Nationwide surveillance program to identify diarrhea-causing Escherichia coli in children in Thailand.

Escherichia coli strains isolated from children with diarrhea were collected from 16 hospitals in different districts in Thailand during 1985 and 1986 and submitted to the National Reference Laboratory. Isolates were identified by serogrouping or as enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC) adhesin factor (EAF) E. coli, or Shiga-like-toxin (SLT)-producing E. coli by DNA hybridization. EPEC strains of known serogroups were isolated from 10%, ETEC strains were isolated from 6%, EAF E. coli strains were isolated from 4%, EIEC strains were isolated from less than 1%, and SLT-producing E. coli strains were isolated from none of 393 children with diarrhea. Among 278 children whose ages were recorded, the highest rate of isolation of EAF E. coli was 11% (9 of 85) from children less than 6 months old. ETEC was isolated from 5% (4 of 85) of children less than 6 months old, from 10% (12 of 118) of children 6 to 23 months old, and from 1% (1 of 75) of children greater than 23 months old. EPEC strains of known serogroups were isolated from 18% (15 of 85) of children less than 6 months old, from 11% (13 of 118) of children 6 to 23 months old, and from 9% (7 of 75) of children greater than 23 months old. E. coli strains that hybridized with the EIEC probe were isolated from three children who were 20, 36, and 48 months old. Examining E. coli for hybridization with DNA probes for virulence determinants is a practical way of conducting nationwide surveillance of diarrhea-causing E. coli. Since only 33% (13 of 39) of EPEC serogroups hybridized with the EAF probe and none hybridized with the SLT probes, identification of EPEC by serogroups analysis, followed by serotyping, should continue to be used in the identification of EPEC.     

Identification of enterotoxigenic Escherichia coli isolated from swine with diarrhea in Thailand by colony hybridization, using three enterotoxin gene probes.

The DNA colony hybridization assay was used to identify enterotoxigenic Escherichia coli among E. coli isolated from 803 swine with diarrhea at 10 farms in Thailand. Between 5 September and 8 December 1981, enterotoxigenic E. coli were identified in 40% of 58 litters of piglets under 10 days old and 17% of 29 litters between 10 and 21 days old with diarrhea at farms at four different locations in Thailand. All E. coli that hybridized with one or more of the three enterotoxin gene probes produced heat-labile or heat-stable toxin or both, as determined by testing culture supernatants in the Y1 adrenal and suckling mouse assays. The DNA colony hybridization technique is a specific method of identifying enterotoxigenic E. coli from swine and can be used to further characterize these enteric pathogens.     

Type II heat-labile enterotoxin-producing Escherichia coli isolated from animals and humans.

Heat-labile enterotoxin (LT)-producing Escherichia coli strains, as identified by the Y1 adrenal cell assay, were examined with a DNA probe coding for type I and type II LTs. Of 236 LT-producing E. coli isolates, 60% hybridized with LT-I, 17% hybridized with LT-II, and 23% did not hybridize with either probe and no longer produced LT as determined by the Y1 adrenal cell assay. These isolates presumably lost plasmids coding for LT-I during storage. A total of 75% of LT-producing E. coli isolates (27 of 36) from cows, 64% of LT-producing E. coli isolates (7 of 11) from buffalo, 31% of LT-producing E. coli isolates (4 of 13) from beef obtained in markets, and 2% of LT-producing E. coli isolates (3 of 168) from humans contained genes coding for LT-II. Genes coding for LT-II were not found in 50 LT-I-producing and heat-stable enterotoxin-producing E. coli isolates from 11 children with diarrhea and 44 LT-nonproducing and heat-stable enterotoxin-producing E. coli isolates from 12 other children with diarrhea. A total of 9% of LT-II-producing E. coli isolates (3 of 34) from cows and buffalo hybridized with DNA probes for genes coding for verocytotoxin 2 (VT2), and 18% (6 of 34) hybridized with a DNA probe coding for enterohemorrhagic E. coli (EHEC) adhesin fimbriae. E. coli SA-53, the original isolate in which LT-II was found, contained genes coding for VT2 and EHEC adhesin fimbriae. Five VT-producing, LT-II-producing E. coli isolates that hybridized with the EHEC probe did not contain DNA sequences coding for VT1 or VT2. LT-II-producing E. coli strains were frequently isolated from cattle and buffalo but were rarely isolated from humans.     

Clinical and Epidemiological Relevance of Quantitating Hepatitis E Virus-Specific Immunoglobulin M

Diagnosis of acute hepatitis E by detection of hepatitis E virus (HEV)-specific immunoglobulin M (IgM) is an established procedure. We investigated whether quantitation of HEV IgM and its ratio to HEV total Ig furnished more information than conventional IgM tests that are interpreted as positive or negative. A previously described indirect immunoassay for total Ig against a baculovirus-expressed HEV capsid protein was modified to quantitate HEV-specific IgM in Walter Reed (WR) antibody units by using a reference antiserum and the four-parameter logistic model. A receiver-operating characteristics curve derived from 197 true-positive specimens and 449 true-negative specimens identified 30 WR units/ml as an optimum cut point. The median HEV IgM level in 36 patients with acute hepatitis E fell from 3,000 to 100 WR units/ml over 6 months, suggesting that 100 WR units/ml would be a more appropriate cut point for distinguishing recent from remote IgM responses. Among three hepatitis E case series, determination of the HEV IgM-to-total-Ig ratio in acute-phase serum revealed that most patients had high ratios consistent with primary infections whereas a few had low ratios, suggesting that they had sustained reinfections that elicited anamnestic antibody responses. The diagnostic utility of the new IgM test was similar to that of a commercially available test that uses different HEV antigens. In conclusion, we found that HEV IgM can be detected specifically in >95% of acute hepatitis E cases defined by detection of the virus genome in serum and that quantitation of HEV IgM and its ratio to total Ig provides insight into infection timing and prior immunity.     

A comparative study of enterotoxin gene probes and tests for toxin production to detect enterotoxigenic Escherichia coli

Escherichia coli isolated from children with diarrhea were tested for enterotoxin production and for hybridization with gene probes for heat-labile (LT) and heat-stable (ST-H and ST-P) enterotoxin. Fecal specimens were also examined directly for genes coding for enterotoxins. E. coli that hybridized with the cloned enterotoxin gene probes was identified by colony hybridization from 46 children, by enterotoxin production from 38 children, and by specimen hybridization from 37 of 304 children examined. Eighty-six percent (473 of 550) of E. coli that hybridized with the cloned DNA probes produced enterotoxins. Four E. coli that hybridized with the LT and 73 E. coli that hybridized with the ST-H probes were nonenterotoxigenic. These isolates were subsequently shown not to hybridize with other constructions of the same probes and did not hybridize with synthetic single-stranded oligonucleotides directed against the LT or ST genes.     

A comparative study of enterotoxigenic Escherichia coli, Shigella, Aeromonas, and Vibrio as etiologies of diarrhea in northeastern Thailand

The incidence of enterotoxigenic Escherichia coli (ETEC), Shigella, Aeromonas, and Vibrio was determined in patients with diarrhea seen at a hospital in northeastern Thailand, and compared with the incidence of these bacteria in household contacts and their neighbors. ETEC was identified in 17%, Shigella in 9%, Aeromonas in 9%, V. parahaemolyticus in 5%, and non-01 V. cholerae in 2% of 299 patients with diarrhea. These five species of bacteria were isolated more often from patients with diarrhea than persons without diarrhea (P less than 0.001). ETEC was found more often in household contacts (22/141) and neighbors (18/147) of index cases than in persons living in homes not associated with ETEC infections (32/1,318; P less than 0.001). While Shigella was isolated less often in family contacts (3/76) and neighbors (4/93) of patients with shigellosis, this enteric pathogen was also isolated more often from contacts than persons not associated with Shigella infection (13/1,437; P less than 0.001). Both Aeromonas and non-01 V. cholerae can also be enteric pathogens; further efforts should be made to define the enteropathogenicity of these bacteria.     

Antibody levels to hepatitis E virus in North Carolina swine workers,non-swine workers,swine, and murids

In a cross-sectional serosurvey, eastern North Carolina swine workers (n = 165) were compared with non-swine workers (127) for the presence of antibodies to hepatitis E virus as measured by a quantitative immunoglobulin enzyme-linked immunosorbent assay. Using a cutoff of 20 Walter Reed U/ml, swine-exposed subjects had a 4.5-fold higher antibody prevalence (10.9%) than unexposed subjects (2.4%). No evidence of past clinical hepatitis E or unexplained jaundice could be elicited. Swine (84) and mice (61), from farm sites in the same region as exposed subjects, were also tested. Antibody prevalence in swine (overall = 34.5%) varied widely (10.0-91.7%) according to site, but no antibody was detected in mice. Our data contribute to the accumulating evidence that hepatitis E may be a zoonosis and specifically to the concept of it as an occupational infection of livestock workers.     

DNA probes to identify Shiga-like toxin I- and II-producing enteric bacterial pathogens isolated from patients with diarrhea in Thailand.

When Shigella species, Escherichia coli, and five other bacterial enteric pathogens isolated from children with diarrhea in Thailand were tested for hybridization under stringent conditions with probes for Shiga-like toxins I and II, only 30 Shigella dysenteriae 1 hybridized with the Shiga-like toxin I probe.     

Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates.     

DNA hybridization in the diagnosis of bacterial diarrhea

DNA hybridization with either cloned genes for enteropathogenic determinants or DNA segments that are species-specific is a valuable tool to identify certain bacterial enteric pathogens. Thus far, only E. coli and V. cholerae enterotoxin gene probes have been used to identify ETEC and V. cholerae in clinical specimens. DNA probes developed for Salmonella, Shigella, Campylobacter, and enteroinvasive and enteropathogenic E. coli need to be evaluated with clinical specimens. The major contribution of this system so far has been to examine large numbers of specimens in epidemiologic studies. Once nonradioactive DNA probes are developed, this system will have potential application in clinical laboratories and in research laboratories in the developing world where diarrheal disease causes its greatest impact.