Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.

This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.

Detection of poliovirus, hepatitis A virus and rotavirus from sewage and water samples.

A modified adsorption-elution technique for concentration of enteric viruses from sewage and water samples was developed. The viruses in water were concentrated by negatively charged membrane filtration, eluted with 2.9% tryptose phosphate broth containing 6% glycine pH 9.0, and reconcentrated using centrifugation by a speedVac concentrator. The presence of poliovirus, hepatitis A virus (HAV) RNA, and rotavirus antigen was determined by cell culture isolation, nested polymerase chain reaction (nested PCR), and enzyme-linked immunosorbent assay (ELISA), respectively. A total of 100 sewage and water samples were collected from various sources in congested communities in Bangkok, concentrated and examined for those enteric viruses. Of 20 surface water samples from canals which located near sewage drains, 15% were positive for HAV RNA by nested PCR. Of 48 domestic sewage samples from man-holes of underground sewers, 8% were positive for rotavirus antigen by ELISA. Even though the samples were concentrated 256-2,000 fold, poliovirus was not found by isolation in cell culture.

Reduction of rotavirus infection in children receiving bifidobacteria-supplemented formula.

This study was conducted at Pakkred Babies Home, Bangkok, Thailand; with the hypothesis that children receiving probiotic-supplemented milk-based formula may be protected from developing diarrheal diseases. Salivary rotavirus-specific IgA antibody was used as an indicator of rotavirus infection. One hundred and seventy-five children, aged 6-36 months, were enrolled in the study. They were divided into 3 groups according to the type of formula given. There were 81 episodes of diarrhea during an 8-month study period, most of which were caused by bacterial enteropathogens. Ninety-seven pairs of salivary samples were adequate for the analysis of rotavirus antibody. Among 23 children receiving milk-based follow-up formula and serving as control group, 30.4 per cent of them had > or = 4-fold increase in the antibody titre, indicating subclinical rotavirus infection. The majority of children in the other 2 study groups, receiving the same formula supplemented with either Bifidobacterium Bb12 alone or together with Streptococcus thermophilus, had no significant change in the antibody titres between the two time points. The results of this study support our hypothesis that children receiving bifidobacteria-supplemented milk-based formula may be protected against symptomatic rotavirus infection.

Dot-ELISA for seroepidemiological study of exposure to Shigella flexneri.

A seroepidemiological study for determining serum antibodies to lipopolysaccharides (LPS) of Shigella flexneri using dot--ELISA was carried out in Krabi Province, Thailand, from January 1989 to December 1990. From 363 serum samples obtained from cord blood and from venous blood of the healthy persons aged from 6 months to over 50 years, 56% and 22%, respectively, were found to be positive for specific IgG and IgM antibodies to S. flexneri LPS. The IgG prevalence was initially detected at 3-4 years of age and then rose sharply with age. In contrast, IgM was detectable earlier, with much lower prevalence than that of IgG. The highest seroprevalence values were in the age groups 30-49 years for IgG and 15-19 years for IgM. The seroprevalence of S. flexneri infection was statistically higher among males, Buddhists, businessmen, and those with elementary education. S. flexneri infection was not associated with family income, home location, eating behaviour or water supply. These seroepidemiologic data demonstrated that most of the population in Krabi Province had been infected with S. flexneri.

Risk factors for childhood diarrhea in an urban community, Bangkok, Thailand.

A follow-up study for diarrheal disease was carried out for a period of one year in children aged 0-5 yrs who lived in a government housing project in Din Daeng community of Bangkok metropolitan area during 1988-1989. The overall incidence was 0.9 episode per child per year with the higher episode of 2 per child per year in children less than 2 yrs. The risk factors of diarrheal occurrence were studies within different age groups. For children less than 6 months the major risk factors were low family income, low education level and unemployed parents. In children 6-11 months the other risk factors added to low income and education in mothers were children who did not live with parents. The risk factors for children 1-2 yrs were non-working mothers, and unhygienic behavior of the child care takers such as no hand washing after toilet use and the method of cleaning milk bottles for children 2-5 yrs. Method of faecal disposal, handwashing, day care and food protection were major sources of diarrheal risk.

Impact of an educational program on the treatment practices of diarrheal diseases among pharmacists and drugsellers.

The impact of an intervention program, measured by changes in the prescription of ORS, antibiotics and antidiarrheal drugs by those pharmacists and drugsellers after administering the proposed educational package was assessed. The results of the study indicated that, before the educational program started, ORS was prescribed by pharmacists for 31.4% and 15.7% of watery diarrhea and dysentery episodes, respectively. Only 18.9% and 13.3% of drugsellers gave ORS to assessors in case of watery diarrhea and dysentery. Antibiotics and antidiarrheal agents were prescribed extensively, watery and dysenteric diarrhea (84% and 56% for watery diarrhea by pharmacists and drugsellers; 92% and 60% for dysentery). Antidiarrheal drugs were used as frequently. After the educational program, the assessment of the prescription behavior of the pharmacists showed no change in ORS, antibiotics and antidiarrheal drugs prescribed to treat watery diarrhea. In dysentery, the effective percent change in prescribing ORS between pre- and post- intervention program was much higher in intervention group than the control group. For drugsellers, effective percent change in ORS usage in treatment of watery diarrhea was 11.8% compared with -7.7% in the control group. No such change was observed in treatment of dysentery. There was a slight significant change in behavior concerning use of antibiotics among subjects getting information by mail, compared to those who got full intervention, when the pre-intervention behavior, store type and treatment type was taken into account.

ELISA for seroepidemiological study of exposure to Vibrio cholerae of population in Krabi Province, Thailand.

Seroepidemiological study of Vibrio cholerae exposure was carried out in Krabi Province during January 1989 to December 1990 using indirect ELISA to determine serum antibodies to lipopolysaccharides (LPS) of V. cholerae. Among 363 serum samples obtained from cord blood and venous blood of healthy persons, aged from 6 months to over 50 years, 65% and 64% were found positive for specific IgG and IgM against LPS of V. cholerae, respectively. The seroprevalence of V. cholerae infection increased with age from that found at 6 months, being highest in the age groups of 30-49 years for IgG and 15-29 years for IgM. The seroprevalence of V. cholerae infection was higher among female Muslims and home-makers, and increased with the family income. The seroprevalence of cholera infection was also influenced by home location, methods of food storage and water supply. These data suggested that a large number of Krabi's population had V. cholerae infection.

Behavior of urban based child caretakers in the home treatment of diarrheal diseases.

Behaviors of low income urban mothers and child caretakers in the treatment of childhood less than 5 year diarrhea were analysed from a surveillance study conducted between August 1988 and July 1989. Help seeking behaviors of mothers and caretakers for 412 episodes of child diarrhea were as follows: investigators 37.1%, drug stores 18.2%, wait and see or self treatment 17.0%, private clinics 12.6%, near by hospital 10.2%, and local health center 4.9%. Major treatment practices included ORT alone (54%) and ORT plus antibiotics and/or antidiarrheal drug (22%). Overall ORT usage was 76%. Twelve percent of diarrheal episodes no treatment was given to the children. Antimicrobials were believed to be essential in addition to ORT especially when diarrhea was associated with fever, vomiting and bloody stools. Thirty-six percent of invasive diarrhea cases (Shigella, Salmonella, Campylobacter) were treated with antibiotics. Only 18.2% of noninvasive diarrhea received antibiotics, most of this antibiotic use being in rotavirus diarrhea where vomiting and some fever are prominent. Availability of oral rehydration salts (ORS) and good experience with ORT were the key to the extensive use or ORT in this study. A surprisingly small number of mothers and child caretakers (4.9%) sought help from the local health center when their children had diarrhea.

Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection.

The incidence of Haemophilus influenzae and Streptococcus pneumoniae in children with acute respiratory infection (ARI) under 5 years was carried out by throat swab culture, blood culture, body fluid or tissue culture in 688 patients from a community, 744 patients from a teaching hospital in Bangkok, 766 normal children from the community and 303 children from a hospital well baby clinic. H. influenzae was found in the throats of 15-20% of patients and in the throats of 4-6% of normal children (p less than 0.001 for both hospital and community patients). Only 12/332 strains (3.6%) of H. influenzae were type b. The rest of H. influenzae were non type b. The most common biotype of H. influenzae non type b was biotype II. S. pneumoniae was found in hospital patients in highly significant numbers compared to the controls (12% vs 4%). No significant difference was observed in strains from the community patients.