to cholera toxin; Act is aerolysin related and has hemolytic, cytotoxic, and enterotoxic activities. We studied the distribution of the alt, ast, and act enterotoxin genes in 115 of 125 aeromonads isolated from 1, 735 children with diarrhea, in all 27 aeromonads isolated from 830 control children (P = 7 x 10(-4) for comparison of rates of isolation of aeromonads from cases versus those from controls), and in 120 randomly selected aeromonads from different components of surface water in Bangladesh. Aeromonas isolates which were positive only for the presence of the alt gene had similar distributions in the three sources; the number of isolates positive only for the presence of the ast gene was significantly higher for the environmental samples than for samples from diarrheal children; and isolates positive only for the presence of the act gene were not found in any of the three sources. Importantly, the number of isolates positive for both the alt and ast genes was significantly higher for diarrheal children than for control children and the environment. Thus, this is the first study to indicate that the products of both the alt and ast genes may synergistically act to induce severe diarrhea. In 26 patients, Aeromonas spp. were isolated as the sole enteropathogen. Analysis of clinical data from 11 of these patients suggested that isolates positive for both the alt and ast genes were associated with watery diarrhea but that isolates positive only for the alt gene were associated with loose stools. Most of the isolates from the three sources could be classified into seven phenospecies and eight hybridization groups. For the first time, Aeromonas eucrenophila was isolated from two children, one with diarrhea and another without diarrhea.

The prevalence of toxin types and colonization factors (CFs) of enterotoxigenic Escherichia coli (ETEC) was prospectively studied with fresh samples (n = 4,662) obtained from a 2% routine surveillance of diarrheal stool samples over 2 years, from September 1996 to August 1998. Stool samples were tested by enzyme-linked immunoassay techniques and with specific monoclonal antibodies for the toxins and CFs. The prevalence of ETEC was 14% (n = 662), with over 70% of the strains isolated from children 0 to 5 years of age, of whom 93% were in the 0- to 3-year-old age range. Of the total ETEC isolates, 49.4% were positive for the heat-stable toxin (ST), 25.4% were positive for the heat-labile toxin (LT) only, and 25.2% were positive for both LT and ST. The rate of ETEC isolation peaked in the hot summer months of May to September and decreased in winter. About 56% of the samples were positive for 1 or more of the 12 CFs that were screened for. The coli surface antigens CS4, CS5, and/or CS6 of the colonization factor antigen (CFA)/IV complex were most prevalent (incidence, 31%), followed by CFA/I (23.5%) and coli surface antigens CS1, CS2, and CS3 of CFA/II (21%). In addition, other CFs detected in decreasing order were CS7 (8%), CS14 (PCFO166) (7%), CS12 (PCFO159) (4%), CS17 (3%), and CS8 (CFA/III) (2.7%). The ST- or LT- and ST-positive ETEC isolates expressed the CFs known to be the most prevalent (i.e., CFA/I, CFA/II, and CFA/IV), while the strains positive for LT only did not. Among children who were infected with ETEC as the single pathogen, a trend of relatively more severe disease in children infected with ST-positive (P < 0.001) or LT- and ST-positive (P < 0.001) ETEC isolates compared to the severity of the disease in children infected with LT only-positive ETEC isolates was seen. This study supports the fact that ETEC is still a major cause of childhood diarrhea in Bangladesh, especially in children up to 3 years of age, and that measures to prevent such infections are needed in developing countries.

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

Vibrio cholerae O139 Bengal initially appeared in the southern coastal region of Bangladesh and spread northward, causing explosive epidemics during 1992 and 1993. The resurgence of V. cholerae O139 during 1995 after its transient displacement by a new clone of El Tor vibrios demonstrated rapid changes in the epidemiology of cholera in Bangladesh. A recent outbreak of cholera in two north-central districts of Bangladesh caused by V. cholerae O139 led us to analyze strains collected from the outbreak and compare them with V. cholerae O139 strains isolated from other regions of Bangladesh and neighboring India to investigate their origins. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA (ribotype) revealed that the recently isolated V. cholerae O139 strains belonged to a new ribotype which was distinct from previously described ribotypes of toxigenic V. cholerae O139. All strains carried the genes for toxin-coregulated pili (tcpA and tcpI) and accessory colonization factor (acfB), the regulatory gene toxR, and multiple copies of the lysogenic phage genome encoding cholera toxin (CTXPhi) and belonged to a previously described ctxA genotype. Comparative analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon downstream of the rfaD gene and the presence of an O139-specific genomic region in all O139 strains. Southern hybridization analysis of the O139-specific genomic region also produced identical restriction patterns in strains belonging to the new ribotype and those of previously described ribotypes. These results suggested that the new ribotype of Bengal vibrios possibly originated from an existing strain of V. cholerae O139 by genetic changes in the rRNA operons. In contrast to previously isolated O139 strains which mostly had resistance to trimethoprim, sulfamethoxazole, and streptomycin encoded by a transposon (SXT element), 68.6% of the toxigenic strains analyzed in the present study, including all strains belonging to the new ribotype, were susceptible to these antibiotics. Molecular analysis of the SXT element revealed possible deletion of a 3.6-kb region of the SXT element in strains which were susceptible to the antibiotics. Thus, V. cholerae O139 strains in Bangladesh are also undergoing considerable reassortments in genetic elements encoding antimicrobial resistance.

Three hundred sixty-two Streptococcus pneumoniae strains were isolated from children under 5 years of age at Dhaka Shishu (Children) Hospital from 1993 to 1997. The strains were isolated from blood (n = 105), CSF (n = 164), ear swab (n = 61), eye swab (n = 20), and pus (n = 12). Of the 362 isolates, 42 (11.6%) showed intermediate resistance (MIC, <0.1 microgram/ml) and only 4 (1.1%) showed complete resistance (MIC, >2.0 microgram/ml) to penicillin. Penicillin resistance exhibited a strong relationship with serotype 14; 47.8% of the penicillin-resistant strains belonged to this type. A remarkably high (64.1%) resistance to co-trimoxazole was observed, along with a significant increase during the time period studied; there was no relationship to capsular type. By way of contrast, penicillin resistance did not show any significant change during the study period. Resistance to chloramphenicol (2.2%) and erythromycin (1.1%) was rare. The high resistance to co-trimoxazole and its increasing trend demand elucidation of the clinical impact of pneumonia treatment by this antimicrobial and reconsideration of the World Health Organization recommendation for co-trimoxazole administration to children with community-acquired pneumonia at the health care worker level in Bangladesh.

One hundred sixty-five invasive Streptococcus pneumoniae strains were isolated from children under five at Dhaka Shishu (Children's) Hospital during the period 1992 to 1995. Ninety-four strains were from cerebrospinal fluid, and 71 were from blood. More than 91% of the strains were isolated from patients aged 24 months or less. Predominant serotypes were, in descending order 7F, 12F, 14, 15B, 18, 5, and 22A. These comprised 70% of all isolates. The marked differences in serotype distribution in different countries indicate the need for a sentinel surveillance study for the countries of South Asia, particularly Bangladesh, China, India, and Pakistan.

A monoclonal antibody-based test, Bengal SMART, was developed for rapid detection of Vibrio cholerae O139 synonym Bengal directly from stool specimens. The test, which takes about 15 min to complete, was used to screen 189 diarrheal stool specimens. The results were compared with those of a monoclonal antibody-based coagglutination test (COAT) and the conventional culture methods used as the "gold standard" for detection of V. cholerae O139. The Bengal SMART test showed a sensitivity of 100% and a specificity of 97% in comparison with the gold standard. It also fared better than COAT, which had a sensitivity of 96% for rapid detection of V. cholerae O139 synonym Bengal. These results show that Bengal SMART is suitable for use in field settings for rapid diagnosis of cholera caused by V. cholerae O139.

A monoclonal antibody-based coagglutination test directly detected Vibrio cholerae O139 synonym Bengal in 83 of 120 watery diarrheal stool specimens; on culture, 90 samples were positive. Thus, with 92% sensitivity, 100% specificity, and 100% positive and 95% negative predictive values, the coagglutination test is a useful rapid test for V. cholerae O139.

Vibrio cholerae O139 synonym Bengal recently caused large epidemics of cholera-like disease in Bangladesh and India. We compared the restriction fragment length polymorphisms of ctxA and rRNA genes (ribotypes) in 27 isolates of V. cholerae O139 from patients in Bangladesh and India with those of 48 isolates of V. cholerae O1 from patients and 21 V. cholerae isolates from surface waters in Bangladesh, which included 2 O139 and 19 other non-O1 isolates. Ribotyping of the isolates with BglI revealed that all 29 isolates of O139 vibrios belonged to a single ribotype, suggesting a clonal nature of the infection. However, the O139 vibrios comprised two ctxA genotypes and carried three or more copies of the ctxA gene, and the chromosomal locations of these copies were unlike those of the El Tor or classical vibrios. Analysis of the restriction fragment length polymorphisms of the rRNA genes suggested that V. cholerae O139 isolates are more closely related to El Tor strains of V. cholerae O1 than were 19 other non-O1 vibrios and 33 classical V. cholerae O1 isolates that were studied. However, further studies are needed to determine whether V. cholerae O139 originated from mutations and genetic changes in a V. cholerae O1 strain or was due to the acquisition of virulence genes by a previously unknown V. cholerae non-O1 strain.

We undertook a controlled study of children younger than 5 years in Bangladesh to determine whether enterotoxigenic Bacteroides fragilis (ETBF) was associated with diarrhea in this population. ETBF was isolated from 22 (6.1%) of 358 patients and 5 (1.2%) of 425 controls (P = 0.0001). In children younger than 1 year, however, low isolation rates (2 to 3%) were found in both patients and controls. In children older than 1 year, the rates were significantly higher in children with diarrhea (16 [9%] of 177) than in controls (2 [1%] of 264; P = 0.00001). When children with mixed infections with other known diarrheal pathogens were removed, the differences in children older than 1 year were still significant (7 [4%] of 177 versus 2 [1%] of 264; P = 0.033). The syndrome associated with ETBF was secretory in nature, with watery diarrhea, and of mild severity. These epidemiological and clinical findings are similar to those from a previous study of White Mountain Apaches in the United States and are the first to suggest that ETBF may also be an important diarrheal pathogen in other geographic areas and in the developing world where diarrhea is highly endemic.

In Bangladesh, the replacement of classical Vibrio cholerae by the E1 Tor biotype in 1968 and the reappearance of the classical biotype and its coexistence with the E1 Tor biotype after 1982 were never adequately explained. We have analyzed 23 classical V. cholerae isolates collected between 1961 and 1968, 14 classical isolates collected between 1982 and 1992 from the capital city, Dhaka, and 6 classical V. cholerae isolates collected from two southern districts of Bangladesh and studied restriction endonuclease cleavage patterns of rRNA genes (ribotypes) to investigate the clonal relationships among the isolates. Southern blots of total DNA digested with restriction enzyme BamHI, BglI, EcoRI, HindIII, or PstI were probed, using a cloned Escherichia coli rRNA operon. While restriction enzymes BamHI, EcoRI, and PstI failed to differentiate the isolates on the basis of ribotyping, BglI and HindIII produced digestion patterns that allowed differentiation. Ribotyping the isolates with BglI and HindIII revealed five different clones (ribotypes IA, IB, IIA, IC, and IIC) of classical vibrios in Bangladesh. Strains belonging to ribotypes IA and IB were isolated in Dhaka before 1968, and one ribotype (IA) was again isolated between 1982 and 1992. Ribotype IIA was isolated in 1988 and 1989, when both clones (IA and IIA) of classical vibrios coexisted with the EI Tor vibrios. Isolates belonging to ribotypes IC and IIC were collected in the southern districts of Bangladesh and were clearly different from those collected in Dhaka between 1968 and 1992 by ribotyping analysis with BglI. These results support the previous assumption that classical vibrios were never completely replaced in Bangladesh and also demonstrate the existence of more than one genetically different clone of classical V. cholerae.

Nine isolates of bacteria biochemically resembling Shigella dysenteriae but not belonging to the 10 recognized serotypes were isolated from patients with diarrhea in Bangladesh. Further studies suggested that two, one, and six isolates belonged to the recently recognized S. dysenteriae serotypes 11, 12, and 13, respectively.

To improve the understanding of the relative importance of serotypes of rotavirus in dehydrating diarrhea, we examined the correlation of clinical characteristics and disease severity with serotype in 2,441 diarrheal episodes among children younger than 2 years of age in rural Bangladesh. Of 764 rotavirus-associated episodes, a single G type (serotype 1, 2, 3, or 4) was determined by oligonucleotide probe in 485 (63%), while 233 episodes were nontypeable. Episodes with G types 2 and 3 were associated with more-severe dehydration than episodes associated with G type 1 or 4 or with nontypeable rotavirus. Episodes did not differ by G type in prevalence of vomiting, copious diarrhea, fever, abdominal pain, or length of treatment center stay. Rotavirus reinfections were detected in seven children, with homologous reinfection (G type 2) in one. Twelve children with diarrhea who died had rotavirus detected in stool specimens within 30 days of death. Children who died were more likely to be malnourished than were surviving children with rotavirus diarrhea. Of 40 specimens tested by polymerase chain reaction, 29 (72.5%) were P type 1, 9 (22.5%) were P type 2, 1 (2.5%) was P type 3, and 1 (2.5%) was nontypeable. One severely symptomatic diarrheal episode was associated with P type 3 rotavirus, a serotype usually found in asymptomatic nursery infections. Although G types 2 and 3 were associated with more-severe dehydration than other serotypes, the differences do not appear to be of major clinical importance. Effective vaccines should protect against all four major G types.

We studied the restriction endonuclease cleavage patterns of rRNA genes (ribotypes) of 72 clinical isolates of Shigella flexneri representing eight serotypes to determine whether ribotyping could be used to distinguish S. flexneri strains and to compare the discriminating ability of the method with that of serotyping. By using a cloned Escherichia coli rRNA operon as the probe, Southern blot hybridization of restriction endonuclease-digested total DNA was carried out. Ribotyping of the isolates with each of the five restriction endonucleases BamHI, EcoRI, HindIII, PstI, and SalI generated reproducible restriction patterns. However, HindIII produced the optimum digestion pattern of the rRNA genes and was more useful than the other enzymes used in differentiating strains. Analysis of the 72 isolates showed 11 different HindIII cleavage patterns of their rRNA genes. Four of these HindIII-generated ribotypes could be further differentiated into two to four subribotypes by using PstI. The results indicate that ribotyping has an application for differentiation of S. flexneri strains and can complement serotyping. Definition of strains in terms of both serotype and ribotype may be of greater use in epidemiological studies.

Rectal swabs from 391 infants less than 18 months of age who were hospitalized with acute diarrhea and from 138 similarly aged healthy infants were examined for the etiologic agents of diarrhea. Aeromonas spp. were recovered from 205 of 391 (52.4%) diarrheic patients, whereas they were recovered from 12 of 138 (8.7%) controls (P less than 10(-11). Among the 205 Aeromonas-positive diarrheic patients, 118 (57.6%) were found to be coinfected with other common enteropathogens. Of the 164 Aeromonas-positive initial diarrheic specimens, 82 (50.0%) had one or more other enteropathogens present; 30 patients were coinfected with rotavirus, 20 with enterotoxigenic Escherichia coli, 16 with Campylobacter spp., 14 with Shigella spp., 13 with enteropathogenic E. coli, 4 with Vibrio spp., 1 with Salmonella spp., and 1 with Plesiomonas spp. of Aeromonas strains from cases compared with that from controls supports an etiologic role for this organism. However, frequent concomitant infections with other well-recognized enteropathogens and a lack of disease correlation with common Aeromonas phenotypes suggest that only a subset of Aeromonas strains may be diarrhea causing and that such strains may be common to several of the existing species.

The intestinal colonization rate of Aeromonas spp. was determined for 52 cesarean-born Peruvian neonates. Rectal swabs were obtained daily from newborns during their postdelivery hospitalization (mean = 5.5 days), and the gross appearances of their feces (blind determinations) were recorded. Aeromonas spp. were recovered from rectal swabs of 12 of 52 (23.1%) infants during their first week of life; the isolates were obtained from 5 of 9 (55.6%) infants with at least one stool with a watery consistency and from 7 of 43 (16.3%) neonates with no watery stools (P = 0.022). None of the infected infants became clinically ill. No other commonly recognized enteropathogens were detected in watery stools. An environmental survey indicated that hospital water was the probable source of infection. These and other data indicated that Aeromonas colonization occurs transiently at a very early age in Peruvian neonates and that in some instances, initial infection may be followed several days later by one or more watery stools of normal volume.

Isolation rates of Aeromonas hydrophila from stool samples of symptomatic and asymptomatic individuals were examined for several common enteric media. Sheep blood agar with 10 micrograms of ampicillin per ml, preceded by overnight enrichment in alkaline peptone water, yielded 2.6 times the number of isolates as the other media examined and is recommended for the isolation of A. hydrophila from humans.

Enterotoxigenic Bacteroides fragilis was isolated from stool specimens of 8 of 44 diarrheic individuals (ages, 4 months to 69 years). The individuals had watery diarrhea and intestinal cramping; and infants had hyperthermia, vomiting, and blood in the stools. No recognized enteric pathogens were detected in seven of the eight diarrheic individuals positive for enterotoxigenic B. fragilis. The bacterium produced an enterotoxin detectable in concentrated broth that supported bacterial growth. Fifteen adult rabbits with ligated ceca developed fatal enteric disease following intraileal injection with 5 x 10(9) CFU of enterotoxigenic B. fragilis. Conversely, eight control rabbits injected with nonenterotoxigenic B. fragilis remained clinically normal. As few as 5 x 10(3) CFU of enterotoxigenic B. fragilis caused fatal enteric disease in the rabbit model. Disease in rabbits was characterized by mucoid, often hemorrhagic, diarrhea. The bacterium colonized the caudal small intestine and the colon of the rabbits and caused moderate to severe necrotizing colitis. Enterotoxigenic B. fragilis is widespread in the intestinal tract of diarrheic humans and is enteropathogenic in adult rabbits with ligated ceca. Its possible role in the enteric disease complex merits further study.

The development of an enzyme-linked immunosorbent assay (ELISA) for the detection of heat-labile Escherichia coli enterotoxin is described. The assay, which is based on the immunological similarity between Vibrio cholerae toxin and heat-labile E. coli enterotoxin, is similar in design to a radioimmunoassay but utilizes enzyme-labeled rather than radioactive isotope-labeled reagents. The ELISA system is as sensitive as both radioimmunoassay and the y-1 adrenal cell assay for the detection of heat-labile E. coli enterotoxin but requires neither radioactive reagents nor tissue culture techniques. The ELISA is easy to perform and is adaptable for use in small laboratories.

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.

With use of the rabbit intestinal loop model, heat-labile enterotoxins from 21 Escherichia coli strains isolated from a wide spectrum of patients with diarrheal diseases were all neutralized to high titer by two antisera prepared against enterotoxins of either E. coli or Vibrio cholerae. These findings suggest marked immunological similarity among heat-labile enterotoxins from a heterogenous group of E. coli.

Diagnosis of enterotoxigenic Escherichia coli diarrhea was made in 109 adult males with an acute dehydrating cholera-like syndrome in Dacca, Bangladesh, by testing 10 colonies isolated from admission stool specimens for production of heat-labile and heat-stable toxins. Toxin testing of one colony yielded a diagnosis in 92% of the cases, testing of two colonies yielded a diagnosis in 95% of the cases, testing of a pool of 5 colonies yielded a diagnosis in 95% of the cases, and testing of a pool of 10 colonies yielded a diagnosis in 96% of the cases. From stool cultures obtained on subsequent days, toxin testing of individual colonies and pools revealed diminished efficacy of pooling with decreasing numbers of enterotoxin-positive isolates in the pool. To detect the presence of enterotoxigenic E. coli in stools, toxin testing of 5 individual isolates and a pool of 10 colonies was found to be almost as effective as the testing of 10 individual isolates.

A laboratory investigation was conducted on cultures collected from travelers before, during, and after a trip to Mexico to characterize the etiology of traveler's diarrhea. Four laboratory methods for detecting enterotoxigenicity of Escherichia coli were evaluated: the infant mouse assay, the Chinese hamster ovary (CHO) cell assay, the Y1 adrenal cell assay, and the rabbit ileal loop. Although a number of common enteric pathogens were identified as a cause of traveler's diarrhea, including six serotypes of Salmonella, two serotypes of Shigella, Vibrio parahaemolyticus, Giardia lamblia, and Entamoeba histolytica, enterotoxigenic Escherichia coli was most commonly isolated. Strains were identified that produced only heat-labile enterotoxin (LT), only heat-stable enterotoxin (ST), or both LT and ST. The infant mouse assay yielded results falling into two distinct groups, providing a clear separation of positive and negative cultures. The CHO assay also formed two groups, with positive cultures producing 11% or more of the elongated cells. There was good agreement between the CHO and the Y1 adrenal cell assays for detection of LT. The adrenal cell system for detection of LT was more suitable than the CHO assay for processing large numbers of specimens because of the miniculture modification of this method utilized in this study. The infant mouse method was a simple and reliable method for detecting ST.