Rapid detection of Haemophilus influenzae by hel gene polymerase chain reaction.

AIMS: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. METHODS AND RESULTS: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65.9%) of 91 samples in contrast to 62 (68.12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. CONCLUSIONS: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. SIGNIFICANCE AND IMPACT OF THE STUDY: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods.

Role of intracellular second messengers and reactive oxygen species in the pathophysiology of V. cholera O139 treated rabbit ileum.

Vibrio cholerae O139 has pandemic potential and it produces copious amounts of fluid secretion. The levels of various second messengers (intracellular Ca2+, cAMP, IP3, PKC) were measured to determine the cause of fluid secretion produced by this strain of V. cholerae. There was a significant increase in the levels of these second messengers in V. cholerae O139 treated ileum as compared to control ileum (enterocytes). Levels of these second messengers were also assessed in V. cholerae 569B induced fluid secretion in rabbit ileum and it was found that the levels were raised more in V. cholerae O139 treated ileum than in V. cholerae 569B treated rabbit ileum. The intestinal damage was assessed by measuring changes in the extent of lipid peroxidation of the enterocytes. Intracellular second messengers are known to raise the extent of lipid peroxidation. In V. cholerae O139 treated loops calcium ionophore A23187 enhanced the extent of lipid peroxidation whereas l-verapamil could only marginally decrease the lipid peroxidation. Dantrolene and H7 significantly decreased the extent of lipid peroxidation of enterocytes in V. cholerae O139 treated rabbit ileum. However, PMA could not enhance further the extent of lipid peroxidation in V. cholerae O139 treated rabbit ileum. So intracellular calcium and protein kinase C appear to be involved in intestinal damage caused by V. cholerae O139. Reactive oxygen species are responsible for causing tissue damage and the extent of oxidative damage depends on the balance between the pro-oxidants and the anti-oxidants. So the changes in the enterocytes' antioxidant level during V. cholerae O139 mediated intestinal infection was estimated. There was a significant decrease in the enterocyte level of the antioxidant enzymes SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase and glucose-6-phosphate dehydrogenase in V. cholerae O139 mediated intestinal infection. So a significant decrease in the levels of antioxidant defenses and a significant increase in the levels of second messengers appear to be important in mediating V. cholerae O139 induced lipid peroxidation which contributes to the changes in membrane permeability and thus to fluid secretion.

Bacterial contamination of reconstituted oral rehydration solution.

The extent and nature of bacterial contamination in oral rehydration solution reconstituted for use by individuals and for group of patients was studied. Twenty three volunteers (all qualified doctors) were asked to reconstitute a packet of prepackaged salt in half litre of clean unboiled water obtained from taps at their residence. Five ml aliquots of ORS were collected at 6, 12 and 24 hours after reconstitution for bacteriologic study. The water used by volunteers to reconstitute the ORS as well as throat swabs, peri-anal swabs and nail clippings of volunteers yielded pathogenic bacteria in all the subjects/samples. All the 23 specimens of ORS prepared by volunteers when cultured at 6 hours after reconstitution yielded pathogenic bacteria. The bacterial colony counts were found to be unacceptably high at 12 hours. Five ml samples of reconstituted ORS prepared in bulk in the children ward of PGIMER, Chandigarh were cultured at 12, and again at 24 hours after reconstitution on 10 different days. These yielded Klebsiella pneumoniae in 8 specimens (80%) and E. coli in 2 (20%). The bacterial colony count was unacceptably high, 12 hours after reconstitution.

Prevalence of biliary tract disease in India: a sonographic study in adult population in Kashmir.

Sonography was used to investigate the prevalence of symptomatic and silent biliary tract disease, in free living urban population in Kashmir. A randomly drawn sample of 1695 subjects aged 15 years or above was interviewed by a questionnaire. Twenty six had previous cholecystectomies, all for gall stones. Ultrasonography was carried out on 1104 (65.1%). The responder rates for ultrasonography in men (64.3%) and in women (66.0%) were similar (p greater than 0.2). Gall stones were detected in 49 adults. Three of these had previous biliary symptoms. The prevalence of gall stones in adult population was 6.12% (men 3.07% and women 9.6%). The prevalence of gall stones rose with age in both sexes to a peak in the sixth decade prevalence of gall stones was significantly higher in age adjusted parous women than in nullipara. There was no correlation with obesity, diet, or socioeconomic status. Five subjects had sonographic appearances of the worm Ascaris lumbricodis in the bile ducts: and had previous biliary symptoms.