An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9 percent tryptose phosphate broth containing 6 percent glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples

Rapid detection of haemophilus influenzae type B by PCR.

Rapid and reliable diagnosis of meningitis caused by Haemophilus influenzae type b (Hib) is essential for early treatment to reduce the mortality rate and neurological damage among survivors. In this study, a PCR assay for Hib was developed as a reliable method in the clinical laboratory. Two methods for DNA extraction from H. influenzae isolates were compared. The extraction by a commercial “DNAzol reagent” was more rapid and convenient than conventional phenol-chloroform extraction. DNA yield from both methods was not significantly different. DNA from standard strains was used for optimizing the PCR reaction with our new designed primers based on the genes coding for capsule type-specific Hib. The sensitivity, specificity and agreement rate of the primers were tested by comparison with one pair of the published primers. There was a perfect agreement between the newly designed and the published primers with K = 1; however, the new primers had higher sensitivity and could detect as low as 1 pg of DNA. When the blind colonies of 187 bacterial meningitis isolates were used in the PCR assay, the sensitivities, specificities and efficiencies of the PCR with both primer sets, comparing the results of the culture and slide agglutination with Hib specific antiserum as the “gold” standard, were 100, 99.32 and 99.47%, respectively. The one capsule-deficient type b mutant strain could be detected by PCR while the serological result with Hib antiserum was negative. The PCR developed in this study shows rapidity, high sensitivity and specificity and may be a useful adjunct to conventional methods for early diagnosis of Hib meningitis in the clinical microbiology laboratory.