Children´s diarrhea in Hanoi, Vietnam : Importance of enteric pathogens

Diarrhea is one of the leading causes of illnesses and death among children in developing countries, where an estimated 1.3 billion episodes and 4 to 10 million deaths occur each year in children less than 5 years of age. In Vietnam, different studies have shown that the frequency of diarrhea is about three episodes for each child in one year and the death rate due to diarrhea is about 1-1.5%. The common pathogens of diarrhea are Group A rotavirus, diarrheagenic Escherichia coli (DEC), Shigella spp. , enterotoxigenic Bacteroides fragilis (ETBF), Salmonella spp. , Campylobacter, and Vibrio cholerae. Our objectives were to perform a detailed microbiological investigation of some potential pathogens associated with diarrhea, to characterize the isolates, and to assess clinical symptoms and the epidemiological factors related to the diarrheal disease in children less than 5 years of age in Hanoi, Vietnam. We have successfully developed a multiplex Polymerase Chain Reaction (PCR) by combining eight primer pairs specific for enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC), enterohemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC), and enterotoxigenic E. coli (ETEC) to facilitate identification of these strains from stool samples in a single reaction. The prevalence of DEC was 22.5% and 12% in the diarrhea group and the control group, respectively, showing a significant difference. Among 587 fecal samples from children with diarrhea, this technique identified 68 samples (11.6%) with EAEC, 12 (2.0%) with EIEC, 39 (6.6%) with EPEC, and 13 (2.2%) with ETEC. Of the 249 age-matched controls, the distribution was 18 (7.2%) with EAEC, 11 (4.4%) with EPEC, and 1 (0.4%) with ETEC. No EHEC, Salmonella or V. cholerae were identified in either of the groups. Shigella spp. has been identified in 4.7% of children with diarrhea. Campylobacter was not investigated. One hundred and sixty-two DEC and 28 Shigella strains isolated from children were tested for the minimum inhibitory concentrations following NCCLS recommendations by the agar dilution method against ampicillin, chloramphenicol, trimethoprim/sulfamethoxazole, imipenem, cefuroxime, cefotaxime, nalidixic acid, and ciprofloxacin. For E. coli strains, the resistance prevalence to ampicillin, chloramphenicol, and trimethoprim/sulfamethoxazole was 86.4%, 77.2%, and 88.3%, respectively. More than 89% of the Shigella strains were resistant to trimethoprim/sulfamethoxazole, 75% were resistant to ampicillin, and 53.6% were resistant to chloramphenicol. Multi-antibiotic resistance was detected in 89.5% of the E. coli and in 78.6% of Shigella strains. Immunoseparation in combination with PCR assay identified ETBF in 7.3% of fecal samples from children with diarrhea and 2.4% of samples from the controls (P < 0.01). Within the diarrhea group, the prevalence was significantly higher in children older than 1 year of age. Three toxin subtypes in the 43 ETBF isolates have been identified with the prevalence of 67.4%, 18.6%, and 16% for bft-1, bft-2, and bft-3, respectively. In the controls, 5 bft-1 subtypes and 1 bft-2 subtype were identified. Group A rotavirus was identified in 46.7% of children with diarrhea and 3.6% of the healthy ones showing a significant difference. Within the diarrhea group, the highest prevalence was seen in children from 13-24 months of age, higher in males compared to females. The symptoms of acute diarrhea caused by rotavirus were watery diarrhea, vomiting, fever and dehydration. Of 587 children with diarrhea, 40.9% were less than 1 year of age, 71.1% were less than 2. Rotavirus, DEC, Shigella, and ETBF have been found in 67.3% of children with diarrhea and 17.3% of the controls (P < 0.00001). 13.5% of children with diarrhea and 0.8% of the controls were infected with two or more potential identified pathogens. Rotavirus infection peaked during the autumn and wintertime, whereas the bacterial infections were predominant in the summertime. The epidemiological factors such as lack of fresh water supply, unhygienic toilet, low living standard, infrequent getting information of health, and low education of parents could give rise to the morbidity of diarrhea in children. This is the first study where several potential enteric pathogens have been identified in relation with clinical symptoms and epidemiology of diarrhea in children less than 5 years of age in Hanoi, Vietnam. The multiplex PCR has now been applied in detection of diarrheagenic E. coli in some microbiology laboratories. Importantly, the results of this study will contribute to the effective diagnosis, treatment, and intervention in order to decrease the morbidity and mortality of diarrhea in children less than 5 years of