Small bowel and fecal microbiology in children suffering from persistent diarrhea in Bangladesh.

BACKGROUND: The etiology of persistent diarrhea in children is multifactorial. The objective of the current study was to ascertain the role of microorganisms in the etiology and pathogenesis of persistent diarrhea in a group of children in Bangladesh. METHODS: Enteric pathogens and total aerobic microflora were studied in the duodenal aspirates of 100 children with persistent diarrhea and compared with those in aspirates of 30 children with acute diarrhea, and those in aspirates of 15 healthy control children. The enteric pathogens in the stools of these children and in stools of an additional 38 patients with persistent diarrhea and 12 with acute diarrhea were also studied. RESULTS: Approximately two thirds of the patients with acute diarrhea and persistent diarrhea, and half of the control subjects had more than 10(5) organisms per milliliter of duodenal fluid. Significantly, more patients with persistent diarrhea had a greater variety of flora than did patients with acute diarrhea and control subjects. The predominant organisms in patients with acute diarrhea and in those with persistent diarrhea were Gram-negative rods, whereas those in control subjects were Gram-positive cocci. Significantly more acute diarrhea patients and persistent diarrhea patients had enteric pathogens isolated from stool than did control subjects. Diarrheagenic Escherichia coli, as a whole, were present in significantly more persistent diarrhea patients than in acute diarrhea patients and control subjects. Among diarrheagenic E. coli, enteroaggregative E. coli were significantly associated only with persistent diarrhea. Other organisms significantly associated with persistent diarrhea were Aeromonas spp. and Klebsiella spp. Some patients in the acute diarrhea and the persistent diarrhea groups had the same pathogens isolated from both the duodenal fluid and stool. CONCLUSIONS: In accordance with results of other studies, an association between enteroaggregative E. coli and persistent diarrhea was found in the present study. This suggests that therapy directed against enteroaggregative E. coli can be evaluated for management of some cases of persistent diarrhea.

Characterization of Aeromonas spp. isolated from humans with diarrhea, from healthy controls, and from surface water in Bangladesh.

Aeromonas isolates from patients with diarrhea in Bangladesh (n = 69), from healthy controls (n = 11), and from surface water (n = 40) were analyzed with respect to their hybridization groups (HGs) by the aid of fatty acid methyl ester (FAME) characterization and DNA fingerprinting by AFLP, biochemical phenotypes (Phe-nePlate [PhP] types), and the production of hemolysin and cytotoxin. The aim of the investigation was to find out whether certain strains carrying virulence factors predominated among patient isolates. According to FAME and/or AFLP analysis, most human isolates were allocated to DNA HGs 4 (Aeromonas caviae) and 1 (A. hydrophila). Most environmental strains were allocated to HG8 (A. veronii biogroup sobria) and HG4 (A. caviae), and only one was of HG1. According to PhP typing, the diversity among patient isolates was lower than that among other strains, and two dominating PhP types (types BD-1 and BD-2) were identified in 29 and 30% of the patient isolates, respectively. PhP type BD-1 was also common among the environmental isolates, whereas PhP type BD-2 was only identified in two of the other isolates. Twenty-five of 26 isolates belonging to HG1 were of the same PhP type (BD-2), whereas isolates of other common HGs were more diverse according to their PhP types. Hemolytic and cytotoxin-producing strains occurred more frequently among the environmental isolates than among patient isolates. However, the hemolytic and cytotoxic activities among human isolates was strongly correlated to the HG1/BD-2 type, which, in addition, showed high cytotoxin titers (median values, 1/512 compared to 1/128 for cytotoxin-positive isolates belonging to other types). Thus, the HG1/BD-2 type may represent a pathogenic A. hydrophila type that is able to produce diarrhea in humans.

Controlled study of cytolethal distending toxin-producing Escherichia coli infections in Bangladeshi children.

The role of cytolethal distending toxin (CDT)-producing Escherichia coli, a newly described category of E. coli, in the causation of diarrhea was studied by screening E. coli isolates from 546 children < 5 years of age with diarrhea and 215 matched controls without diarrhea by using a specific DNA probe. Although CDT-positive E. coli strains were isolated from more children with diarrhea than from healthy controls (3.1 versus 0.93%), this difference did not reach statistical significance (P = 0.082). All CDT-positive strains also possessed the virulence factors of enteropathogenic E. coli or enteroaggregative E. coli isolates.

Detection of provisional serovars of Shigella dysenteriae and designation as S. dysenteriae serotypes 14 and 15.

In initial studies of slide and tube agglutination tests of shigella-like isolates from diarrheal stools from Bangladesh, 10 and 8 strains of Shigella dysenteriae were identified as provisional serovars (serovars that have been defined but not given a number in the antigenic schema) E22383 and E23507, respectively. Further screening of diarrheal stool isolates in a slide agglutination test with antisera (made with the international reference strains) to the two provisional serovars of S. dysenteriae identified an additional 36 isolates as belonging to E22383 and 8 isolates as belonging to E23507. All strains had properties typical of invasive Shigella strains in that they were positive in the Sereny test and had the 120- to 140-MDa plasmid associated with invasiveness. On the basis of these markers of pathogenicity and isolation from various geographical locations, now we recommend that provisional serovars E22383 and E23507 be designated S. dysenteriae serotypes 14 and 15, respectively.

Controlled study of Escherichia coli diarrheal infections in Bangladeshi children.

Diarrheal diseases are highly prevalent in Bangladesh. However, the relative contribution of diarrheagenic Escherichia coli organisms--those that are enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive, enterohemorrhagic, enteroaggregative, and diffuse adherent--to diarrhea in Bangladeshi populations is not known. With DNA probes specific for these diarrheagenic E. coli strains, we analyzed fecal E. coli from 451 children up to 5 years of age with acute diarrhea seeking treatment at a Dhaka hospital and from 602 matched control children without diarrhea from July 1991 to May 1992. Enteroinvasive E. coli was not isolated from any children; enterohemorrhagic E. coli was not isolated from any diarrheal children but was isolated from five control children; enteroaggregative and diffuse adherent E. coli strains were isolated with similar frequencies from children with and without diarrhea, thereby showing no association with diarrhea; ETEC was significantly associated with diarrhea in the diarrheal children as a whole and especially in the age groups of 0 to 24 months and 37 to 48 months (further analysis suggests an association with diarrhea for the heat-stable toxin only and for both heat-labile- and heat-stable-toxin-producing ETEC only); and EPEC was significantly associated with diarrhea in the diarrhea group as a whole and particularly in infants up to 1 year of age. Further analysis suggested that EPEC strains of only the traditional serogroups were significantly associated with diarrhea. ETEC and EPEC infections peaked during warm months. Our data thus suggest that EPEC and ETEC are important causes of acute diarrhea in children in this setting.

Characteristics of invasion of HEp-2 cells by Providencia alcalifaciens.

Previous studies with three isolates from diarrhoeal stools suggested that Providencia alcalifaciens is an invasive enteric pathogen that also causes actin condensation in infected cells. These findings were extended in the present study with a further 14 diarrhoeal stool isolates of P. alcalifaciens and HEp-2 cell monolayers for invasion assays. Studies on invasion characteristics with two selected isolates suggested that P. alcalifaciens required prior growth at 37 degrees C for better invasion. Invasion and actin condensation were inhibited by an agent that inhibits microfilament formation, but not by agents that inhibit receptor-mediated endocytosis, microtubule formation, endosome acidification or receptor recycling. In time-course assays with HEp-2 cell monolayers maintained in medium containing gentamicin, P. alcalifaciens showed a small degree of multiplication after invasion of the cells, but viable bacteria could not be recovered over a 24-h period although the integrity of the cell monolayer was preserved during this period.

Isolation of enterotoxigenic Bacteroides fragilis from Bangladeshi children with diarrhea: a controlled study.

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.

Comparison of a modified adherence assay with existing assay methods for identification of enteroaggregative Escherichia coli.

Localized, diffuse, and aggregative adherence patterns of Escherichia coli identified with specific DNA probes were compared in cell culture adherence assays by using the Center for Vaccine Development, Baltimore, method, the University of Texas Medical School, Houston (UTH), method, and a modified UTH method. Increasing postwash incubation time from 2 to 4 h in the modified UTH method allowed identification of enteroaggregative E. coli, which was otherwise not identified by the original UTH method.

Characterization of Aeromonas caviae antigens which cross-react with Shigella boydii 5.

Live and boiled cells of 16 strains of Aeromonas caviae, isolated from patients with diarrhea, agglutinated with Shigella boydii 5 antiserum in a slide test. Further studies with seven selected strains showed agglutination with boiled cells in a tube test. Lipopolysaccharide antigen extracted from one of these strains cross-reacted with S. boydii 5 in enzyme-linked immunosorbent assay and immunoblot studies. Either all or the majority of the seven strains possessed properties deemed to be diarrheagenic.

Seasonality and toxigenicity ofVibrio cholerae non-01 isolated from different components of pond ecosystems of Dhaka City, Bangladesh.

Diarrhoea due toVibrio cholerae non-01 is common in Bangladesh. Four hundred and eighty samples, including plants, water, phytoplankton and sediment, were collected from five ponds in Dhaka every 15 days for one year.V. cholerae non-01 was isolated from 181 (38%) of the samples. Two peaks were evident: one in April and the other in August/September. Forty-three (23%) of the 181 isolates were examined for toxigenicity and 19% were cytotoxic to Y1 adrenal cells. This study provides evidence of the likely infectious nature of some ponds and may have relevance to the epidemiology of diarrhoea caused byV. cholerae non-01 in Bangladesh.

An ELISA for the detection of localized adherent classic enteropathogenic Escherichia coli serogroups.

An ELISA for the detection of classic enteropathogenic Escherichia coli (EPEC) serogroups was developed. It detected EPEC positive for localized adherence (LA) by HeLa cell assay and EPEC positive for EPEC adherence factor (EAF) by DNA probe assay. A specific antiserum was raised with LA+ EPEC strain E2348/69 (serotype O127:H6) by immunizing rabbits and then absorbing the antiserum with its LA- derivative, MAR20. The absorbed antiserum reacted specifically with all 90 strains of E. coli belonging to eight different EPEC serogroups that were LA+ by HeLa cell assay and EAF+ by DNA probe assay. All E. coli strains including EPEC serogroups that were LA- by HeLa cell assay and EAF- by DNA probe assay were also negative by ELISA. Thus the ELISA is 100% sensitive and specific in detecting LA+ classic EPEC serogroups.

Serotypes of Campylobacter jejuni isolated from patients attending a diarrhoeal disease hospital in urban Bangladesh.

The serotypes of strains of Campylobacter jejuni isolated from patients attending the International Center for Diarrhoeal Disease Research, Bangladesh (ICDDR, B), Dhaka, and from animals were ascertained by Penner and Hennessy's (1980) serotyping scheme. Of 102 isolates from man, 74% were typable and serotypes 53, 15 and 22 predominated. Of 26 isolates from animals, 65% were typable and serotypes 15 and 53 occurred frequently. The diarrhoeal illnesses associated with different serotypes were similar. In one-third of cases other enteropathogens were present. C. jejuni was isolated from 7% of patients tested in a 4% sampling system during 1983, and the prevalent serotypes appeared in most months. It is concluded that C. jejuni is a common enteropathogen in Bangladesh, that a few serotypes predominate among isolates from both man and animals, and that serotype does not determine clinical symptoms.

Yersinia enterocolitica infection; a case report from Bangladesh.

While a large number of confirmed cases of Yersinia enterocolitica have been reported in different temperate zones, there is little information on the incidence of this infection in tropical areas. For the first time in Bangladesh, this organism has been isolated from the stool of a diarrhoea patient and, therefore, is recognized as a new enteropathogen in this country. The strain was characterized as biotype I, serotype 0:8 and phage type X2. The patient was a five-year-old girl from a poor Muslim family, and admitted to ICDDR,B's Treatment Centre with complaints of abdominal pain, low-grade fever and diarrhoea.

Immunobead enzyme-linked immunosorbent assay for quantitating immunoglobulin A in human secretions and serum.

We have developed an enzyme-linked immunosorbent assay for quantitating human immunoglobulin A in human secretions and serum. The procedure uses Immunobeads as the solid phase, has a sensitivity of 10 ng/ml, and can be completed in 1 day. Since there is no diffusion through agar, the assay does not require a correction factor when measuring secretory immunoglobulin A. This method should be useful in studies of local mucosal immunity in humans, especially when a very sensitive assay is needed.

Microtiter ganglioside enzyme-linked immunosorbent assay for vibrio and Escherichia coli heat-labile enterotoxins and antitoxin.

We have developed a microtiter enzyme-linked immunosorbent assay method for detecting the heat-labile enterotoxins of Vibrio cholerae and Escherichia coli using GM1 ganglioside as the base coat. This method compares favorably with a similar assay using anticholera toxin as the base coat, and with the Y1 adrenal cell assay. The assay should be useful in detecting enterotoxin production in E. coli and vibrios (including non-agglutinating Vibrio), in quantitating the toxin, and in determining binding properties of enterotoxins to ganglioside. The assay can also be used to quantitate antibodies which block the attachment of the toxin to the ganglioside.